Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Cytokinesis wikipedia , lookup
Cell growth wikipedia , lookup
Cell nucleus wikipedia , lookup
Endomembrane system wikipedia , lookup
Extracellular matrix wikipedia , lookup
Cell culture wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
Signal transduction wikipedia , lookup
[CANCER RESEARCH54, 6065-6068, December 1, 19941 Advances in Brief Expression of Helix-Loop-Helix Factor Id-i Is Dependent on the Hepatocyte Proliferation and Differentiation Status in Rat Liver and in Primary Culture1 Catherine Le Jossic, Gennady P. Ilyin,2 Pascal Loyer, Denise Glaise, Sandrine Cariou, and Christiane Guguen-Guillouzo INSERM U49. Unite de RecherchesHepatologiques.HôpitalPontchaillou, 35033 Rennes,France Abstract Id proteins are known as negative regulators of differentiation in various cell types. In this report, we show that the Id-i gene was down regulated during the development of rat liver. No Id-i transcripts were detected in terminal differentiated hepatocytes. We have studied Id-i expression in proliferating hepatocytes using an in vivo model of liver regeneration after partial hepatectomy and an in vitro growth factor fetal life; and (c) maturation of neonatal liver and acquisition of a stimulated hepatocyte culture system. Strong activation of Id-i was oh served in mid-late G1 of the hepatocyte cell cycle at a time corresponding to a mitogen restriction point. These observations suggest that Id-i is involved in the control of proliferation and differentiation in liver cells. Introduction vivo with E2A proteins (5, 6). Generally, high levels of Id mRNA in proliferative and undifferentiated cells decrease as they are induced to differentiate (3, 4). On the other hand, the expression of Id genes is rapidly induced in a diverse range of cell lines activated by growth factors or phorbol 12-myristate 13-acetate. Finally, overexpression of human Id-related gene HLH 1r21 produced a morphologically trans formed phenotype in NIH 3T3 cells (7). These observations suggest that Id proteins can act as negative regulators of a differentiation program in various cell types and are involved in the control of cell proliferation and possibly of transformation. The regulation of Id gene expression has been mainly studied in Received 8/18/94; accepted 10!17f94. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. research was supported by lnstitut National de la Santa et de Ia Recherche Médicale, EEC (BIOT-CT9O—0l89)and the Association pour Ia Recherche contre Ic Cancer. 2 To 3 The whom requests abbreviations terminally differentiated phenotype. Quiescent hepatocytes constitute one of the few terminally differentiated cell types in the adult body which retain their proliferating capacity. Both in viva and in vitro models of proliferating hepatocytes are available. In the present re port, we show the sequence of Id-i expression over the course of liver The HLH3 proteins are known to play an important role in the regulation of differentiation in various cell-specific lineages as well as in cell proliferation and transformation. Members of this regulatory gene family share two functional motifs: the region of basic amino acids involved in DNA binding and the HLH domain which is essential for dimerization (1). The HLH proteins recognize the nude otide consensus sequence CANNTG, known as the E-box. It is sug gested that HLH proteins can be divided into different classes. Class A contains the ubiquitously expressed proteins Ei2 and E47. Class B comprises the tissue-specific factors such as the MyoD family, which can form heterodimers with class A molecules (2). A distinct class of HLH transcriptional regulators includes Id pro teins, which lack the basic DNA-binding region, and are able to inhibit the binding of several HLH proteins to DNA by forming biologically inactive hetero-oligomers (3, 4). In particular, Id-i can inhibit differentiation of muscle and myeloid cells by associating in I This tissues and cells of mesodermal origin. To gain insight into the potential role of Id proteins in proliferation and differentiation of endoderm-derived tissues, we determined the expression of the Id-i gene in rat liver in relation to hepatocyte proliferation and differen tiation status. Liver development in mammals involves at least three major steps: (a) commitment of embryonic cells to become hepato cytes; (b) modulation of liver-specific gene expression during late for used reprints are: should HLH, be addressed. helix-loop-helix; P1-fr, partial hepatectomy; EGF, epidermal growth factor; RLEC, rat liver epithelial cells. development and in proliferating adult hepatocytes. Materials and Methods Animals. Pregnantand normalfemale Sprague-Dawleyrats (180—200 g) were obtained from Charles River Laboratories (Cléon,France). Breeding was done by placing female rats with males of the same strain overnight; noon of the next day was considered as 0.5 days postcoitum. On the appropriate days of gestation, rats were anesthetized, embryos were removed, and their livers were minced and washed with PBS to reduce the number of hemopoietic cells. A partial (two-thirds) hepatectomy was performed as described by Higgins and Anderson (8). Control animals underwent a sham operation comprising lapa rotomy and liver manipulation without tissue removal. At different times after PHT, animals were sacrificed, and their livers were washed with PBS by perfusion through the portal vein to eliminate blood cells. Livers were then minced, frozen in liquid nitrogen, and kept at —80°C until further processing. CeHIsolationandCulture. Hepatocytes fromadultmaleSprague-Dawley rats were isolated by the classical two-step collagenase perfusion procedure. Hepatocytes were seeded at 7.5 X 10― cells/cm2on a 75-cm2flask in a mixture of 75% MEM and 25% medium 199, supplemented with 10% FCS and, per ml, 100 units penicillin, 100 mg streptomycin sulfate, 1 mg bovine serum albumin, and 5 mg bovine insulin. After cell attachment (4 h later), the medium was renewed with the same medium deprived of FCS and supplemented with EGF (50 ngfml) and pyruvate (20 mM) or with 1.4 X i0_6 M hydrocortisone hemisuccinate. It was changed every day thereafter. Faza 967 and FAO, well-differentiated rat hepatoma cells derived from H4IIEC3, were maintained in a mixture ofSO% Ham's F-12 and 50% NCI'C 135 with 10% FCS. NIH 3T3 embryonic fibroblasts were grown in MEM supplemented with 10% FCS. RLEC,SDVI, were obtainedas describedpreviously(9), and BRL 3A cells were grown in Williams' E medium supplemented with 10% FCS. RNA Isolation and Northern Blot Analysis. Total RNA was extracted by the thiocyanate guanidium procedure. Twenty ,.@gof RNA were separated by electrophoresis and transferred onto a nylon membrane (Hybond N + ; Amer sham). Hybridization was performed with [ca-32P]dC'I'P-labeledprobe for 16 h at 65°C.A 910-base pair cDNA fragment of clone pRID9.1, spanning the full-length rat Id-i mRNA coding sequence, was used as a probe. This plasmid was kindly provided by Jeremy P. Springhorn (Brigham and Women's Hos pital, Boston, MA) (10). In some experiments, we used a 5'-truncated 550-base pair fragment of rat Id-I cDNA lacking sequences encoding the NH2-terminal 6065 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer Research. ID-i EXPRESSION A region and HLH domain. The blots were rehybridized with rat glyceraldehyde 3-phosphate dehydrogenase IN LIVER RLEC Livar4 6' 6 8 10 12 12 18 24' 24 hours cDNA as a control for gel loading and transfer. .- Gel Mobifity Shift Assay. Preparation of nuclear extracts and gel mobility assay were performed as described by Cereghini et a!. (11), except that the nuclear extracts were not dialyzed. Binding reactions were carried out in a 15-pi volume containing 1 mM sodium phosphate (pH 7.5), 0.1 mM EDTA, . 0.5 mM EGTA, 0.5 mM DTT, 10% (v/v) glycerol, 0.5 ,.@gpolydeoxyinosinic deoxycytidylic acid, 1 mt@iMgC12, 10 m@ spermidine, and 0.1—0.2 ng of 5 ‘-end32P-labeled double-stranded oligonucleotide. The oligonucleotide con taming the E-box was derived from position —618to —598of the 5'-flanking B RLECF.I.H.4 7 17 24 31 48 65 72 80 96 102115l20hours , region ofthe rat a-fetoprotein gene (12) and had the sequence of 5'-ACCCAT GCATCTGTGACATACAT-3'.The oligonucleotide probe, containing the UAE site of the adenovirus major late promoter, had the sequence of 5'CGGTAGGCCACGTGACCGGOT-3'. Five @gof nuclear extracts were added to the reaction mixture and incubated for 10 mm on ice. The DNA C F.I.H.4 18 24 2730 z 42 4$ 54 60 66 l2hours protein complexes were separated by 6% acrylamide gel in 0.5 X ThE (45 mr@i Tris-borate/l.25 mMEDTA). The gel was then fixed, dried, and subjected to autoradiography. N.,,,, Results We have examined the Id-i expression by Northern blot analysis with full-length rat Id-i cDNA probe in several liver-derived cell lines in comparison with proliferating cultures of NIH 3T3 cells. Id-i D 36 48 00 66 l2hosws transcripts were observed in the differentiated rat hepatoma FAO and Faza 967 cells in moderate levels, similar to those found in NIH 3T3 cells, whereas Id-i messengers were abundant in RLEC (Fig. iA). Therefore, RLEC were used as a reference in Northern blot analysis. Fig. 2. Expression of Id-I in proliferating rat hepatocytes. Total RNA was extracted from liver biopsies at different times after PHT (A) and from cultured hepatocytes stimulated with EGF (B and C) or maintained without growth factor (D). Twenty @g of Analysis of Id-i expression over the course of rat liver development revealed the presence of high amounts of Id transcripts in fetal and RNAs were analyzed neonatal rat livers. From 2 weeks after birth, the levels of Id-i RNA by Northern blot hybridization with full-length rat Id-i cDNA probe (A andB) or with probespecificfor Id-I, whichlackednucleotides encodingNH2terminal region and HLH domain (C and D). °,sham-operated animals; FIH. freshly isolated hepatocytes. decreased and were either very low or undetectable after day 28 (Fig. 1B). In the adult rat liver, hepatocytesare blockedin a quiescentstate but can be easily stimulated to proliferate in response to loss of tissue mass. We found a rapid induction of Id-i from 6 h after PHT. mRNA remained through the cell cycle, with a gradual decrease after 80 h (Fig. 28). To define more precisely the appearance ofld-i during the G@phase and to confirm the specificity of hybridization, we Thereafter, the levels of Id-i mRNA increased, reaching a maximum at 18 h. No significant induction of Id-i was observed in sham operated animals (Fig. 14). It was previously shown that EGF/pyru performed a detailed analysis of id-I expression in rat hepatocytes maintained in culture, with and without EGF, using a rat Id-i probe vate-stimulated rat hepatocytes in primary culture represent a suitable model to study mechanisms of cell cycle progression (i3). Only a very lacking nucleotides encoding the NH2-terminal region and conserva tive HLH domain. The strong activation of Id-I occurred at 42 h of culture; it reached a maximum starting at 54 h (Fig. 2C). In contrast, low level of [3Hjthymidine incorporation could be observed in rat hepatocytes, maintained in the basal medium without growth factors. In contrast, DNA synthesis occurred in most of the rat hepatocytes stimulated with EGF/pyruvate, at the third day of culture. No Id-i transcripts were detected in freshly isolated rat hepatocytes and during the first hours in culture. A drastic increase of Id-I transcripts was observed between 31 and 48 h of culture. Then, high amounts of Id-i only low levels of Id-i transcripts were detected in nonstimulated hepatocytes (Fig. 2D). The Id-i protein can selectively inhibit the binding to the E-box oligonucleotide probes of one set of the HLH proteins, such as E12, E47, and MyoD, but not of the other set, including USF and AP-4 (3, A RLEC313 FAZAFAOBRL Fig. 1. Northern blot analysis of Id-i expression @ in various liver-derived cell lines (A) and during development of rat liver (B). Total RNA was cx tractedfromcellsincultureorlivertissue,and20 ,.i.gof RNA were applied to gels. Northem blots were sequentially hybridized with rat Id-i and glyceraldehyde 3-phosphate dehydrogenase cDNA B probes. Idi @EC14.5 16.5 18.5 0.Sd 3d d.p.c. d.p.c. d.p.c. GAPDH • 0 0 0 0 s 3d 7d Sd lid 13d iSd 1$d 21d 24d @d 0 •s•.•.O.O.Oos•ô 6066 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer Research. ID-I EXPRESSION IN LIVER Fig. 3. E-box-binding activities in proliferating hepatocytes.Nuclearextractswerepreparedfrom liver biopsies or from cultured rat hepatocytes stim A I 2 3 comp.- - + + 4 B R.L ulated with EGF. Gel mobilityshift assay was ,._. performed with 32P-labeled E-box probe as de scribed in “Materials and Methods.― A, competition experiments were carried out by addition of 100fold excess of unlabeled E-box probe (Lane 3) or •@s 4 14 24 • % • S • % 48 72 96 @• •@% • . 120 hours • 1$ an unrelated probe containing a UAE site (Lane 4). Lane 1, labeled probe only; Lane 2, labeled probe with rat liver nuclear extracts. B, labeled E-box probe was incubated with nuclear extracts from EGF-stimulated F.I.H. rat hepatocytes at different times in culture. RI.. rat liver; FIll, freshly isolated hepa tocytes. 4). Although it is not known what kind of HLH proteins are present in the hepatocytes, we analyzed E-box binding activity of nuclear extracts prepared from adult rat liver and from cultured hepatocytes stimulated with EGF by gel mobility shift assay. Several E-box-bound complexes were observed in adult liver nuclear extracts. All bindings were efficiently suppressed by addition of 100-fold molar excess of the unlabeled probe. Competition performed with another E-box probe containing the UAE site of adenovirus major late promoter, known to bind the ubiquitous cellular transcriptional factor USF (14), resulted in partial blocking of binding activities of fast-migrating In this laboratory, it has been recently shown that, under the complexes (Fig. 3A). The slow-migrating complexes were drastically reduced in freshly isolated hepatocytes, partly reappeared in hepato cytes from 4—24h of culture, and were again suppressed later through the cell cycle (Fig. 3B). By contrast, fast-migrating complexes were constantly present in proliferating hepatocytes with variations in intensity. conditions used here, an increase of DNA synthesis took place at around 54—60 h and peaked at 72—78h of culture. Furthermore, a restriction point has been defined at 42—45h as a step in G1 beyond which the cells cannot progress without EGF stimulation.4 Activation of Id-i precisely started at 42 h, with the highest levels from 54 h of culture. The fact that the occurrence of Id-i was strictly correlated with the EGF restriction point strongly argues for a particular role of Id-iinlate G1. By gel mobility shift assay, we demonstrated that adult rat liver Discussion Cell proliferation and differentiation are the essential but alternative events of living organisms. For instance, in muscle cells, the differ entiation program is closely coupled to the cell cycle. Identification of the myogenic HLH transcriptional factors has provided insight into the mechanisms of cross-talk between the regulatory pathways that control myoblast proliferation and differentiation (15). Members of the MyoD family of transcriptional regulators can activate the muscle differentiation program as well as induce growth arrest (16, 17). Conversely, Id proteins inhibit myogenesis and, at the same time, act as the essential regulators of cell cycle progression (5, 18). In the liver, cessation of DNA synthesis in postnatal hepatocytes correlates with hepatocyte replicate initially, whereas nonparenchymal cells divide after a lag of 2—3days; and (c) a long-lasting G1 phase characterizes the hepatocyte cell cycle. DNA synthesis begins at 19—20h after PHT and peaks at 22—24h (21, 22). We have observed that ld-i was activated from 6 h after PHT and increased between 12 and 18 h, which corresponded to the mid-late G1 phase. Moreover, analysis of Id-i expression in growth factor-stimulated rat hepatocytes confirmed the beginning of Id-i expression at the mid-late G1 phase of the hepatocyte cell cycle. maturation and acquisition of the adult phenotype. In contrast, stimulation of hepatocyte proliferation by PHT leads to partial retrodifferentiation, which is characterized by reexpression of fetal liver markers, including plasma proteins like a-fetoprotein and intracellular isoenzymes (19, 20). These observations suggest that the molecular mechanisms which link these two biological phenomena might be common in different cell types. Our data demonstrate the presence of Id-i transcripts in fetal and neonatal rat liver. From 2 weeks after birth, the levels of Id-i mRNA decreased, reflecting the gradual maturation of the liver. Terminally differentiated adult rat liver contained very low levels, if any, of Id-i transcripts. To establish the potential role of Id-i in the hepatocyte cell cycle progression, we analyzed the levels of Id-i mRNA in prolifer ating hepatocytes using both an in vivo model of hepatocyte prolifer ation triggered by resection of part of the organ and an in vitro EGF/pyruvate-stimulated rat hepatocyte culture system. Liver regen eration after PHT provides a unique system to study the proliferating response of nontumoral, highly differentiated cells in a solid tissue proteins to the E-box, the activation of Id-i in proliferating hepato cytes would be reflected in the suppression of E-box-binding activi ties. Indeed, slow-migrating complexes were reduced in freshly iso lated hepatocytes and in EGF/pyruvate-stimulated hepatocytes from 48 h in culture, which is correlated with activation of Id-i. However, no Id-i transcripts were detected in freshly isolated hepatocytes, suggesting participation of other proteins in restricting E-box-binding activities. Isolation of hepatocytes was accompanied by a rapid and transient activation of several immediate-early genes, like c-los and c-jun. Expression of these genes was shown to begin during the dissociation of liver cells, to peak in freshly isolated hepatocytes and to decrease thereafter (23). This raises the possibility that c-jun may interact with liver HLH proteins as reported with MyoD (24). Alter natively, other members of the Id family could be involved in regu lation of E-box-binding activities. In line with this possibility, we have found that Id-2 was activated in freshly isolated hepatocytes, decreased thereafter, and also drastically increased in parallel with Id-i in mid-late G@of the hepatocyte cell cycle.5 Taken together, our data suggest a potential role of Id-i in differ entiation and proliferation of hepatic cells. Identification of Id-binding proteins may clarify the molecular mechanisms which couple these mutually exclusive biological phenomena. In addition, since Id-i 4 P. Loyer, and is characterized by several properties: (a) the cell growth response is a perfectly regulated synchronous nuclear extracts contained several proteins that can bind the E-box. At least some of the E-box-bound species were distinct from the ubiq uitous nuclear factor USF. Indeed, an oligonucleotide, known to bind USF preferentially, only partly suppressed fast-migrating complexes. Since the Id-i protein acts as an inhibitor of the binding of HLH process; (b) only hepatocytes S. Cariou, D. Glaise, M. Biodec, G. Baffet, and C. Guguen-Guillouzo. Growth factor dependence of entry and progression through Gi and S phases of adult rat hepatocytes in vitro, submitted for publication. 5 G. P. Ilyin, C. Le Jossic, and C. Guguen-Guillouzo, manuscript in preparation. 6067 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer Research. ID-l EXPRESSION IN LIVER 11. Cereghini, S., Blumenfeld, M., and Yaniv, M. A liver-specific factor essential for appeared to be expressed in various hepatic cell lines with the highest albumin transcription differs between differentiated and dedifferentiated rat hepatoma levels observed in RLEC, which have been reported as a “facultative cells. Genes Dcv., 2: 957—974,1988. hepatocyte stem cell―population (25), the involvement of Id-i protein 12. Nahon, J-L., Danan, J-L., Poiret, M., Tratner, I., Jose-Estanyol, M., and Sala-Trepat, J-M.The rat a-fetoproteinand albumingenes.Transcriptional controlandcompar in liver carcinogenesis might be suggested. ison of the sequence and promoter region. J. Biol. Chem., 262: 12479—12487, 1987. 13. McGowan, J. A. Hepatocyte proliferation in culture. In: A. Guillouzo and C. Guguen Acknowledgments Guillouzo (eds.), Isolated and Cultured Hepatocytes, pp. 13—38.INSERM, Paris and rat Id-i. John Libbey Eumtext, London, 1986. 14. Gregor, P. D., Sawadogo, M., and Roeder, R. G. The adenovirus major late tran scription factor USF is a member of the helix-loop-helix group of regulatory proteins and bind to DNA as a dimer. Genes Dcv., 4: 1730—1740,1990. References 15. Olson, E. L. Interplay between proliferation and differentiation within the myogenic We are grateful to Dr. Jeremy P. Springhorn for supplying cDNA probe to lineage. Dcv. Biol., 159: 261—272, 1992. 16. Weintraub, H., Tapscott, S. J., Davis, R. L, Thayer, M. J., Adam, M. A., Lassar, 1. Voronova, A., and Baltimore, D. Mutations that disrupt DNA binding and dimer formation in the E47 helix-loop-helix protein map to distinct domains. Proc. NatI. A. B., and Miller, A. D. Activation of muscle specific genes in pigment, nerve, fat, Acad. Sci. USA, 87: 4722—4726,1990. 2. Murre, C., McCaw, P. S., Vaessin, H., Caudy, M., Jan, L. Y., Jan, Y. N., Cabrera, liver and fibroblast cell lines by forced expression of MyoD. Proc. Nati. Acad. Sci. USA, 86: 5434—5438,1989. C. V., Buskin, J. N., Hauschka, S. D., Lassar, A. B., Weintraub, H., and 17. Crescenzi, M., Fleming, T. P., Lassar, A. B., Weintraub, H., and Aaronson, S. A. Baltimore, D. Interaction between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell, 58: 537—544, MyoD induces growth arrest independent of differentiation in normal and transformed cells Proc. NatI. Acad. Sci. USA, 87: 8442-8446, 1989. 1990. 18. Hara, E., Yamaguchi, 1., Nojima, H., Ide, T., Campissi, J., Okayama, H., and Otis, K. 3. Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L., and Weintraub, H. The protein Id-related genes encoding helix-loop-helix proteins are required for G, phase pro Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell, 61: 49—59, gression and are repressed in senescent human fibroblasts. J. Biol. Chem., 269: 1990. 2139—2145,1994. 19. Guguen-Guillouzo, C., Szajnert, M-F., Glaise, D., Gregory, C., and Schapira, F. Isozyme differentiation of aldolase and pyruvate kinase in fetal, regenerating, pre 4. Sun, X-H., Copeland, N. G., Jenkins N. A., and Baltimore, D. Id proteins Idi and 1d2 selectively inhibit DNA binding by one class of the helix-loop-helix proteins. Mol. Cell. Biol., II: 5603—5611,1991. 5. Jen, Y., Weintraub, H., and Benezra, R. Overexpression of the Id protein inhibits the muscle differentiation neoplastic Dcv., 6: 1466—1479,1992. 6. Kreider, B. L., Benezra, R., Rovera, G., and Kadesch, T. Inhibition of myeloid differentiation by the helix-loop-helix protein Id. Science (Washington DC), 255: 1700—1702, 1992. 7. Deed, R. W., Bianchi, S. M., Atherton, G. T., Johnston, D., Santibanez-Koref, M., Murfy, J. J., and Norton, J. D. An immediate early human gene encodes an Id-like helix-loop-helix protein and is regulated by protein kinase C activation in diverse cell types. Oncogene, 8: 599—607,1993. 8. Higgins, G. M., and Anderson, R. M. Experimental pathology of liver: restoration of the liver of the white rat following partial surgical removal. Arch. Pathol., 12: and malignant rat hepatocytes during culture. In Vitro, 17: 369—377, 1981. 20. Petropoulos, C., Andrews, G., Tamaoki, T., and Fausto, N. a-Fetoprotein and albumin mRNA levels in liver regeneration and carcinogenesis. J. Biol. Chem., 258: 4901—4906,1983. 21. Fausto, N., and Mead, J. E. Regulation of liver growth: a comparison between liver program: in vivo association of Id with E2A proteins. Genes regenerationandcarcinogenesis. In: P. Bannasch,D. Keppler,andG. Weber(eds.), Liver Cell Carcinoma, pp. 293—303.Dordrecht, the Netherlands: Kiuwer Academic Publishers, 1988. 22. Loyer, P., Glaise, D., Cariou, S., Baffet, G., Meijer, L., and Guguen-Guillouzo, C. Expression and activation of cdks (1 and 2) and cyclins in the cell cycle progression during liver regeneration. J. Biol. Chem., 269: 2491—2500, 1994. 23. Etienne, P. L., Baffet, G., Desvergne, B., Boisnard-Rissel, M., Glaise, D., and Guguen-Guillouzo, C. Transient expression of c-fos and constant expression of c-myc 186—202, 1931. 9. Morel-Chany, E., Guillouzo, C., Trincal, G., and Szajnert M-F. “Spontaneous― neoplastic transformation of epithelial cell strains of rat liver: cytology, growth and enzymatic activities. Eur. J. Cancer, 14: 1341—1352, 1978. 24. 10. Springhorn, J. P., Ellingsen, 0., Berger, H-J., Kelly, R. A., and Smith, T. W. Transcriptional regulation in cardiac muscle. Coordinate expression of Id with neo natal phenotype during development and following a hypertrophic stimulus in adult 25. rat ventricular myocytes in vitro. J. Biol. Chem., 267: 14360—14365,1992. in freshly isolated and cultured normal adult rat hepatocytes. Oncogene Res., 3: 255—262,1988. Bengal, E., Ransone, L., Schafmann, R., Dwarki, V. J., Tapscott, S. J., Weintraub, H., and Verma, I. M. Functional antagonism between c-Jun and MyoD proteins: a direct physical association. Cell, 68: 507—519,1992. Grisham, J. W. Cell types in long-term propagable cultures of rat liver. Ann. NY Acad.Sd., 349: 128—137, 1980. 6068 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer Research. Expression of Helix-Loop-Helix Factor Id-1 Is Dependent on the Hepatocyte Proliferation and Differentiation Status in Rat Liver and in Primary Culture Catherine Le Jossic, Gennady P. Ilyin, Pascal Loyer, et al. Cancer Res 1994;54:6065-6068. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/54/23/6065 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer Research.