role of the uridine diphosphate …

The Pennsylvania State University

The Graduate School

College of Medicine

ROLE OF THE URIDINE DIPHOSPHATE GLUCURONOSYLTRANSFERASE

2A FAMILY IN TOBACCO CARCINOGEN METABOLISM

A Dissertation in

Pharmacology

by

Ryan T. Bushey

©2012 Ryan T. Bushey

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

December 2012

ii

The dissertation of Ryan T. Bushey was reviewed and approved* by the following:

Philip Lazarus

Professor of Pharmacology and Public Health Sciences

Dissertation Advisor

Chair of Committee

Shantu Amin

Professor of Pharmacology

Melvin Billingsley

Professor of Pharmacology

John Ellis

Professor of Psychiatry and Pharmacology

Thomas Spratt

Associate Professor of Biochemistry and Molecular Biology

Kent Vrana

Elliot S. Vessel Professor and Chair of Pharmacology

Head of the Department of Pharmacology

* Signatures are on file in the Graduate School

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ABSTRACT

Tobacco use is considered the most preventable cause of death in the world today,

with tobacco-related cancers causing millions of deaths annually. Environmental and

genetic factors are known to impact cancer susceptibility, and using environmental

exposure and/or genetic information to identify a subset of individuals at high-risk for

developing tobacco-related cancers has the potential to save many lives. Inter-individual

differences in enzymes that activate and metabolize carcinogens are thought to influence

cancer risk. UDP-glucuronosyltransferases (UGTs) are phase II detoxifying enzymes that

play a critical role in the metabolism of endogenous and exogenous compounds,

including multiple classes of tobacco carcinogens. The effects of coding and non-coding

SNPs on UGT activity have been analyzed for many UGT isoforms, and multiple UGT

variants have been determined to be significantly associated with cancer risk.

The entire UGT2A family has been neglected in prior research studies, with

UGT2A tissue expression and enzyme activities relatively unknown. With recent reports

suggesting UGT2A1 expression in the lung and trachea and UGT2A1 glucuronidation

activity against simple polycyclic aromatic hydrocarbon (PAH) substrates, the overall

hypothesis of this research project was that UGT2A1 detoxifies PAH carcinogens in

target organs for tobacco carcinogenesis. Due to the sequence homology between all

UGT2A enzymes, and with little information reported on UGT2A2 or UGT2A3, we also

hypothesized that UGT2A2 and UGT2A3 enzymes are involved in extra-hepatic tobacco

carcinogen metabolism.

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An initial set of experiments was completed to characterize the role of UGT2A1

in tobacco carcinogen metabolism. Quantitative real-time PCR showed highest relative

UGT2A1 expression in the lung, followed by trachea > tonsil > larynx > colon.

Significant UGT2A1 glucuronidation activity was observed against a variety of PAHs,

including the proximate carcinogens benzo(a)pyrene(B(a)P)-7,8-diol, dibenzo(a,l)pyrene-

11,12-diol, and 5-methylchrysene-1,2-diol. No UGT2A1 glucuronidation activity was

observed against additional classes of tobacco carcinogens, including tobacco specific

nitrosamines or heterocyclic amines. In vitro experiments suggested that UGT2A1 over-

expression in a HEK293 cell system prevents B(a)P-mediated cytotoxicity and covalent

binding. These data suggested that UGT2A1 is an important detoxification enzyme in the

metabolism of PAHs within aerodigestive and respiratory tract tissues.

The next set of experiments focused on characterizing two prevalent UGT2A1

non-synonymous coding SNPs, the UGT2A175Lys

and UGT2A1308Arg

variants. The

UGT2A175Arg

variant exhibited a significant (p<0.05) ~25% decrease in glucuronidation

activity (Vmax/KM) against all PAH substrates examined compared to wild-type

UGT2A175Lys

activity, while no detectable glucuronidation activity was observed for the

UGT2A1308Arg

variant against all substrates examined. Results from a lung cancer case-

control study showed the inactive UGT2A1308Arg

variant to be significantly associated

with lung non-small cell carcinoma (p=0.04) and lung squamous cell carcinoma risk

(p=0.02). A significant decrease (p<0.001) in wild-type UGT2A1 activity against

multiple PAH substrates was observed following UGT2A1308Arg

co-expression with wild-

type UGT2A1 at approximately a 1:1 ratio. Co-immunoprecipitation experiments showed

dimerization between wild-type UGT2A1 and UGT2A1308Arg

. The decrease in wild-type

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UGT2A1 activity following UGT2A1308Arg

co-expression is a novel regulatory

mechanism for UGT2A1 and may have implications on cancer risk.

In the initial cloning of UGT2A1 from human lung RNA a novel UGT2A1 exon 3

deletion splice variant was identified (termed UGT2A1Δexon3), and subsequent studies

were completed to characterize the expression and function UGT2A1Δexon3 and the

corresponding UGT2A1_i2 protein. Through qualitative PCR, UGT2A1Δexon3 was

shown to be expressed in various tissues including lung, trachea, larynx, tonsil, and

colon. The ratio of UGT2A1Δexon3:wild-type UGT2A1 expression was highest in colon

(0.79 ± 0.08) and lung (0.42 ± 0.12). An antibody specific to UGT2A1 revealed the ratio

of UGT2A1_i2:UGT2A1_i1 protein expression in lung and colon homogenates to be 0.5-

0.9. UGT2A1_i2 exhibited no glucuronidation activity against a variety of substrates,

including PAHs such as 1-hydroxy-pyrene and B(a)P-7,8-diol. An inducible in vitro

system was created to determine the effect of UGT2A1_i2 co-expression on wild-type

UGT2A1_i1 activity. Increasing UGT2A1_i2 levels resulted in a significant (p<0.01)

decrease in UGT2A1_i1 activity (Vmax) against 1-hydroxy-pyrene, 3-OH-B(a)P and

B(a)P-7,8-diol. Co-IP experiments suggested the formation of UGT2A1_i1 and

UGT2A1_i2 hetero-oligomeric complexes and UGT2A1_i1 homo-oligomeric complexes.

These data suggested that a novel exon 3 deletion UGT2A1 splice variant specifically

regulates UGT2A1-mediated glucuronidation activity via protein-protein interactions,

and that expression of this variant could impact the local detoxification of carcinogens.

The final set of experiments described in this dissertation focused on determining

the functions of UGT2A2 and UGT2A3 in the local metabolism of tobacco carcinogens.

UGT2A2 was determined to be expressed in the trachea and larynx, and a novel splice

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variant of UGT2A2 lacking exon 3 (UGT2A2Δexon3) was identified. UGT2A3 was

determined to be expressed a variety of extra-hepatic tissues, with UGT2A3 well

expressed in the colon, lung, tonsil, trachea, and larynx. Cell homogenates prepared from

HEK293 cells over-expressing UGT2A2Δexon3 had no detectable glucuronidation

activity against all substrates examined. Cell homogenates prepared from HEK293 cells

over-expressing UGT2A2 and UGT2A3 showed activity against simple PAHs. Both

UGT2A2 and UGT2A3 were determined to have no detectable activity against complex

PAH proximate carcinogens, tobacco specific nitrosamines, or heterocyclic amines. Data

presented here suggested UGT2A2 and UGT2A3 are both expressed in various

aerodigestive and respiratory tract tissues; however, these enzymes lack enzyme activity

against PAH proximate carcinogens.

This dissertation has laid the groundwork for understanding the physiological role

of UGT2A enzymes in the local detoxification of PAH tobacco carcinogens. Results

presented in this dissertation suggest that UGT2A1 is a major metabolizer of PAH

carcinogens in the lung and other target tissues for tobacco carcinogenesis. UGT2A1

coding SNPs and a novel UGT2A1Δexon3 splice variant were characterized for the first

time in this study. Results presented here suggest that the UGT2A1308Arg

variant is

associated with increased lung cancer risk, and we propose that inter-individual

variability in UGT2A1Δexon3 expression may also impact cancer risk. Although

additional work is needed to confirm these findings, results presented in this dissertation

suggest that UGT2A1 variants negatively regulate wild-type UGT2A1 activity and may

play a role in tobacco-related cancer susceptibility.

vii

TABLE OF CONTENTS

LIST OF FIGURES ......................................................................................................... ix LIST OF TABLES ........................................................................................................... xi LIST OF ABBREVIATIONS .......................................................................................... xii ACKNOWLEDGEMENTS ............................................................................................. xv

Chapter 1 REVIEW OF THE LITERATURE ......................................................................... 1

1.1 General Background and Significance; Pharmacogenetics and Cancer ..................... 2 1.1.1 Cancer Epidemiology ...................................................................................... 2 1.1.2 Cancer at the Molecular Level ........................................................................ 3

1.2 Introduction to Tobacco Related Cancers .................................................................. 4 1.2.1 Epidemiology of Tobacco Related Cancers .................................................... 5 1.2.2 Constituents of Tobacco Smoke ...................................................................... 6 1.2.3 PAH and TSNA Carcinogenicity in Animal Models and Humans ................. 7 1.2.4 Activation and Detoxification of Tobacco Carcinogens ................................. 8

1.3 Genetics of Lung Cancer Susceptibility ..................................................................... 12 1.3.1 Candidate Gene Studies and Lung Cancer Susceptibility ............................... 13 1.3.2 GWAS and Lung Cancer Susceptibility .......................................................... 14

1.4 UDP-Glucuronosyltransferases .................................................................................. 15 1.4.1 UGT Family Organization and Nomenclature ................................................ 16 1.4.2 UGT Structure and Localization ..................................................................... 20 1.4.3 UGT Tissue Expression .................................................................................. 22 1.4.4 UGT Function ................................................................................................. 24

1.5 UGT Pharmacogenetics ............................................................................................. 27 1.5.1 UGT Pharmacogenetics in the Metabolism of Endogenous Compounds ....... 28 1.5.2 UGT Pharmacogenetics in the Metabolism of Therapeutic Drugs.................. 30 1.5.3 UGT Pharmacogenetics in the Metabolism of Environmental Carcinogens ... 32

1.6 Regulation of UGT Activity....................................................................................... 35 1.6.1 Transcriptional Regulation of UGTs ............................................................... 35 1.6.2 Alternative Splicing of UGTs ......................................................................... 36 1.6.3 UGT Oligomerization ..................................................................................... 38

1.7 Summary of UGT2A Expression and Activity .......................................................... 40 1.7.1 UGT2A1 Expression and Activity .................................................................. 40 1.7.2 UGT2A2 and UGT2A3 Expression and Activity ........................................... 42

1.8 Aims and Hypotheses ................................................................................................. 42

Chapter 2 CHARACTERIZATION OF UGT2A1 EXPRESSION AND THE

POTENTIAL ROLE OF UGT2A1 IN TOBACCO CARCINOGEN METABOLISM .. 45

2.1 Abstract ...................................................................................................................... 46 2.2 Introduction ................................................................................................................ 47 2.3 Methods ...................................................................................................................... 48 2.4 Results ........................................................................................................................ 57 2.5 Discussion .................................................................................................................. 65

viii

Chapter 3 CHARACTERIZATION OF UGT2A1 SNP VARIANTS AND THE

ASSOACIATION OF UGT2A1308ARG

WITH LUNG CANCER RISK ......................... 68


Chapter 4 IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF A

NOVEL UGT2A1 EXON 3 DELETION SPLICE VARIANT ....................................... 105


Chapter 5 CHARACTERIZATION OF THE POTENTIAL ROLES OF UGT2A2 AND

UGT2A3 IN TOBACCO CARCINOGEN METABOLISM ........................................... 146


Chapter 6 FUTURE DIRECTIONS AND FINAL CONSIDERATIONS ............................... 177

6.1 Conclusions ................................................................................................................ 178 6.2 Future Directions ........................................................................................................ 181 6.3 Final Considerations .................................................................................................. 186

Appendix ALTERNATE SUGARS IN UGT2A METBOLISM ........................................... 189

REFERENCES ........................................................................................................................ 195

ix

LIST OF FIGURES

Figure 1.1. Schematic describing the link between smoking and lung cancer. ....................... 9

Figure 1.2. Schematic of B(a)P metabolic activation and detoxification................................. 11

Figure 1.3. Schematic of glucuronidation by UGT enzymes. .................................................. 16

Figure 1.4. Organization of the UGT1A locus. ........................................................................ 18

Figure 1.5. Organization of the UGT2 locus. .......................................................................... 19

Figure 1.6. Structure of UGTs in the ER membrane. .............................................................. 21

Figure 2.1. Qualitative characterization of UGT2A1 tissue expression. ................................. 58

Figure 2.2. Quantitative analysis of UGT2A1 expression in human tissues. .......................... 59

Figure 2.3. Western blot analysis of UGT2A1 protein expression in HEK293 over-

expressing cell line. .......................................................................................................... 60

Figure 2.4. UGT2A1 over-expressing cell line exhibits glucuronidation activity against

PAH substrates. ................................................................................................................ 62

Figure 2.5 UGT2A1 over-expression and UDGPA administration prevent B(a)P mediated

cytotoxicity and covalent binding. ................................................................................... 64

Figure 3.1. Representative Western blot showing relative UGT2A1 protein expression

levels in UGT2A1 over-expressing cell lines. ................................................................. 88

Figure 3.2. UGT2A1308Arg

_FLAG expression is induced by the ecdysone analog PonA. ....... 94

Figure 3.3. Dimerization between wild-type UGT2A1 and UGT2A1308Arg

demonstrated

by co-IP. ........................................................................................................................... 98

Figure 3.4. UGT2A1308Gly

is a conserved residue among all UGT isoforms. .......................... 100

Figure 4.1. Schematic of real-time PCR assay developed to specifically detect either

wild-type UGT2A1 or UGT2A1Δexon3. ......................................................................... 111

Figure 4.2. Determination of UGT2A1Δexon3 expression. .................................................... 123

Figure 4.3. Determination of UGT2A1_i2 expression in a HEK293 over-expressing cell

line and human tissue homogenates. ................................................................................ 125

x

Figure 4.4. UGT2A1_i2 exhibits no detectable glucuronidation activity against multiple

PAHs. ............................................................................................................................... 127

Figure 4.5. UGT2A1_i2 expression is induced by the ecdysone analog PonA. ...................... 129

Figure 4.6. Increasing UGT2A1_i2 expression negatively regulates UGT2A1_i1 activity. ... 131

Figure 4.7. Dimerization between UGT2A1_i1 and UGT2A1_i2 demonstrated by co-IP. ..... 134

Figure 4.8. Homo-dimerization of UGT2A1_i1 demonstrated by co-IP. ................................ 136

Figure 4.9. Level of UGT2A1 over-expression is relatively equal to UGT1A7,

UGT1A10, or UGT2B17 over-expression in stable co-expressed cell lines. .................. 138

Figure 4.10. Glucuronidation activity of UGT1A7, UGT1A10, or UGT2B17 against 3-

OH-B(a)P and 1-naphthol following co-expression with UGT2A1_i1 or

UGT2A1_i2. .................................................................................................................... 140

Figure 5.1. Schematic of real-time PCR assay developed to specifically detect either

wild-type UGT2A2 or UGT2A2Δexon3. ......................................................................... 154

Figure 5.2. Qualitative determination of UGT2A2 and UGT2A3 tissue expression. .............. 162

Figure 5.3. Quantitative determination of UGT2A2 and UGT2A3 expression in multiple

human tissues. .................................................................................................................. 164

Figure 5.4. UGT2A2 and UGT2A3 exhibit glucuronidation activity against simple PAH

substrates. ......................................................................................................................... 167

Figure 5.5. UGT2A1 antibody exhibits no cross-reactivity against UGT2A2 or UGT2A3. ... 168

Figure 5.6. Representative enzyme kinetics curves for UGT2A2_i1 and UGT2A3 activity

against PAH substrates. .................................................................................................... 169

Figure 6.1. UGT2A1 mRNA expression in matched lung and lymphocyte RNA samples. .... 184

xi

LIST OF TABLES

Table 1.1. UGT glucuronidation activity against substrate classes. ........................................ 25

Table 2.1. Enzyme kinetics summary of UGT2A1 activity against PAH substrates. .............. 63

Table 3.1. Prevalence of the UGT2A1*2 and UGT2A1*3 alleles. .......................................... 87

Table 3.2. Enzyme kinetics summary of wild-type UGT2A175Lys

and UGT2A175Arg

activity against PAHs. ...................................................................................................... 89

Table 3.3. Demographics and lung cancer histology of 391 lung cancer cases and 624

controls. ............................................................................................................................ 91

Table 3.4. Distribution of UGT2A1*3 genotypes in lung cancer cases and controls. ............. 92

Table 3.5. Enzyme kinetics summary of wild-type UGT2A1 and wild-type

UGT2A1/UGT2A1308Arg

activity against PAHs. .............................................................. 96

Table 4.1. Kinetic analysis of the effect of UGT2A1_i2 co-expression on UGT2A1_i1

activity against PAH substrates. ....................................................................................... 132

Table 4.2. Glucuronidation activity of homogenates from cell lines co-expressing

UGT2A1_i1 or UGT2A1_i2 with UGT1A7, UGT1A10, or UGT2B17. ......................... 141

Table 5.1. Enzyme kinetics summary of UGT2A2_i1 and UGT2A3 activities against

PAH substrates. ................................................................................................................ 171

xii

LIST OF ABBREVIATIONS

4-MU 4-methylumbelliferone

ABI Applied Biosystems Inc.

AhR aryl hydrocarbon receptor

ATP adenosine triphosphate

BAL bronchoalveolar lavage

B(a)P benzo(a)pyrene

BPDE benzo(a)pyrene-7,8-diol-9,10-epoxide

CNS central nervous system

Co-IP co-immunoprecipitation

CYP cytochrome P450

DMEM Dulbecco’s modified eagle medium

DNA deoxyribonucleic acid

DPBS Dulbecco’s phosphate buffered saline

ER endoplasmic reticulum

FBS fetal bovine serum

G418 Geneticin

GST glutathione S-transferase

GWAS genome wide association studies

HCA heterocyclic amine

HEK human embryonic kidney

HPLC high-pressure liquid chromatography

xiii

HLM human liver microsome

HNF hepatocyte nuclear factor

HRP horseradish peroxidase

MAF minor allele frequency

mRNA messenger ribonucleic acid

NAB N’-nitrosoanabasine

NAT N’-nitrosoanatabine

NNAL 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol

NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

NNN N’-nitrosonornicotine

Nrf2 nuclear factor erythroid 2-related factor 2

NSCC non-small cell carcinoma

NST nucleotide sugar transporter

OH hydroxy

PAH polycyclic aromatic hydrocarbon

PCR polymerase chain reaction

PhIP 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

PonA ponasterone A

RNA ribonucleic acid

RPLPO human large ribosomal protein

RT reverse-transcription

SD standard deviation

SDM site-directed mutagenesis

xiv

SDS sequence detection system

SEER surveillance epidemiology and end results

SNP single nucleotide polymorphism

SULT sulfotransferases

TSNA tobacco specific nitrosamine

UDP uridine diphosphate

UDPGA UDP-glucuronic acid

UGT UDP-glucuronosyltransferase

UGT2A1Δexon3 UGT2A1 exon 3 deletion splice variant

UGT2A2Δexon3 UGT2A2 exon 3 deletion splice variant

UGT2A1_i1 wild-type UGT2A1 protein

UGT2A1_i2 UGT2A1 exon 3 deletion splice variant protein

UGT2A2_i1 wild-type UGT2A2 protein

UGT2A2_i2 UGT2A2 exon 3 deletion splice variant protein

UPLC ultra-pressure liquid chromatography

U.S. United States

XRE xenobiotic response element

xv

ACKNOWLEDGEMENTS

I would like to thank Dr. Philip Lazarus for his support and mentorship

throughout my time as a graduate student. I have improved as a scientist under the

guidance of Dr. Lazarus, and he has given me the experience to be successful as I begin

my post-graduate work. I would also like to thank my committee members, Dr. Amin,

Dr. Billingsley, Dr. Ellis, and Dr. Spratt for giving me helpful advice on my research

project during my time at Penn State. I would like to thank the past and current members

of the Lazarus lab for all their assistance. Finally, I could not have done this without my

family and friends, especially my wife Chrissie. Her support has been invaluable

throughout my entire time in graduate school.

Chapter 1

REVIEW OF THE LITERATURE

2

1.1 General Background and Significance; Pharmacogenetics and Cancer

Cancer is a complex disease characterized by uncontrolled cell proliferation due

to disruptions in normal cell processes. Inter-individual differences in genetic and

environmental factors play a role in cancer initiation, progression, and treatment. While

environmental factors can be controlled, inherited genetic factors are constant. The

identification of genetic alterations that predispose individuals to increased cancer risk, or

a poor response to cancer treatment, is a major focus of current research. The field of

pharmacogenetics attempts to incorporate information on inherited genetic variability to

predict disease risk and treatment response. The field of genomics, including the

complete sequencing of the human genome, has increased the number of genetic factors

determined to be associated with cancer risk, drug response, and drug toxicity. In the

future, progress in our understanding of pharmacogenetics and advances in genomic

sequencing will combine to create a more personalized approach to fight cancer; this

includes personalizing treatment regimens based on the genetic profiles of patients and

identifying individuals at an increased cancer risk based on their genetic profiles and

environmental exposure histories.

1.1.1 Cancer Epidemiology

Cancer is a complex disease that impacts a large proportion of the population

worldwide; approximately 1 in 4 deaths in the United States (U.S.) each year are

attributed to cancer [1]. The Surveillance, Epidemiology, and End Results (SEER)

3

program of the National Cancer Institute estimates that in 2011 there were 1,596,670 new

cases of cancer and 571,950 deaths from cancer in the U.S. alone. Cancers of the prostate,

lung and bronchus, and colorectum were predicted by SEER to account for 52% of all

newly diagnosed cancers in men in the U.S. in 2011, while cancers of the breast, lung and

bronchus, and colorectum were predicted by SEER to account for 53% of newly

diagnosed cancers in women in the U.S. in 2011. Cancers of the lung and bronchus,

prostate, colorectum, and breast were predicted to cause half of total cancer deaths among

men and women in the U.S. in 2011 [1].

1.1.2 Cancer at the Molecular Level

Cancer is a multi-step process characterized by uncontrolled cellular proliferation

due to multiple genetic mutations. The process of carcinogenesis is generally divided into

three distinct stages; initiation, promotion, and progression [2]. Initiation involves the

interaction of a mutagenic agent with deoxyribonucleic acid (DNA), causing an

irreversible change to a single cell [3]. Initiating agents must evade multiple defense

mechanisms to cause DNA damage, including enzymes that metabolize mutagenic agents

and DNA repair enzymes that recognize and correct cellular DNA damage. Promotion is

the next step in carcinogenesis; in this reversible stage an agent supports cellular growth

and/or inhibits apoptosis of initiated cells [2]. Following initiation and promotion, cells

undergo additional changes during the progression state and evolve into malignant cells

with irreversible genetic instability [3]. At the molecular level carcinogenesis in humans

has been determined to include mutations in two classes of genes; mutations in proto-

4

oncogenes cause dominant gain-of-function genetic alterations that lead to positive

proliferative growth signals, while mutations in tumor suppressor genes cause loss-of-

function genetic alterations that impede the ability of the cell to stop cellular division [4].

Cancer is thought to be caused by multiple events, with genetic mutations

initiated by external environmental carcinogens, endogenous genotoxic products, or

inherited in the germline [5]. Knudson’s two-hit hypothesis was developed to explain the

occurrence the hereditary cancer retinoblastoma. Retinoblastoma occurs due to mutations

in both alleles of the RB1 gene; the first “hit” is a hereditary germline mutation in the

RB1 tumor suppressor gene, while the second “hit” is a somatic mutation in the second

copy of RB1 caused by a genetic insult [6]. In more general terms, the two-hit hypothesis

suggests that both alleles of a tumor suppressor gene must be mutated for tumorigenesis

to occur [5, 7]. Most cancers are thought to require multiple genetic changes in proto-

oncogenes and tumor suppressor genes to cause a malignant phenotype; for example, the

adenoma to carcinoma progression in colorectal cancer has been hypothesized to involve

at least five mutational events that affect multiple genes critical for normal cell processes

[5, 8] Mutational alterations in tumor cells range from point mutations to chromosomal

deletions [9].

1.2 Introduction to Tobacco Related Cancers

There is a well-established link between tobacco use and a variety of health

problems, yet it is estimated that approximately 22% of men and 17% of women in the

U.S. smoke cigarettes. In addition to smoking, approximately 7% of men in the U.S. use

5

smokeless tobacco daily [10]. Tobacco use has been estimated to result in $75 billion in

medical costs and an estimated $82 billion in lost productivity annually [11]. Tobacco is

responsible for approximately 30% of all cancer deaths annually, making tobacco use the

most preventable cause of death in our society. Worldwide, it is estimated that roughly

20% of the world’s population are current smokers. In addition to negatively impacting

cardiovascular health, tobacco use also significantly increases the risk for cancer at

multiple sites. Although lung cancer is generally thought of as the major cancer site

associated with tobacco use and will be a focus of this dissertation, tobacco users are also

at an increased risk to develop additional cancers of the mouth, larynx, esophagus, tonsil,

trachea, stomach, pancreas, and colon [10]. In addition to the negative health effects from

first-hand smoke, cancer and heart disease from second-hand smoke are estimated to

cause tens of thousands of deaths annually in the U.S. alone [10].

1.2.1 Epidemiology of Tobacco Related Cancers

Lung cancer is the most prevalent and deadly cancer associated with tobacco

use; it is estimated that lung cancer will cause 29% of cancer deaths in men and 26% of

cancer deaths in women in 2012 [10]. Lung cancer causes more deaths annually than

breast, prostate, and colorectal cancers combined. Although methods for detecting and

treating lung cancer have improved, the five year survival rate for all stages of lung

cancer combined is only 16% [10]. There is a prominent link between lung cancer and

tobacco use, with greater than 85% of lung cancer deaths attributed to cigarette smoking

[12]. It is predicted that only 10-15% of chronic smokers will develop lung cancer over

6

their lifetime [13], suggesting that environmental and/or genetic factors play a role in

lung cancer risk. In addition to lung cancer, other cancers of the upper aerodigestive and

respiratory tract are linked to tobacco use. Tracheal cancer is rare compared to lung

cancer, but estimates suggest that greater than 80% of these cancers occur in current or

former smokers. Similar to lung cancer, tracheal cancer has a high mortality rate, with the

five year survival rate estimated to be only 10-20% [10, 14]. Oral cavity, laryngeal, and

esophageal cancers are also caused by tobacco use, with greater than 75% of these

cancers estimated to be caused by smoking and smokeless tobacco [15]. Tonsil cancer is

heavily linked to tobacco use and the Human Papillomavirus [16]. Long term smoking

has also been determined to increase the risk of developing colorectal and stomach

cancers [10]. Additional risk factors, including diet and alcohol, are known to increase

cancer risk at many sites; for example, tobacco and alcohol use are thought to have a

synergistic and multiplicative effect in the formation of many aerodigestive and

respiratory tract cancers [17, 18].

1.2.2 Constituents of Tobacco Smoke

Cigarette smoke is estimated to contain at least 60 known carcinogens and

approximately 4000 total chemical agents. Although not considered to be carcinogenic

itself, nicotine is the agent in cigarettes that causes addiction [19]. There are 8 separate

classes of carcinogens in cigarette smoke, with polycyclic aromatic hydrocarbons (PAHs)

and tobacco-specific nitrosamines (TSNAs) the two most studied classes of tobacco

carcinogens. TSNAs are formed during the tobacco curing process, while PAHs are

7

present at low levels in unburned cigarettes and generated at much higher levels during

cigarette combustion [20]. In addition to carcinogens, cigarette smoke also contains

agents that aide in tumor promotion [21].

1.2.3 PAH and TSNA Carcinogenicity in Animal Models and Humans

The carcinogenicity of the prototypical PAH, benzo(a)pyrene (B(a)P), has been

analyzed extensively through animal induction studies. B(a)P, administered locally or by

inhalation, has been shown to cause tumors of the lung, larynx, and esophagus in rodent

models [19, 22, 23]. When given systemically through diet or intraperitoneal injection,

B(a)P has been shown to cause tumors of the lung in mice [19, 24]. Though studied less

frequently than B(a)P, additional PAHs, including 5-methylchrysene and

dibenzo(a,l)pyrene, have also been shown to be potent carcinogens in various animal

models [25-27]. Studies analyzing tumor formation in animals and humans following

PAH exposure have suggested that PAHs primarily cause squamous cell carcinomas of

the lung and aerodigestive tract [21, 28-30]. PAH DNA adducts are found in lung tumors

of current and former smokers, and PAH DNA adducts in the tumor suppressor gene p53

are thought be a major cause of tumor initiation [20].

From experiments with rodent models there is also extensive evidence that the

TSNA 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung

carcinogen. NNK has the ability to induce lung tumors following systemic administration

in mice, rats, and hamsters [19]. NNK has been shown to induce lung tumors in mice,

regardless of the route of administration, using a dose of NNK equivalent to the amount

8

that a two pack a day smoker would be exposed to over a lifetime of smoking [31]. In

addition to NNK, the TSNA metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol

(NNAL) has been shown to cause lung adenocarcinoma in rodent models [31, 32]. The

TSNA N’-nitrosonornicotine (NNN), derived from nicotine itself, has also been shown to

be carcinogenic in various rodent models [31]. TSNAs, including NNK, have been

shown to predominantly cause lung adenocarcinoma tumors in animal studies [31, 32].

Increased levels of TSNAs in modern filtered cigarettes manufactured today are thought

to cause an increased percentage of lung adenocarcinomas in smokers, supporting the

notion that TSNAs primarily cause adenocarcinoma tumors [21, 33].

1.2.4 Activation and Detoxification of Tobacco Carcinogens

Tobacco carcinogens require metabolic activation to form DNA adducts and

cause genetic mutations, and the balance between carcinogen activation and

detoxification is hypothesized to impact cancer risk [19]. Figure 1.1 summarizes the link

between smoking and lung cancer. Though not carcinogenic itself, nicotine is the

addictive agent in cigarettes that causes people to smoke continuously. In addition to

nicotine, a mixture of PAHs, TSNAs, tumor promoters, and additional carcinogens are

also found in cigarette smoke. Once in the body, carcinogens take two divergent paths;

metabolic detoxification causes carcinogens to be excreted from the body, while

metabolic activation causes the formation of DNA adducts. Persistent DNA adduct

formation causes mutations in critical cell-cycle control genes, and this leads to cancer

[19]. This dissertation will focus on metabolic detoxification of tobacco carcinogens by a

9

specific class of enzymes, the uridine diphosphate (UDP)-glucuronosyltransferases

(UGTs).

Figure 1.1. Schematic describing the link between smoking and lung cancer. Adapted from

Hecht’s review on lung cancer carcinogenesis [19], this schematic shows the progression from

smoking to cancer. The focus of this dissertation research will be on detoxification of tobacco-

specific carcinogens.

Metabolic activation of tobacco carcinogens predominantly involves the phase I

cytochrome p450 (CYP) enzymes; CYPs form oxidized intermediates that covalently

bind to macromolecules and form DNA adducts. Many tobacco carcinogens, including

PAHs and TSNAs, require phase I bioactivation for DNA adduct formation to occur.

Although CYP enzymes are considered the major phase I metabolizing enzymes in

tobacco carcinogen metabolism, additional enzymes such as lipooxygenases,

cyclooxygenases, myeloperoxidases, and monoamine oxidases are also thought to play a

role in carcinogen activation. Detoxification of tobacco carcinogens occurs through phase

II enzymes, including glutathione S-transferases (GSTs), sulfotransferases (SULTS), and

UGTs [19]. Phase II carcinogen metabolism, and subsequent detoxification, often occurs

following phase I reactions. The balance between carcinogen activation and

detoxification is hypothesized to play a role in cancer risk; for example, genetic

10

polymorphisms causing either efficient phase I activation or inefficient phase II

detoxification of tobacco carcinogens would theoretically increase cancer risk.

Multiple phase I and phase II enzymes are critical in the metabolic pathway of

tobacco carcinogens. The metabolism of the PAH B(a)P to the ultimate carcinogen

B(a)P-7,8-diol-9,10-epoxide (BPDE) will be described below in detail; however, the

schematic of B(a)P metabolism described in Figure 1.2 is valid for all PAHs. B(a)P is

metabolized primarily by CYP1A1, but also CYP1A2 and CYP1B1, to form the epoxide

metabolite B(a)P-7,8-oxide in the first step of metabolic activation [19, 34]. The enzyme

epoxide hydrolase converts the B(a)P epoxide to B(a)P-7,8-diol, considered to be a

proximate carcinogenic form of B(a)P. B(a)P-7,8-diol is metabolized further by

CYP1A1, CYP2C9, and CYP3A4 to create the BPDE ultimate carcinogen [35]. Phase II

enzymes, including GSTs, SULTS and UGTs, metabolize B(a)P intermediates at various

stages in the process to prevent the formation of DNA adducts [19].

11

Figure 1.2. Schematic of B(a)P metabolic activation and detoxification. This dissertation will

focus on UGT-mediated metabolism of PAHs, including PAH-diol and PAH-phenol substrates.

Although not the focus of this dissertation, the stereochemistry of BPDE impacts

DNA adduct formation, with the anti-7R,8S,9S,10R-BPDE metabolite shown though

rodent assays to be the most tumorigenic stereoisomer formed during B(a)P metabolism

[36]. BPDE DNA adduct formation has been studied extensively, and it is know that

BPDE forms N2-deoxyguanosine DNA adducts [37]. DNA adduct levels have been

determined to vary in individuals with similar environmental exposures to PAHs, further

supporting the notion that genetic differences in carcinogen activation and/or

detoxification may have an impact on cancer susceptibility [38]. Other PAHs, including

dibenzo(a,l)pyrene and 5-methylchrysene, are formed during cigarette combustion and

metabolized in a similar manner to B(a)P [19, 39].

12

TSNAs, including the potent lung carcinogen NNK, are activated and detoxified

by phase I and phase II enzymes in a process similar to that described for PAHs. The

majority of NNK is quickly converted to its major metabolite NNAL by carbonyl

reductases, with the conversion of NNK to NNAL a reversible process. Both NNK and

NNAL undergo α-hydroxylation by CYP enzymes, including CYP2A6, to more reactive

intermediates. Hydroxylated NNK and NNAL metabolites have the capability to form

methyl and pyridyloxobutyl DNA adducts. Detoxification of NNAL occurs through

UGT-mediated glucuronidation [19, 31]. As with PAH metabolism, it is hypothesized

that inter-individual variability in the balance between TSNA activation and

detoxification plays a role in cancer risk [19].

1.3 Genetics of Lung Cancer Susceptibility

There is strong evidence that genetic components play a role in the development

of many cancers. Familial clustering studies have suggested that the risk of lung cancer is

1.9-fold greater among first-degree relatives, similar to the increased familiar risk

observed for breast, colon, and prostate cancers [40]. Increased cancer susceptibility

likely involves several genetic variants acting simultaneously, and the association of

genetic variants with cancer risk has most often been tested on a candidate gene basis

[41]. As an alternative to analyzing a single candidate gene, genome-wide association

studies (GWAS) have recently been completed to determine the link between genetic

variants and cancer susceptibility. GWAS require a large sample size and have limited

coverage of rare genetic variants; however, these studies are able to determine the

13

association of common variants with disease risk by analyzing up to a million single

nucleotide polymorphisms (SNPs) in a single study [42]. GWAS have been completed

for a variety of cancer sites but, due to the focus of this dissertation, GWAS analyzing

lung cancer risk will be examined in detail. In addition to the role of genetic variants in

cancer risk, other genetic factors including copy number variation and

insertions/deletions likely impact overall cancer risk.

1.3.1 Candidate Gene Studies and Lung Cancer Susceptibility

Many candidate genes have been determined to be significantly associated with

alterations in lung cancer risk, yet many of these results have not been verified in larger

studies. Two large meta-analyses have analyzed the association of two candidate genes

with lung cancer risk and will be highlighted [41]. The association of the GSTM1 null

genotype with lung cancer susceptibility has been analyzed extensively. A deletion in this

enzyme causes decreased PAH detoxification [43], and a meta-analysis of 43 published

case-control studies found that the homozygotes for the GSTM1 deletion have a slightly

increased risk of developing lung cancer [44]. The same GSTM1 null genotype has also

been determined to be associated with increased risk of bladder cancer [41]. A I157T

polymorphism in a cell cycle control gene CHEK2 has been determined to be associated

with a decreased risk of lung cancer and laryngeal cancer in multi-center case-control

studies [45, 46]. Conversely, the I157T variant of CHEK2 has been found to increase the

risk of developing cancers of the breast, colon, and prostate [47, 48]. Currently the

mechanism by which the CHEK2 I157T variant decreases lung cancer risk is still

14

unknown. Many additional candidate genes have been determined to be associated with

tobacco-related cancer risk in preliminary studies, and larger studies are needed to verify

these findings in additional case-control populations.

1.3.2 GWAS and Lung Cancer Susceptibility

GWAS analyzing lung cancer risk have suggested that multiple chromosomal

regions are associated with altered susceptibility. Three initial studies, using different

lung cancer case and control populations, all determined an inherited susceptibility region

on chromosome 15q25 [49-51]. This region includes three genes encoding nicotinic

acetylcholine receptors that are expressed in neuronal tissues, and the prevailing

hypothesis is that variants in these genes play a role in nicotine addiction and carcinogen

exposure [41]. An acetylcholine receptor gene variant at 15q25 was determined to be

modestly associated with upper aerodigestive tract cancers of the oral cavity, oropharynx,

hypopharynx, larynx, and esophagus, as well as smoking behavior [52]. A region at

chromosome 6p21 was also found to be associated with increased lung cancer

susceptibility [50, 53], and this region includes genes that are involved in apoptosis and

DNA mismatch repair [53]. Multiple studies have also identified the chromosome 5p15

region to also be associated with lung cancer; this region contains the TERT gene,

essential for telomerase activity and telomere maintenance, as well as the CLPTM1L

gene whose function is currently unknown [53, 54]. The 5p15 chromosomal region has

more recently been determined to be specifically associated with increased risk of lung

adenocarcinoma [55], and this region has also been reported to be associated with cancers

15

of the pancreas, bladder, and cervix [56, 57]. Thus far, no chromosomal regions that

include enzymes involved in phase I or phase II carcinogen metabolism have been

identified as being associated with lung cancer risk through GWAS, though a locus that

includes CYP2A6 and CYP2B6 was found to affect smoking behavior [58]. The lack of

association between cancer risk and carcinogen metabolizing enzymes may be due to

poor coverage of SNPs in phase I and phase II enzymes in previous GWAS; for example,

many functional UGT coding SNPs were not covered directly or by tagging SNPs in

previous GWAS. The overall lack of relevant data from GWAS supports the need to

identify pathways important in cancer risk through more targeted approaches.

1.4 UDP-Glucuronosyltransferases

Endogenous and exogenous compounds are typically metabolized in two separate

reactions, resulting in a chemically altered substrate that is more easily excreted in the

urine, bile, or feces. In the initial phase I metabolic reaction a functional moiety, most

commonly a hydroxyl, amine, or sulfhydryl group, is conjugated to the substrate. Phase II

reactions often involve the addition of a polar group to the functional group added during

the phase I reaction. The addition of the polar moiety during phase II metabolism

facilitates excretion by increasing the water solubility of the substrate. There are multiple

families of phase II enzymes, including N-acetyltransferases, SULTS, GSTs, and UGTs.

UGT enzymes catalyze the majority of phase II reactions and will be the focus of this

dissertation. As shown in Figure 1.3, UGT enzymes conjugate a polar glucuronic acid

functional moiety to a nucleophilic substrate, making the substrate more polar and easily

16

excreted [59, 60]. Glucuronidation most frequently occurs at hydroxyl or amine groups,

although glucuronidation can also occur at thiol, carboxyl, or carbonyl moieties [59]. The

co-substrate in the glucuronidation reaction, UDP-glucuronic acid (UDPGA), is

synthesized from UDP-glucose by the enzyme UDP-dehydrogenase. UDPGA is found at

high levels in a variety of tissues, including the liver where the majority of

glucuronidation reactions occur [61].

Figure 1.3. Schematic of glucuronidation by UGT enzymes. The reaction catalyzed UGTs

involves the addition of a glucuronic acid moiety, donated from the co-substrate UDPGA, to a

nucleophilic substrate. Glucuronidation increases the polarity of the substrate and promotes

excretion in the urine, bile, and feces.

1.4.1 UGT Family Organization and Nomenclature

UGT genes are divided into four distinct families based on sequence homology.

The nucleic acid homology of the UGTs is at least 30 percent across all families and at

least 60 percent within a single family [62]. The UGT1, UGT2, and UGT3 families

metabolize various endogenous and exogenous substrates, while the UGT8 family

17

encodes the UDP-galactose:ceramide galactosyltransferase gene involved in cerebroside

and glycosphingolipid biosynthesis [63]. UGT nomenclature is complex, with the first

Arabic number representing the UGT family, a letter denoting the subfamily, and the

second Arabic number representing the specific gene within the subfamily [64].

The UGT1A family gene locus spans approximately a 200 kb region on

chromosome 2q37. Thirteen individual genes are encoded by this locus, with each

UGT1A member containing a unique promoter and exon 1 conjugated to common exons

2-5 [65]. Nine of the UGT1A genes encode active proteins (Figure 1.4), while UGT1A2,

UGT1A11, UGT1A12, and UGT1A13 are pseudogenes. The gene organization of the

UGT1A locus, with unique first exons and shared exons 2-5, allows UGT1A isoforms to

have varying substrate specificities, as the exon 1 sequence and N-terminus of each UGT

enzyme contains the substrate recognition sequence. Tissue-specific UGT1A expression

occurs due to unique promoters for each UGT1A transcript [66]. The process of

transcribing individual genes from the UGT1A locus is often referred to as alternative

splicing; however, recent evidence suggests that UGT1A genes arise from exon sharing

rather than alternative splicing [66, 67]. True alternative splicing occurs when mature or

cryptic 5’ and 3’ consensus splice sites are recognized in a regulated fashion and used in

a spliceosome catalyzed reaction following transcription; this likely does not occur with

UGT1A transcripts [59, 68]. Instead, each UGT1A transcript is produced independently

following transcription initiation due to regulatory sequences flanking each UGT1A exon

1 [59].

18

Figure 1.4. Organization of the UGT1A locus. Alternate first exons are joined to common

exons 2-5 through exon sharing. The location of each UGT1A exon 1 on chromosome 2 is

represented relative to exons 2-5. UGT1A pseudogenes are not shown and the size of the UGT1A

exons are not drawn to scale.

The UGT2B family is divided into the UGT2A and UGT2B subfamilies on

chromosome 4q13 (Figure 1.5). Seven functional UGT2B genes and five UGT2B

pseudogenes have been identified to date. Unlike the UGT1A subfamily described

earlier, UGT2B genes contain 6 exons and are independent genes [63]. The UGT2B

cluster on chromosome 4 is thought to have evolved from whole gene duplication events

[69]. UGT2A family members include UGT2A1, UGT2A2, and UGT2A3; this subfamily

of UGTs, particularly UGT2A1, is the focus of this dissertation. UGT2A1 and UGT2A2

are each comprised of six exons and exon sharing occurs between the two genes; unique

first exons of UGT2A1 or UGT2A2 are joined to common exons 2-6 to create full length

UGT2A1 or UGT2A2 transcripts [63, 70]. UGT2A3 contains six unique exons and does

not participate in exon sharing with UGT2A1 or UGT2A2 [63, 71].

19

Figure 1.5. Organization of the UGT2 locus. Each UGT2B gene and UGT2A3 consists of 6

individual exons. UGT2A1 and UGT2A2 have unique first exons and share common exons 2-6,

similar to the gene structure of the UGT1A family. UGT2B pseudogenes are not shown. UGT2A

genes are shaded grey.

The UGT3 family was recently identified on chromosome 5p13 by sequencing of

human, rat, and mouse genomes. There are only two known enzymes in the UGT3

family, UGT3A1 and UGT3A2, and each enzyme is encoded by seven exons. Instead of

using UDPGA as the preferential sugar donor, UGT3 family members use alternate co-

substrates. UGT3A1 primarily uses N-acetylglucosamine as the co-substrate in the

metabolism of bile acids and estrogens [72]. UGT3A2 uses both UDP-glucose and UDP-

xylose to glycosidate a range of substrates, including 1-hydroxy-pyrene (1-OH-pyrene),

4-methylumbelliferone (4-MU), bioflavones, and estrogens [73]. The UGT8 family

consists of a single gene, UGT8A1, encoded by five exons on chromosome 4q26 [63].

This enzyme does not take part in xenobiotic metabolism, but rather catalyzes the transfer

of galactose from UDP-galactose to ceramide in an important reaction needed for myelin

biosynthesis [74]. UGT8A1 is expressed in oligodendrocytes and Schwann cells of the

20

central nervous system (CNS) [74]. Drugs, carcinogens, and endogenous substrates are

metabolized primarily by the UGT1 and UGT2 families [62], and from this point forward

the abbreviation “UGT” will refer primarily to the UGT1 and UGT2 families unless

otherwise noted.

1.4.2 UGT Structure and Localization

UGT genes encode proteins that are typically 520-540 amino acids in length.

UGT enzymes have a conserved C-terminus region containing the UDPGA binding site,

dilysine motif, and transmembrane domain, while the more variable N-terminus of the

protein contains the substrate binding domain (Figure 1.6) [67]. UGTs are integral type I

trans-membrane proteins that reside in the endoplasmic reticulum (ER), with the

exception of UGT1A10, which likely resides in other organelles such as the nuclear

membrane [59, 75]. All UGTs, with the exception of UGT1A10, contain an N-terminal

ER retention signal peptide that is cleaved following protein insertion into the ER

membrane [67, 76]. The transmembrane domain and dilysine motif cause residency and

retention in the ER, with the dilysine motif located in the cytosol and only a few residues

from the C-terminal end of the UGT protein [77, 78]. Recent reports have suggested that

the dilysine motif alone is sufficient for ER targeting and retention, as deletion constructs

without the ER-retention signal sequence at the N-terminus are still targeted to the ER

[79, 80].

21

Figure 1.6. Structure of UGTs in the ER membrane. The functional domains are not drawn to

scale. The active site includes the more variable N-terminal substrate binding domain and the

more conserved C-terminal UDPGA binding domain, as both are presumed to be located in the

ER lumen. Figure is adapted from Nagar and Remmel’s figure detailing the hypothetical structure

of UGTs [67].

Due to the location of the UDPGA binding domain in the ER lumen, transporters

are needed to shuttle UDPGA from the cytosol, where it is synthesized, to the active site

of the enzyme. Nucleotide sugar transporters (NSTs), including the transporter

hUGTrel7, have been reported to transport UDPGA from the cytosol to the ER lumen, in

a process that is dependent on the presence of N-acetylglucosamine [81, 82]. In addition

to membrane transport proteins such as NSTs, protein-protein interactions between UGT

isoforms have been hypothesized to promote UDPGA transport into the ER lumen by

creating a channel in the ER membrane [83, 84]. Adenosine triphosphate (ATP)-

dependent multi-drug resistant proteins have been shown to transport glucuronides from

the ER lumen to the cytosol following the glucuronidation reaction [85], although it is

also hypothesized that UGT oligomerization may also promote glucuronide export from

the ER [84].

22

Although no complete crystal structure exists for any UGT enzyme, a partial

crystal structure of the C-terminus of UGT2B7 has been completed [86]. The crystal

structure of the UGT2B7 C-terminal domain supports the notion that UGTs belong in the

GT-B family of glycosyltransferases; a hallmark of GT-B enzymes is the presence of two

separate domains (N-terminus and C-terminus) that form a catalytic site at their interface

[86, 87]. The UGT2B7 C-terminus crystal structure also led to the discovery of a

conserved region, similar in sequence to the sugar binding sites of plant and bacterial

glycosyltransferases, where UDPGA is hypothesized to bind [86]. A homology model of

monomeric UGT1A1 suggests that the enzyme is composed of four general domains;

large N-terminal and C-terminal domains, small envelope helices, and a transmembrane

domain that includes the cytoplasmic tail. Of the fifteen known point mutations that lead

to an inactive UGT1A1 protein, the majority were determined using the homology model

to be in the C-terminal domain near the UDPGA binding site [88].

1.4.3 UGT Tissue Expression

The tissue specific messenger ribonucleic acid (mRNA) expression pattern of

each UGT1A and UGT2B gene has been investigated non-quantitatively through reverse-

transcription polymerase chain reaction (RT-PCR) and quantitatively through real-time

PCR studies. Glucuronidation of exogenous substrates primarily occurs in the liver, and

the majority of UGT1A and UGT2B family members have been shown to have hepatic

expression [89-91]. High relative UGT expression in the liver, compared to UGT

expression levels in other organs, also supports the importance of the liver in

23

glucuronidation [90]. There is also significant inter-individual variability in the

expression level of each UGT found in the liver [92, 93], as hepatic UGT levels were

found to vary more than 100-fold between individuals in a study of 25 human liver

samples [93]. Interestingly, mRNA levels of hepatic UGTs have been determined to be

correlated to one another in individual liver samples; for example, UGT1A1 mRNA

levels have been found to be significantly correlated with UGT1A9 and UGT2B7 mRNA

levels in multiple studies [93, 94]. Tissue-specific differences in transcription factor

expression are likely to cause differences in the tissue distribution of UGTs, and

transcriptional regulation of UGTs is an area of research that may have large implications

on human disease and therapeutic response of drugs [95]. Due to the homology between

UGT isoforms, the development of UGT-specific antibodies has been slow and there is

not yet a study completed to date which has exhaustively analyzed tissue-specific UGT

protein expression.

Although the liver is a main site of metabolism, extra-hepatic UGT expression is

important for the local detoxification of endogenous and exogenous substrates. UGT2A1

and UGT2A2, two enzymes that are the focus of this dissertation, are exclusively

expressed in extra-hepatic tissues such as the lung, trachea, and olfactory epithelium [70,

96, 97]. UGT1A7, UGT1A8, and UGT1A10 are also thought to be exclusively expressed

in extra-hepatic tissues [89, 90]. These extra-hepatic UGT1A enzymes have been shown

to be expressed in aerodigestive and respiratory tract tissues that are exposed to chemical

carcinogens, including the larynx, tonsil, trachea, esophagus, and lung [98, 99]. UGTs,

including UGT1A7, UGT1A8, and UGT1A10, are also well-expressed in the stomach

and small intestine [89, 90]. UGT1A8 and UGT1A10 expression in the small intestine is

24

thought to have an impact on drug bioavailability; for example, the drug raloxifene has

been shown to be extensively glucuronidated in the small intestine [100]. Inter-individual

differences in expression and activity of extra-hepatic UGTs would potentially cause

variability in carcinogen detoxification or drug efficacy. Similar to results reported for

the liver, inter-individual differences in UGT expression and activity were reported for 18

small intestine samples [101]. Glucuronidation is also important in the regulation of

estrogen and testosterone levels in steroid responsive tissues, and extra-hepatic UGT

expression has been reported in the prostate, breast, testes, ovaries, and uterus [89, 90].

1.4.4 UGT Function

UGTs play an important role in the metabolism of both endogenous and

exogenous substrates. Endogenous substrates metabolized by UGTs include androgens,

estrogens, bile acids, hormones, fatty acids, retinoic acids, and bilirubin [59]. Adapted

from Tukey et al. [59], Table 1.1 summarizes the activity of UGT isoforms against a

variety of substrate classes; from this table it is clear that there is significant redundancy

in UGT substrate specificity. One exception to this redundancy, highlighted in Table 1.1,

is the metabolism of the heme breakdown product bilirubin. Bilirubin is toxic and

exclusively metabolized by UGT1A1, and mutations in the UGT1A1 gene that lead to

reduced UGT1A1 activity against bilirubin have been found to cause adverse health

consequences [102]. UGTs have to been shown to have stereo-selectivity in their activity

against endogenous compounds. Estradiol metabolism by UGT2B isoforms is a good

example of this stereo-selectivity, as studies by Itaaho et al. have shown that UGT2B4

25

exclusively metabolizes epiestradiol at the 17-OH position while UGT2B17 exclusively

metabolizes β-estradiol at the 17-OH position [103].

Table 1.1. UGT glucuronidation activity against substrate classes.

Substrate Class Major UGTs Responsible for Metabolism

Bilirubin 1A1

Simple phenols 1A1, 1A3, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2A1, 2A2, 2B15, 2B17

Complex phenols 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2A1, 2A2, 2B15

Anthraquinones/flavones 1A1, 1A3, 1A8, 1A9, 2A1, 2B15

Bile acids 1A3, 2A2, 2A3, 2B4, 2B7,

Carboxylic acids 1A3, 1A9, 2A1

Primary amines 1A4, 1A6, 1A9, 2B10

Secondary amines 1A4, 2B10

Tertiary amines 1A4, 2B10

Heterocyclic amines 1A3, 1A6, 1A8, 1A9, 1A10

Opioids 1A3, 1A8, 2A1, 2B7

C18 steroids 1A1, 1A3, 1A8, 1A9, 2A1, 2A2, 2B7

C19 steroids 1A4, 2A1, 2B15, 2B17

C21 steroids 1A4, 2A1, 2B17

Table adapted from Tukey and Strassburg [59]. Activity data for UGT1A5 [104], UGT2B10

[105], UGT2A2 [70], and UGT2A3 [71] was acquired from other sources and incorporated into

the table.

In addition to eliminating endogenous substrates, UGT metabolism of exogenous

substrates is also a critical function. UGTs metabolize a vast number of carcinogens,

environmental toxins, and therapeutic drugs; 35% of all therapeutic drugs undergoing

phase II metabolism are thought to be glucuronidated [106]. In most cases,

glucuronidation causes inactivation of the substrate; however, morphine-6-glucuronide is

an example of a glucuronide metabolite that is twenty times more potent than the parent

26

drug [107, 108]. Recent work by our lab has characterized UGT metabolism of the anti-

cancer drugs tamoxifen [109, 110], exemestane [111], and suberoylanilide hydroxamic

acid (SAHA) [112] , as well as the anti-psychotics olanzapine [113] and clozapine [114].

Carcinogen metabolism is also an important function of UGT enzymes, and many

different classes of carcinogens are known UGT substrates. UGTs are known to

metabolize PAHs, TSNAs, and heterocyclic amines (HCAs), the three major classes of

carcinogens found in tobacco products [115, 116]. Recent studies by our lab have

identified UGT1A4 and UGT2B10 as the UGTs with significant glucuronidation activity

against TSNAs [117, 118], and a separate study has identified UGT1A10 as the major

enzyme active against the HCA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

(PhIP) and its major metabolite N-OH PhIP [75]. UGT expression in extra-hepatic

organs is hypothesized to provide a first-line defense from carcinogens. Recent work by

our lab has also investigated the metabolism of PAHs by extra-hepatic UGTs expressed

in aerodigestive tract tissues, including UGT1A7, UGT1A8, and UGT1A10 [98, 99,

119]. UGTs play a vital role in human health and disease, as thousands of substrates,

including a variety of carcinogens and therapeutic drugs, are metabolized by UGT

enzymes [59].

In vitro studies, in which UGTs are transiently or stably over-expressed in cell

systems, are a commonly used and valuable method to determine UGT function and

substrate specificity [120]. Although rodents and higher order species express UGTs,

they are generally not used as model systems due to significant sequence differences

between human and rodent UGT orthologs. The intra-species differences in UGT genes,

including those between humans and rodents, include significant disparities in expression

27

patterns and substrate specificities [121-123]. Nonetheless, animal models have been

valuable in determining the functional roles of UGT enzymes. Gunn rats, which have no

detectable UGT1A enzyme activity, have been used in experiments to show the

genoprotective effect of UGT expression following carcinogen administration [124, 125]

and have also been used as a model for bilirubin toxicity [126]. Recently a transgenic

mouse over-expressing the human UGT1A locus has been developed [127, 128], and it is

hoped that this animal model may yield more significant results that are applicable to

human UGT function.

1.5 UGT Pharmacogenetics

Defects in the glucuronidation of endogenous or exogenous compounds can cause

serious health consequences. SNPs in promoters of UGT genes have been shown to alter

UGT expression levels, and non-synonymous coding region SNPs have been shown to

cause significant changes in enzyme activity. UGT polymorphisms cause a wide range of

functional changes in enzyme activity; for example, UGT coding SNPs have been

determined to increase enzyme activity, cause no change in enzyme activity, modestly

decrease enzyme activity, or completely knock-out enzyme activity [106]. Due to the

redundancy in UGT function many polymorphisms cause no overt harm under normal

circumstance [59, 84]; however, a subset of UGT polymorphisms has been determined to

play a role in cancer risk and drug response. UGT polymorphisms also cause altered

levels of steroids and other endogenous compounds, and this can lead to health problems

due to a disruption of normal homeostasis.

28

1.5.1 UGT Pharmacogenetics in the Metabolism of Endogenous Compounds

The metabolism of bilirubin by UGT1A1 has been shown to be influenced by

UGT1A1 pharmacogenetics, and a variety of studies have characterized the impact of

UGT1A1 polymorphisms on bilirubin metabolism. Bilirubin, a toxic byproduct of heme

metabolism, becomes toxic to the CNS when it accumulates at high levels in the blood

[129]. UGT1A1 is the sole enzyme responsible for bilirubin metabolism, and functional

polymorphisms in UGT1A1 have been shown to cause bilirubin toxicities in the CNS that

can be fatal [102]. Hyperbilirubinemia is known to be caused by over 60 mutations, point

deletions, and insertions in the UGT1A1 gene, with genetic alterations in both the

UGT1A1 promoter and all five UGT1A1 exons [106]. In the most severe case of

hyperbilirubinemia, Crigler-Najjar syndrome type I, there is a complete knock-out of

UGT1A1 activity, causing infant death due to the accumulation of bilirubin in the CNS

[130, 131]. Crigler-Najjar type II individuals have reduced UGT1A1 activity but a less

severe phenotype [132]. Crigler-Najjar types I and II are relatively rare diseases, yet a

third more minor bilirubin related disease, Gilbert’s syndrome, is estimated to impact up

to 10% of the population [106]. Gilbert’s syndrome is caused by homozygous TATA box

polymorphisms in the UGT1A1 promoter and can lead to intermittent jaundice [133].

Wild-type individuals have six TA repeats in the UGT1A1 TATA box, and functional

studies have revealed that alleles with an increased number of TA repeats, including the

UGT1A1*28 allele with seven TA repeats, have a decreased rate of UGT1A1

transcription initiation [134, 135].

29

The role of UGT pharmacogenetics in steroid metabolism has also been analyzed,

as the development of multiple cancers in steroid responsive organs is thought to be

impacted by aberrant steroid levels. UGT1A1 is the primary UGT responsible for

estrogen metabolism [136], and increased estrogen levels are thought to stimulate the

development of breast cancer [137]. Low activity UGT1A1 genotypes, including

individuals expressing a single copy of the UGT1A1*28 allele and/or the UGT1A1*37

allele (8 TA repeats), were determined to be positively associated with estrogen receptor

negative breast cancer in premenopausal African-American women following an analysis

of 200 breast cancer cases and 200 matched controls [138]. However, in a larger case-

control experiment using Caucasian samples from the Nurses’ Health Study, no

association was observed between the UGT1A1*28 allele and breast cancer risk [139].

Variations in androgen levels have also been hypothesized to play a role in the

development of prostate cancer, and case-control studies have been completed to

determine the association of UGT polymorphisms with prostate cancer risk [106].

Androgens have been determined to be glucuronidated primarily by UGT2B15 [140]. A

small case-control study determined that homozygotes for a low activity UGT2B1585Asp

variant were at an increased risk for prostate cancer [141]; however, this result was

unable to be replicated in a larger case-control study [142]. UGT2B17 also has high

activity against androgens, and a common UGT2B17 whole gene deletion variant, caused

by a 120 kb deletion of chromosome 4, would be predicted to significantly alter androgen

glucuronidation [143, 144]. A case-control study and recently completed meta-analysis

found a significant association between homozygotes for the UGT2B17 whole gene

deletion variant and increased prostate cancer susceptibility in Caucasians [145, 146].

30

Similar to the results observed for the low-activity UT2B15 polymorphism and prostate

cancer risk, case-control studies analyzing the association between the UGT1A1*28

polymorphism and endometrial cancer have also been inconsistent [147-150]. Although

irregular steroid metabolism caused by UGT polymorphisms may impact cancer

development in tissues such as the prostate, breast, and endometrium, it is likely that

environmental and additional genetic factors also impact cancer risk.

1.5.2 UGT Pharmacogenetics in the Metabolism of Therapeutic Drugs

Glucuronidation of pharmacological agents is an important function of UGT

enzymes, as approximately a third of drug metabolism by phase II enzymes occurs by

UGTs [151]. Polymorphisms in UGT genes can cause abnormalities in drug metabolism,

leading to drug induced toxicities or reduced drug efficacy. Individuals with Gilbert’s

syndrome have decreased glucuronidation rates against many therapeutic drugs, including

acetaminophen, tolbutamide, and lorazepam [106, 152, 153]. The impact of UGT

pharmacogenetics on drug metabolism is best exemplified by the glucuronidation of the

chemotherapeutic agent irinotecan. Irinotecan is a topoisomeraise I inhibitor used in the

treatment of colon cancer, and the active intermediate of irinotecan, SN-38, is

metabolized primarily by UGT1A1 [154, 155]. In addition to colon cancer, irinotecan is

also used in the treatment of esophageal, non-small cell lung, and breast cancers [156].

Individuals homozygous for the UGT1A1*28 allele have been determined to be at an

increased risk for irinotecan toxicities, including diarrhea and the much more severe

toxicity of neutropenia [157]. The Federal Drug Administration has changed the package

31

insert for irinotecan to include the UGT1A1*28 allele as a risk factor for developing

severe neutropenia, based on four studies showing that UGT2A1*28 homozygotes have

an increased risk of irinotecan toxicity [156]. A diagnostic test to assess UGT1A1*28

genotype was approved in 2005 [158], and individuals that are candidates for irinotecan

therapy are now routinely genotyped for UGT1A1 prior to treatment.

In addition to the impact of UGT polymorphisms on irinotecan metabolism, UGT

polymorphisms have also been reported to alter the pharmacokinetics of additional

therapeutic agents. SAHA is a histone deacetylase inhibitor used to treat cutaneous T-cell

lymphoma, and multiple UGTs, including UGT1A8 and UGT2B17, have in vitro activity

against SAHA [112]. Human liver microsomes (HLMs) from individuals homozygous

for the UGT2B17 whole gene deletion genotype were determined to have a 45% lower

glucuronidation activity and a 75% higher KM against SAHA compared to HLMs from

wild-type UGT2B17 individuals [112]. SAHA has significant toxicities including

diarrhea, fatigue, nausea, and anorexia [159], and the significance of the UGT2B17

deletion genotype on SAHA metabolism must be analyzed further in a clinical setting.

UGT2B17 is also highly active against metabolites of the aromatase inhibitor

exemestane, and the UGT2B17 whole gene deletion variant may play a role in

exemestane toxicity and response [111, 160]. Active metabolites of the breast cancer drug

tamoxifen are glucuronidated by UGT2B7 in the liver and by UGT1A8 in extra-hepatic

tissues, and the UGT2B7268Tyr

polymorphism has been determined to significantly

decrease tamoxifen glucuronidation in HLMs [161]. This polymorphism, along with

polymorphisms in UGT1A8, may play a role in the inter-individual variability in toxicity

and response to tamoxifen treatment. UGT polymorphisms have also been determined to

32

alter the pharmacokinetics of second generation antipsychotics; UGT1A4 and UGT2B10

variants were determined to significantly alter the in vitro glucuronidation of olanzapine

[113], while UGT1A1 and UGT1A4 variants were determined to significantly alter the in

vitro glucuronidation of clozapine [114]. The pharmacokinetics of additional drugs,

including aspirin and morphine, are also thought to be impacted by UGT

pharmacogenetics [162, 163]. With the exception of UGT1A1*28 and irinotecan, in

many cases the changes in glucuronidation caused by UGT variants must still be verified

in large clinical experiments to validate in vitro results [164].

1.5.3 UGT Pharmacogenetics in the Metabolism of Environmental Carcinogens

UGTs provide protection from environmental carcinogens, and a variety of

studies have analyzed the association of UGT polymorphisms with cancer risk. UGT

isoforms have been shown to metabolize carcinogens in tobacco smoke, including PAHs

and TSNAs [19, 115], and multiple studies have analyzed the association of UGT

variants with tobacco-related cancer risk [165]. UGT1A7 is expressed in extra-hepatic

tissues, including tissues of the aerodigestive and respiratory tract, and has high activity

against B(a)P metabolites found in tobacco [166-169]. Low activity UGT1A7*3 and

UGT1A7*4 genotypes were found to be associated with an increased orolaryngeal cancer

risk in Caucasian and African American smokers, suggesting that the inability of smokers

to metabolize PAHs in local aerodigestive tract tissues leaves these individuals at a

higher risk for developing tobacco-related cancers [169]. Expression of the low-activity

UGT1A7*3 allele was also determined to be a risk factor for colorectal carcinoma; in

33

vitro activity data supports this finding, as the UGT1A7*3 allele has been shown to have

no detectable activity against B(a)P metabolites or the HCA PhIP, both of which are

thought to play a role in colon carcinogenesis [170]. A recent meta-analysis verified an

association between the UGT1A7*3 allele and increased risk of colon cancer [171].

UGT1A7 expression was also found in the pancreas, and the low-activity UGT1A7*3

allele was determined to be associated with pancreatic adenocarcinoma [172]. Although

UGT1A7 is expressed in extra-hepatic tissues, the low activity UGT1A7*3 variant has

been shown to be associated with increased risk of hepatocellular carcinoma [173]. A

more recent meta-analysis has confirmed the association between the low-activity

UGT1A7*2 and UGT1A7*3 alleles and hepatocellular carcinoma risk, with this analysis

also finding a significant association between the UGT1A7*3 variant and overall cancer

risk in Asian individuals [174].

UGT1A0 is also expressed in extra-hepatic tissues and is active against PAH

metabolites, and the UGT1A10139Lys

polymorphism was determined to be associated with

a decreased risk for orolaryngeal carcinoma in African-Americans [175]. A more recent

in vitro activity study has shown the UGT1A10139Lys

variant to have a two-fold decrease

in enzyme activity against PAH substrates, a result that is inconsistent with the protective

effect of UGT1A10139Lys

observed in the orolaryngeal case-control study [98]. The

conflicting results of epidemiological and functional studies could be due to the low

sample size of the case-control study, or the UGT1A10 codon 139 SNP may be linked to

other polymorphisms in the 1A10 promoter or other UGT1A isoforms; additional work

must be done to confirm the association of UGT1A10139Lys

with tobacco cancer risk [98].

34

UGT2B17 is expressed in the liver and extra-hepatic tissues including the lung

[143], and kinetic analyses have suggested UGT2B17 plays a major role in NNAL

glucuronidation [176]. Women homozygous for the UGT2B17 whole gene deletion

variant were found to be significantly associated with increased risk of lung

adenocarcinoma [177]. Gender differences in UGT2B17 expression levels are

hypothesized to cause the female-specific association between the UGT2B17 deletion

variant and lung cancer risk [178]. Benzidine is an environmental carcinogen linked to

bladder cancer [179, 180], and the low activity UGT2B7268Tyr

variant was determined to

be associated with an increased risk of bladder cancer in benzidine-exposed Chinese

workers [181]. Interestingly, the UGT2B7268Tyr

variant was not found to be associated

with increased bladder cancer risk in a Caucasians [182]. Low activity UGT1A8 and

UGT2B4 variants were found to be associated with increased risk of esophageal

squamous cell carcinoma, presumably due to decreased carcinogen detoxification [183].

Originally thought to be only a toxic byproduct of hemoglobin, bilirubin has recently

been reported to be a potent antioxidant that may play a protective role in carcinogenesis

[184, 185]. Individuals with a high activity UGT1A1 genotype, as opposed to individuals

heterozygous or homozygous for the low-activity UGT1A1*28 allele, were determined to

be at an increased risk for head and neck cancer [186].

35

1.6 Regulation of UGT Activity

In addition to coding region SNPs described above, inter-individual variability in

the transcriptional regulation of UGTs impacts the metabolism of drugs, carcinogens, and

endogenous compounds. Polymorphisms in UGT promoters cause inter-individual

variability in glucuronidation; for example, the UGT1A1*28 promoter polymorphism

causes significantly decreased UGT1A1 expression and activity [133, 134]. Alternatively

spliced UGT variants have been discovered to alter glucuronidation activity, adding

complexity to the regulation of UGTs. The modulation of UGT activity by alternatively

spliced products is hypothesized to occur due to protein-protein interactions.

1.6.1 Transcriptional Regulation of UGTs

Hepatic regulation of UGT expression is mediated by liver-enriched transcription

factors, including hepatocyte nuclear factor (HNF) 1α [187]. All UGT1A and UGT2B

gene promoters contain a conserved HNF1α site [188, 189]. HNF4α, HNF3α and octamer

transcription factor 1 are also thought to be major factors in the transcriptional regulation

of UGTs [92, 190]. Polymorphisms in genes that encode transcription factors, as well as

polymorphisms in transcription factor binding sites in UGT promoters, are thought to

cause inter-individual variability in UGT expression and activity [191]. Tissue-specific

UGT expression is thought to occur due to synergism between HNF transcription factors

and additional transcription factors; for example, caudal-related homeodomain protein 2

36

has been shown to bind to UGT1A8 and UGT1A10 promoters and activate transcription

in the presence of HNF1α [188].

The effects of exogenous transcription factors on UGT expression have also been

investigated. Ligand activated transcription factors, including the aryl hydrocarbon

receptor (AhR), nuclear factor erythroid 2-related factor 2 (Nrf2), pregnane X receptor,

and peroxisome proliferator activated receptor are involved in the regulation of UGT

expression [187]. UGT1A genes have been shown to be induced through AhRs following

exposure to tobacco carcinogens [192]. PAHs have been shown to bind to AhRs and

activate xenobiotic response elements (XREs) in the promoters of UGT1A6 and

UGT1A9, causing these genes to be up-regulated [193]. UGT genes are also regulated by

the Nrf2 pathway [192], with this pathway known to be important in sulforaphane-

mediated chemoprevention [194]. UGT2B7 expression is known to be regulated by the

Nrf2 pathway, and a polymorphism in the UGT2B7 Nrf2 binding site was shown to block

UGT2B7 induction following sulforaphane administration [195]. UGTs have been

reported to be involved in autoregulatory negative feedback loops; for example, bilirubin

is a known metabolite of UGT1A1 and bilirubin is also known to induce UGT1A1

expression through an AhR mechanism [196].

1.6.2 Alternative Splicing of UGTs

Alternative splicing is a mechanism by which multiple transcripts are generated

from the same parent mRNA. Greater than 90% of human genes are estimated to undergo

alternative splicing, with alternative splicing thought to be influenced by tissue-specific

37

factors, stress, hormones, and other physiological processes [197]. The spliceosome,

composed of ribonucleoproteins and small nuclear RNAs, is responsible for mRNA

splicing in humans [198]. The recognition of exons and introns in the spliceosome is

controlled primarily by three signals; the 5’ splice site, 3’ splice site, and the branch site

[199]. Cis-acting splicing silencers and enhancers regulate alterative splicing, and these

elements can be either exonic or intronic [200]. The genetic diversity created by

alternative splicing is immense, as exon skipping, intron retention, alternative 3’ splice

sites, and alternative 5’ splice sites are all mechanisms that are known to create novel

protein variants [201]. Alternative spliced mRNA and protein isoforms often differ in

structure, function, and localization from their parent species [201, 202]. Polymorphisms

that alter splice site recognition or cis-acting elements cause inter-individual variability in

alternative splicing by altering the normal function of the splicing machinery [203, 204].

The complexity and genetic diversity of UGT1A gene locus is increased through

alternative splicing at the 3’ end of the transcript. In addition to wild-type UGT1A exon 5

(termed exon 5a) a novel exon 5b has been discovered, with exon 5b found to be

alternatively spliced to exons 1-4 in all nine active UGT1A genes. UGT1A isoform 2

(UGT1A_i2) proteins, translated from mRNA that includes exon 5b at the 3’ end of the

transcript, have been found to lack enzyme activity [205]. Interestingly, in vitro co-

expression studies have shown that UGT1A_i2 variants negatively regulate the activity of

wild-type UGT1A_i1 proteins [205, 206]. The interaction between UGT1A_i1 and

UGT1A_i2 appears to be a regulatory mechanism that specifically alters the Vmax of the

UGT1A_i1 enzyme, as no change in KM is observed [205]. UGT1A_i2 variants represent

an additional form of UGT regulation, and inter-individual variability in UGT1A_i2

38

expression could have major implications on UGT enzyme activity. Novel splice variants

of UGT2B4 [207] and UGT2B7 [208] have recently been discovered. Protein isoforms

translated from UGT2B4 and UGT2B7 splice variants are catalytically inactive, but have

been determined to negatively modulate the activity of wild-type UGT2B4 or UGT2B7.

Splice variants of UGT3A1 and UGT3A2 genes have also been reported, though the

function of these variants is currently unknown [209]. Widespread alternative splicing of

UGT genes appears to exist, and UGT splice variants potentially play a significant role in

the regulation of UGT activity. Inter-individual differences in splice variant expression

may impact glucuronidation, potentially causing variability in cancer susceptibility or

drug response.

1.6.3 UGT Oligomerization

Although knowledge of the oligomeric state of UGTs is limited due to difficulties

in purifying active UGT isoforms from native tissues [83], UGTs are hypothesized to

form dimers and possibly higher oligomers. UGT interactions in the ER have been

reported to include the formation of both homo-dimers and hetero-dimers [83, 210], and

multiple in vitro co-expression studies have concluded that protein-protein interactions

between UGTs affect enzyme activity [211]. The significance of UGT oligomerization is

relatively unknown; one theory that has been suggested is that multiple UGT monomers

in an oligomeric state take part in substrate binding, while another hypothesis is that UGT

oligomers create a channel in the ER membrane that allows UDPGA to access the active

site of the enzyme [84]. Multiple co-expression studies using varying combinations of

39

UGT isoforms have been completed to investigate UGT dimerization. The consensus

from these studies has been that wide-spread UGT homo- and hetero-dimerization occurs

and that dimerization increases or decreases UGT activity in a substrate-specific manner

[212-214]. Hetero-oligomerization of wild-type UGT isoforms with the low activity

UGT1A6486Asp

variant was shown to significantly increase the activity of this UGT1A6

variant, suggesting that protein-protein interactions may attenuate the consequences of

UGT polymorphisms and have widespread functional relevance [215]. Although the

exact domains of UGTs which interact are relatively unknown, experiments using fusion

proteins made up of either amino- or carboxyl-terminal domains have suggested that the

N-terminus is involved in UGT dimerization [216].

In addition to studies analyzing protein-protein interactions between full-length

UGT isoforms, multiple studies have reported interactions between UGT splice variant

isoforms and their wild-type counterparts. Inactive UGT1A_i2 variants described

previously have been shown to dimerize with UGT1A_i1 proteins, and it is hypothesized

that UGT1A_i2 modulation of UGT1A_i1 activity occurs via protein-protein interactions

[217, 218]. An inactive truncated UGT1A1 variant, caused by a UGT1A1Gln331Stop

polymorphism, was determined to cause a dominant-negative effect when co-expressed

with wild-type UGT1A1 [219]. It is hypothesized that this negative modulation of

UGT1A1 activity is caused by the formation of an inactive oligomeric complex. In

addition to the activity changes caused by dimerization of inactive UGT splice variants

with wild-type UGT isoforms, full-length polymorphic UGT isoforms lacking enzyme

activity have also been shown to dimerize with wild-type UGTs and modulate the activity

of the wild-type protein. An inactive UGT1A1Cys127Tyr

isoform, following co-expression

40

with wild-type UGT1A1, was found to negatively regulate wild-type UGT1A1 activity

through dimerization [220].

1.7 Summary of UGT2A Expression and Activity

Although the activity and expression profiles of the UGT1A and UGT2B

subfamilies have been relatively well-characterized, there is considerably less

information known concerning the UGT2A family. Thus far, UGT2A expression has

been examined in a limited number of tissues and the activities of UGT2A isoforms have

been determined against a small subset of substrates which, for the most part, has not

included drugs or carcinogens. The following is a summary of activity and expression

data reported for the UGT2A subfamily.

1.7.1 UGT2A1 Expression and Activity

Two novel glucuronosyltransferase transcripts were initially cloned from bovine

and rat olfactory epithelium tissues [221, 222]. The rat olfactory-specific enzyme was

later named Ugt2a1, based on sequence alignment and apparent divergent evolution from

other Ugt enzymes [63, 64]. Ugt2a1 was initially hypothesized to protect the central

nervous system from neurotoxic compounds, as rat Ugt2a1 expression was reported in

both the olfactory epithelium and olfactory bulb neurons located at the junction between

the nasal epithelium and the brain [223]. Human UGT2A1, with 91% sequence similarity

41

to rat UGT2a1, was later cloned from human olfactory epithelium [97]. In addition to

being neuroprotective, UGT2A1 was hypothesized to play a role in olfactory perception

and the termination of odorant signals, due to olfactory expression and activity against a

broad range of phenol and alcohol odorants [97]. UGT2A1 was also speculated to be

involved in the metabolism of exogenous and endogenous substrates during development,

as UGT2A1 expression was reported to be significantly higher in fetal tissues than adult

tissues [70, 224]. Though UGT2A1 was initially cloned from nasal epithelium, UGT2A1

expression is not confined to olfactory tissue. A study analyzing tissue-specific

expression levels of phase I and phase II enzymes reported UGT2A1 to be extra-hepatic

and well-expressed in the lung and trachea [96]. In an additional study analyzing overall

UGT expression in the lung and liver, UGT2A1 was shown to be highly expressed in

lung parenchymal cells but not expressed in human hepatocytes [225]. UGT2A1

expression was also observed in lung cells obtained from bronchoalveolar lavage (BAL)

fluid and bronchial biopsies, with UGT2A1 expression significantly lower in BAL fluid

from smokers than from non-smokers [226].

In the initial characterization of UGT2A1 activity, UGT2A1 was found to have

activity against phenols, coumarins, flavonoids, steroids, and alcohols [97]. UGT2A1 was

also shown to metabolize a small number of drugs including morphine, ibuprofen, and

indomethacin [97, 227]; however, UGT2A1 activity has not been determined against

additional drugs and carcinogens. UGT2A1 activity has been determined against a variety

of estrogens and androgens [103, 228, 229]. UGT2A1 has been reported to be less

affected by substrate stereochemistry than other UGT isoforms. For example, UGT2A1

has been shown to metabolize epiestradiol and β-estradiol [103], as well as testosterone

42

and epitestosterone [228], while other UGT isoforms have stereo-selectivity for only one

estrogen or testosterone diastereomer.

1.7.2 UGT2A2 and UGT2A3 Expression and Activity

In comparison to UGT2A1, even fewer studies have characterized the expression

and activities of UGT2A2 and UGT2A3. UGT2A2 expression was observed primarily in

fetal and adult nasal mucosa [70]. UGT2A2 activity was reported against estrogen and

phenolic substrates, although UGT2A2 glucuronidation rates were generally found to be

lower than rates observed for UGT2A1 [70]. Additional studies have reported relatively

low UGT2A2 activity against epiestradiol and β-estradiol [103], as well as testosterone,

epitestosterone, and androsterone [103, 228, 229]. Unlike UGT2A1 and UGT2A2,

UGT2A3 is expressed hepatically, with UGT2A3 expression also reported in the small

intestine, colon, kidney, and pancreas [70, 71]. UGT2A3 was reported to specifically

metabolize bile acids, including chenodeoxycholic acid, deoxycholic acid,

hyodeoxycholic acid, and ursodeoxycholic acid [70, 71]. A prevalent UGT2A3

Thr497Ala polymorphism was determined to cause no significant change in bile acid

glucuronidation when compared to wild-type UGT2A3 activity [71].

1.8 Aims and Hypotheses

Tobacco-induced cancers are a major global health problem today, and a better

understanding of the mechanisms involved in tobacco-related carcinogenesis is needed to

43

help identify individuals predisposed to developing cancer and promote earlier detection.

UGTs are critical enzymes in the metabolism of tobacco carcinogens, and inter-individual

variability in carcinogen metabolism is thought to have significant implications on cancer

susceptibility. Determining which UGTs are involved in carcinogen detoxification,

including the pharmacogenetics of UGT-mediated carcinogen metabolism in extra-

hepatic target tissues, will help identify populations at an increased risk for developing

tobacco-related cancers. The focus of UGT research during the last 30 years has centered

on the UGT1A and UGT2B subfamilies. Only a few studies have analyzed the expression

and activities of UGT2A enzymes, and the physiological roles of the UGT2A enzymes

are still relatively unknown. Due to the lack of research focusing on the UGT2A

enzymes, many tissues have never been analyzed for UGT2A expression and the

importance of UGT2A enzymes in the glucuronidation of many substrates has never been

determined.

Studies have reported UGT2A1 expression in the lung and UGT2A1 activity

against a broad range of substrates, including simple phenols. Based on this data, the

central hypothesis of this dissertation research was that UGT2A1 is expressed in

aerodigestive and respiratory tract tissues and exhibits activity against tobacco

carcinogens. The tissue expression and role of UGT2A2 and UGT2A3 in carcinogen

metabolism was also relatively unknown, and due to the sequence homology between

UGT2A family members it was hypothesized that UGT2A2 and UGT2A3 are also

involved in tobacco carcinogen metabolism. During the initial investigation of UGT2A1

expression, a novel UGT2A1 exon 3 deletion (UGT2A1Δexon3) splice variant was

discovered. The UGT2A1Δexon3 variant and prevalent non-synonymous UGT2A1

44

coding SNPs were hypothesized to cause alterations in UGT2A1 activity and this

hypothesis was examined as a secondary goal of the project. The role of the UGT2A

enzymes in tobacco carcinogen metabolism was investigated through four major aims: 1)

characterize the tissue expression of UGT2A1 and glucuronidation activity of UGT2A1

against a panel of tobacco carcinogens, 2) investigate UGT2A1 SNPs to determine their

prevalence and the functional impact of SNPs on UGT2A1 enzyme activity and cancer

risk, 3) determine the expression, enzyme activity, and functional role of the

UGT2A1Δexon3 splice variant, and 4) determine UGT2A2 and UGT2A3 tissue

expression and the glucuronidation activities of UGT2A2 and UGT2A3 against tobacco

carcinogens.

45

Chapter 2

CHARACTERIZATION OF UGT2A1 EXPRESSION AND THE POTENTIAL

ROLE OF UGT2A1 IN TOBACCO CARCINOGEN METABOLISM

46

2.1 Abstract

UGTs play an important role in the metabolism and excretion of various

endogenous and xenobiotic compounds, including carcinogens and chemotherapeutic

agents. The goal of the present study was to examine UGT2A1 expression in human

tissues and determine the glucuronidation activity of UGT2A1 against tobacco

carcinogens. As determined by RT-PCR, UGT2A1 was expressed in multiple tissues

including trachea, larynx, tonsil, lung and colon; no expression was observed in breast,

whole brain, floor of mouth, cerebral cortex, pancreas, prostate, kidney, liver or

esophagus. Real-time PCR suggested that UGT2A1 exhibited highest expression in the

lung, followed by trachea > tonsil > larynx > colon > olfactory tissue. Cell homogenates

prepared from human embryonic kidney (HEK) 293 cells over-expressing wild-type

UGT2A1 showed significant glucuronidation activity, as observed by reverse-phase ultra-

pressure liquid chromatography (UPLC), against a variety of PAHs including B(a)P-7,8-

diol and 5-methylchrysene-1,2-diol. No activity was observed in UGT2A1 over-

expressing cell homogenates against substrates that form N-glucuronides, including

NNAL, nicotine, or N-OH-PhIP. In vitro experiments suggested that UGT2A1 over-

expression is cytoprotective and genoprotective following administration of activated

B(a)P metabolites in cell culture. These data suggest that UGT2A1 is an important

detoxification enzyme in the metabolism of PAHs within target tissues for tobacco

carcinogenesis.

47

2.2 Introduction

UGT enzymes catalyze the glucuronidation of a variety of compounds, including

endogenous compounds such as hormones and bilirubin, as well as xenobiotics such as

drugs and carcinogens [230-232]. Based on sequence and structural homology, UGTs are

classified into several families and subfamilies [233]. In comparison to the UGT1A and

UGT2B families, relatively no information has been reported on the tissue expression and

glucuronidation activity of UGT2A enzymes. This portion of the dissertation focused on

UGT2A1 expression and activity in relation to tobacco carcinogen metabolism. The roles

of UGT2A2 and UGT2A3 in tobacco carcinogen metabolism were also investigated and

will be covered in Chapter 5 of the dissertation.

UGT2A1 was originally cloned from human olfactory epithelium tissue, and the

physiological role of UGT2A1 was hypothesized to be in the initiation and termination of

olfactory stimuli [97]. In non-quantitative RT-PCR expression studies, UGT2A1 was

determined to be extra-hepatic, with expression observed in the olfactory epithelium,

fetal lung, and brain [97]. In more recent quantitative real-time PCR studies comparing

the expression of various phase I and phase II enzymes, including eight individual UGTs,

UGT2A1 was shown to be well-expressed in both the lung and trachea [96]. A limited

number of studies have been completed examining UGT2A1 enzyme activity and

xenobiotic metabolism. UGT2A1 was reported to exhibit activity against a range of

steroids, drugs, and phenol odorants, [97]. UGT2A1 was also recently reported to exhibit

activity against the PAH 1-OH-pyrene [70].

48

Given the activity of UGT2A1 against the simple PAH 1-OH-pyrene and other

phenols, and its reported expression in lung and trachea, the goal of the present study was

to more fully investigate the role of UGT2A1 as a potentially relevant enzyme involved

in tobacco carcinogen metabolism. A more complete expression profile of UGT2A1 was

completed by screening aerodigestive and respiratory tract tissues never before

investigated for UGT2A1 expression. UGT2A1 enzyme activity was determined for the

first time against multiple classes of tobacco carcinogens, including PAHs, TSNAs and

HCAs.

2.3 Methods

Chemicals and materials. 1-OH-B(a)P, 3-OH-B(a)P, 7-OH-B(a)P, 8-OH-B(a)P, 5-

methylchrysene-1,2-diol, dibenzo(a,l)pyrene-11,12-diol, B(a)P-7,8-diol, and N-OH-PhIP

were synthesized in the Organic Synthesis Core Facility at the Penn State College of

Medicine (Hershey, PA). UDPGA, alamethicin, -glucuronidase, nicotine, 4-MU, 1-OH-

pyrene, and 1-naphthol were purchased from Sigma-Aldrich (St. Louis, MO). NNAL,

NNN, N-nitrosoanabasine (NAB), N-nitrosoanatabine (NAT), PhIP, cotinine, and 1-

naphthol-glucuronide were purchased from Toronto Research Chemicals (Ontario,

Canada). High pressure liquid chromatography (HPLC)-grade ammonium acetate,

acetonitrile, and agarose were purchased from Fisher Scientific (Pittsburgh, PA). Gene

expression assays were acquired from Applied Biosystems Inc. (ABI), Life Technologies

(Carlsbad, CA). Dulbecco’s modified eagle medium (DMEM), Dulbecco’s phosphate-

49

buffered saline (DPBS) (minus calcium-chloride and magnesium-chloride), fetal bovine

serum (FBS), penicillin-streptomycin, and Geneticin (G418) were purchased from Gibco,

Life Technologies (Grand Island, NY). The Platinum Pfx DNA polymerase,

pcDNA3.1/V5-His-TOPO mammalian expression vector, and Superscript II RT kit were

obtained from Invitrogen, Life Technologies (Grand Island, NY). The BCA protein assay

kit was purchased from Pierce (Rockford, IL). The RNeasy kit, QIAquick gel extraction

kit, Plasmid Mini kit, and Plasmid Maxi kit were all purchased from Qiagen (Valencia,

CA). All PCR primers were purchased from IDT (Coralville, IA).

Determination of UGT2A1 tissue expression. Tissue-specific UGT2A1 mRNA

expression was determined by RT-PCR using pooled RNA from multiple tissues. RNA

was obtained from lung, larynx, trachea, breast, whole brain, cerebral cortex, prostrate,

kidney, and pancreas tissues from Clontech (Mountain View, CA) or Agilent (Santa

Clara, CA). A sample of human olfactory tissue RNA was purchased from Biochain

Institute (Hayward, CA). Tonsil, colon, mouth, esophagus, and liver RNA was extracted

using an RNeasy kit from normal tissue obtained from the Penn State College of

Medicine Tissue Bank (Hershey, PA). All RT-PCR assays were performed using pooled

RNA from at least three samples for each tissue. Two μg of RNA was used for RT using

a Superscript II RT kit following the manufacturer’s protocol. cDNA corresponding to

100 ng of RNA was used to PCR-amplify UGT2A1 with Pfx Polymerase and sense (5’-

CTGCATCAAGCCACATCATG-3’) and anti-sense (5’-TCCCATGATTTCCAAA-

GAGT-3’) primers corresponding to nucleotides -17 to +3 and nucleotides +692 and

+673, respectively, relative to the UGT2A1 translation start site as previously described

50

[97]. PCR reactions were performed in a Bio-Rad MyCycler (Hercules, CA) with an

initial denaturing temperature of 94°C for 2 min, 40 cycles of 94°C for 30s, 55°C for 30s,

and 68°C for 2 min, followed by a final cycle of 10 min at 68°C. RNA from a HEK293

cell line over-expressing UGT2A1 was used as a positive control for PCR amplification

(see below for Methods), while water was used as a negative control. PCR products were

gel-purified using a QIAquick gel extraction kit and sequenced by the Penn State College

of Medicine Functional Genomics Core Facility (Hershey, PA). To verify UGT2A1

expression in tissues analyzed, PCR reactions were run multiple times with positive and

negative controls.

Real-time PCR was performed to quantitatively assess UGT2A1 expression in

tissues determined to express UGT2A1 from RT-PCR. Real-time PCR was performed

using a UGT2A1-specific TaqMan gene expression assay (Hs00792016_m1) using the

standard assay protocol with human large ribosomal protein (RPLPO) as the

housekeeping gene (Hs99999902_m1). RPLPO expression exhibits little inter-individual

variability in lung and aerodigestive tract tissues (P. Lazarus, unpublished data). cDNA

corresponding to 20 ng RNA was used for each reaction. Real-time PCR experiments

were carried out in the Penn State College of Medicine Functional Genomics Core

Facility (Hershey, PA) using an ABI 7900 HT thermal cycler, and data was analyzed

using ABI Sequence Detection System (SDS) 2.2 software. Relative expression of

UGT2A1 in different tissues was calculated using the delta delta (ΔΔ) Ct method, relative

to the tissue which had the highest expression of UGT2A1 (lung).

51

Generation of UGT2A1 over-expressing cell lines. A cell line over-expressing

wild-type UGT2A1was generated by RT-PCR using pooled lung RNA. Two μg of RNA

was used for the RT reaction, and cDNA corresponding to 100 ng lung RNA was used

with Pfx Polymerase for the PCR amplification of full-length UGT2A1. The primers used

to amplify UGT2A1 from lung cDNA were 5’-CATCAAATCTTCTGCATCAAGC-

CAC-3’ (sense) (UGT2A1_S1) and 5’-TGACAGGAAGAGGGTA-TAGTCAGC-3’

(anti-sense) (UGT2A1_AS1), corresponding to nucleotides -28 to -4 and +1834 to +1811,

respectively, relative to the UGT2A1 translation start site. PCRs were performed with an

initial denaturing temperature of 94°C for 2 min, 40 cycles of 94°C for 30s, 56°C for 45s,

and 68°C for 2 min, followed by a final cycle of 10 min at 68°C. UGT2A1 sequences

were verified by dideoxy sequencing of the PCR-amplified product, performed using the

same PCR primers and an UGT2A1-specific internal sense primer (5’-TGAAGTCC-

TGGTGTCTGATTCAGT-3’, corresponding to nucleotides +432 to +455 relative to the

UGT2A1 translation start site) and compared with that described for UGT2A1 in

GenBank (NM_006798). The fully-verified wild-type UGT2A1 cDNA was cloned into

the pcDNA3.1/V5-His-TOPO vector using standard protocols using One Shot TOP10

competent E. Coli. After a large-scale plasmid prep, electroporation was used to generate

the HEK293 cell line over-expressing wild-type UGT2A1 using 10 μg of pcDNA3.1/V5-

His-TOPO_UGT2A1 vector. Cells were grown in DMEM supplemented with FBS and

G418 to 70% confluence. Cell homogenates were prepared essentially as previously

described [98]. Total RNA was extracted using the RNeasy Mini kit using manufacturer’s

protocols. Total homogenate protein concentrations were determined using the BCA

protein assay.

52

Determination of UGT2A1 antibody specificity. The specificity of the anti-

UGT2A1 antibody was determined by Western blot analysis using protein from the

UGT2A1-over-expressing cell line and protein from HEK293 cell lines over-expressing

additional UGT isoforms. An antibody specific for UGT2A1 was designed by Open

Biosystems (Huntsville, AL), using a peptide unique to the N-terminal regions of

UGT2A1 (ELTDQMSFTDRIRNFISYHL) as an antigen in rabbits. UGT2A1 protein in

the HEK293 over-expressing cell line was measured using the anti-UGT2A1 antibody at

a 1:500 dilution, as recommended by the manufacturer. In order to determine antibody

specificity, 100 μg of total protein homogenate from the UGT2A1 cell line and protein

from HEK293 cells over-expressing UGT1A1 and UGT2B7, was loaded into each lane.

The monoclonal β-actin antibody (Sigma-Aldrich) was used as a loading control.

UGT2A1 glucuronidation assays. Glucuronidation assays using homogenates

from HEK293 cell lines over-expressing wild-type UGT2A1 were performed essentially

as previously described [234, 235]. Briefly, after an initial incubation of total cell

homogenate protein (100 μg) with alamethicin (50 μg/mg protein) for 15 min on ice,

glucuronidation reactions were performed in a final reaction volume of 25 μL at 37°C

with 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 4 mM UDPGA, and between 6 and 750

μM of substrate. For each substrate, the glucuronidation rate was determined at 8

concentrations that encompassed the experimentally determined KM of the substrate.

Reactions were terminated by the addition of 25 μL cold acetonitrile on ice. Reaction

mixtures were centrifuged for 10 min at 16,100 g prior to the collection of supernatant.

For glucuronidation rate determinations, cell homogenate protein levels and incubation

53

times for each substrate were determined experimentally to ensure that substrate

utilization was less than 10% and to maximize levels of detection while in a linear range

of glucuronide formation.

Levels of glucuronide formation were determined using a Waters Acquity UPLC

System (Milford, MA) as previously described [110, 112, 234, 236]. The flow rate was

maintained at 0.5 mL/min and a reverse phase Acquity UPLC BEH C18 - 1.7 μm - 2.1 x

100 mm column was used to separate free substrate and the conjugated glucuronide. A

gradient of solution A (5 mM NH4OAc (pH 5.0), 10% acetonitrile) and solution B (100%

acetonitrile) was used to elute the glucuronide and substrate from the column. The initial

solvent gradient used to detect glucuronidation of 1-naphthol was 80% solution A/20%

solution B for 2 min, a linear gradient to 25% solution A/75% solution B from 2 to 4 min,

and re-equilibrium to the initial condition from 4 to 6 min. For other substrates, a similar

gradient was used, but the initial ratio of solution A to solution B was varied slightly. The

initial condition for B(a)P-7,8-diol, 5-methylchrysene-1,2-diol, dibenzo(a,l)pyrene-11,12-

diol, 1-OH-B(a)P, 3-OH-B(a)P, 7-OH-B(a)P, and 8-OH-B(a)P was 85% A and 15% B.

The initial condition for 1-OH-pyrene and 4-MU was 90% A and 10% B, while the initial

condition for NNAL, nicotine, PhIP, N-OH PhIP, NNN, NAB, NAT, and cotinine was

99% A and 1% B. The UV absorbance wavelength determined experimentally to detect

each substrate and glucuronide was as follows: 1-OH-pyrene and 1-naphthol were

detected at 240 nm; 5-methylchrysene-1,2-diol, B(a)P-7,8-diol, NNAL, NNN, NAT,

NAB, nicotine, and cotinine were detected at 254 nm; 1-OH-B(a)P, 3-OH-B(a)P, 7-OH-

B(a)P, 8-OH-B(a)P, dibenzo(a,l)pyrene-11,12-diol, and 4-MU were detected at 305 nm;

and PhIP and N-OH-PhIP were detected at 316 nm.

54

Quantification of glucuronide formation for each substrate was determined

essentially as previously described [75, 112, 118, 161]. Briefly, the amount of

glucuronide formed was determined based on the ratio of glucuronide versus

unconjugated substrate after calculating the area under the curve for the substrate and

glucuronide peaks, using the known amount of substrate in each reaction as the reference.

This quantification method was validated for 1-naphthol-glucuronide formation, as this

was a glucuronide of a substrate tested in the present study that was also available

commercially. Validation was performed by constructing a 1-naphthol-glucuronide

standard curve and comparing the levels of 1-naphthol-glucuronide formation calculated

using the peak area ratio method described above with the values from the standard

curve. This was performed for 10 independent glucuronidation reactions using a 10-fold

range of 1-naphthol concentrations, and, in all cases, levels of 1-naphthol-glucuronide

formation were within 5% of the level predicted from the standard curve. Glucuronides

were confirmed by sensitivity to β-glucuronidase, by mass spectrometry analysis, and in

the case of 1-naphthol, by comparison to an authentic 1-naphthol glucuronide standard.

Reactions with non-transfected HEK293 cell homogenate, no substrate added to the

reaction mixture, or only substrate and no homogenate in the reaction mixture were used

as negative controls. UGT2A1 activity against 4-MU, a common UGT substrate, was

used as positive control for UGT2A1 activity [237]. The lower limit of glucuronide

detection was determined experimentally using a 1-naphthol glucuronide standard.

Cytoprotective and genoprotective effects of UGT2A1 over-expression. 3H

labeled B(a)P (specific activity 50 mCi/mmol) was used to determine whether UGT2A1

55

over-expression prevents 3H B(a)P covalent binding, while unlabeled B(a)P was used to

determine the cytoprotective effect of UGT2A1 over-expression. Radioactively labeled

3H B(a)P, 0.2 μCi (final concentration 16 μM), and 10 μM unlabeled B(a)P were used

in two separate pre-incubation reactions to create oxidized B(a)P intermediates. Each

B(a)P compound was incubated with 500 μg of HLM for 120 min, in a buffered solution

containing 130 mM NaCl, 5.2 mM KCl, 1.3 mM KH2PO4, 10 mM Na2HPO4, 1.3 MgSO4

(pH 7.4), and 10 mM NADPH as described previously [238]. Control pre-incubation

reactions without B(a)P added to the reaction mixture were also completed. Each pre-

incubation reaction was terminated by the addition of an equal volume of cold

acetonitrile. The samples were centrifuged at 16,000 x g for 15 min at 4°C and the

supernatant was vacuum centrifuged for 30 min. The aqueous phase of the supernatant,

containing oxidized B(a)P metabolites, was used in the next step of experiments.

An equal amount of 3H B(a)P pre-incubation supernatant was added to HEK

cells over-expressing UGT2A1 at 80% confluence, in either the presence or absence of

10 mM UDPGA. Cells were incubated for 4 h at 37°C and 5% CO2 to determine 3H

B(a)P covalent binding. Cells were centrifuged at 10,000 x g for 5 min following the 4 h

incubation and cell pellets were washed with 56°C methanol to eliminate removable

radioactivity. Cell pellets were then incubated overnight at 40°C in 0.5 mL of 1.0 M

NaOH and neutralized the following day with an equal volume of 1.0 M HCl.

Radioactivity was determined using a liquid scintillation spectrometer and standard liquid

scintillation cocktail. 3H B(a)P covalent binding was compared in HEK293 cells over-

expressing UGT2A1 in the presence of absence of UDPGA. 3H B(a)P covalent binding

results were replicated in three independent experiments.

56

An equal amount of B(a)P pre-incubation supernatant was added to HEK cells

over-expressing UGT2A1 at 80% confluence, in either the presence and absence of 10

mM UDPGA, to determine the cytoprotective effect of UGT2A1 over-expression. Cells

were incubated for 16 h at 37°C and 5% CO2. Following the incubation, a 100 μL aliquot

of cells was mixed with an equal volume of 0.2% trypan blue in normal saline solution.

At least 200 cells were counted for each experimental condition using a hemocytometer.

Trypan blue exclusion was used to determine the percentage of viable HEK293 cells

over-expressing UGT2A1 following B(a)P treatment. B(a)P cytotoxicity results were

confirmed through the completion of three independent experiments. For both the

cytotoxicity and covalent binding protocols, supernatants from the control reactions

lacking B(a)P were added to additional plates of cells as negative controls.

Data analysis and statistics. Three independent experiments were performed for

kinetic analyses of UGT2A1 over-expressing cell homogenate against the various

substrates tested. GraphPad Prism 5 software was used to calculate kinetic values. Kinetic

constants Vmax and KM for all substrates were calculated by graphing the rate of product

formation versus substrate concentration and then using the Michaelis-Menten equation.

For visualization as the whether the kinetics data was consistent with the simple

Michaelis-Menten mechanism, the data were transformed into linear Eadie-Hofstee plots.

The Student’s t-test was used to compare covalent binding and cytotoxicity in HEK293

cells over-expressing UGT2A1, with and without the addition of UDPGA, following

B(a)P administration.

57

2.4 Results

Expression of UGT2A1 in human tissues. Previous studies demonstrated that

UGT2A1 is expressed primarily in olfactory epithelium, with expression also observed in

the brain, lung and trachea [96, 97]. In the present study a more comprehensive analysis

of UGT2A1 expression was completed. In an initial screening, pooled RNA samples

were obtained and probed non-quantitatively for UGT2A1 expression by RT-PCR

(Figure 2.1). UGT2A1 was well-expressed in the lung, larynx, trachea, tonsil, and colon.

Using quantitative real-time PCR (Figure 2.2), the relative level of UGT2A1 expression

was demonstrated to be the highest in lung (used as the reference at 1.0 ± 0.03) followed

by the trachea (0.91 ± 0.04) > tonsil (0.61 ± 0.07) > larynx (0.51 ± 0.07) > colon (0.33 ±

0.05) > olfactory epithelium (0.19 ± 0.04). No UGT2A1 expression was detected after

multiple RT-PCR attempts or by real-time PCR in prostate, liver, floor of mouth, or

pancreas (Figure 2.1), or esophagus, whole brain, cerebral cortex, kidney or breast

(results not shown).

58

Figure 2.1. Qualitative characterization of UGT2A1 tissue expression. Initial characterization

of UGT2A1 expression in multiple human tissues using RT-PCR. RNAs pooled from at least

three individuals were used to assess overall UGT2A1 tissue expression. RNA from HEK293 cell

lines over-expressing wild-type UGT2A1 was used as a positive control; water in place of cDNA

was used as a negative control.

59

Figure 2.2. Quantitative analysis of UGT2A1 expression in human tissues. RNAs from

tissues exhibiting UGT2A1 expression in Figure 2.1 were used in conjunction with a UGT2A1-

specific real-time PCR assay (ABI) to quantitatively assess UGT2A1 expression. Relative

UGT2A1 mRNA tissue expression was determined by comparing mRNA levels in each tissue to

that observed in the tissue with the highest UGT2A1 expression (i.e., lung). Results, expressed as

the mean ± SD of triplicates, were normalized to RPLPO expression in each tissue.

Specificity of UGT2A1 antibody. As shown in Figure 2.3, Western blot analysis

using an anti-UGT2A1 antibody showed a high level of UGT2A1 protein expression in a

HEK293 cell line over-expressing UGT2A1. No cross-reactivity was observed with other

UGT1A and UGT2B isoforms, including when protein homogenates from UGT1A1 or

UGT2B7 over-expressing cell lines were used (Figure 2.3).

60

Figure 2.3. Western blot analysis of UGT2A1 protein expression in HEK293 over-

expressing cell line. An antibody against UGT2A1 was analyzed for specificity and cross-

reactivity using homogenate from HEK293 cell lines over-expressing UGT1A1, UGT2B7, and

UGT2A1.

Kinetic studies of UGT2A1 carcinogen metabolism. With UGT2A1 shown to be

well-expressed in lung and a variety of aerodigestive tract tissues, the next experimental

aim was to examine the glucuronidation activity of UGT2A1 against a panel of tobacco

carcinogens. Using homogenates from a HEK293 cell line over-expressing wild-type

UGT2A1, in vitro glucuronidation assays demonstrated UGT2A1 activity against the

simple PAH 1-naphthol, with a naphthol-1-O-glucuronide peak observed at 1.3 min and a

1-naphthol substrate peak observed at 4.0 min by UPLC (Figure 2.4, Panel A). The

naphthol-1-O-glucuronide peak was sensitive to treatment with β-glucuronidase (Figure

2.4, Panel B). Similarly, UPLC results suggested that the proximate carcinogen 5-

methylchrysene-1,2-diol was also glucuronidated by UGT2A1, as shown by the

glucuronide of 5-methylchrysene-1,2-diol at 3.2 min and a 5-methylchrysene-1,2-diol

substrate peak at 4.0 min (Figure 2.4, Panel C). Representative Michaelis-Menten (Figure

61

2.4, Panel D) and Eadie-Hofstee (Figure 2.4, Panel E) plots are shown for UGT2A1

activity against 5-methylchrysene-1,2-diol. Kinetic analyses demonstrated similarly high

glucuronidation activity for wild-type UGT2A1 against all other PAHs tested including

1-OH-pyrene, 1-OH-B(a)P, 3-OH-B(a)P, 7-OH-B(a)P, 8-OH-B(a)P, dibenzo(a,l)pyrene-

11,12-diol, and B(a)P-7,8-diol (Table 2.1). The lower limit of glucuronide detection for

the Waters Acquity UPLC System was determined to be 20 pmol, using a 1-naphthol-

glucuronide standard. UGT2A1 exhibited no detectable activity against several TSNAs

(NNAL, NNN, NAT or NAB), nicotine, or its major metabolite cotinine. In addition,

UGT2A1 exhibited no glucuronidation against the HCA, PhIP, or its major metabolite,

N-OH-PhIP (results not shown).

62

Figure 2.4. UGT2A1 over-expressing cell line exhibits glucuronidation activity against PAH

substrates. (A) UPLC trace of 1-naphthol-0-glucuronide formation following incubation with

UGT2A1 cell homogenate. (B) UPLC trace of reaction from Panel A following treatment with

1000 units of E. Coli β-glucuronidase. (C) UPLC trace of 5-methylchrysene-1,2-diol glucuronide

formation following incubation with UGT2A1 cell homogenate. (D) Representative Michaelis-

Menten plot of 5-methylchrysene-1,2-diol glucuronidation by UGT2A1. (E) Representative

Eadie-Hofstee plot of 5-methylchrysene-1,2-diol glucuronidation by UGT2A1.

63

Table 2.1. Enzyme kinetics summary of UGT2A1 activity against PAH substrates.

KM Vmax Vmax/KM

Substrate (μM) (pmol/min/mg)a

(μL/min/mg)a

1-OH-pyrene 91 ± 7.2 300 ± 17 3.3 ± 0.2

1-naphthol 30 ± 2.9 185 ± 11 6.2 ± 0.4

5-methylchrysene-1,2-diol 270 ± 24 100 ± 7.2 0.37 ± 0.04

dibenzo(a,l)pyrene-11,12-diol 284 ± 27 26 ± 1.0 0.09 ± 0.004

B(a)P-7,8-diol 397 ± 33 109 ± 14 0.28 ± 0.04

1-OH-B(a)P 247 ± 12 70 ± 4.4 0.28 ± 0.01

3-OH-B(a)P 271 ± 15 84 ± 3.6 0.31 ± 0.008

7-OH-B(a)P 261 ± 35 58 ± 4.6 0.22 ± 0.01

8-OH-B(a)P 308 ± 20 76 ± 6.6 0.25 ± 0.008

a Data expressed as mg of total protein homogenate.

KM, Vmax, and Vmax/KM represent the mean of three independent experiments.

UGT2A1 modulation of B(a)P covalent binding and cytotoxicity. The ability of

UGT2A1 to prevent B(a)P mediated cytotoxicity was determined using supernatant from

B(a)P and HLM pre-incubation reactions. As shown in Figure 2.5, Panel A, there was a

significantly higher percent (p<0.001) of viable HEK293 cells over-expressing UGT2A1

when UDPGA was added during the 16 h incubation with B(a)P supernatant, as opposed

to when B(a)P was added without UDPGA. The ability of UGT2A1 to prevent 3H B(a)P

mediated covalent binding was also determined using supernatant from the 3H B(a)P and

HLM pre-incubation. As shown in Figure 2.5, Panel B, there was significantly less 3H

B(a)P covalent binding (p<0.001) in HEK293 cells over-expressing UGT2A1 when

UDPGA was added during the 4 h incubation of 3H B(a)P supernatant, as opposed to

when 3H B(a)P was added without UDPGA. The control supernatant lacking B(a)P had

minimal effects on cell viability (Figure 2.5, Panel A), and only background radioactivity

64

was observed when the control supernatant was used in place of 3H B(a)P supernatant

(Figure 2.5, Panel B).

Figure 2.5 UGT2A1 over-expression and UDGPA administration prevent B(a)P mediated

cytotoxicity and covalent binding. (A) The percentage of viable HEK293 cells over-expressing

UGT2A1was significantly higher when UDPGA was added with B(a)P for the 16 h incubation.

(B) 3H B(a)P covalent binding in HEK293 cells over-expressing UGT2A1 was significantly

lower when UDPGA was added with B(a)P for the 4 h incubation. In both cases the addition of

control supernatant had a minimal effect on cytotoxicity or covalent binding.

65

2.5 Discussion

This study is the first to demonstrate that UGT2A1 is expressed in a variety of

tissues that are target sites for tobacco carcinogenesis and that UGT2A1 exhibits

glucuronidation activity against the PAH class of tobacco carcinogens. Results presented

here confirmed previous studies reporting UGT2A1 expression in the lung and trachea

[96], and for the first time demonstrated expression in other tissues including the larynx,

tonsil, and colon. The expression of UGT2A1 in multiple aerodigestive and respiratory

tract tissues, including the lung, and the activity of UGT2A1 against PAH carcinogens

suggest that UGT2A1 may play a role in the local detoxification of tobacco carcinogens.

UGTs are known to have overlapping substrate specificities, and multiple UGTs

are expressed in the lung and other aerodigestive tract tissues. No comprehensive analysis

of UGT tissue-specific expression completed to date has included UGT2A1, and tissues

such as the larynx, tonsil, and trachea have often been overlooked in these studies.

UGT1A7 and UGT1A10 are known to be well-expressed in multiple aerodigestive tract

tissues and have active against complex PAH metabolites [98, 99]. The relative

expression and glucuronidation activity of UGT2A1 compared to UGT1A7 or UGT1A10

is currently unknown. The present study confirmed previous results indicating relatively

high UGT2A1 expression in lung [96]. UGT1A10 is expressed in lung but at relatively

low levels [89, 98, 99], while UGT1A7 is thought to be primarily expressed in oral,

laryngeal, gastric, and small intestinal tissues [239, 240]. While UGT1A6 was expressed

at a higher level than UGT2A1 in lung in a previous study [96], this enzyme exhibits

limited activity against simple B(a)P metabolites such as 7-OH B(a)P, and no reported

66

activity against more complex activated B(a)P metabolites, such as B(a)P-7,8-diol [99,

241, 242]. These results, combined with data from the current study, suggest that

UGT2A1 is potentially the only UGT well-expressed in the lung and active against

complex PAH proximate carcinogens. A comprehensive study comparing UGT2A1

expression levels relative to other UGTs in the lung and aerodigestive tract is needed

clarify the importance of UGT2A1 in these tissues.

UGT catalyzed glucuronidation of bioactivated PAHs protects the cell from PAH-

induced DNA adduct formation. B(a)P was added, following a pre-incubation step to

create reactive B(a)P intermediates as previously described [238, 243], to HEK293 cells

over-expressing UGT2A1 in order to determine the cytoprotective and genoprotective

effect of UGT2A1 over-expression. UGT2A1 over-expression in the presence of UDPGA

was shown to prevent B(a)P mediated covalent binding and cytotoxicity through in vitro

experiments. This in vitro data supports the hypothesis that UGT2A1 is protective against

PAH induced cellular damage. Similar experiments will be completed in the future using

HEK293 cells over-expressing other UGT isoforms to determine the cytoprotective and

genoprotective effect of UGT2A1 over-expression compared to other UGT enzymes.

Together, the in vitro data presented in this study suggest that UGT2A1 may play

an important role in PAH metabolism in multiple target organs for tobacco

carcinogenesis. Interestingly, UGT2A1 exhibited similar glucuronidation rates against the

proximate carcinogens B(a)P-7,8-diol and 5-methylchrysene-1,2-diol, while significantly

lower UGT2A1 glucuronidation of dibenzo(a,l)pyrene-11,12-diol was observed. The low

UGT2A1-mediated glucuronidation of this carcinogenic substrate may leave the lung and

other target tissues vulnerable to DNA adduct formation. No UGT2A1 glucuronidation

67

activity was observed against carcinogens other than PAHs, including TSNAs NNAL,

NNN, NAB, or NAT or the HCA PhIP or its major metabolite N-OH PhIP. TSNAs and

HCAs are primarily glucuronidated at Nitrogen moieties, which suggests that UGT2A1 is

not an efficient enzyme for N-glucuronidation. Additional work is needed to determine

the expression level and activity of UGT2A1 compared to other UGT isoforms in these

tissues. The creation of a UGT2A1-specific antibody was described for the first time in

this study, and UGT2A1 protein levels in tissue homogenates will be assessed in the

future using this antibody Genetic alterations that decrease UGT2A1 activity may play a

role in PAH related cancer susceptibility in the lung and aerodigestive tract. UGT2A1

was also found to be expressed in colon, where dietary PAH exposure is a known risk

factor for colorectal cancer [244, 245], and UGT2A1 polymorphisms may also be

involved in colon cancer risk.

68

Chapter 3

CHARACTERIZATION OF UGT2A1 SNP VARIANTS AND THE

ASSOACIATION OF UGT2A1308ARG

WITH

LUNG CANCER RISK

69

3.1 Abstract

UGT2A1 is a phase II detoxifying enzyme expressed in respiratory and

aerodigestive tract tissues with activity against PAH metabolites implicated in tobacco

carcinogenesis. Prevalent non-synonymous SNPs in the UGT2A1 coding region were

characterized in this set of experiments, with the primary goal to assess the functional

changes in UGT2A1 activity caused by these SNPs. A significant (p<0.05) ~25%

decrease in glucuronidation activity (Vmax/KM) was observed against all PAH substrates

tested for the UGT2A175Arg

variant, as compared to homogenates from wild-type

UGT2A175Lys

. No detectable glucuronidation activity was observed for cell homogenates

over-expressing the UGT2A1308Arg

variant for all substrates examined. Both the

UGT2A1*2 (encoding the UGT2A175Arg

variant) and UGT2A1*3 (encoding the

UGT2A1308Arg

variant) alleles were determined to be prevalent in multiple populations, as

the allelic prevalence of both alleles was found to be at least 4% in Caucasians, African-

Americans, and Asians. Results from a lung cancer case-control study showed that the

UGT2A1308Arg

variant is associated with lung non-small cell carcinoma (NSCC) (p=0.04)

and lung squamous cell carcinoma (p=0.02). A statistically significant decrease (p<0.001)

in UGT2A1 activity against multiple PAH substrates was observed following

UGT2A1308Arg

co-expression in a 1:1 ratio with wild-type UGT2A1. Co-IP results

suggested that dimerization occurs between wild-type UGT2A1 and UGT2A1308Arg

. The

association of the UGT2A1308Arg

variant with NSCC and in particular squamous cell

carcinoma is consistent with the hypothesis that UGT2A1 metabolism of PAHs protects

the lung and other extra-hepatic tissues from tobacco carcinogenesis. The decrease in

70

wild-type UGT2A1 activity following UGT2A1308Arg

co-expression is a novel regulatory

mechanism for UGT2A1 that may have implications on cancer risk.

3.2 Introduction

Lung cancer is the leading cause of cancer mortality in the United States, with the

annual number of lung cancer deaths greater than deaths from colon, breast, and prostate

cancer combined [12]. There is an indisputable link between tobacco smoking and lung

cancer, with greater 90% of lung cancer deaths in men and 80% of lung cancer deaths in

women attributed to cigarette smoking; however, it is estimated that only 15% of lifetime

smokers develop lung cancer [246]. Variability in the susceptibility of smokers to

develop lung cancer suggests that inter-individual differences in heritable traits may

predispose individuals to developing tobacco-induced lung cancer [247, 248]. PAHs,

including the well-studied compound B(a)P, make up a subset of the more than 60

carcinogens in tobacco smoke [19]. PAH compounds have been implicated in the

formation of squamous cell carcinoma in both animal and human studies [21, 33, 249].

PAHs require metabolic activation prior to the formation of DNA adducts, and both CYP

and epoxide hydrolase enzymes convert pro-carcinogenic PAHs to their carcinogenic

form. In competing detoxification pathways, phase II enzymes such as GSTs, SULTS,

and UGTs metabolize activated PAHs so that they are eliminated from the body [19, 116,

250]. The balance between PAH activation and detoxification is thought to play a role in

cancer risk [19], and polymorphisms in phase I and phase II enzymes impact the balance

of carcinogen activation and detoxification.

71

Polymorphisms have been previously characterized for many UGT genes, and

several studies have examined the role of UGT SNPs in tobacco carcinogen metabolism

[98, 106, 118, 177]. UGT2A1 exhibits two known non-synonymous coding

polymorphisms with a >1% minor allele frequency (MAF) as determined by a review of

HapMap [251]. A SNP (rs1347046) at base +224 (encoded by what we refer to as the

UGT2A1*2 allele) results in a conservative lysine to arginine amino acid change at

codon 75. This SNP, according to HapMap, has a relatively low MAF of 1.1% in Han

Chinese and is not reported in Caucasian, Yoruban, or Japanese populations [251]. A

second SNP (rs4148301) at base +922 (encoded by what we refer to as the UGT2A1*3

allele) results in a non-conservative glycine to arginine amino acid change at codon 308.

This SNP, according to HapMap, exhibits a MAF of 13% in Caucasians and at least 4%

in all other populations analyzed [251]. The effects of these amino acid changes on

UGT2A1 activity were not previously investigated.

UGT polymorphisms have been implicated in orolaryngeal, gastrointestinal,

colorectal, lung, breast, pancreatic and prostate cancer risk [67]. Work by our lab has

shown that multiple UGT polymorphisms are associated with changes in lung and

orolaryngeal cancer risk. UGT2B17 has been shown to be expressed in the lung and to

metabolize the TSNA metabolite NNAL, and a prevalent UGT2B17 deletion phenotype

was determined to be associated with an increased risk for lung adenocarcinoma in

women [177]. UGT1A7 is expressed in the aerodigestive tract and has reported

glucuronidation activity against both PAH and TSNA substrates, and low-activity

UGT1A7 polymorphisms were determined to be associated with increased risk for

orolaryngeal cancer in both light and heavy smokers [169]. UGT1A10 is expressed in the

72

upper aerodigestive tract and has reported activity against PAH metabolites, and a

polymorphism at codon 139 in UGT1A10 was found to be associated with decreased

orolaryngeal carcinoma risk in black individuals [175]. Additional cancers have also been

found to be associated with low-activity UGT polymorphisms, including UGT1A7

variants with pancreatic cancer [172] and UGT1A1 variants with breast cancer [138].

Recent studies have suggested that UGTs form dimers and potentially higher

order oligomers in the membrane, and multiple studies have analyzed the implications of

UGT oligomerization on enzyme structure and function [83]. Both homo-dimerization

and hetero-dimerization have been reported for UGT1A and UGT2B isoforms through in

vitro co-expression studies, with the effects of dimerization on enzyme activity complex

and substrate specific [212-214]. Homo-dimerization of truncated or inactive UGT

isoforms with their wild-type counterpart has also been reported for multiple UGTs. A

Cys127Tyr polymorphism in UGT1A1 leads to an inactive protein isoform that was

reported to dimerize with wild-type UGT1A1 and inhibit UGT1A1 activity against

bilirubin [220]. Similarly, a Gln331Stop polymorphism in UGT1A1, leading to a

truncated and inactive protein isoform, was shown to inhibit wild-type UGT1A1

glucuronidation activity when both variants were co-expressed in the same cell system

[219]. UGT1A_i2 variants containing an alternate exon 5b were also shown to negatively

regulate wild-type UGT1A glucuronidation through protein-protein interactions [218].

The primary goal of the current study was to determine the effect of non-

synonymous coding SNPs on UGT2A1 activity, with a focus on the metabolism of

tobacco carcinogens, and to investigate the potential associations of functional UGT2A1

SNPs with lung cancer risk. Once it was determined that UGT2A1308Arg

had no detectable

73

glucuronidation activity, a secondary goal was to characterize the potential negative

regulation of wild-type UGT2A1 activity by the UGT2A1308Arg

variant, a mechanism that

had been described previously for other UGT isoforms.

3.3 Methods

Chemicals and Materials. The SNP genotyping assays to detect the UGT2A1*2

and UGT2A1*3 alleles were purchased from Applied Biosystems Inc., Life Technologies

(Carlsbad, CA). UDPGA, alamethicin, -glucuronidase, 4-MU, 1-OH-pyrene, and 1-

naphthol were purchased from Sigma-Aldrich (St. Louis, MO). 1-OH-B(a)P, 3-OH-

B(a)P, 7-OH-B(a)P, 8-OH-B(a)P, 5-methylchrysene-1,2-diol, dibenzo(a,l)pyrene-11,12-

diol, and B(a)P-7,8-diol were synthesized in the Organic Synthesis Core Facility at the

Penn State College of Medicine (Hershey, PA). HPLC-grade ammonium acetate, agarose,

and acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA). The

QuikChange® site directed mutagenesis (SDM) kit, Complete Control Inducible

Mammalian Expression System kit, and Pfu High Fidelity DNA Polymerase were

purchased from Agilent (Santa Clara, CA). All oligonucleotides for PCR were purchased

from IDT (Coralville, IA). The pcDNA6.2/V5/GW/D-TOPO mammalian expression

vector kit, Superscript II RT kit, Lipofectamine 2000, and ampicillin were acquired from

Invitrogen, Life Technologies (Grand Island, NY). The BCA protein assay kit was

purchased from Pierce (Rockford, IL). The RNeasy kit, QIAquick gel extraction kit,

Plasmid Mini kit, and Plasmid Maxi kit were all obtained from Qiagen (Valencia, CA).

74

DMEM, DPBS (minus calcium-chloride and magnesium-chloride), FBS, penicillin-

streptomycin, blasticidin, and G418 were purchased from Gibco, Life Technologies

(Grand Island, NY).

Genotyping assays to determine allelic prevalence of UGT2A1*2 and

UGT2A1*3. The UGT2A1*2 (Lys75Arg) and UGT2A1*3 (Gly308Arg) SNPs were

genotyped using TaqMan drug metabolism genotyping assays. Control genomic DNAs

from 187 Caucasians, 112 African Americans, and 30 Asian individuals were used to

verify the allelic prevalence of both SNPs. Briefly, controls were recruited as part of

previous case-control studies examining risk factors important in oral cancer risk.

Controls were self-reported to have no previous diagnosis of cancer and were recruited

between 1994 and 2000 from the Temple University Hospital (Philadelphia, PA) and the

New York Eye and Ear Infirmary (New York, NY) [169, 175].

For each genotyping assay, 10 ng of genomic DNA was used for each reaction

and water was used as a negative control. To analyze the UGT2A1*2 SNP, a

commercially-available drug metabolism genotyping assay (C_8851830_30) was used to

determine the allelic frequency of the A and G alleles at base 224. For the UGT2A1*3

SNP, a custom ABI genotyping assay (AH5H88C) was designed, with sense (5’-

GGAAGAATTTATCCAGAGCTCAGGTAA-3’) and anti-sense (5’-TGAGGCAATA-

AGATTGGCCTTTTCT-3’) primers corresponding to nucleotides +870 to +897 and

+973 to +948, respectively, relative to the UGT2A1 translation start site. The probe used

in this assay was TGTTTTCTCTG[G/A]GATCAA, corresponding to nucleotides +911 to

+929 relative to the UGT2A1 translation start site [the bracketed nucleotides represent

75

the UGT2A1*1 (G) and UGT2A1*3 (A) alleles at base 922]. The probe for the wild-type

G allele was labeled with VIC, and the probe for the variant A allele was labeled with

FAM. Genotyping assays were completed in the Penn State College of Medicine

Functional Genomics Core Facility (Hershey, PA), using an ABI 7900 HT thermal cycler

with data analyzed using SDS 2.2 software. Automatic calls were generated using the

SDS software, and calls were verified by analyzing the absolute quantification plots for

each sample. Genotype frequencies in each population were checked for Hardy-Weinberg

Equilibrium.

Creation of HEK293 cell lines over-expressing UGT2A1 variants. The variant

UGT2A1*2 and UGT2A1*3 alleles were created by SDM of the pcDNA3.1/V5-His-

TOPO plasmid expressing the wild-type UGT2A1*1 allele (described in Chapter 2). The

SNPs were induced using the QuikChange® SDM kit as described previously [98]. The

primers used to change base +224 from an A (UGT2A1*1) to G (UGT2A1*2) were:

sense, 5’-CATTTGAAATATATAGGGTGCCCTTTGGC-3’, and anti-sense, 5’-

GCCAAAGGGCACCCTATATATTTCAAATG-3’, both corresponding to nucleotides

+209 to +237 from the translation start site. The primers used to change base 922 from G

(UGT2A1*1) to A (UGT2A1*3) were: sense, 5’-GTGGTGTTTTCTCTGAGATCAAT-

GGTCAAAAAC-3’, and anti-sense, 5’-GTTTTTGACCATTGATCTCAGAGAAAA-

CACCAC-3’, both corresponding to nucleotides +907 to +940 from the translation start

site. The underlined base in each primer denotes the base pair change. The UGT2A1*2

and UGT2A1*3 cDNA sequences were confirmed by dideoxy sequencing. After a large-

scale plasmid prep, electroporation was used to generate the HEK293 cell lines over-

76

expressing either UGT2A175Arg

or UGT2A1308Arg

, using 10 μg of the pcDNA 3.1/V5-His-

TOPO_UGT2A1*2 or pcDNA 3.1/V5-His-TOPO_UGT2A1*3 vectors respectively.

Cells were grown in DMEM supplemented with FBS and G418 to 70% confluence. Cell

homogenates were prepared essentially as previously described [98]. Total RNA was

extracted using the RNeasy Mini kit using manufacturer’s protocols. Total homogenate

protein concentrations were determined using the BCA protein assay.

Determination of UGT2A1 over-expression in HEK293 cell lines. The specificity

and design of a UGT2A1 specific antibody was described previously (Chapter 2). The

levels of UGT2A1 protein in the wild-type UGT2A1, UGT2A175Arg

, and UGT2A1308Arg

over-expressing cell lines were measured using the anti-UGT2A1 antibody at a 1:500

dilution. Fifty μg of total protein homogenate from each UGT2A1 cell line was loaded

into each lane and the monoclonal β-actin antibody (Sigma-Aldrich) was used as a

loading control. The intensity of UGT2A1 signal was measured with the ImageJ program

(NIH). As a UGT2A1 standard is not commercially available, the relative expression of

UGT2A1 in homogenate from each cell line was calculated relative to the cell line with

the highest UGT2A1 expression (wild-type UGT2A1). The relative UGT2A1 protein

levels for all three cell lines were expressed as the mean of three independent

experiments, and enzyme kinetics were normalized based on relative UGT2A1 protein

expression in each UGT2A1 over-expressing cell line.

Glucuronidation assays to determine functional effects of UGT2A1 SNPs.

Glucuronidation assays using homogenates from HEK293 cell lines over-expressing

77

UGT2A175Arg

and UGT2A1308Arg

were performed essentially as previously described

[234]. Briefly, after an initial incubation of total cell homogenate protein (100 μg) with

alamethicin (50 μg/mg protein) for 15 min on ice, glucuronidation reactions were

performed in a final reaction volume of 25 μL at 37°C with 50 mM Tris-HCl (pH 7.4), 10

mM MgCl2, 4 mM UDPGA, and between 6 and 750 μM of substrate. For each substrate,

the glucuronidation rate was determined at 8 concentrations that encompassed the KM of

the substrate. Reactions were terminated by the addition of 25 μL cold acetonitrile on ice.

Reaction mixtures were centrifuged for 10 min at 16,100 g prior to the collection of

supernatant. For glucuronidation rate determinations, cell homogenate protein levels and

incubation times for each substrate were determined experimentally to ensure that

substrate utilization was less than 10% and to maximize levels of detection while in a

linear range of glucuronide formation.

Levels of glucuronide formation were determined using a Waters Acquity UPLC

System (Milford, MA) as previously described [110, 112, 234, 236]. The flow rate was

maintained at 0.5 mL/min and a reverse phase Acquity UPLC BEH C18 - 1.7 μm - 2.1 x

100 mm column was used to separate glucuronide and substrate. A gradient of solution

A (5 mM NH4OAc (pH 5.0), 10% acetonitrile) and solution B (100% acetonitrile) was

used to elute the glucuronide and substrate from the column. The initial solvent gradients

and UV absorbance wavelengths for each substrate were described previously (Chapter

2). Quantification of glucuronide formation for each substrate was completed essentially

as previously described [75, 112, 118, 161]. Briefly, the amount of glucuronide formed

was determined based on the ratio of glucuronide versus unconjugated substrate after

calculating the area under the curve for the substrate and glucuronide peaks using the

78

known amount of substrate in each reaction as the reference. Glucuronides were

confirmed by sensitivity to β-glucuronidase, by mass spectrometry analysis, and in the

case of 1-naphthol, by comparison to an authentic 1-naphthol glucuronide standard.

Reactions with non-transfected HEK293 cell homogenate, no substrate added to the

reaction mixture, or only substrate and no homogenate in the reaction mixture were used

as negative controls. 4-MU, a common UGT substrate [237], was used as positive control

for UGT2A175Arg

or UGT2A1308Arg

activity

Study population for case-control study. Genomic DNA used for the lung cancer

association study was obtained from a case-control study completed at the H. Lee Moffitt

Cancer Center (Tampa, FL) between 2000 and 2003. Caucasian lung cancer cases

(n=391) were histologically confirmed, and these subjects were reported to have no prior

history of tobacco-related cancer. Caucasian controls (n=624) were randomly selected

from community residents involved with the Lifetime Cancer Screening facility at the H.

Lee Moffitt Cancer Center. This facility conducted community outreach and education

programs in the Tampa area, and control subjects commonly underwent prostate specific

antigen testing, skin examinations, endoscopies, and mammograms. A list of control IDs

was matched with a hospital database to identify any subject that developed cancer

following control sample collection, and all control subjects with a new cancer diagnosis

were excluded from the control population. All study subjects signed a consent form

approved by the institutional review board, and a trained interviewer delivered a

questionnaire that covered lifestyle questions including smoking status, education,

occupation, and cigarettes smoked per day. For smoking status, a current smoker had

79

smoked at least 1 cigarette a day for the past year. Smoking status information for a

subset of cases and controls was unknown, and lung cancer histology for a small subset

of cases was also not determined. The questionnaire used was based off of a previously

validated questionnaire used by investigators at the American Health Foundation in a

large hospital-based case-control study [252]. Medical charts of lung cancer case subjects

were reviewed to determine diagnostic and pathology records.

Association of UGT2A1308Arg

with lung cancer cases. To determine the allelic

prevalence of the UGT2A1*3 SNP in case and control populations, the custom TaqMan

drug metabolism SNP genotyping assay (AH5H88C) was used as previously described.

SNP genotyping assays were completed at the Penn State University Genomics Core

Facility (State College, PA) using an ABI 7900HT thermal cycler. Thermal cycling

conditions used were 50°C for 2 min, 95°C for 10 min, and then 40 cycles at 92°C for 15

s and 60°C for 1 min. A post-amplification allelic discrimination run was used to

determine the genotype of each sample based on VIC or FAM fluorescence. Data was

analyzed using SDS 2.4 software. Genotype calls were verified by analyzing the absolute

quantification plots for each sample and DNAs from cell-lines over-expressing

UGT2A1*1 or UGT2A1*3 were used as positive controls.

Creation of wild-type UGT2A1 and UGT2A1308Arg

co-expressed cell line. The

creation of pcDNA3.1/V5-His-TOPO_UGT2A1*1 or pcDNA3.1/V5-His-TOPO_

UGT2A1*3 vector constructs was previously described. Both vectors were used in

conjunction with alternate PCR primers to make additional vector constructs. UGT2A1*1

80

was PCR amplified from the pcDNA3.1_UGT2A1*1 vector using a sense primer 5’-

CACCATGTTAAACAACCTTCTGC-3’ (UGT2A1_S2) and anti-sense primer 5’-

TTCTCTTTTTTTCTTCTTTCCTATCTTACC-3’ (UGT2A1_AS2), corresponding to

nucleotides +1 to +19 and +1581 to +1552, respectively, relative to the UGT2A1

translation start site. This primer pair amplified the entire coding region of wild-type

UGT2A1 minus the stop codon (underlined nucleotides in sense primer indicate CACC

anchor), and following this amplification the PCR product was then cloned into a

pcDNA6.2/V5/GW/D-TOPO vector using the standard protocol. UGT2A1*3 was PCR

amplified from the pcDNA3.1_UGT2A1*3 vector and then cloned into the pEGSH

vector, using a similar primer set to that described above but containing a Xho I

restriction site (underlined) on the 5’ end of the sense primer 5’-GCACTCGAGATGTT-

AAACAACCTTCTGC-3’ (UGT2A1_S3) and a Xba I restriction site (underlined) on the

5’ end of the anti-sense primer 5’GATTCTAGACGTTCTCTTTTTTTCTTCTTTCCT-

ATCTTACC-3’ (UGT2A1_AS3). Vector sequences were verified and vectors were

amplified as described previously.

The pEGSH vector is part of an inducible mammalian expression system, and this

system was used to control UGT2A1308Arg

expression levels. The pEGSH_UGT2A1*3

vector contains hygromycin resistance and has the capability to create a FLAG tagged

protein, while the regulatory pERV vector necessary for the inducible expression system

has G418 resistance. The pcDNA 6.2_UGT2A1*1 vector contains blasticidin resistance

and has the capability to create a V5 tagged protein. Eight μg each of the

pEGSH_UGT2A1*3 and pERV regulatory vector, comprising the inducible system, and

the pcDNA 6.2_ UGT2A1*1 vector were stably transfected into HEK293 cells using a

81

standard lipofectamine protocol. Selection of HEK293 cells over-expressing the three

vectors was completed using a combination of 400 μg/mL G418, 9 μg/mL blasticidin,

and 75 μg/mL hygromycin B in DMEM media with 10% FBS. Multiple clones were

analyzed for inducible gene expression, and a stable cell line over-expressing all three

vectors simultaneously was chosen based on the efficiency of UGT2A1308Arg

induction.

After the creation of the stable UGT2A1308Gly

_ V5/UGT2A1308Arg

_FLAG/pERV

cell line, UGT2A1308Arg

_FLAG expression was induced through addition of the ecdysone

analog Ponasterone A (PonA). HEK293 cells at 50% confluence were treated with 10μM

of PonA in ethanol for 12 h. Ethanol alone at 0.01% was added as a negative control.

Cells were harvested and homogenates were made as previously described [75, 112].

Protein homogenates from the untreated control and 10 μM PonA treatment groups were

screened for wild-type UGT2A1308Gly

_V5 and UGT2A1308Arg

_FLAG expression through

Western blots. 50 μg of total protein was loaded in each lane and induction of

UGT2A1308Arg

_FLAG and protein quantification was completed in triplicate. Wild-type

UGT2A1308Gly

_V5 expression was determined using a monoclonal mouse horseradish

peroxidase (HRP) conjugated V5 antibody (Invitrogen) at a 1:5000 dilution, while

UGT2A1308Arg

_FLAG expression was determined using a monoclonal mouse anti-Flag

antibody (Sigma-Aldrich) at a 1:1000 dilution. The monoclonal β-actin antibody was

used as a loading control. With two different antibodies used to determine relative wild-

type UGT2A1308Gly


_FLAG expression levels, a Western blot was

also completed using the anti-UGT2A1 antibody. This was completed to confirm the

levels of UGT2A1308Arg

_FLAG induction and correct for any differences in V5 and

FLAG antibody affinities. Fifty μg of protein from the untreated control group and 10μM

82

PonA treatment group was loaded into each lane, and an anti-UGT2A1 antibody at a

1:500 dilution was used as described previously.

Glucuronidation assays to determine the effect of UGT2A1308Arg

co-expression on

wild-type UGT2A1 activity. Following verification of wild-type UGT2A1308Gly

_V5 and

UGT2A1308Arg

_FLAG protein levels, homogenates were prepared and used for activity

assays as previously described [75, 110, 112]. Cell lysates were homogenized for 10

seconds on ice using a Bio-Vortexer (Biospec Products, Bartlesville OK).

Glucuronidation activity was determined against four PAHs which were previously

shown to be substrates of UGT2A1, with glucuronidation assays using homogenates from

HEK293 cells over-expressing wild-type UGT2A1308Gly

_V5 alone and co-expressing

wild-type UGT2A1308Gly


_FLAG completed as previously

described [234, 235]. Briefly, after an initial incubation of 100 μg protein homogenate

with alamethicin (50 μg/mg protein) for 15 min on ice, glucuronidation reactions were

performed in a final reaction volume of 25 μL at 37°C with 50 mM Tris-HCl (pH 7.4), 10

mM MgCl2, 4 mM UDPGA, and substrate. Reactions were terminated by the addition of

25 μL cold acetonitrile on ice. Reaction mixtures were centrifuged for 10 min at 16,100 g

prior to the collection of supernatant.

Glucuronide formation was determined using a Waters Acquity UPLC System

(Milford, MA) as previously described [75, 110, 112, 234, 236]. The flow rate was

maintained at 0.5 mL/min and a reverse phase Acquity UPLC BEH C18 - 1.7 μm 2.1 x

100 mm column was used to separate free substrate and the conjugated glucuronide. A

gradient of solution A (5 mM NH4OAc pH 5.0, 10% acetonitrile) and solution B (100%

83

acetonitrile) was used to elute the glucuronide and substrate from the column. The initial

solvent gradients and UV absorbance wavelengths used to detect glucuronidation of

various substrates were described previously (Chapter 2). Activity assays were completed

in triplicate for each substrate examined, using homogenate from the control and 10 μM

PonA treatment groups. For each substrate, the glucuronidation rate was determined at

the previously established KM for each substrate. For glucuronidation rate determinations,

cell homogenate protein levels and incubation times for each substrate were determined

experimentally to ensure that substrate utilization was less than 10% and to maximize

levels of detection while in a linear range of glucuronide formation. Cell lines over-

expressing either wild-type UGT2A1308Gly

_V5 or UGT2A1308Arg

_FLAG alone were

created, and activity of homogenates prepared from these cell lines was compared to

activity of homogenates from cell lines over-expressing wild-type UGT2A1 or

UGT2A1308Arg

with no C-terminal tags.

Co-IP assays to investigate dimerization of wild-type UGT2A1 with

UGT2A1308Arg

. HEK293 cells over-expressing UGT2A1308Gly

_ V5/UGT2A1308Arg

_FLAG

were treated with 10uM PonA for 12 h. Cells were washed with PBS then lysed for one

hour on ice and homogenized as described previously, in a lysis buffer containing 0.05 M

Tris-HCl, pH 7.4, 0.15M M NaCl, 0.3% deoxycholic acid, 1% Igepal, and 1 mM EDTA

[218]. A Dynabead Protein G Immunoprecipitation Kit (Invitrogen) was used to

determine potential protein interactions between wild-type UGT2A1 and UGT2A1308Arg

.

3 μg of mouse monoclonal anti-FLAG antibody or 1.5 μg of mouse monoclonal V5

antibody (Santa Cruz Biotechnology; Santa Cruz, CA) was incubated with the

84

Dynabeads. 2.5 μg/μL lysate from the UGT2A1308Gly

_ V5/UGT2A1308Arg

_FLAG

inducible cell line was incubated with the Dynabeads for 30 min. Following various

washes, the immunoprecipitated protein complex was eluted and heated at 90°C for 10

min in a sodium dodecyl sulfate loading buffer. The resulting co-IP supernatant was run

on an acrylamide gel, and samples were analyzed for the presence UGT2A1308Gly

_V5

with a monoclonal mouse V5-HRP antibody at a 1:5000 dilution or

UGT2A1308Arg

_FLAG with a mouse FLAG-HRP antibody (Cell Signaling Technology;

Beverly, MA) at a 1:1000 dilution. Co-IP was repeated multiple times to confirm wild-

type UGT2A1 and UGT2A1308Arg

dimerization.

Data analysis and statistics. Western blots to detect levels of UGT2A1 over-

expression in wild-type UGT2A1 and UGT2A1 variant over-expressing cell lines were

completed in triplicate. The Student’s t-test was used to compare the KM, Vmax, and

Vmax/KM of glucuronide formation between the wild-type UGT2A175Lys

and

UGT2A175Arg

cell lines. For in vitro co-expression and glucuronidation activity studies,

Western blots to determine wild-type UGT2A1308Gly


_FLAG

levels and glucuronidation assays were completed in triplicate using homogenate from

each independent experiment. Levels of wild-type UGT2A1 and UGT2A1308Arg

were

determined through ImageJ, with UGT2A1308Arg

_FLAG expression represented as mean

± standard deviation (SD) relative to wild-type UGT2A1308Gly

_V5 expression (set to 1.0

as a reference). The Student’s t-test was used to compare glucuronidation rates using

protein homogenate when wild-type UGT2A1was over-expressed alone and when wild-

type UGT2A1 was co-expressed with UGT2A1308Arg

at approximately a 1:1 ratio.

85

Subject characteristics for the lung cancer case-control association study were

described by means, SDs, and percentages as calculated in SPSS 15.0 (SPSS, Inc.,

Chicago, IL). The association of the UGT2A1308Arg

polymorphism (UGT2A1*3 allele)

with lung cancer risk was modeled using an unmatched logistic regression analysis,

adjusting for known lung cancer risk factors including age, body mass index, education

level, and pack years of smoking. The control group was used as the reference group for

all analyses. The association between UGT2A1308Arg

and lung cancer risk was determined

using a dominant model and Fisher’s exact test, with the assumption that one copy of the

UGT2A1*3 allele increases lung cancer risk. The association of the UGT2A1308Arg

SNP

with lung cancer cases was also analyzed following stratification by lung cancer

histology type.

3.4 Results

Allelic prevalence of UGT2A1*2 and UGT2A1*3 SNPs. The prevalences of the

UGT2A1*2 and UGT2A1*3 alleles were examined in multiple populations. The

UGT2A1*2 allele (adenine to guanine at base +224) encodes a lysine to arginine change

at codon 75 and was previously reported by HapMap to only be expressed in a Han

Chinese population, at a MAF of 1.1% [251]. The UGT2A1*3 allele (guanine to adenine

at base +922), encodes a glycine to arginine amino acid change at codon 308 and was

reported by HapMap to be found in all populations examined, with the highest allelic

prevalence being 13.1% in Caucasians [251]. In the current study, the UGT2A1*2 allele

was determined to have an allelic frequency of 8.3% in Asians (n=30 subjects) and 4.0%

86

in both Caucasians (n=186 subjects) and African Americans (n=111 subjects; Table 3.1).

The UGT2A1*3 allele was found to have an overall allelic prevalence of 5.0% in Asians

(n=30 subjects), 10.4% in Caucasians (187 subjects), and 4.5% in African Americans

(n=112 subjects; Table 3.1). None of the subjects expressing the UGT2A1*2 allele also

expressed the UGT2A1*3 allele. For both variant alleles, the genotype distributions

followed Hardy-Weinberg equilibrium for all three populations examined.

UGT2A1 protein levels in over-expressing cell lines. To examine the effects of

two prevalent coding SNPs on UGT2A1 glucuronidation activity, the UGT2A175Arg

and

the UGT2A1308Arg

variants were cloned into HEK293 cell lines and their activities were

compared to the that of wild-type UGT2A175Lys308Gly

. Western blots were completed to

determine the relative level of UGT2A1 protein in each stable cell line using a UGT2A1

specific antibody, with relative UGT2A1 protein expression in each cell line used for

normalization of kinetic data. In the representative Western blot shown in Figure 3.1,

homogenates from the UGT2A175Arg

and UGT2A1308Arg

over-expressing cell lines were

determined to express slightly less UGT2A1 protein relative to the level of UGT2A1

expression in the wild-type UGT2A1 over-expressing cell line (set to 1.0 as the

reference). Western blot protein quantification was completed in triplicate; UGT2A175Arg

homogenate was determined to have a relative protein level of 0.94 ± 0.03 relative to

wild-type UGT2A1 expression, while UGT2A1308Arg

homogenate was determined to have

a relative protein level of 0.89 ± 0.04 relative to wild-type UGT2A1 expression. For

enzyme kinetics calculations, the glucuronidation rate at each substrate concentration was

normalized to the relative UGT2A1 protein expression in each UGT2A1 cell line.

87

Table 3.1. Prevalence of the UGT2A1*2 and UGT2A1*3 alleles.

a Informative results were obtained for 30 Asians, 186 Caucasians, and 111 African Americans for the UGT2A1*2 allele,

and 30 Asians, 187 Caucasians, and 112 African Americans for the UGT2A1*3 allele.

UGT2A1 Allele Genotype Caucasiansa

African Americansa

Asiansa

UGT2A1*2 AA 172 102 26

AG 13 9 3

GG 1 0 1

MAF 4.0% 4.0% 8.3%

UGT2A1*3 GG 151 102 27

GA 33 10 3

AA 3 0 0

MAF

10.4% 4.5% 5.0%

88

Figure 3.1. Representative Western blot showing relative UGT2A1 protein expression levels

in UGT2A1 over-expressing cell lines. The protein level of wild-type UGT2A1 homogenate was

set to 1.0 as a reference. UGT2A175Arg

homogenate had a relative protein level of 0.94 ± 0.03 and

UGT2A1308Arg

homogenate had a relative protein level of 0.89 ± 0.04, compared to the wild- type

UGT2A1 homogenate. The relative UGT2A1 protein expression in each cell line was determined

using the mean of three independent Western blot experiments, and β-actin was used as a loading

control.

Functional effects of UGT2A1 SNPs on glucuronidation activity. Homogenate

from HEK293 cells over-expressing the UGT2A175Arg

variant exhibited glucuronidation

activity against all PAH substrates examined. Kinetic analysis demonstrated that the

UGT2A175Arg

variant exhibited a significantly (p<0.05) lower Vmax/KM as compared to

wild-type UGT2A175Lys

for all nine PAH substrates examined (Table 3.2). For both 1-

naphthol and 5-methylchrysene-1,2-diol there was a statistically significant difference in

KM (p<0.05) between wild-type UGT2A1 and the UGT2A175Arg

variant, while for

dibenzo(a,l)pyrene 11,12-diol, B(a)P-7,8-diol, 1-OH-B(a)P, 3-OH-B(a)P, 7-OH-B(a)P,

and 8-OH-B(a)P there was a statistically significant difference in Vmax (p<0.05) between

wild-type

89

Table 3.2. Enzyme kinetics summary of wild-type UGT2A175Lys

and UGT2A175Arg

activity against PAHs.

Wild-type UGT2A175Lys

UGT2A175Arg

KM Vmax Vmax/KM KM Vmax Vmax/KM

Substrate (μM) (pmol/min/mg)a

(μL/min/mg)a

(μM) (pmol/min/mg)a

(μL/min/mg)a

1-OH-pyrene 91 ± 7.2 300 ± 17 3.3 ± 0.2 111 ± 19 269 ± 18 2.4 ± 0.2b

1-naphthol 30 ± 2.9 185 ± 11 6.2 ± 0.4 44 ± 11b

177 ± 20 4.0 ± 0.7b

5-methylchrysene-1,2-diol 270 ± 24 100 ± 7.2 0.37 ± 0.04 381 ± 37b

87 ± 7.4

0.23 ± 0.02b

dibenzo(a,l)pyrene-11,12-diol 284 ± 27 26 ± 1.0 0.09 ± 0.004 307 ± 8.9 22 ± 0.4b

0.07 ± 0.001b

B(a)P-7,8-diol 397 ± 33 109 ± 14 0.28 ± 0.04 426 ± 53 86 ± 8.1b

0.20 ± 0.009b

1-OH-B(a)P 247 ± 12 70 ± 4.4 0.28 ± 0.01 263 ± 29 61 ± 1.4b

0.23 ± 0.02b

3-OH-B(a)P 271 ± 15 84 ± 3.6 0.31 ± 0.008 278 ± 21 74 ± 1.6b

0.27 ± 0.014b

7-OH-B(a)P 261 ± 35 58 ± 4.6 0.22 ± 0.01 334 ± 20 49 ± 2.4b

0.15 ± 0.004b

8-OH-B(a)P 308 ± 20 76 ± 6.6 0.25 ± 0.008 368 ± 51 60 ± 5.1b

0.16 ± 0.02b

a Data expressed as mg of total protein homogenate, corrected for relative UGT2A1 protein from Western blot. KM, Vmax, and Vmax/KM

represent the mean of three independent experiments. b Denotes p<0.05 versus corresponding value for wild-type UGT2A1.

90

UGT2A1 and the UGT2A175Arg

variant. The UGT2A1308Arg

variant exhibited no

detectable glucuronidation activity against all of the PAHs tested and was inactive against

all other substrates examined in this study, including TSNAs, HCAs, and 4-MU, a known

UGT2A1 substrate (Chapter 2) and a common substrate of most UGT isoforms [237]

(using up to 400 µg cellular homogenate and 750 μM substrate in a 12 h incubation;

results not shown).

Association of UGT2A1308Arg

with lung cancer risk. The basic demographic

characteristics of subjects in the lung cancer case-control study are summarized in Table

3.3. Forty-four percent of cases and 48% of controls were female, while the mean ages of

cases and controls were 64.6 ± 9.9 and 58.1 ± 11, respectively. A significantly higher

(p<0.01) percentage of cases than controls were classified as current smokers (42% vs.

30%). Smoking history, expressed by the number of mean pack years, was also

significantly higher (p<0.01) for cases than controls (56.2 years vs. 24.9 years). Forty

percent of controls and 22% of cases had at least a college education. The mean BMI for

cases and controls was 27.0 ± 5.0 versus 27.1 ± 4.9. Stratifying by histology in the case

group, the most frequent histology was adenocarcinoma (36%) followed by squamous

cell carcinoma (21%). There was no observed interaction between smoking status and the

UGT2A1308Arg

SNP, and smoking (pack-years) was adjusted as a covariate because it is a

known significant independent predictor of lung cancer risk.

91

Table 3.3. Demographics and lung cancer histology of 391 lung cancer cases and 624

controls.

Cases [n (%)] Controls [n (%)] p value

Mean age*

Women (%)

Mean BMI*

Current Smoker

Yes (%)

No (%)

Pack years*

Years of education

<High school degree (%)

High school degree (%)

College degree (%)

Postgraduate degree (%)

Histology

Squamous cell-carcinoma (%)

Adenocarcinoma (%)

Undifferentiated NSCC (%)

Small-cell carcinoma (%)

Large cell carcinoma (%)

Other/mixed (%)

64.6 ± 9.9

171 (44)

27.0 ± 5.0

149 (42)

203 (58)

56.2 ± 39

60 (15)

245 (63)

58 (15)

28 (7)

83 (21)

139 (36)

78 (20)

30 (8)

28 (7)

32 (8)

58.1 ± 11

298 (48)

27.1 ± 4.9

123 (30)

280 (70)

24.9 ± 32

24 (4)

349 (56)

151 (24)

100 (16)

<0.01

0.21

0.89

<0.01

<0.01

<0.01

* Mean ± SD

Table 3.4 summarizes the distribution of UGT2A1*3 genotypes by lung cancer

case-control status, as well as the genotypes of the cases stratified by lung cancer

histology. The prevalence of the UGT2A1*3 allele in the control population was

consistent with Hardy-Weinberg equilibrium (p=0.18). The overall allelic prevalence of

the UGT2A1*3 allele in controls was determined to be 8.4%, similar to the allelic

prevalence previously described for UGT2A1*3 in Caucasians (Table 3.1). Using a

92

dominant model, in which only one copy of the UGT2A1*3 allele is needed to increase

risk [253], and the genotypes of the control population as a reference, no association was

detected between the UGT2A1*3 allele and overall lung cancer risk (p=0.09). However,

a significant (p=0.02) association was observed between the UGT2A1*3 allele and

overall risk for NSCC. Following stratification by lung cancer histology, there was a

significant association (p=0.04) between the UGT2A1*3 allele and squamous cell

carcinoma risk. No association was observed between the UGT2A1*3 allele and

adenocarcinoma (p=0.31) or large cell carcinoma risk (p=0.79). Using a recessive model,

in which two copies of the UGT2A1*3 allele are needed to increase risk [253], no

significant associations were found between the UGT2A1*3 allele and overall lung

cancer risk or lung cancer risk stratified by histology

Table 3.4. Distribution of UGT2A1*3 genotypes in lung cancer cases and controls.

* p values adjusted for age, BMI, education level, and pack years of smoking

UGT2A1 Genotype

G/G

G/A

A/A

UGT2A1*3

prevalence

p value *

Controls

526

91

7

8.4%

REF

All Cases

313

73

5

10.6%

0.09

All NSCC

256

67

5

11.7%

0.02

Squamous Cell Carcinoma

62

20

1

13.3%

0.04

Adenocarcinoma

112

24

3

11.6%

0.31

Large Cell Carcinoma

23

5

0

8.9%

0.79

93

Effect of UGT2A1308Arg

co-expression on wild-type UGT2A1 enzyme activity.

An association between the UGT2A1308Arg

variant and lung cancer risk was observed

using a dominant association model. Previous studies have suggested that inactive UGT

variants negatively regulate wild-type UGT isoforms through dimerization [218, 220].

To explore the possibility that the inactive UGT2A1308Arg

directly inhibits wild-type

UGT2A1 activity, an inducible co-expression system was generated. Wild-type

UGT2A1was over-expressed at a constant level using a stable promoter, while

UGT2A1308Arg

expression was regulated by the ecdysone analog PonA. In order to more

easily differentiate between the UGT2A1 isomers and to also perform co-IP experiments,

wild-type UGT2A1 was V5-tagged while UGT2A1308Arg

was FLAG-tagged, both at the

C-terminus of the protein. As shown by Western blot analysis (Figure 3.2, Panel A), no

detectable expression of UGT2A1308Arg

_FLAG was observed in the group not treated

with PonA. UGT2A1308Arg

_FLAG expression was induced following the addition of 10

10 μM PonA , while wild-type UGT2A1308Gly

_ V5 expression remained relatively

constant. UGT2A1308Arg

_FLAG induction and protein quantification were completed in

triplicate experiments; treatment with 10 µM PonA was shown to induce

UGT2A1308Arg

_FLAG expression to 1.12 ± 0.05 relative to wild-type UGT2A1308Gly

_V5

expression (set to 1.0 as a reference). Relative wild-type UGT2A1308Gly

_V5 protein levels

were determined for the untreated control and PonA treatment groups, and enzyme

kinetic data was normalized to the amount of relative wild-type UGT2A1308Gly

_V5

expression in each group. Expression of wild-type UGT2A1308Gly

_V5 in the untreated

control group was determined to be 0.93 ± 0.06 that of wild-type UGT2A1308Gly

_V5

expression in the 10 μM PonA treatment group (set to 1.0 as a reference). Expression

94

levels of wild-type UGT2A1308Gly


_FLAG were confirmed by

Western blot using an anti-UGT2A1 antibody (Figure 3.2, Panel B). Using this UGT2A1-

specific antibody, the total amount of UGT2A1 protein (wild-type UGT2A1308Gly

_V5 +

UGT2A1308Arg

_FLAG) following 10 μM PonA treatment was determined to be 2.14, as

compared to wild-type UGT2A1308Gly

_V5 expression in the untreated control group (set

to 1.0 as a reference). This confirmed the roughly 1:1 ratio detected between wild-type

UGT2A1308Gly


_FLAG expression in the 10 μM PonA treatment

group using the anti-V5 and anti-FLAG antibodies.

Figure 3.2. UGT2A1308Arg

_FLAG expression is induced by the ecdysone analog PonA.

(A) Representative Western blot showing stable expression of wild-type UGT2A1308Gly

_V5 and

induction of UGT2A1308Arg

_FLAG expression in HEK293 cells. UGT2A1308Arg

_FLAG expression

was induced through addition of 10 μM of Ponasterone A (PonA) for 12 h. The expression level

of wild-type UGT2A1308Gly

_V5, as well as the relative ratio of wild-type UGT2A1308Gly

_V5:

UGT2A1308Arg

_FLAG expression, was determined for the untreated control cells and cells treated

with PonA. Induction of UGT2A1308Arg

_FLAG expression and Western blots were completed in

triplicate. β-actin was used as a loading control. (B) A Western blot with 50 μg protein from the

untreated control and 10 μM PonA treatment groups was completed using an anti-UGT2A1

antibody to confirm expression levels detected by the anti-V5 and anti-FLAG antibodies. The

anti-UGT2A1 antibody was used at a 1:500 dilution and β-actin was used as a loading control.

95

Activity assays and enzyme kinetics were completed to determine the impact of

UGT2A1308Arg

co-expression on wild-type UGT2A1 enzyme activity. The effect of

UGT2A1308Arg

_FLAG co-expression on wild-type UGT2A1308Gly

_V5 glucuronidation

activity was determined using the following PAHs as substrates: 1-OH-pyrene, 1-OH-

B(a)P, B(a)P-7,8-diol, and 5-methylchrysene-1,2-diol. These four substrates represent

PAHs of varying complexity and were all previously shown to be substrates of UGT2A1.

Glucuronidation rates were corrected for the relative level of wild-type

UGT2A1308Gly

_V5 in the untreated control and PonA treatment groups. As shown in

Table 3.5, kinetic analyses suggested that UGT2A1308Arg

_FLAG co-expression with wild-

type UGT2A1308Gly

_V5 at approximately a 1:1 ratio caused a statistically significant

decrease in the rate of wild-type UGT2A1308Gly

_V5 glucuronidation of the four PAH

substrates analyzed (p<0.001 for 1-OH-pyrene, 1-OH-B(a)P, and B(a)P-7,8-diol; p<0.005

for 5-methylchrysene-1,2-diol). PonA treatment alone had no effect on wild-type

UGT2A1308Gly

_V5 enzyme activity (data not shown). The C-terminal V5 tag also caused

no significant changes in UGT2A1 enzyme activity, as the glucuronidation rates of wild-

type UGT2A1308Gly

_V5 in the untreated control group were similar to glucuronidation

rates reported previously for UGT2A1 against PAH substrates (Chapter 2). PAH

glucuronidation rates measured over a range of substrate concentrations suggested that

co-expression of UGT2A1308Arg

caused no significant changes in the wild-type UGT2A1

KM (data not shown).

96

Table 3.5. Enzyme kinetics summary of wild-type UGT2A1 and wild-type UGT2A1/UGT2A1308Arg

activity against PAHs.

For glucuronidation rate determinations, substrate concentrations at the experimentally determined UGT2A1 KM were used

** p<0.001; * p<0.005

aGlucuronidation rates were corrected for relative levels of wild-type UGT2A1 expression in each treatment group

Glucuronidation rate (pmol/min/mg)a

UGT(s) over-expressed

1-OH-pyrene

1-OH-B(a)P

B(a)P-7,8-diol

5-methylchrysene-

1,2-diol

Wild-type UGT2A1

307 ± 13 104 ± 4.7 151 ± 5.9 94 ± 10

Wild-type UGT2A1/UGT2A1308Arg

232 ± 19**

76 ± 4.4**

108 ± 12**

68 ± 5.8*

97

UGT2A1308Arg

and wild-type UGT2A1 dimerization. To test whether a protein-

protein interaction occurs between UGT2A1308Arg

and wild-type UGT2A1, protein lysate

following 10 μM PonA treatment was used in co-IP experiments. Using an anti-FLAG

antibody to immunoprecipitate UGT2A1308Arg

_FLAG and an anti-V5-HRP antibody to

detect wild-type UGT2A1308Gly

_ V5 by Western blot analysis, a band corresponding to

wild-type UGT2A1308Gly

_V5 (~57 kDa) was detected (Figure 3.3, Panel A; lane 5). As

expected, wild-type UGT2A1308Gly

_V5 was detected by Western blot analysis when an

anti-V5 antibody was used to pull down wild-type UGT2A1308Gly

_V5 as positive control

(Figure 3.3, Panel A; lane 4), while no bands were detected when no antibody was added

to the immunoprecipitation lysate (Figure 3.3, Panel A; lane 3) and when lysate was used

from HEK293 cell lines over-expressing either wild-type UGT2A1308Gly

_V5 or

UGT2A1308Arg

_FLAG alone (Figure 3.3, Panel A; lanes 1-2). Similarly, a band

corresponding to UGT2A1308Arg

_FLAG (~57 kDa) was detected when an anti-V5

antibody was used to immunoprecipitate wild-type UGT2A1308Gly

_V5 and an anti-Flag-

HRP antibody was used to detect UGT2A1308Arg

_FLAG by Western blot (Figure 3.3,

Panel B; lane 5). A UGT2A1308Arg

_FLAG band was observed by Western blot analysis

when an anti-FLAG antibody was used to pull down UGT2A1308Arg

_FLAG as a positive

control (Figure 3, Panel B; lane 4), while no bands were observed when no antibody was

added to the immunoprecipitation lysate (Figure 3.3, Panel B, lane 3) and when lysate

was used from HEK293 cell lines over-expressing either wild-type UGT2A1308Gly

_V5 or

UGT2A1308Arg

_FLAG alone (Figure 3.3, Panel B; lanes 1-2).

98

Figure 3.3. Dimerization between wild-type UGT2A1 and UGT2A1308Arg

demonstrated by

co-IP. (A) The wild-type UGT2A1308Gly

_V5/UGT2A1308Arg

_FLAG complex was

immunoprecipitated with an anti-FLAG antibody, then visualized with a HRP labeled V5

antibody. Homogenates from cells over-expressing wild-type UGT2A1308Gly

_V5 (lane 1) or

UGT2A1308Arg

_FLAG (lane 2) alone were used as negative controls. Dynabeads without antibody

conjugation were used as an additional negative control (lane 3). The wild-type

UGT2A1308Gly

_V5:UGT2A1308Arg

_FLAG complex was immunoprecipitated with an anti-V5

antibody, and then visualized with an anti-V5-HRP antibody as a positive control (lane 4). Co-IP

suggested that the wild-type UGT2A1308Gly


_FLAG isoforms dimerize, as

shown by the signal observed in lane 5 when the complex was immunoprecipitated with an anti-

FLAG antibody and probed with an anti-V5-HRP antibody. (B) In order to verify the interaction

between wild-type UGT2A1308Gly


_FLAG, co-IP was also completed by

immunoprecipitating the complex with an anti-V5 antibody visualizing the complex with an anti-

FLAG-HRP antibody. Positive and negative controls identical to those used in Panel A were

used. Once again, results shown in lane 5 suggested a protein-protein interaction between wild-

type UGT2A1308Gly


_FLAG. Co-IP results were repeated multiple times to

verify the protein-protein interactions between wild-type UGT2A1 and UGT2A1308Arg

.

99

3.5 Discussion

Functional polymorphisms have been characterized for many UGT genes, and

multiple UGT polymorphisms have been shown to significantly alter enzyme activity and

impact cancer risk. Results from the present study demonstrate that UGT2A1

polymorphic variants exhibit decreased glucuronidation activity against PAHs. In

comparison to wild-type UGT2A175Lys

activity, the UGT2A175Arg

variant exhibited a

~25% decrease in enzyme activity against multiple PAH substrates. Conversely, the

UGT2A1308Arg

variant did not exhibit detectable glucuronidation activity against any

substrate tested in the present study. The glycine to arginine amino acid change at codon

308 is a non-conservative amino acid change in the C-terminal region of the UGT

protein. This region is conserved between UGT isoforms and thought to contain the

UDPGA binding pocket [67]. Analysis of a crystal structure of the UDPGA-binding

domain of UGT2B7 suggests that a glycine residue at codon 310 is critical for the

creation of the UDPGA-binding pocket [86]. This glycine residue is conserved between

all UGT2B isoforms and is found in the common region shared by all UGT1A enzymes.

Interestingly, this same glycine residue corresponds to the UGT2A1 codon 308 glycine

variant upon amino acid sequence alignment (Figure 3.4). Although the complete crystal

structure of UGT2A1 is unknown, there is a high likelihood that UGT2A1 and UGT2B7

have similar UDPGA-binding regions due to high (70%) amino acid homology between

these two enzymes [97]. A recently completed homology model of UGT1A1 also

predicts the Gly308 residue in UGT1A1 to be critical for UDPGA binding [88].

Therefore, the non-conservative glycine to arginine change in this highly conserved UGT

100

region could inhibit UDPGA-binding by altering the conformation of the UDPGA

binding pocket.

Figure 3.4. UGT2A1308Gly

is a conserved residue among all UGT isoforms. Alignment of the

UDPGA-binding region domain for UGT2A1, UGT2B enzymes, and the common UGT1A

region, including the glycine residue at codon 308 in UGT2A1. The conserved serine and glycine

residues indicated form a portion of the hypothetical UDPGA-binding domain in the crystal

structure of UGT2B7 [86].

As shown in Table 3.4, results from this study suggest that UGT2A1*3 is

significantly associated with NSCC of the lung, and in particular squamous cell

carcinoma of the lung. The association of the inactive UGT2A1308Arg

variant with

increased risk of squamous cell carcinoma is consistent with experimental studies

implicating PAHs as a major cause of squamous cell carcinomas of the lung in animals

and humans [21, 28, 29, 33, 249]. The null association of the UGT2A1308Arg

SNP with

adenocarcinoma is consistent with in vitro activity studies; UGT2A1 has been shown to

have no detectable activity against TSNAs (Chapter 2), with TSNAs implicated in the

formation of adenocarcinomas [31, 32]. Large-scale GWAS analyzing lung cancer risk

101

have included the UGT2A1308Arg

SNP as well as many other UGT coding SNPs through

the sequencing of tagging SNPs. Thus far, no UGT SNPs have been determined to be

associated with lung cancer following the completion of multiple GWAS [254]. This may

be due to a lack of stratification of lung cancer by histology in previous GWAS [41, 55],

or the fact that rarer SNPs in carcinogen metabolizing enzymes are not always covered by

GWAS.

The association of UGT2A1308Arg

with lung cancer risk was analyzed using a

dominant model. This model was used with the assumption that individuals with even

one copy of the variant allele, in this case UGT2A1*3 heterozygotes, are at an increased

risk for developing lung cancer compared to wild-type UGT2A1*1 homozygotes [253].

While a UGT2A1*3 heterozygote would theoretically have less active UGT2A1 protein

and lower glucuronidation activity, in vitro activity data and co-IP experiments suggest

that the UGT2A1308Arg

variant negatively regulates wild-type UGT2A1308Gly

activity

through a protein-protein interaction. This is a novel finding for UGT2A1 and validates

the use of the dominant model in the lung cancer association study performed in this

study, suggesting that UGT2A1*3 heterozygotes would be at an increased cancer risk.

A limitation of the present study is a low sample size; the association of the

UGT2A1308Arg

variant with lung NSCC and squamous cell carcinoma must be validated

in a larger case-control study in the future.

Genotyping results from this study suggest that UGT2A1 SNPs may be more

prevalent than HapMap data suggests. The UGT2A1*2 SNP was reported by HapMap to

have a low allelic prevalence of 1.1% in Han Chinese individuals and was not observed

in Caucasian or African-American populations [251]. The data presented here suggest

102

that this SNP may have a greater allelic prevalence in an Asian population (~8%) and

may also be found in a significant proportion of Caucasians and African Americans (~4%

allelic prevalence for both groups). Not as large of a discrepancy was observed between

our results and HapMap data for the UGT2A1*3 SNP, yet there were minor differences

in the allelic prevalence of this SNP in all three groups examined. Differences observed

between our study and that reported in HapMap may be due to low subject numbers; a

larger genotyping study may be warranted to validate the allelic frequencies of the

UGT2A1*2 and UGT2A1*3 SNPs in multiple racial groups, particularly in Asians as

only 30 individuals were genotyped in this study.

A Cys127Tyr polymorphism in UGT1A1, causing an inactive protein isoform,

was previously shown to inhibit wild-type UGT1A1 activity through in vitro co-

expression studies [220]. Inactive splice variants of UGT1A1 [206], UGT2B4 [194], and

UGT2B7 [195] were also previously shown to negatively regulate their corresponding

wild-type UGT1A or 2B proteins. Inactive UGT1A isoforms, encoded by UGT1A splice

variants expressing a novel exon 5b, were also previously shown to negatively regulate

wild-type UGT1A activity [192, 205]. Based on these previous reports, we hypothesized

that UGT2A1308Arg

variant negative regulates wild-type UGT2A1 activity in a similar

fashion. In vitro activity studies were completed to determine the effects of

UGT2A1308Arg

co-expression on wild-type UGT2A1 activity. UGT2A1308Arg

_FLAG

expression was induced so that wild-type UGT2A1308Gly


_FLAG

were co-expressed at approximately a 1:1 ratio. This 1:1 expression ratio of wild-type

UGT2A1 to UGT2A1308Arg

created an in vitro representation of a UGT2A1*3

heterozygote. Using this system, the activity of wild-type UGT2A1 against several PAH

103

metabolites was shown to be significantly decreased following co-expression with the

UGT2A1308Arg

variant. These co-expression data suggested that UGT2A1308Arg

heterozygotes would have a significantly diminished glucuronidation capacity compared

to homozygous wild-type UGT2A1 individuals. Future studies are planned to examine

lung homogenates from UGT2A1*1/*1, UGT2A1*1/*3, and UGT2A1*3/*3 individuals

to confirm the in vitro co-expression results presented here.

The inactive UGT1A1Cys127Tyr

variant was shown to homo-dimerize with wild-

type UGT1A1 and negatively regulate wild-type UGT1A1 activity [220]. Inactive

UGT1A isoforms caused by the alternative splicing of a novel exon 5b were also shown

to modulate wild-type UGT1A activity through protein-protein interactions [218]. Based

on the previous reports of UGT dimerization, we speculated that the negative modulation

of wild-type UGT2A1 activity following UGT2A1308Arg

co-expression was caused by

dimerization. The Co-IP experiments performed in the present study suggested that

UGT2A1308Arg

and wild-type UGT2A1 dimerize in our in vitro co-expression system.

UGT2A1308Arg

is hypothesized to decrease wild-type UGT2A1 activity through the

formation of an inactive complex in the ER membrane; the interaction between

UGT2A1308Arg

and wild-type UGT2A1 may disrupt the normal conformation of UGT2A1

in the membrane, including the formation of wild-type UGT2A1 homo-dimers in the ER.

UGT2A1 and UGT2A2 transcripts are comprised of individual first exons joined

to common exons 2-6 [63, 70]. The UGT2A1*2 SNP is located in exon 1, making the

functional effects of this SNP unique to UGT2A1. The UGT2A1*3 SNP lies within the

common region shared by UGTs 2A1 and 2A2, but the effect of this codon 308 SNP on

UGT2A2 activity has not yet been determined. Another non-synonymous missense

104

coding SNP in UGT2A1, a valine to isoleucine change at codon 391 (rs4148304), is

reported by HapMap to only be expressed in Caucasians at a low allelic frequency of

0.8% [251]. This SNP was not analyzed in our study due to its low allelic frequency;

however, it may warrant further investigation if the allelic frequency of this SNP is

determined to be higher.

UGT2A1 was previously shown to be well-expressed in the lung and capable of

metabolizing PAH proximate carcinogens. Results from this study suggest that two

prevalent UGT2A1 variants, UGT2A175Arg

and UGT2A1308Arg

, exhibit decreased

glucuronidation activities, with the UGT2A1308Arg

SNP determined to have no detectable

glucuronidation activity against all substrates examined. The inactive UGT2A1308Arg

variant was determined to be associated with increased risk for NSCC and squamous cell

carcinoma of the lung; this finding is supported by the relatively high UGT2A1

expression in the lung [96] (Chapter 2) and wild-type UGT2A1 activity against PAH

proximate carcinogens (Chapter 2). Not only is the Gly to Arg change at codon 308 a

functional knock-out of UGT2A1 glucuronidation activity, the UGT2A1308Arg

variant was

found to negatively regulate wild-type UGT2A1 activity through dimerization. Future

experiments are needed to examine the association of UGT2A1308Arg

with cancer risk in

additional target organs for tobacco carcinogenesis where UGT2A1 is also expressed

(e.g., head and neck, colon). Results from this study suggest that UGT2A1308Arg

heterozygotes likely have a significantly decreased glucuronidation capacity compared to

wild-type UGT2A1 individuals, potentially identifying a relatively large subset of

individuals at increased risk for developing tobacco-related cancers.

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Chapter 4

IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF A

NOVEL UGT2A1 EXON 3 DELETION SPLICE VARIANT

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4.1 Abstract

UGT2A1 is a respiratory and aerodigestive tract-expressing phase II detoxifying

enzyme that plays an important role in the metabolism and excretion of various

xenobiotics including PAHs. In the present study, a novel exon 3 deletion splice variant

was identified for UGT2A1 (termed ‘UGT2A1Δexon3’). As determined by RT-PCR,

UGT2A1Δexon3 was shown to be expressed in various tissues including lung, trachea,

larynx, tonsil, and colon. The ratio of UGT2A1Δexon3:wild-type UGT2A1 expression

was highest in colon (0.79 ± 0.08) and lung (0.42 ± 0.12) as determined by real-time

PCR; an antibody specific to UGT2A1 showed UGT2A1_i2:UGT2A1_i1 ratios in the

range of 0.5-0.9 in these tissues. Using UPLC, cell homogenates prepared from

UGT2A1_i2-over-expressing HEK293 cells exhibited no glucuronidation activity against

a variety of substrates, including PAHs like B(a)P-7,8-diol. An inducible in vitro system

was created to determine the effect of UGT2A1_i2 expression on UGT2A1_i1 activity.

Increasing UGT2A1_i2 levels resulted in a statistically significant (p<0.01) decrease in

UGT2A1_i1 Vmax against 1-OH-pyrene, 3-OH-B(a)P and B(a)P-7,8-diol; no significant

changes in KM were observed for any of the three substrates. Co-IP experiments

suggested the formation of UGT2A1_i1 and UGT2A1_i2 hetero-oligomers and

UGT2A1_i1 homo-oligomers; co-expression of UGT2A1_i1 or UGT2A1_i2 with other

UGT1A or UGT2B enzymes caused no change in UGT1A or UGT2B glucuronidation

activity. These data suggest that a novel UGT2A1 splice variant regulates UGT2A1-

mediated glucuronidation activity via UGT2A1-specific protein-protein interactions, and

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that expression of this variant could play an important role in the local detoxification of

carcinogens within target tissues for tobacco carcinogenesis.

4.2 Introduction

UGT2A1 was previously shown to have high expression in lung, trachea, tonsil,

larynx, and colon tissues, as well as significant enzyme activity against PAHs implicated

in tobacco carcinogenesis (Chapter 2). UGT2A1 is hypothesized to be the only UGT

well-expressed in both respiratory and aerodigestive tract tissues with significant

glucuronidation activity against pro-carcinogenic PAHs and their metabolites [96, 98, 99,

241], suggesting that this under-studied enzyme potentially has a critical role in the local

detoxification of activated PAHs in target tissues for tobacco carcinogenesis.

Through the process of alternative splicing, single genes can produce multiple

mRNA and protein isoforms with varying functions. Alternative splicing was initially

proposed to affect half of all genes, but recent reports suggest that over 90% of genes

undergo alternative splicing [197, 255]. Alternative splicing creates increased UGT1A

diversity through the alternative splicing of a novel exon 5b to exons 1-4 of UGT1A

transcripts [205, 206]. The UGT1A mRNAs containing exon 5b have been shown to have

widespread tissue expression, and functional assays have shown that the proteins

translated from exon 5b splice variants lack glucuronidation activity [205]. These exon

5b containing protein variants have also been shown to interact with wild-type UGT1A

proteins and negatively modulate wild-type UGT1A activity through dimerization [205,

206, 218]. UGT2B4 and UGT2B7 splice variant transcripts have been found to be well-

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expressed in multiple tissues, and these splice variant proteins have been shown to be

inactive negative modulators of wild-type UGT2B4 and UGT2B7 activity [207, 208].

Described in this chapter are studies examining the expression and function of a

novel UGT2A1Δexon3 variant, including the potential regulation of wild-type

UGT2A1_i1 activity through a UGT2A1_i2-mediated protein-protein interaction. The

hypothesis that protein-protein interactions between UGT2A1_i2 and UGT2A1_i1 cause

alterations in UGT2A1_i1 activity was based on previous studies reporting that inactive

UGT variants negatively modulate wild-type UGT activity through dimerization [218-

220]. Additionally, previous work investigating the UGT2A1308Arg

variant suggested

dimerization and negative regulation occurs between wild-type UGT2A1 and the

UGT2A1308Arg

variant (Chapter 3). In addition to determining the interaction between

UGT2A1_i2 and UGT2A1_i1, we also investigated whether UGT2A1_i2 or UGT2A1_i1

could modulate the activity of UGT1A or UGT2B isoforms expressed in aerodigestive or

respiratory tract tissues.

4.3 Methods

Materials. UDPGA, alamethicin, -glucuronidase, 4-MU, 1-OH-pyrene, and 1-

naphthol were purchased from Sigma-Aldrich (St. Louis, MO). 1-OH- B(a)P, 3-OH-

B(a)P, 7-OH-B(a)P, 8-OH-B(a)P, 5-methylchrysene-1,2-diol, dibenzo(a,l)pyrene-11,12-

diol, and B(a)P-7,8-diol were synthesized in the Organic Synthesis Core Facility at the

Penn State College of Medicine (Hershey, PA). HPLC-grade ammonium acetate,

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acetonitrile, and agarose were purchased from Fisher Scientific (Pittsburgh, PA). Real-

time PCR probes and gene expression assays were acquired from Applied Biosystems

Inc., Life Technologies (Carlsbad, CA). Complete Control Inducible Mammalian

Expression System kits, PonA, and Pfu Polymerase were obtained from Agilent (Santa

Clara, CA). DMEM, DPBS, FBS, penicillin-streptomycin, G418, and blasticidin were

purchased from Gibco, Life Technologies (Grand Island, NY). The pcDNA3.1/V5-His-

TOPO and pcDNA6.2/V5/GW/D-TOPO mammalian expression vectors, the Superscript

II RT kit, Dynabead Protein G Immunoprecipitation kit, and Hygromycin B were

obtained from Invitrogen, Life Technologies (Grand Island, NY). The BCA protein assay

kit was purchased from Pierce (Rockford, IL). The RNeasy kit, QIAquick gel extraction

kit, Plasmid Mini kit, and Plasmid Maxi kit were all purchased from Qiagen (Valencia,

CA). All oligonucleotides were purchased from IDT (Coralville, IA).

Determination of UGT2A1Δexon3 tissue expression. The UGT2A1 exon 3

deletion splice variant was initially discovered following RT-PCR using pooled lung

RNA. The primers used to amplify full length UGT2A1Δexon3 from lung cDNA were

5’-CATCAAATCTTCTGCATCAAGCCAC-3’ (sense; UGT2A1_S1) and 5’-

TGACAGGAAGAGGGTATAGTCAGC-3’ (anti-sense; UGT2A1_AS1), corresponding

to nucleotides -28 to -4 and +1834 to +1811, respectively, relative to the UGT2A1

translation start site. For all PCR reactions, RNA was acquired and RT-PCR was

completed as previously described. Briefly, 2 μg of RNA from each tissue was used with

oligo dTs for RT, with cDNA corresponding to 100 ng of RNA used in the subsequent

PCR reactions. Unless otherwise noted, all PCRs were performed using Pfu Polymerase,

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with an initial denaturing temperature of 94°C for 2 min, 40 cycles of 94°C for 30s, 58°C

for 45s, and 72°C for 2 min, followed by a final cycle of 10 min at 72°C.

Following the initial discovery of UGT2A1Δexon 3 from pooled lung cDNA,

additional pooled RNAs from human tissues were screened for UGT2A1Δexon3 RNA

expression by RT-PCR. Primers specific to exon 1 5’-GACATGGCTGGAAAAT-

AGACC-3’ (sense) and exon 5 of UGT2A1 5’-CCATAGGGACTCCGTGGTAAAT-3’

(anti-sense) of UGT2A1, corresponding to nucleotides +276 to +296 and +1165 to

+1144, respectively, relative to the translation start site were used to screen for

UGT2A1Δexon3 expression. RT was completed using RNAs from HEK293 cell lines

over-expressing wild-type UGT2A1 or UGT2A1Δexon3 (see below for Methods), and

cDNAs following RT were used as positive controls for PCR amplification while water

was used as a negative control. PCR products were gel-purified using a QIAquick gel

extraction kit and sequenced by dideoxy sequencing at the Penn State University Nucleic

Acid Facility (State College, PA) and compared to the UGT2A1 sequence in GenBank

(NM_006798.3). To verify UGT2A1Δexon3 expression in tissues analyzed, PCR

reactions were run multiple times with all positive and negative controls.

A real-time PCR assay was developed to quantitatively assess relative levels of

wild-type UGT2A1 and UGT2A1Δexon3 transcripts in tissues that were previously

determined to have UGT2A1 expression. Separate assays were designed to specifically

amplify wild-type UGT2A1 or UGT2A1Δexon3 (Figure 4.1).

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Figure 4.1. Schematic of real-time PCR assay developed to specifically detect either wild-

type UGT2A1 or UGT2A1Δexon3. Identical forward primers and probes were used to detect

both transcripts, while reverse primers were designed to specifically recognize either wild-type

UGT2A1 or UGT2A1Δexon3. Assay specificity was confirmed by running real-time PCR

products on an agarose gel and sequencing the products.

A sense primer specific to exon 1 (5’-CTACATGTTTGAAACTCTTTGGAAATC-3’)

and a 5’ labeled VIC probe (ABI) specific to exon 2 (5’-TCCGAACATATTGGGATT-

3’), corresponding to nucleotides +660 to +686 and +767 to +784, respectively, relative

to the UGT2A1 translation start site, were used to detect both wild-type UGT2A1 and

UGT2A1Δexon3. An anti-sense primer specific to UGT2A1 exon 3 (5’-TTACCT-

GAGCTCTGGATAAATTCTTC-3’), corresponding to nucleotides +896 to +871 relative

to the UGT2A1 translation start site, was used to specifically amplify wild-type UGT2A1

transcript, while an anti-sense primer specific to the UGT2A1Δexon3 exon 2 and 4

junction (5’-TTTCCTTTGTATCTCCATAAAACCTTAG-3’), corresponding to

nucleotides +887 to +860 relative to the UGT2A1Δexon3 translation start site, was used

to specifically amplify the UGT2AΔexon3 transcript. Reactions were completed using

the standard ABI thermal cycling parameters, with RPLPO (Hs99999902_m1) used as a

housekeeping gene. cDNAs, corresponding to 20 ng RNA, were used for each real-time

assay and reactions were performed in triplicate. Real-time PCR experiments were

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carried out in the Penn State College of Medicine Functional Genomics Core Facility

(Hershey, PA) using an ABI 7900 HT thermal cycler and data was analyzed using SDS

2.2 software. Assay specificity for wild-type UGT2A1 or UGT2A1Δexon3 transcripts

was confirmed through agarose gel electrophoresis and dideoxy sequencing of real-time

PCR products. Real-time PCR data was corrected to account for the amplification

efficiency of each real-time PCR assay, as described previously [256]. The relative tissue

expression of UGT2A1Δexon3 transcript was calculated using the ΔΔCt method relative

to the amount of wild-type UGT2A1 transcript for each tissue specimen examined.

Generation of an UGT2A1_i2 over-expressing cell line. A HEK293 cell line over-

expressing wild-type UGT2A1 (termed ‘UGT2A1_i1’) was previously established

(Chapter 2). A HEK293 cell line over-expressing the isoform corresponding to

UGT2A1exonΔ3 (termed ‘UGT2A1_i2’) was created using a similar protocol. Briefly,

UGT2A1exonΔ3 was cloned from pooled lung RNA using Pfu Polymerase and the

UGT2A1_S1 and UGT2A1_AS1 primers. Following gel extraction and sequencing of the

PCR product of the appropriate size, the verified UGT2A1exonΔ3 cDNA was cloned into

the pcDNA3.1/V5-His-TOPO vector using standard protocols and grown in One Shot

TOP10 competent E. Coli. After direct dideoxy sequencing for sequence confirmation

and a large-scale plasmid preparation, electroporation with 10 μg of the pcDNA3.1/V5-

His-TOPO_UGT2A1exonΔ3 vector was used to generate the HEK293 cell line over-

expressing UGT2A1_i2. Cells were grown in DMEM supplemented with 10% FBS, 1%

penicillin-streptomycin, and 400 μg/mL G418 to 75% confluence. Cell homogenates

were prepared essentially as previously described in 1X Tris-buffered saline (25 mM Tris

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base, 138 mM NaCl and 2.7 mM KCl; pH 7.4) [98, 109]. Total RNA was extracted using

the RNeasy Mini kit following the manufacturer’s protocols. Homogenate protein

concentrations were determined using the BCA protein assay.

Determination of UGT2A1_i2 protein expression. An antibody designed to

recognize UGT2A1 protein was previously created using a peptide encoded by exon 1, a

region common to both UGT2A1_i1 and UGT2A1_i2. Levels of UGT2A1_i1 and

UGT2A1_i2 protein were determined by Western blot analysis using the anti-UGT2A1

antibody at a 1:500 dilution (as recommended by the manufacturer; Open Biosystems,

Huntsville, AL) and 50 μg of UGT2A1-over-expressing cell homogenate protein. The

monoclonal β-actin antibody (Sigma-Aldrich; St. Louis, MO) was used as a loading

control and the Western blot was done in triplicate. The intensity of UGT2A1 signal was

measured with the ImageJ program (NIH). Since a UGT2A1 standard is not

commercially available, the relative protein expression of UGT2A1_i1 and UGT2A1_i2

in homogenate from each cell line was calculated relative to the β-actin loading control.

UGT2A1 protein expression was also analyzed in three normal human lung and three

normal human colon tissue homogenates (obtained from Banner Sun Health Research

Institute, Sun City, AZ) by a similar Western blot procedure, using 250 μg tissue

homogenate prepared using an Omni TH rotor-stator homogenizer in 50 mM Tris pH 7.5,

1.15% KCl, and 1 mM disodium EDTA. For the determination of UGT2A1 expression in

colon and lung tissue homogenates, protein from cell lines over-expressing UGT2A1_i1

and UGT2A1_i2 was mixed in a 1:1 ratio and loaded (60 μg total protein) as a positive

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control. The relative amount of UGT2A1_i2 to UGT2A1_i1 in each lung or colon

specimen was determined relative to β-actin as described above.

Glucuronidation assays. Glucuronidation assays using homogenate from HEK293

cells over-expressing UGT2A1_i1 and UGT2A1_i2 were completed essentially as

previously described [234, 235]. Briefly, after an initial incubation of 100 μg protein

homogenate with alamethicin (50 μg/mg protein) for 15 min on ice, glucuronidation

reactions were performed in a final reaction volume of 25 μL at 37°C with 50 mM Tris-

HCl (pH 7.4), 10 mM MgCl2, 4 mM UDPGA, and between 6 and 800 μM of substrate.

Reactions were terminated by the addition of 25 μL cold acetonitrile on ice. Reaction

mixtures were centrifuged for 10 min at 16,100 g prior to the collection of supernatant.

Glucuronide formation was determined using a Waters Acquity ultra-pressure liquid

chromatography (UPLC) System (Milford, MA) as previously described [75, 112, 234,

236, 257]. The flow rate was maintained at 0.5 mL/min and a reverse phase Acquity

UPLC BEH C18 - 1.7 μm 2.1 x 100 mm column was used to separate free substrate and

the conjugated glucuronide. A gradient of solution A (5 mM NH4OAc pH 5.0, 10%

acetonitrile) and solution B (100% acetonitrile) was used to elute the glucuronide and

substrate from the column. The initial solvent gradients and UV absorbance wavelengths

used to detect glucuronidation of various substrates were described previously. Reactions

with non-transfected HEK293 cell homogenate, no substrate added to the reaction

mixture, or only substrate and no homogenate added to the reaction mixture were used as

negative controls. Homogenate from a HEK293 cell line over-expressing UGT2A1_i1

was used as a positive control.

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Creation of an UGT2A1_i2 inducible system. Wild-type UGT2A1 was cloned

into the V5-tagged, blasticidin resistance gene-containing pcDNA6.2/V5/GW/D-TOPO

vector using a sense primer 5’-CACCATGTTAAACAACCTTCTGC-3’ (UGT2A1_S2)

and anti-sense primer 5’-TTCTCTTTTTTTCTTCTTTCCTATCTTACC-3’

(UGT2A1_AS2) corresponding to nucleotides +1 to +19 and +1581 to +1552,

respectively, relative to the UGT2A1 translation start site, amplifying the entire coding

region of wild-type UGT2A1 minus the stop codon (underlined nucleotides in sense

primer indicate an CACC anchor). An ecdysone-analog inducible mammalian expression

system was used to regulate UGT2A1_i2 levels, with UGT2A1Δexon3 cloned into the

FLAG-tagged, hygromycin resistance gene-containing pEGSH vector using a similar

sense primer to that described above but containing a Xho I restriction site (underlined)

on the 5’ end (5’-GCACTCGAGATGTTAAACAACCTTCTGC-3’; UGT2A1_S3), and a

similar anti-sense primer to that described above with a Xba I restriction site (underlined)

on the 5’ end (5’GATTCTAGACGTTCTCTTTTTTTCTTCTTTCCTATCTTACC-3’;

UGT2A1_AS3). After plasmid preparations for each clone, vector sequences were

verified by direct dideoxy sequencing. Eight micrograms each of the

pEGSH_UGT2A1Δexon3 vector and the G418 resistance gene-containing pERV

regulatory vector, comprising the inducible system, as well as the pcDNA6.2/V5/GW/D-

TOPO_wtUGT2A1 vector, were stably transfected simultaneously into HEK293 cells

using a standard lipofectamine protocol. Selection of HEK293 cells over-expressing the

three vectors was completed using a combination of 400 μg/mL G418, 9 μg/mL

blasticidin, and 75 μg/mL hygromycin B in DMEM containing 10% FBS. Multiple

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clones were analyzed for inducible gene expression, and a stable clone over-expressing

all three vectors simultaneously was chosen based on the efficiency of UGT2A1_i2

induction.

After the creation of the stable UGT2A1_i1_V5/UGT2A1_i2_FLAG/pERV-over-

expressing cell line, UGT2A1_i2 expression was induced with varying levels of the

ecdysone-analog PonA. HEK293 cells at 50% confluence were treated with 2 μM, 6 μM,

or 10 μM of PonA in ethanol for 12 h. Vehicle (0.01% ethanol) was added to these cells

as a negative control. Cells were harvested and homogenates were made as previously

described [98, 109]. Protein homogenates from the various treatment groups were

screened for UGT2A1_i1_V5 and UGT2A1_i2_FLAG expression by Western blot

analysis using 50 μg of total protein per sample, with the analysis of UGT2A1_i2_FLAG

induction performed in triplicate. UGT2A1_i1_V5 expression was determined using a

monoclonal mouse V5-HRP antibody (Invitrogen; Grand Island, NY) at a 1:5000

dilution, while UGT2A1_i2_FLAG was determined using a monoclonal mouse anti-Flag

antibody (Sigma-Aldrich; St. Louis, MO) at a 1:1000 dilution. UGT2A1_i1_V5 and

UGT2A1_i2_FLAG expression levels were also confirmed using the anti-UGT2A1

antibody described above, with 100 μg of protein homogenate from both the control and

10 μM PonA treatment groups used with the anti-UGT2A1 antibody at a 1:500 dilution.

In all cases, the monoclonal β-actin antibody was used as a loading control.

Following verification of UGT2A1_i1 and UGT2A1_i2 protein levels,

homogenate was prepared and used for activity assays as previously described [75, 110,

112, 257, 258]. Cell lysates were homogenized for 10 sec on ice using a Bio-Vortexer

(Biospec Products, Bartlesville, OK). Activity was determined against three PAHs which

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were previously shown to be substrates of UGT2A1 (Chapter 2). Activity assays were

completed in triplicate for each substrate examined, using the control cell line and cell

lines treated with 2 μM, 6 μM, or 10 μM of PonA. For each substrate, the glucuronidation

rate was determined at 8 concentrations that encompassed the KM of the substrate. For

glucuronidation rate determinations, cell homogenate protein levels and incubation times

for each substrate were determined experimentally to ensure that substrate utilization was

less than 10% and to maximize the levels of detection while in a linear range of

glucuronide formation. Cell lines over-expressing either UGT2A1_i1_V5 or

UGT2A1_i2_FLAG alone were also created using an identical protocol, and cell

homogenates were prepared as described above.

UGT2A1_i1 and UGT2A1_i2 co-IP assays. HEK293 cells over-expressing

UGT2A1_i1_V5/UGT2A1_i2_FLAG were treated with 10 μM PonA for 12 h prior to

washing with DPBS and homogenate preparation as described previously [218]. A

Dynabead Protein G Immunoprecipitation Kit was used to determine potential protein

interactions. Three μg of a mouse monoclonal anti-FLAG antibody or 1.5 μg of mouse

monoclonal V5 antibody (Santa Cruz Biotechnology; Santa Cruz, CA) were incubated

with Dynabeads for 15 min with rotation at room temperature. Lysates (2.5 μg/μL) from

vehicle- or PonA-treated UGT2A1_i1_V5/UGT2A1_i2_FLAG inducible cells were then

incubated with the same Dynabeads for 30 min with rotation at room temperature.

Following three washes in PBS at room temperature, immunoprecipitated proteins were

eluted, heated at 90°C for 10 min in loading buffer, and subjected to Western blot

analysis using either a monoclonal mouse V5-HRP antibody at a 1:5000 dilution or a

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mouse FLAG-HRP antibody (Cell Signaling Technology; Beverly, MA) at a 1:1000

dilution. All co-IP experiments were repeated three times to verify results.

Homo-dimerization of UGT2A1_i1 by co-IP. An inducible co-expression system,

similar to that used to investigate UGT2A1_1:UGT2A1_i2 hetero-dimerization, was used

to examine potential UGT2A1_i1 homo-dimerization. Creation of the pcDNA

6.2/V5/GW/D-TOPO_wtUGT2A1 vector was described above. Wild-type UGT2A1 was

cloned into the FLAG tagged, hygromycin resistance containing pEGSH vector, using

UGT2A1_S3 and UGT2A1_AS3 primers as described above. A HEK293 cell line stably

expressing the pcDNA6.2/V5/GW/D-TOPO_wtUGT2A1, pEGSH_wtUGT2A1, and

pERV vectors was created as described above. UGT2A1_i1_FLAG expression was

induced by treating HEK293 cells at 50% confluence with 10 μM of PonA (in ethanol)

for 12 h. Vehicle (0.01% ethanol) was added to HEK293 cells as a negative control.

Determination of UGT2A1_i1_V5 and UGT2A1_i1_FLAG expression levels using the

anti-V5 and anti-FLAG antibodies, the use of the anti-UGT2A1 antibody to confirm

UGT2A1_i1_V5 and UGT2A1_i1_FLAG levels, and co-IP experiments were completed

using identical conditions to that described above.

Co-expression of UGT2A1_i1 and UGT2A1_i2 with other UGT isoforms.

UGT2A1Δexon3 was cloned into the pcDNA6.2/V5/GW/D-TOPO vector using

UGT2A1_S2 and UGT2A1_AS2 primers as described above. The newly-created

pcDNA6.2/V5/GW/D-TOPO_UGT2A1Δexon3 vector was transfected using a standard

lipofectamine protocol into previously-established stable HEK293 cell lines over-

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expressing UGT1A7, UGT1A10, or UGT2B17 [98, 109, 118, 230]. The

pcDNA6.2/V5/GW/D-TOPO_wtUGT2A1 vector described above was also transfected

into UGT1A10 and UGT2B17 over-expressing cell lines using a standard lipofectamine

protocol. Selection of HEK293 cells co-expressing UGT2A1_i1 or UGT2A1_i2 and

UGT1A7, UGT1A10, or UGT2B17 was completed using blasticidin (9 μg/mL) and G418

(400 μg/mL).

Following RNA extraction and RT of RNA from the co-expressing cell lines,

real-time PCR was performed to determine the relative level of UGT1A7, UGT1A10, or

UGT2B17 expression versus wild-type UGT2A1 or UGT2A1Δexon3 expression in each

co-expressed cell line. ABI gene expression assays for UGT1A7 (Hs02517015_s1),

UGT1A10 (Hs02516990_s1), UGT2B17 (Hs00854486_sH), and UGT2A1

(Hs00792016_m1) were used to determine relative transcript levels. The UGT2A1 ABI

gene expression assay is specific for UGT2A1 exon 1, enabling the assay to detect

transcripts from both the wild-type UGT2A1 and exon 3-deleted UGT2A1 splice variant.

Reactions were completed using the standard ABI protocol, with RPLPO used as a

housekeeping gene. cDNA corresponding to 20 ng RNA was used for each real-time

reaction and reactions were performed in triplicate using standard thermal cycling

parameters. Real-time-PCR data was corrected to account for the amplification efficiency

of each real-time PCR assay, as described previously [256]. The relative level of

UGT2A1 transcript in each cell line was calculated using the ΔΔCt method, relative to

the amount of UGT1A7, UGT1A10, or UGT2B17 in each co-expressed cell line. An anti-

UGT1A antibody (BD Biosciences; San Jose, CA) at 1:3000 dilution and the V5-HRP

antibody described above at a 1:5000 dilution were used in Western blot analyses to

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verify that co-expression had no impact on UGT1A7, UGT1A10, or UGT2A1_V5

expression.

Homogenate was created from each of the HEK293 co-expressed over-expressing

cell lines and glucuronidation assays were completed as described above.

Glucuronidation assays were performed using homogenates from HEK293 over-

expressing either UGT1A7/ UGT2A1_i2, UGT1A10/UGT2A1_i2, or

UGT2B17/UGT2A1_i2, with 3-OH-B(a)P and/or 1-naphthol as substrates, as PAHs were

previously shown to be glucuronidated by these UGTs [98, 99, 259]. Since wild-type

UGT2A1 exhibits activity against PAHs , homogenates from HEK293 cells over-

expressing UGT1A10/UGT2A1_i1 and UGT2B17/UGT2A1_i1 were used in

glucuronidation assays with the TSNA NNAL, which is not a substrate for UGT2A1 but

is metabolized by both UGTs 1A10 and 2B17 [176, 258]. As previous co-expression

studies have reported activity changes to be substrate dependent [83, 213], additional

activity reactions were performed using other non-UGT2A1 substrates, including the

flavonoid chrysin for UGT1A7/UGT2A1_i2 [260], the HCA metabolite N-OH PhIP for

UGT1A10/UGT2A1_i1 and UGT1A10/UGT2A1_i2 [75], and ibuprofen for

UGT2B17/UGT2A1_i1 and UGT2B17/UGT2A1_i2 [259]. For chrysin, N-OH PhIP, and

ibuprofen glucuronidation rate determinations, substrate concentrations were chosen

which approximated the reported KM from previous experiments.

Data analysis and Statistics. Three independent experiments were performed for

kinetic analyses and GraphPad Prism 5 software was used to calculate kinetic values.

Kinetic constants Vmax and KM for all substrates were calculated using the Michaelis-

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Menten equation for the rate of product formation versus substrate concentration, with

data transformed into linear Eadie-Hofstee plots. An ANOVA, followed by a post test for

linear trend, was used to compare the KM and Vmax of glucuronide formation for the

various UGT2A1_i1 /UGT2A1_i2 PonA treatment groups. The Students t-test was used

to compare mRNA expression levels and enzyme kinetics of homogenate activities

following co-expression of UGT2A1 with UGT1A7, UGT1A10, or UGT2B17.

4.4 Results

Relative UGT2A1Δexon3 expression in multiple tissues. Since its original

discovery in olfactory epithelium [97], UGT2A1 has been shown to be expressed in

multiple respiratory, digestive, and aerodigestive tract tissues. In the present study, RT-

PCR amplification of UGT2A1 from pooled lung RNA yielded two distinct products;

wild-type UGT2A1 and a novel variant of UGT2A1, which upon direct sequencing was

shown to be a splice variant lacking exon 3 (UGT2A1Δexon3; Figure 4.2, Panel A).

Following the discovery of this splice variant in lung tissue, additional aerodigestive tract

tissues known to express wild-type UGT2A1 were screened for UGT2A1Δexon3

expression. As shown in Figure 4.2 Panel B, UGT2A1Δexon3 was also expressed in the

trachea, larynx, tonsil, and colon; no UGT2A1Δexon3 expression was observed in pooled

olfactory RNA. Using a custom real-time PCR assay (Figure 4.1), the relative expression

levels of UGT2A1Δexon3 were determined relative to wild-type UGT2A1 in each tissue,

with wild-type UGT2A1 expression in each tissue set to 1.0 as a reference. The relative

UGT2A1Δexon3 expression was demonstrated to be the highest in colon (0.79 ± 0.08),

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followed by lung (0.42 ± 0.12) > larynx (0.39 ± 0.05) > trachea (0.27 ± 0.07) > tonsil

(0.10 ± 0.02) (Figure 4.2, panel C). No UGT2A1Δexon3 expression was detected after

multiple RT-PCR attempts or by real-time PCR in tissues of the prostate, liver, pancreas,

kidney, esophagus, whole brain, cerebral cortex, floor of mouth, olfactory, or breast

(results not shown).

123

Figure 4.2. Determination of UGT2A1Δexon3 expression. (A) Full-length UGT2A1 was PCR-

amplified following RT of pooled lung RNA. A UGT2A1 mRNA variant lacking exon 3

(UGT2A1Δexon3), in addition to wild-type UGT2A1 mRNA, was discovered following gel

extraction and dideoxy sequencing of the PCR products. (B) A sense primer specific to exon 1

and an anti-sense primer specific to exon 5 of UGT2A1 were used in RT-PCR to determine

tissue-specific expression of UGT2A1Δexon3 in tissues that were previously determined to

express wild-type UGT2A1. For both RT-PCR experiments (panels A and B), the cDNA

equivalent of 100 ng RNA was used. RNAs from HEK293 cell lines over-expressing wild-type

UGT2A1 or UGT2A1Δexon3 were used as a positive control, while water in place of cDNA was

used as a negative control. (C) Quantitative real-time PCR was completed to determine

UGT2A1Δexon3 expression levels relative to wild-type UGT2A1 expression in human tissues.

cDNAs, corresponding to 20 ng of RNAs from tissues exhibiting UGT2A1Δexon3 expression

(panels A and B), were used in conjunction with the custom-designed real-time PCR assay

described in Figure 4.1. Relative UGT2A1Δexon3 expression in each tissue was determined by

comparing UGT2A1Δexon3 mRNA levels to wild-type UGT2A1 mRNA levels, set to 1.0 as a

reference. Results, expressed as the mean ± SD of triplicates, were normalized to RPLPO RNA

expression for each tissue.

124

UGT2A1_i2 protein expression in a HEK293 over-expressing cell line and tissue

homogenates. An antibody against UGT2A1 was previously created and used to assess

wild-type UGT2A1 (UGT2A1_i1) protein levels in HEK293 over-expressing cell lines.

Similarly, a stable HEK293 cell line transfected with UGT2A1Δexon3 demonstrated

UGT2A1_i2 over-expression using the same antibody (Figure 4.3, Panel A), with the

mean level of UGT2A1_i1 protein expression calculated to be 0.92 ± 0.07 versus that

observed for UGT2A1_i2 (set as 1.0 as a reference) in the two UGT2A1 over-expressing

cell lines. Expression of both UGT2A1_i1 and UGT2A1_i2 were shown in protein

homogenates made from normal lung and colon tissues (Figure 4.3, Panel B). For the

lung and colon specimens analyzed, UGT2A1_i1 was expressed at marginally higher

levels than UGT2A1_i2, with the ratios of UGT2A1_i2:UGT2A1_i1 ranging from 0.6 –

0.9 for lung and 0.5 – 0.7 for colon.

125

Figure 4.3. Determination of UGT2A1_i2 expression in a HEK293 over-expressing cell line

and human tissue homogenates. (A) Representative Western blot showing UGT2A1_i2

expression in an over-expressing HEK293 cell line. 50 μg of total protein homogenate from cell

lines over-expressing UGT2A1_i1 or UGT2A1_i2 were loaded to each lane for a Western blot,

and expression was determined using an anti-UGT2A1 antibody. β-actin was used as a loading

control. The relative ratio of UGT2A1_i1 or UGT2A1_i2 protein to β-actin was used to

determine relative protein expression in each over-expressing cell line. (B) Lung and colon tissue

homogenates (250 μg) were screened for UGT2A1_i1 and UGT2A1_i2 expression; 60 μg of

protein (30 μg of each UGT2A1 isoform) from HEK293 cell lines over-expressing UGT2A1_i1

or UGT2A1_i2 was mixed in a 1:1 ratio and used as a positive control. β-actin was used as a

loading control.

Enzymatic activity of UGT2A1_i2. As UGT2A1Δexon3 RNA and UGT2A1_i2

protein were shown to be expressed in lung and a variety of other aerodigestive tract

tissues, and because UGT2A1_i1 was previously shown to be active against PAHs

involved in tobacco carcinogenesis , UGT2A1_i2 activity against PAHs and other

tobacco carcinogens was investigated. Homogenate from UGT2A1_i2-over-expressing

HEK293 cells, which was shown to express UGT2A1_i2 protein at slightly higher

relative levels than the stable HEK293 cell line over-expressing UGT2A1_i1 (see Figure

4.3, Panel A), was used in glucuronidation activity assays to determine the enzymatic

activity of UGT2A1_i2. While cell homogenates over-expressing UGT2A1_i1

126

demonstrated glucuronidation activity against both 3-OH-B(a)P (Figure 4.4, panel A) and

5-methylchrysene-1,2-diol (Figure 4.4, panel C), no glucuronide was detected when

UGT2A1_i2 protein homogenate was used in the glucuronidation assay (Figure 4.4,

panels B and D). No detectable glucuronidation activity was observed for UGT2A1_i2-

over-expressing cell homogenates against all other PAHs examined including 1-OH-

pyrene, 1-naphthol, 1-OH-B(a)P, 7-OH-B(a)P, 8-OH-B(a)P, B(a)P-7,8-diol, and

dibenzo(a,l)pyrene-11,12-diol, using up to 400 µg cellular homogenate and 750 μM

substrate in a 18 h incubation (results not shown). The UGT2A1_i2 variant also lacked

activity against 4-MU, a known UGT2A1 substrate and a common substrate of most

UGT isoforms [237]. UGT2A1_i1 was previously shown to have no detectable

glucuronidation activity against TSNAs and HCAs (Chapter 2); UGT2A1_i2 also had no

detectable glucuronidation activity against these substrates (results not shown).

127

Figure 4.4. UGT2A1_i2 exhibits no detectable glucuronidation activity against multiple

PAHs. UGT2A1_i2 activity was determined against PAHs that were previously determined to be

substrates of wild-type UGT2A1. Shown are representative UPLC chromatograms of (A)

UGT2A1_i1 activity against 3-OH-B(a)P, (B) UGT2A1_i2 activity against 3-OH-B(a)P, (C)

UGT2A1_i1 activity against 5-methylchrysene-1,2-diol, and (D) UGT2A1_i2 activity against 5-

methylchrysene-1,2-diol.

UGT2A1_i2 modulates UGT2A1_i1 activity. To examine the potential effects of

increasing levels of UGT2A1_i2 expression on UGT2A1_i1 activity, a co-expression

system was generated to allow for stable UGT2A1_1 protein levels and UGT2A1_2

expression levels regulated by the ecdysone analog PonA. In order to more easily

differentiate between the UGT2A1 isomers in co-IP experiments, UGT2A1_i1 was V5-

tagged and UGT2A1_i2 was FLAG-tagged, both at the C-terminus of the protein. As

128

shown by Western blot analysis (Figure 4.5, Panel A), no detectable expression of

UGT2A1_i2_FLAG was observed in the vehicle control group. Following the addition of

increasing dosages of PonA there was a corresponding increase in UGT2A1_i2_FLAG

levels while UGT2A1_i1_V5 levels remained relatively constant. The UGT2A1_i2

induction and corresponding protein quantification was completed in triplicate: 2 μM

PonA treatment induced the mean UGT2A1_i2 levels to 0.28 ± 0.05 relative to

UGT2A1_i1 (set as 1.0 as a reference in all cases), 6 μM PonA treatment induced the

mean UGT2A1_i2 levels to 0.76 ± 0.06 relative to UGT2A1_i1, and 10 μM PonA

treatment induced the mean UGT2A1_i2 levels to 1.18 ± 0.09 relative to UGT2A1_i1.

Relative UGT2A1_i1_V5 protein levels were calculated for each treatment group and

used for normalization of kinetic data, with UGT2A1_i1 expression in the control group

set as the reference at 1.0. UGT2A1_i1 expression was relatively consistent in all

treatment groups (Figure 4.5, Panel A). Mean UGT2A1_i1_V5 levels were determined to

be 0.89 ± 0.08 in the 2 μM PonA treatment group, 0.95 ± 0.06 in the 6 μM PonA

treatment group, and 0.94 ± 0.04 in the 10 μM PonA treatment group. UGT2A1_i1_V5

and UGT2A1_i2_FLAG expression levels were confirmed by Western blot using the

anti-UGT2A1 antibody (Figure 4.5, Panel B). Using this antibody, no UGT2A1_i2 was

detected in the control group and the ratio of UGT2A1_i2:UGT2A1_i1 in the 10 μM

treatment group (1.1) was approximately equal to that observed using the anti-FLAG and

anti-V5 antibodies (1.18 ± 0.09) described above. In addition, the relative expression of

UGT2A1_i1 in the 10 μM PonA treatment group using the anti-UGT2A1 antibody (0.97)

was similar to that observed using the anti-FLAG and anti-V5 antibodies (0.94 ± 0.04)

described above.

129

Figure 4.5. UGT2A1_i2 expression is induced by the ecdysone analog PonA.

(A) Representative Western blot showing stable expression of UGT2A1_i1_V5 and induction of

UGT2A1_i2_FLAG expression in HEK293 cells. UGT2A1_i2_FLAG expression was induced

through addition of increasing doses of PonA for 12 h; 50 μg of total protein homogenate from

each PonA treatment group was loaded per lane for each Western blot. (B) A Western blot with

100 μg protein from the control and 10 μM PonA treatment groups was performed with an anti-

UGT2A1 antibody to confirm UGT2A1 expression levels detected by the anti-V5 and anti-FLAG

antibodies. For both panels A and B the relative expression of UGT2A1_i1, as well as the relative

UGT2A1_i2:UGT2A1_i1 ratio, was determined for each treatment group. Induction of

UGT2A1_i2 expression and Western blots were performed in triplicate. In all cases, β-actin was

used as a loading control.

Activity assays and enzyme kinetics were completed to determine the potential

impact of UGT2A1_i2 expression on UGT2A1_i1 enzyme activity. The effects of

UGT2A1_i2 expression on UGT2A1_i1 activity were assessed in glucuronidation assays

using the PAH substrates 1-OH-pyrene, 3-OH-B(a)P, and B(a)P-7,8-diol. These

130

substrates represent PAHs of varying complexity and were all shown to be substrates for

UGT2A1_i1 in previous studies. Michaelis-Menten kinetics curves for three PAHs

examined (Figure 4.6) show that increases in UGT2A1_i2 expression caused a decrease

in the rate of UGT2A1_i1 glucuronide formation. Upon kinetic analysis, significant

(p<0.01) trends were observed between increasing levels of UGT2A1_i2 expression and

decreasing PAH-glucuronide formation as determined by Vmax for each of the three

substrates analyzed (Table 4.1). Homogenate from cells with the highest UGT2A1_i2

expression, which exhibited a UGT2A1_i2:UGT2A1_i1 ratio of approximately 1.2, had a

~50% reduction in glucuronide formation for all three substrates examined. No

significant changes in KM values were observed when UGT2A1_i2 expression was

induced, regardless of UGT2A1_i2 expression levels. PonA treatment had no effect on

the glucuronidation activity of homogenates expressing UGT2A1_i1 against 1-OH-

pyrene (results not shown). The C-terminal V5 tag caused no significant changes in

UGT2A1 enzyme activity (data not shown), with the enzyme kinetics (Vmax and KM) for

the control UGT2A1_i1_V5-over-expressing cell homogenates in these experiments

similar to those reported previously for untagged wild-type UGT2A1 against PAHs in

Chapter 2.

131

Figure 4.6. Increasing UGT2A1_i2 expression negatively regulates UGT2A1_i1 activity.

Michaelis-Menten kinetic curves for 1-OH-pyrene, 3-OH B(a)P, and B(a)P-7,8-diol

glucuronidation by UGT2A1_i1_V5, showing the decrease in glucuronidation activity by

UGT2A1_i1_V5 following UGT2A1_i2_FLAG co-expression.

132

Table 4.1. Kinetic analysis of the effect of UGT2A1_i2 co-expression on UGT2A1_i1 activity against PAH substrates.

a Data expressed as mg of total protein homogenate, corrected for relative UGT2A1_i1 protein expression. KM and Vmax represent the

mean of three independent experiments.

* p trend<0.01

1-OH-pyrene 3-OH-B(a)P B(a)P-7,8-diol

Treatment KM Vmax

KM Vmax

KM Vmax

(μM) (pmol/min/mg)a

(μM) (pmol/min/mg)a (μM) (pmol/min/mg)

a

Vehicle Control 159 ± 16 319 ± 12* 256 ± 32 82 ± 4.3* 230 ± 22 86 ± 3.6*

2 μM PonA 140 ± 16 261 ± 11 276 ± 20 74 ± 2.2 253 ± 37 70 ± 4.6

6 μM PonA 145 ± 17 219 ± 8.8 261 ± 24 64 ± 2.4 224 ± 19 53 ± 2.0

10 μM PonA 138 ± 12 153 ± 5.6 238 ± 24 47 ± 1.9 205 ± 32 41 ± 2.6

133

UGT2A1_i1 and UGT2A1_i2 hetero-dimerization. To test whether UGT2A1_i2

potentially modulates UGT2A1_i1 glucuronidation activity by direct protein-protein

interactions, protein lysate from cells treated with 10 μM PonA was used in co-IP

experiments. Using an anti-FLAG antibody to immunoprecipitate UGT2A1_i2_FLAG

and an anti-V5 antibody to detect UGT2A1_i1_V5 by Western blot analysis, a band

corresponding to UGT2A1_i1_V5 (~57 kDa) was observed (Figure 4.7, Panel A; lane 5).

As expected, a UGT2A1_i1_V5 band was observed using the anti-V5 antibody by

Western blot analysis when an anti-V5 antibody was used to pull down UGT2A1_i1_V5

(Figure 4.7, Panel A; lane 4); no bands were observed when no antibody was added to the

immunoprecipitation lysate (Figure 4.7, Panel A; lane 3) or when lysate was used from

HEK293 cell lines over-expressing either UGT2A1_i1_V5 or UGT2A1_i2_FLAG alone

(Figure 4.7, Panel A; lanes 1-2). Similarly, a band corresponding to UGT2A1_i2_FLAG

(~52 kDa) was detected when an anti-V5 antibody was used to immunoprecipitate

UGT2A1_i1_V5 and an anti-Flag antibody was used to detect UGT2A1_i2_FLAG by

Western blot (Figure 4.7, Panel B; lane 5). Again as expected, an UGT2A1_i2_FLAG

band was observed by Western blot analysis when an anti-FLAG antibody was used to

pull down UGT2A1_i2_FLAG (Figure 4.7, Panel B; lane 4); no bands were observed

when no antibody was added to the immunoprecipitation lysate (Figure 4.7, Panel B; lane

3) or when lysate was used from HEK293 cell lines over-expressing either

UGT2A1_i1_V5 or UGT2A1_i2_FLAG alone (Figure 4.7, Panel B; lanes 1-2).

134

Figure 4.7. Dimerization between UGT2A1_i1 and UGT2A1_i2 demonstrated by co-IP.

(A) The UGT2A1_i1_V5/UGT2A1_i2_FLAG complex was immunoprecipitated with an anti-

FLAG antibody, then visualized with a HRP-labeled V5 antibody (lane 5). Homogenates from

cells over-expressing UGT2A1_i1_V5 (lane 1) or UGT2A1_i2_FLAG (lane 2) alone were used

as negative controls. Dynabeads without antibody conjugation were used as an additional

negative control (lane 3). The UGT2A1_i1_V5:UGT2A1_i2_FLAG complex was

immunoprecipitated with an anti-V5 antibody, and then visualized with an anti-V5-HRP antibody

as a positive control (lane 4). (B) The UGT2A1_i1_V5/UGT2A1_i2_FLAG complex was

immunoprecipitated with an anti-V5 antibody, then visualized with a HRP-labeled FLAG

antibody (lane 5). Homogenates from cells over-expressing UGT2A1_i1_V5 (lane 1) or

UGT2A1_i2_FLAG (lane 2) alone were used as negative controls. Dynabeads without antibody

conjugation were used as an additional negative control (lane 3). The

UGT2A1_i1_V5:UGT2A1_i2_FLAG complex was immunoprecipitated with an anti-FLAG

antibody, and then visualized with an anti-FLAG-HRP antibody as a positive control (lane 4). All

co-IP experiments were repeated at least 3 times to verify the protein-protein interactions between

UGT2A1_i1 and UGT2A1_i2.

UGT2A1_i1 homo-dimerization. As UGT2A1_i1 and UGT2A1_i2 were shown to

hetero-dimerize, experiments were conducted to determine whether UGT2A1_i1 homo-

dimerization also occurs. As described in the Methods section, a stable HEK293 cell line

was created to co-express UGT2A1_i1_V5 and UGT2A1_i1_FLAG. Following

treatment with 10 μM PonA, UGT2A1_i1_FLAG expression was induced to

approximately the same level as UGT2A1_i1_V5, with UGT2A1_i1_FLAG expression

determined to be 0.95 relative to UGT2A1_i1_V5 expression (set to 1.0 as a reference).

135

No UGT2A1_i1_FLAG expression was observed in the vehicle control group (Figure

4.8, Panel A). UGT2A1_i1_V5 and UGT2A1_i1_FLAG expression levels were

confirmed to be relatively equal following Western blot analysis using the anti-UGT2A1

antibody (Figure 4.8, Panel B), as 10 µM PonA treatment induced UGT2A1 expression

(UGT2A1_i1_V5 + UGT2A1_i1_FLAG) to 1.86 relative to UGT2A1_i1_V5 expression

in the untreated control (set to 1.0 as a reference). Using an anti-FLAG antibody to

immunoprecipitate UGT2A1_i1_FLAG and an anti-V5-HRP antibody to detect

UGT2A1_i1_V5 by Western blot analysis, a band corresponding to UGT2A1_i1_V5 was

detected (Figure 4.8, Panel C; lane 5). Similarly, a band corresponding to

UGT2A1_i1_FLAG was detected when an anti-V5 antibody was used to

immunoprecipitate UGT2A1_i1_V5 and an anti-Flag-HRP antibody was used to detect

UGT2A1_i1_FLAG by Western blot (Figure 4.8, Panel D; lane 5). Positive and negative

controls, identical to those described in detail for UGT2A1_i1 and UGT2A1_i2 hetero-

dimerization, were used in this set of experiments.

136

Figure 4.8. Homo-dimerization of UGT2A1_i1 demonstrated by co-IP. (A) Representative

Western blot showing stable expression of UGT2A1_i1_V5 and induction of UGT2A1_i1_FLAG

expression in inducible co-expressed cell line. UGT2A1_i1_FLAG expression was induced

through addition of 10 μM PonA for 12 h. 50 μg of control and PonA treated protein was used for

each Western blot, with the ratio of UGT2A1_i1 to UGT2A1_i1 expression determined to be

approximately 1:1. (B) A Western blot using 100 μg protein from the control and 10 μM PonA

treatment groups was performed using an anti-UGT2A1 antibody to confirm UGT2A1 expression

levels detected by the anti-V5 and anti-FLAG antibodies in panel A. (C) The

UGT2A1_i1_V5/UGT2A1_i1_FLAG complex was immunoprecipitated with an anti-FLAG

antibody, then visualized with a HRP-labeled V5 antibody (lane 5). Homogenates from cells

over-expressing UGT2A1_i1_V5 (lane 1) or UGT2A1_i1_FLAG (lane 2) alone were used as

negative controls. Dynabeads without antibody conjugation were used as an additional negative

control (lane 3). The UGT2A1_i1_V5:UGT2A1_i1_FLAG complex was immunoprecipitated

with an anti-V5 antibody, and then visualized with an anti-V5-HRP antibody as a positive control

(lane 4). (D) The UGT2A1_i1_V5/UGT2A1_i1_FLAG complex was immunoprecipitated with an

anti-V5 antibody, then visualized with a HRP-labeled FLAG antibody (lane 5). Homogenates

from cells over-expressing UGT2A1_i1_V5 (lane 1) or UGT2A1_i1_FLAG (lane 2) alone were

used as negative controls. Dynabeads without antibody conjugation were used as an additional

negative control (lane 3). The UGT2A1_i1_V5:UGT2A1_i1_FLAG complex was

immunoprecipitated with an anti-FLAG antibody, and then visualized with an anti-FLAG-HRP

antibody as a positive control (lane 4). All co-IP experiments were repeated 4-6 times to verify

UGT2A1_i1 homo-dimerization.

137

Effect of UGT2A1_i1 or UGT2A1_i2 co-expression on the glucuronidation

activity other UGT enzymes. To examine whether UGT2A1 isoforms potentially

modulate the activity of other UGTs, studies were performed co-expressing either

UGT2A1_i1_V5 or UGT2A1_i2_V5 with UGT1A7, UGT1A10, or UGT2B17. These

UGTs were chosen because they are expressed in the respiratory and/or aerodigestive

tract and are active against tobacco carcinogens [98, 99, 177]. As determined by real-time

PCR, the relative levels of transcript in each UGT-co-expressing cell line were not

significantly different for the UGT2A1 isoform as compared to its UGT1A or 2B

counterpart (Figure 4.9). Western blots demonstrated that UGTs 1A7 and 1A10 were

expressed at similar levels when over-expressed alone or when co-expressed with

UGT2A1, and that similar levels of UGT2A1_i1_V5 and UGT2A1_i2_V5 protein were

observed in each co-expressed over-expressing cell line (data not shown).

138

Figure 4.9. Level of UGT2A1 over-expression is relatively equal to UGT1A7, UGT1A10, or

UGT2B17 over-expression in stable co-expressed cell lines. Real-time PCR was used to

determine approximate mRNA levels of each UGT in stable co-expressed HEK293 over-

expressing cell lines. ABI gene expression assays were used to quantitatively detect levels of

UGT2A1, UGT1A7, UGT1A10, or UGT2B17 transcript. Relative UGT2A1Δexon3 levels were

determined by comparing mRNA levels of UGT2A1Δexon3 transcript with UGT1A7,

UGT1A10, or UGT2B17 mRNA using the ΔΔCt method. Similar experiments were completed to

determine wild-type (wt) UGT2A1 mRNA levels relative to UGT1A10 or UGT2B17 in

additional co-expressed cell lines. Data are expressed as the mean ± standard deviation of

quadruplicate experiments and were normalized to RPLPO protein levels in each cell line and

corrected for differences in assay efficiencies.

Glucuronidation assays were performed using protein from cells over-expressing

UGT1A or UGT2B enzymes alone or after co-expression with UGT2A1 isoforms.

Representative Michaelis-Menten curves for UGT1A7- and UGT1A7/UGT2A1_i2 -over-

expressing cell homogenates show similar kinetics against 3-OH-BaP (Figure 4.10, Panel

A). A similar pattern was observed for UGT1A10- and UGT1A10/UGT2A1_i2 -over-

expressing cell homogenates against 3-OH-BaP and for UGT2B17- and

139

UGT2B17/UGT2A1_i2 -over-expressing cell homogenates against 1-naphthol (Figure

4.10, Panels B and C). Glucuronidation assays also showed no significant changes in

glucuronidation activity for UGT1A10- and UGT1A10/UGT2A1_i1 -over-expressing

cell homogenates and UGT2B17-and UGT2B17/UGT2A1_i1 -over-expressing cell

homogenates against the non-UGT2A1 substrate NNAL (results not shown). No

significant differences in KM or Vmax were observed for any substrate analyzed after

UGT1A or 2B co-expression with either UGT2A1_i2 or UGT2A1_i1 (Table 4.2). In

addition, no significant differences in glucuronidation rates of additional substrates were

observed using homogenates from cells co-expressing UGT1A7, UGT1A10, or

UGT2B17 and UGT2A1; this included glucuronidation of the flavonoid chrysin by the

UGT1A7 co-expressing cell line, glucuronidation of the HCA metabolite N-OH PhIP by

the UGT1A10 co-expressing cell lines, and glucuronidation of ibuprofen by the

UGT2B17 co-expressing cell lines (data not shown).

140

Figure 4.10. Glucuronidation activity of UGT1A7, UGT1A10, or UGT2B17 against 3-OH-

B(a)P and 1-naphthol following co-expression with UGT2A1_i1 or UGT2A1_i2. Representative Michaelis-Menten kinetics curves summarizing activity data using homogenates

from HEK293 cell lines over-expressing UGT1A7 or UGT1A7 + UGT2A1_i2 against 3-OH-

B(a)P, UGT1A10 UGT1A10 + UGT2A1_i2 against 3-OH-B(a)P, and UGT2B17 or UGT2B17 +

UGT2A1_i2 against 1-naphthol.

141

Table 4.2. Glucuronidation activity of homogenates from cell lines co-expressing UGT2A1_i1 or UGT2A1_i2 with UGT1A7,

UGT1A10, or UGT2B17.

3-OH-B(a)P 1-naphthol NNAL

UGT(s) over-expressed KM Vmax

KM Vmax

KM Vmax

(μM) (pmol/min/mg)a (μM) (pmol/min/mg)

a (mM) (pmol/min/mg)

a

UGT1A7 120 ± 12 2074 ± 69 28 ± 5.7 745 ± 47 Not performed

UGT1A7/UGT2A1_i2_V5 142 ± 16 1998 ± 78 22 ± 4.3 796 ± 41

UGT1A10

UGT1A10/UGT2A1_i2_V5

25 ± 5.7

21 ± 3.9

652 ± 22

677 ± 17

17 ± 3.4

12 ± 2.7

574 ± 38

528 ± 26 Not performed

UGT2B17

UGT2B17/UGT2A1_i2_V5

BLD

BLD

BLD

BLD

200 ± 34

243 ± 29

382 ± 25

438 ± 28

Not performed

UGT1A10 Not performed

6.7 ± 1.7 34 ± 4.8

UGT1A10/UGT2A1_i1_V5 5.9 ± 1.3 37 ± 2.5

UGT2B17 Not performed

2.9 ± 0.9 11 ± 2.7

UGT2B17/UGT2A1_i1_V5 3.3 ± 0.7 10 ± 3.3

a Data expressed as mg of total protein homogenate. Kinetics data represents the mean of three independent experiments.

BLD, below limit of detection – UGT2B17 does not exhibit detectable glucuronidation activity against 3-OH B(a)P.

142

4.5 Discussion

The present study is the first to identify and characterize a novel splice variant of

UGT2A1, demonstrating its expression in multiple tissues at both the level of mRNA and

protein and its functional importance as an inhibitor of wild-type UGT2A1 activity. This

variant consists of a deletion of exon 3 that results in an inactive UGT2A1 isoform.

UGT2A1 exon 3 is a conserved region throughout all UGT family members [67] and is

also highly conserved with the corresponding region of the mouse ortholog UGT2a1

following sequence alignment. The exon 3-encoded protein region of all UGTs is

hypothesized to form a portion of the UDPGA co-substrate binding pocket necessary for

UGT function, and similar to the activity results for the splice variant UGT2A1 isoform

in the present study, amino acid point mutations in this region have been previously

shown to ablate UGT enzyme activity [86, 88].

In addition to possessing no detectable glucuronidation activity, UGT2A1_i2 was

shown negatively regulate wild-type UGT2A1_i1 activity. This negative inhibition

appears to be due to direct binding of the inactive UGT2A1_i2 variant with UGT2A1_i1,

a mechanism similar to that observed for other UGTs including the UGT1A exon 5b

variant [218]. Further, a Gln331Stop polymorphism in UGT1A1 leads to a truncated and

inactive protein, and this protein isoform has been reported to bind to and inhibit wild-

type UGT1A1 activity [219]. Ghosh et al. showed that an inactive mutant form of

UGT1A1, caused by a single amino acid change at codon 127, could bind to and inhibit

wild-type UGT1A1 activity in a dominant-negative manner [220]. Also, recent work has

143

identified inactive UGT2B4 splice variant isoforms that negatively regulate wild-type

UGT2B4 activity [207], and inactive UGT2B7 splice variant isoforms negatively

modulate wild-type UGT2B7 activity [208].

In the present study, a PonA-inducible co-expression system was designed to

control UGT2A1_i2 expression so that its effects on UGT2A1_i1 activity could be

examined. A linear correlation was observed between the ratio of

UGT2A1_i2:UGT2A1_i1 expression and overall glucuronidation activity of

homogenates from co-expressed cells. At the highest level of UGT2A1_i2 induction,

with the expression of UGT2A1_i2 approximately equal to that of UGT2A1_i1, there

was an approximately 50% reduction in the rate of glucuronide formation for three PAH

substrates examined. As the UGT2A1_i2:UGT2A1_i1 ratio decreased, proportional

increases in UGT2A1_i1 activity were observed against all three PAH substrates. These

data suggest that UGT2A1_i1 interacts with UGT2A1_i2 in a 1:1 stoichiometry, likely as

a dimer as proposed for other UGTs [83, 218]. The in vitro inducible

UGT2A1_i2:UGT2A1_i1 ratios observed in this study approximated the ratios observed

physiologically in several human tissues, including the lung and colon. These

UGT2A1_i2:UGT2A1_i1 ratios corresponded with 10-50% decreases in the Vmax for

UGT2A1_i1-mediated glucuronidation activities against PAH substrates; no significant

change in UGT2A1_i1 KM due to UGT2A1_i2 co-expression was observed. These data

suggest that the inhibitory effect of UGT2A1_i2 expression is mediated by removal of

UGT2A1_i1 from the active protein pool, rather than an effect on UGT2A1_i1 enzyme-

substrate affinity.

144

UGTs have been shown to dimerize in the ER to form both homo- and hetero-

oligomers [83, 213, 214]. In vitro UGT co-expression and dimerization studies have

shown that UGT interactions are complex, with kinetic changes dependent on the specific

UGT isoforms interacting and the substrates undergoing glucuronidation [83, 213, 214].

The co-IP experiments presented in this study suggested hetero-dimerization occurs

between the UGT2A1_i1 and UGT2A1_i2 isoforms. UGT2A1 homo-dimerization was

also investigated for the first time in this study, and in vitro co-IP experiments showed for

the first time that UGT2A1_i1 homo-dimerization does occur. UGT2A1 co-expression

studies were completed with two members of the UGT1A sub-family, UGT1A7 and

UGT1A10, and UGT2B17 to determine any UGT2A1-mediated changes in UGT1A or

UGT2B glucuronidation activity. Like UGT2A1, UGT1A7, UGT1A10, and UGT2B17

are expressed in certain tissues of the respiratory and aerodigestive tract and exhibit

glucuronidation activity against multiple tobacco carcinogens [75, 98, 99, 177]. UGT2A1

was determined to have no effect on the glucuronidation activity of UGT1A7, UGT1A10

or UGT2B17 against tobacco carcinogens, including PAHs, TSNAs and HCAs,

suggesting that UGT2A1 does not affect the ability of these UGTs to detoxify tobacco

carcinogens in vivo and that UGT2A1_i2 regulation of UGT activity is UGT2A1-

specific.

The entire UGT2A family has been under-studied, with limited information

reported on expression and activity of these enzymes. UGT2A3 has been shown to be

well expressed in the colon, small intestine and liver, with activity reported against bile

acids [71]. UGT2A2 has been reported to be expressed in the nasal mucosa, with broad

substrate selectivity reported against various estrogen and phenylphenol metabolites [70].

145

Neither of these enzymes has been carefully investigated for aerodigestive and

respiratory tract expression or metabolism against carcinogens. UGT2A1 and UGT2A2

have both been shown to exhibit glucuronidation activity against similar substrates, with

UGT2A1 generally having higher glucuronidation rates [70]. Based on exon sharing of

exon 1 of UGT2A1 or UGT2A2 to common exons 2-6, the same exon 3 deletion splice

variant for UGT2A2 may exist. The importance of UGT2A2 and UGT2A3 in tobacco

carcinogen metabolism will be investigated in future studies.

We have previously characterized UGT2A1 expression and activity, including the

discovery of a prevalent UGT2A1308Arg

polymorphism that ablates UGT2A1 enzymatic

activity. The identification of the UGT2A1_i2 splice variant isoform presented here

further complicates the regulation and functional significance of UGT2A1. The novel

UGT2A1_i2 regulatory mechanism described here likely allows for tighter control of

UGT2A1 glucuronidation activity. With expression of both UGT2A1_i1 and

UGT2A1_i2 in lung and colon tissue homogenates, as well as UGT2A1Δexon mRNA

expression in additional aerodigestive tract tissues, it is likely the in vitro data presented

here has widespread physiological significance. We hypothesize that the balance between

active UGT2A1_i1 and inactive UGT2A1_i2 could influence carcinogen metabolism in

local tissues susceptible to tobacco induced carcinogenesis. Individuals expressing higher

amounts of UGT2A1_i2 could potentially be at a greater risk to develop cancers in target

organs for tobacco carcinogenesis. Together the data presented in this study suggest that

UGT2A1_i2 may play an important role in modulating PAH metabolism in multiple

target organs for tobacco carcinogenesis.

146

Chapter 5

CHARACTERIZATION OF THE POTENTIAL ROLES OF UGT2A2 AND

UGT2A3 IN TOBACCO CARCINOGEN METABOLISM

147

5.1 Abstract

UGT2A1, a member of the under-studied UGT2A family, was previously shown

to be expressed in the lung and other aerodigestive tract tissues and have glucuronidation

activity against PAH substrates. The goal of this set of experiments was to investigate the

importance of two additional UGT2A enzymes, UGT2A2 and UGT2A3, in tobacco

carcinogen metabolism. Quantitative real-time PCR suggested that wild-type UGT2A2

had highest expression in the breast, followed by trachea > larynx > kidney. A novel

splice variant of UGT2A2 lacking exon 3 (UGT2A2Δexon3) was also discovered, with

UGT2A2Δexon3 expression determined to be 30-50% that of wild-type UGT2A2

expression in all tissues examined. UGT2A3 was determined to be expressed a variety of

tissues, with UGT2A3 well-expressed in the liver and colon, followed by pancreas >

kidney > lung > tonsil > trachea > larynx. Cell homogenates prepared from HEK293 cells

over-expressing wild-type UGT2A2 showed significant glucuronidation activity against

1-OH-pyrene, 1-naphthol, and hydroxylated B(a)P metabolites, while cell homogenates

prepared from HEK293 cells over-expressing UGT2A2Δexon3 had no detectable

glucuronidation activity against any substrate examined. Cell homogenates prepared from

HEK293 cells over-expressing UGT2A3 showed activity only against simple PAHs,

including 1-OH-pyrene and 1-naphthol. Both UGT2A2 and UGT2A3 were determined to

have no detectable activity against more complex PAH proximate carcinogens, TSNAs,

or HCAs. Data presented here suggest UGT2A2 and UGT2A3 are both expressed in

various aerodigestive and respiratory tract tissues and have activity against simple PAH

148

substrates; however, both enzymes were determined to lack enzyme activity against PAH

proximate carcinogens found in tobacco smoke.

5.2 Introduction

While the human UGT1A and UGT2B subfamilies have been characterized

extensively, a limited number of studies have analyzed UGT2A tissue expression and

activity against endogenous and exogenous substrates. One study previously examined

UGT2A2 expression and enzyme activity in comparison to UGT2A1 and UGT2A3.

UGT2A2 mRNA expression was reported in fetal and adult nasal mucosa tissues;

however, no UGT2A2 expression was observed in liver, fetal liver, lung, or fetal lung

tissues [70]. Histidine-tagged UGT2A2 was over-expressed in a baculovirus-insect cell

system to investigate enzyme activity, and UGT2A2 was determined to have

glucuronidation activity against a range of phenolic and estradiol substrates [70].

UGT2A1 and UGT2A2 were determined to have detectable activity against a similar

panel of substrates, though UGT2A2 was less active than UGT2A1 against the majority

of substrates examined [70]. In the same study, UGT2A3 was determined to only have

activity against the bile acid metabolite hyodeoxycholic acid [70]. An additional study

focused primarily on characterizing UGT2A3, with UGT2A3 expression determined to

be highest in the small intestine, followed by liver, colon, adipose and pancreatic tissue

[71]. A baculovirus-insect cell system was used to determine UGT2A3 enzyme activity

and UGT2A3 activity was only observed against bile acid substrates, including

149

hyodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and chenodeoxycholic

acid [71].

UGT2A1 was initially cloned from olfactory epithelium tissue and shown to

exhibit activity against common odorants; based on this data the physiological role of

UGT2A1 was hypothesized to be in the initiation and termination of olfactory stimuli

[97]. Studies recently conducted in our lab more fully characterized UGT2A1, with

UGT2A1 expression reported in the lung, trachea, tonsil, larynx and colon. Following

over-expression in a HEK293 mammalian cell system, UGT2A1 was determined to

exhibit activity against PAH proximate carcinogens such as 5-methylchrysense-1,2-diol,

B(a)P-7,8-diol, and dibenzo(a,l)pyrene-11,12-diol. Expression and activity data together

suggested that UGT2A1 potentially plays a role in the local metabolism of PAHs at target

tissues for tobacco carcinogenesis. In additional experiments with UGT2A1, a novel exon

3 deletion splice variant of UGT2A1was identified and determined to be an inactive

modulator of wild-type UGT2A1 activity.

Functional protein domains have been elucidated for the UGTs, with the substrate

binding domain more variable between UGT isoforms and found at the N-terminus and

the UDPGA binding site conserved between UGT isoforms and found at the C-terminus

of the protein [67, 78, 261]. With different substrate recognition sites due to unique first

exons, UGT2A1 and UGT2A2 would be hypothesized to have divergent substrate

specificities. However, one previous study suggested that UGT2A1 and UGT2A2 have

overlapping substrate specificities, as both enzymes had detectable O-glucuronidation

activities against similar estradiol and phenolic substrates [70]. A sequence alignment of

the UGT2A enzymes shows the exon 1 nucleotide sequences of UGT2A1 and UGT2A2

150

to be 66% similar, with an 84% similarity between the corresponding amino acids.

Although no exon sharing occurs between UGT2A3 and the other UGT2A enzymes,

UGT2A1 and UGT2A3 also have a high degree of sequence similarity. Exons 1 of

UGT2A1 and UGT2A3 have a 56% similarity in nucleotide sequence and a 70%

similarity in amino acid sequence. The high degree of sequence similarity between the

UGT2A enzymes in the substrate recognition region of the enzymes supports the

possibility of an overlap in substrate specificity.

The primary goal of the present study was to examine the potential roles of

UGT2A2 and UGT2A3 in tobacco carcinogen metabolism. We hypothesized that

UGT2A2 and UGT2A3 would have activity against tobacco carcinogens, in particular

PAH metabolites, based on UGT2A1 activity against these substrates and the sequence

homology between UGT2A enzymes. In this study, the activities of UGT2A2 and

UGT2A3 were tested against tobacco carcinogens using homogenates from a HEK293

mammalian over-expression system. In addition, mRNA expression levels of UGT2A2

and UGT2A3 were qualitatively and quantitatively examined in a large panel of human

tissues, many of which were never previously analyzed for UGT2A2 or UGT2A3

expression. As a secondary goal of this study, the tissue-specific expression and

enzymatic activity of a novel exon 3 deletion splice variant of UGT2A2

(UGT2A2Δexon3) were also investigated.

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5.3 Methods

Chemicals and Materials. Pfu High Fidelity DNA Polymerase was purchased

from Agilent (Santa Clara, CA). All oligonucleotide PCR primers were purchased from

IDT (Coralville, IA). Probes for real-time PCR experiments and gene expression assays

were acquired from Applied Biosystems Inc., Life Technologies (Carlsbad, CA). The

pcDNA3.1/V5-His-TOPO mammalian expression vector kit, Superscript II RT kit,

Lipofectamine 2000, and ampicillin were acquired from Invitrogen, Life Technologies

(Grand Island, NY). The BCA protein assay kit was purchased from Pierce (Rockford,

IL). The RNeasy kit, QIAquick gel extraction kit, Plasmid Mini kit, and Plasmid Maxi kit

were all obtained from Qiagen (Valencia, CA). DMEM, DPBS, FBS, penicillin-

streptomycin, and G418 were purchased from Gibco, Life Technologies (Grand Island,

NY). UDPGA, alamethicin, β-glucuronidase, 4-MU, 1-OH-pyrene, and 1-naphthol were

all acquired from Sigma-Aldrich (St. Louis, MO). NNAL, NNN, NAB, NAT, nicotine,

and PhIP were purchased from Toronto Research Chemicals (Ontario, Canada). 1-OH-

B(a)P, 3-OH-B(a)P, 7-OH-B(a)P, 8-OH-B(a)P, 5-methylchrysene-1,2-diol,

dibenzo(a,l)pyrene-11,12-diol, B(a)P-7,8-diol, and N-OH-PhIP were synthesized in the

Organic Synthesis Core Facility at the Penn State College of Medicine (Hershey, PA).

HPLC grade ammonium acetate, acetonitrile, and agarose were purchased from Fisher

Scientific (Pittsburgh, PA).

Qualitative determination of UGT2A2 and UGT2A3 expression in tissues. RT-

PCR was completed to determine tissue specific UGT2A2 and UGT2A3 expression.

152

Pooled RNAs from lung, larynx, trachea, breast, whole brain, prostate, kidney, cerebral

cortex and pancreas were obtained from Clontech (Mountain View, CA) or Agilent

(Santa Clara, CA). Adjacent normal liver, colon, floor of mouth, tonsil, and esophagus

tissues were obtained from the Penn State College of Medicine Tissue Bank (Hershey,

PA), and RNAs were extracted using an RNeasy kit. All qualitative RT-PCR experiments

were performed using pooled RNA from at least three individuals for each tissue

examined. RT reactions were completed using 2 μg of RNA and a Superscript II RT kit,

with cDNAs corresponding to 100 ng of RNA then used for each subsequent PCR

reaction.

Tissues were screened for full-length UGT2A2 expression using Pfu Polymerase

and sense (5’-CCATAAGGGATTTTACCATGCCTAAG-3’) (denoted as UGT2A2_S1)

and anti-sense (5’-TTCTCTTTTTTTCTTCTTTCCTATCTTACC-3’) primers

corresponding to nucleotides -17 to +9 and nucleotides +1584 to +1555, respectively,

relative to the UGT2A2 translation start site. The nucleotide locations of all UGT2A2

primer sequences in this study are designated assuming that the UGT2A2 protein is eight

amino acids shorter at the N-terminus than the updated sequence described in GenBank

(NM_001105677.2). This discrepancy was described previously by Sneitz et al. and

likely has no consequence on the mature UGT2A2 protein sequence, due to the post-

translational cleavage of the signal sequence at the N-terminus [70]. PCR parameters

used to amplify UGT2A2 were as follows; an initial denaturing temperature of 94°C for 2

min, then 40 cycles of 94°C for 30 s, 58°C for 40 s, and 72°C for 1.5 min, followed by a

final step of 72°C for 10 min. All PCR reactions were completed in a Bio-Rad Mycycler

(Hercules, CA, USA). RNAs from HEK293 cell lines over-expressing wild-type

153

UGT2A2 and UGT2A2Δexon3 (See Methods blow) were used as positive controls, while

water was used as a negative control. Tissues were screened for UGT2A3 expression

using Pfu Polymerase and exon 1 specific sense (5’-CATGAGGTCTGACAAGTCA-

GCTT-3’) and anti-sense (5’-CCTGTAGCTTCTTCATAAGCGTCTG-3’) primers

corresponding to nucleotides -1 to +22 and nucleotides +418 to +394, respectively,

relative to the UGT2A3 translation start site. PCR parameters used to amplify UGT2A3

were as follows; an initial denaturing temperature of 94°C for 2 min, then 40 cycles of

94°C for 30 s, 60°C for 40 s, and 72°C for 1.5 min, followed by a final step of 72°C for

10 min. RNA from a HEK293 cell line over-expressing UGT2A3 (See Methods below)

was used as a positive control, while water was used as a negative control. UGT2A2 and

UGT2A3 PCR products were gel purified using a QIAquick gel extraction kit and

sequenced by dideoxy sequencing at the Penn State University Nucleic Acid Facility

(State College, PA), with sequences compared to that described for UGT2A2

(NM_001105677.2) and UGT2A3 (NM_024743.3) in GenBank. To verify UGT2A2 or

UGT2A3 expression in tissues by RT-PCR, reactions were run multiple times with

positive and negative controls.

Quantitative determination of UGT2A2 and UGT2A3 tissue expression.

Quantitative real-time PCR experiments were completed to determine relative UGT2A2

and UGT2A3 RNA expression levels in tissues which were determined to express

UGT2A2 or UGT2A3 by RT-PCR. Separate real-time PCR assays were designed to

specifically and quantitatively detect wild-type UGT2A2 or UGT2A2Δexon3 transcripts

(Figure 5.1).

154

Figure 5.1. Schematic of real-time PCR assay developed to specifically detect either wild-

type UGT2A2 or UGT2A2Δexon3. Identical forward primers and probes were used to detect

both transcripts, while reverse primers were designed to specifically recognize either wild-type

UGT2A2 or UGT2A2Δexon3. Assay specificity was confirmed by running real-time PCR

products on an agarose gel and sequencing the products.

A sense primer specific to UGT2A2 exon 1 (5’-GGAGAATGGAATTCATA-

CTATAGCAAA-3’) and a 5’ labeled VIC probe specific to UGT2A2 exon 2 (5’-TCCG-

AACATATTGGGATT-3’), corresponding to nucleotides +685 to +711 and +770 to

+787, respectively, relative to the UGT2A2 translation start site, were used to detect both

wild-type UGT2A2 and UGT2A2Δexon3 transcript expression. An anti-sense primer

specific to UGT2A2 exon 3 (5’-TTACCTGAGCTCTGGATAAATTCTTC-3’),

corresponding to nucleotides +899 to +874 relative to the UGT2A2 translation start site,

was used to specifically detect wild-type UGT2A2. An anti-sense primer specific to the

UGT2A1Δexon3 exon 2 and 4 junction (5’-TTTCCTTTGTATCTCCATAAAACC-

TTAG-3’), corresponding to nucleotides +890 to +863 relative to the UGT2A2Δexon3

start site, was used to specifically detect UGT2AΔexon3. Following RT, cDNA

corresponding to 20 ng of pooled RNA was used for each reaction. Assay specificity for

wild-type UGT2A2 or UGT2A2Δexon3 was confirmed through agarose gel

electrophoresis and dideoxy sequencing of PCR products following amplification. The

efficiency of each UGT2A2 real-time PCR assay was determined, and relative levels of

155

wild-type UGT2A2 or UGT2A2Δexon3 were corrected for differences in assay

efficiency, as described previously [256]. Experiments to investigate relative UGT2A3

tissue expression were completed using an ABI UGT2A3 specific gene expression assay

(Hs00226904_m1). cDNA corresponding to 20 ng of pooled RNA was used for each

UGT2A3 real-time PCR reaction. For both UGT2A2 and UGT2A3 experiments, standard

thermal cycling parameters were followed and RPLPO was used as a housekeeping gene

(Hs99999902_m1). The RPLPO gene has been shown to have relatively low inter-

individual variability in lung and aerodigestive tract tissues (Jones et al), and multiple

studies analyzing mRNA expression in human tissues have used RPLPO as an

endogenous control [262, 263]

All real-time PCR experiments were completed in triplicate at the Penn State

College of Medicine Functional Genomics Core Facility (Hershey, PA) using an ABI

7900HT Thermal cycler and data was analyzed by SDS 2.2 software. Relative wild-type

UGT2A2 or UGT2A3 expression levels were calculated using the ΔΔCt method, relative

to the tissue which had the highest expression of wild-type UGT2A2 (breast) or UGT2A3

(liver). The relative expression of UGT2A2Δexon3 transcript was calculated using the

same method relative to the amount of wild-type UGT2A2 transcript in each tissue, with

wild-type UGT2A2 expression determined to be higher than UGT2A2Δexon3 expression

in all tissues analyzed. The relative expression levels of wild-type UGT2A2 and

UGT2A3 in larynx, trachea, and kidney were also compared using the ΔΔCt method

relative to wild-type UGT2A2, following a correction for differences in assay efficiency

as described previously [256].

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156

Generation of HEK293 cell lines over-expressing UGT2A2 and UGT2A3. Cell

lines over-expressing wild-type UGT2A2 and UGT2A2Δexon3 were generated by RT-

PCR using pooled trachea RNA. The cDNA equivalent of 100 ng RNA was used with

Pfu Polymerase for UGT2A2 PCR amplification. The entire UGT2A2 coding region was

amplified using the UGT2A2_S1 primer, described previously, and an anti-sense (5’-

TGACAGGAAGAGGGTATAGTCAGC-3’) primer corresponding to nucleotides +

1837 to +1814 relative to the UGT2A2 translation start site. UGT2A2 PCR reactions

were completed with an initial denaturing temperature of 94°C for 2 min, then 40 cycles

of 94°C for 30 s, 60°C for 40 s, and 72°C for 2 min, followed by a final step of 72°C for

10 min. Both wild-type UGT2A2 and UGT2A2Δexon3 PCR products were gel purified

using a QIAquick gel extraction kit and sequenced by dideoxy sequencing at the Penn

State University Nucleic Acid Facility (State College, PA), using the forward and reverse

PCR primers and an internal sense primer (5’-GCTCACTGACCAGATGACCTTTG-3’),

corresponding to nucleotides +600 to +622 relative to the UGT2A2 translation start site.

Full-length UGT2A3 was amplified following RT-PCR from pooled pancreas

RNA. The cDNA equivalent of 100 ng RNA was used with Pfu polymerase to amplify

full length UGT2A3. UGT2A3 was amplified using sense (5’-TTGCAGATCAG-

TGTGTGAGGGAACTG-3’) and anti-sense (5’-CCCCATCAGGTCTTTCTTGA-

ATTTGG-3’) primers corresponding to nucleotides -31 to -6 and +1616 to +1591,

respectively, relative to the UGT2A3 translation start site. UGT2A3 PCR reactions were

completed with an initial denaturing temperature of 94°C for 2 min, then 40 cycles of

94°C for 30 s, 59°C for 40 s, and 72°C for 2 min, followed by a final step of 72°C for 10

min. Following gel extraction, the UGT2A3 PCR product was sequenced using the

157

previously described UGT2A3 primers and an internal sense primer (5-GAGAGC-

TTTATCTACAATCAGACGC-3’), corresponding to nucleotides +376 to +400 relative

to the UGT2A3 translation start site.

Fully verified wild-type UGT2A2, UGT2A2Δexon3, and UGT2A3 cDNAs were

cloned into a pcDNA3.1/V5-His-TOPO vector using the standard protocol, and

transformation was completed using OneShot TOP10 Competent E. Coli. Following large

scale plasmid preparations, the standard Lipofectamine protocol was used to generate

HEK293 cell lines over-expressing the protein corresponding to wild-type UGT2A2

(defined as UGT2A2_i1), the protein corresponding to UGT2A2Δexon3 (defined as

UGT2A2_i2), or UGT2A3. Cells were grown up in DMEM, supplemented with 10%

FBS, 1% penicillin-streptomycin, and G418 (400 μg/mL), to 75% confluence. Cell

homogenates were prepared as previously described in 1X Tris-buffered saline (25 mM

Tris base, 138 mM NaCl, and 2.7 mM KCl; pH 7.4) [98, 110, 207]. Total RNA was

extracted from each cell line using an RNeasy Mini kit following manufacturer’s

protocols. Total protein concentrations from cell line homogenates were determined

using a BCA protein assay.

Examination of levels of UGT2A2 or UGT2A3 over-expression. Currently no

readily available antibody exists for quantification of UGT2A2 or UGT2A3 protein. A

custom antibody specific for the UGT2A1 N-terminus was designed previously to have

no cross-reactivity against other UGT isoforms. In the current study, the UGT2A1

antibody was tested for cross-reactivity against UGT2A2 and UGT2A3 protein for the

first time. Up to 150 μg of UGT2A2_i1 or UGT2A3 protein lysates were used in Western

158

blots with the anti-UGT2A1 antibody (Open Biosystems, Huntsville, AL) at a 1:500

dilution. 50 μg of UGT2A1 protein was loaded as a positive control, while 150 μg of

HEK293 protein lysate was loaded as a negative control.

To examine relative wild-type UGT2A2, UGT2A2Δexon3, and UGT2A3 mRNA

expression levels in HEK293 over-expressing cell lines, real-time PCR was completed

using ABI gene expression assays (Hs04195512_s1 for determination of UGT2A2 or

UGT2A2Δexon3 expression, Hs00226904_m1 for UGT2A3 expression). The UGT2A2

gene expression assay is specific for UGT2A2 exon 1, enabling the assay to detect both

wild-type UGT2A2 and UGT2A2Δexon3 transcripts. GAPDH (Hs99999905_m1) was

used as a housekeeping gene, and all assays were completed as described previously

using standard thermal cycling parameters. Relative levels of UGT2A over-expression in

each stable cell line were calculated using the ΔΔCt method, after corrections were made

to account for differences in the amplification efficiency of each assay [256].

Glucuronidation Activities of UGT2A2_i1, UGT2A2_i2, and UGT2A3.

Glucuronidation assays using homogenates from HEK293 cell lines over-expressing

UGT2A2_i1, UGT2A2_i2, and UGT2A3 were completed as previously described [98,

110, 234]. After an initial incubation of 200 μg total cell homogenate protein with 10 μg

alamethicin for 15 min on ice, glucuronidation reactions were done in a final reaction

volume of 25 μL at 37°C with 50 mM Tris-HCl, pH 7.5, 10mM MgCl2, 4 mM UDPGA,

and between 6μM and 1mM of substrate.. Reactions were terminated by the addition of

25 μL acetonitrile and reaction mixtures were centrifuged at 16100 g for 10 min prior to

the collection of supernatant.

https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=ABAssayDetailDisplay&assayID=Hs04195512_s1&Fs=y&adv_phrase3=EXACT&adv_phrase2=EXACT&adv_phrase1=EXACT&assayType=GE&catID=601267&adv_kw_filter3=ALL&srchType=keyword&adv_kw_filter2=ALL&SearchRequest.Common.QueryText=UGT2A2&kwdropdown=ge&adv_kw_filter1=ALL&species=Homo+sapiens&adv_query_text3=&searchType=keyword&adv_query_text2=&adv_query_text1=&uploadType=ID+List&adv_boolean3=AND&adv_boolean2=AND&adv_boolean1=AND&chkBatchQueryText=false&kwfilter=ALL&SearchRequest.Common.PageNumber=1&msgType=ABGEKeywordResults

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159

Glucuronides were detected using a Waters Acquity UPLC System (Milford, MA)

as described previously [110, 112, 257]. Briefly, a flow rate of 0.5 mL/min was used with

a reverse phase Acquity BEH C18-1.7 μM 2.1 X 100 mm column to detect free substrate

and conjugated glucuronides. The glucuronides and substrates were eluted from the

column using substrate specific gradients that differed in the proportion of solution A

(5mM NH4OAc pH 5.0, 10% acetonitrile) and solution B (100% acetonitrile). The initial

solvent gradients and UV absorbance wavelengths used to determine the glucuronidation

of various substrates were described previously (Chapter 2). Quantification of

glucuronide formation was completed as described previously [75, 98, 110, 112, 118].

Briefly, the amount of glucuronide was determined based on the ratio of the area under

the curve of the glucuronide peak versus substrate peak, with the concentration of the

substrate in each reaction used as a reference. The glucuronidation rate for each substrate

was determined using at least eight substrate concentrations encompassing the

experimentally determined KM. β-glucuronidase treatment and mass spectrometry

analysis were used to confirm all glucuronides. Various negative controls were used,

including reactions with empty HEK293 cells and reactions with no substrate added to

the reaction mixture. Substrates of UGT2A2 and UGT2A3 that had glucuronide

formation below the limit of detection were incubated for 18 h with 750 μg of total

protein homogenate to confirm the lack of glucuronidation activity. For glucuronidation

rate determinations, cell homogenate protein levels and incubation times for each

substrate were determined experimentally to ensure that substrate utilization was less

than 10% and to maximize levels of detection while in a linear range of glucuronide

formation.

160

Data analysis and statistics. Three independent experiments were completed for

UGT2A2 and UGT2A3 kinetic analyses in this study. Kinetic constants were calculated

using GraphPad Prism 5 software (San Diego, CA). Vmax and Km values for each substrate

examined were calculated by graphing the glucuronide product formed versus the

substrate concentration and then using the Michaelis-Menten equation. All data was

transformed into a linear Eadie-Hofstee plot to visually confirm that a simple Michaelis-

Menten mechanism was followed. Enzyme kinetics of UGT2A isoforms were compared

through use of the Student’s t-test, after correcting for relative UGT2A over-expression

in each stable cell line.

5.4 Results

Qualitative expression of UGT2A2 and UGT2A3 in human tissues. A previous

study reported full-length UGT2A2 expression in fetal and adult nasal mucosa tissues

[70]. In the current study a more comprehensive analysis of UGT2A2 expression was

performed, with a focus on enzyme expression in the aerodigestive and respiratory tracts.

Initially, pooled RNA samples from various tissues were analyzed qualitatively for

UGT2A2 expression. As shown in Figure 5.2 (Panel A) UGT2A2 was found to be well-

expressed in the breast, larynx, trachea, and kidney, and two distinct UGT2A2 products

were amplified using PCR primers specific for the amplification of full-length UGT2A2.

Following gel extraction and sequencing it was determined that, in addition to wild-type

UGT2A2 (NM_001105677.2), a novel UGT2A2 transcript lacking exon 3 was also

161

amplified. No UGT2A2 expression was observed following multiple RT- PCR attempts

in any of the other tissues examined, including whole brain, lung, tonsil, colon, pancreas,

prostate, cerebral cortex, floor of mouth, esophagus, and liver (data not shown).

Earlier studies analyzing UGT2A3 expression reported the enzyme to be

expressed predominantly in the small intestine, but also in the liver, colon, pancreas,

kidney and stomach [70, 71]. In the current study additional tissues were analyzed for

UGT2A3 expression. As shown in Figure 5.2 (Panel B) RT-PCR using primers specific

for the amplification of exon 1 showed UGT2A3 to be expressed in the trachea, larynx,

tonsil, lung, colon, liver, kidney, and pancreas. Full-length UGT2A3 (NM_024743.3)

was able to be amplified from these tissues and no novel UGT2A3 splice variants were

observed (data not shown). No UGT2A3 expression was observed following multiple

RT- PCR attempts in whole brain, breast, cerebral cortex, floor of mouth, esophagus, and

prostate (data not shown).

162

Figure 5.2. Qualitative determination of UGT2A2 and UGT2A3 tissue expression. (A) Initial

characterization of UGT2A2 expression in multiple human tissues using RT- PCR and primers

specific for the full-length UGT2A2 transcript. RNAs from HEK293 cell lines over-expressing

wild-type UGT2A2 and UGT2A2Δexon3 were used as positive controls. As a negative control,

water was used in the place of cDNA. Two distinct transcripts were amplified using primers

specific for UGT2A2, and following sequencing it was determined that both wild-type UGT2A2

and a novel UGT2A2 exon 3 deletion splice variant were amplified. (B) Initial screening of

UGT2A3 expression in multiple human tissues using RT-PCR. Primers specific for exon 1 of

UGT2A3 were used to determine tissue expression. RNA from a HEK293 cell line over-

expressing UGT2A3 was used as a positive control and water was used in place of cDNA as a

negative control. For the determination of UGT2A2 and UGT2A3 tissue expression, pooled

RNAs from at least three individuals were used.

Quantitative assessment of UGT2A2 and UGT2A3 expression. A custom

UGT2A2 real-time PCR assay (Figure 5.1) was developed to specifically and

quantitatively detect wild-type UGT2A2 or UGT2A2Δexon3 expression in tissues which

were shown to express UGT2A2 through RT-PCR. As shown in Figure 5.3 (Panel A)

wild-type UGT2A2 expression was highest in breast (1.0 ± 0.09, set as a reference),

followed by trachea (0.57 ± 0.10) > larynx (0.51 ± 0.11) > kidney (0.05 ± 0.02). The

custom UGT2A2 real-time PCR assay was also used to determine the relative level of

UGT2A2Δexon3 compared to wild-type UGT2A2 expression in each tissue. In each

tissue analyzed, wild-type UGT2A2 expression (set at 1.0 as a reference) was greater

than UGT2A2Δexon3 expression. As shown in Figure 5.3 (Panel B) UGT2A2Δexon3

163

expression relative to wild-type UGT2A2 expression was highest in the larynx (0.51 ±

0.06) followed by trachea (0.47 ± 0.05), breast (0.39 ± 0.08), and kidney (0.25 ± 0.02).

Real-time PCR was also used to quantitatively analyze relative UGT2A3 expression in

tissues where UGT2A3 expression was previously detected by RT-PCR. As shown in

Figure 5.3 (Panel C) relative UGT2A3 expression was found to be highest in the liver

(used as a reference at 1.0 ± 0.12) followed by colon (0.65 ± 0.09) > pancreas (0.17 ±

0.05) > kidney (0.15 ± 0.03) > lung (0.14 ± 0.02) > tonsil (0.06 ± 0.01) > trachea (0.04 ±

0.01) > larynx (0.02 ± 0.01). Expression levels of wild-type UGT2A2 and UGT2A3 were

directly compared in the larynx, trachea, and kidney (Figure 5.3, Panel D), with wild-type

UGT2A2 expression set as the reference (1.0). UGT2A3 expression was determined to be

lower than wild-type UGT2A2 expression in the larynx (0.36 ± 0.09) and trachea (0.22 ±

0.05), while UGT2A3 expression was determined to be higher than wild-type UGT2A2

expression in the kidney (1.85 ± 0.12).

164

Figure 5.3. Quantitative determination of UGT2A2 and UGT2A3 expression in multiple

human tissues. (A) RNAs from tissues exhibiting UGT2A2 expression in Figure 5.2 (Panel A)

were used with a wild-type UGT2A2 specific real-time PCR assay to determine relative wild-type

UGT2A2 expression. Wild-type UGT2A2 tissue expression was calculated using the ΔΔCt

method, relative to wild-type UGT2A2 expression in the breast. (B) Relative UGT2A2Δexon3

mRNA expression in each tissue was determined using the assays described in Figure 5.1.

Expression data was corrected to account for differences in the amplification efficiency of each

assay. Relative UGT2A2Δexon3 expression in each tissue was determined, using the ΔΔCt

method, by comparing levels of UGT2A2Δexon3 to levels of wild-type UGT2A2 expression.

(C) RNAs from tissues exhibiting UGT2A3 expression in Figure 5.2 (Panel B) were used with a

UGT2A3 specific real-time PCR assay to quantitatively determine UGT2A3 tissue expression.

UGT2A3 tissue expression was calculated using the ΔΔCt method, relative to UGT2A3

expression in the liver. (D) Expression levels of wild-type UGT2A2 and UGT2A3 were

compared in the three tissues that were determined to express both enzymes. Expression levels

were normalized to wild-type UGT2A2 (set to 1.0 as a reference) following correction for the

amplification efficiency of each assay. For all real-time PCR data, results are expressed as the

mean ± SD of triplicates and results were normalized to RPLPO expression in each tissue. cDNA

corresponding to 20 ng RNA was used for each reaction.

165

Enzyme activity of UGT2A2 and UGT2A3 against tobacco carcinogens. With

mRNA expression of both UGT2A2 and UGT2A3 in target tissues for tobacco

carcinogenesis, and UGT2A1 previously shown to exhibit activity against PAH

metabolites, experiments were completed to investigate the activities of UGT2A2 and

UGT2A3 against various PAHs, TSNAs, and HCAs.

The glucuronidation activities of UGT2A2_i1 and UGT2A2_i2 were determined

against various PAH metabolites using homogenates from a stable HEK293 over-

expression system. As shown in Figure 5.4 (Panel A), UGT2A2_i1 was shown to exhibit

activity against 1-naphthol, with a 1-naphthol-O-glucuronide peak observed at a retention

time of 1.8 min and a 1-naphthol substrate peak observed at 4.8 min by UPLC. Activity

assays using homogenates from HEK293 cells over-expressing UGT2A2_i1 were

completed for a panel of additional PAH substrates. In addition to activity against 1-

naphthol, UGT2A2_i1 was determined to have glucuronidation activity against 1-OH-

pyrene, 1-OH-B(a)P, 3-OH-B(a)P, 7-OH-B(a)P, and 8-OH-B(a)P. No UGT2A2_i1

activity was detected against more complex PAH proximate carcinogens, including 5-

methylchrysene-1,2-diol, B(a)P-7,8-diol, or dibenzo(a,l)pyrene11,12-diol. As shown in

Figure 5.4 (Panel B), UGT2A2_i2 exhibited no detectable glucuronidation activity

against 1-naphthol, with only a 1-naphthol substrate peak observed at 4.8 min and no 1-

naphthol-O-glucuronide peak detected. UGT2A2_i2 was also found to exhibit no

detectable glucuronidation activity against any PAH tested, including the six PAH

substrates determined to be substrates of UGT2A2_i1. UGT2A2_i2 also had no

detectable activity against the common UGT substrate 4-MU [237] (data not shown).

166

Activity assays using homogenates from HEK293 cells over-expressing UGT2A3

were also performed. UGT2A3 was determined to have glucuronidation activity against

the simple PAH 1-OH-pyrene, with a pyrene-1-O-glucuronide peak observed at 1.1 min

and a 1-OH-pyrene substrate peak observed at 3.7 min on UPLC (Figure 5.4, Panel C).

UGT2A3 was also found to have activity against the simple PAH 1-naphthol; however,

no UGT2A3 activity was detected against the remainder of the PAHs analyzed, including

1-OH-B(a)P, 3-OH-B(a)P, 7-OH-B(a)P, 8-OH-B(a)P, 5-methylchrysene-1,2-diol, B(a)P-

7,8-diol, and dibenzo(a,l)pryrene-11,12-diol.

The glucuronidation activities of UGT2A2_i1, UGT2A2_i2, and UGT2A3 against

multiple TSNA and HCA substrates were also investigated. UGT2A2_i1, UGT2A2_i2,

and UGT2A3 all exhibited no detectable glucuronidation activity against TSNAs

(including nicotine, NNAL, NAB, NAT, and NNN) or HCAs (including PhIP and N-OH-

PhIP) (data not shown).

167

Figure 5.4. UGT2A2 and UGT2A3 exhibit glucuronidation activity against simple PAH

substrates. (A) UPLC trace of 1-naphthol-O-gluronide formation following 1-naphthol

incubation with UGT2A2_i1 over-expressing cell homogenate. A naphthol-1-O-glucuronide peak

was observed at 1.8 min, while a 1-naphthol substrate peak was observed at 4.8 min. (B) No

detectable glucuronide was observed following incubation of UGT2A2_i2 over-expressing cell

homogenate with 1-napthhol, as shown by representative UPLC chromatogram with only a 1-

napthol substrate peak at 4.8 min. (C) UPLC trace of pyrene-1-O-glucuronide formation after 1-

OH-pyrene incubation with UGT2A3 over-expressing cell homogenate. A glucuronide peak was

observed at 1.1 min and a 1-OH-pyrene peak was observed at 3.7 min.

Enzyme kinetics of UGT2A2 and UGT2A3 activity. No readily available

common antibody currently exists to detect UGT2A2 or UGT2A3 protein, and a custom

designed UGT2A1 antibody was shown to exhibit no cross-reactivity towards UGT2A2

or UGT2A3 (Figure 5.5). Real time-PCR was used to approximate UGT2A2 or UGT2A3

mRNA levels in each UGT-over-expressing HEK293 cell line. The cell-line over-

expressing wild-type UGT2A2 exhibited the highest relative mRNA expression (set as

168

1.0 as the reference), while the cell line over-expressing UGT2A3 exhibited a relative

mRNA level of 0.84 ± 0.08. The cell-line over-expressing UGT2A2_i2 had a slightly

lower mRNA level of UGT2A2Δexon3 (0.91 ± 0.11) relative to wild-type UGT2A2 in

the UGT2A2_i1 over-expressing cell line. The enzyme kinetics for UGT2A2 and

UGT2A3 were corrected based on the relative mRNA expression of each enzyme in each

HEK293 over-expressing cell line.

Figure 5.5. UGT2A1 antibody exhibits no cross-reactivity against UGT2A2 or UGT2A3.

Protein homogenates from HEK293 cells over-expressing UGT2A1_i1 (lane 1), empty HEK293

cells (lane 2), HEK293 cells over-expressing UGT2A2_i1 (lane 3), and HEK293 cells over-

expressing UGT2A3 (lane 4) were used to determine cross-reactivity of an anti-UGT2A1

antibody against other UGT2A isoforms. No cross-reactivity was observed for the anti-UGT2A1

antibody against UGT2A2 or UGT2A3 following multiple Western blots, including when up to

150 μg of protein was loaded per lane. An antibody specific to B/actin was used as a loading

control.

UGT2A2_i1 enzyme kinetics against six PAH substrates and UGT2A3 enzyme

kinetics against two PAH substrates were determined. Representative Michaelis-Menten

and Eadie-Hofstee kinetics curves for UGT2A2_i1 activity against 7-OH-B(a)P are

shown in Figure 5.6 (Panels A and B, respectively). The Eadie-Hofstee transformation

169

was completed in order to ensure that the reactions followed simple Michaelis-Menten

kinetics, as shown by the linearity of the plot. Similar curves were generated to analyze

UGT2A3 activity, and representative Michaelis-Menten and Eadie-Hofstee kinetics

curves for UGT2A3 activity against 1-naphthol are shown in Figure 5.6 (Panels C and D,

respectively). Additional Eadie-Hofstee transformations suggested that all UGT2A2 and

UGT2A3 enzyme kinetics followed a simple Michaelis-Menten mechanism (data not

shown).

Figure 5.6. Representative enzyme kinetics curves for UGT2A2_i1 and UGT2A3 activity

against PAH substrates. (A) Representative Michaelis-Menten curve for UGT2A2_i1 activity

against 7-OH-B(a)P. (B) Eadie-Hofstee transformation of UGT2A2_i1 activity against 7-OH-

B(a)P. (C) Representative Michaelis-Menten curve for UGT2A3 activity against 1-naphthol. (D)

Eadie-Hofstee transformation of UGT2A3 activity against 1-naphthol.

170

The enzyme kinetics of UGT2A2_i1 and UGT2A3 glucuronidation activities

against PAH substrates are summarized in Table 5.1. UGT2A2_i1 exhibited

approximately 10 fold higher overall activity (Vmax/KM) against the simple PAH

substrates, 1-OH-pyrene and 1-naphthol, as compared to hydroxylated B(a)P metabolites.

A comparison between UGT2A2_i1 and UGT2A3 activities against common substrates

1-OH-pyrene and 1-naphthol suggests that UGT2A2_i1 has significantly higher activity

against 1-OH-pyrene (p<0.005) and 1-naphthol (p<0.01), with UGT2A2_i1 exhibiting an

approximately 40-fold higher Vmax/KM against 1-OH-pyrene and a 10-fold higher

Vmax/KM against 1-naphthol.

171

Table 5.1. Enzyme kinetics summary of UGT2A2_i1 and UGT2A3 activities against PAH substrates.

UGT2A2_i1

UGT2A3

KM Vmax Vmax/KM KM Vmax Vmax/KM Substrate (μM) (pmol/min/mg)

a (μL/min/mg)

a (μM) (pmol/min/mg)

b (μL/min/mg)b

1-OH-pyrene 100 ± 15 38 ± 2.1 0.38 ± 0.03 363 ± 38 5.5 ± 0.2 0.01 ± 0.001*

1-naphthol 106 ± 15 26 ± 1.5 0.25 ± 0.04 148 ± 18 3.1 ± 0.2 0.02 ± 0.003*

1-OH-B(a)P 279 ± 40 8.2 ± 0.8 0.03 ± 0.004 BLD

3-OH-B(a)P 332 ± 54 7.4 ± 0.5 0.02 ± 0.002 BLD

7-OH-B(a)P 240 ± 29 8.5 ± 0.4 0.04 ± 0.008 BLD

8-OH-B(a)P 287 ± 37 9.3 ± 0.6 0.03 ± 0.002 BLD

5-methylchrysene-1,2-diol BLD BLD

B(a)P-7,8-diol BLD BLD

Dibenzo(a,l)pyrene-11,12-diol BLD BLD

a

Data expressed as mg of total protein homogenate, corrected for relative UGT2A2 expression as determined by real-time PCR.

b Data expressed as mg of total protein homogenate, corrected for relative UGT2A3 expression as determined by real-time PCR.

KM, Vmax, and Vmax/KM represent the mean of three independent experiments.

BLD; below limit of detection

*p<0.01 compared to UGT2A2_i1 activity

5.5 Discussion

The UGT2A subfamily has been relatively under-studied when compared to

members of the UGT1A and UGT2B families, with a limited amount of information

reported concerning the expression and activity of UGT2A family members. It was

previously reported that UGT2A1 potentially plays a critical role in PAH metabolism in

target tissues for tobacco carcinogenesis. The current study expands on this work by

examining the potential role of two additional UGT2A family members, UGT2A2 and

UGT2A3, in tobacco carcinogen metabolism. The physiological relevance of UGT2A

enzymes in local carcinogen metabolism was determined by screening a large subset of

tissues for UGT2A2 and UGT2A3 expression and analyzing the activity of UGT2A2 and

UGT2A3 against tobacco carcinogens.

This study is the first to investigate and report UGT2A2 and UGT2A3 expression

in respiratory and aerodigestive tract tissues, including UGT2A2 expression in the larynx

and trachea and UGT2A3 expression in lung, trachea, larynx, colon, and tonsil. A

previous study investigating UGT2A2 expression in a limited number of tissues reported

UGT2A2 expression only in fetal and adult nasal mucosa tissues [70]. Adult lung and

liver tissues were previously screened for UGT2A2 expression, and in agreement with

our results no UGT2A2 expression was observed in these tissues [70]. High UGT2A3

expression was observed in liver, colon, pancreas and kidney, which is consistent with

results reported in one previous study analyzing UGT2A3 expression in human tissues

[71]. Our results suggest modest UGT2A3 expression in the lung, using RNA pooled

173

from five Caucasian individuals; this contradicts previous results suggesting no

detectable UGT2A3 mRNA expression using individual lung tissues [70, 71]. These

conflicting results could be due to inter-individual variability in UGT2A3 expression or

RNA quality issues. Additional experiments directly comparing UGT2A1, UGT2A2, and

UGT2A3 mRNA levels would help further clarify the tissue specific expression level of

each UGT2A enzyme, and this comparison will be completed in future studies.

The present study was the first to identify and investigate a novel UGT2A2 splice

variant, UGT2A2Δexon3, which was expressed in the same tissues as wild-type

UGT2A2. Activity assays using homogenate from HEK293 cells over-expressing

UGT2A2_i2 suggested that this UGT2A2 splice variant lacks enzyme activity. A similar

exon 3 deletion splice variant was recently discovered for UGT2A1, and subsequent

activity studies suggested that the UGT2A1_i2 isoform lacks also glucuronidation

activity but modulates wild-type UGT2A1_i1 enzyme activity through a protein-protein

interaction (Chapter 4). The protein expression and potential regulatory role of

UGT2A2_i2 will be addressed in future studies. In addition, it is currently unknown

whether UGT2A2_i2 can alter UGT2A1_i1 activity and vice versa; these studies will also

be completed in the future.

The glucuronidation activities of UGT2A2_i1 and UGT2A3 were determined

using homogenates from a HEK293 mammalian over-expression cell system; previous

studies analyzing UGT2A2 or UGT2A3 activity used baculovirus insect cell systems [70,

71]. In the current study UGT2A2_i1 and UGT2A3 were both found to have activity

against PAH substrates. UGT2A2_i1 exhibited activity against simple PAHs such as 1-

OH-pyrene and 1-naphthol, which is in agreement with previously published activity data

174

[70]. In the current study, it was also shown for the first time that UGT2A2_i1 is active

against hydroxylated B(a)P metabolites. UGT2A3 was determined to have very limited

activity against only two PAH substrates, 1-OH-pyrene and 1-naphthol. In the current

study, both UGT2A2 and UGT2A3 were found to have no detectable glucuronidation

activity against PAH proximate carcinogens. UGTs 2A2_i1, 2A2_i2 and UGT2A3 were

also determined to have no detectable glucuronidation activity against TSNAs and HCAs.

UGT2A1 and UGT2A2 have common exons 2-6 and a high degree of sequence

similarity in exon 1. The KM’s of UGT2A1_i1 and UGT2A2_i1 are similar for 1-OH-

pyrene (UGT2A1 91 μM vs. UGT2A2 100 μM) and hydroxylated B(a)P metabolites

(UGT2A1 247-308 μM vs. UGT2A2 240-332 μM), using activity data reported

previously for UGT2A1 (Chapter 2). The KM’s observed for UGT2A3 activity against 1-

OH-pyrene (363 μM) and 1-naphthol (148 μM) are significantly greater (p<0.001) than

the KM observed for UGT2A1 against these substrates (91 μM 1-OH-pyrene, 30 μM 1-

naphthol). After using real-time PCR expression data to correct for relative UGT2A over-

expression in each HEK293 cell line, UGT2A1_i1 was determined to have a significantly

higher (p<0.01 for all substrates) glucuronidation activity (Vmax/ KM) against the six

common PAH substrates which UGT2A1_i1 and UGT2A2_i1 are both active against.

UGT2A1_i1 also has a significantly higher (p<0.001) glucuronidation activity (Vmax/

KM) than UGT2A3 against 1-OH-pyrene and 1-naphthol. UGT2A1 is the only UGT2A

isoform active against complex PAH proximate carcinogens and UGT2A1 has higher

relative glucuronidation activity than UGT2A2 and UGT2A3 against PAH substrates,

suggesting that UGT2A1 is the most important UGT2A isoform in extra-hepatic tobacco


175

In previous work with UGT2A1, we identified two prevalent non-synonymous

polymorphisms that cause significant changes in enzyme activity, including a non-

conservative UGT2A1Gly308Arg

polymorphism in a conserved region of the enzyme. With

UGT2A1 and UGT2A2 sharing common exons 2-6, the same polymorphism at codon

309 of UGT2A2 has been reported in HapMap [251] and would be hypothesized to

significantly alter UGT2A2 activity. The functional effects of this polymorphism on

UGT2A2 activity will be addressed in future studies. Additional UGT2A2 SNPs from

HapMap with a minor allele frequency greater than 1.0%, including the UGT2A2Ala58Val

polymorphism, will also be analyzed in future studies [251]. One prevalent (>1%) non-

synonymous SNP, Thr497Ala, has been identified for UGT2A3. This SNP was

previously reported by Court et al. to cause no significant change in UGT2A3 activity

against bile acids [71]. A variety of low prevalence UGT2A polymorphisms identified

through the 1000 genomes project have never been tested functionally and may cause

large changes in UGT2A enzyme activities.

This study for the first time shows UGT2A2 and UGT2A3 expression in target

tissues for tobacco carcinogenesis such as the lung, larynx, trachea, tonsil, and colon.

Additionally, this study is the first to identify and characterize the expression and activity

of a novel UGT2A2 splice variant, UGT2A2Δexon3, which was determined to be

expressed in the same tissues as wild-type UGT2A2 mRNA. Similar to that observed for

the UGT2A1Δexon3 splice variant, homogenate from a cell line over-expressing

UGT2A2Δexon3 had no detectable glucuronidation activity against any substrate

examined; however, it is unknown at this time whether the UGT2A2_i2 isoform could be

a modulator of UGT2A2_i1 activity. This study is also the first to show UGT2A2 activity

176

against hydroxylated B(a)P metabolites, suggesting that UGT2A2 may play a role in the

local detoxification of PAH substrates in aerodigestive tract tissues such as the larynx and

trachea. Unlike UGT2A1, UGT2A2 expression was not detected in the lung and

UGT2A2_i1 exhibited no detectable activity against three proximate PAH carcinogens.

Although UGT2A3 was more widely expressed in tissues such as the lung, colon and

tonsil, this enzyme had relatively low glucuronidation activity against only the most

simple PAH substrates analyzed. None of the UGT2A family of enzymes (UGTs 2A1,

2A2, or 2A3) were active against TSNAs or HCAs, suggesting that the importance of

UGT2A enzymes in tobacco carcinogen metabolism is restricted to PAHs.

Results from this study suggest that UGT2A1 plays a more vital role than

UGT2A2 or UGT2A3 in tobacco carcinogen metabolism, due to UGT2A1 mRNA

expression in the lung and other aerodigestive tract tissues, UGT2A1 activity against

PAH proximate carcinogens, and higher UGT2A1 glucuronidation activity in comparison

to UGT2A2 and UGT2A3 against PAHs. Nonetheless, this study adds to the relatively

small amount of data on UGT2A2 and UGT2A3 expression and activity, for the first time

showing UGT2A2 and UGT2A3 expression in multiple aerodigestive tract tissues and

UGT2A2 and UGT2A3 activity against PAH simple substrates.

177

Chapter 6

FUTURE DIRECTIONS AND FINAL CONSIDERATIONS

178

6.1 Conclusions

The overarching goal of this research study was to investigate UGT2A tissue

expression and activity in order to determine the potential role of UGT2A enzymes in

local carcinogen metabolism. Two pieces of preliminary data suggested that UGT2A

enzymes might be relevant in tobacco carcinogen metabolism. Nishimura et al. reported

UGT2A1 to have high mRNA expression in the lung and trachea, compared to the

expression of three UGT1A and five UGT2B transcripts [96]. Initial studies investigating

UGT2A1 activity reported broad activity against multiple classes of substrates [97], and a

later study reported UGT2A1 activity against the simple PAHs 1-OH-pyrene and 1-

naphthol [70]. The data presented in this dissertation suggest that UGT2A enzymes,

particularly UGT2A1, play a critical role in PAH metabolism in target tissues for tobacco

carcinogenesis.

The experiments described in this dissertation represent the most comprehensive

analysis of UGT2A enzymes to date. UGT2A1 expression was investigated qualitatively

and quantitatively for the first time in a large panel of human tissues, and through these

studies UGT2A1 was determined to be expressed in multiple tissues including the lung,

larynx, trachea, tonsil, and colon. UGT2A1 glucuronidation activity was assessed against

multiple classes of tobacco carcinogens, with glucuronidation activity assays suggesting

that UGT2A1 is active against PAH metabolites implicated in tobacco carcinogenesis.

In subsequent studies, two prevalent non-synonymous UGT2A1 coding SNPs were

investigated for the first time. Both the UGT2A175Arg

and UGT2A1308Arg

variants had

lower glucuronidation activities than wild-type UGT2A1 against PAH substrates, with

179

the UGT2A1308Arg

variant exhibiting no detectable glucuronidation activity against all

substrates examined. Due to UGT2A2 expression in the lung, UGT2A1 activity against

PAHs, and the significant functional change caused by the UGT2A1308Arg

variant, we

hypothesized that the UGT2A1308Arg

variant plays a role in lung cancer susceptibility.

Case-control studies showed that UGT2A1308Arg

is significantly associated with lung

NSCC and lung squamous cell carcinoma. Co-expression studies in particular suggested

that UGT2A1308Arg

negatively modulates wild-type UGT2A1 activity through

dimerization, a novel mechanism for UGT2A1 that is similar to the regulatory

mechanism described for other inactive UGT variants. These in vitro results suggested

that UGT2A1308Arg

heterozygotes may be at an increased risk to develop PAH-related

cancers, validating the dominant model used to analyze the lung cancer case-control

association study. Additional studies are warranted to confirm this association in a larger

lung cancer case-control population, yet the data presented in this dissertation suggest the

UGT2A1 pharmacogenetics may play a role in cancer risk.

In addition to functional changes in enzyme activity caused by coding SNPs,

alternative splicing has been hypothesized to contribute to inter-individual differences in

drug and carcinogen metabolism. The discovery of a UGT2A1 exon 3 deletion splice

variant adds complexity to the function and regulation of UGT2A1 activity.

UGT2A1Δexon3 mRNA and UGT2A1_i2 protein were determined to be expressed, at

lower levels than wild-type UGT2A1, in multiple human tissues. The UGT2A1_i2

variant was found to exhibit no detectable glucuronidation activity itself and was

determined to negatively modulate wild-type UGT2A1 activity through a direct protein-

protein interaction. In addition to co-IP experiments showing UGT2A1_i1:UGT2A1_i2

180

hetero-dimerization, in vitro co-expression results suggested that UGT2A1_i1 homo-

dimerization also occurs. This result was consistent with previous data suggesting that the

UGT2A1308Arg

variant dimerizes with wild-type UGT2A1. Interestingly, UGT2A1_i2

mediated regulation of UGT activity was found to be UGT2A1-specific, as neither

UGT2A1_i1 nor UGT2A1_i2 were found to alter the glucuronidation activity of UG1A7,

UGT1A10, or UGT2B17 against multiple substrates.

Following extensive work characterizing the tissue expression of UGT2A1 and

the activity of UGT2A1 against tobacco carcinogens, the next goal of the research project

was to investigate the involvement of UGT2A2 and UGT2A3 in tobacco carcinogen

metabolism. Virtually no expression or activity data was previously reported for

UGT2A2 or UGT2A3 at the onset of these studies. A comprehensive analysis of UGT2A

expression in human aerodigestive and respiratory tract tissues showed UGT2A2

expression in the larynx and trachea, and widespread UGT2A3 expression in the trachea,

larynx, tonsil, and lung. Similar to that described for UGT2A1, a novel exon 3 deletion

splice variant was also identified for UGT2A2, and the mRNA of this inactive UGT2A2

variant was determined to be expressed in the same tissues as wild-type UGT2A2.

Glucuronidation assays completed with UGT2A2 and UGT2A3 homogenate showed

activity against simple PAH substrates though, unlike UGT2A1, neither enzyme was

determined to be capable of metabolizing complex PAH proximate carcinogens.

181

6.2 Future Directions

A comprehensive study determining the relative mRNA expression levels of

UGT1A, UGT2B, UGT2A, and UGT3 in human tissues is necessary to better assess the

importance of each UGT isoform in human health and disease. Only a few studies to date

have focused on determining the relative tissue-specific mRNA expression level of each

UGT transcript [59, 89, 90]; however, these studies and others have neglected to include

UGT2A enzymes and have also failed to investigate UGT expression in various human

tissues, including many tissues of the aerodigestive and respiratory tracts. Whereas the

UGT expression profile in the liver is well-established [93], there is a surprising lack of

data published on UGT expression in the lung. One previous study analyzed the

expression of multiple phase I and phase II enzymes in lung tissue, but failed to include

UGT enzymes [264]. Pulmonary metabolism of drugs and environmental carcinogens is

known to be an important function in the body [265-267], and a comprehensive analysis

of UGT expression in the lung would be helpful in determining the importance of specific

UGT isoforms in this tissue. Environmental carcinogens have been shown to up-regulate

the transcription of phase I and phase II metabolizing enzymes [268-270]. Promoter

studies have reported that UGT1A enzymes are induced via PAHs binding to AhRs and

activating XREs in UGT promoters. UGT1A1, UGT1A6, and UGT1A9 have been shown

to be induced through this mechanism, and, interestingly, all of these UGTs are known to

metabolize PAHs [271-275]. UGTs have also been shown to be up-regulated by the nrf2

transcription factor, often due to the actions of chemopreventative agents [195, 276].

UGT2A promoter studies are needed to determine whether UGT2A expression is induced

182

via these mechanisms, as this could have significant implications on UGT2A-mediated


The lack of an antibody specific for each UGT isoform is a major problem in the

UGT field, as the development of UGT antibodies is limited by the sequence homology

between UGT isoforms [84]. No UGT2A2 or UGT2A3 specific antibodies are currently

commercially available. We designed an antibody to specifically detect UGT2A1 protein;

this antibody was determined to have no cross-reactivity against UGT2A2 or UGT2A3.

Most UGT expression studies to date have relied on UGT mRNA measurements, yet

mRNA levels have been shown to correlate poorly with UGT protein expression in

human liver specimens [93]. The activities of UGT2A enzymes against PAHs were

compared in this dissertation using real-time PCR to approximate the level of UGT2A

mRNA over-expression in each stable cell line. The use of a more general UGT2A

antibody that recognizes all three UGT2A isoforms would allow for the determination of

the relative expression of each UGT2A isoform in over-expressing cell lines, enabling a

true comparison of UGT2A activities to be completed. Also, the development of

UGT2A2 and UGT2A3 specific antibodies would allow for the determination of UGT2A

protein expression in tissue homogenates to be investigated. The trans-membrane nature

of UGT enzymes causes difficulties in creating full-length UGT purified protein

standards, making the absolute quantification of UGT protein expression technically

challenging [277]. The creation UGT2A protein standards would enable the levels of

UGT2A over-expression and the levels of UGT1A and UGT2B over-expression to be

compared in stable cell lines, allowing UGT2A activity to be compared to that of other

UGT isoforms.

183

There are numerous studies describing the impact of UGT pharmacogenetics on

cancer susceptibility [60, 67, 106]. Results presented in Chapter 3 suggest that the

UGT2A1308Arg

variant is significantly associated with lung NSCC and lung squamous cell

carcinoma in particular. The association of UGT2A1308Arg

with increased lung cancer risk

must be verified in a larger lung cancer case-control sample set, including a sample set

containing an increased number of squamous cell carcinoma cases. Activity assays will

be completed in the future using lung homogenates from wild-type UGT2A1 individuals,

UGT2A1308Arg

heterozygotes, and UGT2A1308Arg

homozygotes to validate in vitro co-

expression results suggesting that UGT2A1308Arg

negatively regulates wild-type UGT2A1

activity. The association of UGT2A1 SNPs with cancer risk at other sites where PAHs are

thought to induce carcinogenesis, including the colon and oral cavity, will also be

completed in future studies.

The discovery of a novel UGT2A1Δexon3 splice variant adds to the complexity

of how UGT2A1 is regulated. As the UGT2A1_i2 variant was determined to negatively

regulate wild-type UGT2A1 activity through in vitro co-expression studies, our working

hypothesis is that inter-individual variability in UGT2A1_i2 expression could impact

cancer risk. Lung tissue samples from lung cancer cases and controls will be analyzed for

UGT2A1 expression in order to determine whether low ratios of wild-type UGT2A1

expression to UGT2A1Δexon3 expression are associated with increased cancer risk. The

epigenetic modification of multiple genes, including the expression of genetic variants

caused by alternative splicing, has been determined to be associated with increased

cancer risk [278, 279]; however, the impact of UGT alternative splicing on cancer risk

184

has not previously been investigated. This is a highly novel idea that requires further

exploration.

In order to determine whether UGT2A1 lymphocyte expression could be used as a

marker for UGT2A1 expression in the lung, preliminary experiments have been

conducted analyzing UGT2A1Δexon3 splice variant expression in lymphocyte samples.

Preliminary results using matched lymphocyte and lung RNA suggest that, although

UGT2A1 is expressed in lymphocytes, the pattern of UGT2A1 expression lymphocyte

cDNA does not reflect the pattern of wild-type UGT2A1 and UGT2A1Δexon3

expression in lung cDNA from the same individual, with only UGT2A1 Δexon3 observed

in lymphocytes (Figure 6.1).

Figure 6.1. UGT2A1 mRNA expression in matched lung and lymphocyte RNA samples.

Wild-type UGT2A1 and UGT2A1Δexon3 expression were observed in all three human lung

samples investigated, while matching lymphocyte RNA from the same individuals had either no

detectable UGT2A1 expression or only UGT2A1Δexon3 transcript expression. cDNA from wild-

type UGT2A1 or UGT2A1Δexon3 over-expressing ell homogenates were used as a positive

control.

185

At this time the mechanism behind UGT2A1Δexon3 expression in lymphocytes is

unknown. UGT2A1 protein expression has also yet to be investigated in lymphocytes,

and though lymphocyte protein extraction protocols exist [280, 281], this may be a more

technically challenging procedure.

The expression and regulatory role of UGT2A1_i2 was well characterized in this

dissertation. A similar UGT2A2 exon 3 deletion splice variant was discovered by RT-

PCR, and relative UGT2A2Δexon3 mRNA expression was determined through real-time

PCR experiments. UGT2A2_i2 tissue expression has yet to be explored, and at this time

it is unknown whether UGT2A2_i2 modulates UGT2A2_i1 activity similar to the

mechanism described for UGT2A1. Co-expression studies suggested that UGT2A1_i1 or

UGT2A1_i2 co-expression does not affect the enzyme activities of UGT1A or UGT2B

isoforms. With mRNA expression of UGT2A1and UGT2A2 reported in the larynx and

trachea, the potential exists for UGT2A1 and UGT2A2 hetero-dimerization; this will be

addressed in future studies. This interaction could provide another mechanism of UGT2A

regulation; for example, UGT2A1_i2 may be able to hetero-dimerize with UGT2A2_i1

and alter wild-type UGT2A2_i1 activity in these tissues. UGT2A1 and UGT2A2 hetero-

dimerization with UGT2A3 will also be investigated in future experiments, as UGT2A3

is expressed in many of the same tissues as UGT2A1 and UGT2A2.

The characterization of two prevalent non-synonymous UGT2A1 coding SNPs

was described previously. Multiple prevalent coding SNPs have been reported for

UGT2A2 and UGT2A3 through HapMap [251]. The inactive UGT2A1308Arg

variant

described in Chapter 3 is translated from the common region shared between UGT2A1

and UGT2A2; however, the functional effects of this SNP on UGT2A2 activity have not

186

been determined. Functional studies characterizing prevalent UGT2A2 and UGT2A3

coding SNPs will be completed in the future, including the effect of UGT2A2 and

UGT2A3 SNPs on PAH metabolism. Non-coding UGT2A SNPs, including those in

UGT2A promoters or UGT2A 3’UTRs, will also be investigated in order to determine the

effect of these SNPS on UGT2A expression and activity.

6.3 Final Considerations

Results presented in this dissertation suggest that UGT2A enzymes play an

important role in the metabolism of PAH tobacco carcinogens in the lung and other target

tissues for tobacco carcinogenesis. There is strong in vitro evidence that individuals

expressing the inactive UGT2A1308Arg

variant are at an increased risk for developing lung

cancer. Although additional work is needed to confirm this association, the

UGT2A1308Arg

SNP may also be a susceptibility marker for tobacco-related cancers of the

trachea, larynx, tonsil, and colon. In vitro studies characterizing the UGT2A1Δexon3

splice variant suggest that inter-individual variability in UGT2A1Δexon3 expression may

also influence cancer risk, though additional studies are needed to confirm this

hypothesis. Future work will focus on determining whether lymphocytes can be used as a

biomarker to predict the ratio of wild-type UGT2A1 to UGT2A1Δexon3, as this ratio

could potentially be used as a susceptibility marker for lung cancer risk.

UGT2A1 locally detoxifies proximate PAHs in target organs for tobacco

carcinogenesis. Results from a case-control study suggest that the UGT2A1-mediated

187

local detoxification of activated PAH carcinogens is an important mechanism to prevent

tobacco-induced lung cancer, as the inactive UGT2A1*3 allele was found to be

associated with increased NSCC and squamous cell carcinoma risk. Although the liver is

the major site of phase I and II metabolism, results from this dissertation suggest that

UGT2A1 activity in the lung and other target tissues also protects these organs from

DNA adduct formation. A lack of UGT2A1-mediated PAH detoxification, due to the

UGT2A1 SNP variants or the UGT2A1Δexon3 splice variant, would allow activated

PAH diols to remain in target tissues for longer periods of time and eventually be

converted to their more carcinogenic diol-epoxide form. The local metabolism of tobacco

carcinogens is often over-looked; however, results from this study suggest that UGT2A1

metabolizes proximate carcinogens locally prior to their conversion to ultimate

carcinogens in target tissues. Additional research into the non-hepatic metabolism of

PAHs and other tobacco carcinogens is needed to fully understand the importance of

phase I and II metabolism in these tissues.

Tobacco use is a global health epidemic, and identifying individuals with a

greater susceptibility to developing tobacco-related cancers has the potential to save

many lives. Lung cancer treatment success is known to be heavily dependent on the time

of diagnosis; however, currently-used diagnostic tools are expensive and cannot detect

early stage lung cancer [282, 283]. Results presented in this dissertation suggest that the

UGT2A1308Arg

and UGT2A1_i2 variants could be candidates for targeted tobacco-related

cancer prevention strategies. The use of genetic biomarkers to identify populations at

high risk for developing tobacco-related cancers is an alternative approach to the standard

detection and treatment regimens used today. In the future, targeted cancer screening and

188

personalized cessation techniques should be the standard of care for high-risk

populations. The goal of a targeted screening approach would be to identify cancer early

in high-risk smokers; for example, it might be more cost-effective for these individuals to

be screened more routinely for cancer than the normal population of smokers.

The UGT field has been saturated in recent years with studies focusing on

UGT1A and UGT2B enzymes; yet relatively little information has been published on the

UGT2A family. This dissertation has laid the groundwork for understanding the

physiological role of UGT2A enzymes in the local detoxification of PAH tobacco

carcinogens. Initially, human tissues were screened for UGT2A mRNA expression and

multiple classes of tobacco carcinogens were examined as potential substrates for

UGT2A enzymes. Once it was determined that UGT2A enzymes, in particular UGT2A1,

metabolize PAHs and are expressed in the lung and other target tissues for tobacco

carcinogenesis, the focus of the project shifted to characterizing the expression and

activity of UGT2A1 coding SNPs and a novel UGT2A1Δexon3 splice variant. Together

the data presented in this dissertation have significantly improved our knowledge of the

physiological role of the UGT2A family, including how inter-individual differences in

UGT2A1 activity caused by UGT2A1 SNPs and a novel UGT2A1 splice variant may

impact carcinogen detoxification and play a role in cancer risk.

189

Appendix

ALTERNATE SUGARS IN UGT2A METBOLISM

190

Introduction

The relatively well characterized UGT1 and UGT2 families primarily use

UDPGA as the sugar donor for xenobiotic metabolism [209]. Multiple UGTs have been

shown to use alternate sugars other than UDPGA as co-substrates. For example, UGT2B7

has been reported to use UDP-glucose and UDP-xylose in the metabolism of

hyodeoxycholic acid [284]. UGT3A1 has been reported to use N-acetylglucosamine as its

preferential sugar donor and UGT3A2 has been reported to strictly use UDP-glucose and

UDP-xylose in the metabolism of multiple substrates [72, 73]. Co-substrates in addition

to UDPGA, including N-acetylglucosamine, UDP-glucose and UDP-galactose, were used

for the first time in this study to determine whether UGT2A enzymes could use alternate

co-substrates in the metabolism of tobacco carcinogens.

Materials and Methods

UDP-glucose, UDP-galactose, and N-acetylglucosamine were purchased from

Sigma-Aldrich (St. Louis, MO). Glucuronidation assays using homogenates from

HEK293 cell lines over-expressing UGT2A enzymes were performed essentially as

previously described [234, 235]. Briefly, after an initial incubation of total cell

homogenate protein (200 μg) with alamethicin (50 μg/mg protein) for 15 min on ice,

glucuronidation reactions were performed in a final reaction volume of 25 μL at 37°C

with 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 4 mM co-substrate, and 500 μM of

substrate. Alternate co-substrates used included UDP-glucose, UDP-galactose, or N-

191

acetylglucosamine. Following a 2 hour incubation, reactions were terminated by the

addition of 25 μL cold acetonitrile on ice. Reaction mixtures were centrifuged for 10 min

at 16,100 g prior to the collection of supernatant. Activity assays were completed using

50 µg HLM in place of UGT2A protein as a positive control. Levels of glucuronide

formation were determined using a Waters Acquity UPLC System (Milford, MA) as

previously described [110, 112, 234, 236]. The flow rate was maintained at 0.5 mL/min

and a reverse phase Acquity UPLC BEH C18 - 1.7 μm - 2.1 x 100 mm column was used

to separate free substrate and the conjugated glucuronide. A gradient of solution A (5

mM NH4OAc (pH 5.0), 10% acetonitrile) and solution B (100% acetonitrile) was used to

elute the glucuronide and substrate from the column. The gradients of solution A and B

used were described earlier in the dissertation.

Results

UGT2A activity assays were completed using UDP-glucose, UDP-galactose, or

N-acetylglucosamine in place of UDPGA. As shown in Appendix Figure 1 (Panel A), no

UGT2A1 activity was observed against the substrate 1-naphthol when UDP-galactose

was used in place of UDPGA. A 1-naphthol-galctoside was detected when HLM was

used in place of UGT2A1 protein (Panel B). UGT2A1 lacked detectable enzyme activity

against all PAH, TSNA, and HCA substrates examined when alternate sugars were used

in place of UDPGA. UGT2A1 also lacked detectable activity against the common UGT

substrate 4-MU when alternate sugars were used in place of UDPGA (data not shown).

192

Appendix Figure 1. Homogenate from UGT2A1 over-expressing cell line exhibits no activity

against 1-napthol using UDP-galactose as the co-substrate. (A) UPLC trace of UGT2A1

activity against 1-naphthol with UDP-galactose used as the co-substrate. Only UDP-galactose and

1-naphthol peaks were observed. (B) UPLC trace of HLM activity against 1-naphthol with UDP-

galactose used as the co-substrate. A 1-naphthol-galactoside peak was observed at 2.7 min.

The enzyme activities of UGT2A2 and UGT2A3 were also determined using

UDP-glucose, UDP-galactose, or N-acetylglucosamine in place of UDPGA. As shown in

Appendix Figure 2 (Panel A), no UGT2A2 activity was observed against the substrate 1-

OH-pyrene when UDP-glucose was used in place of UDPGA as a co-substrate.

Additionally, no UGT2A3 activity was observed against 1-OH-pyrene when UDP-

glucose was used (Panel B). A 1-OH-pyrene-glucoside peak was observed when HLM

was used in place of UGT2A protein as a positive control (Panel C). Similar to results for

UGT2A1, both UGT2A2 and UGT2A3 lacked detectable enzyme activity against all

193

PAH, TSNA, and HCA substrates examined when alternate sugars were used in place of

UDPGA.

Appendix Figure 2. Homogenates from UGT2A2 and UGT2A3 over-expressing cell lines

exhibit no activity against 1-OH-pyrene using UDP-glucose as the co-substrate. (A) UPLC

trace of UGT2A2 activity against 1-OH-pyrene with UDP-glucose used as the co-substrate. Only

UDP-glucose and 1-OH-pyrene peaks were observed. (B) UPLC trace of UGT2A3 activity

against 1-OH-pyrene with UDP-glucose used as the co-substrate. (C) UPLC trace of HLM

activity against 1-OH-pyrene with UDP-glucose used as the co-substrate. A 1-OH-pyrene-

glucoside peak was observed at 2.5 min.

194

Discussion

UGT3A enzymes have been reported to only use alternate sugars other than

UDPGA as co-substrates in the metabolism of various compounds [209]. Additional

UGT isoforms that primarily use UDPGA as the co-substrate have also been shown to

use alternate sugars in metabolic reactions; for example, UGT1A1 has been shown to use

UDP-xylose and UDP-glucose in the metabolism of bilirubin [136]. The use of alternate

sugars by UGT2A enzymes was never previously examined. In the current study, we

determined that no detectable UGT2A activity was observed against 4-MU or tobacco

carcinogens when alternate sugars, including UDP-glucose, UDP-galactose, or N-

acetylglucosamine, were used in place of UDGPA. These results suggest that UGT2A

enzymes predominantly use UDPGA as the co-substrate in the metabolism of

xenobiotics.

195

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CURRICULUM VITAE

NAME

Ryan T. Bushey

POSITION TITLE

Graduate Assistant, Department of Pharmacology

Penn State College of Medicine EMAIL ADDRESS

[email protected]

EDUCATION/TRAINING INSTITUTION AND LOCATION DEGREE YEAR(s) FIELD OF STUDY

James Madison University, Harrisonburg, VA B.S. 2002-2006 Biology

Pennsylvania State University, Capital College

(Harrisburg), Middletown, PA

M.B.A. 2009-2011 Business Administration

Pennsylvania State University, College of

Medicine, Hershey, PA

Ph.D.

2007-2012 Pharmacology

A. Positions and Honors

Positions and Employment

2008-2012 Graduate Assistant, Laboratory of Dr. Philip Lazarus, Department of Pharmacology, Penn

State College of Medicine

2006-2007 Research Technician, SAIC-Frederick Inc., Contractor for National Cancer Institute

2004-2006 Undergraduate Researcher, Laboratory of Dr. Terrie Rife, Department of Biology, James

Madison University

Honors

2007-2010 Fund for Excellence in Graduate Recruitment Award, Penn State College of Medicine

2006 Excellence in Biology Award, James Madison University

2005 Tri-Beta Outstanding Junior Biology Student, James Madison University

B. Publications

1. Bushey RT, Chen G, Blevins-Primeau AS, Krzeminski J, Amin S, Lazarus P. Characterization of UDP-

glucuronosyltransferase 2A1 (UGT2A1) variants and their potential role in tobacco carcinogenesis.

Pharmacogenet Genomics, 2011, 21(2):55-65.

2. Bushey RT, Lazarus, P. Identification and functional characterization of a novel UGT2A1 splice variant:

Potential importance in tobacco-related cancer susceptibility. (Accepted: J Pharmacol Exper Ther)

3. Bushey RT, Berg, A, Lazarus, P. The UDP-Glucuronosyltransferase (UGT) 2A1308Arg

polymorphism:

Association with lung cancer risk and functional implications of homo-dimerization. (In preparation)

4. Bushey RT, Dluzen, DF, Lazarus, P. Characterizing the expression and activity of UDP-

Glucuronosyltransferases 2A2 (UGT2A2) and 2A3 (UGT2A3) and their potential role in tobacco carcinogen

metabolism. (In preparation)

C. Conference Presentations

1. Bushey RT, Chen G, Blevins-Primeau AS, Lazarus P. Characterization of the Activity and Expression of UDP-

Glucuronosyltransferase 2A1 Variants and Potential Role in Tobacco Carcinogenesis. AACR Annual

Meeting, 2010.

role of the uridine diphosphate …

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