CELL-CYCLE GENE-REGULATION IN REVERSIBLY DIFFERENTIATED NEW HUMAN HEPATOMA-CELL LINES

Several novel differentiated cell lines have been derived from a human hepatocarcinoma named HBG. Analysis of their functional properties ev idenced 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, Demo nstration that the differentiated hepatic cells were growth-arrested i n G(1) phase was provided by the increased number of cells with 2C DNA content and decreased expression of S-phase markers, Characteristic f eatures of oncogenes and cell cycle genes were defined during the diff erentiation process: (a) a biphasic expression of c-myc, with the latt er 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 t he cyclin D1-associated kinase activity, The mechanisms by which quies cent differentiated cells might reinitiate active proliferation were a nalyzed by studying several genes involved in cell growth and death re gulation, We found: (a) a point mutation and loss of the specific acti vity of the tumor suppressor gene p53 without alteration of the apopto tic response to transforming growth factor beta 1; (b) a gradual decre ase of retinoblastoma protein, which was constantly present, mainly in a hyperphosphorylated form; and (c) an increase of cyclin-dependent k inase inhibitor p27 expression in confluent differentiating cells, as expected, whereas, surprisingly, a disappearance of the p21 protein wa s observed in parallel, These data may reflect specific mechanisms of cell cycle regulation in liver parenchymal cells through which these c ells can proceed to control their reversible differentiation program.