l

l

THE ROLE OF CARBOHYDRATES IN THE I:NTERCBLLULAR J~HES::rON

MEDIATBD BY CARCINOEMBRYONIC ANTIGBN USING CHO MUTANT CBLL

LINBS

by

Julie Charbonneau

A Thesis submitted to the Faculty of Graduate Studies and

Research, McGill University, in partial fulfillment of the

requirements for the degree of Master of Science

Department of Biochemistry

McGill University

Montreal, Quebec

CANADA

Julie Charbonneau Pebruary 1992

.

i

ABSTRACT

Carcinoembryonic antigen (CEA) is a highly glycosylated cell

surface glycoprotein which has recently been demonstrated to

behave as a Ca++-independent intercellular adhesion molecul~.

In order to study the effects of carbohydrates on the

intercellular adhesion funcl._on of CEA, we l,ave transfected

the functional cDNA of CEA into wilù type and glycosylation

mutant cells Lecl, Lec2, and Lec8. Aggregation assays of cells

in suspension were performed with stable CEA transfectants of

these cell lines and showed that aIl of the aberrant CHA

glycofo~s could still mediate adhesion, and that the

specificity of adhesion of these glycoforms was not altered.

Lecl transfectants did, however, show an increased speed and

final extent of aggregation, which suggests that, although

carbohydrates can modulate the strength of adhesion, they do

not determine the adhesion property; this property must

therefore reside in the CEA protein backbone itself •

-

RESUME

L'antigène carcinoembryonnaire

surface cellulaire, est glycosylé

(CEA) ,

à 60%

ii

exprimé à la

de son

moléculaire et possède 28 sites

l'asparagine.

de glycosylation

poids

liée à

Nous avons récemment démontré que CHA peut agir comme

molécule d'adhésion intercellulaire indépendemment de la

température et de la concentration en calcium. Afin d'étudier

l'effet des glycanes sur la fonction d'adhésion

intercellulaire de CHA, le cDNA de cn a été transfecté dans

les cellules CHO mutantes Lecl, Lec2, LeeS. Celles-ci sont

déficientes en enzymes impliquées dans la modification de la

structure des sucres des glycoprotéines. Des études

d'aggrégation en suspension, à l'aide des transfectants ci­

haut mentionés, ont démontré que l' adhésion él~it encore

possible malgré les glycoformes abbérantes de CEA. De plus.

les essais d'aggrégation homotypiques et hétérotypiques ont

démontré que la spécificité d'adhésion n'est pas influencée

par les glycoformes de CHA. La comparaison des courbes

d'adhésion des différentes glycoformes de CZA, a révélé que

celle du transfectant Lecl (CEA) était nettement plus

prononcée, ce qui signifie une adhésion plus rapide et un

pourcentage d'aggrégation plus élevé pour ce transfectant.

puisque Lecl possède le glycane le plus siDriile par rapport aux

autres mutants, nos résultats sont en accord avec le

iii

modèle qui suggère que les glycanes empêchent l'adhésion

entre leu domaines protéiques.

Nous concluons donc, Que les sucres de CBA ne sont pas

essentiels comme tel, mais participent à la force d'adhésion

intercellulaire. Les éléments essentiels à l'adhésion

intercellulaire de CBA devraient donc être dU à la ~tructure

de la protéine, i.e., le code spécifique des acides aminés.

~-

r 1

iv

PRIU'ACE

The work presented here has been sent for publication

to the Journal of cell Biology. The expression vector p91023B

cOlltaining the CEA cDNA was provided by Sarita Benchimol, the

antibodies to CEA were provided by Abe Fuks, the ELISA assaye

were performed by Aurora Labitan. The rest of the work was my

own achievement.

1 :

Il' ,

f f. ~ , L Il

t il l, ~ .~~ . ~ ~

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TABLE OF CONTENT

Abstract

Résumé

Préface

Table of contents

List of tables

List of figures

List of abbreviations

Acknowledgemellts

CHAPTER 1 :INTRODUCTION

1.1 Cell Adhesion Molecules

1.2 C~inical Significance of Carcinoemb~onic

Ar,~igen (CHA)

1.3 Molecular Biology of CHA

1.4 CD Pamily

1.4.1 Holecular Biology

1.4.2 Normal and Tumor Localization of

CBA Family Hembers

1.5 Possible Punctions of CHA

1.6 Adhesion Function of CHA and pamily Hembers

1.7 Role of Sugars in CD Mediated Adhesion

1.8 Carbohydrates in Cancer

1.9 Glycosylation of Glycoproteins

1.10 Hethods for Neasuring Cellular Adhesion

i

ii

iv

v

vii

vii

ix

xi

1

2

6

7

8

8

12

12

15

17

19

20

21

v

'1 ~

~ 1

j

J

j

r vi

CHAPTBR II MATBRIALS AND MBTHODS 23

II.l Cell Culture and Transfections 24

II.2 Aggregation Assay 25

II.3 Cell Sorting Assay 26

11.4 Western Blot Analysis 27

II.5 FACS analysis 28

CHAPTER III RESULTS 29

III.l Isolation of Glycosylation Defective CEA

transfectants 30

111.2 Homotypic aggregation of tranfectants 35 " r III.3 Specificity of cn Adhesion 38 , , 1 t 111.4 Capacity of Adhesion 43 .

CHAPTER IV DISCUSSION 51

CHAPTBR V REFERBNCBS 62

<.

Table 1

FIGURB 1

FIGURB 2

FIGURB 3

FIGURB 4

FIGURB 5

FIGURB 6

(

LIST OF TABLES

LISTS OF FIGURES

CHA family

Bxpected carbohydrate structures

of glycoproteins produced by the

various wild type and Lee mutant

PAGE

42

11

cell lines 32

Western blot analysis of CHA

transfectants 34

Kinetics of aggregation of

transfectants producing different

cn glycoforms

Speeificity of adhesion mediated by

different CHA glycoforms

Aggregation kineties for CHA

transfectants of different wild type

37

40

CHO cells 45

vii

r r 1

, i. î

FIGURE 7

viii

Comparative capacities of adh'9sion for

different CEA glycofonns. 49

a.a,

ASN, N

BGP

Cau

CEA

cDNA

CHO

DNase

ELISA

ER

FACScan

FBS

FITC

:t-CAM

:tg

:t.V.

kD

LecCAM

LFA-l

L-MAG

M

MAb

MEM

LIST OF ABBREVIATIONS

amine acid

asparagine

biliary glYCopI'otein

calcium

ix

carcinoembryonic antigen

complementary deoxyribonucleic acid

chinese hamster ovary

deoxyribonuclease

enzyme-linked immunosorbent assay

endoplasmic reticulum

fluorescence associated cell scan

fetal bovine se~

fluorescein isothyocyanate

intercellular cell adhesion molecule

immunoglobulin

int:ta venous

kilodalton

lectin cell adhesion molecule

lymphocyte function-associated

antigen 1

myelin associated glycoprotein

molar

monoclonal antibody

Bagle's minimum essential medium

NCA

N-CAM

NH2

PBS

PSG

sns PAGE

THR

X

x

Magnesium

non-specifie cross-reacting antigen

neural celI adhesion molecule

amino

phosphate-buffered saline

pregnancy-specifie glycoprotein

sodium dodecyl sulfate polyacrylamide

gel electrophoresis

threonine

any amino ae id

xi

ACKNOWLEDGBMENTS

1 would like to express my deepest gratitude to my

supervisor, Cliff Stanners, whom 1 greatly respect for his

love of research and hio shrewd scientific vision, for his

sup~ort and guidance through my gradua te studies. Thank you

for your patience and for giving me the freedom to develop my

skills and discover the true ups and doWDs of research, and

especially for showing faith in me, and making me realize that

l was the limiting factor of my achievements.

1 am sincerely grateful to my coworkers, who have made me

feel more a part of a family than a member of the laboratory.

A special thanks to Wendy Hauck, Hua Zhou and Mercedes Rojas,

my big sisters in the lab, for their friendship, honesty,

constant encouragement and help, and for many pleasant

analytical discussions, and to Chris Ilantzis, the man of the

lab, for his challenging research discussions, technical

advice, helpful literature references, and his immediate

attention in times of personal crisis. l thank Sarita

Benchimol, for her help, generosity, care and diplomacy

through the good and the bad times.

1 would also like to thank Nicole Beauchemin, Mireille

Cartier, Frank Bidelman, Karin sadoul, Prance Garnier, Claire

Turbide, ~lriel Chamoux, Philippe Gros and André Veillette

xii

for their support and resources, and the McGill Biochemistry

department for a good academic program and an outgoing staff

and student group.

1 cannot thank my family and Michael Adelman enough for

their constant encouragement throughout my studies. Their

presence and understanding has, and will always be, a major

driving force in my life.

In addition, 1 wish to thank Dr. Shore for the use of his

computer, Dr. Frojmovic of the Physiology department for the

use of the FACScan apparatus as well as Truman Wong and Aurora

Labitan for technical assistance. The open-door policy of the

Biochemistry department and the Cancer centre was very

beneficial to me. 1 feel that 1 have made many friends in the

different laboratories, and was able to develop new techniques

from the experts themselves.

This work was supported by an International Studentship

from the Paculty of Medicine of McGill university.

1

(

CHAPTBR I

INTRODUCTION

:2

1.1 Cell Adhesion Molecules

Cell adhesion molecules are cell surface receptors which

function to form and maintain tissue structure through either

of two different mechanisms, Ca++-dependent or Ca++­

independent. Thus far, four families of widely accepted

adhesion molecules have been described: the integrin family,

the immunoglobulin (Ig) supergene family, the cadherins and

the selectins.

Integrins consist of a family of highly versatile cell surface

adhesion receptors which can transmit signaIs into and out of

cells. Twenty integrins have been characterized thus far in

this rapidly growing field (see 1, for a review). Integr:f.ns

~re believed to be the major receptors by which cells attach

to extracellular matrices, and some particular integrine also

play a role in cell-cell adhesion. AlI integrins are composed

of an a and a 8 subunit which come together to form a non­

covalently associated heterodimer. The a subunits have

putative divalent cation binding motifs and require Ca·· or

Mg" for their function (2). The N-terminal domains of the Cl

and 8 subunits are believed to confer the ligand binding site.

Tbis binding site is connected by the two stalks to the

transmembrane domains, and tbus to the cytoplasmic tails

wbich interact with the cytoskeleton (3). Integrins can be

classified into three subfamilies according to tbeir shared 8

•

(

3

subuni t • These subf ami 1 ies are known as 81, 82, and 83

integrins. 81 integrin subfami1y includes receptors which bind

to extracellular matrix components such as fibronectin,

laminin and collagen and are expressed on leukocytes as weIl

as non-haemopoietic cells (4}. 82 integrins are expressed only

on white blood cells and are important for endothelium binding

of neutrophils and monocytes prior to extravasation through

blood vessel walls at sites of inflammation (5). 82 integrin

LFA-l (lymphocyte function-related antigen) which is expressed

on T-Iymphocytes can interact with antigen presentiûg cells

through direct interactions with Ig superfamily members I-CAM

1 or I-CAM 2 (intercellular adhesion molecule) and thus

mediates cell-cell adhesion (6).83 integrins are distributed

on endothelial cells and on platelets and are involved in

substrate binding auch as fibronectin, fibrinogen and

thrombospondin by cross-linking to Arg-Gly-Asp (RGD) sequences

on the substrate (7).

The immunoglobulin supergene family includes a wide range of

cell surface glycoproteins involved in cellular recognition

and adhesion (8). These molecules can interact homophilically

or heterophilically with other Ig family members, usually on

opposing cell surfaces. Ig molecules share primary sequence

homology as weIl as structural homology such as disulfide

bonds at conserved cysteine residues, Ig variable regions (V)

and Ig conserved regions (C). Ig molecules are either anchored

4

to the membrane through a transmembrane domain followed by a

cytoplasmic domain or via a phosphatidylinositol glycan. The

I~ family can be further subdivided into Cl-set and C2-set

molecules. The Cl-set subgroup includes Molecules involved in

cell-cell recognition proceBses of the immune system such as

TcR, CD4, CD8 and the C2-set subgroup includes moiecules

involved in Ca++-independent:. homophilic intercellular adhesion

such as N-CAM and MAG which are important for tissue

organization during development (9).

Cadherins are another family of cell-cell adhesion receptors

which can mediate selective homophilic intercellular adhesion

in a Ca++ and temperature dependent fashion. The cadherin

family is divided into Many subclasses that show different

tissue distribution patterns, e.g., E-cadherin (epithelial

cadherins or uvomorulin), p-cadherin (placental cadherins), N­

cadherin (neural cadherins) and L-CAM (chicken liver cell

adhesion molecule) (10). Populations of cells expressing

different subclassed of cadherins, when mixed together, sort

out into homotypic aggregates, which suggests that cadherins

are invoived in celi adhesion selectivity (11). The

intracellular portion of cadherins has been demonstra~ed to

interact with the cytoskeleton, and this interaction appears

to be essential for the cell-cell adhesion function of

cadherins (12). The activity of cadherins influence the

formation of junctional complexes snch as t ight, gap and

l 5

desmosomes (13) and may also be involved in establishing

cellular polarity (14). Furthermore, the regulation of

cadherin expression, i.e., the on and off switching of

expression, may be involved in the segregation of cell layers

during development (15).

Selectins or Lec-CAMs (lectin cell adhesion molecules) are a

family of three related adhesion molecules including Mel 14

(homing receptor, LAM-1) , endothelial leukocyte adhesion

molecule (ELAM-1) and platelet activation dependent granule-

external membrane protein (PADGBM, CD62, GMP-140), which are

involved in leukocyte binding to the endothelium at

inflammatory sites (16). AlI selectins have a specifie N-

terminal lectin-like domain with homology to the calcium-

dependent carbohydrate binding family (17), an epidermal

growth factor domain (egf), and a variable number of short

('-,nsensus repeats (csr) also found in members of the

complement regulatory protein family, followed by a membrane

anchor and a cytoplasmic domaine The lectin-like domain ie

believed to directly mediate a Ca++-dependent prote in­

carbohydrate interaction for specifie cell adhesion to

endothelium during various types of inflammation. More

specifically, selectins are thought to mediate the initial

unstable interaction of leukocytes to ftndothelium, preceding

the activation of 82 integrins which mediate the stronger

adhesions necessary for leukocyte arrest and extravasation

6

(18). Mel 14 is expressed on lymphocytes and la invo1ved in

lymphocyte··endothe1iwn binding (19). ELAM-1 is transient1y

expressed on activated endothe1ium and is be1ieved to mediate

the rapid influx of neutrophi1s at the site of inflammation

(20). PADGEM is expressed at the surface of activated

p1atelets and endothe1ia1 ce11s (21) as .) response to products

of the clotting cascade such as thrombin, and bind to

neutrophils and monocytes (22).

X.2 Clinical Significance of Carcinaa.bryonic ADtigen

Carcinoembryonic antigen, first described by Gold and Freedman

in 1965 (23), is a cell surface glycoprotein of 180 kD present

in elevated amounts on the surface of tumour cells as well as

in the blood of patients with colon, breast and 1ung cancer

(24). CHA was first be1ie7ed to be expressed during

embryogenesis and reappear specifically in colon cancers as an

oncofetal antigen and seemed an ideal candidate as a tumor

marker. It was soon realized however, that CAA overexpression

was not perfectly correlated with cancer. First of a11, with

the use of more sensitive immunoassays, it was demonstrated

that elevated CHA blood concentrations were detected in other

malignancies and rlon-ma1ignant diseases such af} a1coho1ic

cirrhosis, gastrointestinal inflammatory diseases and in

smokers (25, 26). Furthermore, only 60% of colonic tumors were

7

found to give elevated levels of blood CHA, depending on the

state of differention of the carcinoma (24). It is now known

that CBA iu normally expressed early during embry~genesis in

tissues derived from aIl three germ layers and persists mainly

in entodermal tissues in adult life, e.g., on the brush border

of colon:1.c epithelial cells (27). CD is nevertheless the most

widely used tumor marker, as a diagnostic tool and for the

clinical management of colon carcinoma. Thus CHA levels are

measured in the blood of cancer patients ta monitc,r their

progress following chemotherapy; arise :lll CHA blood

concentrotion above the normal 5 ng/ml concentration post-

operatively can represent an early indication of tumor

recurrence or metastasis formation.

1.3 Molecular Biology of CHA

The gene and cDNA corresponding to CHA have recent ly been

cloned and characterized by our laboratory (28) and by others

(29, 30, 31). Sequence analysis revealed a processed leader

sequence of 34 amine acids (a.a.), followed by an amino­

terminal domain of 108 a.a., three 178 8.a. internaI domains

with 68-72.5% amine acid sequence homology to one another,

each containing 4 cysteine residues bound by disulfide

bridges, followed by a short carboxy-terminal tail of 27 a.a.;

the latter domain is posttranslationally processed, to he

, 8

rep1aced by a phosphatidy1 inosito1 glycan 1inking CBA to the

cytoplasmic membrane. There are 28 potential asparagine-linked

glycosy1ation sites (ASN-x-Thr/Ser) in the protein, 2 in the

N-termina1 domain, 11 in the first internaI domain, 7 in the

second internaI domain and 8 in the third internaI domaine The

predicted molecular weight of the mature protein is 72.8 kD

and the predicted total glycosylation weight ia approximately

100 kD for a biantennary complex carbohydrate structure, which

is in reasonable agreement with the experimental molecu1ar

weight obtained on SDS polyacrylamide gel electrophoresis

(PAGE) of 180 kD.

1 .4 CHA Paaily

1.4.1 Molecular Biology

Nine CEA-1ike genes have been dlacovered, aIl of which are

clustered on human chromosome 19 (32, 33). Fig. 1 shows the

proteins which have so far been characterized. The CEA family

is divided into two subfamiIiea including the CHA subgroup,

containing CHA itself, Non-specific Cross-reacting Antigen

(ReA), different splice variants of Biliary Glycoprotein

(BGP), and CGM6; and the pregnancy specific glycoprotein

subgroup containing at least Il different gene products

(PSGs). These molecules all share a processed leader sequence,

-

9

an NHa-terminal domain of 108-110 amine acids, followed by 2

to 6 repeated Ig-like C2-set domains, consisting of either 92

or 96 a.a. (A domain) or 86 a.a. (B domain) and each

containing at least 2 cysteine residues forming a disulfide

bridge. Carboxy-terminal tails of CD, NCA and CGM6 are

processed to leave the molecules membrane-bound by a

phosphatidyl inositol anchor (34, 35, 36). AlI BGP splice

variants have transmembrane domains and either a short or long

cytoplasmic tail (37, 38). PSGs do not have a membrano anchor

and are secreted from the plasma membrane.

10

FIGURE 1

CD. faaily

CEA FAMILY

1 CEA Subgroup

L N Al BI A2 B2 A3 B3 M

CEA ~t e e e e c e e e e e e e • '.1 l')f: ("j'L 86 92 86 92 86 26

L N A B M NCA l@ e CGM6

e e e • 34 loe 92 86 24/29

L N Al BI A2 TM CYT

BGPa @ e e le ccl c c ~ 34 108 02 86 96 43 18 49

L N Al BI ,.\2 TM CYl

BGPc 34 108 92 86 96 43 5

L N Al BI TM CYr

BGPb IZ], " e e le cc ~

34 108 (~2 86 43 18 49

L N Al BI TM CYr

BGPd E?ttfi"il e c le cc = 34 108 92 86 43 5

PSG Subgroup

PSG lb L N Al A2 B2 c PSG la ~ • C c e c e c 1 PSG le PSG Id 34 108/110 92 92 86 2/4111/13

L N Al 62 e

PSG 2

34 110 92 86 13

1 L N A2 B2 e

PSG 5 c c c c 1 34 110 92 86 13

1 12

I.4.2 Normal and Tumor Localization of CHA Faaily Meabers

As weIl as having a normal site of expression in the body,

each CEA family member has been shown to be associated with

malignancy. CEA is normally expressed at low levels on

intestinal mucosa and reappears on colon, breast and lung

tumor cells (24). NCA is norme.lly expressed in lung (39),

spleen (40), colon mucosa, granulocytes and monocytes (41),

and has been detected in breast and colon cancer cells (42).

BGP can normally be detected in bile canuliculi (43) and

mucosa of gal1 bladder (44), and transcripts can be found in

colorectal cell lines (38). CGM6 has been detected in normal

peripheral leukocytes (36) and in blast cells in peripheral

blood of patients with leukocytes of chronic myelogenous

leukemia (45, 42). PSGs are usually expressed in placental

tissues (46), and have been used as markers for

choriocarcinoma (47).

l . 5 POflsible Punctions of CHA

sequence analysis of CEA has also revealed a degree of

homology with the immunoglobulin (Ig) supergene family. Ig

family members can be classified according to their content of

constant-like 1 (Cl) or constant-like 2 (C2) domains. Cl-set

like molecules are involved in recognition processes between

{

13

free molecules whereas C2-set like molecules are involved in

recognition processes occuring at the cellular membrane. The

C2-set subgroup includes neural cell adhesion molecule (R­

CAM), intercellular adhesion molecule (I-CAM), mye 1 in

associated glycoprotein (L-MAG) and CD2. (8). CHA family

members showed sequence homology to the Ig C2-set domains,

which suggested a possible role for CHA in intercellular

adhesion. Cell adhesion assays were designed in our laboratory

based on the work of Brackenbury et al. (48) and Orushihara et

al. (49), to study the function of CHA in vitro. To this

effect, LR-73, a line derived from Chinese hamster ovary (CHO)

cells, were transfected with the functional cDNA of CHA.

Positive transfectants in our adhesion assays demonstrated

Ca++-independent homotypic intercellular adhesion (50). These

results have since been confirmed by others (51). Adhesion

molecules are believed to be involved in cell-cell and cell­

substrate interactions during development, and therefore play

an important role in tissue organization during embryogenesis

and tissue regeneration (9, 15, 52). Hence, CHA expression

during embryogenesis could participate in the organization of

cells of the gastro-intestinal tract. Furthermore, CHA

reappearance in tumor cells could generate new associations

between cells and thus cause disruption of normal tissue

architecture (50). Evidence for homotypic adhosion of CHA in

vivo has been provided by Hostetter et al. (53). They reasoned

that CHA being cleared from the blood by the uptake of Itupffer

"

14

ce1ls in the liver, and thus present at the surfaces of

Kupffer cells and hepatocytes, could act as a homing receptor

for metastatic cells disseminating trom a primary CBA­

producing colonie tumor to the liver, via the portal

circulation. Their results showed that increased CBA blood

concentrations produced by 1:. V. injections of mice with

purified CHA correlated with an increased metastatic potential

of CEA producing tumor cells to the liver (53).

A second function for CHA was later proposed by Leusch et al..

(54). This group suggested a potential role for cn family

members in the recognition of bacteria and the regulation of

bacterial colonization of the human intestine. This hypothesis

stems from the observation that certain strains of bacteria

bind to immobilized purified preparations of CHA, NCA and BGP

on nitrocellulose. Furthermore, bacterial binding to CEA

family members appears to occur in a carbohydrate-specific

manner by interactions between oligosaccharides on CHA, NCA-55

and BGP-8S (specifie glycoforms of NCA and BGP) with lectins

on the bacteria. A comparative study between two NCA molecules

originating from the same cDNA but differing in their

carbohydrate structure showed different binding affinities to

bacteria (Leusch et al., personal communication). Thus NCA-SS

which contains 30-50% high-mannose type carbohydrate

structures readily binds bacteria, whereas TBX-7S which

contains only a few high-mannose type structures binds at a

1

-

15

much lower affinity. Binding of these molecules to bacteria

can be competed out using various a-glycosidases of D-mannose,

which suggests that the bacterial lectin recognizes the

mannose residues on carbohydrate structures of CHA family

members.

A third function for CHA has been proposed by Pignate1li et

al. (55). This group suggested that CBA plays an accessory

role in the in vitro binding of a human colon carcinoma cell

line (SW1222) to type 1 collagen matrix of collagen coated

culture dishes. This work has yet to be confirmed.

1 .6 Adhesion functioD of CBA aDd f_ily I1811bers

Subsequent to the f inding of the role of CD in homotypic

intercellular adhesion, similar adhesion experiments were

performed on other CHA family members. The cDRA of ReA was

transfected into LR-73 cells as previously described and was

also shown to act as a Ca++ - independent, homotypic

intercellular adhesion molecule. CBA and RCA transfectants

were tested for their specificity of adhesion by mixing

experiments. As a negative control, LR-73 parental cells were

mixed with either CHA or NCA transfectanta and were

demonstrated to segregate from homotypic aggregates of CD or

r r t' ~

1

1 f ~

16

NCA transtactants, indicating that the intercellular adhesion

is specifically due to CW',,-CEA or NCA-NCA interactions and not

to CU or NCA interactions with other cell surface molecules.

Heterotypic adhesion between CHA and ~CA transfectants was

demonstrated as well as cell sorting between either CEA or NCA

transfectants and transfectants of other adhesion molecules of

the Ig family such as N-CAM or L-MAG. thus showing adhesion

specificity between cn family members but not between Ig

supergene family members (56). CGN6 has been reported to be

incapable of homotypic adhesion and yet was capable of

performing heterotypic adhesion with NCA (57), suggesting

binding interactions between these two molecules. Chimeric

mo].ecules have been constru(!ted using N-CAM cDNA and CU cDNA,

and LR-73 transfectants of these chimeric molecules have Deen

tested in adhesion experiments to define the domains of cn

involved in binding. CD homotypic adhesion was deduced to

depend on two essential binding sites, involving the N-

terminal domain of one molecule binding to one of the three

homologous internal domains of another molecule (Zhou, H. et

al., in preparation). BGP adhesion, unlike CD and NCA, was

demonstrated to mediate CaH-dependent homotypic adhesion (58)

as seen for another non-J:g class of adhesion molecules. the

cadherins (15), and in preliminary heterotypic adhesion

assays, BGP transfectants segregated fl"Om either NCA or cn

transfectants (Rojas and Stanners, unpublished), thUB

suggesting different mechanisms of adhesion between certain

17

CHA family members.

1.7 Role of SUgara in CHA llediated Adhesion

As described above, cn is a highly glycosylated molecule with

60% of its molecular weight due to oligosaccharidee.

Glycosylation analysis has been achieved using purified CBA

preparations (rom liver metastases deriveil from primary tumors

of colon and breast origins. Glycosylation was deduced to be

mostly asparf.\gine linked with 80% tetra-antennary structures,

15 tc, 20% tri-antennary structures and 5 to 10% diantennary

structures (59). Microheterogeneity of CBA carbohydrate

composition has been observed from one tissue preparation to

another. The above study revealed the presence of only complex

type carbohydrates, while "lnother study demonstrated 10% high

mannose type structures combined with 90% complex type

structures with various levels of sialylation and varying

percent age of branched structur\!s ( 60) •

Sugars have been shown to be important for protein stability,

for protein conformation and as a 1180rting signal/l for

cellular targeting of proteins to cellular organelles (61).

Recent evidence has invo1ved sugars in cellular compaction (

(62) and direct binding of glycoprotein through fts

18

carbohydrate structure to cell surface receptors (63). In

addition, cell adhesion molecules known as LecCAMS were

recently shown to bind to carbohydrate ligands (64, 65) using

lectin-like N-terminal domains (4, 66). Other evidence of

carbohydrate participation in adhesion has been demonstrated

for N-CAM embryonic (E) and adult (A) forme In this system,

the B-form which is heavily polysialy1ated is 1ess adhesive

than the A-form \'Ihich contains two thirds less sia1ic acid

residues. These N-CAM differences in adhesion abilities have

been attributed to ste rie hindrance and charge repu1sion

through sialic acid residues (67). N-CAM carbohydrates were

a1so demonstrated to assist the homophilic binding of L1 cell

adhesion moleculetl on one cell surface, to other L1 molecules

on an opposing cell surface (68). I-CAM-1, another member of

th~ 19 family, has been demonstrated to perform heterotypic

adhesion with LFA-1 and Mac-1 integrins. Interestingly, the 1-

CAM-l/Mac-l interaction is modulated by the level of I-CAN-l

glycosylation (69). The unusually high level of glycosylation

on CHA glycoprotein compared to other 19 family members has

led us to investigate a role for carbohydrates in the function

of this molecule, namely, homotypic intercelluldr adhesion.

(

19

1.8 Carbohydratea in Cancer

Correlations have been demonstrated between altered

glycosylation structures on cell surface glycoproteins and

malignant transformation. The more common sugar alterations

include increased branching, sialylation and fucosylation of

N-Linked carbohydrate structures (61). High levels of

polyslalylatlon and high levels of CBA at the cell surface

have b~en demonstrated to enhance the metastatic potential of

colorectal carcinoma cells injected Into the spleen of rodents

(53). In assaying the role of carbohydrates in CBA-mediated

adhesion, we hope to better understand the role of CBA in

development and carcinogenesis. If sugars are demonstrated to

be important for specificity and strength of CBA-mediated

adhesion, then it would imply that the adhesion function of

CHA has another level of regulation, i.e. in its glycosylated

structure. :It is thus possible that the various CBA glycoforms

with different physical and biochemical properties may each

have a unique set of biological activities important for

tissue architecture during development, tumorigenesis and

metastases formation.

1 20

1.9 GlycoaylatioD of GlycoproteinB

ASN-linked Glycosylaticn of proteins occurs in the rough BR as

proteins are translated and secreted through the ER membrane.

On the luminal side of the ER, oligosaccharyltransferase

enzyme tranafers a nine unit carbohydrate structure endhlg in

dolichol phosphate to the ASN residue of the proteine Three

basic oligosaccharide structures have been characterized for

N-linked glycoproteins: high-mannose, hybrid, and complex

types (for a review see 70). In the hybrid and complex type,

there are alternate branching possibilities such as bi, tri

and tetra antennary as weIl as bisecting structures. To this

level of complexity is added the options of fucosylation,

polylactosamination and polysialylation, ail of which are

regulated by the various processing enzymes in the BR and the

different biochemical compartments of the Golgi. apparatus.

Different glycoforms of the same molecules expressed in

different tissues or at different developmental stages have

been reported (71). These slight variations of carbohydrate

structures on the same molecules but in different environments

could be an efficient way to modulate the function in a subtle

fashion to bring more functional divecsity.

CHO mutant cell lines have been generated which are deficient

for certain enzymes in this processing pathway (72). In

transfecting these cells with the functional cDNA of CHA, we

(

1(

21

were able to produce CHA molecules bearing specifically

truncated oligosaccharide structures. Osing these

transfectants, we assessed the role of carbohydrates in CHA­

mediated adhesion. (Jar results show that sugars have no

influence on adhesion specificity but do, however, ne~atively

modulate the speed and extent of CHA-mediated adhesion.

1.10 Methods for Measuring Cellular Adhe8ion

Different assays for measuring cellular adhesion have been

described over the year8. One 8uch assay con8ists of removing

cells from a monolayer culture with a mild treatment of

trypsin, and forcing the suspension through a syringe to break

up any remaining cellular aggregates in order to get a single

cell suspension. Single cell suspensions are then incubated at

37°C for a two hour period with stirring, and aliquots are

taken at various time points and assayed for the percent age of

single cells relative to the total number of cells. Rates and

extent of aggregation reflect the adhesion properties of the

cell line (50).

A variation of this type of experiment measures the adhesion

of a radioactively labelled cell sU8pension directly on a

confluent monolayer of cells (51). In this assay, the cell

mixture is incubated 15 to 80 min to allow the two cel1

22

populations to come into contact by gravity. The radioactivity

remaining after washing the cells is counted by liquid

scintillation methods to measure the degree of adhesion

between the two cell populations. Washing away the unbound

cells is a very critical step in these experiments as

undesired shear forces Can make the results poorly

reproducible and thq force of adhesion impossible to measure.

McClay et al. (73) have introduced a centrifugaI force-based

assay which Can quant if y the forces of adhesion between cells

and their substrates. In this assay, microtiter plates are

coated with substrates and a radioactively labelled cell

suspension is added to the chambers which is then sealed with

a second microtiter plate. The microtiter plates are

centrifuged briefly to bring the cells into contact with the

substrate and then immediately centrifuged in the opposite

direction to separate unbound cells at different centrifugaI

forces. Both substrate coated (bound) and opposing wells

(free) are clipped and counted by liquid scintillation

methods. The strength of adhesion between the cells and their

substrates is provided by the centrifugaI force required to

separate them.

Another group has modified the McClay assay to measure cell­

cell adhesion. This assay is basically the same except that

the microtiter plates are initially coated with a confluent

monolayer of cells (74).

1

CHAPTBR II

MATBRIALS AND METRODS

23

24

II.1 Cell CUlture and Tr~ectioD8

Wild type chinese hamster ovary (CHO) cell lines LR-73 (7S)

and Pro-S, and CHO glycosylation deficient mutants Lec1, Lec2

and Lec8 cells (72) were grown in monolayer in (X-MEN (76)

supplemented with 10% fetal bovine serum (FBS) at 37°C in a

humidified, S% carbon dioxide atmosphere. CHA transfectants

were obtained by the lipofection technique of BRL (Bethesda

Research Laboratories, Life Technologies, Inc. Gaithersburg),

except for the use of dioleoylphosphatidyl­

ethanolamine/dioleoyloXY-3(trimethylammonio)propane, chloride

salt liposomes (a generous gift from Dr. John Silvius, McGill

university), or by calcium phosphate mediated coprecipitation

(77). For lipofect ions , cells were incubated for 16 hours in

10 ml of (X-MEN + 10% FBS plus a li90some-DNA mixture composed

of 40 ~g liposomes, 10 ~g of the P9l023B expression vector (R.

Kaufman, Genetic« Institute, Boston, MA) containing CHA cDNA,

and 1 Jlg of the dominant selectable marker, pSV2 -AS (78).

Cells transfected by the calcium phosphate method were

obtained by coprecipitation of S Ilg of CEA cDNA, O. S Jlg of

pSV2-AS and 4.5 Jlg of CHO-N3 (79) carrier DNA per 3xlO li cells.

Transfected clones were selected with 2mM albizziin in

asparagine free medium as previously described (78), picked

and maintained in selective medium. Clones producing levels of

CHA exceeding 50 ng/mg celi protein, as assessed by a double

monoclonal clinica1 assay for CBA (Abbott, Mississauga), were

25

selected for further experimentation. The numbers after each

transfectant designate the clone number. Control transfectant

clones which did not produce CEA were obtained by tranafection

with the same expression vector containing en oDNA in the

antisense orientation. Antisense clones are referred to, in

the text and figures, as "anti".

%I.2 AggregatioD Assay

Cells were removed from plastic surfaces with 0.06% trypsin

(Difco: Bacto trypsin) in phosphate buffered saline (PBS)

containing 15 mM sodium citrate and resuspended in (X-MBM +

0.8% FBS + 10 ~g/ml DNase I. Aggregation assays were carried

out in suspension at 10' cells/ml in 17 x 100 mm polypropylene

round bot tom Falcon tubes (Becton, Dickinson labware) at 37°C

with stirring (80-100 rpm) using a 3 x 10 mm stir bar, as

previously described (50). In aIl separate experiments, the

seme initial cell count was used and all cell populations

showed a single fluorescent peak as determined by their

FACScan profiles. 10 ~l aliquots were gently removed from

stirring cell suspensions at varioulY time points using a

pipetman and a 200 ~l plastic pipet tip (Sarstedt). The

percentage of single cells relative to the total number of

cells was counted on a haemocytometer and was plotted as a

function of time. Sinee the probability of aggregation

r 1

t 1

• 26

decreases as a function of the concentration of single cells

in suspension, the initial rates of aggregation are rapid and

the curves level off to a certain percent age within the l hour

incubation period.

1:1.3 Cell Sorting Assay

Cell sorting assays in mixtures of two transfectant cell

populations were carried out as previously described (50).

Brief1y, 2x104 cells of one ce11 population were labelled with

50 ~1 of a 0.5 mg/ml stock solution of fluorescein-

isothiocyanate (Sigma, St.uouis) in 100 ~l Puck's saline + 2%

FBS. Cells were washed 01'1: excess FITe by centrifugat ion

through a column of FBS prior to mixing with 2xlO li unlabelled

cells of another cell population in a final volume of 3 ml (l­

MEIl + 0.8% FBS + 10 ~g/ml DNase I. Slidei! were made from

aliquots taken after a 75 min incubation at 37°e with stirring

and the aggregates were visualized under light and fluorescent

microscopy. FITe can 1eak out of cells after prolonged

incubation times, however, our experiments were done within

relatively short periods and as demonstrated by the negative

controis of Fig. 5 and Table 1, and the ability to visualize

mixed aggregates, FITC leakage was not a problem. FITC

analogues which get trapped within the cells are available and

shou1d be used in experiments with longer incubation periods.

(

27

II.4 We8tern Blot Analysis

Confluent cel18 were washed, removed with PBS containing 15 mM

sodium citrate, eentrifuged and resuspended in 0.5 ml PBS.

Celi suspensions were then sonicated at 4°C. The protein

content of the supernatant was measured using the Bio Rad

assay for protein determination (Bio-Rad laboratories,

Richmond, CA). 50 ~g of protein was boiled for 5 min in 0.4%

sodium dodecyl sulphate (SDS): 1% 28-mereaptoethanol sample

buffer, and electrophoresed on a 7.5% polyaerylamide gel

eontaining 0.1% SDS under standard conditions (80). The

separated proteins were transferred to a nitrocellulose

membrane and probed with a goat gamma-eut polyclonal anti-CBA

antibody (obtained from Dr. A. Puks, McGill University) and

subsequently probed with a rabbit anti-goat antibody

eonjugated to alkaline-phosphatase (Alkaline Phosphatase AP

F(ab')2 Pragment Rabbit Anti-Goat IgG (H&L), Jackson Immuno

Research Laboratories, Inc.). The CBA proteins were detected

by the reaetion of alkaline-phosphatase with its sub8trates,

5-bromo-4-ehloro-3-indolyl-phosphate and nitro blue

tetrazolium, in carbonate buffer (0.1 M NaHC03 - 1 mM "gCla• pH

9.6), (Promega, Madison) which produces a visible dark blue

band.

-

28

II.5 PACS ADalysis

Cell surface CD levels in transfectants were measured by flow

cell fluorimetry <:)f whole cells treated with fluorescent anti­

CD antibody. Cell monolayers were rendered single cell

suspensions by a 2 min incubation with 0.06% trypsin in PBS­

citrate, a treatment which did not affect the level or the

molecular weight of cell associated cn, as assessed by

Western blot analysis. 2.5xl05 cells were resuspended in 0.5

ml PBS + 2% FBS, labelled first with a mouse monoclonal anti­

CD antibody, B18 (81), at 40 ~g/ml and second with an FITC-

conjugated goat anti-mouse antibody for 30 min at 4°C.

Labelled cells were washed with 2 ml PBS + 2% FBS, resuspended

in 0.75 ml of the same solution, and their fluorescence

intensity measured using the FACScan fluorimeter (Becton

Dickinson, Canada, Inc. ). Comparison of CBA levels for

different cn glyeoforms determined with one antibody assumes

an equal degree of labelling per molecule of CBA, independent

of the level of glycosylation. Since the same quantitative

relationship between the mean fluorescent labelling for

different Lee mutant transfectant clones was obtained using

two antibodies which recognize different epitopes, this

assumption seems justified.

(

CHAPTBR III

RBSULTS

29

30

III.1 Isolation of Glycoaylation Defe,~tive CHA 'l'ransfectanta.

To examina the effects of the carbohydrate structure on the

intercellular adhesion function of CHA, CHO derived cell lines

(LR-73, Pro'S, Lecl, Lec2, Lec8), which yield different

glycosylation structures, were transfected with the functional

cDNA of CHA. The CHO Lec mutants have been shown to be

defective in various steps of their N-glycosylation processing

pathway (72). Fig. 2 displays the predicted final

glycosylation states of glycoproteins produced by the

different cell lines. LR-73 cells and the direct parent of the

lec mutants, pro'S, do not have defects in their N­

glycosylation pathways and thus are capable of producing

complete glycosylation structures terminating in sialic acid.

Lec2 cells, which are defective in the translocation of CMP­

sialic acid across the gOlgi membrane, are believed to

terminate glycosylation with galactose. Lec8 cells, which are

defective in the translocation of UDP-Galactose into the golgi

apparatus, result in sugar structures ending with N­

acetylglucosamine residues. Lecl cells are defective for the

enzyme N-acetylglucoaaminyltransferase-I and express

glycoproteins which bear 5 mannose residues as terminal

carbohydrate structures. Stable CHA cDNA transfectant clones

of these normal and glycosylation deficient cell lines

producing levels of CHA exceeding 50 ng per mg of cell prote in

were aelected for further study. In order to characterize the

.

31

FIGURB 2

Ibr:peCtad carbohydrate 8tructure8 of glycoprotelna

producad b.Y the .arlou8 .114 type aDd Lac .utant call

I1D88

<.'

1

, .-, "

f ~

r t

l .~

1 ~,

'.1'

SA SA

1 1 Gal Gal

1 GleNac GleNac

1 Man Man -- ......­Man

(GleNAc) 2

1 ASN

1 WddType 1

Gal Gal

1 1 GleNac GleNac

1 1 Man Man -- ....­Man

(GleNAc) 2

1 ASN

GleNac GleNac

Man Man -- ....­Man

(GlcNAc)2

1 ASN

32

Man Man -- ......-Man Man -....... ......­Man

(GJCNAc)~

1 ASN

33

PJ:GURB 3

Western blot aaalysis of CIA traDSfactaDts

CHA proteiD.8 were detected iD eell lysates from various

transfeetants by immunoblotting witb goat polyelonal

anti-CHA antibody. Tbe numbers after each transfeetant

designate the clone number. The molecular weights in

kilodaltoDs of marker proteiDS are given at the left.

l

_ nrn.~-~~~-:;-........ ~~.=-, .. "~ ... ,,...., .... ~.---., ,!"",.;r ... ~ ... "''"-'''- - .. ~~-- ,... .... ~ ... __ ~~_~ .. r ~ ... ,.- -,-- ..

, ?

en ..... CD ..... en ...... 0)

1 1 1

•

N

8

or ..

,--~ , ... "<

1

LR-73

LR(CEA)-6

Pro -5 (CEA)-7

Lec2 (CEA)-3

LeeS (CEA}-4

Lec1 (CEA)-15

lec1 (anti)

w ...

--~--

1 35

CHA glycoforms produced by these transfectants, Western b10t

ana1ysis of total cellular proteins using po1yc1ona1 anti-CBA

antibody was performed (Fig. 3). The different migration

patterns seen for CHA produced by the various cell 1ines are

consistent with the predicted glycosy1ation deficiencies;

i.e., CHA with the apparent mo1ecu1ar weight (180 kD) of the

fu11y glycosy1ated mo1ecule was produced by wi1d type LR-73

transfectant ce11s, and a more heterogeneous and slight1y

lower mo1ecular weight CHA glycoform by wi1d type Pro-5

transfectant ce11s, while CBA with successive1y lower

mo1ecular weights down to 135 kD was produced by the Lec

mutants giving successive1y more truncated sugar structures.

III.2 H~ty'pic AggregatioD of Transfectants

To study the involvement of carbohydrate structures in the

interce11u1ar adhesion mediated by CHA, we performed homotypic

adhesion assays using the various transfectants. The resu1ts

are shown in Fig. 4. In each experiment, the kinetics of

aggregation of the transfected mutant were compared with its

antisense transfectant and to the fu11y glycosy1ated LR-73

(CHA) transfectant as negative and positive contro1s,

respectively. In every case the aggregation of the antisense

transfectant was minimal.

36

FIGURE 4

Kinetics of aggregatioD of transfectants producing different CHA glycoforms

Homotypic aggregation assays of mutant cells t.ransfected with the functional cDNA of CEA in the sense and antisense orientation. In all separate experiments, the kinetics of aggregation of a low (0) and a high (A) CEA producing transfected mutant clone are eompared to the antisense transfectant (D) and wild type LR (CEA) transfectant (+) as negative and positive controls respeetively. A, homotypic aggregation of suspensions of Lec2 (CEA)-7 (0) and Lec2 (CEA)-3 (A) low and high producers, respeetively. B, homotypic aggregation of suspensions of LeeS (CEA) -3 (0) and LeeS (CEA) -4 (~ low and high produeing transfectants, respectively. C, homotypic aggregation of cell suspensions of Lee! (CEA) -9 (0) and Lee! (CEA) -40 (A) low and medium produeing transfectants, respectively. Three sets of experiments showed similar kineties of a~gregation. These data represent results from a typieal experiment.

t

" i

-

37

1

100

80

60

40

.!!!

1f 20

j!

~ 1/)

ë 0 15 30 4S 60

~ ~ lime (min)

20

o~--~----~--~----~~ o 30 eo 10 120

Time(min)

(

38

Aggregation studies of the Lec mutant CHA transfectants

demonstrated homotypic adhesion of low and high producing

clones, with more rapid aggregation and lower final levels of

single cells for the high producers (Fig 4 A, B, and C).

The above aggregation studies thus show that CEA transfectants

with an altered glycosylation structure are capable of

homotypic adhesion and that this adhesion is direetly related

to the expression level of CEA by the transfeetant clones;

this suggests that carbohydrate residues are not directly

involved in intercellular adhesion mediated by CEA.

111.3 B.Pecificity of CHA Adhesion

To examine the question of specificity of adhesion mediated by

the different eBA glycoforms 1 we assessed the ability of mixed

populations of transfectants displaying CHA moleeules with

different earbohydrate structures to sort themsel ves into

homotypic aggregates. Specifie cell sorting has been observed

for transfectants bearing different adhesion molecules (56),

and would be expected if sugar structures were capable of

conferring adhesive specificity. Fig. 5 shows the results of

an aggregation experiment in which equal concentrations of LR

(CBA)-6 cells labelled with FITC were mixed with unlabelled

Leel (CHA) -40 cells in suspension. After a suitable incubation

39

PIGtJRB 5

s.pecificity of adbeaioD .adiated bY different CHA

glycofoD18

lqual amounts of cells of two different populations, one

labelled with PITe (*) and the other not labelled, were

mixed and incubated at 37°C for a 75 min periode Aliquots

were Acored for the percent age of labelled cells per

aggregate and plotted as a function of the total amount

of aggregates visualized. A, control experiment between

non-transfected wild type cella, LR-73, and Lecl (CHA) -40

shows cell sorting. B, heterotypic aggregation of PITe

labelled LR (CD) -6 cells with Lecl (CHA) -40 transfectant

cells is observed. Insert shows light and fluorescent

micrographs of the aggregates.

40

60~----------------------------------------,

70 "LR·73 vs Lee1 (CEA)-40

60

50

40

30

20

10

40 "LR (CEA)-6 vs Lee1 (CEA)-40 B

30

10

o 10 20 30 40 50 60 70 80 90 100

Percent labelled cells per aggregate

-

-

41

period, a1iquots were visua1ized under 1ight and fluorescent

microscopy and the number of aggregates with a given

percent age of 1abe11ed ce11s per aggregate p10tted as a

histogram. The resu1ts show that 90% of the aggregates were

heterogeneous in nature (Fig. 5 B). Control mixing experiments

between Lecl (CHA) -40 and the parental ce11s demonstrated that

the parental LR-73 ce11s remained as single ce11s (Fig. 5 A).

Thus the observed adhesion in Fig. 5 B was presumab1y due to

CD-CHA interaction and not to CD binding to some other

mo1ecu1e on the parental ce11s. Table l summarizes the resu1ts

of mixing experiments with a11 the transfectants. Results with

LS-180, a human colon cancer ce11 1ine which produces high

endogenous 1eve1s of CD are inc1uded and, from the

heterotypic aggregates observed, demonstrate that CD

mo1ecu1es with a human carbohydrate structure can mediate

adhesion with CHA mo1ecu1es with a hamster structure. A11 CBA

producing ce11s tested in these experiments, regard1ess of

their considerable differences in glycosy1ation, showed an

abi1ity to mediate heterotypic interce11u1ar adhesion. We

conclude that carbohydrates do not influence the specificity

of CD-mediated interce11u1ar adhesion. This specificity

therefore probab1y lies within the protein backbone itse1f.

, ,

42

Table 1. Specificityof Adhesion of Various CEA Glycoforms

Expressed on the Cell Surface of CHO Cells

Cell populations Aggregates visualized

FITC labelled Unlabelled Homotypic Heterotypic Homotypic unlabelled mixed labelled

% % %

LR-73 Lec1 (CEA) 82 17

LR (CEA) Lec1 (CEA) 3 90 7

LR (CEA) Lec2 (CEA) 7 90 3

Lec8 (CEA) LA (CEA) 14 86 0

Lec1 (CEA) Lec2 (CEA) 7 92

LA (CEA) LS-180 4 94 2

Summary of heterotypic experiments between two

populations of cells, one labelled with FITe and the

other unlabelled. Large percentages of mixed

aggregates indicate heterotypic adhesion whereas low

percentages suggest cell sorting.

( 43

111.4 Capacityof Adhesion.

previous experience with transfectants producing variable cell

surface amounts of various Adhesion molecules, including eEA

family members (CD and NCA), N-CAM and B-cadherin has shown

that the kinetics and final levels of aggregation provide a

semi-quantitative measure of the strength of intercel1ular

adhesion (56). Thus en transfectants producing increasing

levels of cn at the cell surface showed progresf ively faster

kinetics of aggregation and higher percentages of aggregated

cells (50, 56) • 'l'he kinetics of aggregation of CBA

transfectants of the two CHO derived wild type cell lines were

compared in Fig. 6, taking into account the cell surface CBA

production of each transfectant as measured by FACS. Under the

conditions of this experiment, the untransfected LR-73

parenta1 cell line was shown to aggregate to the same low

extent as a pra·s CD transfectant clone with a fluorescent

mean of 590, whereas the Pro-S parental cell line showed

virtually no aggregation. An LR-73 CD transfectant with a low

cell surface mean fluorescence of 128 also demonstrated a

greater level of aggregation than a second pro·S CBA

transfectant with a much higher cell surface mean fluorescence

of 798. These results clearly demonstrate that the cellular

background can influence intercellular adhesion; the extent of

Adhesion in cn transfectants of LR-73 cells is presumably

FIGURE 6

AggregatioD ld.neticl! for CM trlUl8fectant. of different

.i14 type CHO cella

Comparison of the kinetics of aggregation of non­

transfected clnd CHA transfected wild type CHO cell line.

LR-73 and Pro·S. At the right. the PACS distributions.

relating the number of cells with a given fluorescence

and the mean fluorescence, are shawn. The dashed-line

distribution8 represent the anti8ense mutant clone

labelled in the 8eme way as the CHA tran8fected mutant

clone, and used a8 a negative control for fluorescence

measurement8. These data repre8ent 8ingle point8 from a

typical experiment.

45

1

1°Oi==~~&-~==~=====;======l Pro"S (MIl)

10 Ut-73

LR-73 Pnl"'5 (CEA)-20

!! B ..... tcf.N40 QI Ct · c: '. 581 iii , . ë Pro"'5 (CEA)-22 ' . , . ~

, · 1 • · QI ..... tcIA)oa Q.

LR (CEA)-7

· '. 1118 , . , . , . , • 1 • · 20 UttcEAt-7 . '. 128 '. " •• , . , . • • / 1 • \

0 0 ., 10 120 1r1 101 ~ttf

TimI(,,*,)

46

facilitated by the presence of other endogeneous molecules at

the cell surface. For quantitative compariBon of adhesive

strengths produced by the various CEA glycoforms it is thus

apparent that the Pro·5 wild type CHO cell line, the direct

parent of the Lee mutants, must be used.

To define a potential role for carbohydra":es in the ability to

recruit the maximal percentage of cells into aggregates, the

kinetics and final levels of aggregation were measured for

transfectants producing similar cell surface levels of various

cn glycoforms as determined by FACS analysis. The Lecl (CHA)-

6 transfectant, with the most severely truncated sugar

structures terminating in 5 mannose residues and a lower mean

fluorescence of 350, showed more aggregation than either Lec2

(cn) -5 lacking only sialic acid residues, Lec8 (CU)-4

lacking both galactose and sialic acid residues or a

transfectant of its direct parental cell line, Pro-5 (CEA) -22,

wit.h a complete carbohydrate structure, aIl at mean

fluorescence values of 800 and above (Fig. 7). Lec2, Lec8 and

pro·S transfectants, aIl at comparable levels of CHA

production show decreasing degrees of adhesion, in that order.

The rates of aggregation in Fig. 6 and 7 for the various

" . .. transfectants can be misleading in their similar appearances f:-T , f

when comparing profiles of single point experiments. However, t t k

when the profilt!s of the mean of three sets of experiments for

~ /~ . the various transfectants are compared, the initial rates of ~

l l ~

47

aggregation tend to be more pronounced for clones with greater

extent of aggregation. This order in capacity of adhesion:

Lec1 > Lec2 > LecS > pro·5 was repeatedly observed with

transfectants of mean fluorescence approximating 500 and 300

in value (data not shown). The Lecl (CD) carbohydrate

structure thus appears to favor CBA-mediated adhesion most,

perhaps due to the greatest exposure of binding sites on the

proteine Lec2 (CBA)-5 showed a better aggregation than LecS

(CD) -4 which suggests that adhesion is not necessarily

related to the number of carbohydrate residues removed, but

more likely related to the overall conformation of the protein

modulated by the carbohydrate structure. These results suggest

that carbohydrates negatively modulate the capacity of CBA

mediated adhesion.

48

PIGUR. 7

Ca.parativa capacities of adhesioD for different CBA

glycoforas

The kinetics of aggregation of transfectants bearing CHA

with different glycosylation structures are shown for

wi1d type Pro"S, Lec2 and Lec8 transfectants producing

simi1ar leve1s of CHA, and Lec1 transfectant with a lower

CHA 1evel of production. These data represent single data

points from a typical experiment. PACS profiles for each

transfectant cel1 population (solid line) are shown

together with profiles for populations of their

respective parental cell 1ine (dashed llnes).

49

(

PrG"51ŒA) -22

l' 1 I, 118

80 I, • 1 l ,

/ ' LeclIŒA)-4

Jll

1i 60 PIQ-S (CEA)-22 .' l ,

l ,

~ g' iii

• , Lec8 (CEA)-4

• \ 1 ,

E

~ Q.

40 Lec2 (CEA)·S

" " " " 1 1

• , 1 ,

LeelIŒAH

20 Lee1 (CEA)~

3110

0 104 0 30 60 90

Tune (min)

(

50

.......

f

(

CHAPTBR IV

DISCUSSION

51

52

Carbohydrate structures on prote in molecules have enormous

potential for variability and therefore for the storage of

information (see introduction). Such information could be used

for molecular recognition as required, for example, in

specifie interactions between extracellular adhesion

molecules. The extremely high level of glycosylation of CHA

and the biological variability observed in the structure of

ite sugar chains (59) lend credence to the view that these

structures could play a significant role in the intercellular

adhesion function of CEA. The specificity of adhesion observed

between cells bearing CBA, 1. e., the failure of these cells to

bind to the parental cells or cells presenting other adhesion

molecules of the immunoglobulin supergene family (56), makes

it unlikely that en adhesion is simply due to the non­

specifie stickiness of its carbohydrate chains. In addition,

highly glycosylated cn constructs lacking relatively short

regions of domains essential for adhesion do not mediate

intercellular adhesion (Bidelman and Stanners, unpublished

results) .

The results obtained in this work show that functional CHA

cDNA transfectants of mutant cells with different defects in

their ability to glycosylate proteins are still capable of

specifie aggregation. Even the most severely truncated

structures terminating in mannose residues do not affect the

ability of the CHA moleeules to mediate intereellular adhesion

53

and do not affect the specificity of adhesion. There is,

however, the question of effects of these structures on the

strength of adhesion, a parameter which can be important in

the function of adhesion molecules during development (82) and

which, under extreme conditions, can even produce cell sorting

of strongly adhering cells in the presence of weakly adhering

cells (83). OUr ::-esults suggest that the CHA carbohydrates may

be more important in modulating adhesion than in determining

its essential features. Thus the faster kinetics and increased

extent of aggregation of CHA transfecants of Lecl mutant cells

may in fact be due to a carbohydrate structure which changes

the secondary structure of the CHA molecule, a conformational

change which enhances the access to binding sites on the

prote in backbone. A reduction in charge, due to the absence of

sialic acid residues on CBA glycoforms produced by certain of

the Lec mutant cells, could remove a repulsive component in

intercellular interactions. This charge effect has been

suggested to account for the reduced adhesion observed for

polysialylated N-CAM relative to the adult less sialylated

glycoform (67). Since our desialylated mutant transfectants

were more adhesive than fully sialylated wild type

transfectants, our findings support the charge hypothesis; the

large differences in the capacity of adhesion observed for

transfectants bearing different desialylated sugar structures,

however, indicates that the role of carbohydrate structures in

CHA at least is more complex.

1, f, 1

54

The very different tinetics of aggregation of CHO wild type

Pro-S and LR-73 transfectants has demonstrated that the cel1

surface background can influence the analysis of cn function

in relation to its adhhesive capacity, Although both of these

wild type cell lines are deri ved from CHO cells, the LR-73

line shows a more normal flattened morphology in monolayer

culture (75). Presumably, the presence of higher levels of

other adhesion molecules in LR-73 relative to Pro-5 cells has

synergistic effects with the intercellular adhesion mediated

by CHA. Alternatively the Pro-S cells could express anti-

adhesive molecules on their surfaces. These considerations

should be taken into account in the Interpretation of results

in which the adhesive properties of transfectants of a CHO Lec

mutant are compared with those of a CHO wild type control

different from Pro-S, the direct parent of the mutants, aB in

the recent report for Po transfectantB of CHO cell lines (84) •

Although Pro-S cells would have been a better wild type

control than LR-73 cells in determining the role of

carbohydrates in the specif icity of CBA-mediated adhesion

(Fig. 5 and Table 1), our results suggest that LR-73 and LR

(CBA) transfectants were Buitable cell lines to assess this

question for the following reasons. Firstly, although the

above results are consistent with the suggestion that LR-73

cells were observed to express background endogeneous adhesion

molf/cules, when mixed with CHA transfectants, LR-73 cell.s

:cemained essent ially aingle cella while Lee (CHA)

( 55

transfectants for.med homotypic aggregates. Furthermore, in LR

(CD) transfectants, these putative background endogeneous

molecules did not Interfere with CBA-mediated Adhesion between

LR (CBA) and Lee (CHA) transfectants, as demonstrated by their

ability to form mixed aggregates.

In general, our results support the findings of others for

1ess extensively glycosylated adhesion molecules of the

immunoglobulin supergene family; Le. that the information for

the specificity of Adhesion does not reside in the

carbohydrate chains on glycoprotein adhes ion molecules but

that these structures can negatively modulate the rate and

extent of cell aggregation (67, 70).

An altered carbohydrate structure on glycoproteins is a weIl

characterized phenomenon in cancer cells. The most common

changes include increased fucosylation, increased sialic acid

content and increased branching (for a review see 61).

Preparations of CBA from serum of normal patients and patients

with benign disease showed carbohydrate structures of the

complex biantennary and hybrid type without internaI fucose,

whereas CD preparations from the serum of patients with

colorectal cancer showed more heterogeneity of glycoforDls with

increased branching and increased fucosylation (85). CD

preparations from liver metastases derived from primary

colonie carcinomas showed carbohydrate structures of mostly

56

complex-type with bi, tri, and tetra-antennary structures and

pOlys ialylat ion (59). Thus CHA carbohydrate structures

isolated from the serum of cancer patients and from liver

metastases of colon primary tumor are consistent with the

pattern observed in other malignancies. Recent studies

revealed that an increased blood concentration of CBA favors

metastasis formation to the liver possibly by homophilic

intercellular interactions between Kupffer cells of t,he Iiver,

which have bound circulating CBA, and CBA expressing tumor

cells (53). Furthermore, an increased sialic acid content in

the CBA on tumor cells increased the colonization process of

metastasis in the liver, presumably through an

oligosaccharide-lectin-type of recognition event (86).

OUr results have shown that sugars affect the capacity of cell

recruitment into aggregates in transfectants in vitro. During

embryogenesis and carcinogenesis, when CBA may play a role as

an intercellular Adhesion molecule, different glycosylated

structures could modulate these functional roles.

Glycoproteins have been shawn to have different glycoforms,

with distinct biochemical and biophysical properties leading

to functional diversi~y (71). Par example N-CAM sialic acid

content regulates its affinity ir! homophilic intercellular

Adhesion during development (67). Insulin shows reduced

binding affinity for Lecl cell- expressing insulin receptor

when compared to that of wild type cells, which suggests that

glycosylation is involved in the

hormone/receptor binding affinity (87).

57

regulation of

CHA, with its 28

potential glycosylation sites, has a tremendous potential for

glycoform variability, i.e., glycosylation at Any of the 28

sites coupled with heterogeneity of the carbohydrate

structures at each glycosylation site. Thus, the glycoform

diversities of cn found in metastasis preparations and in the

serum of cancer patients may effect subtle variations in the

conformation of the molecule leading to functional diversities

which are not present in normal tissues. It is thus possible

that the overexpression of CD, coupled to an abnormal

carbohydrate processing mechanism in the cell, could result in

the disruption of normal cellular interactions.

Our results have also shown that CHA retains its specificity

of Adhesion, regardless of the struC"ture of its carbohydrates,

but its Adhesive capacity is affected. In CHA expressing

tumors, where CHA is heterogeneous in its glycoforms,

intercellular Adhesion could differ depending on the CHA

glycoform expressed by the cells. Thus, subpopulations of

cells within the tumor may interact with more or less

strength, creating regions of stronger adhering cells and

weaker adhering cells. These subpopulations may break away

from the primary tumor and form metastases. As metastases were

found to express CHA with highly branched, fucosylated and

polysialylated carbohydrate structures which, by our results

, "

58

are predicted to form less Adhesive intercellular

interactions~ we would suggest that subpopulations of weakly

adhering cells dissociate from the primary tumor and form

metastases. Since less glycosylated carbohydrate structures

were shown to be more adhesive, it is possible that tightly

adhering cells of primary tumors could express more high

mannose-type CBA, whereas less adhering cells could express

more highly branched structures. Opdyke and Nicholson (88), on

the other hand, have predicted that metastasis is favored by

increased homotypic intercellular Adhesion. It is possible

that carbohydrate structures of fer a subtle variation in the

strength of Adhesion so that aggregates of cells bearing

complex structu~es in the primary tumor are weak enough in

their intercellular adhesion to break away from it, and yet

strong enough to remain in an aggregated form for a better

chance of survival in the circulation and for lodging and

growth in other organs. A comparison of glycoforms expressed

by cells of metastases and the primary tumor they arose from

may shed some light on this hypothesis. As our results suggest

that CHA Adhesion is dependent on its carbohydrate structure,

its level of expression and its cellular background, the role

of CBA glycoforma in carcinogenesis and metaatasea fo~tion

may be complex.

The role of carbohydrates in the intercellular Adhesion of

other CBA family members remains to be investigated. NCA

l 59

shares many common features with cn including sequence

homology, sites of asparagine-linked glycosylation and percent

carbohydrate content, and would be expected to behave like CBA

in these experiments. BGP, however, was demonstrated to

mediate intercellular Adhesion by a different mechanism than

that of CD and RCA, and might respond differently in

glycosylation experiments. BGP Adhesion, unlike that of. CBA

and NCA is calcium and temperature dependent and has a reduced

speed and extent of Adhesion (58). lt would be interesting to

study these particular features of BGP Adhesion i. e., Ca++ and

temperature dependence, with respect to the carbohydrate

structures on BGP using the Lec mutant celi lines.

Lecl cells which express glycoproteins with high-mannose type

carbohydrate structures could represent a good experimental

system for the bacterial binding experiments described by

Leusch et al. (54, and l.5). CHA, BGP, and RCA transfectants

of Lecl cells may be used to produce high-mannose type

molecules, or used directly in experiments to investigate the

importance of mannose residues on CD family members in the

interaction with lectins of type l-fimbriated bacteria.

Oitawa et al. (in press) have reported that the R-terminal

portion of CD is essential for homotypic intercellular

Adhesion. In these experiments 2/3 of the N-terminal domain of

CHA, including two R-linked glycosylation sites, were deleted.

, r

1 60

Transfectants of LR-73 cells with this CBA mutant (AN) were

demonstrated to lose their adhesive function (Kidelman et al. 1

in preparation). It would be interesting to evaluate the

importance of these glycosy1ation sites on the interce11u1ar

adhesion function of CBA by "knocking them out" using site-

directed mutagenesis.

OUr Lec (CBA) ce11 1ines cou1d a1so be used to verify our

hypothesis that aggregates of cells m,pressing CBA molecules

with complex type carbohydrates [Pro"S (CBA)] wou1d be favored

to metastasize from an aggregate composed of a mixture of

cells expressing CBA glycoforms of high-mannose type [Lecl

(CBA)] and complex type carbohydrates. To this effect, mixed

aggregates of CBA transfectants of Lec mutants and wi1d type

cells could be implanted into nude mice, as out1ined by

Hostetter et al. (53). If tumors develop and give rise to

metastases, then the cellular composition of the primary tumor

and derived metastases could be assayed with regards to the

CBA glycoforms expressed.

The work presented in this thesis has given us more insight

into the mechanisms of CBA mediated interce1lu1ar adhesion.

The carbohydrate strucure of CBA, which we predicted would be

important for its function, has been demonstrated to

negatively modulate the capacity to recruit cells into

aggregates but not the specificity of homotypic intercellular

( 61

interactions mediated by CBA. Although the measure of capacity

to recruit cells into aggregatos clearly suggests that

carbohydrates can modulate CEA-mediated adhesion function, a

more in depth characterization of the strength of adhesion

would be greatly beneficial to our understanding of the

mechanisms involved in cell-cell interactions during

development and carcinogenesis. McClay type assays described

in the Introduction cou),\! be usod to measure strength of

adhesions between CBA family membera with and without

molecular modifications such as glycosylation variations. To

reduce the effects of variable cell surface context, which can

Interfere with Interpretation of data, purified CHA molecules

could be coated onto plastic microtiter plates thus serving as

a direct a~esive subatrate for the various CHA transfectants

in these assaya. Alternatively, CHA transfectants could be

replaced by CBA-coated latex beads (89) and thus eliminate

non-specifie adhesion mediated by unknown adhesion molecules.

pinally, the Lee cell linea appear to represent a good system

for future functional experiments involving the members of the

CBA family.

CHAPTBR V

RBFBRBNCBS

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