CLONAGE D ’ADNc SPECIFIQUEMENT IMPLIQUES DANS LA VOIE

Afin d’identifier et de caractériser des nouveaux gènes (et les protéines codées par ces gènes)

dont l’expression est modulée lors de l’activation de la cascade mitogénique de l’AMPc nous

avons :

• construit plusieurs banques d’ADNc. En effet, ce travail peut être réalisé au départ de

cellules thyroïdiennes en culture primaire stimulées par divers agents mitogènes, ou encore

au départ de thyroïdes stimulées in vivo.

• mis au point la technique de criblage différentiel à l’aide de sondes marquées non

radioactivement, par la digoxygénine.

• décidé de cribler différentiellement l’une des trois banque d’ADNc : la banque préparée au

départ de thyroïdes de chien stimulées in vivopar la TSH.

Ce projet est présenté et discuté dans l’article 2 : “Cloning of cDNA specifically

involved in the thyroid cAMP mitogenic pathway

RESULTATS

Hormone Research

Editer: J. Girard, Basel

Reprint

Publisher: S. Karger AG, Basel

Printed in Switzerland Hortn Res 1994;42:27-30

F. Miot

F. Wilkin

S. Dremier

N. Uyttersprot

F. Lamy

J.E. Dumont

C. Maenhaut

Cloning of cDNA Specif ically

Involved in the Thyroid cAMP

Mitogenic Pathway

Institute of Interdisciplinary Research,

University of Brussels, School of Medicine,

Campus Erasme, Brussels, Belgium

Key Words

Thyroid

Prolifération

Cyclic AMP

Cloning

Abstract

The activation of the cyclic AMP cascade in dog and human thyroid cells in

primary culture induces the expression of differentiated gene expression,

hyperfunction and prolifération. These programs are developed simultaneous-

ly in quiescent dedifferentiated cells. In this paper the strategy followed by our

group to define the genes involved in the cAMP mitogenic cascade is out-

lined.

Introduction

Tumorigenesis, i.e. increased and abnormal cell multi­

plication, results from an altered balance between cell pro­

lifération and cell death. Cell prolifération itself is regu-

lated positively by protooncogenes and negatively by an-

tioncogenes. Any constitutive activation of protoonco­

genes or inhibition of antioncogenes or cell death genes,

causes an imbalance, which after several steps may lead to

tumorigenesis. The définition of such genes, of the proteins

they code, of their function and of their alterations in

tumors is therefore the main objective of fundamental can­

cer research. The success of this strategy leads to better

diagnosis, classification and understanding of the physio-

pathology of the varions cancer diseases, and to new thera-

peutic approaches of these diseases. Such research has

already gone a long way in the élucidation of the genes

involved in two major classes of mitogenic pathways: the

growth factor receptor tyrosine protein kinase and the

phorbol ester protein kinase C cascades. Most of the known

oncogenes and antioncogenes belong to these cascades. The

purpose of our présent work is to define genes involved in a

less-studied cascade: the cyclic AMP mitogenic pathway.

We shall concentrate on early and cell-specific steps of

this latter pathway for 2 reasons: (l)The basic mecha-

nisms controlling directly prolifération are probably

simi-lar in ail cells. They are therefore now rapidly delineated

by the study of cells which can easily be manipulated

genetically: the yeast. (2) Tumors are very cell spécifie,

therefore presumably dérivé from alterations in the ini­

tial, cell-specific, part of the regulatory cascades. It is of

course realized that there may be many or only a few

unknown genes of interest in the process we study but we

believe that the cloning and characterization of even one

new oncogene or antioncogene would justify our effort.

Genes of the Mitogenic Cyclic AMP Cascade

Only 10% of growth control studies are devoted to the

differentiated épithélial cells which produce 90% of can­

cers. Using our model Systems of thyroid épithélial cells in

primary culture, we hâve demonstrated that cAMP con-

stitutes a sufficient mitogenic signal responsible in thy­

roid for the TSH-dependent goitrogenesis and tumor pro­

motion. The cAMP pathway is compatible with différen­

tiation expression, and hyperactivation of adenylate cy-

clase is found in some hyperfunctional autonomous ade-

nomas in thyroid and other endocrine tissues. In thyroid

cells, the cAMP-dependent mitogenic pathway strikingly

diverges from the mitogenic pathways elicited by EGF

and tumor-promoting phorbol esters, which are

associât-Carme Maenhaut, PhD © 1994 S. Karger AG, Basel

Institute of Interdisciplinary Research (IRIBHN), ULB 0301-0163/94/0422-0027

School of Medicine, Campus Erasme, Bldg C $8.00/0

ed with cell dedifferentiation. It does not involve the

phosphorylation and nuclear translocation of MAP ki­

nases [1] and the early transcription of c-jun. The kinetics

of c-myc mRNA accumulation, PCNA synthesis and of

cell cycle progression are very different [2], We can there-

fore hope reasonably to find new protooncogenes and

antioncogenes in the cyclic AMP mitogenic pathway.

Détection and Cloning

The genes will first be searched for in a System for

which material is not too difficult to obtain and which has

been thoroughly studied and well defmed in our group:

dog thyrocytes in primary culture. Corresponding cDNA

from human cells will be cloned immediately thereafter

from our new human thyroid cDNA library.

Several approaches will be used to clone and identify

new protooncogenes and antioncogenes of the cyclic AMP

mitogenic cascade.

(1) In the no a priori approach, no assumption on the

nature of interesting genes is made. Differential screening

of cDNA libraries constitutes a systematic approach

which could lead to the discovery of new genes [3]. This

methodology consists in constructing a cDNA library of

mRNA expressed in stimulated cells, and in screening it

by single-stranded cDNA of mRNA derived from quies­

cent or stimulated ceUs. Overexpression (protooncogenes)

and underexpression (antioncogenes) will both be looked

for. We hâve constructed three A, ZAP II cDNA libraries.

Two of them hâve been constructed starting from mRNA

expressed in early (1 h) or late (16 h) G1 in response to a

stimulation of the dog thyroid cells by EGF, TSH and

sérum. This combination of three mitogenic agents was

chosen because of its synergistic effect on cell proliféra­

tion (98% (3H)thymidine-labelled nuclei). Stimulation by

TSH alone would hâve led to less prolifération (40%

(3H)thymidine-labelled nuclei) but also to the expression

of différentiation genes. The third A ZAP II cDNA library

dérivés from in vivo TSH-stimulated dog thyroid cells:

the dogs were treated during 4-6 weeks with the antithy-

roid drugs methimazole and propylthiouracil to increase

their circulating TSH levels [4]. To differentially screen

this library, single-stranded cDNAs hâve been synthe-

sized by reverse transcription from mRNA extracted

from quiescent or TSH-stimulated thyroid tissue. For the

in vitro libraries, the probes were originated from quies­

cent or TSH, EGF and serum-stimulated cells (1 or 16 h).

However, under these conditions, if the potentially inter­

esting clones are effectively characteristic of the proliféra­

tion, they are not spécifie for the cAMP-dependent path­

way. These last ones will then be picked out by creating an

ordered sublibrary with the positive clones, and by differ­

entially screening it using probes derived from TSH or

EGF and sérum stimulated cells. To perform the difîer-

ential screening, we first developed a methodology based

on nonradioactive differential colorimétrie détection: hy­

bridization to target nucleic acids is performed simulta-

neously with nuclear probes labelled with digoxigenin,

fluorescein, or biotin, and subsequently detected by alka-

line phosphatase conjugates: each label is visualized by a

different color precipitate (green, red, blue) [5]. However,

this method is rapid but appeared not sensitive enough

and was thus abandoned on behalf of a chemilumines-

cent-based détection procedure, achieved with digoxige-

nin-labelled probes in combination with a secondary en-

zyme-labelled antibody complex and a chemiluminescent

substrate (AMPPD). In comparison to radioactive label-

ling techniques, chemiluminescent détection allows a

much more rapid détection of nucleic acids, and appears

at least as sensitive [6]. When starting the differential

screening, our first choice between the three libraries

tumed to the in vivo library, i.e. prepared from in vivo

TSH-stimulated thyroids, mainly for the following rea-

son: in this case, the screening occurs in one step, between

quiescent but highly differentiated thyroid tissue and 4

weeks-TSH-stimulated tissue, where only the cAMP-

dependent pathway is activated. As control tissue is well

differentiated, more than control cells in culture, this

should avoid the isolation of clones linked to différentia­

tion rather than prolifération. On the other hand, the

library from in vivo-stimulated thyroids might be poorly

enriched in cDNA corresponding to shortly expressed

genes (e.g. immédiate early genes) and even less so of

cDNA corresponding to genes with biphasic expression in

time with a longer downregulation than upregulation (e.g.

as c-myc). It might be more enriched in cDNA corre­

sponding to enzymes of the basic machinery of réplication

(e.g. DNA polymerases). It might also reveal differentia-

tion-related genes whose expression is further increased in

stimulated glands (e.g. iodide transport). Moreover, we

expected only a few positives by comparison with the

screening of the in vitro libraries from thyroid cells sub-

mitted to the combined EGF, sérum TSH treatment.

Of course differential screening in this way requires pos­

itive signais in the phage library with the population of

cDNA of one of the probes, i.e. will select phages expressing

mRNA which are more frequent in this population. Less-

frequent mRNA détection might require substractive clon­

ing. In a parallel approach at the protein level, proteins

which are phosphorylated or synthesized in response to the

TSH cyclic AMP pathway will be defined by comparison

RESULTATS

with cells in different conditions (vide supra). Microse-

quencing of these proteins or antibody génération will allow

the cloning of the corresponding cDNAs from our libraries.

(2) Search for new members of known families of

genes. A certain number of protooncogenes and antionco-

genes of the growth factor and phorbol ester cascades as

well as genes involved in the basic control of cell proliféra­

tion in yeasts are known. The mitogenic cAMP pathway

might use some common steps of these cascades or, even

more probably, related proteins. Indeed, we realize more

and more that in régulation pathways, each known pro-

tein in fact represents a member of an ever-expanding

family of isozymes. The specificity of a pathway in a cell

results from the use at each of the N steps of one of the M

proteins or of a particular combination of these proteins.

The use of common enzymes in the cAMP pathway, and

in the better known pathways, can be suggested by the

kinetics of appearances, translocation (e.g. nuclear) dur-

ing the prereplicative phase; by immunocytochemistry,

Northern and Western blotting, the kinetics of the phos­

phorylation of proteins by Western blotting and 2D gel

electrophoresis and autoradiography. By such techniques

we hâve just demonstrated that MAP kinases (p42 and

p45) activation which is obligatory in the growth factor

pathway does not take place in the cAMP pathway in the

thyroid [1]. The proof that a gene or a protein is involved

in the pathway can be given by the inhibition of this path­

way by microinjected antisense oligonucleotides or inhib-

itory antibodies. New members of the known families of

proteins involved at the successive steps will be cloned by

PCR methodology. We shall first study the known cAMP

responsive transcription factors (CREB, CREM, ATF

family, TTFl, PAX2, etc.) [7]. Of course the différentia­

tion may lie further in the growth pathways (e.g. at the

level of cyclins). These phases will be investigated later.

This last approach has been developed first on the tran­

scription factors CREB and CREM. CREB and CREM iso-

forms found in several tissues are generated by alternative

splicings of one gene, respectively. Our strategy is to com­

pare the expression of the genes in the thyroid of the dog

and the pig. It is established that TSH activâtes the prolif­

ération of the dog thyrocytes, however this effect has not

been observed in pig thyroid cells; it may even inhibit it.

On the other hand, EGF is a potent mitogenic dedifferen-

tiating agent of the thyroid cells of both species. The meth­

odology we followed is based on PCR amplification of

cDNA obtained by reverse transcription of mRNA pre-

pared from dog or pig thyroid cells in primary culture in

control conditions. The comparison of the base sequences

of the cloned genes coding from CREB and CREM has lead

us to choose spécifie primers able to achieve an amplifica­

tion of the two distinct genes. The PCR products are sepa-

rated by electrophoresis on agarose gel and transferred by

Southern blotting on a nylon membrane. This membrane

is then hybridized with a CREB or CREM probe.

(a) Expression of CREB transcripts: The primers cho-

sen to amplify specifically the CREB gene correspond on

the one hand to the 5' end of the CREB gene and on the

other to a zone coding for the leucine zipper of the pro­

tein. We obtain by PCR after hybridization a main band

of approximately 1,000 bp corresponding to the fragment

of the described CREB 341 (341aa) starting from dog and

from pig thyroid mRNA. As in other tissues, the expres­

sion of CREB seems to be ubiquitous.

(b) Expression of CREM transcripts: From the five

CREM isoforms known, four of them (a, (3, y. S) are

repressors of the transcription of certain genes and one,

CREM T, is an activator of the transcription [8]. This last

form is expressed more specifically in the brain and the

testis. Recently, it has been shown that the expression of

CREM T is switched on by FSH during spermatogenesis

[9]. We chose primers to amplify ail the isoforms (a, P, y,

t) like in the brain and the testis.

The repressor forms a, P, y seem to be amplified from

dog and pig thyroid mRNA. The activator form

t

was

présent in the dog thyroid cells. The absence of CREM x

in the pig thyroid was suggested by a PCR achieved with

primers spécifie of this form. The preliminary results sug-

gest that the presence of cyclic AMP-dependent activation

factors of the transcription could explain at least partially

why the dog thyrocytes proliferate under TSH activation

through the cyclic AMP cascade and the pig thyroid cells

missing such a factor do not. They should be confirmed

and extended to the thyroids of other species.

Identification

Once partial cDNA of supposedly important regulatory

proteins hâve been cloned, their rôle should be proven.

Sequencing may allow to relate them to a known family.

Kinetics of Northern blotting and/or in situ hybridization

in the prereplicative phase should demonstrate a regulated

expression. Microinjection of antisense oligonucleotides

should impair (for protooncogenes) or mimic (for antion-

cogenes) the TSH cAMP prolifération pathway allowing in

some cases the démonstration of a rôle of the gene. This

does not require prior détermination of the full sequence of

the cDNA. Criteria of interest of a cDNA will be its novelty

and the démonstration of a rôle in the prolifération. Full

transcripts of the selected cDNA should then be cloned and

sequenced. The sequence might suggest a function.

29

Définition of the Rôle ofidentifîed Genes in the

Prolifération in vitro and in vivo

Study of the Protein. To examine in detail the function

of the protein, it should be expressed in one of the Systems

used in the laboratory (Escherichia coli or Baculo virus).

This will allow the study in the relevant acellular Systems

(e.g. footprinting and gel retardation for transcription fac­

tors, proteins phosphorylation for kinases), the génération

of polyclonal antibodies for immunohistochemical local-

ization and coprécipitation experiments.

Rôle in the Intact Cell. Overexpression of the protein in

dog thyroid cells, microinjected with a suitable expression

vector or RNA, should allow either to mimic the effects of

activation of the pathway or inhibit this pathway in stim-

ulated cells. This approach allowed our laboratory to

demonstrate the acute mitogenic rôle of the adenosine A2

receptor [10].

Rôle in vivo. Démonstration of the putative oncogenic

or antioncogenic rôle of a gene or a protein in vivo

requires a System in which expression of the gene can be

stimulated chronically in spécifie cells in vivo, the trans-

genic mice. Transgenic mice with spécifie thyroid consti­

tutive expression of the SV40LT gene and the adenosine

A2 receptor gene hâve allowed our group to demonstrate

the rôle of these genes [4,12]. The hereditary transmission

of the oncogenic disease insures a stable supply of tumor

models for physiopathology and therapeutics. The Cross­

ing of the varions models allows in vivo chronic oncogene

complémentation to be studied.

Rôle in Human Tumorigenesis

Cloning of the Human Forms of the cDNA. A library of

cDNA from differentiated TSH-stimulated human thy­

roid cells has been constructed. Screening by homology of

this library should allow us to select and clone the human

counterparts of the dog cDNAs of interest.

Study of Expression in Normal Tissue and Tumors.

The expression in normal human tissue and in tumors of

the genes of interest will be studied by Northern blotting

and in situ hybridization. This should allow one to suggest

a rôle of overexpression or underexpression in tumorigen­

esis.

Search for Mutations in Tumors. When the rôle of a

gene of interest has been established, we shall systemati-

cally search for mutations in the genes by PCR synthesis

of fragments of putative crucial domains of the cDNA,

and by sequencing of these cDNAs. This approach has

now allowed us to demonstrate the first G protein coupled

receptor mutation leading to tumorigenesis (a mutation in

the TSH receptor in autonomous thyroid adenomas).

We hope this strategy will help develop our knowledge

of the oncogenic properties of the cyclic AMP cascade.

Acknowledgements

Supported by the Ministère de la Politique Scientifique (PAI), the

Communauté Française (Action Concertée), the Fonds National de

la Recherche Scientifique (FNRS, Fonds de la Recherche Scientifi­

que Médicale, Télévie and Association contre le Cancer). C. Maen-

haut is Chargé de Recherche du Fonds National de la Recherche

Scientifique, F. Wilkin and S. Dremier are fellows of IRSIA.

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