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• W2007455340 abstract "Finkel-Biskis-Reilly (FBR) osteosarcoma virus v-Fos causes tumors of mesenchymal origin, including osteosarcomas, rhabdomyosarcomas, chondrosarcomas, and liposarcomas. Because the cell of origin in all these tumors is a pluripotent mesenchymal cell, the variety of tumors seen in mice which express FBR v-Fos implies that FBR v-Fos inhibits multiple differentiation pathways. To study the mechanism of FBR v-Fos' inhibition of mesenchymal differentiation, we utilized an in vitro model of adipocyte differentiation. We show by both morphological and biochemical means that FBR v-Fos inhibits adipocyte differentiation in vitro. This inhibition is due to FBR v-Fos' inhibition of the growth arrest characteristic of terminal differentiation and FBR v-Fos' inhibition of the expression and activity of a key regulator of this growth arrest, C/EBPα. The in vitro inhibition of adipogenesis by FBR v-Fos has in vivo significance as immunostaining of FBR v-Fos-induced tumors shows no CCAAT/enhancer binding protein (EBP)-α expression. These data implicate C/EBPα as a protein involved in the generation of liposarcomas. Finkel-Biskis-Reilly (FBR) osteosarcoma virus v-Fos causes tumors of mesenchymal origin, including osteosarcomas, rhabdomyosarcomas, chondrosarcomas, and liposarcomas. Because the cell of origin in all these tumors is a pluripotent mesenchymal cell, the variety of tumors seen in mice which express FBR v-Fos implies that FBR v-Fos inhibits multiple differentiation pathways. To study the mechanism of FBR v-Fos' inhibition of mesenchymal differentiation, we utilized an in vitro model of adipocyte differentiation. We show by both morphological and biochemical means that FBR v-Fos inhibits adipocyte differentiation in vitro. This inhibition is due to FBR v-Fos' inhibition of the growth arrest characteristic of terminal differentiation and FBR v-Fos' inhibition of the expression and activity of a key regulator of this growth arrest, C/EBPα. The in vitro inhibition of adipogenesis by FBR v-Fos has in vivo significance as immunostaining of FBR v-Fos-induced tumors shows no CCAAT/enhancer binding protein (EBP)-α expression. These data implicate C/EBPα as a protein involved in the generation of liposarcomas. Cellular differentiation and cancer are tightly linked. Clinically, cancers are graded according to their degree of differentiation. A high degree of differentiation of a cancer indicates a favorable prognosis while a low degree of differentiation indicates a poor prognosis (1Cotran R.S. Kumar V. Robbins S.L. Pathological Basis of Disease. W. B. Saunders, Philadelphia1994: 153-200Google Scholar). Despite the well established clinical relationship between differentiation and cancer, the molecular mechanisms by which this lack of differentiation occurs in high grade cancers are unknown. A model system to study this phenomenon has emerged through the use of the retroviral oncogene FBR v-fos. 1The abbreviations used are: FBR v-Fos, Finkel-Biskis-Reilly osteosarcoma virus v-Fos; C/EBP, CCAAT/enhancer binding protein; CAT, chloramphenicol acetyltransferase;neo R, neomycin resistance gene; G3PDH, glycerol-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; CRP, C-reactive protein; RSV, Rous sarcoma virus; MSV, murine sarcoma virus. FBR v-Fos is a fusion protein containing a retroviral gag gene, the mouse c-fos proto-oncogene, and the mouse c-fox gene (Fig. 1) (2Finkel M.P. Reilly Jr., C.A. Biskis B.O. Front. Radiat. Ther. Oncol. 1975; 10: 28-39Crossref Google Scholar, 3Lee C.K. Chan E.W. Reilly C.A. Pahnke V.A. Rockus G. Finkel M.P. Proc. Soc. Exp. Biol. Med. 1979; 162: 214-220Crossref PubMed Scopus (10) Google Scholar, 4Curran T. Verma I.M. Virology. 1984; 135: 218-228Crossref PubMed Scopus (68) Google Scholar). The retroviral gag sequence contains a myristylation site which alters FBR v-Fos' carcinogenic potential (5Jotte R.M. Holt J.T. J. Cell Biol. 1996; 135: 457-467Crossref PubMed Scopus (10) Google Scholar). When FBR v-Fos is myristylated, transgenic mice which express this oncogene develop multiple undifferentiated tumors of mesenchymal origin, including rhabdomyosarcomas, chondrosarcomas, osteosarcomas, and liposarcomas (5Jotte R.M. Holt J.T. J. Cell Biol. 1996; 135: 457-467Crossref PubMed Scopus (10) Google Scholar). When the myristylation site of FBR v-Fos is altered in such a way that myristylation cannot occur (G2A v-Fos; Fig. 1), mice expressing this mutant retroviral oncogene develop only lipomas (5Jotte R.M. Holt J.T. J. Cell Biol. 1996; 135: 457-467Crossref PubMed Scopus (10) Google Scholar). The studies in the transgenic mice indicate that in vivo, FBR v-Fos can disrupt normal cellular differentiation pathways, and that since the effect on adipocytes is independent of the myristylation state of v-Fos, adipogenesis is particularly sensitive to inhibition by the Fos proteins. FBR v-Fos is thought to cause carcinogenesis via a two-step mechanism (6Abbott D.W. Holt J.T. J. Biol. Chem. 1997; 272: 14005-14008Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). First, transformation must occur. Second, genomic instability must be created. Our laboratory has recently shown that while FBR v-Fos and G2A v-Fos both cause transformation, only cells which express FBR v-Fos are highly susceptible to DNA damage caused by ionizing radiation. Cells which express G2A v-Fos show normal sensitivity to ionizing radiation (6Abbott D.W. Holt J.T. J. Biol. Chem. 1997; 272: 14005-14008Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). These data imply that the difference between benign tumors and malignant tumors lies in genomic instability. However, to reach the malignant phenotype, transformation must also occur. Because either FBR v-Fos or G2A v-Fos expression leads to tumor formation (either liposarcomas or lipomas, respectively) both FBR v-Fos and G2A v-Fos must be inhibiting adipocyte differentiation, and studying this step may give insight into the first step of FBR v-Fos-induced carcinogenesis. The study of adipocyte development has been made possible through the use of a pre-adipocyte cell line (NIH 3T3-L1) (8Green H. Kehinde O. Cell. 1974; 1: 113-116Abstract Full Text PDF Scopus (746) Google Scholar, 9Green H. Kehinde O. Cell. 1975; 5: 19-27Abstract Full Text PDF PubMed Scopus (1097) Google Scholar, 10Green H. Kehinde O. Cell. 1976; 7: 105-113Abstract Full Text PDF PubMed Scopus (616) Google Scholar). When exposed to isobutylmethylxanthine, dexamethosone, and insulin, confluent NIH 3T3-L1 cells begin to change their cytoskeletal shape and eventually become adipocytes (10Green H. Kehinde O. Cell. 1976; 7: 105-113Abstract Full Text PDF PubMed Scopus (616) Google Scholar, 11Russell T.R. Ho R.-J. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 4516-4520Crossref PubMed Scopus (157) Google Scholar). Adipogenesis is initiated and maintained by the sequential expression of three tissue-specific transcription factors; C/EBPβ, PPARγ2, and C/EBPα (12MacDougald O.A. Lane M.D. Annu. Rev. Biochem. 1995; 64: 345-373Crossref PubMed Scopus (943) Google Scholar, 13Yeh W.-C. McKnight S.L. Curr. Opin. Cell Biol. 1995; 7: 885-890Crossref PubMed Scopus (19) Google Scholar, 14Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3133) Google Scholar, 15Samuelsson L. Stromberg K. Vikman D Bjursell G. Enerback S. EMBO J. 1991; 10: 3787-3793Crossref PubMed Scopus (140) Google Scholar). PPARγ2 is thought to be the major transcription factor responsible for the initiation of adipogenesis (14Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3133) Google Scholar) while C/EBPα is thought to be the major protein involved in the maintenance of adipogenesis (13Yeh W.-C. McKnight S.L. Curr. Opin. Cell Biol. 1995; 7: 885-890Crossref PubMed Scopus (19) Google Scholar, 15Samuelsson L. Stromberg K. Vikman D Bjursell G. Enerback S. EMBO J. 1991; 10: 3787-3793Crossref PubMed Scopus (140) Google Scholar). In the differentiated adipocyte, C/EBPα is the factor responsible for the expression of many fat cell-specific genes (16Christy R.J. Yang V. Ntambi J. Geiman D. Landschulz W.H. Friedman A. Nakabeppu Y. Kelly T. Lane M.D. Genes Dev. 1989; 3: 1323-1335Crossref PubMed Scopus (467) Google Scholar) including PEPCK (17Park E.A. Roesler W.J. Liu J. Klemm D.J. Gurney A.L. Thatcher J.D. Shuman J.D. Friedman A. Hanson R.W. Mol. Cell. Biol. 1990; 10: 6264-6272Crossref PubMed Scopus (174) Google Scholar), and ap2 (18Cheneval D. Christy R.J. Geiman D. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8465-8469Crossref PubMed Scopus (49) Google Scholar). Expression of C/EBPα has been shown to be both sufficient and necessary for adipocyte development as ectopic expression of C/EBPα will convert pre-adipocytes to adipocytes in the absence of hormonal stimulation (19Freytag S. Paielli D.L. Gilbert J.D. Genes Dev. 1994; 8: 1654-1663Crossref PubMed Scopus (393) Google Scholar, 20Lin F.-T. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8757-8761Crossref PubMed Scopus (383) Google Scholar), and antisense inhibition of C/EBPα expression inhibits adipocyte development (21Lin F.-T. Lane M.D. Genes Dev. 1992; 6: 533-544Crossref PubMed Scopus (275) Google Scholar). Expression of C/EBPα also causes growth arrest (19Freytag S. Paielli D.L. Gilbert J.D. Genes Dev. 1994; 8: 1654-1663Crossref PubMed Scopus (393) Google Scholar, 21Lin F.-T. Lane M.D. Genes Dev. 1992; 6: 533-544Crossref PubMed Scopus (275) Google Scholar, 22Hendricks-Taylor L.R. Darlington G.J. Nucleic Acids Res. 1995; 23: 4726-4733Crossref PubMed Scopus (113) Google Scholar, 23Timchenko N.A. Wilde M. Nakanishi M. Smith J.R. Darlington G.J. Genes Dev. 1996; 10: 804-815Crossref PubMed Scopus (346) Google Scholar), a key characteristic of terminally differentiated cells. C/EBPα's maintenance of the characteristic growth arrest of terminally differentiated adipocytes is implicated in the formation of liposarcomas. CHOP, a member of the C/EBP family of proteins, is an antagonist of C/EBP-mediated transactivation (24Ron D. Habener J. Genes Dev. 1992; 6: 439-453Crossref PubMed Scopus (986) Google Scholar). In a myxoid liposarcoma, CHOP was identified in a chromosomal translocation fused to the Ews RNA-binding protein (25Crozat A. Aman P. Mandahl N. Ron D. Nature. 1993; 363: 640-644Crossref PubMed Scopus (762) Google Scholar, 26Rabbitts T.H. Forster A. Larson R. Nathan P. Nat. Genet. 1993; 4: 175-180Crossref PubMed Scopus (484) Google Scholar). This fusion protein allows constitutive activation of CHOP, thereby activating an inhibitor of C/EBPα activity (27Batchvarova N. Wang X.Z. Ron D. EMBO J. 1995; 14: 4654-4661Crossref PubMed Scopus (207) Google Scholar). The CHOP-Ews fusion protein is sufficient for transformation of fibroblasts (28Zinszner H. Albalat R. Ron D. Genes Dev. 1994; 8: 2513-2526Crossref PubMed Scopus (243) Google Scholar), implying that inhibition of C/EBP-mediated transactivation can subvert the C/EBPα-mediated growth arrest and lead to liposarcomas (29Hunter T. Cell. 1997; 8: 333-346Abstract Full Text Full Text PDF Scopus (629) Google Scholar). Because FBR v-Fos and G2A v-Fos give rise to either liposarcomas or lipomas in transgenic mice (5Jotte R.M. Holt J.T. J. Cell Biol. 1996; 135: 457-467Crossref PubMed Scopus (10) Google Scholar), we sought to study the mechanism of this inhibition of adipocyte development in vitro. We show that pre-adipocytes which express FBR v-Fos or G2A v-Fos do not differentiate into adipocytes. These cells begin the adipocyte differentiation pathway as they express normal levels of C/EBPβ, but later stages of differentiation are faulty as these cells do not express C/EBPα. These cells also do not undergo the growth arrest which is characteristic of terminally differentiated cells. We further show that expression of FBR v-Fos or G2A v-Fos causes decreased transcriptional activity of C/EBPα but not C/EBPβ. Lastly, by utilizing tumors from the FBR v-fos transgenic mice, we show that the expression pattern of the C/EBP proteins in vivomatches that seen in vitro. Thus, the use of FBR v-Fos to induce liposarcomas further implicates the C/EBP family of proteins as key factors in the generation of liposarcomas. NIH 3T3-L1 cells and HeLa cells were obtained from the American Type Tissue Collection (ATCC). These cells were grown in 10% fetal bovine serum (Sigma), 2 mml-glutamine (Sigma), and 1 × antimycotic/antibiotic (Sigma). RSV-neo, FBR v-Fos, and G2A v-Fos stable cell lines were generated by transfecting NIH 3T3-L1 cells with empty vector, FBR v-fos or G2A v-fos and the neomycin resistance gene (neo R) in a 9:1 ratio using calcium phosphate precipitation. Cells were then selected for 2 weeks in 1 mg/ml G418 (Sigma). Approximately 500–1000 colonies were pooled and shown to express FBR v-Fos or G2A v-Fos by reverse transcription-polymerase chain reaction (5Jotte R.M. Holt J.T. J. Cell Biol. 1996; 135: 457-467Crossref PubMed Scopus (10) Google Scholar). CRP-CAT cells, CRP-CAT + FBR v-Fos cells and CRP-CAT + G2A v-Fos cells were generated in a similar manner, using a 9:9:1 ratio of CRP-CAT:empty vector:neo R, FBR v-fos or G2A v-fos:CRP-CAT:neo R. HeLa cells were used for transient transfection assays. These cells were transfected with the indicated amounts of CRP-CAT, FBR v-Fos, G2A v-Fos, C/EBPα, or C/EBPβ and β-galactosidase. Cells were transfected with a total of 23 μg of DNA, using Pgem 7Z(+) to standardize to equal amounts of DNA. Cells were transfected as described previously (30Graham F. van der Eb A. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6499) Google Scholar). MSV-C/EBPα, MSV-C/EBPβ, FBR v-Fos, G2A v-Fos, and β-galactosidase were used as described previously (5Jotte R.M. Holt J.T. J. Cell Biol. 1996; 135: 457-467Crossref PubMed Scopus (10) Google Scholar,31Kamata N. Jotte R.M. Holt J.T. Mol. Cell. Biol. 1991; 11: 765-772Crossref PubMed Scopus (22) Google Scholar, 32Kamata N. Holt J.T. Mol. Cell. Biol. 1992; 12: 876-882Crossref PubMed Scopus (15) Google Scholar, 33Jotte R.M. Kamata N. Holt J.T. J. Biol. Chem. 1994; 269: 16383-16396Abstract Full Text PDF PubMed Google Scholar). CRP-CAT was obtained from Christian Trautwein and Michael Manns (Medizinishe Hochschule Hannover) (34Trautwein C. Walker D.L. Plumpe J. Manns M.P. J. Biol. Chem. 1995; 270: 15130-15136Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Initiation and maintenance of differentiation was performed as described previously (35Yeh W.-C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (813) Google Scholar). All experiments were performed on low passage-number cells. Briefly, at confluence (day 0 of differentiation), cells were fed with medium containing 10% fetal bovine serum, 0.5 mmisobutylmethylxanthine (Sigma), 10 μg/mL insulin (Sigma), and 1 μm dexamethosone (Sigma). Two days later, the medium was replaced with medium containing 2.5 μg/ml insulin and 10% fetal bovine serum. The cells were refed with this medium every 2 days for the remainder of the differentiation period. Cells were fixed in 10% formaldehyde, and Oil Red O staining was performed as described previously (35Yeh W.-C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (813) Google Scholar). Cells were washed twice with ice-cold phosphate-buffered saline and scraped into a buffer containing 50 mm Tris-HCl (pH 7.5), 1 mm EDTA, and 1 mmβ-mercaptoethanol. Cell suspensions were allowed to sit on ice for 10 min and then sonicated for 10 s. Cells were centrifuged for 10 min, and the protein concentration of the supernatant was determined using the Bio-Rad protein assay. G3PDH activities were then performed as described elsewhere (36Kozak L.P. Jensen J.T. J. Biol. Chem. 1974; 249: 7775-7781Abstract Full Text PDF PubMed Google Scholar, 37Wise L.S. Green H. J. Biol. Chem. 1979; 254: 273-275Abstract Full Text PDF PubMed Google Scholar). 10 μg of total cell protein were added to a buffer containing 50 mm Tris-HCl (pH 7.5), 5 mm EDTA, 0.16 NADH (Fluka Chemical Corp.), and 0.8 mm dihydroxyacetone phosphate (Fluka) in a total volume of 500 μl. This reaction was allowed to incubate at 30 °C for 30 min. Activity was measured using absorbance at 340 nm on a spectrophotometer. The reaction was linear for both time and enzyme concentration. Cells from each line were plated a day prior to the commencement of the differentiation protocol. For each cell line, 12 plates were used. At day 0 of differentiation, half the plates were subjected to the differentiation protocol and half were fed with normal medium (refed every 2 days). Cells were harvested and counted at the times indicated. Cells were differentiated for the time periods listed. Whole cells lysates were prepared as described above for the G3PDH assays. Equal amounts of lysates were treated with SDS-polyacrylamide gel electrophoresis, 12% gel. Lysates were transferred to nitrocellulose filters (Amersham Corp.) for 2 h at 40 mV. To assure equal loading of proteins, the membranes were stained with Ponceau S prior to blocking. The nitrocellulose membrane was then blocked using 5% nonfat dry milk, 1% bovine serum albumin, 0.1% Tween 20 for 1 h. The blot was washed three times with Tris-buffered saline with 0.1% Tween 20 and exposed to a 1:1000 dilution of the chosen antibody for 1 h. After washing, the blot was then exposed to a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) at a dilution of 1:2500 for 1 h. Bands were visualized using the ECL detection system (Amersham) using the manufacturer's procedures. Antibodies used (anti-C/EBPα and anti-C/EBPβ) were purchased from Santa Cruz Biotechnology. For CAT assays on the CRP-CAT, FBR v-Fos + CRP-CAT and G2A v-Fos + CRP-CAT stable cell lines, cells were differentiated for the time periods indicated. Cells were harvested and lysed by four cycles of freezing and thawing. Protein concentrations were standardized. CAT activity was determined as described previously (31Kamata N. Jotte R.M. Holt J.T. Mol. Cell. Biol. 1991; 11: 765-772Crossref PubMed Scopus (22) Google Scholar). For CAT assays on the transiently transfected HeLa cells, cells were harvested 48 h after transfection. Cells were lysed by four cycles of freezing and thawing. Transfection efficiency was then standardized using β-galactosidase activity by methods previously described (38Kerr L.D. Holt J.T. Matrisian L.M. Science. 1988; 242: 1424-1427Crossref PubMed Scopus (210) Google Scholar). CAT activity was assayed as described previously (31Kamata N. Jotte R.M. Holt J.T. Mol. Cell. Biol. 1991; 11: 765-772Crossref PubMed Scopus (22) Google Scholar). RSV-neo cells and FBR v-Fos cells were subjected to the differentiation protocol for 2 days. Nuclei were isolated and proteins extracted by the method described in Rana et al.(39Rana B. Xie Y. Mischoulon D. Bucher N.L. Farmer S.R. J. Biol. Chem. 1995; 270: 18123-18132Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Protein concentrations were standardized, and EMSA was performed as described previously (39Rana B. Xie Y. Mischoulon D. Bucher N.L. Farmer S.R. J. Biol. Chem. 1995; 270: 18123-18132Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Briefly, 10 μg of nuclear extract from the indicated cell line was incubated with 3 mg of poly(dI-dC) in 25 μl of buffer containing a final concentration of 150 mmKCl, 25 mm Hepes (pH 7.6), 7.5% glycerol, 0.1 mm EDTA, and 2.5 mm MgCl2. A double-stranded oligonucleotide probe containing the C/EBP consensus site 5′-GATCCGCGTTGCGCCACGATG-3′ was end-labeled with [γ32P]ATP. Protein binding to the oligonucleotide was determined by electrophoresis on a nondenaturing 6% polyacrylamide gel in TBE buffer (0.09 m Tris borate, 2 mm EDTA (pH 8.0)). In supershift studies, 5 μl of appropriate antibody were preincubated with the protein mixture for 1 h at room temperature prior to the addition of the probe. After 8 days of differentiation, total RNA was harvested using the RNAeasy kit (Promega). Pgem7z-C/EBPα was digested with EcoRI/BamHI, and the resulting 1.4-kilobase pair fragment was isolated. This fragment was then random prime-labeled with [α-32P]dATP using the Prime-It II kit (Stratagene). 30 μg of RNA were electrophoresed on a formaldehyde gel and transferred to GeneScreen Plus. Prehybridization was performed using 200 μg/ml salmon sperm DNA, 6 × SSC, 50% formamide. Hybridization was then allowed to proceed overnight. The blot was washed five times in 2 × SSC, 1% SDS and subjected to autoradiography. Tumors were harvested from transgenic FBR v-Fos. Generation of these transgenic mice has already been reported (5Jotte R.M. Holt J.T. J. Cell Biol. 1996; 135: 457-467Crossref PubMed Scopus (10) Google Scholar). Processing of the tumor was conducted according to standard procedure by the Vanderbilt Cancer Center Human Tissue Acquisition and Pathology Shared Resource. Immunohistochemistry was conducted on an automatic stainer (model Techmate; Biotech Research Labs Inc., Rockville, MD) using a universal ABC detection kit with diaminobenzidine (DAB) chromagen using the following antibodies: anti-C/EBPα and anti-C/EBPβ (Santa Cruz Bioctechnology). Both antibodies were diluted 1:100. The transgenic mouse model suggested that both FBR v-Fos and G2A v-Fos inhibited adipocyte differentiation (5Jotte R.M. Holt J.T. J. Cell Biol. 1996; 135: 457-467Crossref PubMed Scopus (10) Google Scholar). To study this phenomenon further, we utilized an in vitro model of adipocyte differentiation. NIH 3T3-L1 cells are a pre-adipocyte cell line which will differentiate into adipocytes upon exposure at confluence to insulin, isobutylmethylxanthine, and dexamethosone for 2 days followed by exposure to insulin for the remainder of the differentiation period. By day 8 of the differentiation protocol, greater than 90% of the cells show overt morphological signs of adipocyte differentiation (8Green H. Kehinde O. Cell. 1974; 1: 113-116Abstract Full Text PDF Scopus (746) Google Scholar, 9Green H. Kehinde O. Cell. 1975; 5: 19-27Abstract Full Text PDF PubMed Scopus (1097) Google Scholar, 10Green H. Kehinde O. Cell. 1976; 7: 105-113Abstract Full Text PDF PubMed Scopus (616) Google Scholar, 11Russell T.R. Ho R.-J. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 4516-4520Crossref PubMed Scopus (157) Google Scholar). 3T3-L1 cell lines were made which stably expressed G2A v-Fos or FBR v-Fos. These cell lines were shown to constitutively express FBR v-Fos or G2A v-Fos by reverse transcription-polymerase chain reaction (5Jotte R.M. Holt J.T. J. Cell Biol. 1996; 135: 457-467Crossref PubMed Scopus (10) Google Scholar) and Western blotting (data not shown). The RSV-neo cells, FBR v-Fos cells, and G2A v-Fos cells were then allowed to grow to confluence and exposed to differentiation media for 8 days. The control cell line (RSV-neo cells) accumulated lipid droplets and showed morphological signs of adipocyte differentiation (Fig. 2). In contrast to the RSV-neo cells, neither the FBR v-Fos or G2A v-Fos cells showed any morphological signs of adipocyte differentiation (Fig. 2). The lack of morphological evidence of adipocyte differentiation in FBR v-Fos and G2A v-Fos cells was consistent as two additional cell lines derived independently from those shown in Fig. 2 gave similar results. In addition to morphological study of these stably transfected cell lines, we wanted to establish by biochemical means that FBR v-Fos and G2A v-Fos cells do not differentiate into adipocytes. G3PDH is an enzyme whose activity increases greatly as a cell differentiates into an adipocyte (36Kozak L.P. Jensen J.T. J. Biol. Chem. 1974; 249: 7775-7781Abstract Full Text PDF PubMed Google Scholar, 37Wise L.S. Green H. J. Biol. Chem. 1979; 254: 273-275Abstract Full Text PDF PubMed Google Scholar, 40Serrero G. Mills D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3912-3916Crossref PubMed Scopus (75) Google Scholar). Measuring the biochemical activity of this enzyme is a well established method to assay adipocyte differentiation (41Serrero G. Lepak N. Int. J. Obes. 1996; 3: 58-64Google Scholar). After 8 days of differentiation, RSV-neo cells, FBR v-Fos cells. and G2A v-Fos cells were lysed, and biochemical activity of G3PDH was assayed. RSV-neo cells showed over 50-fold greater G3PDH activity when compared with FBR v-Fos and G2A v-Fos cells (TableI), consistent with the fact that the RSV-neo cells differentiate to adipocytes, while neither the FBR v-Fos cells nor the G2A v-Fos cells differentiate to adipocytes.Table IGlycerophosphate dehydrogenase activity after 8 days of differentiation to adipocytesCell lineSpecific activity ± S.E.nmol NADH/mg protein/minRSV-neo1180 ± 9.40FBR v-Fos22.9 ± 7.201-ap values are <0.001 by Student's t test when compared to the RSV-neo cell line.G2A v-Fos23.8 ± 16.11-ap values are <0.001 by Student's t test when compared to the RSV-neo cell line.1-a p values are <0.001 by Student's t test when compared to the RSV-neo cell line. Open table in a new tab To begin to determine the molecular mechanism for this inability to differentiate, growth curves of RSV-neo cells, FBR v-Fos cells, and G2A v-Fos cells were measured. In normal adipocyte differentiation, cells must be growth-arrested before they began to show overt evidence of adipocyte differentiation (12MacDougald O.A. Lane M.D. Annu. Rev. Biochem. 1995; 64: 345-373Crossref PubMed Scopus (943) Google Scholar, 42Umek R. Friedman A. McKnight S.L. Science. 1991; 251: 288-292Crossref PubMed Scopus (573) Google Scholar). Growth curves of differentiating pre-adipocytes should become relatively flat as the cells differentiate. To show that this is the case in our RSV-neo control cells, two plates of RSV-neo cells were plated at equal cell density for each of the six time points. At day 0, one plate of cells per time point began the differentiation protocol, while the other plate of cells per time point remained in normal media, maintaining a normal growth rate. Cell counts were taken 0, 1, 2, 3, 5, and 8 days after the beginning of the experiment. The RSV-neo cells remaining in normal medium showed exponential growth (Fig.3 A), while those undergoing adipogenesis showed a significantly lower rate of growth (Fig.3 B), consistent with differentiation to adipocytes. Both FBR v-Fos cells and G2A v-Fos cells showed a growth curve similar to the RSV-neo cells in normal media (Fig. 3 A), showing that the expression of these oncogenes does not increase the proliferation of these cells. Upon exposure to differentiation media, however, the G2A v-Fos cells showed no growth arrest, and the FBR v-Fos cells growth-arrested only slightly (Fig. 3 B). These results imply that FBR v-Fos and G2A v-Fos do not inhibit adipocyte differentiation by stimulating cell growth, but by inhibiting the growth arrest which is required for terminal adipocyte differentiation. The C/EBP family of transcription factors regulates fat cell-specific genes and is important for the initiation and maintenance of adipocyte differentiation (15Samuelsson L. Stromberg K. Vikman D Bjursell G. Enerback S. EMBO J. 1991; 10: 3787-3793Crossref PubMed Scopus (140) Google Scholar, 42Umek R. Friedman A. McKnight S.L. Science. 1991; 251: 288-292Crossref PubMed Scopus (573) Google Scholar). In an undifferentiated preadipocyte, C/EBPβ is expressed at a low level. One day after the initiation of the differentiation protocol, expression of C/EBPβ is greatly induced before decreasing at later differentiation stages (35Yeh W.-C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (813) Google Scholar). C/EBPα is not expressed in the undifferentiated preadipocyte, but as the cell begins to show overt signs of adipocyte differentiation (days 3–5), C/EBPα begins to become expressed (35Yeh W.-C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (813) Google Scholar). Since both C/EBPβ and C/EBPα are involved in a cascade regulation of adipocyte differentiation, we wanted to determine whether these proteins were expressed in FBR v-Fos and G2A v-Fos cells. By Western blot analysis, the RSV-neo cells show normal patterns of both C/EBPα and C/EBPβ expression. C/EBPβ is expressed early in differentiation, and expression lowers as the cell continues differentiating (Fig. 4,left/lower panel). In addition, the alternatively spliced C/EBPβ, liver inhibitory protein (43Descombes P. Schibler U. Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (862) Google Scholar), which this antibody also recognizes, shows a similar expression pattern. C/EBPα begins to be expressed in the RSV-neo cells as the cell begins to reach the later stages of differentiation (Fig. 4, left/top panel). The expression pattern of these proteins is different in the FBR v-Fos and G2A v-Fos cells. In both cell lines, C/EBPβ is expressed at levels indistinguishable from RSV-neo cells (Fig. 4, compare lower panels). Since C/EBPβ expression is one of the earliest indicators of the initiation of adipogenesis (35Yeh W.-C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (813) Google Scholar), this implies that the early stages of adipogenesis are functioning normally in FBR v-Fos and G2A v-Fos cells. While the initiation of adipogenesis is unaltered, the expression pattern of C/EBPα shows that later stages of adipogenesis are faulty. Neither the FBR v-Fos cell line nor the G2A v-Fos cell line expresses C/EBPα (Fig. 4, upper middle and upper right panels; a positive control is shown in the far left lane of each blot). This expression pattern of C/EBPα is consistent with the fact that these cells do not differentiate. Moreover, the expression pattern of C/EBPβ and C/EBPα suggests that FBR v-Fos and G2A v-Fos affect adipo" @default.
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• W2007455340 title "Finkel-Biskis-Reilly Osteosarcoma Virus v-Fos Inhibits Adipogenesis and Both the Activity and Expression of CCAAT/Enhancer Binding Protein α, a Key Regulator of Adipocyte Differentiation" @default.
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