Cell cycle gene regulation in reversibly differentiated new human hepatoma cell lines.
Glaise D. Ilyin GP. Loyer P. Cariou S. Bilodeau M. Lucas J. Puisieux A. Ozturk M. Guguen-Guillouzo C.
INSERM U49, Hopital Pontchaillou, Rennes, France.
Several novel differentiated cell lines have been derived from a human hepatocarcinoma named HBG. Analysis of their functional properties evidenced a gradual differentiation process as they became confluent and a remarkable stability of the whole quiescent population for at least 6 weeks. However, when replated at low density after several weeks of quiescence, the differentiated cells were able to rapidly reverse to active proliferation, accompanied by transient dedifferentiation. Demonstration that the differentiated hepatic cells were growth-arrested in G1 phase was provided by the increased number of cells with 2C DNA content and decreased expression of S-phase markers. Characteristic features of oncogenes and cell cycle genes were defined during the differentiation process: (a) a biphasic expression of c-myc, with the latter wave covering the quiescence period; (b) opposite kinetics of c-Ki-ras and of N-ras expression with a pattern of changes paralleling that of c-myc; and (c) a decrease of cyclin D1 protein expression and of the cyclin D1-associated kinase activity. The mechanisms by which quiescent differentiated cells might reinitiate active proliferation were analyzed by studying several genes involved in cell growth and death regulation. We found: (a) a point mutation and loss of the specific activity of the tumor suppressor gene p53 without alteration of the apoptotic response to transforming growth factor beta1; (b) a gradual decrease of retinoblastoma protein, which was constantly present, mainly in a hyperphosphorylated form; and (c) an increase of cyclin-dependent kinase inhibitor p27 expression in confluent differentiating cells, as expected, whereas, surprisingly, a disappearance of the p21 protein was observed in parallel. These data may reflect specific mechanisms of cell cycle regulation in liver parenchymal cells through which these cells can proceed to control their reversible differentiation program.
Enhanced expression of a new class of liver-enriched b-Zip transcription factors, hepatocarcinogenesis-related transcription factor, in hepatocellular carcinomas of rats and humans.
Kishimoto T. Kokura K. Ohkawa N. Makino Y. Yoshida M. Hirohashi S. Niwa S. Muramatsu M. Tamura T.
Department of Biology, Faculty of Science, Chiba University, Japan.
Rat hepatocarcinogenesis-related transcription factor (HTF) was earlier identified as a b-Zip transcription factor in chemically induced rat hepatocellular carcinoma (HCC) by cDNA subtraction, and its structure was found to be different from that of the conventional b-Zip proteins. We investigated htf gene expression in rat tissues by Northern analysis and found that HTF expression was ubiquitous but was enriched in the liver. HTF expression increased concomitantly with HCC development in rat liver, and the HTF-containing DNA-binding factor also increased. Stimulated HTF gene expression also was observed in rat regenerating livers. From the results of various assays, X-box-binding protein 1/Tax-response element binding factor 5 was suggested to be a human homologue of rat HTF. In humans, HTF gene expression was also abundant in the liver and was revealed to be specifically stimulated in HCCs, but not in other types of cancers. To our knowledge, HTF is the first example of a liver-enriched transcription factor that exhibits HCC-associated gene expression. Injection of anti-HTF antibody decreased the growth rate of cultured HCC cells. Consequently, HTF is thought to participate in hepatocyte growth as well as in hepatocarcinogenesis.