) 3. to'

ftsnnt{Y'?3

IN CANCER AND IMMUNE SUPPRESSION.

. DEVELOPMENTAL ANTIGENS

by

B.sc. (HONS)

Ross Samuel Savvas '

.¡lhesissubmittedtotheUniversityofAdelaideinfulfilmentofthe requi"*tt" for the *"t of Doctor of Philosophy'

FebruarY 1977

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TABLE OF CONTENTS

DEDICATION ..

TABLE OF CONTENTS

SUMMARY

DECLARATION

ACKNOl^lLEDGEÞlENTS

PUBLICATIONS

ABBREVIATIONS

PREFACE

CHAPTER I

SECTION I

SECTION II

Page

1

iivl'l

ix

X

xi

xlt

xtlì

I

i

t.'

1

1

2

LITERATURE REVIEt,l

General CharaÇteri sti cs of Cancer

1. The di sease

2. Tumour de'¡eìoPment

3. Genome alterations and phenotypic

expressi on

4. SummarY

Acqu'i si ti on of Foetal Propert'ies

1. Introduction

2. Cell surface characteristics and

di vi si on

3. DNA production

4. 'Foetal ' RNA

5. Enzyme Patterns

6. Devel oPmenta'l anti gens

7. SurunarY

3

5

6

6

I

I

cel J

7

7

B

8

9

9

t

l'tl

Onco-deveìoprnentaì Antigens - Foetal .:

1. Introduction

2. Foetal antigens - classificat'ion and

nomencl ature

3. Spec'ies speci f i ci tY

4. cr1-foetoprotein (AFP) ..

(a) definition

(b) carcinoma sPecificitY

(c) in the non-malignant state ..

(d) summary and conclusions

5. Carcino-embrYonic antigen

(a) definitjon

(b) human carcinoma

(c) and the non-maìignant state

(d) summary .u

Onco-deve'lopmentaì Antigens - Placental

1. Introduction

2. Carcìno-placental molecules

(a) Regan isozYme

(b) human p'lacental ìactogen

(c) human chori oni c gonadotroph'in ' '

(d) pyruvate kinase

(e) histaminase

3. Concl usions

Page

12

t2

l

)

,1

II

SECTION III

SECTION IV

13

16

18

18

1B

18

2L

22

22

22

22

24

26

27

27

27

29

29

29

29

r

tv.

SECTI0N V : Concl usi ons

SECTION VI : Introduction to Proposed Study

Page .

33

36

36

36

36

36

37

37

37

37

37

38

39

39

39

39

30

CHAPTER I I

SECTION I

SECTION II

CHAPTER IIISECTION I

MATERIALS AND METHODS

Materi al s

1. Animals .

(a) rats

(b) mi ce

(c) rabbits :.

2. Reagents ..

Methods

1. Preparation of antigen extracts

(a ) sa] i ne sol ubl e extract

(b) cel'l surface bound anti gens

(c) whol e p'lacental cel I s

2. Antisera

3. Immunol og'i caì tests

(a) immunodiffusion

(b) immunoel ectroPhoresis

MURINE FOETAL ANTIGENS

Rationa'le of tlt,-' Proposed Study 41

V

SECTION ÏI The System

1. The anti gen

2. Tumours .

SECTI0N III : Characterization of Anti-foetal Serum

SECTION IV Concl usions

1. The antibody

2. Time course studY ..

Page

42

42

42

43

44

44

48

CHAPTER IV

SECTION I

ONCO.PLACENTAL ANTI GENS

Introducti on

Rationale for the Appearance of Placental

Antigens

Saline-soluble Cytoplasmic Placental

Antigens .. ..

1. Immunodiffusion studies

2. Immunoelectrophoretic studies

Membrane-bound Placental Antigens

1. Introduction

2. Intact Placental cells

3. Placental pìasma membrane fraction

50

50

SECTION II

SECTION III

SECTION ÏV

53

53

54

59

59

59

60

64SECTION V : SummarY

vt.

CHAPTER V :

SECTION I

FOETAL ANTIGENS AND II4MUNE REPRESSION

I ntroducti on

Pagg

66

SECTION II

SECTION III

SECTION IV

Suppression of Antibody Synthesis to

Foetal Extracts

1. Rabbit antibody

2. Murine antibody

3. Conclusions

Immunogenicity of Murine Foetal Extracts

1. Cel I organe'l 1e anti gens

2. Antigen modifier

3. Conclusions

Inununol ogi caì SuPPressi on

I . Rati ona'l e .

2. Methodology

3. Results

67

67

69

73

75

75

76

7B

BO

BO

BO

B1

SECTION V : Discussion

CHAPTER VI : CONCLUDING REMARKS

ADDENDA

APPENDI CES

85

92

97

98

105REFERENCES

v]1.

SUMMARY

The malignant transformation, and the relevance of developmental

ant.igens to the cancer process, is broadly reviewed. The two

developmental antigens - foetal and placental - are then examìned

in experimental mouse and rat tumour systems'

An attempt to prepare a specific arltìserum to foetal antigens

is described. Thìs type of antiserum, reported by Stoneh-ill and

Bendi ch ( 1970) as bei ng ab'le to detect muri ne onco-foetal ant'igens '

could not be reproduced in these studies. Further examinatìon

revealed that the antìserum used by Stonehill was not detecting an

onco-foetal anti gen, but rather a ci rcul at'ing normal adul t ant'igen '

A number of attempts were al so made to demonstrate the

appearance of p'lacental antìgens in rat tumours. Although a model

vras devel oped whereby at I east 6 sal 'ine-sol ubl e carci no-p'lacental

antìgens cou'ld be detected, this system was found unsu'itable for

general laboratorY use.

The reappearance of membrane-bound placental antìgens 'in a

chemically induced hepatoma was also examjned' Ìlowever, due to the

inabiìity to produce a spec'ific anti-pìacental serum, these experiments

were unsuccessful.

During the course of these studies, it was found that rabbits

and mice were unable to raise antibod'ies to heterologous foetal

extracts unless the immunogen was freeze-dried prior to inoculation'

This unusual situation was unexpected, as lyophìlizaLion should not

si gni f .icant'ly al ter the immunogenì cì ty of the anti gens. By

viii.

experimental'ly eìiminating ail other likely possibiìities' it was

proposed that the non-freeze dried foetal extract contained a

repressor which spec,ifìcally inhjbited the production of antibodies

to foeta'l antigens. Further, it was a]so postulated that the

repressor may be a lyophilization-labile antigen-antibody complex.

The relevance of these repressor studies to foetal and t:lmour

immunology is discussed.

IX.

DECLARATION

I declare that this thesis does not contain any material which has

been submitted previously fon any degree or diploma to any university;

and to the best of my knowledge it does not contain any material

previously published or written by another person, except where due

reference is made in the text.

ss Savvas

AC KNOl^lLE DG EMENTS

I am sincereìy indebted to my supervisor, Dr. J'R. Sabine, for his

gu'idance and encouragement through this study. I would also like to

thank Professor l,l.V. Macfarlane for many hours o'f stiniulat'ing

djscussion. I am grateful to Mr. J. Zupp and the Animal House staff

for thei r di I 'igent mai ntenance of experimentaì an'imal s , and to

I'lisses D. Gibson and J. Scott for techn'ical ass'istance. Many ideas

are generated by djscussion with other laboratory members, and for

their thoughts I would like to thank Dr. R. Bais and the other

graduate students with whom I have worked.

The photograph'ic expert'ise of Mr. B.A. Pal k, the xeroxi ng work of

Mrs. B. Steedman, and the typ'ing of M'iss Pat Watson is also

appreciated. Finally, I w'ish to thank my w'ife, whose perseverance

and solid encouragement have assisted me during this research

pr0granrne .

xi.

PUBLI CATI ONS

Aspects of this study have been presented at scientific meet'ings 'in

Australia. The abstracts appear in the folìow'ing journals.

Sabine, J.R., Savvas, R.S., Murray, 4.l,l., Verma, A.K. and

Ruoslahti, E. (1975). Time course of cellular changes

during spontaneous hepatic-carcinogenesis in mice.

CLin. Eæp. Pharrn. Phas. 2 : 85.

Savvas, R.S., Bajs, R.

pì acentaì antigens' .

and Sabine,

CLin. Eæp.

J.R. (1e76).

Phøîtn. Phys.

'Carci no-

3 i 2Bl.

xt1.

AFP

BSA

CEA

DNA

FCA

FD

FIA

HCG

IEP

MeDaB

NFD

RNA

ABBREVIATIONS

q-foeto prote'in

bovi ne serum al bumi n

carci no-embryonic antigen

deoxyri bonucl ei c ac'id

Freund's complete adjuvant

freeze dried

Freund's incomplete adiuvant

human chorionic AonadotroPhin

i nmunoel ectrophores'i s

3-methyl-4-d'imethyl amino azo benzene

non freeze drì ed

ribo nucleic acid

xt11.

In this thesis, I have attempted to broadly revìew the current

concepts of cancer research, With particular enrphasis on tumour

immunology. More specifically, I have concentrated on two emerging

fields - carcinofoetal and carcinoplacental antigens.

Much of the current research is masked in a confusion of

termino'logy. Further, there has been only a small attempt to answer

the question, 'why do foetal and placental ant'igens re-appear in

tumours?' - perhaps a fundamental quest'ion ìn the understand'ing of

the cancer process. Part of the prob'lem ljes in the fact that the

foetal antigens have not been characterized in terms of a specific

non-immunol ogi ca'l functi on . The exper"imental work presented i n thi s

thesis is to some degree also gui'lty of this. However, my approach

from the start has been three-fold:

1. Demonstrate the presence of these onco-developmental antigens

in exPerimental tumours.

2. Determine at which point of t'ime folìowing carcinogen

appl i cati on theY aPPear.

3. Attempt to assign a non-immunological, biologìcaì funct'ion

to these ant'igens.

Thus, wh'ile still keeping the third area of study in mind, it was

first important to demonstrate the presence of these antigens in the

tumours, and then to answer the questìon, 'What are they doing there?''

Xi V.

In the final chapters of this thesis, I describe a new

phenomenon - that of antibody synthesis repressÍon by foetal

antigens. I say new, not because the idea is new, but its concept

and relationship to spec'ific biological problems is now presented

in the form of models as a basis for solving these problems.

l

I

I

1

G

SECTION I : GENERAL CHARACTERISTICS OF CANCER

1. The Di sease

Canceris a disease of multi-cellular organìsms. It is

characterized by uncontrolled cellular d'ivision, resulting in the

production of tumours. lrlany such cancerous grovtths are abìe to

spread to other tissues, often resulting'in death of the host due to

direct impairment of essential funct'ion, and/or wasting.

Although studies of cancer have been a'lmost exclusiveìy in

mammalian systems, the disease has also been shown to occur in

vertebrates (Halver, 1965), insects (Harshbarger and Taylor, 1968),

and plants (El Khalifa and Lippincott, 1968). In this revjew, only

mammalian cancer is considered, in order to integrate the many and

diverse studies of oncology into a broad Summary of cancer. These

studies are presented at three organ'izational levels:

(a) tumour development

(b) genetic changes which injtiate tumours and those which

result from tumour develoPment

(c) phenotypic products of these gene changes.

This thesis is principally concerned with a particular phenotypic

chafacteristic of the tumour - the reappearance' in carcinomas, of

properties normally associated with embryonic development.

il

z2

.\Ftr Et4

WAITE IN

2

2. Tumour Development - the clonal evolution of tumour cells

Before discussing any phenotypic changes which occur durìng

the malignant transformation, it is necessary to describe the

mechanism of tumour development from an initial cancerous cell.

Such a d'iscussion is warranted because many tumours share a number

of phenotypic products, and yet still maintain their indivìduaf ity.

It is the path of tumour development which may determ'ine the

characteristics of a tumour.

Nowell (1976) has formulated a model which describes the clonal

evol utì on of cancerous cel I s , and the'ir subsequent devel opment 'into

a tumour. In this model, tumour initiat'ion occurs by an induced

change in a single, previously normal , cel l. 'Neoplastic proìiferatjon

then proceeds either immediately or after a latent period. This small

number of cel 1s may escape immunol ogi ca'l surve'i I I ance due to

insufficient stimulation of the immune system (Bonmasser et aL.,1976), and

hence the number of cancer cells grows. As the mass of cells increases

some may ultimately be destroyed by the immune system. Others, however,

undergo mutation due to genet'ic instability in the tumour cell

popuìation. Although many of these mutant cells are destroyed, either

immunolog'ically or by metabolic disadvantages, one mutant may arise

which has a selective advantage and hence survjves. This cell then

becomes the precursor of a new preciom'inant sub-popu'lat'ion of cancer

cells. This sequent'ia1 selection process continues over a period of

time, with the cells becoming increas'ingly abnormal both genetìcaì'ly

and metabo'lically. However, it is important to note that thìs sequence

3

of events is not comp'leteìy random. Consequently, d'ifferent tumours

may acquire certain similarities. The divergence or tumour-specific

characteristics develop due to the effect of local cond'itions

influencing the emergence of variant sub-lines. Ultimately, the

ful ly devel oped maì i gnancy as i t appears cl i ni ca'l 'ly , has a geneti c

make-up uniquely different from the origìnal non-neopìast'ic celj.

Associated with this is an aberrant metabo'lic behaviour and "new"

ant'igeni c properti es .

3. Genomi c Al terations Durinq Transformat'ion

In the model presented above, a change in the normal cell was

required in order to initiate the neoplastic process. Except in the

case of viral transrnission, the initiator of the transformatìon has

yet to be isolated. However, it is general'ly considered that tumour

initiation occurs due to some degree of escape from nonnal controls

(e.g. local ,chalones,, hormonal, intracellular). The cause of

this "escape" may be due to an ajteration in the intracellular DNA,

e.g. a number of workers have shown that ionizing radiation, carcino'

genic chemicals, and oncogenic viruses can react with the host cell

genome in a variety of ways, in order to bring about the required

al terati ons i n gene funct'ion (El ki nd and bJhi tmore , L967; Tem'in , 1971 ;

Mi I I er and M'il I er, 1974). Phenotypi c stud'ies of f ul ly devel oped tumours

have resulted in the followìng c'ìassificatìon of genome changes

(reviewed by Anderson and Coggin, 1971):

(a) new DNA is added

(b) some DNA ìs lost

4

(c) nucleotide sequences are misread or not read

(d) DNA which is norrnal]y not read, is now coding for mRNA.

Although any one of these changes may have the potential to initiate

carcinogenesis, they may however be a product of the rselective

evolution', i.e. not necessariìy a cause of the loss of control,

but rather a secondary effect caused by the clonal evolution of the

initial cancer cells.

(a) New DNA (req:gerg:)

Due to a ,'mutation" (induced by carcinogen, vìrus, radiatjon

or by vertica'l transmission (Huebner and Todaro, 1967) ), new Dl'lA

nucleotide sequences appear and an extragenomic molecule appears.

Thus the observed phenotypìc effect is an a'ltered structuraì protein

or a neo-ant'igen.

(b) Loss of DNA

Aga.in due to a mutation, certain nucleotide sequences are

lost, resuìting 'in proteìn deletions. Consequentìy enzymes, antigens

and other molecules characteristic of the normal cell are lost.

(c) Ui:lqe9lrg-gl-rvgleellde-:esse!ee:

A'l though not speci f i cal'ly 'invol vì ng a nucl eoti de change , thi s

genetic abberation results in certain sequences being misread or not

read at all. Such a defect may be caused by producer gene expression

, be'ing al tered.

(d ) Bg:srP19::19!-gI-crgbeg:991e:

Genes which are expressed only during the embryonic

5

development of the cell, are once again 're-activatedr. These genes

begin coding for phenotypic products generally on'ly found associated

with normal embryon'ic development.

These genome changes are reflected in the phenotyp'ic alterations

known to occur in expe¡imental and human cancers. The phenotypic

profile of tumour cells reveals that there'is a loSS of certaln

"normal" cell structures and functions, and a concommitant acquisition

of 'new' cancer-character j sti c pr'operti es . Thi s i s consì stent w'ith

the genomi c prof i I e which essentì a1 ly 'invol ves a I oss or mi sread'ing

of DNA, and a gain or react'ivation of existing DNA. This loss and

acquisition of properties has been demonstrated in studies on tumour

antigens (Ingleton, t97I; Kuusel a et aL., 1965; Coll'ins and Black'

1973; Kyriazjs and Wi ssl er, 1972; Bal dwi n and Gl aves , 1972) ,

metabolism and enzymology (reviewed by Weinhouse, 1973) and cell

receptors (Sharon and Lis, 1972; Fox et aL.' I97l; Garrjdo et aL.,

re74).

4. Summary

The ultimate phenotyp'ic expression of a tumour may be influenced

by the genet'ic "evolution" of cancerous cel1s during tumour development.

Nowell's model pred'icts that tumours possess a number of tumour-spec'if ic

characteristics wh'ich have arisen due to these evolutionary processes.

The production of tumour specific ant'igens (Kyriazis and Wissler' I97L

Baldwin and Glaves, Ig72) is an examp'le of such specificity. However,

6

NowelI's model also env'isages simiIarities between a number of tumours

clue to the non-randomness of the evolution" Such sirnilarit'ies include

many common enzymes (l,.¡einhouse , Ig73) , metabol'ism features such as h'igh

g'lycolytic rates (Warburg, 1923, 1956), and the acquìsition of

embryonic-associated properties (i.e. foetal and p'lacental ). It ìs

this acquisition of embryonic-associated properties, demonstrated in

many human and experìmenta'l tumours, which may be an ìmportant event

in the sequentia'l evolut'ion, or even the initiation, of tumour

development. Alternat'ively, the acquisition of these properties may

be a consequence of the carcjnogenic state ('i.e" a secondary effect).

These two alternatiVes are exam'ined below, by consìdering foetal

properties in cancer, the non-ntalignant and healthy states.

SECTION II : ACQUISITION OF EMBRYONIC-ASSOCIATED PROPERTiES

1. Introduction

The relationsh'ip between embryonic development and neopìasia

has been a central theme of cancer research s'ince 1901' when Ehrlich

first formulated the concept of "nrisplaced cel'l rests" (see Alexander,

lg72). Embryonic and cancerous cells share many properties' These

include a number of b'ioìogical processes' ranging from DNA synthes'is

enzymes, general synthes'is of proteins, ant'igens, and of cell division.

diû+

I

t

7

2. Cell Surface Characteristics and Cell Division

The fundamental similarìty between the cancerous and

embryonic cell is their abiìity'to div'ide rapid'ly. D'ifferentiated

adult cells do not genera'liy divide, and are djffìcult to culture

in uitz,o. Ëmbryon'i c and cancerous cel I s , however, ffiâY be cul tured

with relative ease (Elkind and hlhitmore, L967).

The abiìity of cells to stop d'ivjding, when a certain cell mass

has been reached, is thought to be dependent on cell-cell surface

membrane interactions (Burger, 1973). The ab'ility of cells to bind

the plant lect'in, concanavalin A, is dependent on membrane receptors.

Becker (1974) has found that mal'ignant and foetal hepatocytes were

able to bind thjs lectin, whereas non-dividing ljver cells did not.

This differential lectin binding suggests that there may be changes

in cell surface receptors of tumour cells which are common to

embryoni c cel'l recePtors.

3. DNA Production

Stafford and Jones (tglZ) and Piddington et aL. (tglï) have

shown that thym'idine k'inase' an enzyme involved in DNA synthesis'in

the normal cell, differs to the kinase found in the cancer cell'

They have reported the presence of a foetal-specific isozyme of

thymidine kinase in human He La and KB tumour cells. This partìcular

isozyme, which catalyses the production of the DNA precursor, thymjdine

tri phosphate, i s the predom'i nant vari ant i n the devel op'ing foetal cel I ,

and is generally in very low quantit'ies in the normal cell.

I

#I

I

i

!

B

,T

ÊJ

'04

4. RNA

Aìthough there has not been any ev'idence that the enzymes

controlling mRNA synthesis in cancer cells differ to those of the

normal cell,'it has been shown that tumour cells produce a different

type of tRNA.

Gonano et aL. (I97I) have studied the tRNA species in 5123c

Morris hepatomas, and have found that these substances have different

chromatographìc characteristjcs than those of the adult liver.

Further, they have also shown that the chromatograph'ic pattern of

the tumour pheny'lalanine accept'ing tRNA (tRURphe) is the same as

the tRNAPhe isolated from rat embryonic livers (Gonano et aL,, 1973).

Similarly, Yang (1971) has reported the presence of foetal tyrosine

acceptìng IRNA in Reuber hepatoma. Again, this form of tRNAtyt was

not detectable 'in the adult liver.

5. Enz.yme Patterns

Foetal metabolism is poor'ly understood. However, it is clear

that foetal tissues contain a large number of enzymes which, although

performing the same function, vary slightly in amino acid composition

and tissue specjficity to the homologous adult enzyme. Many of these

foetal isozymes have also been found in the tumour cell. Together

with this reappearance of foetal enzyme forms, there is a simultaneous

loss or decrease ìn the activity of the adult isozyme (Knox, 1972).

The summary of a few of these foetal enzymes (Table 1.1) suggests

that there is a broad enzyme pattern established during carcinogenes'is,

i.e. there is a shift from adult to foetal isozyne synthes'is. It is

I

tII

I

t

9

I

not clear what special advantage these 'isozyres may provide to

the rnalignant cell. Maugh (7974) has suggested that they may be

better suìted to the more rap'id sustained growth of tumour and

foetal tjssues. However,'it may be equa'l1y possible that no

sel ect.i ve advantage i s ga ì ned , and the'i r appea rance , as w'i th other"

foetal properti es , i s a s'impl e consequence of the carci nogen'i c

state rather than a speci f i c necessi ty. ' ''' '

6. Developmental Antiqens

The antigenic structure of cancer cells also exhibits a

reappearance of characterjstics noimally associated wìth embryonic

development. l,lany workers have reported the appearance of antigens

which are found'in foetal cells, but not in the correspond'ing normal

adult tissues (see Sections 2 and 3). However, it is'important to

note that the presence of these antigens'in tumours ìs onìy one

exampìe of the reappearance of developmenta'l ant'igens. The other

types of developmental antigens are those whìch are found in the

placenta and tumour, but not in foetal and normal adult cells

(revìewecl by Bagshawe , 1974) -

7. Summary

From the data presented above, it can be seen that the

acquisition of embryonic properties is associated with, perhaps a

necessary association With, the malìgnant transformation' The

mechanism and reason for the re-expression of the controììing genes'

however, 'is not we'11 understood. Potter (1969, 1970) has proposed

I

10.

TABLE 1.1

SPECi FICITY

TUMOUR

REFERENCEENZYME

FOETAL ADULT

Alanine aminotransferase

Aspartate aminotransferase

Hexoki nase

Pyruvate Ki nase

DNA polymerase

Gl utami nase

Aryl esterase

M

M

H

M

T1

G

S & 1'1 Nakata et aL. (1964)

s&14 Nakata et; o.L. (1964 )

Shied et eL. (1965)

G Sato et aL. (1969)Sug'imura et aL. (1969

a,b)

Tanaka et aL. (1967)

}ve et aL. (1969)Ono & Umehara (1968)

Knox et aL. (1969)Horowitz & Knox (1968)

Mu

D

Mu

D

L

N

*

K

A

K

A

L

FaillUri e

y -CrepinI (1e73)

&

Enzymes which exhibit foetal ìsozymes during carcinogenesis'

M = membrane bound

$ = soluble, cYtoPlasmic

G = TYPe IV isozYme

tl = TYPe I, II or III isozYme

Mu = muscl e ti ssue sPec'i f i c

| = liver tissue sPecific

K = kidneY tissue sPecìfic

A = ôrYlesterase

* absent from healthy adu'lt tissues

11.

that fol 'lowì ng carc'inogen appl ì cati on , a i'ìorma'l popul ati on of cel I s

is damaged. Many of these damaged cells are replaced by "new",

rapi dìy-di vi d'ing ce'l 'l s . The cancer cel l s ari se from the di vi di ng

cel I s whi ch fai I to respond to normal d'if f erent'iati on control

mechanisms, possibìy due to some block in one or more ontogenic

pathways. Thus these cells remain essentially in the embryonic

Stage of the dìfferentiation program. Consequent'ly' cancer cells

would be expected to exhjbit a range of foetal properties, inc'luding

biochemjcal functions, antigens' and cell division propert'ies' The

information descrìbed above confjrms this expectation.

Potter's hypothesìs'is also consistent vlith the tumour, clonal

evolut'ion concepts of Nowell (1976). In this theor¡ the initial

cancer cel'l vrould exhjb'it embryonic properties, and these would

subsequentìy be passed on to future generations. During further

evolution, some of these archeo-genes would be lost due to the

"mutations" wh'ich occur. Those which rema'in act'iVe may produce

mol ecul es whi ch gi ve the tumour cel I some sel ect'ive advantage (Matlgh ,

t974; l,leinhouse, 1973). The tumour which ultimately develops, would

hence exhibit a number of embryon'ic properties, some unique to a group

of tumours and others shared by aì I tumours '

The reappearance of developmenta'l antigens in tumours is an

examp'le of such embryonic properties exhibited by a wìde range of

species. These ant'igens have been studied extensively by many

workers. It will be seen in the following sect'ion that the

reappearance of these antigens occurs 'in many tumours' This onco-

foetal associat'ion may be an important factor in understandìng the

carcinogenic Process.

t2.

SECTI()N III : ONCO-DTVELOPMENTAL ANTIGENS - FOETAL ORIGIN

1. Int roduct'ion - Tumour Cell Antiqens

Three main grori¡t:; of onco-associated ant'igens have been found

i n the ful 1.y devel oped bumour:

(i ) tumour specific or transp'lantation-associated antigens

(rnn)

(iì ) foetal antigens

( i i i ) pl acental ant'i gens .

The tumour-specific antigens differ from the other two types because

each antigen Ís specific to the particular tumour from wh'ich it has

been isolated. This type of antigen has been demonstrated jn a

variety of tumours, and is often found to be intimately assoc'iated

with the cell membrane (W'issler, 1956; Baldwi n et aL., 1971; Baldwjn

et aL., !972; Prehn, 1967).

Foetal and placental antigens may be found in cancerous and

embryonic tissue (foetus or placenta), bul: not 'in apprec'iab]e

quantitìes in normal adult cells.

In this section, the association between foetal antigens and

cancer cel I s 'is exp'lorecl by exami nì ng the nomencl ature and cl assi -

fication of these antigens, and the types of tumours which exhib'it

them

13.

2. Foetal Antiqens - Cl assi fication and Nomenclature

The first significant attempt to classify foetal ant'igens

was by Alexander (1972). His system, (Tabl e I.2),was based on three

.immunol og.ical propert'ies of the onco-foetal mol ecul e:

(i) immunogenicity in homologous and heteroìogous hosts

(i j ) cytotox.i c.i ty i nductì on i n tumour-beari ng hosts

(i'i i ) cel I ul ar I ocal i zati on '

It can be seen from Table I.2 that these properties are studied by

employing two basic techn'iques. Specifically, these are:

(a) Using heterologous antiserum to detect foetal antigens

(i ) in tumour-bearer' s serum (e.g. a-foetoprotei n (Abel ev et aL' ,

1963); carcino-embryonic antigen (Gold and Freedman, 1965) )

and tumour extracts (Stoneh'ill and Bendich, 1970). In

Alexander's system these are termed onco-foetal associated

ant'igens ;

(ii) on the cell surface membrane of tumour cells (Ishimoto ¿¿

aL., 1974; Cauchi et aL', 7974)'

(b) Examjning animals immunized w'ith either foetal extracts or cell

suspensions for their abì1ìty to resìst challenge against a

tumour forming dose of cancer cells or virus' e'g' such onco-

foetal associated transpìantation antigens have been demonstrated

by Cogg'i n et aL. (1970)'

Immunogeni ci tY

Heterologous Homologous

TABLE 1.2

CytotoxicitY Suggested NameExampìe and

ReferenceMolecules in foetusand tumour

and some normal adultti ssues

absent from al I norma'lt'issues but i nducetolerance because theYoccur in late foetallifeon'ly occur in ear'lYfoetal life, or in a

cryptic form, andhence are recognizedas foreign(a) cytoplasmic

+ 0nco-foetal associatedanti gens (OFA-assoc. )

0nco-foetal sPecificheteroanti gen(OFA-speci fi c )

co-foetal specificto antìgen (OFA-auto

p

)

e antigen

CEA (Gold andFreedman, 1965) - -^- 1\

AFP (Abelev,et F+ t teb s)

Stonehi I I andBendich (1970)

Ishimoto et aL.(re74)

Cauchi et aL. (1974)+

++ 0nau

Chemi cal l) hepatoma

-i nducedBal dwi n72)

v(

(b) plasma membrane-bound + Onco-foetal trans-p'l antati on-tY(OFA-transp'l .

++

et aL., 19

Hamster, viraltumours (Cogginet aL., 1970)

The classification of onco-foetal antigens as proposed by Alexander (lgVZ)

15.

Although Alexander's nomenclature'is specific about the

jmmunological nature of the foetal molecule, 'it does not assign

a spec'if ic functjon or role to the ant'igen under study' Further

characterization of the antigen is desirable because

(a) much of the confus'ing termÍnology could be eliminated

if each foetal antigen could be referred to by its

functional name;

(b) a knowledge of its function may provìde a clearer

understanding of the biochem'ical and physìolog'ical

processes which occur in the tumour'

spasmod'ic attempts have been made to achieve th'iS, €.9. Raftell

et aL. (1974) have detected and assigned a funct'ion to onco-

foetal ant'igens in rat tumours. Ruoslaht'i et aL. (1'976) have

demonstrated that the hepatoma-teratoma associated foetal ant'igen'

a1-foetoprotein, shares amino acid sequence homology w'ith albumin'

Thus jt may be poss'ible that alpha-foetoprotein'is a "foetal"

albumin. However, desp'ite these attempts, and a current trend

towards elucidating the functional role of foetal antigens, there

stilt remain a number of misnomers in foetal antigen terminology'

e.g. carcino-embryonìc antigen (Go]d and Freedman, 1965), is a

general term which is used to describe a specific colon carcinoma-

associated antigen.

16.

3. The Rearrpearance of Foetal Antiqens in Tumours

The reappearance of foetal antìgens in tunlours is not

restricted to one partìcular animal specìes. From Table 1.3,

it can be seen that a wide varjety of tumour types in d'ifferent

species produce these ant'igens.

It is ìmportant to ¡ote bhat one certain tumour may produce

two or three d'ifferent foetal antigens, e.9. chem'ica'l1y-jnduced

hepatomas produce cr-foetoprotein, membrane-bound and cytoplasm'ic

foetal antigens (Cauchi et aL., 7974). Converse'ly, a variety

of different tumours may produce the same foetal antìgens

(Stoneh'i11 et aL.,1971), while others have been shown to produce

only one (Banwo et aL., !974; Mukherii ancl Hìrshaut, 1973).

Thus it can be seen that the reappearance of foetal ant'igens

is a characterjstic of a large number of tumours. Perhaps the

most Studied onco-foetal ant'igens are cx1-foetoprotein and carcino-

embryonic antìgen. These antigens have been examined princ'ipally

as a di agnost j c and prognost'i c parameterin the cl i ni cal management

of cancer (rev'iewed by Laurence and Nev'ille, L972; l'laugh , 1974).

These turo a.ntigens are brief'ly djscussed in the next section in

order to clemonst.rate that the reappearance of foetal antigens 'in

the diseased state is not necessarily specific for cancer. This

observati on i s of parti.ul ul_.'importance as i ts non-speci f i ci ty

for cancer suggests that their presence may S'imply be a function

of the rapìdly-d.ividing state rather than the maf ignant

transformati on.

18.

4. a,-Foetoprotein (AFP)

(a) Defi ni ti on

AFP has been defined as the first cr-gìobulin to

appear in mammal'ian sera du¡ing development, and the dominant

serum prote'in during embryonic life (Annuaì Report of the

International Agency for Research into Cancer, 1969). The

foetal liver and yo]k sac are the primary sites of synthesis

durìng embryogenesìs. After birth, smal'l quantities of AFP are

detected only by radio-imnìunoassay, and ìts synthesis is

restrj cted to parenchyma'l I i ver cel I s (Abel ev , 1971 ) .

( u ) ôil-cl9 -scrsilere -:Peeili g1!.v

AFP is also synthesìzed by primary and metastat'ic

hepatomas (Abel ev et aL., 1963; Abelev, 797I). From Table 1.4,

it can be seen that AFP has been detected in the sera of a variety

of hepatoma-bea¡ing hosts. However, it has also been found in a

few sera of other non-hepatic carcinoma hosts (Table 1.4). From

this table it is concluded that, other than hepatonlas, only

teratomas synthesize AFP to any sign'ificant degree.

(c) App and the non-malignant state

(i) normaL sev'um uaLues

AFP has been detected in the serum of apparently

heal thy humans (Ruos'lahti and seppäl ä , I971.), mi ce (pi hko and

Ruoslahtì, 1973), and rats (Sell and Gord, 1973). The level of

AFP in these cases can only be detected by the sensit'ive radio-

immunoassay technique. However, the tumour-associated levelS may

19.

TABLE 1.4

SPEC i ES TUMOUR TYPES AFPINC I DENCE

REFERENCE

Human

Rats

Mi ce

Dogs

Monkeys

10 hepatoma

10 teratoma

other non-hepatì c

L0 hepatoma

asci tes hepatoma

transpl antable sol idnon-hepati c

1.o hepatoma

transplantablehepatoma

ascites hepatoma

non-lrepa ti c

10 hepatoma

1o hepatoma

Laurence & Neville (1972)

Laurence & Neville (1972)

Laurence & Nev'ille (I972)

Kroes et aL. (1972)

llatabe et aL. (1972)

Sell and Morris (tgz+)

Abelev et aL. (1963)

Koda et aL. (1973)

Shìnomiya et aL. (1973)

Hull et aL. (1969)

68%

45%

0.03%

n.a

n.a

a

a

a

e

a

a

a

e

n

n

n

n

n

n

n

n

Spec.ies in which the presence of AFP-bearing carcinomas have been

demonstrated.

(It is important to note that the references cited, and the specific

tumours studied, are only a representative sample of a large number

of publications in which AFP-product'ion has been demonstrated' using

a number of carcinogens to induce these tumours'

n . a. = not aPP'l i cabl e

n.e. = not examined

2t.

be measured by relatively insensitìve methods such as counter-

immunoel ectrophores i s , and immunodi ffusi on (Abel ev , 1971 ) .

(ii) rhe regenerating and eæperimentaLLy iniut'ed Liuer

Conflict'ing data has been collated concerning

AFP synthesis in the regenerating l'iver. Perova (tgAg) and

Stanislawski-Birrencwaig (1967) failecl to demonstrate the

appearance of AFP in partìally hepatectomized animals. Holever,

further investigation by Perova et aL. (tgZt) using 'immuno-

autoradiography, showed that AFP could be secreted by partially

hepatectomized rats. These results have since been confirmed

in mice by Pihko and Ruoslahti (1973).

Ruosl ahti et aL. (1973 ) have found a sharp increase in

serum AFP levels in mice with carbon tetrachloride-induced liver

injury. They have concluded that even in the notr-malignant liver,

regeneration depends on a relatively primitìve (or embryonic)

celì population which produces or passes through an AFP-producìng

stage during the differentiation phase of regeneratìon.

(i i i ) Non-maLignant diseases

Elevated serum levels of AFP have been shown to

be associated with non-cancerous hepatic diseases. Abelev (tgZi)

and Silver et aL. (1973) have shown such increases in serum hepatitis

occurring durìng the recovery phase of the disease (Ruos'lahti ei

aL., 1973). Elevated levels have also been associated with

alçoholic hepat'it'is (Meshkiqeour et aL., L974), cirrhosis and

2I.

trauma (Ruosì ahti et aL., I973; N'ishi and Hirai, 1973)'

Ruoslahtt et aL, (I974a) have shown that these AFP levels are

generally'lower than those associated with primary ìiver

carcinoma. These auLhors have further suggested that 'in some

cases of hepatic coma, caused by acute hepat'it'is, the h'igh

level nay be due to the regeneration of damaged l'iver t'issue.

These results further support Ruoslahti's concept that

regeneratìng 'liver celìs pass through an embryonic AFP-produc'ing

stage, i.e. in non-ma'lìgnant diseases, AFP productìon ìs a

f uncti on of the d j v'idi ng state.

(¿) lyrycly

There are two inrportant conclus'ions that may be drawn

from the AFP stud'ies Presented:

(i) AFP secretion is not spec'ific for hepatomas;

(ii) AFP synthesis 'is also associated with other non-

cancerous diseases.

These conclusions suggest that AFP synthesis may be a function of

the rap.idly-dìviding state (i.e. ejther due to maf ignancy or

regenerat'ion), rather than a spec'if ic property of the malignant

state.

Th.is observation has a particular signjficance in determin'ing

whether foetal antigens are a necessary pre-requisite for cancer'

or s'impìy a secondary consequence.

2?.

5. Carc'ino-emb ryonic Antiqen (CEA)

(a) Definition

ctAisdef.inedastheperch]oricacid-so]uble

gìycopr.ctein antigen fourrd in the human foetal gut between

2 and 6 months of gestatìon, but not in significant quantjties

in healthy adult gastro-'intestinal tracts'

(b) CEA and human carc'inoma

Usingthedoubleimmunodiffusiontechnique,Go]d

and Freedman (1965) found CEA'in 12 cases of colonic adeno-

carcinoma. The cEA synthesized by the tumours ìs secreted into

the serum of tumour-bearing hosts.

From Table 1.5 it can be seen that cEA secretion occurs ìn

a majori ty of gastroi ntest'i nal carci nomas . Al though 'inì ti al ly

thought to be a spec'ific product of these tumours and theìr

metastases, it may also be assoc'iated with other maf ignancìes

(Table 1.5). However, it has been proposed by warner (1976) tnat

colonic, breast and bronchial tumour cEA',s may be d'ifferent to

the others. Th'is is supportecl by e'ridence from col igan et aL'

(fgZ¡), who showed that CEA was a heterogeneous mixture rather

than a sing'le well-defined molecule'

(c) 9EA-cnd-!he-ler:[q].Isnqn!-:!q!q

l,lith the advent of the sensitive radio-jmmunoassay,

there has been a concentrated study of cEA levels in tjssues and

blood of patients with non-malignant diseases'

23.

TABLE 1.5

DISORDER N0. ¡tr CEA +\rE

PLASI4AS

/oS ITE

Gastroi ntes-ti nal tract

Breast

Respi ratorYtract

Carcinoma of:Colon and Rectum

Pancreas

Li ver

0thers

Mal i gnant

Ben i grí

Carcinoma of:Bronchus

Upper resPi ratorYtract

230/316

48/s2

72/LB

51/85

82/rsetl24

65/90

8/t5

73

92

67

60

52

4

72

53

0.3Healthy Individuals t/335

plasma cEA leveìs 'in gastrointestinal , bronch'ial and mammary

disorders. A serum is classified as CEA +ve if it contains

nore than 10 ng/ml of ant'igen'

(collated from Zamcheck et aL., Ig72; LoGerfo et aL.' t972;

Reynso et aL., Lg72; Laurence et a'L'' 1972')

24.

(i) wor:maL serum

Li ke AFP, CEA has been detected 'i n the sera of

apparently healthy adults. However, these values are very low

(.10 nglml, Laurence et aL., Lg72)' Any appearance of CEA in

the dìseased state is classified as an elevation in Serunl

quantìties above an arbitrary normal range'

(ti) cga and non-malignant diseases

CEA has also been found in the sera of patients

with non-malignant diseases (fa¡le 1.6). However, the highest

incidence of plasma CEA elevation is amongst djseases of the

gastro.intest'inal tract. The levels of cEA in these sera vary'

and are often in the range characterist'ic of colonic carcinona

(Laurence et aL., 1972).

(¿) lyryer.y

Fromthedatapresentedhere,jtcanbeseenthatCEA

is also produced in pat'ientswith a number of non-mafignant gastro-

i ntesti nal di seases , as wel I as those w'ith gastroì ntest'inal

carcinoma. This lack of specificìty for the ma'lìgnant state

suggests that the reappearance of cEA i s due to the presence of

rapidìy-dividing cells.

25;

TABLE 1.6

DI SORDER N0. 0F cEA +VE

PLASMAS

ollo

S ITE

Gastroi ntest'inaltract

Breast

Respi ratorYtract

Polyps of:Colon and Rectum

Infl ammatorY:

Ul cerati ve col i ti s 'Crohn's di sease

Di verti cul i t'i s n

peptic ulcer

Ci rrhos'is

Alcohol ic Pancreatìti s

0thers

Infl ammatorY;

PulmonarY tubercul osi s

Chronic bronchitis &

emphysema

6/67

20/95

7 /33

28/66

L7 /322/43

2t

2L

42

53

5

38

25

9

Reactìve fibroadenosì s 5170 7

Bl21

16/63

PlasmacEA]evelsinpatientswithnon'malignantdiseases

(Laurence and Nev'il1e, 1972) '

#rút

26

SECTION IV : ONCO-DEVELOPMENTAL ANTIGENS - PLACENTAL ORIGIN

1. Introducti on

The second type of developmenta'l "antigen" produced

by the cancer cell is of placental orìg.in, i.e. synthesized by

the placenta, and not foetal or healthy adult tissues' The methods

used to study p'lacental-tumour relationships d'iffer from those

used in onco-foetal antìgen studies. Foetal nlolecules have been

studied as a function of their antìgenìc'ity. Placental molecujes

have, however' been demonstrated in tumours by conventional

biochem.ical assays, rather than antigen'ic behaviour' There has

not been a spec'ific rationale for the study of onco-placental

antigens - most of these molecules were discovered because elevated

serum levels were found in tumour-bearing patients'

Theonco-placentalmo]eculessofarreportedjntheliterature

are:

(a) Regan isozYme

(b) Human chorionic gonadotrophin

(c) Placental lactogen

(d) Pyruvate kinase

(e) Hìstaminase

Thesesubstances,andtheirappearanceinhumancarcinoma'are

l"

r

discussed below.

27.

2 0nco-pl acen tal Molecules

( a ) Bggel_i:elyue-(pleçellcj-cLl:stlle-P!eePhq!i:e)

Placentala]kal.inephosphatase.isproducedby

trophoblastic cells and is secreted jnto the serum. It is not

produced by foetal or non-trophob'lastic adult cells. Its

associatìon wìth human cancer was first reported by Fishman et aL-

(1968), and has since been studied by a number of other workers

(Doellgast , Ig76; Suzuki et aL., !976; Laurence and Nevjlle'

Lg72). Regan isozyme production has sìnce been demonstrated in

a number of human tumours (fable 1.7). From Table 1.7, it can

beseenthatonlyasma]lpercentageoftumourtypesproduce

thi s enzyme. Further, the 'inc'idence wi thi n one part'i cul ar group

of tumours is low compared to CEA (c'f'Table 1'5) incidence in

colonic carcinoma, and AFP ìn hepatomas (".f. Table 1'4)'

(u) !vml-Plqeellsl-leçlgge!-(llL)

Placentalce]lsproducehumanp.lacentallactogen

durìngtheth.irdtr.imesterofpregnancy.ithasalsobeen

detected in elevated levels in the sera of patients with

trophob'last'ic tumours (Samaän et aL., 1966) and in 8% of patients

w.ith a va¡iety of non-gonadal tumours (Weintraub and Rosen' 1971)'

Aga.in the incidence of this part'icular antigen' as compared to

foetal ant'i gens , i s verY 1 ow '

driÈ

I

I

t

28

TABLE 1.7

CARCINOFIA INCI DENCEollo

I

l,

Bronchus

Mammary

Geni to-uri narY

Lymphoma

l4el anoma

Gastroi ntesti nal

7 l5L

6/ 49

10/ 5s

2/25

015

10/81

13.7

L2.24

18. 18

8.00

0

t2.35

,i,j

I

I

il'

I

Ir,

I'II

r

Regan isozyme incidence amongst cancer patients showing an

elevated serum alkaline phosphatase level (from Laurence and

Neville, 7972).

29.

(c) !stcl-sbqlielig-gsledg!rsp!il-(U99)

HCGisahormonewhichhasalsobeenfound.inthe

serum of pat'ients with teratoma, seminoma, insuloma and gastro-

intestinal carcinoma (Braunstein et aL.' 1973). It is important

tonote,however,+,hatthesynthesisofthishornronebymalìgnant

cells may be a case of ectop.ic hormone synthesjs, and not

necessarily a true reappearance of a pìacental antìgen, due to a

genera'l de-repression of archeogenes'

(d) P uvate k'inase

I

Spellman and Fottrell (1973) have examined the isozyres

ofpyruvatekinaseinplacental,foetal,adult'andfivetumour

tìssues. They found the placental-specific isozyme only ìn

extracts of 1ung, stomach and jejunum carcinoma'

(e) Histaminase

cheì-l^Je'i and Kìr'ley (1976) have shown that ascites

fluid obtained from human ovarian cancers contains a placental-

specific isozyme of histaminase. Although elevations of

histam.inase were also associated with other tumours, on]y the

ovarian carcìnoma produced the placentaf isozyme of thìs enzyme'

3. Concl usi ons

The appearance of p'lacental -spec'i f i c mol ecul es i n human

tumours supports the concept that p'lacental ant'igen reappearance

i s assoc'iated w'i th the mal'ignant state ' Hov¡ever, the l ow

r

I

30.

'incidence amongst certa'in tunlours' and complete absence 'in many

others, suggests that the reappearance of placental antigens

may not be a maior characteristic of carcinomas. Comparatively'

the association of foetal antigens and cance¡is very Strong'

The establishment of suitable animal models may provide the

necessary methodology to determine whether the appearance of

placentaì antigens is associated with cancer to the same degree

as foetal antigens.

SECTION V : CONCLUSIONS

1. Introduction

In the preceding sections, three important probìems in

tumour immunology have been ra'ised.

(a) fne current nomenclature is unsuitable and often mis-

lead'ing when discussing embryonic-associated tumour

ant'igens .

(b) There has not been a significant attempt to define the

metabolic or physiolog'ica1 functjon of foetal antìgens

i n tumours.

(c) Is the presence of these antigens a pre-requisite for

a cell to undergo mal'ignant transformation?

31.

The first quest'ion has been exam'ined by many workers, and many

meetings have been convened to exam'ine the problem (c'f' Ëirst

Conference on tmbryonic and Foetal Antigens in Cancer, Qak R'idge'

Tennessee, u.s.A., lgTI). This prob'lem may be cons'iderab'ly reduced

if a spec'ific funct'ion could be assigned to the antìgens.

The third problem is pos:'ibly of highe¡importance as it may

provide a basis for an understand'in9 of the many compiex events

which occur durìng malignant transfonnation. Essential'ly, it is

important to determ'ine whether the appearance of embryonic properties

in general is necessary before transformatìon may occur' 0r

whether these antigens appear later during the evolution of the

first cancer cell clones.

The presence of AFP in patients with non-maf ignant d'iseases,

suggests that the reappearance of this embryonìc property 'in

these cases'is probably a function of rapidly-d'iv'iding ce'l'ls

(i .e. the anti gen 'i s produced by und'if ferenti ated, embryonì c cel I s

which replace those destroyed by the disease agent). By extra-

po'lation,AFPproductionintumourslstbeproducedby

"embryonic-type" celIs wh'ich repìace adult celIs destroyed by

carcinogens. However, in this case, these rep'lacement ceìls

faìl to respond to normal differentiation control mechanìsms'

This argument has been postulated by Potter (1969, 1970)' Thus,

this proposition would support the concept that the reappearance

of embryonic properties is an 'integral characteristic of the

first cancer cell (s) which ultimately undergo a selective evolutjon'

and hence develoP into a tumour'

3?.

However, there is some evidence to suggest that' the embryonic

propert'ies may be acquired during the sequent'ia1 evolution of

the first cancer cells. Becker (L974) has shown that foetaj and

tumour cells exhibit differential lectin binding, whereas adult

and div.idìng hepatocytes did not. Thus, thìs property is not

s impty a f uncti on of the undi f ferent j ated, rap'i dly-d'i vi di ng state '

Rather it is a spec'ific characteristic of embryonìc and tumour

cells. From these results it may be concluded that this

particular onco-foetal characteristic does not necessarì'ly need

to be associated with the cells which replace those damaged by

carcinogens. If this theory'is val'id, then thìs property, and

possibly other onco-foetal characteristics, lilâY have appeared

du¡ing the sequential evolution of the first cancer cells'

The evidence in favour of either alternative, however, 'is

insufficient to produce a definjte conclusigll' It would be useful

to determìne whether there are any cancer-characteristic propert'ies

which precede and poss'ib'ly contribute to the appearance of

embryon'ic characteristics in the developing tumour. Such studies

may he]p establish a sequence of the events which occur during

tumorigenesis.

JJ.

SECTION VI : INTRODUCTION TO PROPOSED STUDY

Perhaps the fundamental questìon in experirnenta] oncoìogy

is 'What is cancer?'. At the molecular level ' cancelis often

defined as a mass of cells that have lost a number of mechanisms

which control normal cellular funct'ions. However, it has yet to

be established whether many of these lost control mechanjsms are

a prìmar"y event of carcinogenes'is ' or s'imply a secondary

consequence of the malignant transformat'ion. The appearance of

developmenta'l antigens in tumours may be an example of Such a

secondary event. The initjal obiective of the proposed study

was to heìp resolve this questìon by determinìng the role of

these ant'igens in experimental tumour systems, j,e. why do they

appear? The following quest'ions vrel"e asked in an attempt to

characterize and to determine the role of onco-developmenta'l

ant'i gens i n the ma'l ì gnant transf ormati on .

(a) At what point of time after the application of the

carcinogenìc stimulus do the developmental antigens

fi rst appear?

(b) Is their appearance corre]ated with the appearance or

d.isappearance of other parameters known to be indicatìve

of the carc'inogenic state?

(c) Do these antigens have a biological funct'ion other than

iust being antigenic?

(d) what is the celIular Iocalization of these antigens?

34.

By follow'ing the sequence of events wh'ich lead up to the first

appearance of developnrenta] antigens, it was hoped to determine

whether there was a correlation between th'is appearance and other

events which are characteristìc of the cancerous state'

The foetal antigens chosen for this study were 'tirst described

by Stoneh'iìt and Bendich (1970). They were chosen because they

could be detected by relatively simple means (ìmmunodjffusion)'

and as their appearance WaS demonstrated in 72 different tumours

of mice, this permitted theìr use in virtually any tumour system'

The p'lacental antigens of rat tumours were exarrined by raising

antisera to both saline soluble and cell-membrane bound antigens'

Before these antìgens could be studìed, it was necessary to

raise the appropriate antisera, and to detect developnlental

antigens'in the tumour systems ava'ilabte in our'laboratory'

The anti-foetal serum was not able to detect onco-foetal molecules

'in the tumours exam'ined - a dìrect contrad'iction of the work of

Sab'ine and Stonehill (tglZ). It was concluded from these studies

that stonehi I I ' s ori gi naì anti serum was probably detect'ing ant'igens

of adul t and not foetaì ori g'in. Al though the anti serum ra'ised

against saline-soluble placental ant'igens could detect these

ant'igens in rat tumours, the antigen extraction and detect'ion

methods were unsu.itable for stud.ying the probìems proposed above'

Attempts at rais'ing an antiserum to membrane bound placental

antigens were unsuccessful.

35.

The inability to produce an antiserum and a system which

could detect developmenta'l ant'igens in tumours resulted ìn the

proposed questions remaìn'ing unanswered. consequently, the

methodol ogy of thi s study was criti cal'ly re-exam'i ned, and

modifications to these techniques theoretìcally evaìuated'

As a consequence of the study on the onco-foetal antigens,

an interestìng and unusua'l observation Was made' It was found

that non-freeze-dried foetal extracts Were unable to raise

antjbodies ìn rabb'its, an effect easily reversed by lyophilization'

It was subsequent]y proposed that the foetal extracts contained

a lyophilization-labile immune repressor whìch specifìcal'ly

inhibited the product'ion of antibody to foetal antigens. This

propos'itìon was examined further and confirmed' The releVance

of these studies to tumour immunology was also discussed in the

form of a model.

36.

SECTION 1 : MATERIALS

1. Animals

(a) Bs!:

The rats used were of the Buffalo strain which had been

bred by the University's Central Animal House. Tumours of thìs strain

were obtained originaìly from Dr. Harold P. Morris of Harvard

universìty, l,lashington D.c., and subsequently ma'intained in our

Anjmal House by se¡ial transplantation into animals of the same

strai n.

Other rats used were 3 month old male and female Hooded Wistars'

also obtained from the Univers'ity's Animaì House' The rat (Wistar)

beari ng the 3-methyl -4-d'imethyì am'inoazobenzene-i nduced hepatoma

was a g.ift from l4r. K. l4atthaei of the Australian Natjonal un'iversity'

Sw1ss Albino and C3H.AvY mice were used in th1s study'

The latter were obtained originally from Dr. ld.E. Heston of the

National Cancer Institute, Bethesda, Maryland' and have since been

bred and maintained in our Animal House on a cedar-shaving bed' as

described by Sabine et aL. (igZ:).

(c) Rabb'its

All rabbit ant'isera were ra'ised in New Zealand l^Jhites'

purchased from the univers'ity of sydney veterinary Patho'logy An'imal

( Uigeb

House.

37

2. Reaqents

Co'llagenase - Sigma Chemical Co', St' Louis' Mo" U'S'A'

Sephadex G-200 - Pharmac'ia Fine chemìcals, uppsa'la, sweden'

Freund's acljuvants - Difco Laboratories, Detroit, l4ichìgan' U'S'A'

Bovine Serum Album'in - Sjgma chemicaì co., St. Louis, Mo., u's'A'

Agarose - BDH Chem'icals, Poole, Dorset, England'

SECTI0NII : METI'{0DS

1. Pre aration of anti ens

la) Saline-soluble extracts\--, -----

Soluble extracts were prepared by homogenizing the

appropriatetissuewithanequalvolumeof0.B5%saljne,inan

Ultra-turrax homogenizer at maximum speed'for 1"5 mins' The

homogenate was then centri fuged at 1500 g for 15 m'i ns, and the

supernatant dia'lysed agaìnst several changes of physiologicaì saline'

In one study with murine foetal material (Chapter III)' extracts were

first freeze-dried and then reconstituted to 15 mg solid matter per

ml, wì th d.isti t I ed water (Stoneh'il I and Bend'ich, 1970) .

(b) Cel I surface bou l9-cl!igel:

AcrudeSuspens.ionofplasmamembranesWaspreparedfrom

normal adult t'issues (heart, ìung, k'idney, spleen' liver and

intestine),byhomogen.iz.ingeachtissuewithphosphate-buffered

38

saljne jn a tefIon hontogenizer. The homogenate was cent¡ifuged at

1500 s for 30 mins and the pellet resuspended'in PBS and washed

three t'imes. Fol'lowing the f inal wash, the memþrane pel let (and some

nucle'i) was centrifuged at 105,000 g for 60 mins'

solubilìzation of the membrane-bound antigens was achieved by

the add'ition of 1 ml of 0.5% sodium deoxycholate and 1% Triton x-100

to the membrane pe'l1et and resuspendìng the peìlet' Folìowing a 5 min

incubation at 370C, the mixture was centrifuged at 105,000 g for

60 mifìSrâfìd the supernatant dialysed aga'inst PBS for 24 hours' The

presence of solubilized antigens in this extract was verified by

doubl e immunod'i ffusi on.

cellular membranes and solubiìized ant'igens were simi'la11y

prepared from rat placenta, foetus and MeDaB-induced hepatoma'

( c) I,Jhol e Pl acental cel I s

S.ingìecel.lsuspensionswerepreparedfromratplacenta

using the method detaiìed by þluller et aL. (1972), with the

fol I owi ng mod'i f i cations:

(i) The dissoc.iation medium outlined by Berry (1973) was found to

give a better yietd and viability of cells'

(ii) centrifugation at 800 g for 60 seconds was performed following

eachdìssociatjon.Eachpeìletwasresuspendedin0.5mlof

phys'io'ìog'ical sal i ne, and pool ed '

cell counts were carried out in a Neubauer haemocytometer' and viability

establ.ishedus.ingthetrypanblueexc]usiontest.

Priortoinjection,thece]lswerekilledwithl0mMsodjum

39

arsenate, and then washed three times with 0.85% sal'ine.

2. Antisera

Antisera were raised according to the schedules described in

Appendices I-V. Blood was taken from the marg'inal ear vein of

rah'bits. lulice were given live tøycobacter'íum phLei during the final

injections in order to develop an ascites fluid rich in antibodies

(Sommerville, 1967). Thjs flujd was tapped 7 days after the final

jnjection, allowed to coagulate, and centrjfuged to obtain a clear

antibody sol ution.

3 . Immunoloqical Tests

(a) Irugre9llfs:igl

This was performed on 1..5% agarose slides us'ing the double

di ffusion pattern descri bed by Quchterl ony ( 1968) . Fol I owi ng the

addjt.ion of ant'iserum and antigen (adjusted to 10 mg protein/ml ) to

the wells, the slides were incubated at room temperature for 72 hrs'

washed, dried, and stained in 0.3% Ponceau S'

(b) luuvreeles!rePhere:i:

1.0%,agarose sl'ides were used as the electrophoresing

medium. 0.04 M -do¿lrt barbitone buffer, pH 8.2, was used as the

electrode buffer, and 0.08 M sodium barbìtone, pH 8'2, as the gel

buffer . 2 v1 of antigen (concentrated to 10 mg protein/ml ) was

e'lectrophoresed for 2.25 hrs at 6 volts per cm of ge]. Care was taken

not to allow the current to exceed 20 rnAmps at any stage durìng the

run. 0n comp'letion of the eìectrophoresis, antibody was added to the'

40.

central trough and the slides allowed to incubate for 72 hrs at' room

temperature, washed, dried, and stained'in 0.3% Ponceau S.

4r.

SECTION I : RATIONAI.E OF THE PROPOSED STUDY

The b'iochemjcal and physìological events which occur in the

cancerous and pre-cancerous cell have been reported by a number of

workers in different fields. Tjme course studies before and during

the pre-cancerous stage, and ult'imately in the fully developed tumour'

have met with ljm'ited success. Such a study would be useful because

it would tead to some understandìng of the time sequence of events

which occur during the malìgnant transformation process.

The purpose of th'is study was to deternline at what stage during

the pre-cancerous stage did foetal antigens first appear' It has

been mentioned previously,that these antigens may on]y be an effect

rather than a cause of the cancerous state. To help in the

clarification of this theory, known cancer associated parameters

were examìned by other workers to determ'ine whether foetal antigens

reappeared before or after these markers of the carcìnogenic state

(SaUi ne et aL., 1975). These parameters included:

(a)AFP(Dr.E.Ruoslahti,UniversityofHe]sinki);

(b) adenyl cyclase (Professor A.l^1. Murray, Fl inders university of

South Austral i a) ;

(c) DNA polymerase II (Dr. L.A. Burgoyne, Flinders University of

South Austral ì a) ;

(d) histology (Dr. R. Rac, Institute of Medical & veterinary

Science' Adelaide) -

42

SECTION II : THE SYSTEM OF STUDY

1. The anti gen

Perhaps one of the simp]est systems of study of murìne foetal

ant'igens is that reported by Stonehil'l and Bendjch (1970)' They

produced an antiserum whìch was capable of detecting the pres'ettce of

foetal antigens 'tn 72 different tumours, from a variety of mice

strains. These onco-foetal associated antigens were chosen for this

study because such an antiserum allows a comprehensive examinat'ion

of any tumour sYstem available.

2. The tumours

We have maintained in our laboratories, a strain of mice wh'ich

have a 100% jncidence of cancer when housed under the appropriate

condj tions (Sabi ne et aL., 1973 ) . Thi s IO0% inci dence may be al tered

almost to zero if the animals are born and bred on a different beddìng'

Th'is presents an excellent test and control system for a unified study

of the malignant transformation process'

A furtherimpetus to the proposed study has been presented by

Sabine and Stonehill (Ig72). They have shown that both mammary and

'liver cancers from these tumour-prone C3H.Avy m1ce produce foetal

antigens of the stonehill type. They have also shown that these

ant.igens could be detected jn the livers of animals between 6 and 7'5

months of age, even though they were completely devoid of any obvious

signs of tumours.

IIi FIGURE 3.1

Immunodiffusion examination of unadsorbed serum from a rabbit

immunized with murine foetal extracts.

= foetal

= composite adult

= skin

= mammary tumour

= hepatoma

= unadsorbed anti-foetal serum

FIGURE 3.2

Immunodiffusion examination of exhaustively adsorbed rabbit

anti-foetal serum.

foetal

composite adult

NaCl control

skin

C3H:AvY mammary tumour

caH.AvY hepatoma

F

a

S

T

H

A

,l

l

i

¿,

Ì

{

I

i

i

IitI.

IrtII

I

F

a

c

S

T

H

3- I

H F

¡

3-2

a )

ú

C

?

I

s

þ

'iif''tj

o

a

'1,I'

FIGURE 3.3

Immunodiffusion examination of rabbit anti-mouse foeta'l extract.

This serum was given a primary adsorption with 3 mg of a composite

mouse adul t extract.

F

A

c

= foeta'l

= adul t= NaCl control (band associ ated wi th thi s we'l I i s

an artefact produced during the drying procedure)

= C3H.AvY mammary tumour

= C3H.AvY hepatoma

= skin

= Lo adsorbed anti-mouse foetal serum

T

H

S

AI

1

FIGURE 3.4

Inrnunodiffusion examination of rabbit anti-mouse foetal extract.

Thìs serum has been adsorbed twice with 3 mg of a composite mouse

adu'lt eitract.

f = foetal

c = NaCl control

. t = C3H.AvY mammary tumour

a = composite adul tþ = caH.AvY hepatoma

s = skin

t = 20 adsorbed anti-mouse foetal serum

I

i

í

I

It,

I'I

I

I

is

c'tt.s, A

ì/

^

H ì F

\

A1

I

1

I3.4

m ì ryt

"a-ffi Oa

t

3

tI

{l

!

I

a

43.

This study was desìgned to determine the poìnt of time during

carcjnogenesìs at which foetal ant'igens first reappear. This was to

be achieved by following the loss of foetal ant'igens from the neo-rlatal

liver, and the'ir subsequent reappearance in the lìvers of adult mjce

which had been maintained on the 100% tumour incìdence environment'

SECTION III : CHARACTERIZATION OF THE FOETAL ANTISERUM

Since Sabine and stonehill (Ig72) had shown that a number of

foetal antìgens reappeared in C3H.AvY hepatomas, an antibody to a

foetal extract was raised ìn our ìaboratory, in the same manner as

Stonehill's. The unadsorbed antiserum was found to react strongly

against foetal, skin, mammary and liver cancer, and a composìte

adult extract (Figure 3.1). Follow'ing exhaustìve adsorption with the

composite adult extract (i.e. 15 mg adsorbent per ml of antiserum)'

the residual serum only continued to produce double diffusion

prec.ipitati on I i nes wi th the foetal extract ( F'igure 3'2) ' Thi s res ul t

differed to that of stonehill and Bendjch (i970), who found that their

adsorbed serum reacted with skin and tumour extracts as well'

To further exam.ine thjs discrepancy, a sequentjal adsorption v'|as

performed by adding 3 mg of adsorbant to each ml of antiserum' removing

the immune precipìtate and testing the resìdual serum against the

antigens used prevjously (Figure 3.3). This adsorption and retesting

was repeated a second time (Figure 3'4)'II

I

44

Following the first adsorption, the antiserum st'ill reacted

against all the ext.racts. More importantly, the band'indicated with

an arrOw on the precìp'itation pattern has a line of identity w'ith a

band found in all the other extracts. Since there is a l'ine of

ident'ity with the adult extract, it may be concluded that it

represents a precip'itat'ion of adult antigens which may also be found

in the foetus. This type of precipìtat'ion band is similar to that

found by Stonehiì'l and his colleagues. In their system, this band

represented the tumour-associated foetal antigens (i.e' they were not

found in the adult extracts; Stonehill et aL., I97I). When a

secondary adsorption WaS performed on the Serum raised 'in our

laboratory (Figure 3.4), the arrowed band slowly faded until it

di sappeared fol ì owi ng exhausti ve adsorpt'ion ( Fi gure 3.2) .

Thus, aìthough a foetal-specific antiserum was obtained, it

failed to react aga'inst either of our two tumour extracts - a direct

contradiction of the work of Stonehill and h'is assoc'iates'

SECTION IV : CONCLUSIONS

1. The anti bod.Y

It is d.ifficult to explain the discrepancy between the results

described here and those obtained by Stonehill. There are a number of

conclusions, however, wh'ich may be drawn from the adsorpt'ion studies'

and from Stonehi 1 1 's pubì i shed materi al . These suggest that hi s

anti serum was detect'ing normal adu] t ant'i gens whi ch c'i rcul ate i n

healthy adult mice, and which are adsorbed by rapidly d'iv'id'ing cells

such as skin, tumour, and intestìne'

45.

( a) AÊs-erelier-:!s{ie:

S.ince there is a Iine of identity between a foetal

prec'ipitin band ancl the other itnmune precipìt'in bands in Figure 3'3'

it may be conclucled that the foeta'l , adult, skin, and tumour extracts

all share a comnlon antìgen(s). Further, since thjs common band

disappears from all the extracts after adsorpt'ion with adult adsorbant

(Figure 3.2), the antigens wh1ch prec'ipìtate in this band must be

normal adul t anti gens. Accord'ing to Stonehì'l 1 ' s system, antì gens

which precip'itate in th'is cross band contajn the tumour-associated

foetal antìgens. Assuming that the cross banci found in our study

detects the same ant'igen as that found by Stonehì'll and Bendjch (1970)'

and there ìs no reason to assume otherwise s'ince the same precìpitation

patterns were obta'ined, it is proposed that stonehill's antiserum was

on]y detect'ing adult antigens, and that these may have been of

jntestinal onig'in. This concl us'ion is drawn after cons'ideration of

the adul t adsorb'ing mjxtures used i n both our I aboratory and

Stonehill,s. 0ur composite m'ixture conta'ined extracts of heart, lung'

spleen, k.idney, liver and intestine. stonehill, however, did not add

intestjne to hìs adsorbant. Thus it may be that Stoneh'ill's antiserum

was detecting an antìgen(s) which is shared by both tumours and adult

intestine, i.e. a normal adult antigen(s), and one wh'ich is also

expressed in tumours-

(b) Reacti v ì tv wi th s k'i n extracts

Another possible expìanation of this d'iscrepancy l'ies in

the abil'ity of Stoneh'ill's antiserum to react against skin tissue'

Boone, commenting On stonehìII's antibody (stoneh'iII et aL" I97I)'

46

suggested that perhaps Stonehill's ant'iserum was detecting collagen

or connective tissue antigens, which, in addition to being found in

skin extracts, may also be found in tumours. in reply. Stonehill

adm.itted that the on]y dìfference he found between the tumour and

skin antjgens whjch were detected by hiS serum' was in molecular

weight. The sk'in antigen had a molecular weight of 15,000, and the

tumour antìgen, 67,000. It 'is, however, guite possjble that the

larger molecule may consist of a number of sub-unìts of the smaller

molecule. This would make the two ant'igens immunologically

indistinguishable, and hence corroborate Boone's idea that the

tumour-assocìated antigen was merely collagen' or a connective tissue-

associ ated ant'i gen.

(c) C'i rcul at'in q adul t anti S

Toexp'ìaintheappearanceofaskinantigenintumour

ce]ls, it is proposed that the skin antigen circula'tes in the tumour

bearing an.imal and ìs absorbed by the tumour cells. A similar ana'logy

exists with the Lewis blood group antigens, which are absorbed by

human red blood cells. Ev'idence to support this concept again comes

from Stonehill's own published material. Stonehill et aL' (1971)

found that freshly excised tumour cells were ant'igen positive' After

some time in tissue culture, however, they no longer produced any

anti gens capabl e of react'ing wjth thei r anti -serum' l'Jhen the cel I s

were reinjected into non-tumour bearìng mice,they aga'in began to

,produce' the antigen(s). The concept of a circulating ant'igen would

explain this phenomenon. In further corroborat'ion of this idea'

Stonehill et aL (1971), reported that on examination of healthy

mouse sera, they detected an antigen which reacted aga'inst their ant'i-

47.

serum. They dism'issed thjs as being a d'ifferent antigen than the

tumour assocìated foetal ant'igen, because it had a molecular weight

of 250,000 - 350,000. The presence of this high molecular weight

ant.igen in the serum has not been explained by stonehil'l ' It is

poss.ible that the tumour, skin and serunl antÍgens are the same' the

difference in molecular weìghts be'ing a reflectìon of the degree of

sub-unì t d'issoc'iatì on. In other words, the anti gen detected by

Stonehìll is a norma'l adult antìgen of high molecular weight. Tltis

antigen circulates in the host, and is adsorbed by rap'id]y dìv'iding

cells such as tumour, skin and intestine. Adsorption may be jn the

sub-uni t form, or al ternati ve]y, these cel I s may absorb the hi gh

molecular weìght antigen 'in the multìple aggregate form, and on

extraction of the tissue the ant'igen breaks down into the sub-unit

structure. (It is ìnteresting to note that sk'in tissue requ'ires a

more vigorous honrogenìzation than tumour, and hence a h'igh molecujar

weight ant'igen would be subiected to a greater degree of breakdown of

sub-unìt structure. This is 'in keeping wìth the observed results

that the skìn ant.igen has a molecular weight of approx'imately 15'000,

and the tumour antìgen, 67,000')

Nevertheless, whatever the explanatìon for the discrepancy between

the results presented here and those of Stonehill, 'it 'is clear that his

antìserum system could not be reproduced, and this could not be used in

th'i s s tudY.

4B

2. The time course studY

The doubtful nature of Stonehi I I 's antì gen system rai sed a

number of interest'ing points concerning the use of a heterologous

antibody. The work of Stonehìll and his col'leagues has been

extensive'ly cìted in the literature as the principal examp'le of foetal

antigen reappearance in tumours of the mouse. The'ir method, hurwêvê1"'

of inject'ing a mouse foetal extract into heterologous hosts, such as

rabbits, results in the maiority of the antibody response bejng

directed to the high concentration of adult antigens'in the extract'

The foetuses used by both Stonehill, and in the study presented here,

were of late gestation (16-19 days). A foetus of this age would be

expected to have a low foetal-specific component and a relatively h'igh

concentrat'ion of adult antìgens. Perhaps the most su'itable method of

overcoming thìs probìem would be to immunize heteroìogous hosts with

a purified foetal antigen(s). This has been done with AFP (Savvas'

unpublished results) and CEA (Gold, 1965). For this proposed study'

however, the only mouse antìgen that has been purifieci sufficient'ly

is AFP, but the levels of this were measured in Dr' Ruoslahtirs

laboratory (Sab'ine et aL.' 1975) '

Another possible substitute for Stoneh'ill rs system would be to

use membrane-bound foetal antigens as a parameter of study. This would

involve the use of early gestation foetal cells to raise an antiserum

in homologous hosts. This would ensure that there would be a relatively

high concentration of foetal antigens on the cell membrane' and by

using homologous hosts, eliminate the product'ion of antibodìes to the

49

adult antigens. Further, since membrane-bound ant'igens would be used

as the immunogen, thìs shou'ld facjljtate a strong jmmunological

reaction.

In view of the inval'idity of the Stonehill antigens, and the

unavailability of new cancerous and pre-cancerous tissue' it was

decided to abandon this parameter in the time course study' anC seek

a ne\{| system for study. carcìno-placental antigen production 'in rat

tumours was examined.

50

SECTION I : INTRODUCTION

Examples of the appearance of carcino-p'lacental nlolecules in human

carcinoma have been clescribed 'in Chapter I. The maiority of research

on these substances has been limited to the use of HCG and Regan

isozyme in the clin'ica'l management of cancer. There has been I ittle

research into the use of these substances as bio'logìcal, as opposed to

clinical, markers of the malignant transformation. Perhaps the reason

for this,ìs that these carc'ino-placental 'antigens'have so far been

found only in human tumours, and hence ethical considerations have

placed a lim'it on the expe¡imental studies that may be done.

In this chapter, a hypothesis 'is presented to exp'lain the

appearance of these antigens, and attempts to demonstrate the presence

of these antigens ìn experìmental animals are descrjbed.

SECTION II : RATIONALE FOR THE REAPPEARANCE I:i PLACENTAL ANTIGENS

An explanation for the appearance of foetal antigens in cancer

cells is summarized diagrammat'ica1]y in Figure 4.1. In terms of gene

expression, it is proposed that during the malìgnant transformat'ion

certain genes wh'ich once appeared early 'in the developmenta'l program

of the an.imal, and have since been inactive, are once again re-expressed.

Thìs gives rise to the many foetal characteristjcs known to prevail

in tumours. In arbìtrary terms, the cell has undergone a

" de-di f ferenti at'ion" .

FIGURE 4.1

The developmenta'l sequence of mammalians. The blastocyst goes

on to develop into the embryo and pìacenta. At each stage of

development, there are certain genes that are active. As

development proceeds, some genes are "swÍtched off" and new genes

acti vated.

SPERIl

ZYGOTE

BLASTOCYST

EI4BRY(]

I1ATURE ADULT

TUI'IOUR BEARING ADULT

oOVUI\1

PLACENTA

SINCE THE TUMOUR TISSUEREVERTS BACK TO AN EMBRYONICSTATE. PERHAPS THEDEDI FFERENTIATION GOESBACK FURTHER TO THEBLASTOCYST STAGE WHEREPOTENTIAL PLACENTAL ANTIGENSMAY BE FOUND

X

,l

51.

Th.is concept may also be usecl to explain the mechanism of

appearance of p'lacental ant'igens. It i s hypothesi zed that the

de-differentiation of the tumour cell goes back further, past the

embryo/foetal stage, to the blastocyst. At this stage of development,

there are four gene types wh'ich are active:

(a) blastocyst onlY

(b) pl acenta'l onlY

(c) foetal only

(d) foetal and adult

Each of these gene types code for a specifìc type of phenotypic

product. consequentìy, the placental cells which develop from the

blastocyst will synthesize all four phenotypìc products' Thus the

de-different'iation of the normal cell to the cancerous could result

ìn the reappearance of phenotypic products of all the blastocyst

gene types. It is difficult to look for blastocyst-specific products

in tumours, since th'is tissue'is so smalì in experimental an'imals

that accurate immunologìca'l stud'ies are ìmposs'ib1e' However' s'ince

foetal and placental products do appear in human tumours, there is

considerable support for this theory of de-different'iation'

In the proposed study, a d'i rect 'immunol ogi ca1 exanti ttati on of

rat tumours for the presence of placentaì ant'igens is descfiibed'

The immunologìcal approach was chosen ratl'rer than the biochemical '

because the currently known onco-placental molecules (e.g. alkaline,

phosphatase, pyruvate kjnase),may not necessarily be the placental

antigens re-expressed 'in the rat tumours. Further, s'ince the onco-

52.

placental antigens may be membrane bound, a study of cel'l-surface bound

and cytoplasmic antigens was undertaken.

The experirnents presented in this chapter have two distinct but

rel ated aims:

1. To determine whether soluble and membrane-bound p'lacental

ant'igens reappeared during carcinogenesis'

2. If so, to use these ant'igens as markers of the transition

of normal liver cells to the cancerous state, and in

particular to deter"mine the earl'iest tìme of appearance

following the application of chemical carcinogens'

Attempts to demonstrate the reappearance of these ant'igens in rat

tumours were only partìa1ìy successful. The presence of soluble

p'lacental antigens was demonstrated in three rat tumours. However,

the complexity of this system prevented its use as a means of

study'ing pl acental anti gens i n cancer.

Membrane-bound placental antigens were then examined to determine

whether these ant'igens reappeared in rat tumours'

53.

SECTION III : SALINE-SOLUBLE CYTOPLASMIC PLACEI.ITAL ANTIGENS

1. Immunodif'Fusion stud'ies

(a) Uelbsg

The methodology of th'is study was sim'ilar to that of the

foetal ant'igen program, described in the prev'ious chapter. Rat

placentae (Br¡ffalo strain) were homogen'ized 'in 0.85% sal ine, and the

soluble extract prepared as outl'ined in the Materjals and Methods.

Antibodies to this extract were raised in rabbits. Since th'is

antiserum would contain antibodies to foetal, adult and placental

antìgens, it was adsorbed fìrst with a composite adult extract and

then with a foetal extract 'in an attempt to rendelit p'lacental-

spec'ifìc. It was then planned to use this antiserum to detect

pl acenta'l anti gens 'in tumour extracts usì ng doubl e immunodì ffus i on

pl ates.

(b) Resul ts

From Figure 4.2 1t can be seen that the unadsorbed antiserum

reacted very strongly against the placenta'l, foetal, aduìt and tumour

(5123c Morrìs hepatoma) extracts. Follow'ing exhaustive adsorption wjth

a composite adult extract the serum failed to react with the adult and

tumour antigens, with only the pìacental and foetal extract exh'ibìtìng

precipìtin bands (F'igure 4.3). Further, since the foetal and pìacenta'l

'bands exhibited a react'ion of Identity (Ouchterlony' 1968 ) it seems

lìkely that the adsorbed antiserum was detectìng only the foetal

component of the placenta. Thus in th'is system, the antibody which

FIGURE 4.2

Immunodiffusion pattern of unadsorbed anti-placental serum tested

against tumour, foetal, placental and normal adult extracts'

p=p=

H=K=l=T=ap=

pl acental

foetal

heart/l ung

ki dney/spl een

I iver/intestine

transpìantable hePatoma

anti-Placental serum

= pl acental

= foetal

= heart/l ung

= kidneY/sPleen

= I iver/intestine

= Morris 5123c transplantable hepatoma

= anti-placental serum adsorbed with adult tissue

extracts

FIGURE 4.3

Irrrmunodiffusionpatternofanti-placentalserumtested

againstpìacental,foeta'l,tumourandnormaladultextracts'

P

F

H

K

L

TI

ap1

54

was raised ìn rabbits seemed to be directed aga'inst the adult and

foetal anti gens , and not aga'inst the p1 acenta'ì -spec'if i c mol ecul es '

A major consideratìon here, however, is the effect that adsorptÌon

has on the antiserum, and thìs'is considered below'

2. Immunoelectro horetic studies

(a) Rati onal e

Inthepreviousstu<ly,thereWasaposs.ibilitythat

placental-spec'ific antibodies may have been removed during adsorpt'ion

of the antìserum with adult ant'igens. This might occur, for jnstance'

if the part'icular ant'ibody jn the serum was directed agaìnst a

pìacental enzyme wh'ich has an adult isozymel Should the ant'ibody

bì nd to the antì gen.ic determi nant on both i sozymes , adsorpti on w'ith

an adult extract would remove the antiplacental activìty from the

serum. To examine this particular exampìe of adsorption effects

placental, tumour, foetal and aclult extracts were to be individually

immunoelectrophoresed. Antìgens wh'ich were found in the p'lacenta

were visualìzed by addìng unadsorbed antiplacental serum to the IEP

trough.Bycomparingtheprec.ipitinbandsofthefourextracts,it

was hoped to locate molecules which are conmon to the tumour and

placenta, but not found in either the adult or foetus' This technique

would eliminate any problems which may have been caused by adsorpLìon'

D.iscrjm'inat j on of the adul t and pl acental i sozyme shoul d occur

.because d.ifferent forms of the same enzyme often have different

el ectroPhoreti c mobi I 'i tì es '

*Such sharing of antigenic determinants has been demonstrated in the

case of ptacental and adult (intestinal) alkaline phosphatase

(Doel I gast , L976) .

55.

(b) Di rect Imnlunoel ect'ro Phoresis

Firstly, only p'lacenta'ì and foetal extracts were loaded

d.irectly on the gel and electrophoresed, 'in an attempt to determine

which of the pìacental ant'igens detected by the ant,'iserum were not

present in the foetus. A similar method has been used extensively

in the .identìfication of abnormal serum proteins (0uchterlony, 1968)'

Figure 4.4 demonstrates the unsuitabì1ìty of the system as applied here'

since there was ineffective separation of the extracts' Th'is was

probably due to steric hjndrance' caused by the'large number of molecules

which constitute the extracts exam'ined. This idea of ineffectìve

separat.ion 'is supported by the immunodiffusion patterns seen in

Figure 4.2. The precipitation bands on that plate suggest that the

unadsorbed antìserum should detect at least 6 distinct precipitation

bands when the ant'ibody is reacted aga'inst the p'lacentaì extract'

However,theìmmunoelectrophores.ispatternofF.igure4.4showsthe

presence of onìy 4 bands. (Unpublished results from our laboratory

have shown a similarly poor separation when whole placental, tumour

and foetal extracts are electrophoresed on polyacrylamide gels')

(c) Col umn Se rati on / Immunoel ectroP hores i s

(1) Method

In order to reduce the large number of molecules bejng

appl.ied to the ge]s, each extract was first fractionated on Sephadex

G-200 columns, before electrophoresis. A protein profile of the

column eluant was obtained (Figure 4'5)' and then each peak was poo'led

and adjusted to a protein concentration of 10 mg/ml' The poo'ls were

examined for the presence of placental antigens by testing aga'inst

I,ll]È

!

II

;

t

"i

FIbURE 4.4

Direct immunoelectrophoresis of pìacental and foeta'l extracts.

p

f

p'lacental

foetalill1,1

j central trough conta j ns unadsorbed anti -p'ì acenta'l serum.

II

I

þ

4-4

I

j

I

-. --.^ /^

&tiü

,tl

Ù

a'¿

I

II

;

I

I

FIGURE 4.5

Gel filtration of lrlorris transpìantable tumours, foeta'l and adult

extracts on 40 cm x 2.5 cm Sephadex G-200 col umns. Each extract was

adjusted to 50 mg protein/m1,3 mls applied to the column, and eluted

with phosphate buffered saline. 2 n\ fractions were collected and

assayed for protein by the method of Lowry et aL. (1951)' Shaded areas

represent those fractions which reacted positively agaìnst unadsorbed

anti -pì acenta'l serum.ì

i

',1

;l

I

l

V voi d vol ume

pooled fractionso

PL'P2

I

I

itr

I

lt,

I

T

I

FETAT

ADU tT

M.K 2kidney

HIGH Hb

v7794A

hepntomo

Vov5123Chepoüorro

co tu M N CHRO'I'IAT-o-GBAPHY DATA- -futvtoR - FETAT - ADutïSEPHADEX G-2OO

I

,l

I

!Fo{gz!rTl7

=l-

1

PI

FRACTION NO

56.

unadsorbed anti-placental serum, in 0uchterlony double-diffsuion p'lates.

To demonstrate that some of the antigens found in the tumour were

pì acenta'l -specì f i c, pì acenta'l -pos i ti ve peaks f rom each tumour were

immunoelectrophoresed alongs'ide the corresponding peaks from the foetal

and adult extracts (ì.e. Peak P, from each tumour, foetal and adult,

then P, from the same extracts - Figure 4.5). By comparing the precìpit'in

bands, placenta'l-specific antigens were des'ignated as those wh'ich were

found'in tumours, but not jn the foetal nor adult extracts.

(ii) ResuLts and Discussion

The precipitätion bands which formed after e'lectro-

phoresis are shown in Fìgures 4.6 (Pool 1) and 4.7 (Pool 2)- The

number of separate placental-spec'ific ant'igens (i.e.specific by the

above criteria) and their relative electrophoretic mob'ilities, are

summarized in Tables 4.1 and 4.2. Direct comparisons of e'lectrophoretic

mobilit'ies is valid because all the samples were run on the same sljde.

By comparìng the eìectrophoretic mobilities summarized in Table

4.1, it can be seen that the 5I23c and 77944 tumours share an antigen

with a common mobility and molecular vleight range (Pool 1, CPA-I and

CPA-$). This antjgen is absent, however, from the MK-2 tumour.

S'imilarly, the 5123c and MK-Z tumours share another antìgen (CPA-2 and

CPA-4), wh'ich is absent from the 7794A. In addition to these antigens'

the l4K-2 tumour also produces an additional antigen, which is not found

in either of the two hepatomas.

The electrophoretic patterns of the second pool of the tumour's

column profi'le (Table 4.2) reveal that each tumour produces a specific

FIGURE 4.6

Immunoelectrophoresis of Morris transplantable tumour, foetal and

adult extracts. Poo'l L from each of these extracts were electrophoresed

alongs'ide each other and placental antigens visualized immuno'logically

by adding unadsorbed ênti-placental serum to the central trough.

antigens founC in tumours but not adult or foetalCPA

CPA

FIGURE 4.7

Immunoelectrophoresis of l4orri.s transplantable tumour, foetal

and adult extracts. Pool 2 from each of these extracts were

electrophoresed alongside each other and pìacental ant'igens visualized

immunologically by addìng unadsorbed anti-placental serum to the

central trough

antigens found in tumours but not adult and foetal

.L'þ

z,

t1

57.

placental ant'igen wh'ich is not shared wi'L'h any of the other tumours

examjned here (Cpn-6, CPA-7, CPA-B). Such tumour specìfìcity for

developmental antigens has been described previousiy for a number of

foetal antìgens (Kroes et aL., lg72; Häkkjnen, 1974), and is

consiclered a valid conclusìon'in these studies.

Thus , based on el ectrophoreti c mob'i I i ty compari sons , 6 di sti nct

p'lacenta'l antigens have been shown to reappealin l4orris transp'lantable

tumours.

(iii) ConcLusions

Although the methodology employed here is suitable

for an initial demonstration of the presence of carcìno-placental

ant.igens, it ìs, however, unsuitable if these ant'igens are to be

monitored in a large number of samp'les. This ìs because the method of

column separatìon, followed by'immunoelectrophoresìs is a long and

tedious procedure, requ'irìng approxìmately 3 weeks to compìete per

sample. The technique also requìres a 'large quantity of tissue

(approximate'ly 1.0 gm wet weight), which is generaì]y unavailable in

the cancerous and pre-cancerous stages. Therefore, a technique was

sought wh'ich would enable':

(a) a small quantity of tissue to be utilized'

(b) simPle extractìon of tissue'

(c) detection of the antigens with relative ease'

,Membrane-bound antigens conform with these criteria' Consequentiy' thìs

typeofantìgenwaschoseninafurtherattempttodemonstratethe

reappearance of placental antigens in rat tumours'

58.

TABLE 4.1

TUMOUR ELECTROPHORETI C ÌIIOBILITY GRADIENT

ANTIGEN NO.

SLOl^l FAST

512 3c

(Li ver)

MK-2

(ri ¿ney)

77944

(Lì ver)

CPA-3

CPA. l

CPA.5

CPA-2

CPA.4

TABLE 4.2

TUMOUR ELECTROPHORETIC MOBILITY GRADIENT

FASTSLOt^l

ANTIGEN NO.

5123c

(Li ver)

MK-2

( Ki dney )

77944

(Li ver)

CPA.6

CPA-7

CPA.B

59.

SECTI0NIV:MEI4BRANE-BOUNDPLACENTALANTIGENS

1. Introductìon

l,lembrane.bound placental antìgen reappearance in rat tumours

was examined using a similar experimental approach to that previously

described for sal'ine-soluble ant'i3ens. In thìs studvr âh antiserum

was rai sed to the membrane bound anti gens, and attempts were made to

renderit pl acental -specì f i c by adsorbi ng the ant'iserum wi th adul t

tissue membrane extracts'

Brìefìy,itwasfoundthatthetwojmmunogen.s(i.e.whole

pìacental cells and a crude preparation of their cell membranes)'

stimulated the product'ion of an antiserum with activity directed

pr.incipa.llyagainsttheadu]tantigencomponentoftheplacenta,and

to a lesser degree, the foetal portion. In both cases' a placental-

specific antìbody was unobtainable. These attempts are described

bel ow.

2. Intact Pl acental Cells

(a ) lrspelcliel-ef -el!lÞedY

Placentasfrompregnantrats(Wistar)ofgestationage

13-lgdays,weremincedintosmal'lpieces.Isolatedcellswere

prepared as outlined in the lnlaterials and Methods, and antibod'ies

to the cells raised in rabb'its. Antibod'ies in the rabb'it serum were

detected by testing against antigens which had been solubìlized front

tumour (i,leOag induced hepatoma), placental and adult membranes'

60.

From Figure 4.8(a), it can be seen that the ant'iserum reacted strongìy

against all the antìgens examined.

( u ) Adsorpti on w'i th a{ul !-q!!i99!9-----L---

Antibody against adult ant'igens was removed by incubatìng

L ml of immune serum for 30 mjns at room temperature, with 50 ul of

packed, normal , adul t red bl ood cel I s. Thi s adsor'ptì on v'ras repeated

a further two times. 0n re-testing the adsorbed serum, all reactiv'ity

against the tumour, p'lacental and adult antigens had disappeared

(Fisure 4.9).

(c) Concl us i on

Sì nce al I i rrmune react'i vi ty wa s removed by adsorpt'i on

with adult red blood cells, it was concluded that all the ant'ibody

rai sed aga.inst pì acentaì cel I s was di rected agai nst e'i ther anti gens

of the adult red blood cells, or adult isozymes of p'lacental-specific

enzymes. Thjs result was sjm'i1ar to that obtained follow'ing

adsorpti on of anti serum ra'ised agai nst sal 'ine-sol ubl e extracts of

rat pìacenta (Chapter IV, Sect'ion III)'

3. Placental Plasma Memb rane Fraction

The previous experiments were repeated with an ant'iserum

raised aga.inst a crude preparation of placental cell-surface

membranes, rather than whol e cel I s '

Heterol eeev:-s!!iÞsdv(a)

(i) Pxeparation of antibodY

FÏGURT 4.8

Immunodiffusion s'lide of antiserum from rabbits immunized with whole

placental cells. The peripheral wells contain the test antigens, ì.e.

ahtigens solubilized off the appropriate tissue cell membranes, and the

central wells contain the antiserum.

(a) P

N

H

= p'lacental

= NaCl control

= heart

A=

[tl = NaCl control

| = ìung

I = intestine

anti-whole placental cells

(b) T

c

S

A

tumour

NaCì control

spl een

Anti serum

A=

Jrl =

(=

Li=

NaCl controi

kidney

liver

unadsorbed anti -whol e pl acenta'l ce'l I s

4-8a

4_ gb

N

eì

1ii

;l

I)î

o

II

¡

þ

IIGURE 4.9

Immunodiffusion slide of antÍserum from rabbits immunized with whole

placental cells. The serum had been adsorbed three times with packed rat

red blood cells before testing. The peripheral wells contain the test

antigens, i.e. antigens solubilized off the appropriate tissue cell

membranes, and the central weìls contain the adsorbed antiserum.

(a) = placental N - NaCl negative control

= NaCl negati ve control L = I ung

=heartl-intestine= adsorbed anti-whoìe p'lacental ce'lls (concentrated x10)

(b) tumour [rl = NaC] negati ve control

NaCl negative controt K = kidney

spleen Li = 'liver

adsorbed anti-whole placental ceìls (concentrated x10)

P

N

H

A

T

c

s

A

4,ga

4.9b

Na

¡

I

I

TI

Nt lt¿

N s

61.

Washed pìacenta'l membranes were prepared from 13-19

day gestational pregnant l^listar rats. Rabb'its were inttnunized with

thi s membrane preparat'ion , and the presence of anti bod'ies aga'in

verified by testing the rabbit serum agaìnst membrane solub'ilized

MeDaB hepatoma, p'lacental and adult antigens. The antibody reacted

strongly aga'inst al'l the anti gens exanl'ined by doubì e immunodi f f usi on

(Figure 4.10) .

(ii) adsorption uith aduLt antigens

1 ml of immune serum was adsorbed w'ith 50 ul of

packed normal adult red blood cells as described prev'iously' The

adsorpt.ion markedly reduced the i ntensi ty of precì p'itat'ion of the

antìgens by the immune serum (Figure 4.11). However, a residual anti-

adul t acti vi ty st'iì 1 remained i n the serum'

1 ml of this antiserum was then further adsorbed with a composÍte

mixture of adult tissue membranes. 0n repeated adsorption, all

reactivity with the adult and tumour disappeared from the antiserum

(Figure 4.I2). The resultant serum still reacted with the placenta'l

extract, although the degree of activ'ity was cons'iderabìy dim'inished.

This adsorbed antiserum was tested to determine whether the renraìn'ing

antibody was placental-specìfic, or simply directed agaìnst the foetal

component of the p'lacental cell membrane. This was done by testìng

the residual antiserum by 'immunodiffusion, against a solubil'ized

membrane extract from hlistar rat foetus. This antiserum was able to

precipitate antigens from the rat foetus (Figure 4.13). Further,

since the precipit'in bands of the placentaì and foeta'l antigens

FIGURE 4.10

Immunodiffusìon slide

membrane fractjon prepared

contajn the test antigens,

tissue cell membranes, and

anti serum.

of antiserum from rabbits immunized with a cell

from rat placental cells. The peripheral wells

i . e. anti gens sol ubi I i zed off the appropri ate

the central we]ls contain the adsorbed

(a) P

N

H

= placental N - NaCl negative control

= NaC'l negati ve control L = ì ung

= heart l= intestine

A = unadsorbed anti-placental cell membranes

=spleenK=kidney= NaCl negati ve contro'l Li = I i ver

= tumour

= NaC'l negati ve control

A = unadsorbed anti-placental ce'll membranes

(b) s

N

T

c

a

N

I

4.10a

4-l0b

i$)

a

II

I a

N\ O

a

a

Na

I

-

a

FIGURE 4.11

Immunodiffusion slide of antiserum from rabbits immunized with a cell

membrane fraction prepared from rat placental cel'ls. The serum had been

adsorbed three times wìth packed rat red blood cells prior to testing.

The peripheral wells contain the test antigens, i.e. antigens solubilized

off the appropriate tissue cel'l membranes, and the central we'lls contain

the adsorbed antiserum.

(a) = placental N = NaCl control

= NaCl control | = lung

= heart I = int,estine

A = RBC adsorbed anti-placental membranes

(b) = spl een lrl = NaCl negati ve control

= NaCl negative control K = kidney

= tumour Li = liver

A = RBC adsorbed anti-pìacental membranes

P

N

H

S

N

T

4-lla

N

Ë¡ I

t

a

4 b

N

N

-ll

a

IL

¡

a

aN

FIGURE 4.12

Immunod'iffusion slide of antiserum from rabbits immunized with rat

placental cell membrane fraction. The antiserum had been adsorbed

three times, with packed rat red blood cells, and then three ti¡nes with

a composite mixture of membrane from normal adult tissues. The

peripheral wells contain the soiubil'ized test antigens, and the

central wells the adsorbed ant'iserum.

ti

h

p

S

k

= pl acenta

= spìeen

= lung

= tumour

= i ntesti ne

= heart

FIGURE 4.13

Immunod'iffusion slide of antìserum from rabbits immunized with

placenta'l cell membrane fraction. The antiserum has been adsorbed

with rat red blood cells and a composite adult membrane suspension.

P = pì acentaì

f' = foetal

4-12

hI

a

Ða

t

4.13

P,¡

\ol

)f

p

ro

t

,lp\,f'"JT tI

o

I

o

62.

exhibited a reaction of identity, the antiserunl activ'ity was

exclusiveìy directed aga'inst the foeta'l component of the placenta'

(iii) ConcLusion

The results obtained here ind'icate that the

antiserum which was raised against the p'lacental membrane fractìon

was directed aga'inst the foetal and adult (or adult ìsozyme of a

placental enzyme) component on'ly. Th'is effect is agaìn similar to

that which occurred durìng the preparatìon of an antibody to'saline-

soluble extracts of the rat p'lacenta. consequent'ly, thjs method of

raising an ant'ibody was also unsuitable. A method of eliminatìng

the production of antibody to the large quantity of adult antigens

was thus sought.

(b) Homol ous anti bo dy

(t) Introduction

The attempts to raise an antibody to pìacenta'l-specific,

membrane-bound ant'igens had been unsuccessful so fat" since most of

the immune response was directed aga'inst the high concentration of

adult antigens on the cell surface membrane, and to a jesser degree

against those of foetal origin. In an attempt to overcome this problem'

rats were immunized with homologous pìacental nlembranes. s'ince the

rats shou'ld not recogn'ize the adult antigens as foreìgn, the ant'ibody

response to these antigens should be elìminated. This would allow the

product.ion of pl acental and foetal -spec'if i c antì sera. The amount of

antibody to these antÍgens should also be cons'iderably higher because

63.

the'immunol ogicaì response to the adult antigens (principaì'ly, the

highly immunogen'ic transplantation antigens), which would normally

"swanrp" the host's immune system, would be el imirrated.

(ii) ResuLts and discussion

The attempt to imnlunjze rats with homo'logous placentaì

membranes Was unsuccessful. The sera from the 12 immunized rats

fai I ed to react agai nst the sol ub'il i zed p'lacental membrane extracts

(F'igure 4.14). This inabi'lity to raise an antibody to the p'lacental

antigens was probab'ly due to either the small quantity of p'lacental-

specìfic antigens which constjtute the placental cell membrane, or

to the host's immunolog'icaì tolerance to these antigens. In the

first circumstance, this could possibly be overcome by cont'inuaì

jmmunization over periods of 6 months. Chang et aL. (1976) have

overcome thìs problem in such a manner, and have successfully

rai sed, i n rats , an anti body to two so'l ubl e rat p'lacenta'l enzymes .

The second prob'l em of immunol ogi cal tol erance woul d be d'i ff icul t to

sol ve. Thi s woul d requ'ire the formati on of p1 acentaì ant'igen

haptens, to facilitate their recognition by the rat's 'immune

system. A sim'ilar method has been used by Ruoslaht'i et aL. (1974)

to break homol ogous tol erance to AFP . |l|'ith pl acental ant'i gens ,

however, many technical difficultjes prevail, since a pure so'lution of

placentaì-specific antigens is not available. Any attempt to haplen'

an 'impure mixture of placental, foetal and adult antigenS' would

probably result in the product'ion of antibodies to the adult ant'igens

. as wel1, since adult antigens would be haplenated to such an extent'

ji¡

ilr-r

FIGURI 4.14

Immunodiffusion slides of sera taken from [^listar rats immunized

with homo'logous rat pìacental membranes. The peripheral wells

contain the antisera and the central wells contaìn an antigen extract

prepared from rat placental cell membranes

p = placental membrane antigens

1 ... 72 = rat sera numbers

I

4.14 a

3

-ñ.

trl46

a

a

t4-l4b

I

9

loi

a

f.,

..t

',j

iÌ

64

that i mtnunol ogi ca'l tol erance to these woul d be broketl , resul ti ng 'in

the unwanted production of ant'ibodies to adult molecules.

SECTION V SUMMARY

The aim of thjs study had been to

(a) demonstrate the appearance of placental antigens in experimental

rat tumours;

(b) to use these antigens in a time course study as markers of the

mal i gnant state.

The first aim has been achieved. However, the second has not been

attenrpted because of the inability to raise an anti-placental serum,

which could be used to detect placental antigens in cancerous tissues.

In the majority of attempts to obtajn such a Serum, two'important

observations were noted:

(i) adsorpt'ion of an anti-placental serum with adult tissues

removed most of the antibody activjty;

(ii) any residua'l activity after such adsorption was directed

against the foetal component of the placenta.

The first observat'ion was examined further and it was found that the

unadsorbed ant'iserum to saline-solubìe pìacental extracts could be

used to detect p'lacental ant'igens in tumour extracts. However, the

particular antigens against which this activity was d'irected probably

.T

¡,{

II

I

I

tII

I

!

nlì,Ë

,.1

65

had adult jsonlers, hence their detect'ion with an absorbed antiserum

was not possible because absorption with adult extracts removed the

antibodies to these antigens. Although a similar s'ituat'ion was not

shown in the membrane stud'ies, this may have occurred here also'

Thus, although placental antigen reappearance in rat tumours was

demonstratecl by indirect means, it was not possible to raise an

antibody to a placental-specìfìc antigen (j.e. one without a similar

adult form). There nray be two reasons for the jnabiljty to do this:

(a) they do not exist;

(b) aìtlrough synthes'ized by placental cells, they ex'ist in

small quantìties and are unable to raise a sufficient

ìmmune response from the inocculated host'

The complete absence of such molecules would linlit the use of placental

ant.igens i n experimental studi es of the mal 'ignant transformati on.

such studies would emp'loy placental molecules which also have an adult

form, e.g. the Regan ìsozyme, HCG, etc' of humans'

If the presence of placental-spec'ific antigens is to be verified,

however, the methods used'in these studies requ'ire considerable

modifications. The alterat'ions to this methodo'logy are revjewed

furtherin ChaPter VI.

i

I

{

t

66.

SECTION I : INTRODUCTION

Consìderable difficuìty was observed in raisìng ant'ibody to a

1500 g extract of mouse foetal homogenates (see Chapter^ III). This

difficu'lty was overcome by freeze-drying the foetal material, and

reconstituting prior t.o immunizat'ion - an unusual situation because

freeze-drying is not normally associated wìth any change in the

immunogenicìty of an antigen.

To exp'lain this unusual phenomenon, the presence, in the foetal

extract,of a'lyophilizatjon-lab'ile ìmmune repressor was proposed.

A'lthough this possib'il'ity was initia'l'ly thought to be unl i kely, the

importance of this find'ing was such as to warrant further invest'igation.

Duri ng th'i s study:

(1) the ìmmunogenìc'ity of the foetal extracts was examined;

(2) the'inabilìty to rajse antibodies to foetal extracts vras

a'lso repeated in mice immunized with non-freeze-dried rat foetal

extracts; and

(3) it was found that th'is repressìon of ant'ibody synthesis

was specific for foetal antigens on'ly.

From the data presented, it has been proposed that the inab'ilìty to

raise antibod'ies to non-freeze-drìed foetal extracts was due to the

act'ion of a'lyoph'ilization-labile repressor' possibly an ant'igen-

antibody complex.

I

67.

SECTION II : SUPPRESSION OF ANTIBODY SYNTHESIS TO FOETAL EXTRACTS

1. Rabbit Immunjt.y

(a) Ait_cld Methods

During the attempts to raise an antibody to mouse foetal

extracts, with wh'ich foetal antigens of murine tumours could be

detected (Stonehill and Bendich, 1970), two groups of rabbìts were

immunized - one with non-freeze-d¡ied (NFD) foetal extract, and the

other with freeze-dried and reconstituted (FD) extract.

Inrnunizatjons were given to the animals via a number of iniection

routes, using three different concentrations of antigen (faUles 5.1

and 5.2). Ant'ibody production was monitored by double immunodìffusion,

using non-freeze-dried foetal extracts as the precìpitating antigen.

(b) Res ul ts

The raisìng of antibodies to non-freeze-dried foetal

extracts is summarized in Table 5.1. All attempts, usìng a variety

of inoculation protocols, and antigen concentrations, were unsuccessful.

In contrast to these results, antibody product'ion was induced in the

second group of animals which had been g'iven freeze-dried extract

(Table 5.2). In this case, the response varied but was present in

all animals examined.

The d'ifference'in the responses of the two groups of animals is

difficult to expla'in since both groups apparently received the same

ant'igen, and in four cases, in comparable concentrations. It is

unusual that freeze-drying of the antigens should alter the

'immunogenicìty of the extract, since ì.yophiljzation is a standard

b'iophysicaì technique which rare'ly al ters the chemical 'integrity of

6B

TABLI 5. 1

Immune responses of rabbits inoculated with murine

foetal extracts

1 2 3RABBIT NO. ANTIGEN CONC. S ITE RESPONSE

1

2

3

4

5

6

7

I9

10

I3

3

10

10

3

3

8

3

3

s.c./i.m.i.v./s.c./i.m.i.v./s.c./i.m.

fpfplm1m

s.c./i .m.

s.c./i.m.s. c. /i .nl.

2the route of administration of the antigen

where multiple s'ites were used, the extract was divided into equal

Rabbits were immunized with various concentrations of non-freeze-dried

murine foetal extracts and their ability to raise antibodìes to the

extract examined. Protocols are given ìn Appendix IV.

lmg protein/ml Per iniection

s.c. = subcutaneous

i.v. ='intravenous

i.m. = intramuscular

f.P. = footpad

porti ons .

3The humoral immune response was measured by testing the rabbit serum

for antibodies in immunodiffus'ion pìates. The antigen used was non-

freez-e-dried mouse foetal extnact, at a concentratjon of 10 mg prote'in

per m'l .

69.

any molecule

It was decided to examine this unusual situation further, and

in particular to see if these results could be repeated usjng other

an'imal speci es .

. Muri ne Immun i t.Y2

(a) A'im and Methods

To determ.ine whether non-freeze-dried foetal extracts are

able to raìse antibodies 'in other heterologous spec'ies, m'ice were

immun.ized with NFD and FD rat (|¡listar) foetal extracts and antìbody

production examined by immunodiffusion. This system was chosen for

three reasons:

(i) pregnant rats are available at regular intervals from

the Uni versi tY Anima'l House;

(ii) the rat foetus is relatively ìarge' and a considerable

quantityoftjssuecanbeobta.inedfromasingìe

pregnancy;

(iìi) previous'ly unpubìished results from this laboratory

showed that NFD rat foetal extracts were unable to induce

antibody production in rabbits. (Immunìzation with freeze-

dried rat foeta] extracts had not, however, been

attempted, and hence a comparison of these results with

the murine foetal results was not cons'idered valid')

The rat foetal extracts were prepared as described in Chapter II and

70.

TABLE 5.2

1 2 3RABBIT NO. ANTIGEN CONC. SITE RESPONSE

1

2

3

4

5

6

I8

3

3

3

3

i.v./s.c./i.m.i.v./s.c./i.m.i.v./s.c./'i .m.

i.v./s.c./i.m.i.m.i.m.

+

++

++

++

++++

+++

lmg protein per mì (8 mg protein per ml per iniection)'

Concentration was ca]culated from a freeze-dried extract reconstituted

to 15 mg solid matter per ml with distilled water'

2

3as in Table 5.1

7r.

inoculat'ion of Swiss Albino mice performed accord'ing to the protocol

des cri bed i n ApPend'ix V -

(b) Resul ts

Two groups of animals were immunized with either freeze-

dried or non-freeze-drìed rat foetal extracts. The ascites fluid of

each anjmal was examined for the presence of antibodies to rat foetal

antigens by 'immunodiffusion" and the results and statistical analysis

recorded ìn Table 5.3. The x2 analys1s of this data showed a

signifjcant difference in the ability of the two groups to raise

antibodies to foetal antigens, Ðiz. the group gìven freeze-dried

extract contained twice the number of animaìs which produce antìbody

to the foetal extract

These results are sim'ilar to those obtained in the rabbit/murine

foetal system. However, repression of antibody production in the

rabbits occurs in alI the an.imals given non-freeze-d.ied foetal extract'

whereas repress jon ìs not comp'lete in mice g'iven rat foetus' The

statjstical anaìyses, however, do show that the difference between

both groups of mice ìs significant'

There are two possible explanations for the incomplete repression

of the murine irnrnune sYstem:

(a) fhe degree of repression may vary amongst different animal

speci es .

(b) Incomplete repressìon may aìso occur in rabbits given

NFDfoetalextracts.However,becauseoftherelative]y

72

TABLE 5.3

Freeze dried Non-freeze-dri ed

No. anjmals positive

Total no. of animalsper group

% no. raising anti-bodi es

18 6

30

?9 20

62.07

x2 Analysis : p < 0.05, therefore difference is significant'

Number and percentage of antibody positive animals inrnunized with

either freeze-dried or non-freeze-dried rat foetal extract'

73-

smal I number of rabbi ts , animal s wi th pos'it'ive antì body

productjon may not have been isolated'

Whatever the explanation for this difference, it is clear that

repression of antibody Synthesis to rat foetal extracts in mice may

only occur if the lyophilization procedure is om'itted during the

preparation of the extract.

3. Concl usions

From the results presented here,'it is clear that NFD murine

foetal extracts were unable to evoke a humoral jrnmune response in

rabbits. Similarly, a statisticalìy significant number of mice were

unable to raìse ant'ibodies to NFD rat foetal extracts. In both cases

this negatìve ant'ibody response was reversed by freeze-drying the

foetal extract prior to inoculatìon'

Four possible explanations of this phenomenon are proposed here:

( j ) Immunoqen'ic'ity of the foeta'l extract

The extract used to raise the antibody was a 1500 g supernatant

of whole foetus homogenate. consequently, the extract contains many

small cellular organelles which may not have been disrupted durìng

homogenizatjon. Followjng lyophilization, these organelles may have

been broken, hence releasing new immunogenic material ' It is proposed

here that it is this material whjch causes the production of ant'ibody

in the host an'imals. Conversely, if lyophil'izatìon is omitted' the

organelles remain intact and no immunogenjc molecules are available

to cause antibodY Production.

74.

(ii) Immunocompetence of the animals

The raìs'ing of antibodies in the FD group of animals and

not in those given NFD foetal extracts may have been due to the non-

immuno-competence of the animals inoculated with NFD extracts' This

is, however, very unlikely because product'ion of rabbjt and mouse

antisera to other antigens studied 'in th'is laboratory has been

Successful (antigens include AFP, gamma globu'l'ins, placental extracts

and membrane-bound antigens).

( i i i ) Anti qen modi fì er

The third poss'ible explanation of the observed phenomenon

is that the foetus contains a modifier which renders the ant'igen non-

imrnunogenic. Further, thìs modifier may be destroyed by freeze-drying'

permi tt'ing the resumpti on of ant'ibody synthesi s '

(iv) Immunol oqical repressor

The foetus produces, or contains' an immunologìca'l suppressor

wh'ich is found in the 1500 g extract of the t'issue homogenate' This

repressor acts on the rabbit's humora'l 'immune system, preventing the

production of antibodies to the foetal antigens. The ìyoph'ilìzation

of the extract destroys the repressor, and antibody synthesis now

occurs.

In the following sections, these four concepts are exam'ined

further,to determine which may su'itably expla'in th'is unusual phenomenon

75.

SECTION III : IMMUNOGENICITY OF MURINE FOETAL EXTRACT

In the previous section, it was proposed that freeze-dryìng

may cause the djsruptìon of ce'll organeì1es, hence reìeasing rnewl

ìmmunogenic material. Alternatively, it was also theorizeC that

lyoph'i I i zatì on may destroy a modi f i er , whi ch somehow pre'rents ant'igens

from being immunogenic.

These two possibilities were examined by characterizing the

anti gens , both quanti tati vely and el ectrophoretì ca'l 'ly. Muri ne

foetal extracts were chosen for these experiments because these NFD

preparations were unable to evoke antìbody production in any

immunized rabbits, whereas rat foetal extracts did produce Some

humoral response i n i nocul ated m'i ce.

1. Cell 0rqanelle Ant'igens

To determine whether lyoph'ilization s'ignìfjcantly adds to

the immunogen poo'l by disrupting ce'|1 organe'lìes, fresh mouse

foetuses were homogenized and centrifuged at 105,000 g for one hour

to remove all und'isrupted cell organelles. The resultant supernatant

was dialysed and examined for the foetal antigens which are capable

of evoking humora'l 'immunìty. The ant'ibody used to detect these

antigens was raised to FD foetal extracts. If the concept that

immunogenjc materjal is released only after" freeze-dry'ing is correct'

then ant.igens should not be detected in the 105'000 g supernatant

since their source (the cell organel'les) has been removed. The

76.

immunodìffusion patterns of Figure 5.1, however, show that the

105,000 g extract does contain antigens which are capable of evokìng

immun'ity. Further, these antigens exhibit a reaction of identity

w'ith both FD and NFD 1500 g extracts. It may be argued that. altltough

the antigens are present'in the 105,000 g extract, theìr presence

may be due to earl'ier disrupt'ion of the organelles, and lyoph'iljzation

results in comp'lete breakage, causing the quant'ity of immunogens to

rise to a'level high enough to evoke humoral immunity. This is'

however, unlikely as the quantity of immunocompetent ant'igens in

both the 105,000 g extract and the FD,1500 g extract is comparabìe

(raute 5.4).

Thus it would seem that the breakage of cell organe'lles would

not signifìcant'ly add any more antigens to the immunogen poo'l .

2. Antiqen Modifier

The presence of an antigen modifier was exarnjned by

determining the number of ant'igens in the foetal extract, which

could be detected by the anti-foetal serum.

If an ant'igen mod'ifie¡is active'in these extracts, then it is

unlikeìy that one such molecule wjll alter the jmmunogen'ic'ity of a

variety of antigens. From Figure 5.2, it can be seen that there

are at least four, possìbly five, antigens which by their reactìv'ity

aga'inst this antiserum are able to ra'ise an ant'ibody'in rabbits.

Further, these antigens have a diverse range of electrophoret'ic

mobilities, some also being found 'in adult t'issues as well '

IGURE 5.1

Immunodiffusion examination of 105,000 g extract of murine foetus

r the presence of foetal antigen.

þ = 105,000gextract

d = 1500 g non-freeze-dried extract

f = 1500 g freeze-dried extract

a = adutt tissue extracts (This extract was used as a

positivecontrol.Previousstudiesfromthislaboratoryhaveshownthatfoetatextractsdocontain some adult antigens' )

{

p

-

I-ç

77.

TABLE 5.4

Ant'ibody Preci Pi tati onResponse of:

ANTIGEN DILUTION*

NEAT 2 4 B 16 32 64 r2B 256

**105,000 g

1500 s (FD)

***t +++ +++ +++ ++ ++ ++ +

+++ +++ +++ +++ ++ ++ ++ +

The ability of a constant concentration of anti-foetal serum to

precipitate various concentrations of antigens from a 105,000 g and

1500 g extract of mouse foetal extract'

*The antigen dilution is recorded as a reciproca'l dilution

(e.g. 2 -- >").

**Each extract was initìally adiusted to 10 mg of proteìn per ml'

f *** + r indicates the degree of precip'itation'

FTGURE 5.2.

Tmmuno-electrophoresis of murine foetal, and

The central trough contains anti-serum raisedextracts.

fcetal extracta = adult extract

adult extracts

against F. D. foet_a1

f : N.F.D.

N

ùs,

f,

z'9

78.

ilid

I

It'is unlikely that a sìng]e mod'ifier can alter the immuno-

gen.icity of such a d'iverse range of antigens. It is equally

unl.ikely that the foetus needs to produce a large number of

modifiers, and more ìmportantly, each one being 'lyophilization-

labile. The concept that freeze-dry'ing makes the antigens more

.immunogenìc would requ'ire a large number of antigens to be

lyophìlizatìon-labile - an improbable situation as such antìgens

are rare.

3. Conclusions

From these results ìt l¡||as concluded that the inab'ility of

NFD foetal extracts to raise an antibody in rabbits was not due to

the presence of an antìgen mod'ifier or to the lack of immunogen'ic

materi al due to the undi srupted state of cel 1 organel I es ' l'li th

the further improbability that the animals used in th'is study were

non-immunocompetent, it was proposed that the foetal extract contained

an immunolog'ical repressor, i.e. a substance which would 'inhibit the

productionofantibod'iestothefoetalextracts.

To accurate'ly determine whether such a repressor was present'

an isolation or partiaì purificatìon of the med'iatjng mo'lecule(s)

wasrequired.Twosuchmethodswerecons.idered:

(a)Co]umnchromatographyonSephadexG-200usingan0.85%

saline solvent.

(b) Ammonium sulphate fractionation'I

r

i

.I

n{,',,

,l

79.

The first technique'is probab'ly the better of the two, because the

inert Sephadex and saline would not be expected to alter the

activity of the repressor. The second method, although considered

"Safe" by biochemists, may have a similar effect aS freeze-drying -

particular]y since both of these techniques remove water' Thus the

,,denaturation,'of the repressor(s) by the ammonium ions may Oerntit

the production of antjbodies to non-freeze-dried foetal extracts -

a negatìVe result which would neither confirm nor reiect the presence

of a repressor.

Attempts at partialìy purifying murine foetal extracts by column

chromatography were not successful. consequent'ìy, the only remainìng

method for direct'ly showing that the foetus produced an 'immune

repressor was by using the foetal extract to 'impair antibody

production to an antigen' not produced by the foetus'

Because such an experiment required a 'large number of animals'

it was decided to inoculate m'ice with rat foetal extracts' rather

than rabbits with murine foetus. The use of a dìfferent animal

system was considered as valid because both rabbit and murine

antibody production to NFD foetal extracts was r'epressed'

tII

I

r

\

80

SECTION IV : IMMUNOLOGICAL REPRESSION

f. introduction

The presence of a lyoph'ilizatjon-labi'le immunological repressor

was examìned by immuniz'ing mice with bovine serum album'in (gSR) ' and

non-freeze-clrìed foetal extracts. If an ìmmune repressor was present

in the foetal extracts, then there should have been a repression of

antibodY Production to BSA'

2. Methodology

Threegroupsofmicewereinocu]atedwitheitherBsAand

saline(groupl),BsAandNFDratfoetalextract(group2),orBSA

and FD foetal extract (group 3). Antibody product'ion by the first

group w.iìì confirm the use of BSA as a suitable antìgen' The th'ird

group of animals serves as a control to determine whethelinject'ing

BSA and foetal extracts overloads the immune system, resutlting ìn

immune paralys'is. If thi s shoul d occur, there woul d be no ant'ibody

synthesis in the second group, causìng confusion between inh'ib'ition

by a repressor, and immune paralysis med'iated repression'

TheBsAwasmixedwiththefoetalextractandgivenintra-

peritonealìy according to the dosage and ìnoculation routes descflibed

in Appendix V.

HÌ

tÌI

I

t

81.

3. Resul ts

The antjbody product'ion by animals in the three groups is

presented in Table 5.5. All animals of the group given BSA and

saline (group 1) were able to raise ant'ibodies to this antigen. Thus

the concentration of ant'igen g'iven is suffic'ient to rajse antibodies

in mìce. The an'imals given BSA and NFD foetal extract were also able

to raise antibodies to the antigen. Conversely, the animals inlmunized

with FD foetal extract did not develop antibodies to the BSA.

Initial ly, it was thought that 'immune para'lysis had occurned in the

animals of the third group. However, this is unlikely because the

group given BSA and NFD foetal extract had the same quantìty of

antigenic material jniected as the group given BSA and FD foetal

extract.

These results are a direct contradiction of the supposition

that the NFD foetal extract contains an active immunologìcal suppressor

which should prevent the product'ion of antibodies to BSA.

Th.is apparent contradìctjon could be exp'lained by propos'ing that

the repressive process still occurred, but ìts inhibitive action was

specific only for foeta'l antigens and did not signìficantly alter the

antibody productìon to BSA. Under these condit'ions, it would be

expected that group 2 animals would raise an ant'ibody to BSA on'ly

and not foetal extracts. From Table 5.6 it can be seen that th'is

situation did occur. Similarly, group 3 animals would be expected

to ra.ise antibodies to both BSA and foetal extracts, as repressìon

has now been eliminated by freeze-dry'ing. This, however, did not occur'I

t

82.

TABLE 5.5

1

BSA + Saljne( coNTRoL )

2

BSA + NFD

3

95¡ + FD

No. positive

Total No. animals

% positive

t2

16

75

9

72

75

0

12

0

x2 Ana'lYs i s p < 0.05, therefore difference between groups 2 & 3

is sign'ificant.

Percentage number of animals able to raise antibodies to BSA, when

given concurrent immunizat'ions of saline, non-freeze-dried or freeze-

dried foetal extracts.

ANTI-BSAÃffiTVTTY

No. positiveTotal No. animals

% positive

ANTI-FOETALACTIV TY

No. pos'itì ve

Total No. animals

% Pos'i ti ve

TABLE 5.6

Group 2

BSA + NFD FoetAI

9

12

75

2

t2

t7

83.

GrouP 3

BSA + FD FoetAI

0

L2

0

11

12

92

STATIST I CS:

(f) nnti-BSA activitY :

(2) Anti-foetal activìtY :

(see Tabl e 5. 5) .

x2 analysis - p < 0.05, therefore the

two groups are si gn'if icantly di f ferent '

Percentage of animals inoculated with BSA and foetal extracts'

able to develop humoral immunity to these ant'igens.

B4

The aninrals of group 3 only raised antibody to the foetal antigens

and not BSA - an unusual result as both antìgens should have raised

an ant'ibody ì n mi ce.

A'lthough these resul ts conf i rm that repress'ion i s sti I I occurrì ng

in animals given NFD foetal extrac'L,s, it is unusual that antibodies

to BSA djd not develop in animals immunized with BSA and FD foetal

extracts (group 3). A poss'ibìe explana't'ìon is that an antigen 'in

the FD foetal extract competed wìth the BSA for the productjon of

antibodjes. Antr'genic competìtion between BSA and othet^ antigens has

been extensivel.y reported in the literature (see Liacopoulos and

Ben-Efraim, 1975). These authors have concluded that competition may

occur betvleen two antigens gìven simultaneously ìn the same Freund's

adjuvant emulsion. In the experiments desc¡ibed in thìs chapter'

the BSA and foetal extracts were m'ixed together and emulsified with

Freund's adjuvant.

Aìthough this hypothesis 'is tenable, it would be expected that

the NFD foetal antigens would also compete with the BSA' hence

elìminat'ing the latter immune response. However, this did not occur

in these experiments (Table 5.6). To expìa'in why competìtion did not

occur in thìs case, it is necessary to consider the mechanism of antigen

competit'ion. In the mouse experiments described here, ìt has been

proposed that a specific repressor prevents the production of antibody

to foetal antigens. In the next section of this chapter, it is

further proposed that this repression occurs at the macrophage site'

Gottl ieb et aL. (tglz) have reported that antigen competit'ion probably

occurs at the macrophage site, cluring ant'igen hand'ling and/or

85.

processing. consequently, when BSA is given together wjth the foetal

antigens, the immune reaction to the latter antìgens is suppressed'

hence elimjnating the conrpet'itjon wìth BSA. Thus BSA antjbody synthesis

now occurs. l,Jhen FD foetal extract is given with the BSA, however'

repression no longer ex'ists, competition l:etween both antìgen types

is inìtiated, and an immune response to the foetal ant'igens only occurs'

The concept of a competition between BSA and a foetal ant'igen'is

further supported by evìdence from Ruoslahti et aL. (1976) who have

shown that human albumin, and the foetal antigen AFP' shol considerable

amino acid sequence homology. (AFP is present in large quantìties in

rat foetal homogenates.) Further, Harel et aL. (tglz) have shown that

another globu'lin, bov'ine gamma gìobu1in, is able to compete with BSA'

resulting'in eliminat'ion of the latter's antibody production'

Thus, from these reports, jt 'is probable that ant'ibody production

aga'inst BSA when i niected simul taneously wi th FD 'îoetal extracts ' i s

suppressedbecauseofantigencompetition.Theresultspresented

here suggest that repression of antibody synthesis is specìfic for

antigens found in the foetal extract'

SECTTON V : DISCUSSION

The inability of rabb'its and mice to raise ant'ibod'ies to

heterologous foetal extracts has been demonstrated. After elimjnat'ing

al l othen 'l'ikely expl anati ons of thi s phenonlenon, 'it was proposed

that the foetal extracts contained a substance wh'ich was able to

B6

specifically repress the product'ion of antìbodies to the foetal

antigens. Further, this repressor was lyophìljzat'ion-labile'

Three .important questions have arisen from this work:

(i) What 'is the nature of the repressor?

(ii) llhat type of immune inhjbition does th'is molecule(s) exhib'it?

(ii.i) How does freeze-drying destroy the repressor(s)?

It is difficult to answer these questìons without further experi-

mentation. Holever, there is a considerable amount of information

in the lìterature from which a model of the nature and mechanism of

the repression may be formulated, and ultimately examined experimentally'

(i ) Nature of the rePt9::9r

To answer the fjrst quest'ion, it is desirable to list two

Ímportant criteria which the repressor(s) must fulfil:

1. Since the repression is specific for ant'igens in the foetal

extracts, then the antigens themselves must somehow be

medìatorsoftherepression.Itìsunìikelythatthe

antigens are immunosuppressive.* Rather' another molecule(s)

directly interacts with the ant'igens and th'is blocks the

humoral immune system from raising ant'ibodjes to these

anti gens .

2.Sincetherearea.largenumberofantjgens'eachw.itha

different electrophoretìc mobil ity (see Chapter V, Section

II), there must also be an equa'l number of repressors' each

*Th... have been a number of studies reported' wh'ich suggest that

cr1-foetoprotei n may be an 'imtnuno-regul ator (Tomasi et aL' ' I976;

Sheppard et aL., !g76; Goeken and Thompson, I976). However, these

stud'ies have been i nconcl us i ve , as yet '

87.

beìng lyophilization-labìle, and spec'ific for one ant'igen.

consequent]y, the repressors should be within one class of

chemical substance, i.ê. sharing many characterìstics, but

still able to confer specificity on a particular ant'igen - in

this case, foetaì antìgens.

Antibodies are molecules which conform to both of these criteria'

Although a different antigen specificity exists, the chemical compos'itjon

of the antibod.ies ìs essentìa'l1y the same. It'is difficult to envjsage

any other class of molecules wh'ich may conform to these establ'ished

crì teri a. Assumi ng that ant'ibocly mol ecul es may be the repressors '

it is then poss'ible to consider the mechanism of theìr actìon and

their labilitY to freeze-drying.

(i i ) Mechan'ism of action

It is unl.ikely that repress'ion is caused by antibody mask'ing

every antìgenìc determinant on all molecules 'in the extracts' A more

plaus'ible explanation is that only some of the antigens are bound to

antìbodies, and these compìexes specifically block the product'ion of

"new" antibodies. A similar situatìon has been reported by Mishell

and Dutton (tgoz), who showed that when anti-sheep red blood cell was

mjxed wjth an imrnunogen'ic dose of sheep red blood cells, thjs conjugate

was able to repress the appearance of antibody-form'ing cells to this

antigen. Nossal and Ada (197i), rev'iewjng this area of immunology'

have concluded that the repression occurs early in the immune process'

poss i b'ly at ant'igen-macrophage i nteractì on '

B8

The complexitrg ant'ibody must be of either foetal or maternal

origin. Although some specific globulìn synthesis by the foetus has

been reported (Solomon, 1971), it is improbable that this antjbody

forms the anti gen-anti body compl exes . consequent'ly, th'is ant'i body

ntay be of maternal orig'in. A number of workers have reported the

presence of maternal antibody against antjgens of the developing

foetus. Further, they have shown that antìbody synthesis in neonatal

animals may be repressed by the presence of ìarge quantities of

spec.ifìc maternal antibody (Greengard and Bernstein, i935; Cooke

et aL., 1948; Barr et aL., 1953). The proposed mechanism of action

of the repressor found ín the foetal extracts descrìbed in this chapter

is consistent w'ith the results reported by other workers, i'e' the

maternal antibody in the foetus forms antigen-antìbody complexes'

vrh'ich may prevent the productìon of ant'ibody to these antigens by

heterol ogous hosts.

(ì i i ) Lyssþill:e!ie!:liÞlulv-gf ress 1 0n

The experiments described here have shown that the

repression phenomenon is labile to freeze-drying- To explain this

labiìity, in terms of an active antigen-antìbody repressor, freeze-

dry'ing must either destroy the antibody portìon of the immune complex

or its bond with the ant'igen be broken. It is unlìke'ly that

lyophilizatìon destroys the antìbody 'itself , because freeze-dryìng is

rout.inely used to preserve antisera from degradat'ion. Thus the

- lab.ile po'int must be the bond between the ant'igen and antibody'

B9

There has not been any report in the literature which describes

a dissociation of antibody-antigen compìexes by freeze-dryjng'

There have, however, been numerous reports of high salt concentrations

caus'ing the dissocjation of these complexes into free antibody and

anti gen (Hei del berger et aL., 1936; He j de1berger ancl Kendal I , 1936) '

It is possìble that the foetal extracts used'in these studjes had a

hypertonic salt content after freeze-dryìng and subsequent

re-constitut'ion. This would result in a breakjng of the ant'igen-

antibody bond, and the subsequent elìminat'ion of the repressive

phenomenon. The valjdity of thìs observat'ion is clearer ìf the

equiì.ibrium reaction for antigen-antibody complex formation js

cons i dered:

'isotonic salt -tsiz Ag

(antigen)

Ab

( anti bo

+ Ag*Ab

(complex)ìypertonic salt

dv)

During the preparat'ion of murine foetal extracts, for rabbit inoculation'

the freeze-dried'inocu'lum was re-constituted to 15 mg of solid materials

per ml of d.ist.illed water. This results 'in the salt concentration of

the inocul um bei ng e'levated, hence shi f ti ng the equ'i ì ì bri um reacti on

to the left. Raffel (tgOt) has reported that only a 15% increase ìn

salt content is sufficient to cause this sh'ift' Consequently' it

may be poss.ible that a smalj elevation in salt concentration'in the

murjne foetal ext,racts was able to destroy the repressive antìbody-

antigen comPlexes.

90

Foetal rat extracts, for inoculatìon 'into mjce, were re-

const'ituted to their pre-'lyoph'iÌization volume. Thus they would not

be expected to contajn a sìgnificant quantity of dissociated immune

compìexes, because the concentration of the salt has not been altered.

Horvever, during the 'lyoph'il'ization procedure, there 'is a

concentrating of the salt that ìs present, and hence dissociation

may occur here. However, on reconstitution, reassociation would be

expected over a period of time because the extract solutìon has been

restored Lo isotortìc strength (see equilibrium reactìon) ' This

si tuati on .i s probab'ly ref I ec'ted experimental 1y i n Tabl e 5. 3, where

37.5% of an.imals given FD foetal extracts unexpectedly dìd not raise

antibodies. This may have been due to re-aggregat'ion of antigen and

ant'ibody i nto jrnmune comp'lexes.

Thus it is proposed here that the immune repress'ion demonstrated

in these studies is not specifical'ly reversed by lyophil'izatìon.

Rather it.is the eleva.tion of salt concentrations brought about by

lyoph'i I i zat'ion/reconsti tuti on procedure that reverses the repressi on '

( i v) !yuugl.y

In thjs chapter it has been demonstrated that antibody

synthesìs against foetal extracts occurs when 'lyoph'il i zation

is included in the preparative procedure. This phenomenon is thought

to be due to the action of an ant'igen-specific humoral immune system

- repressor. A theoretical model, formulated to expìain the mode of

repression, has been presented. However, it'is'important to note that'

'raldeqo lxau aql u! rêtlunJ passnlslp s! Lapou pasodord aql Jo 6utlsal

LPluaullJadxaaql.|.apouaqxJosldacuocaqlsl¡oddnsÁ¡6uo"t1s

p[e!J aq1 6uLra^oc arnlerallL aql 'a^!1e¡noads s¡ Lapoul stLll a[lqm

'I6

92

SECTION I : DEVELOPMENTAL ANTIGIN REAPPEARANCE IN CANCER

In this thesjs, the methorlology for the preparation of antibody

to both foetal and placental antigens has been the same, i'e' an

anti serum was rai sed to each extract, and then an attempt was made to

renderit speci f ic for ei ther foetal or pl acental antì gens by

absorptìon w'ith aclult tjssue powders. Thìs technìque was desìgned

to detect a range of foetal and placental antìgens, without

specifying the t'issue of o¡igìn of the antigen. However' one maior

prob'lem prevented the effective use of these systems to detect

developmental antigens in tumours - both foetal and placental extracts

corrtained a high concentration of adult antigens' Thus the antisera

to these extracts had a h'igh proportion of antibody whjch was

directed against the normal adult antigens' e.g. the antiserum to

intact placenta'l cells was exclusively reactive against adult red

blood cells, and not placental ant'igens'

For this type of system to be used successfully, the'immune

response to the adult antigens should be el'iminated or significantly

reduced. This may be done in one of two ways:

(a) Immun'ization of homologous hosts instead of heterologous

anìmal s.

(u) prior purif.ication of the extracts, resulting in a

relatively higher concentration of developmental antigens

be'ing available for stimulation of the immune system'

93

Both methods have been briefly d'iscussed in the earlier chapters.

The second concept of purification, however, requires Some further

discussion as immunizat'ion of homologous hosts may not always result

in antibody production (see Chapter IV).

The purification of developmental tissue extracts may be

achjeved by eìther:

(a) fractionat'ing the extract into smaller pools' or

(b) selecting a specific foetal organ or placental tissue.

The first method of purification may involve either Sephadex G-200

or ammonium sulphate fractionation. These two techniques may ensure

that each fraction contains a higher proportion of certain foetal

antigens than the unfractionated extract. Thus the immunizing host

would receive a larger quantity of foetal antigens. This method may

al so be appl i ed to p'lacental anti gens .

The second method involves the selection of one particular

foetal organ and rais'ing an antibody Eo either a soluble or membrane

extract of thjs tissue. Again, this may eliminate excess antigens'

and in particular, d large quantity of adult antigens. This type

of approach has been used successfully in thjs laboratory for the

preparatìon of anti-AFP,

tsiz, rather than used whole foetus, amnìotic fluid was used as

the vaccine for the preparation of the ant'iserum (Okita et aL., I974).

A simiìar approach may also be adopted to rajse antibody to both

membrane and cytoplasm'ic extracts of the placenta. In this method'

it is desirable to firstly perfuse the placenta with jsotonìc saline

94

in order to remove as much blood as possible. Following this' a

particular area of the placenta is selected and an extract (either

membranous or cytoplasmic) prepared for immunization. Sjm'ilarly'

other areas of t'issue may be chosen. Thus there is an eliminatìon

of excess antigen, and an effectìve increase in the concentration of

p'lacental specific antigen enter.ing the immuniz'ing host.

In conclusion, although the methods proposed above may st'i11

be effectìve as a means of preparing spec'ific antibody for detecting

developmental antigens jn tumours, it is not possible to produce a

single antiserum wjth multiple foetal and p'lacental antigen

specific'ity.

SECTI0N II : IMI'IUN0L0GICAL REPRESSI0N

Thea.imofth.isstudyhasbeentodeterm'inewhetherthefoetus

produces a 'lyophilization-labile immune repressor or antigen

mod.ifier. It has been proposed in this thesis that the rat and

mouse foetal extracts conta.in ,modi f i edr anti gens wh'ich are abl e

to specifìcally inhibjt the production of ant'ibodies to these

antigens'in xenogenic hosts. Further, this modificatjon process is

an attachment of antibod'ies to the foetal antjgens' and it is this

antigen-ant.ibody complex wh'ich represses antibody product'ion to the

foetal ant'igens.

'However,itisimportanttonotethatthisconceptislargely

specu'lative, and aìthough supported by a number of experiments

95.

reported jn the literature, no direcÈ experimental evidence has been

presented in this thesis. Further, a direct contradiction of this

theory is evident from the results of tlishi (1970), who has been able

to raise an antibody to AFP by imrnunizing horses with AFP-anti-AFP

compìexes. This evidence emphas'izes the need to establjsh the vaf idity

of the theory presented'in Chapter V and to use th'is to possibly

determine Nishi' s d'iscrepancy.

There are three important aspects of this theory which may be

examined experimental'ly:

(a) Are there sìgnifìcant numbers of antigen-antibody complexes

in the NFD foetal extracts?

(b) Can these complexes' if they exist, be dissociated during

freeze-drying and further reconstitution wjth distilled

water?

(c) l,.lill the addition of antigen-antibody complexes to a FD

foetal extract result in the inhibition of ant'ibody

product'ion to the foetal antigens?

These experiments may assist jn confirm'ing or rejectìng the proposed

theory.

The relevance of the proposed model to tumour immunity may be

considered in view of the many similarit'ies between foetal and

cancerous cells (Chapter I). Currie (fg0g) has shown that maternal

T-lymphocyte attack against the developing foetus may be repressed

by bl ock'ing factors whi ch cons i st of anti gen-ant'i body factors . Such

96

blocking factors have also been impl'icated as a defense mechanjsm

which tumours possess to prevent destruct'ion by the hostrs immune

system (Sjogren et aI 1971 Chalmers et aL., 1976).

Evidence in favour of B-ìymphocyte (i.e. ant'ibody production)

suppressi on by ant'ibody-foetal ant'igen compl exes has been I imi ted

to a few studies from our laboratory. Sjx attempts have been made

to raise antibody to NFD 1500 g extracts of hepatoma tissue. Each

attempt was found to be unsuccessful. By repeatìng the experiments

in the previous chapter with tumour rather than foetal extracts, the

possible role of antibody-foetal antigen complexes in the reguìation

of B-lymphocyte immunity to the developing tumour may be determined.

The model of B-'lymphocyte suppression presented in this thesis,

requires further examination and verification. However, ìts

importance in tumour, foetal and transplantation inrnunology, is such

that the basic concepts of this model may prov'ide an impetus for

further investigation.

97.

CHAPTER IV

Sabìne(personaìcommunjcatìon),us.ingthesamemethods

descrjbed in this chapter, has also been unable to produce a

specific antìserum capable of detecting carcino-p'lacental antigens

in syngeneic rat tumours.

CHAPTER V

An ìmportant observation has been made by a number of

coìleagues, during d'iscussions of the results presented in thìs

Chapter.Theyhavenotedthatiftherepressorisspecìfìcfor

foetalantìgensonly,thentherabbitsandmiceshouldhave

raised antìbodies to the adult component of the foetal extract'

However, it ìs 'important to note that the adult component

consists of both maternal and paternal antigens. consequently'

the foetal extract would contajn paternal antigen-ant'ibody

complexes, because these antigens would be recognized as foreign

by the maternal immune system' Hence antibody synthesis to

these ant'igens in heterologous hosts would be inhibited'

The maternal antigens should stimulate antibody product'ion in

heterologous hosts, as there shoujd be no antigen-antibdoy complexes

present.However,the]eve]oftheseantigensìnthefoetalextracts

isprobablytoolowtoproduceenoughantibodywhichcouldbe

.detected by the insensitive immunodiffus'ion system emp'loyed in

il'G

I

II

r

these studies.

*.'{

Ê+

âæ

98.

APPTNDIX I

IMMUNIZATION SCHEDULE FOR THE PRODUCTION OF RABBIT ANTI-SãRUM

TO MOUSE FOETAL EXTRACTS.

Day Iniection No. Inocul um Dose

gen Adiuvant)* TYPe : (ml )

si te***

Anti(ml

1

B

L5

29

1

2

3

4

1.0

1.0

1.0

1.0

FCA

FCA

FCA

FIA

1.0

1.0

1.0

1.0

lm

tm'lm

1m

*Antigen concentration was 15 mg solid matter (after

lyophilization).

**FCA : Freund' s ComP'l ete Adj uvant

FIA : Freund's IncomPlete Adiuvant

**:k5. ç. : SUbCutaneggs

i.m. : intramuscular

Animals were bled 10 days after the final inoculation'{l

Ir'

IT

I

I

rI

99

APPENDIX II

IMMUNIZATION SCHEDULE FOR THE PRODUCTION OF RABBIT ANTISERUM TO

SALINE SOLUBLE EXTRACTS OF RAT PLACENTA.

Day Injection No. Inocul um Dose

Anti gen* Adiuvant(mÍ) Type : (m])

Si te

I

2

3

4

1

8

15

29

1.0

1.0

1.0

1.0

FCA

FCA

FCA

FIA

1.0

1.0

1.0

1.0

s. c.

s.c

s.c

tm

*antigen concentration l,las I mg protein per ml '

Animaìs were bled 10 days after the final inocu'lation.

100.

APPENDIX III

IMMUNIZATION SCHEDULE FOR THE PRODUCTION OF ANTISERUM TO RAT

PLACENTAL ANTIGENS.

1. HETEROLOGOUS ANTISERUM TO l^lHOLE

PLACENTAL CELLS.

Day Injection No. Inocul um Dose*

(ml )

Si te**

1

6

11

2

2

2

1

2

3

nprlil'tv

IV

*concentration of cells was 3.6 x 108 per ml'

**n. p.

i.m.

i.v.

nape of neck

i ntramuscul ar

i ntravenous

Animals were bled at 7 day intervals after the final

i nocul ati on

101.

2. HETEROLOGOUS ANTISËRUM TO CRUDE

PLACENTAL CELL MEMBRANE EXTRACT

Day Iniect'ion No. Inocul urn Dose

Anti gen Adi uvant(mi ¡* TYPe : (ml )

Si te

I

2

3

4

I

15

29

43

1.0

1.0

1.0

1.0

FCA

FCA

FCA

FIA

1.0

1.0

1.0

1.0

i.m

i.m

i.m

i.m

*concentration of antigen was 10 mg prote'in per m] '

Animals were bled 14 days after the finaj inoculation'

3. HOMOLOGOUS ANTISERUM TO CRUDE

PLACINTAL MEMBRANE EXTRACTS

Day Iniection No. Inocul um Dose

Anti gen Adi uvant(ml )* Type : (ml )

Si te**

1

15

22

43

57

1

2

3

4

5

0.5

0.5

0.5

0.5

0.5

FCA

FCA

FCA

FCA

FIA

0.5

0.5

0.5

0.5

0.5

i.p

i.p

i.p

i.p

i.p

*ant'igen concentration was 10 mg protein per ml '

**i . p. : i ntraPeni toneal '

Animals were bled 14 days after the fjnal jnoculation.

102.

APPENDIX IV

IMMUNIZATION SCHEDUI-ES FOR THE PRODUCTiON OF ANTIBODY TO NFD

MURINT FOETAL EXTRACTS.

1. FOOTPAD PROTOCOL

Day Iniection No. Inoculum Dose

Anti gen Adi uvant(ml )* Type : (ml )

Si te

1

2

1

11

0.5

0.5

FCA

FiA

0.5

0.5

f.p

f. p.

*concentration of the ant'igen was 10 mg prote'in per m] .

Animals were bled 10 dals after the final jnoculation.

2. SUBCUTANEOUS. INTRAMUSCULAR

PR0T0C0L (i.e. s.c./i.m. )

Day Iniection No. Inocul um Dose

Antìgen Adiuvant(ml )* Type : (m] )

Si te

I

2

3

4

1

I15

29

1.0

1.0

1.0

1.0

FCA

FCA

FCA

FIA

1.0

1.0

1.0

1.0

s.c.

s.c

s.c.

l.m

*concentration of the antigen was either 8 or 3 mg

protein Per ml (see Table 5'1)'

Animals were bled 10 days after the final inoculation'

103

3. INTRAI'IUSCULAR PROTOCOL

Day Iniectjon No. Inocul um Dose

Antigen Adiuvant(ml )" Type : (ml )

Si te

1

2

3

1

15

29

1.0

1.C

1.0

FCA

FCA

FIA

1.0

1.0

1.0

'l

j

i

m.

m.

m.

*concentration of the ant'igen was B mg protein per m] .

Animals were bled 10 clays after the finaì inoculation.

4. INTRAVTNOUS-SUBCUTANEOUS-

INTRAMUSCULAR PROTOCOL

Day Iniection No. Inocul um Dose Si te

Anti(ml

qen Adi uvant)* Tvpe : (ml )

1

2

3

4

5

1

1

B

15

29

0.5

0.5

1.0

1.0

1.0

FCA

FCA

FCA

FIA

0.5

1.0

1.0

1.0

ì.v

s.c

s.c

s.c.

i.m

*concentration of the ant'igen was 3 mg protein per m1.

An-imals were bled 10 days after the final inoculation'

The same immun'izat'ion schedules were employed when raìsing

antibodies to FD extracts vlhich use these protocols (Table 5'2)'

104.

APPENDIX V

IMMUNIZATION SCHEDULE FOR THE PRODUCTION OF MURINE ANTISERUM TO

RAT FOETAL EXTRACTS, AND BOVINE SERUM ALBUMIN.

Day Injection No. Ant'igen

Type : Dose(ml )

Adj uvant

Type : Dose(nrl )

Site

1

15

21,

25

30

37

1

2

3

4

5

6

Sal i ne 0.2

Foetal * 0.2

Foetal 0.2

Foetal 0.2

Foetal 0.2

Foetal 0.2

FCA 0.2

FiA O.?

FIA 0.2

FIA 0.2

FCAM** 0.2

FCAM 0.2

rprprp1p

rp1p

*concentration of foetal inoculum was 2.7 ng protein per ml '

Where BSA was given also, it was added to the foetal extract (or

saline) prior to inoculation, to give a final concentration of

15 mg per ml.

**FCAM : Freund's comp'lete Adjuvant with 5 mg per cent live

Mycobacter,iunphLeiadded(Sommervi.l1e,1967).

Ascites fluid from these an'imals was tapped B days after the

final inocul ation.

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