Proc. Nati. Acad. Sci. USAVol. 90, pp. 89-93, January 1993Cell Biology

A hepatitis B virus pre-S-retinoic acid receptor f3 chimeratransforms erythrocytic progenitor cells in vitro

(nuclear receptor/hematopoiesis/tumorigenicity)

MADELEINE GARCIA*, HUGUES DE THOt, PIERRE TIOLLAISt, JACQUES SAMARUT*, AND ANNE DEJEANt

*Ecole Normale Superieure de Lyon, Laboratoire de Biologie Moldculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de laRecherche Agronomique, 46, Allde d'Italie, 69364 Lyon Cedex 07, France; and tUnitd de Recombinaison et Expression Gdndtique, Centre National de laRecherche Scientifique, Institut National de la Sante et de la Recherche MWdicale, Institut Pasteur, 28, Rue du Docteur Roux, 75724 Paris Cddex 15, France

Communicated by Etienne Baulieu, September 4, 1992

ABSTRACT In this report, we investigated the transform-ing properties of retinoic acid receptor 13 (RARfi). The v-erbAprotein, which is the viral oncogenic homologue of the thyroidhormone receptor, was replaced by either the complete RAROB(fIR) or a hepatitis B virus pre-S-RAR13 (HIR) hybrid productin an avian erythroblastosis virus-based vector. In chickenhematopoietic cells, the HfR protein was able to transformerythroid progenitor cells, whereas no such transformation wasobserved with the wild-type PR protein. Moreover, the fullytransformed phenotype was observed even in the absence ofv-erbB, and HfiR-transformed erythroid cells grew indepen-dently ofgrowth factors and transforming growth factor a. Theanalysis of erythrocytic-specific proteins revealed that thetransformed cells were blocked at the colony-forming unit-erythroid stage and that the expression of the carbonic anhy-drase II gene, a gene normally regulated by thyroid hormones,was repressed by the HP3R protein. Finally, hepatocarcinomasrapidly developed in some chickens infected in ovo with virusesencoding either the normal or the hybrid HfIR, suggesting thatan inappropriate expression of the RARi3 gene may representan important event in oncogenesis.

Retinoic acid receptors (RARs) are members ofa large familyof ligand-regulated transcriptional factors that includes thethyroid hormone receptors (TRs) and steroid hormone re-ceptors. This nuclear receptor family has a modular structureconsisting essentially of a DNA-binding domain defined bytwo zinc finger motifs and a carboxyl-terminal hormone-binding domain. Upon binding of their respective ligands,these receptors interact with specific DNA sequences andregulate the transcription of adjacent genes (1). Three differ-ent RAR subtypes (a, /3, and y) have been isolated in humansand other animal species. They might exert their functions byregulating the transcription of various genes at different timesof development and in specific cells (2). Interestingly, RARaand RARP8 are more homologous to TRa and TRp, respec-tively, than to any other members of the nuclear receptorfamily (3, 4). Moreover, their similarities are not only struc-tural, but also functional: the facts that RARa binds to andactivates transcription from thyroid responsive elements (5)and that TRa and TRP can both form heterodimers withRARa (6) suggest that retinoic acid (RA) and thyroid hor-mones, acting through their respective receptors, could con-trol overlapping gene networks involved in the regulation ofvertebrate morphogenesis and homeostasis.The v-erbA protein is the viral oncogenic homologue of

c-erbA, the a form of the nuclear receptor for the thyroidhormone 3,3',5-L-triiodothyronine (T3). It contains an intactDNA-binding domain but has lost its ability to bind T3 (7, 8).Recently, it was proposed that v-erbA can act as a constitu-

tive repressor against both c-erbA and RAR (9-11). In thisstudy, we have investigated the possibility that other nuclearreceptors may be implicated in tumorigenesis. So far, twoobservations have raised the possibility that altered retinoidreceptors may have oncogenic properties: (i) in one humanhepatocellular carcinoma, the RARE3 gene is rearranged as aresult ofa hepatitis B virus (HBV) integration (12); and (ii) inacute promyelocytic leukemias, the RARa subtype is trun-cated by the (15:17) translocation (13-15).We have analyzed the transforming potential of RAR,3 by

using avian erythroblastosis virus (AEV) as a model system.AEV induces erythroleukemias and fibrosarcomas in sus-ceptible chickens and transforms erythroid progenitor cellsand fibroblasts in culture (16). The transforming properties ofAEV are determined by two host-derived oncogenes, v-erbAand v-erbB (17). The v-erbB product is a transmembraneglycoprotein showing extensive homology with the receptorof epidermal growth factor and transforming growth factor a(TGF-a) (18, 19). In this report, we constructed AEV-basedretroviral vectors in which the v-erbA sequences were re-placed by either the entire RAR/3 coding sequence or achimeric HBV-RARS3 form derived from a human hepato-cellular carcinoma (12). We show that the HBV-RAR,8 form,but not its wild-type counterpart, was able to transformerythroid progenitor cells. The transforming potential ofRAR,8 was further confirmed after in vivo injection, sincesome infected chickens developed hepatocarcinomas.

MATERIALS AND METHODSViruses. A partially digested Sau3A-Mae I fragment, 1360

base pairs long and corresponding to the complete codingregion of the human RAR,8 gene positions 318-1677 (3), wastreated with Klenow polymerase and inserted in place ofv-erbA, in phase with the gag sequences, in the genome ofAEV (20). Thus, the resultant retroviral vector genome,DR-B, contained, in addition to the v-erbB gene, a fusedgag-RAR,8 sequence, which is expressed from the viral longterminal repeat as a genomic RNA encoding a gag-RARPfusion protein of approximately 82 kDa. The PR-N retroviralvector has the same structural organization as pR-B exceptthat the v-erbB sequences have been replaced by the neo-mycin resistance gene (neo) from TnS (20). The HJBR-B andH/3R-N retroviral vectors were constructed similarly to ,R-Band PR-N, respectively, except that in the HpR-expressingviruses, the 74 N-terminal residues of RAR,8 were replacedby the first 30 amino acids of the pre-S1 envelope protein ofHBV as it occurred in the initial hepatocarcinoma (21). All

Abbreviations: RA, retinoic acid; RAR, RA receptor; TR, thyroidhormone receptor; HBV, hepatitis B virus; AEV, avian erythroblas-tosis virus; TGF-a, transforming growth factor a; CFU(s)-E, colony-forming unit-erythroid; BMC, bone marrow cell; T3, 3,3',5-L-triiodothyronine.

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Aviruses were rescued as Rous-associated virus 1 (RAV1)pseudotypes by transfection on chicken embryo fibroblasts(CEFs) as described (22).The pR-BN and H,3R-BN constructions are similar to the

,pR-B and Hp8R-B retroviruses, respectively, except that aneo gene was inserted downstream from the v-erbB se-quences (23). The pR-BN and Hp8R-BN retroviruses wereproduced as helper-free retroviral vectors in an avian leuko-sis virus-based packaging cell line: the QT6 Isolde clone (24).The viruses AEV(v-erbA, v-erbB), XJ12 (v-erbA, neo), and

AEV-H (v-erbB) have been described (19, 22).Protein Analysis. Cultured cells were lysed in 1% SDS lysis

buffer, and protein extracts were analyzed by electrophoresison SDS/12.5% polyacrylamide gels. After electrotransfer,the Western blots were incubated with specific antibodies,and detection of alkaline phosphatase was performed in thepresence of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate substrates (Promega).

Infection ofBone Marrow Cells (BMCs). BMCs from 1-day-old chickens were infected and grown in Methocel as de-scribed (25). After 7 days, colonies were picked and seededin liquid culture containing a-MEM supplemented essentiallywith 20% fetal bovine serum and 5% chicken serum.

RESULTSGenomic Structures of the Viruses. In one case of human

hepatocellular carcinoma following HBV infection that wehave studied, HBV integration generated a virus-host hybridsequence in which the first codons of the viral pre-S1 enve-lope gene were inserted in-frame, six codons upstream of theDNA-binding domain of the RARP gene (12). The resultingchimeric protein, presumably expressed from the viral pre-S1promoter (26), was postulated to participate in the cellulartransformation process, but so far there is no direct experi-mental data supporting this hypothesis. Fig. 1A outlines thejunction site between HBV and RARl protein in the originalhuman hepatocellular carcinoma (patient D). The predictedhybrid retained most of the receptor part except for the first74 amino acids, which had been substituted by the 30amino-terminal residues of pre-S1.

In this report, we investigated the transforming capacity ofboth the HBV-RARp hybrid (HPR) and its wild-type RARBcounterpart (J3R) and analyzed their possible cooperationwith v-erbB. For that, we constructed avian retroviral vec-tors expressing one or the other protein based on the struc-tural organization of the AEV genome. Fig. 1B shows theoutline of the vectors used. OR-B carries both RARI3 andv-erbB sequences, whereas BR-N contains only RARP cod-ing sequences associated with the neo gene. pR-BN wassimilar to PR-B except that an additional neo gene wasinserted downstream from the v-erbB sequences. In theseviruses, the RARB sequences are translated from a genomicRNA as a gag-RAR/3 fusion protein. In the gag-RARP gene,765 nucleotides of the gag gene were fused in-frame with theintact RARP coding sequence. The v-erbB and neo proteinsencoded in /R-B and ,BR-N, respectively, are translated fromsubgenomic RNAs. The HPR constructions were essentiallyidentical to the pR-containing proviruses except that in thesecases the RAf3 gene was fused to the HBV pre-S, sequenceas previously mentioned. All these viruses were rescued onchicken embryo fibroblasts (CEFs) as RAV-1 pseudotypes oras helper-free retroviruses in Isolde cells (24).The Chimeric HflR Protein Can Transform Erythroid Ceils.

The v-erbA oncogene is responsible for blocking the differ-entiation program of erythroid progenitor cells at the colony-forming unit-erythroid (CFU-E) stage (25). Therefore, weinvestigated whether RARp8 was able to interfere similarlywith any differentiation process in hematopoietic cells.

HBV large envelopeDrotei n

1 129 184

I 1 147 193RAR I A 18n c D

DNA

409

413 448

HORMONE

HBV-RAR prs c D E F

|/ ~~~~~3075Met Ala Ph. Arg Ala Asn Thr Ala Asn Pro Pro Arg Va1 Tyr Lys PrATO GCCTTCAGAGCAAACACCGCAAATCCtCGTCGAGTCTACAAACCC

pre Si RAR

BSa Ps Xh Ea Xb Xb]Gr{( \) jf= z=

LTR I gag RAR I No. LTRSD SA

Sa Ps Xh E Xb Xbr ,]g ) 'Nf Q f

LTR I gag T RAR I No LTRSD HBV SA

Sa Ps Xh Ec=n)f. I ! ".

LTR T, gag RARSD

Sa Ps Xh E¢

LTR I gagT RARSD HEW

SaPsXh Ec

LTR I gag RARSD

S PsD X E

LTR gag T RAR'SDHBV

pBa Ec Xb XbI r I If1C , =31LYerb B Neo LTRSA SA

ApBa Ea Xb Xb

A ASA SA

|v orb B LTRSA

I-r B LTRSA

PR.N

HP R.N

PR.BN

HPR.BN

PR.B

HPR.B

FIG. 1. (A) Schematic structure of the HBV large envelopeprotein, RARj3, and the predicted HBV-RARB gene products. Thelarge envelope protein of HBV is divided into pre-S1, pre-S2, and Sregions. Amino acid positions are indicated (26). The RARB productis divided into six regions, A-F; the C and E regions correspond,respectively, to the DNA- and hormone-binding domains. Numbersdenote amino acid positions (12). The chimeric protein, presumablyproduced in the original liver tumor of patient D, is represented (12).The DNA sequence of the pre-S-RARf3 junction is shown. (B)Genomic structures of the different viruses. In all constructions, thegag-RAR or gag-HBV-RAR fusion proteins are translated fromgenomic RNA. The v-erbB and the neo genes are transcribed fromthe viral long terminal repeat (LTR) as subgenomic RNAs. Ap, ApaI; Ba, BamHI; Ec, EcoRI; Ps, Pst I; Sa, Sac l; Xb, Xba 1; Xh, XhoI; SD, splice donor; SA, splice acceptor.

Since the v-erbB oncogene cooperates with v-erbA byinducing a mitogenic stimulation and releasing the cells fromtheir dependence on specific growth factors (27), we firsttested the RARP-containing viruses carrying the v-erbB on-cogene. BMCs from 1-day-old chickens were infected in vitrowith the j3R-B and HR-B retroviruses and their respectivehelper-free retroviral forms 8R-BN and H,8R-BN. The cellswere then seeded in semisolid methylcellulose cultures con-taining essentially 20% fetal bovine serum and 5% normalchicken serum and G418 in the case of PR-BN and HPR-BNinfections. This medium supports the growth of several dif-

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ferent lineages including erythroid, myeloid, and megakaryo-cytic lineages. Although no transformed colony was observedwith the AR-B or pR-BN viruses, colonies oftransformed cellsrapidly developed in BMCs infected with Hf3R-B andH,8R-BN viruses. These colonies were compact, were made ofround cells, and were very similar to the erythroid coloniesdeveloped from BMCs infected with AEV (28). Cells isolatedfrom these colonies and grown in liquid culture exhibited thesame cytological features as AEV-transformed erythroid cells(data not shown). The transformation was directly mediatedby the RAR-containing retrovector since the helper-free viruscould also transform these cells.The same transformation assays were then performed

using the retroviral constructions expressing the HBV-RARB hybrid product in the absence of the v-erbB oncogene.For that, the growth conditions applied were the same asthose described for Hf3R-B and Hf3R-BN infections. Again,identical erythrocytic-transformed cells developed in semi-solid methylcellulose using the HpR-N retrovirus, and thesecells could be grown in liquid culture in the presence of G418(data not shown; see Fig. 4). Moreover, the transformingefficiency of H,8R-N viruses, as assessed by the number oftransformed colonies, was 7 times higher than that of theXJ12 retrovirus expressing only the v-erbA oncogene (datanot shown).To check that retroviruses expressing the HBV-RARj3

protein have not undergone rearrangements in transformedBMCs,- the synthesis of the appropriate H.SR protein waschecked in HSR-B-transformed hematopoietic cells. By im-munoblot analysis, these cells expressed an 82-kDa proteinspecifically recognized by a monoclonal antibody directedagainst an HBV pre-Si epitope (Fig. 2A and ref. 29).

Expression of Proteins Characteristic of the ErythrocyticLineage. HpR-N- and HR-B-transformed BMCs were furthercharacterized by analyzing the expression of proteins specificto the erythrocytic lineage. Expression of histone H5 isrestricted to the avian erythroid lineage. It is normally ex-pressed in v-erbA- and AEV-transformed CFU-E. Immuno-blot analysis of protein extracts from HpR-N- and HPR-B-transformed BMCs revealed that these cells expressed highlevels of H5 (Fig. 2B). The amount of H5 was similar to thatobserved in AEV-transformed BMCs and in 6C2 cells, anAEV-transformed erythroid cell line. As expected, H5 was notfound in HBR-B-transformed chicken embryo fibroblasts.The erythrocytic nature of the transformed BMCs was

further confirmed by the presence of the Im antigen on thecellular membrane, suggesting that HR-transformed BMCsrepresented cells blocked at the CFU-E stage (data notshown; ref. 28).

Repression of the Carbonic Anhydrase I Gene by theHBV-RARM Hybrid Protein in Erythrocytic Ceils. The v-erbAoncogene blocks erythrocytic differentiation at the CFU-Estage (25), and it has been shown that the v-erbA productspecifically represses the gene encoding the carbonic anhy-drase II in these cells (30, 31). To test whether regulation ofthe carbonic anhydrase II gene is also impaired by the mutantRA receptors, immunoblot analyses were performed onHpR-transformed erythroid cells. Like 6C2, Hf3R-N-transformed CFU-E showed a barely detectable level ofcarbonic anhydrase II (Fig. 3). In this case, erythroid cellswere grown for only a few generations before protein extrac-tion, and very little spontaneous erythroid differentiationoccurred. In contrast, when primary erythroid cells trans-formed by either H,3R-N or AEV were grown longer, thepresence of the carbonic anhydrase II product in these cellscould be detected resulting from some spontaneous differ-entiation during their expansion.Growth Characteristics of Hf3R-N-Transformed BMCs.

v-erbA-transformed CFU-E express functional TGF-a recep-tors and require TGF-a to grow in culture (27). We thus

200 _,

92.5 _

69

46 -

30 -

2 00

92.5

69

46

30

21 5

FIG. 2. (A) Detection of the gag-HBV-RARj3 fusion protein inBMCs transformed by H,(R-B virus. (B) Detection of histone H5 intransformed BMCs. The size markers indicated on the left aremyosin (200 kDa), phosphorylase b (92.5 kDa), bovine serum albu-min (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), andtrypsin inhibitor (21.5 kDa).

further checked whether TGF-a might control the growth ofHf3R-transformed erythroid cells. Growth curves analyseswere performed in the absence or presence of recombinanthuman TGF-a at 1 ng/ml. As shown in Fig. 4, HfR-N-transformed erythroid cells grew independently of TGF-awith a doubling time of -21 hr. In contrast, XJ12-transformed erythroid cells were strongly dependent onTGF-a for their growth (27). As expected, AEV erythroleu-kemia cells did not require TGF-a for growth.

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92.5

69

4 _

30

21.5

CARB ANHYDRASE

FIG. 3. Repression of the carbonic anhydrase lI gene in H.3R-N-transformed CFU-E. Size markers are the same as in Fig. 2.

Because of the presence of an intact hormone-bindingdomain in the receptor part of the hybrid HBV-RAR/3proteins, growth curve analyses were also performed in thepresence of 1 ,M retinoic acid. Again, no effect of RA wasobserved, and HBR-N-transformed BMCs grew similarly inthe presence or absence of RA (data not shown).In Vivo Tumorigenicity of the Viruses. The tumorigenic

potential of the ,8R and H.3R proviruses was assessed in vivoby infection of 3- or 10-day-old embryos. Approximately100-200 ,ul of viral suspension was inoculated near theblastoderm or into the chorioallantoic veins. Major patho-logical lesions in the hatched chickens were found in the liver(Table 1). Chickens infected as 3-day-old embryos with the,BR-N and H(3R-N viruses rapidly developed bile duct hyper-plasia and died a few days after birth. Animals infected as10-day-old embryos were sacrificed at 4 weeks posthatchingand carefully examined macroscopically; tissues were pro-cessed for histological examinations. As a whole, 12% of theinjected chickens developed lesions histologically classifiedas hepatocarcinomas, whereas 13% showed hepatic hyper-plasia. Moreover, a slight erythroblastosis also appeared assoon as 1 week after birth in the chickens born from embryosinfected at 10 days with eitherBR orHBR viruses. As control,10-day-old chicken embryos injected with AEV-Pst 124, a

to

20x

a)

E10z

0)0

0 AEV -

- AEV +- HPR -

-0 HPR +

1 2 3 4 5Days

FiG. 4. Growth characteristics of HfBR-N-transformed BMCs.The cells were seeded at 2 x 105 cells per dish in the presence (+)or absence (-) of TGF-a (1 ng/ml), and each day the cells werecounted. Each point represents the mean of duplicate cultures.

Table 1. Tumorigenicity assay after in ovo inoculationNumber withpathological Aealesions/total* Age atautopsyt Apparent tumors

Virus Day 3 Day 10 days or affections

,3R-N 1/1 3 Hepatitis3/3 3 Hepatic steatosis and

bile ducthyperplasia

27 Hepatocarcinoma28 Cholangiosarcoma

Hf3R-N 3/3 3 Hepatic steatosis3 Hepatic steatosis and

bile ducthyperplasia

6 Hepatic steatosis3/6 28 Hepatocarcinoma

31 Hepatosarcoma39 Bile duct hyperplasia

13R-B 3/11 29 Hepatic steatosis andbile ducthyperplasia

29 Hepatic steatosis30 Hepatocarcinoma

H,3R-B 2/15 29 Hepatocarcinoma30 Ellipsoid hyperplasia

*Viral suspensions were injected either in the chorioallantoic veinsof 10-day-old chicken embryos or near the blastoderm of 3-day-oldchicken embryos.

tAfter birth the animals were checked periodically for signs ofdisease and sacrificed for histological examinations. Apparentlyhealthy animals were autopsied after 4 weeks.

virus carrying the v-erbB oncogene as the sole oncogene (22),developed erythroblastosis between 10 and 17 days of age,with no other type of detectable tumors.

DISCUSSIONBoth RA and T3 are important modulators of developmentand morphogenesis (32, 33). Direct evidence for the onco-genic potential of nuclear hormone receptors comes fromanalysis of the properties of the v-erbA oncogene, a naturalretroviral oncogene encoding an altered version of thechicken TR (7, 8). v-erbA contributes to cellular transforma-tion by inhibiting the function of its normal T3 and RAreceptors (9, 11). The finding that in one human hepatocel-lular carcinoma the HBV genome was inserted in the RARBgene first raised the possibility that altered retinoid receptorsmight exhibit oncogenic properties (12). Moreover, it hasbeen reported recently that the translocation breakpoint inhuman acute promyelocytic leukemia fuses the RARa gene toa novel locus, named PML (13, 15, 34), and that the resultingPML-RARa hybrid exhibits altered transactivating proper-ties (35, 36). Hence, the possibility that RAR genes could beinvolved in oncogenesis would not be surprising in view ofthese observations and due to the fundamental role played byvitamin A derivatives in differentiation and cell proliferation(33). We have investigated the oncogenic potentialities of theRAR13 gene by transferring RAR,8 coding sequences derivedeither from the normal human cDNA (,BR) or from therearranged form (H3R) found in the original human hepato-cellular carcinoma (12) into avian cells by retrovirus vectors.In HPR, the amino terminus of RARS was replaced by acoding sequence from the pre-S1 large envelope protein ofHBV. To mimic the structure of the v-erbA oncogene, thesetwo RARP-derived sequences were fused in-frame with gagretroviral sequences.

In chicken erythrocytic progenitor cells, the v-erbA onco-gene blocks the differentiation program at the CFU-E stage

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(25) and inhibits the transcription of erythrocyte-specificgenes (30, 31). In these cells, the v-erbA oncogene cooperateswith the v-erbB oncogene to induce full leukemogenic trans-formation in vitro and in vivo (37). In this report, we showthat, like v-erbA, the H,3R form was able to block thedifferentiation of chicken erythrocytic cells at the CFU-Estage, whereas the wild-type RAR,8 was not. The H,8R-expressing virus induced the growth of the blocked CFU-E inthe absence of TGF-a, a growth factor of chicken erythro-cytic progenitor cells normally required for the growth ofv-erbA-transformed CFU-E (27). When tested in vivo, boththe intact and the HBV-RAR,8 induced hepatocellular hy-perplasias and carcinomas in chickens. Such tumors neverarise in AEV-infected chickens. The slight erythroblastosisobserved in RAR,3-injected animals was never as dramatic asthe severe erythroblastosis usually induced by AEV. Thisfinding together with the low frequency ofappearance of livertumors in BR- and Hf3R-infected chickens may reflect therequirement of a second genetic event involving the activa-tion of one or more additional oncogenes. Our results allowus to establish a direct link between an inappropriate expres-sion of the RAR,8 gene and the development of liver carci-noma. Altogether, our data demonstrate that the RAR/3 andHBV-RARf3 proteins are both tumorigenic by themselvesand that the mechanisms of cellular transformation by RAR/3may be different from that of v-erbA. The difference betweenthe normal and the mutant forms of RARI3 used here relies onthe presence of a peptide sequence derived from the HBVlarge envelope protein fused at the amino terminus of RARp.It is then likely that this viral sequence strongly enhances theoncogenic potential of the rearranged RAR in erythroid cells.What are the molecular mechanisms governing the oncogeniceffect of HBV-RAR,8? It is surprising that this form isoncogenic even in the presence of RA, although it contains anormal carboxyl-terminal hormone-binding domain. v-erbAoncoproteins in which the normal hormone-binding domain isrestored display their oncogenic effect only in the absence ofthyroid hormone T3. In the presence of T3, the chimericoncoprotein binds the hormone and is no longer oncogenic(30, 31). We may imagine that the HBV pre-S1 sequence in thechimeric receptor induces strong modifications in the struc-ture and function of the receptor, which remains oncogeniceven in the presence of RA.The role of retinoids in avian erythropoiesis has not been

established. However, recent results suggest that v-erbA maytransform erythrocytic cells by interfering with a retinoid-mediated differentiation process (11). At least two explana-tions could be put forward to explain erythroid transforma-tion by HBV-RAR,3. First, one may imagine that the mutantRAR,8 binds competitively with normal RAR,8 to the specificresponse element, thereby arresting transcription of genesnormally required for erythrocytic differentiation. The sec-ond possibility is the formation of inactive heterodimersbetween c-erbA and HBV-RARj3 that could not activate thegenes required for the differentiation of CFU-E. In conclu-sion, this work demonstrates that a RA receptor can beactivated into an oncogene and suggests that rearrangementsof RARl might contribute to the development of cancers.

We are grateful to W. Gerlich for the pre-Si monoclonal antibodyand to M. A. Maillard for typing the manuscript. This work wassupported by grants from Association pour la Recherche contre leCancer and Federation Nationale des Centres de Lutte Contre leCancer.

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