novel pathways associated with bypassing cellular ... · downregulated in immortalized hpecs...

Novel Pathways Associated with Bypassing Cellular Senescence in Human Prostate

Epithelial Cells

Steven R. Schwarze1, Samuel E. DePrimo2*, Lisa M. Grabert1, Vivian X. Fu1, James D. Brooks2,

and David F. Jarrard1†

1Department of Surgery, Division of Urology, University of Wisconsin Medical School,

Molecular and Environmental Toxicology and the University of Wisconsin Comprehensive

Cancer Center, Madison, WI 53972 and 2Department of Urology, Stanford University, Stanford,

CA 94305

*Current address: SUGEN, Inc., South San Francisco, CA 94080

Running title: “GENE EXPRESSION PROFILES IN SENESCENCE AND

IMMORTALIZATION”

This work was supported by: National Institutes of Health (CA76184-01) (D.F.J), the University

of Wisconsin Comprehensive Cancer Center (D.F.J.), the Doris Duke Foundation (J.D.B.), and

the Kovitz Foundation (J.D.B.).

†To whom correspondence should be addressed: David F. Jarrard, M. D. Department of Surgery,

University of Wisconsin, 600 Highland Avenue, K6/530, Madison, WI 53792

Ph. 608-265-2225; Fax: 608-265-8133; E-mail: [email protected]

The abbreviations used are: SA-ß-gal, senescence-associated ß-galactosidase; HPEC, human

prostate epithelial cell; EST, expressed sequence tag; qRT-PCR, quantitative reverse

transcriptase-PCR.

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on February 8, 2002 as Manuscript M200373200 by guest on M

ay 31, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Gene expression profiles in senescence and immortalization

2

SUMMARY

Cellular senescence forms a barrier that inhibits the acquisition of an immortal phenotype, a

critical feature in tumorigenesis. The inactivation of multiple pathways that positively regulate

senescence are required for immortalization. To identify these pathways in an unbiased manner,

we performed DNA microarray analyses to assess the expression of 20,000 genes in human

prostate epithelial cells passaged to senescence. These gene expression patterns were then

compared to those of HPECs immortalized with the Human Papilloma Virus 16 E7 oncoprotein.

Senescent cells display gene expression patterns that reflect their non-proliferative, differentiated

phenotype and express secretory proteases and extra-cellular matrix components. A comparison

of genes transcriptionally upregulated in senescence to those whose expression is significantly

downregulated in immortalized HPECs identified three genes: the chemokine BRAK, DOC1,

and a member of the insulin-like growth factor axis, IGFBP-3. Expression of these genes is

found to be uniformly lost in human prostate cancer cell lines and xenografts and previously

their inactivation has been documented in tumor samples. Thus, these genes may function in

novel pathways that regulate senescence and are inactivated during immortalization. These

changes may be critical in not only allowing cells to bypass senescence in vitro, but in the

progression of prostate cancer in vivo.

by guest on May 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


3

INTRODUCTION

Normal primary cells proliferate in culture for a limited number of population doublings

prior to undergoing terminal growth arrest and acquiring a senescent phenotype (1). Senescent

cells are resistant to mitogen-induced proliferation, express senescence-associated ß-

galactosidase (SA-ß-gal), and assume a characteristic enlarged, flattened morphology (reviewed

in (2)). With progressive cell divisions, errors in DNA replication, oxidative metabolism and

environmental insults generate somatic mutations and place cells at risk for oncogenic

transformation. Accumulating data suggest that the terminal arrest associated with cellular

senescence represents a major barrier, analogous to apoptosis, that cells must circumvent to

become malignant (3). Research into the pathways that positively regulate senescence and ways

cells bypass senescence is therefore critical in understanding carcinogenesis.

In part, senescence is mediated by loss of telomere length that occurs with progressive

divisions of mortal cells (4). Cellular senescence also can be induced prematurely by a variety of

stimuli including DNA damage, perturbations of chromatin structure, and overexpression of

mitogenic signals including E2F1, oncogenic H-ras, Raf or MEK (5-8). Two genes implicated

in and critical for the induction and maintenance of senescence are pRb and p53 (3).

Overexpression of downstream members of these pathways including p16, the p53 effector

molecule p21, and p14ARF can elicit cell cycle arrest and senescence (5,9,10). These gain of

function experiments demonstrate senescence can be prematurely induced, however do not

directly address the complex pathways regulating senescence in unmanipulated, serially-

passaged, normal primary cells. These mechanisms may significantly differ. For example in

fibroblasts, the overexpression of E2F1 leads to senescence, however E2F1 expression is actually

repressed as unmanipulated cells senesce (11).


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


4

Inactivation of the p16/pRb and p53 pathways is necessary to bypass senescence,

however is insufficient for immortalization. Telomerase activation is also required, although the

overexpression of telomerase alone is not sufficient to confer immortalization in epithelial cells

(12). In addition, genetic analyses of immortalized prostate epithelial and other cell types reveal

a number of consistent genetic alterations are required including the amplification of

chromosome 20q and 8q (13,14). Based on these findings, and other microcell transfer

experiments, it has been proposed that alterations in multiple pathways are required for the

acquisition of the immortal phenotype (15). In a series of classical experiments the fusion of

different immortalized cell lines could restore senescence and these cell lines could be sorted into

four complementation groups. This work implies that there are a limited number of pathways

critical to senescence, that these pathways must be activated to initiate senescence, and that they

can override other transforming genetic events to yield a senescent phenotype (16).

Genes that have been implicated in senescence are also targeted in the development and

progression of human prostate and bladder cancer. The p16/pRb pathway is altered in 85% of

primary prostate cancers and predicts adverse clinical outcome (17). The tumor suppressor p53

is one of the most commonly mutated genes in human cancer and is altered frequently in

advanced prostate and bladder cancers (18,19). Several lines of evidence suggest that bypassing

senescence may also be important in cancer progression in vivo. Immortalized colon cancer cell

lines can only be established from large, pathologically advanced lesions (20). When non-

invasive papillary bladder neoplasms are grown in culture, they uniformly senesce and maintain

an intact p16/pRb pathway (19,21). In contrast, cells cultured from myoinvasive, aggressive

transitional cell bladder carcinomas do not senesce, have alterations of the p53 pathway and

inactivate either p16 or pRb. Furthermore, prostate tumors that grow as xenografts have been


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


5

generated solely from advanced, metastatic cancers (22). These results suggest that bypassing

senescence is important in cancer progression, and occurs late in the progression of cancers in

vivo.

To gain insight into novel pathways involved in bypassing senescence, we developed a

model in which transcript levels for over 20,000 unique sequences were tracked in replicate cultures

as human prostate epithelial cells senesced or were immortalized. A major advantage of this

approach is that it avoids exogenous gene expression for the induction of premature senescence that

can result in biased or aberrant results. Using this strategy, we have identified several genes, BRAK,

DOC1, and IGFBP-3, which are upregulated in senescence and inactivated during immortalization.

We find utilizing quantitative RT-PCR, that these genes are significantly downregulated in human

prostate cancer cell lines and xenografts. Interestingly, all of these genes have been previously

implicated in the tumorigenic phenotype (23-25). These data support the concept that pathways

activated at senescence and selectively inactivated with immortalization are important in

carcinogenesis and tumor progression. Furthermore, these results provide an important effort in

cataloging genes altered in senescence, a biologically relevant model of aging.

MATERIALS AND METHODS

Cell Culture—Prostate tissue was obtained under an approved IRB protocol from men (ages

44-66) undergoing cystoprostatectomy for bladder cancer at the University of Wisconsin Hospitals

and Clinics. Histology confirmed that no bladder or prostate cancer was present in the tissues

harvested for our studies. Prostate epithelial cultures were established as described previously (13).

Prostate tissues were minced with a scalpel and digested in a solution containing 500 U/ml

collagenase (Sigma) and plated on collagen-coated plates. Cells were maintained in Ham’s F-12


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


6

media (Life Technologies, Inc.) supplemented with regular insulin, 0.25 units/ml; hydrocortisone, 1

µg/ml; human transferrin, 5 µg/ml; dextrose, 2.7 mg/ml; non-essential amino acids, 0.1 mM;

penicillin, 100 units/ml; streptomycin,100 µg/ml; L-glutamine, 2 mM; cholera toxin, 10 ng/ml;

bovine pituitary extract, 25 µg/ml and 1% FBS (26). Cells were passaged when confluent after

incubation with trypsin-EDTA. Proliferating cells were harvested 2 days following the first passage.

Pre-senescent cells were harvested when cell growth ceased, 1-5% of all cells appeared

morphologically circular, flattened and enlarged, and SA-ß-gal expression was negative.

Terminally-senescent cells were harvested when >60% appeared morphologically senescent and

expressed high levels of SA-ß-gal.

Transformation of HPECs with HPV16 E6 and/or E7—Infections and characterization of

HPEC cell lines was carried out as described (13). We used a retrovirus construct carrying either the

HPV16 E6 and/or E7 gene(s) as well as a gentamicin resistance cassette (received from Dr. D.

Galloway, Seattle, WA) (27). Sub-confluent proliferating HPECs were infected with 103-105

infectious viral units at pre-passage (~5 X 105 cells per 100-mm dish) in 3 ml of 1% FBS, F-12+

containing 4 µg/ml polybrene (Sigma). After 6 hours, the virus containing media was exchanges and

infected cells were selected with 50 µg/ml of G418 (Life Technologies, Inc.) for a minimum of 7

days. Immortalized cell lines were screened for HPV16 E6 and/or E7 protein expression and loss of

p53 and pRB by western blot analysis. All immortalized lines were passaged in 1% FBS F-12+ for

well over 20 times to confirm their immortality.

Microarray Hybridizations and Data Analysis—Microarray manufacture and

hybridizations were carried out in accord with previously published methods available at (28).

For this set of experiments we used DNA microarrays with either 41,000 or 47,000 spots

representing over 20,000 unique human genes and expressed sequence tags (ESTs). For each


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


7

hybridization, two micrograms of poly(A) mRNA from each sample were reverse-transcribed

and labeled with fluorescence-tagged nucleotides (Cy3 for the reference sample, Cy5 for the

experimental sample). Samples were hybridized against a common reference pool of mRNA

derived from a panel of human cell lines, as described previously (29). Hybridizations were

carried out for 16-18 hours at 65°C and the arrays were washed. After drying, the microarrays

were scanned with a confocal laser GenePix microarray scanner (Axon Instruments) and were

analyzed with Genepix software. After visual inspection, spots of poor quality were flagged and

excluded from analysis. Data files containing fluorescence ratios were entered into the Stanford

Microarray Database, and compiled experiments were further analyzed with hierarchical

clustering software and visualized with Treeview software (30). Prior to cluster analysis, gene

entries were filtered to select those with >2.5-fold changes in signal intensity when comparing

proliferating cells to pre-senescent, terminally-senescent and immortalized cell lines on at least

1/3 of the array samples, with fluorescent intensity in each channel that was greater than 1.6

times the local background. Identities of spotted cDNAs are based on information available in

UniGene cluster Build 137 (June, 2001).

Statistical Analysis of Microarray Data—Genes with significant expression changes in

response to senescence and immortalization in normal prostate epithelial cells were identified

using the Significance Analysis of Microarrays (SAM) procedure (31). Changes in gene

expression for any single gene as measured in several array experiments provide a statistically

testable measure of robustness, regardless of the magnitude of change in expression. The SAM

procedure computes a two-sample T-statistic (e.g. proliferating vs. senescent HPECs) for the

normalized log ratios of gene expression levels for each gene. It thresholds the T-statistics to


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


8

provide a “significant” gene list and provides and estimate of the false discovery rate (the percent

of genes identified by chance alone) from randomly permuted data.

For the experiments described in our study, the raw expression ratio dataset was filtered,

using the program Cluster, for genes whose transcript levels differ from their median value by at

least 1.5-fold in androgen treated cells compared to controls in at least 2 experiments (with not

more than 30% of measurements discarded due to poor data quality for each entry). We selected

a dataset in which 0.5 is the median number of likely false positive genes (false detection rate of

0.088%) for further analysis. We selected this value because it provided statistical assurance that

most genes in the set were significantly altered, but was not so stringent as to exclude genes of

biological interest.

Quantitative RT-PCR analysis—Total RNA was isolated using the RNeasy RNA

isolation kit (Qiagen), DNAse treated and 1 µg was used to prepare cDNA. Quantitative RT-

PCR was performed by monitoring in real time the increase in fluorescence of the SYBR Green

dye as described using a iCycler detection system (Bio-Rad) (32,33). For comparison of

transcript levels between samples, a standard curve of cycle thresholds for several serial dilutions

of a cDNA sample was established. This value was then used to calculate the relative abundance

of each gene. These values were then normalized to the relative amounts of 18S cDNA. All

PCR reactions were performed in duplicate. Sequences of primers used for PCR analysis are

available upon request.

RESULTS AND DISCUSSION

Analysis of Senescence Pathways in Human Prostate Epithelial Cells—Genome-wide

RNA expression analysis of serially passaged primary human prostate epithelial cells (HPECs)


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


9

and HPV16 E7 immortalized HPECs was performed with DNA microarrays representing over

20,000 distinct human genes and ESTs. In order to identify genes induced at senescence and

inactivated at immortalization, HPEC cultures derived from three separate patients were

analyzed at three defined stages in their lifespan. Cells harvested in their first passage and

growing exponentially were defined as proliferating. Pre-senescent cells were harvested when

growth was halted, but the cells had not yet expressed the morphologic characteristics of

senescence and were not yet SA-ß-gal positive. We have previously shown that this is a distinct

point at which cells transiently elevate protein levels of selected cell cycle inhibitory genes, and

it appears critical in signaling the onset of senescence (34). At terminal-senescence, > 60% of

HPECs expressed SA-ß-gal and had acquired an enlarged, flattened morphology (13,34).

Three independent proliferating HPEC cultures were then immortalized utilizing HPV16

E7 retrovirus. We have previously used this molecular tool to dissect the genetic events involved

in bypassing senescence (13,35). The E7 oncoprotein acts, in part, by inactivating the pRb/p16

pathway, and via the binding of p21, the p53 pathway, thus recapitulating several genetic

alterations commonly observed in prostate tumors (13,18). HPV E7 immortalized cells are non-

tumorigenic (36) making them an applicable model with which to isolate genes associated with

the immortalization phenotype. However, because of some of the diverse activities of the HPV

oncoproteins, we note not all targets relevant to bypassing senescence will be identified utilizing

this approach. Poly(A)+ mRNA was isolated from each of these experimental samples, reverse

transcribed, labeled with Cy-5, and hybridized in comparison with a common reference pool of

cDNA (labeled with Cy-3) (29).

Determination of genes that significantly differed in expression levels between cellular

phenotypes was made utilizing the analytical tool Significance Analysis of Microarrays (SAM)


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


10

(31). Comparison of proliferating to terminally-senescent HPECs generated 262 unique entries

using SAM, of which 93 were induced at senescence (51 of known function) and 159 were

repressed (77 of known function). Complete details and transcript identities for all outputs can be

accessed in the supplemental data (Supplemental Fig. 1-2). SAM outputs comparing

proliferating to pre-senescent cells identified no genes with significant induction in expression in

pre-senescent cultures, but did find 101 genes whose expression levels were repressed. These

101 genes also displayed repressed transcript levels in terminally-senescent cultures. The results

generated by SAM were filtered to remove ESTs and genes of unknown function. The

remaining genes were then classified based on their cellular function and placed into one or more

related groups.

Consistent with a non-proliferative senescent state, 28 of the 77 known genes that were

repressed at senescence are positive growth regulators or transcription factors involved in promoting

cell cycle progression. An additional 28 repressed genes are directly involved in DNA replication,

repair or mitosis. Of greater interest to us were those genes induced as cells progressed to terminal-

senescence as these genes may lend insights into the induction of senescence. It has been postulated

that p16 and p53 function as tumor suppressors, in part, by inducing senescence pathways (37). We

have previously reported at the cdk inhibitor p16 protein level, a known marker of senescent cells, is

elevated at senescence (34). In this study, we find ~50-fold increase in p16 mRNA at senescence,

suggesting that p16 protein increases are due, at least in part, to increased transcription. Transcript

levels of other negative growth regulators were also increased including the p53-regulated targets

BTG2, p21, IGFBP-3 and mdm2 (Fig. 1A), suggesting elevated p53 activity at senescence.

Overexpression of BTG2 (38) , p21 (39), IGFBP-3 (25) and even mdm2 in normal diploid cells (40)


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


11

can result in G0/1 cell cycle arrest. These p53-inducible genes may reflect the participation of p53

in regulating the onset of senescence and lend further insight into senescence-associated p53 targets.

Senescent cells show permanent growth arrest and express markers associated with terminal

differentiation. Consistent with this phenotype, 15 of 51 genes induced in senescent cells are

associated with differentiation, 10 of which are commonly expressed in neuronal tissue (Fig. 1A).

These genes may be indicative of the terminally differentiated phenotype rather than the induction of

a neuronal phenotype as senescent HPECs do not resemble elongated, spindle-shaped cells

characteristic of neuronal cells in culture (13). Senescent HPECs also acquire a phenotype in which

extracellular matrix degrading proteases, growth factors and other inflammatory cytokines are

produced (41). 16 of the 51 genes induced at senescence code for extra-cellular matrix (ECM)

proteins and matrix proteases (Supplemental Fig. 1). This group includes transcripts for

chondroitan sulfated proteoglycan 2, matrix metalloproteinase 2 and micofibrillar-associated protein

2 and their respective increases (5.3, 4.9 and 4.3 fold by cDNA array, respectively) are among the

highest we observed. Several other transcripts encoding ECM proteins (CSPG3, COL1A1, and

fibronectin) and proteases (PAPPA and cathepsin F) are also induced (2.3 and 4.3 fold by cDNA

array, respectively). Senescent cells produce extracellular proteases that promote matrix and

basement membrane degradation, and these changes are thought to contribute to the aging process in

vivo (42). Our data suggest that senescence is associated with significant alterations of the ECM,

and provides some of the molecular underpinnings for these matrix changes.

The finding that the ras homolog gene family member B (RhoB) was induced at senescence

is novel. RhoB mRNA levels were induced 3.6-fold by cDNA array and confirmed with real-time

quantitative PCR (17-fold increase, Fig. 2A). It has been demonstrated that the overexpression of

oncogenic H-ras promotes senescence in primary, mortal epithelial cells and fibroblasts (6);


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


12

however we did not observe changes in H-ras expression during senescence in our HPEC expression

profiles. RhoB appears to have several functions in the cell including negative growth regulation,

the induction of cytoskeletal changes, and sensitizing cells to apoptosis (43,44). In aging mice,

RhoB expression was found to be induced in skeletal muscle and this alteration was attenuated with

caloric restriction, the only known mechanism for retarding the aging process (45). Collectively,

these data suggest that RhoB may be integral to both senescence and the aging process, potentially

contributing to both the growth arrest and the characteristic morphology observed in senescent cells.

To validate the alterations in gene expression observed with senescence and immortalization,

we performed real-time quantitative PCR (qRT-PCR) in additional HPEC cultures on 20 genes that

had high (> 3 fold) or low (< 3 fold) level expression changes (Fig. 2). The pattern of gene induction

or repression observed on the microarrays correlated with that seen by qRT-PCR consistent with

other validation experiments performed using this array system (46). However, microarrays tended

to underestimate changes in transcript levels when genes were induced or repressed over ~3 fold.

Many of the gene expression changes we observed are also observed in primary fibroblasts

undergoing senescence (Table 1). However, in contrast to senescent fibroblasts, HPECs show

increased transcript levels of the retinoic acid receptor α, interleukin 1α, interleukin 6, and IGF-I at

terminal-senescence. These differences in expression may be cell-type specific or may reflect

differences between epithelial cells and fibroblasts (29). Comparison of limited expression profiles

from fibroblasts to our data derived from HPECs are extraordinarily consistent suggesting that

pathways that regulate senescence are conserved among cells derived from different tissues.

Identification of pathways and genes altered following HPEC immortalization—To aid

in defining pathways important in bypassing senescence, we immortalized HPEC cell cultures

with the HPV16 E7 oncoprotein. Comparison of genes expressed by these cells to proliferating


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


13

non-immortalized cells revealed 229 significant alterations in expression of which 92 were

induced (40 of known function) and 137 repressed (70 of known function) (for a complete list

see Supplemental Fig. 3-4). In bypassing senescence, HPECs appear to lose many specialized

functions associated with proliferating, mortal prostate epithelial cells. Of 70 genes

downregulated upon immortalization, ~29 are associated with an epithelial cell function. For

example, HPV16 E7 immortalized HPECs lose expression of prostate epithelial cell specific

kallikreins 10 and 11, two secreted proteases potentially important in reproduction (47). Not

surprisingly, immortalized HPECs activate genes participating in pathways that promote

proliferation, including transcription factors and signaling proteins (Fig. 1B). In addition, genes

involved in DNA damage response were also upregulated during immortalization (Fig. 1B). It is

unclear whether induction of these repair genes reflects increased DNA surveillance, or an

enhanced DNA repair capacity.

Identification of genes associated with bypassing senescence in prostate cancer

cells—We hypothesized that a gene whose expression is induced during senescence (activation)

and repressed following immortalization (inactivation) could be targeted during prostate cancer

progression. Hierarchical clustering analysis was performed on genes whose expression changed

greater than 1.5-fold as cells progressed to senescence or following immortalization to identify a

group of genes with elevated transcript levels at senescence that returned to levels below or

comparable to normal proliferating cells upon immortalization (Fig. 3). To further identify genes

that may be inactivated during carcinogenesis, we focused on transcripts induced in senescent

HPECs and downregulated in the immortalized HPECs below levels found in HPECs

(determined using SAM). This comparison revealed that only BRAK, downregulated in ovarian


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


14

cancer (DOC1), and IGFBP-3, were significantly induced with senescence and repressed in

immortalized cells markedly below (>10-fold) levels in proliferating HPECs.

To extend these findings, we analyzed the expression of BRAK, DOC1, and IGFBP-3 levels

in the human prostate cancer cell lines LNCaP, Du145, TSU, PPC-1, PC-3 and DuPro using qRT-

PCR. In 6/6 tumorigenic, immortalized prostate cancer cell lines we found DOC-1 to be

downregulated roughly 23 fold, and BRAK and IGFBP-3 expression to be undetectable (Fig. 4). We

analyzed the expression levels of these three genes in two additional human prostate cancer

xenografts LAPC4 and LAPC9 to evaluate whether their expression is also lost in vivo. Similar to

the prostate cancer cells, expression of BRAK, DOC1 and IGFBP-3 were all reduced markedly in

these models compared to normal HPECs (Fig. 4). These prostate cancer xenografts have not been

passaged in vitro, suggesting that the decreased expression of BRAK, DOC1 and IGFBP-3 is not due

to artifacts introduced by growth in vitro (22). Our results indicate that genes and pathways

activated at senescence and repressed during HPV16 E7 immortalization are also downregulated in

human prostate cancer cell lines and xenografts and may have a role in human prostate cancer.

Previous reports suggest that BRAK, DOC1, and IGF family members have roles in the

progression of other tumor types in vivo. BRAK is a member of the CXC chemokine family that is

widely expressed in normal tissues, while commonly repressed in head and neck squamous cell

carcinomas (23). Its location on 5q harbors a putative susceptibility gene for prostate cancer based

on familial linkage studies (48). As its name implies, DOC1 was discovered as a transcript

downregulated in ovarian cancers (24). Its function remains unknown. The IGF axis includes

several ligands (IGF-I, IGF-II), cell-surface receptors (IGF-R) and binding proteins (IGFBPs) that

regulate cell growth and differentiation (reviewed in (49)). IGFBP-3 modulates interaction of the

IGFs to their receptors and also possesses IGF-independent functions (50). Decreased IGFBP-3


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


15

expression is associated with prostate cancer progression, demonstrating more frequent loss of

expression in advanced disease, in both human and mouse models (25,51,52). Our data suggest that

this may be due to bypassing senescence pathways. Another factor implicating IGFBP-3 in a

senescence pathway is that the HPV16 E7 oncoprotein targets IGFBP-3 for inactivation (53).

Further study of prostate cancer specimens will be necessary to evaluate the role of BRAK and

DOC1 specifically in prostate cancer. These findings underscore the potential importance of

inactivation of senescence in carcinogenesis and tumor progression.

In summary, using DNA microarray analysis, we have identified genes participating in

normal human prostate epithelial cell senescence. We suspect these pathways participate in the

induction of senescence in many other cell types and these genes are members of a limited number

of pathways that can be inactivated to bypass senescence during tumorigenesis (15). We also have

identified novel markers for the senescent phenotype that could assist in an analysis of the role of

senescence in normal and pathologic states in vivo (54). Future studies will explore whether

reactivation of these pathways in immortalized cells can also re-establish cellular senescence and

whether this may represent a novel therapeutic approach to prostate cancer.

Acknowledgements

We would like to thank Dr. Rob Reiter for the xenografts and Dr. John Svaren for the critical

reading of this manuscript.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


16

REFERENCES

1. Hayflick, L. (1965) Exp Cell Res 25, 585-621

2. Campisi, J. (2000) In Vivo 14(1), 183-8

3. Campisi, J. (2001) Trends Cell Biol 11(11), S27-31.

4. Campisi, J., Kim, S., Lim, C. S., and Rubio, M. (2001) Exp Gerontol 36(10), 1619-37.

5. Dimri, G. P., Itahana, K., Acosta, M., and Campisi, J. (2000) Mol Cell Biol 20(1), 273-

85.

6. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D., and Lowe, S. W. (1997) Cell

88(5), 593-602

7. Lin, A. W., Barradas, M., Stone, J. C., van Aelst, L., Serrano, M., and Lowe, S. W.

(1998) Genes Dev 12(19), 3008-19

8. Zhu, J., Woods, D., McMahon, M., and Bishop, J. M. (1998) Genes Dev 12(19), 2997-

3007.

9. Vogt, M., Haggblom, C., Yeargin, J., Christiansen-Weber, T., and Haas, M. (1998) Cell

Growth Differ 9(2), 139-46

10. Brown, J. P., Wei, W., and Sedivy, J. M. (1997) Science 277(5327), 831-4

11. Dimri, G. P., Hara, E., and Campisi, J. (1994) J Biol Chem 269(23), 16180-6.

12. Kiyono, T., Foster, S. A., Koop, J. I., McDougall, J. K., Galloway, D. A., and

Klingelhutz, A. J. (1998) Nature 396(6706), 84-8.

13. Jarrard, D. F., Sarkar, S., Shi, Y., Yeager, T. R., Magrane, G., Kinoshita, H., Nassif, N.,

Meisner, L., Newton, M. A., Waldman, F. M., and Reznikoff, C. A. (1999) Cancer Res

59(12), 2957-64


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


17

14. El Gedaily, A., Bubendorf, L., Willi, N., Fu, W., Richter, J., Moch, H., Mihatsch, M. J.,

Sauter, G., and Gasser, T. C. (2001) Prostate 46(3), 184-90.

15. Sasaki, M., Honda, T., Yamada, H., Wake, N., Barrett, J. C., and Oshimura, M. (1994)

Cancer Res 54(23), 6090-3.

16. Pereira-Smith, O. M., and Smith, J. R. (1988) Proc Natl Acad Sci U S A 85(16), 6042-6.

17. Jarrard, D. F., Modder, J., Sandefur, C., Shi, Y., Heisey, D., Schwarze, S., and Friedl, A.

(submitted)

18. Heidenberg, H. B., Sesterhenn, I. A., Gaddipati, J. P., Weghorst, C. M., Buzard, G. S.,

Moul, J. W., and Srivastava, S. (1995) J Urol 154(2 Pt 1), 414-21.

19. Yeager, T. R., DeVries, S., Jarrard, D. F., Kao, C., Nakada, S. Y., Moon, T. D.,

Bruskewitz, R., Stadler, W. M., Meisner, L. F., Gilchrist, K. W., Newton, M. A.,

Waldman, F. M., and Reznikoff, C. A. (1998) Genes Dev 12(2), 163-74

20. Newbold, R. F., Cuthbert, A. P., Themis, M., Trott, D. A., Blair, A. L., and Li, W. (1993)

Toxicol Lett 67(1-3), 211-30.

21. Sarkar, S., Julicher, K. P., Burger, M. S., Della Valle, V., Larsen, C. J., Yeager, T. R.,

Grossman, T. B., Nickells, R. W., Protzel, C., Jarrard, D. F., and Reznikoff, C. A. (2000)

Cancer Res 60(14), 3862-71.

22. Klein, K. A., Reiter, R. E., Redula, J., Moradi, H., Zhu, X. L., Brothman, A. R., Lamb, D.

J., Marcelli, M., Belldegrun, A., Witte, O. N., and Sawyers, C. L. (1997) Nat Med 3(4),

402-8.

23. Frederick, M. J., Henderson, Y., Xu, X., Deavers, M. T., Sahin, A. A., Wu, H., Lewis, D.

E., El-Naggar, A. K., and Clayman, G. L. (2000) Am J Pathol 156(6), 1937-50.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


18

24. Mok, S. C., Wong, K. K., Chan, R. K., Lau, C. C., Tsao, S. W., Knapp, R. C., and

Berkowitz, R. S. (1994) Gynecol Oncol 52(2), 247-52.

25. Grimberg, A., and Cohen, P. (2000) J Cell Physiol 183(1), 1-9.

26. Reznikoff, C. A., Loretz, L. J., Pesciotta, D. M., Oberley, T. D., and Ignjatovic, M. M.

(1987) J Cell Physiol 131(3), 285-301.

27. Halbert, C. L., Demers, G. W., and Galloway, D. A. (1991) J Virol 65(1), 473-8.

28. DeRisi, J., Penland, L., Brown, P. O., Bittner, M. L., Meltzer, P. S., Ray, M., Chen, Y.,

Su, Y. A., and Trent, J. M. (1996) Nat Genet 14(4), 457-60.

29. Ross, D. T., Scherf, U., Eisen, M. B., Perou, C. M., Rees, C., Spellman, P., Iyer, V.,

Jeffrey, S. S., Van de Rijn, M., Waltham, M., Pergamenschikov, A., Lee, J. C., Lashkari,

D., Shalon, D., Myers, T. G., Weinstein, J. N., Botstein, D., and Brown, P. O. (2000) Nat

Genet 24(3), 227-35.

30. Eisen, M. B., Spellman, P. T., Brown, P. O., and Botstein, D. (1998) Proc Natl Acad Sci

U S A 95(25), 14863-8.

31. Tusher, V. G., Tibshirani, R., and Chu, G. (2001) Proc Natl Acad Sci U S A 98(9), 5116-

21.

32. Wittwer, C. T., Herrmann, M. G., Moss, A. A., and Rasmussen, R. P. (1997)

Biotechniques 22(1), 130-1, 134-8

33. Morrison, T. B., Weis, J. J., and Wittwer, C. T. (1998) Biotechniques 24(6), 954-8, 960,

962

34. Schwarze, S. R., Shi, Y., and Jarrard, D. F. (2002) Oncogene 20, 8184-8192


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


19

35. Reznikoff, C. A., Belair, C., Savelieva, E., Zhai, Y., Pfeifer, K., Yeager, T., Thompson,

K. J., DeVries, S., Bindley, C., Newton, M. A., and et al. (1994) Genes Dev 8(18), 2227-

40.

36. Puthenveettil, J. A., Frederickson, S. M., and Reznikoff, C. A. (1996) Oncogene 13(6),

1123-31.

37. Reznikoff, C. A., Yeager, T. R., Belair, C. D., Savelieva, E., Puthenveettil, J. A., and

Stadler, W. M. (1996) Cancer Res 56(13), 2886-90

38. Guardavaccaro, D., Corrente, G., Covone, F., Micheli, L., D'Agnano, I., Starace, G.,

Caruso, M., and Tirone, F. (2000) Mol Cell Biol 20(5), 1797-815.

39. McConnell, B. B., Starborg, M., Brookes, S., and Peters, G. (1998) Curr Biol 8(6), 351-4

40. Brown, D. R., Thomas, C. A., and Deb, S. P. (1998) Embo J 17(9), 2513-25.

41. Krtolica, A., Parrinello, S., Lockett, S., Desprez, P. Y., and Campisi, J. (2001) Proc Natl

Acad Sci U S A 98(21), 12072-7.

42. Campisi, J. (1997) J Am Geriatr Soc 45(4), 482-8.

43. Fritz, G., and Kaina, B. (1997) J Biol Chem 272(49), 30637-44.

44. Liu, A., Cerniglia, G. J., Bernhard, E. J., and Prendergast, G. C. (2001) Proc Natl Acad

Sci U S A 98(11), 6192-7.

45. Lee, C. K., Klopp, R. G., Weindruch, R., and Prolla, T. A. (1999) Science 285(5432),

1390-3.

46. Iyer, V. R., Eisen, M. B., Ross, D. T., Schuler, G., Moore, T., Lee, J. C., Trent, J. M.,

Staudt, L. M., Hudson, J., Jr., Boguski, M. S., Lashkari, D., Shalon, D., Botstein, D., and

Brown, P. O. (1999) Science 283(5398), 83-7.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


20

47. Saitoh, S., Kumamoto, Y., Shimamoto, K., and Iimura, O. (1987) Arch Androl 19(2),

133-47

48. Witte, J. S., Goddard, K. A., Conti, D. V., Elston, R. C., Lin, J., Suarez, B. K., Broman,

K. W., Burmester, J. K., Weber, J. L., and Catalona, W. J. (2000) Am J Hum Genet 67(1),

92-9.

49. O'Dell, S. D., and Day, I. N. (1998) Int J Biochem Cell Biol 30(7), 767-71.

50. Ferry, R. J., Jr., Cerri, R. W., and Cohen, P. (1999) Horm Res 51(2), 53-67

51. Kaplan, P. J., Mohan, S., Cohen, P., Foster, B. A., and Greenberg, N. M. (1999) Cancer

Res 59(9), 2203-9.

52. Hampel, O. Z., Kattan, M. W., Yang, G., Haidacher, S. J., Saleh, G. Y., Thompson, T. C.,

Wheeler, T. M., and Marcelli, M. (1998) J Urol 159(6), 2220-5.

53. Mannhardt, B., Weinzimer, S. A., Wagner, M., Fiedler, M., Cohen, P., Jansen-Durr, P.,

and Zwerschke, W. (2000) Mol Cell Biol 20(17), 6483-95.

54. Chang, B. D., Xuan, Y., Broude, E. V., Zhu, H., Schott, B., Fang, J., and Roninson, I. B.

(1999) Oncogene 18(34), 4808-18


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


21

Fig. 1. Identity and cellular role of specific categories of genes induced in terminally-senescent

HPECs (A) and repressed in immortalized HPECs (B). Transcripts of named genes were selected

from the SAM outputs and placed into functional categories. Included in (A) are genes induced at

senescence associated with differentiated cell or neuronal functions and those associated with growth

regulation. Genes repressed in immortalized cells that are involved in signaling, transcription and

DNA repair are listed in (B). The Genbank accession number and fold change from normal

proliferating HPEC proliferating values are listed for each entry (+ indicates gene induction and –

indicates repression).

Fig. 2. Quantitative RT-PCR validation of cDNA microarray results. Real-time PCR following the

incorporation of SYBR green into DNA was performed on cDNA derived from three independent

HPEC cultures at in the proliferating, pre-senescent, terminally-senescent stages and in HPV16 E7

immortalized HPECs. Relative expression levels were normalized to the 18S content, also

determined by RT-PCR, and plotted on a log scale as fold change compared to proliferating cells.

Expression levels from three separate microarray experiments were averaged and plotted with the

qRT-PCR data for comparison. Good correlation was observed for all transcript levels, although the

DNA microarrays tended to under estimate changes in transcript levels at higher magnitudes.

Fig. 3. Cluster analysis of transcripts from senescent and HPV16 E7 immortalized HPECs identified

by the SAM procedure. Experiments are grouped to show transcripts that are induced at pre- and

terminal-senescence and at or below levels of proliferating cells in the immortalized HPECs. Red

squares indicate expression levels higher than that of proliferating HPECs, green squares indicate

expression levels lower than that of proliferating HPECs, black squares indicate transcript levels


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


22

approximately equal to those in proliferating HPECs; gray squares indicate data of insufficient

quality. NP, normal prostate; HPVE7, HPV16 E7 immortalized HPECs; prol, proliferating; Pre-

sen., pre-senescence; T-sen., terminally-senescent.

Fig. 4. Expression of BRAK (A), DOC1 (B), and IGFBP-3 (C) is very low in immortalized human

prostate cancer cell lines. qRT-PCR was performed on 6 prostate cancer cell lines (LNCaP, Du145,

TSU, PPC-1, PC-3 and DuPro) and two xenografts (LAPC4 and LAPC9) and normalized to

proliferating HPEC values. Expression levels in all samples were standardized to 18S RNA levels.

Prol, proliferating; Pre-sen., pre-senescent; T-sen., terminally-senescent.

Supplemental Fig. 1. Identity and cellular role of specific categories of genes induced in terminally-

senescent HPECs. Transcripts of named genes were selected from the SAM outputs and placed into

functional categories. The Genbank accession number and fold change from normal proliferating

HPEC values are listed for each entry (+ indicates gene induction).

Supplemental Fig. 2. Identity and cellular role of specific categories of genes repressed in

terminally-senescent HPECs. Transcripts of named genes were selected from the SAM outputs and

placed into functional categories. The Genbank accession number and fold change from normal

proliferating HPEC values are listed for each entry (– indicates gene repression).

Supplemental Fig. 3. Identity and cellular role of specific categories of genes induced in

immortalized HPECs. Transcripts of named genes were selected from the SAM outputs and placed


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


23

into functional categories. The Genbank accession number and fold change from normal

proliferating HPEC values are listed for each entry (+ indicates gene induction).

Supplemental Fig. 4. Identity and cellular role of specific categories of genes repressed in

immortalized HPECs. Transcripts of named genes were selected from the SAM outputs and placed

into functional categories. The Genbank accession number and fold change from normal

proliferating HPEC values are listed for each entry (– indicates gene repression).


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


24

Table 1. Comparison of HPEC senescence-associated gene mRNA levels

to those reported in the literature (fibroblasts). Fold-change was calculated

by comparing relative transcript levels in proliferating and terminal-senescent

HPECs. Genes are grouped according to the following cellular functions:

(A) Growth regulatory; (B) Cytokine/growth factor; (C) Structural. Values

listed were determined by cDNA microarray. NC, no change.

Gene Reported change HPEC changeId-1 repressed repressed 1.25XId-2 repressed repressed 1.25Xc-fos repressed repressed 1.45XE2F1 repressed repressed 1.5XE2F4 repressed repressed 1.3XE2F5 repressed repressed 1.2Xp33ING induced induced 1.8Xhic-5 induced induced 1.25XDHFR repressed repressed 3.8Xthymidine kinase repressed repressed 9.5Xthymidylate synthase repressed repressed 2.6XDNA polymerase α repressed repressed 1.6Xcyclin A repressed repressed 5.4Xcyclin B repressed repressed 5.2Xcdk2 repressed repressed 2.5Xcdc2 repressed repressed 11.3Xp16INK4a induced induced 49Xp21WAF1/CIP1 induced induced 2.3Xp53 induced induced 2.0XRARα induced NCInterleukin 1α induced repressed 2XInterleukin 6 induced repressed 1.9XIGF-I repressed NCIGFBP-3 induced induced 2.6Xfibronectin 1 induced induced 2.9Xcornifin induced induced 2.4Xcollagenase (MMP2) induced induced 4.9Xstromelysin (MMP3) induced induced 1.6XPAI-1 induced induced 2.5XTIMP1 repressed repressed 4X

C

B

A


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Figure 1

A

Differentiation/neuronalNM_017459 +4.3 microfibrillar-associated protein 2X07868 +4.4 insulin-like growth factor 2NM_006426 +3.5 dihydropyrimidinase-like 4NM_005461 +3.5 Kreisler maf-related leucine zipperNM_002391 +3.3 midkine (neurite growth-promoting)NM_013372 +2.9 melanoma adhesion moleculeAF183421 +2.8 RAB31, ras GTP exchangerNM_013372 +2.7 BMP antagonist 1AL136179 +2.7 SOX4NM_006763 +2.7 BTG2AF231124 +2.7 testicanNM__004112 +2.4 fibroblast growth factor 11NM_032088 +2.4 protocadherin gamma subfamily A, 8NM_004986 +2.3 chondroitan sulfate proteoglycan 3NM_018901 +2.2 protocadherin alpha 5

Growth regulator/tumor associationNM_000077 +49 p16 INK4a

NM_005672 +4.8 prostate stem cell antigenX07868 +4.4 insulin-like growth factor 2NM_014890 +3.2 downregulated in ovarian cancer 1NM_006763 +2.7 BTG2NM_002392 +2.4 MDM2NM_000389 +2.3 p21 WAF1/CIP1

NM_002430 +2.3 meningioma 1

AF144103 +5.3 BRAK

M35878 +2.3 IGFBP-3

B

DNA Repair

subunit 2 homolog

+7.8 proliferating cell nuclear antigenNM_002592+7.2 p53-inducible ribonucleotide small AB036063

+5.7 p53 regulated PA26 nuclear proteinNM_014454+4.5 mdm2NM_002392+4.2 damage-specific DNA binding proteinNM_000107

Signaling

NM_012324 +2.3 JNK-interacting protein 2 (JIP2)

+12 interleukin 1, betaNM_000576+7.2 TNF ligand, member 7NM_001252+6.5 epiregulinNM_001432+6.0 placental growth factorNM_002632+5.4 LRDDNM_018494+4.8 coagulation factor II (thrombin) receptorNM_001992+3.6 ras homolog, family member HNM_004310+3.4 inositol polyphosphate-5-phosphataseNM_005541

+2.4 c-jun amino terminal kinase 1 (JNK1)NM_002750+3.2NM_014288 integrin beta 3 binding protein

Transcription factors+7.6 forkhead box D1NM_004472+7.2 HOXB7NM_004502

S53374 +4.2 transcription factor 19


25


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

A

B

C

Figure 2 Gene expression profiles in senescence and immortalization

26


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Figure 3

UteroglobinEVG1 proteinSRY (sex determining region Y)-box 4ESTshypothetical protein FLJ20059matrix metalloproteinase 2cDNA DKFZp56402364cathepsin FCdc42 effector protein 3sema domain, immunoglobulin domainprotocadherin gamma subfamily A, 8matrix Gla proteinhypothetical protein DKFZp434J0617hypothetical protein FLJ14213ESTsDKFZP564I1922 proteinESTsepoxide hyrolase 1ESTsS100 calcium-binding protein A9S100 calcium-binding protein A8STAT4ESTsESTs, similar to p53 regulated PA26-T2aldo-keto reductase family 1, member C2

downregulated in ovarian cancer 1small inducible cytokine subfamily, BRAKnicotinamide N-methyltransferasecytochrome P450, polypeptide

NP

47-P

rol

NP

104-

Pro

lN

P49

-Pro

l

NP

47-P

re-s

en.

NP

104-

Pre

-sen

.N

P49

-Pre

-sen

.

NP

47-T

-sen

.

NP

104-

T-se

n.N

P49

-T-s

en.

HP

VE

7-9

HP

VE

7-15

HP

VE

7-14

P311 proteinATP-binding cassette, sub-family A, 1ras homolog gene family, member B (rhoB)gap junction protein, alpha 1, 43kDmicrofibrillar-associated protein 2milk fat globule-EGF factor 8 proteinchondroitan sulfate proteoglycan 2


27


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

-100 -80 -60 -40 -20 1 20

RNA abundance(fold change from prol. HPEC)

HPEC-prol.

HPEC-Pre-sen

HPEC-T-sen.

Du145

DuPro

LNCaP

PC-3

PPC-1

Tsu

LAPC4

LAPC9

HPEC-immortal

BRAKDOC1IGFBP-3

Figure 4 Gene expression profiles in senescence and immortalization

28


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Motility/MigrationNM_004040 +3.6 RhoBNM_004772 +2.6 P311AL136842 +2.4 Cdc42 effector protein 3

Stress/Injury/XenobioticNM_002964 +5.5 S100 calcium-binding protein A8NM_001354 +3.9 aldo-keto reductase family 1, C1NM_002965 +3.7 S100 calcium-binding protein A9NM_001992 +3.0 coagulation factor II (thrombin) receptorNM_005627 +2.9 serum/glucocorticoid regulated kinaseNM_005527 +2.9 heat shock 70kD protein-like 1NM_000120 +2.7 epoxide hydrolase 1, microsomalNM_006169 +2.6 nicotinamide N-methyltransferase

OtherNM_005518 +4.8 HMGCS2-ketone body synthesisNM_000165 +2.8 gap junction protein (connexin 43)U10991 +2.8 G2 protein-glucosidaseNM_005502 +2.7 ABC1-cholesterol metabolism NM_006763 +2.4 milk fat globule-egf factor 8 protein

Supplemental Figure 1

Differentiation/neuronalNM_017459 +4.3 microfibrillar-associated protein 2X07868 +4.4 insulin-like growth factor 2NM_006426 +3.5 dihydropyrimidinase-like 4NM_005461 +3.5 Kreisler maf-related leucine zipperNM_002391 +3.3 midkine (neurite growth-promoting)NM_013372 +2.9 melanoma adhesion moleculeAF183421 +2.8 RAB31, ras GTP exchangerNM_013372 +2.7 BMP antagonist 1AL136179 +2.7 SOX4NM_006763 +2.7 BTG2AF231124 +2.7 testicanNM__004112 +2.4 fibroblast growth factor 11NM_032088 +2.4 protocadherin gamma subfamily A, 8NM_004986 +2.3 chondroitan sulfate proteoglycan 3NM_018901 +2.2 protocadherin alpha 5

Growth regulator/tumor associationNM_000077 +49 p16 INK4a

NM_005672 +4.8 prostate stem cell antigenX07868 +4.4 insulin-like growth factor 2NM_014890 +3.2 downregulated in ovarian cancer 1NM_006763 +2.7 BTG2NM_002392 +2.4 MDM2NM_000389 +2.3 p21 WAF1/CIP1

NM_002430 +2.3 meningioma 1

AF144103 +5.3 BRAK

M35878 +2.3 IGFBP-3

Cytokine/growth factorNM_003357 +5.5 uteroglobin

NM_001874 +3.1 carboxypeptidase M

AF144103 +5.3 BRAKX07868 +4.4 insulin-like growth factor 2NM_002391 +3.3 midkine (neurite growth-promoting)

NM__004112 +2.4 fibroblast growth factor 11

Extracellular matrixU16306 +5.3 chondroitan sulfate proteoglycan 2NM_004530 +4.9 matrix metalloproteinase 2NM_017459 +4.3 microfibrillar-associated protein 2NM_002581 +3.5 pregnancy-associated plasma protein ANM_003793 +3.0 cathepsin FNM_000088 +3.0 collagen, type I, alpha 1X02761 +2.9 fibronectinNM_004986 +2.3 chondroitan sulfate proteoglycan 3

TranscriptionAF055376 +5.9 v-maf musculoaponeurotic fibrosarcomaNM_005461 +3.2 Kreisler maf-related leucine zipper NM_003151 +3.0 STAT4AF129290 +2.8 Cbp/p300 interacting transactivator, 2

NM_000168 +2.2 GLI3AL136179 +2.7 SOX4


29


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

DNA Replication-9.5 thymidine kinase 1NM_003258-9.2 ribonucleotide reductase M2NM_001034-6.8 uridine phosphrylaseNM_003364-5.6 CDC45NM_003504-4.7 topoisomerase II, alphaNM_001067

-4.2 DNA polymerase, epsilon 2NM_002692-3.8 dihydrofolate reductaseNM_000791-2.8 replication factor CNM_002915-2.6 primase, polypeptide 1NM_000946

-4.6NM_001254 Cdc6

DNA Repair-4.7 RAD51-interacting proteinNM_006479-4.2 translinNM_004622-3.3 RAD51NM_002875-3.0 BRCA1 associated RING domain 1NM_000465

Transcription factor-5.8 transcription factor 19S53374-4.4 v-myb oncogene homologNM_002466-4.3 forkhead Box M1NM_021953-3.2 pituitary tumor transforming 1NM_004219-3.1 prostate epithelium-specific EtsNM_012391

Apoptosis/Stress Response-5.9 BCL2-related protein A1NM_004049-5.5 transgluaminase 1NM_000359-4.0 catenin, alpha-like 1NM_003798-3.7 alcohol dehydrogenase 4NM_000670-2.8 biliverdin reductase BNM_000713-2.7 tumor rejection antigen 1NM_003299-2.6 CSR1 proteinNM_016240

Positive Growth regulator-11 cdc2NM_001786-11 PDZ-binding kinaseNM_018492-10 CDKN3NM_005192-7.9 polo-like kinaseNM_005030-7.9 HBP17NM_005130-7.3 G0/G1 switchNM_015714-7.2 claspinNM_022111-6.1 RHAMMNM_012484-5.4 cyclin A2NM_001237-4.8 serine/threonine kinase 15NM_003600-4.3 forkhead box M1NM_021953-4.4 v-myb oncogene homologNM_002466-4.2 pro-platelet basic proteinNM_002704-3.9 thyroid hormone receptor interactor 13NM_004237-3.7 anterior gradient 2 homologNM_006408-3.6 CDC28 protein kinase 2NM_001827-3.1 MAPK 13NM_002754-2.8 notch homolog 4NM_004557-3.2 pituitary tumor-transforming 1NM_004219-3.0 insulin-induced gene 1NM_005542-3.0 GRO3 oncogeneNM_002090-2.6 CDC28 protein kinase 1NM_001826-2.2 cdc25ANM_001789

Signaling-4.1 rho GDP dissociation inhibitor betaNM_001175-4.0 catenin, alpha-like 1NM_003798

-2.9 integrin, alpha 4NM_000885-2.9 small inducible cytokine A20NM_004591

-3.9NM_012242 dickkopf homolog 1

Mitosis-11 anillinNM_018685-9.1 NUF2RNM_031423-8.6 ubiquitin protein E2-CNM_007019-7.3 centromere protein FNM_005196-6.8 centromere protein ENM_001813-6.2 NIMA-related kinase 2NM_002497-5.9 BUB1NM_004336-5.9 kinesin-like 5NM_004856-4.2 ZW10 interactorNM_007057-3.2 MAD2-like 1NM_002358-3.0 insulin-induced gene 1NM_005542-2.9 survivinNM_001168-2.8 MCM6NM_005915-2.7 SMC4AL136877 Other

-8.5 follistatinNM_006350-6.9 ADAM8NM_001109-6.7 SLC14A1NM_015865-5.1 L2DTLNM_016448

-4.5 UAP1NM_003115

-4.1 EFEMP1NM_004105

-3.6 diazepam binding inhibitorNM_020548

-3.3 coagulation factor IIINM_001993

-3.1 TROAPNM_005480-3.2 MYT1NM_004203

-3.4NM_004631 apolipoprotein E receptor

-3.6NM_005573 lamin B1

-4.4AB032261 steoaroyl CoA desaturase

-4.2NM_000963 prostaglandin-endoperoxide synthase 2

Supplemental Figure 2 Gene expression profiles in senescence and immortalization

30


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


Metabolism/Transport+5.1 solute carrier 26NM_000112+5.1 solute carrier 10NM_003049

+4.7 fucosidase, alphaNM_000147+4.6 L2DTL proteinNM_01448+4.0 aldehyde dehydrogenase 7NM_001182

+3.8 ferredoxin reductaseNM_004110+3.8 synoaptosomal-associated proteinAF278704+2.9 sorting nexin 5NM_014426

+4.0NM_000067 carbonic anhydrase II

+4.9NM_004117 FK506-binding protein

Proliferation+7.8 proliferating cell nuclear antigenNM_002592+4.3 Cdc7-like 1NM_003503

+3.2 S-phase kinase-associated protein 2NM_005983+3.1 dihyrofolate reductaseNM_000791


Transcription factors+7.6 forkhead box D1NM_004472+7.2 HOXB7NM_004502


DNA Repair

subunit 2 homolog

+7.8 proliferating cell nuclear antigenNM_002592+7.2 p53-inducible ribonucleotide small AB036063

+5.7 p53 regulated PA26 nuclear proteinNM_014454+4.5 mdm2NM_002392+4.2 damage-specific DNA binding proteinNM_000107

Growth factor/Cytokine+12 interleukin 1, betaNM_000576

+6.5 epiregulinNM_001432+6.0 placental growth factorNM_002632

Signaling

NM_012324 +2.3 JNK-interacting protein 2 (JIP2)

+12 interleukin 1, betaNM_000576+7.2 TNF ligand, member 7NM_001252+6.5 epiregulinNM_001432+6.0 placental growth factorNM_002632+5.4 LRDDNM_018494+4.8 coagulation factor II (thrombin) receptorNM_001992+3.6 ras homolog, family member HNM_004310+3.4 inositol polyphosphate-5-phosphatase (SHIP)NM_005541

+2.4 c-jun amino terminal kinase 1 (JNK1)NM_002750+3.2NM_014288 integrin beta 3 binding protein

Apoptosis+7.2 TNF ligand, member 7NM_001252+5.4 TNF receptor, member 10c, decoyNM_003841

Other+8.2 fibrillin 2NM_001999+6.3 IGF2 mRNA-binding protein 3NM_006547+4.9 protocadherin, alpha 5NM_018901+3.6 NCAMNM_005010+3.4 NPATNM_002519

+9.1NM_001874 carboxypeptidase M


31


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


Signaling/growth factor/cytokine-27 small inducible cytokine A2NM_002982

-16 interleukin 11NM_000641-10 TGF§2NM_003238-11 pro-platelet basic proteinNM_002704-8.9 BRAKAF144103-7.1 interleukin 6NM_000600-7.0 IGFBP-3M35878-6.6 TNF ligand superfamily, 10NM_003810-5.6 PDGF§NM_002608-4.9 osteonectinNM_003118-4.8 IGFBP-5NM_000599-4.7 CEACAM6M18216-4.5 integrin, beta 8NM_002214-3.9 CEACAM5NM_004363-3.8 GRO1 oncogeneNM_001511-3.5 WNT5bNM_030775-3.4 interleukin 8NM_000584-3.2 neurotrophin-1NM_013246-3.1 PLAURNM_002659-3.1 guanylate binding protein 1NM_002053-2.8 CTGFNM_001901-2.8 protein kinase H11NM_014365-2.7 enhancer of filamentation 1NM_006404

-21 RAR responder 1NM_002888

ECM-component-58 matrix metalloproteinase 7NM_002423-30 collagen, type III, alpha 1NM_000090-15 collagen type IV, alpha 3NM_004369-10 clade B, member 3NM_006919-14 collagen type IV, alpha 1BC005159-11 hexabrachionNM_002160-7.6 kallikrein 10NM_002776-6.6 collagen type I, alpha 1NM_000088-5.3 tryptase, alphaAK027693-4.5 ADAMTS1NM_006988-4.2 collagen, type V, alpha 2NM_000393-3.4 kallikrein 11NM_006853-3.4 protease, serine, 23NM_007173-3.2 biglycanNM_001711-2.6 thrombospondin 1NM_003246

Cytoskeletal/adhesion-10 keratin 4X07695-12 keratin 13NM_002274-9.5 transgelinNM_003186 -4.9 myosin VBAB032945-4.5 integrin, beta 8NM_002214-4.4 keratin 6ANM_005555-4.2 myosin regulatory light chain 2AC074331-3.2 contactin 1NM_001843

Calcium regulation-6.3 S100 calcium-binding protein PNM_005980-5.6 calmodulin-like 3M36707-3.8 calcium channel, voltage-dependent, beta 2NM_000724-3.6 annexin A6NM_001155-2.9 tumor-associated calcium signal transducer 1 NM_002354

Other-8.1 anterior gradient 2NM_006408-7.0 odd Oz/tenAB032953-5.9 protein kinase inhibitor betaNM_032471-5.5 cerebellar degeneration related proteinNM_004065-4.8 downregulated in ovarian cancer 1NM_014890-4.2 sulfotransferase 2B, member 1NM_004605-3.8 netrin 4AF278532-3.3 solute carrier family 14NM_015865-3.0 transcription elongation factor BNM_003198-2.8 transcript release factorBC004295-2.7 purinergic receptorNM_005767

Stress/Xenobiotic-12 glutathione peroxidase 2NM_002083-6.8 cytochrome p450, subfamily 1NM_000104-6.8 nicotinamide N-methyltransferaseNM_006169-4.3 glutathione S-transferase theta 1NM_000853-3.0 aryl hydrocarbone receptorNM_001621


32


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

and David F. JarrardSteven R. Schwarze, Samuel E. DePrimo, Lisa M. Grabert, Vivian X. Fu, James D. Brooks

epithelial cellsNovel pathways associated with bypassing cellular senescence in human prostate

published online February 8, 2002J. Biol. Chem.

10.1074/jbc.M200373200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M200373200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M200373200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2002/02/08/jbc.M200373200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2002/02/08/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2002/02/08/jbc.M200373200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

novel pathways associated with bypassing cellular ... · downregulated in immortalized hpecs...

Documents