P53 but not p16INK4a induces growth arrest in retinoblastoma-deficient hepatocellular carcinoma cells.
OBJECTIVE
Both p16INK4a and p53 proteins are negative regulators of the cell cycle. In human hepatocellular carcinomas (HCC), the loss of function of p53, retinoblastoma (pRb) and pl6INK4a genes by different mechanisms has been largely documented, but their hepatocellular effects are poorly known. We compared the growth-inhibitory effects of p16INK4a and p53 proteins in Hep3B cell line-derived clones.


METHODS
Cells were transfected with inducible p16INK4a and p53 expression vectors, and stable clones were analyzed for transgene expression by Western blotting and immunoperoxidase staining. Effects on cell growth were analyzed by in vitro growth assay, thymidine incorporation and flow cytometry. Biochemical effects of p53 were tested by Northern blotting of p21Cip1 transcripts and by Western blotting of p21Cip1, mdm-2, bax, cyclin-dependent kinase 2 and cyclin E proteins. The pRb protein was studied by Western blotting and immunoprecipitation assays.


RESULTS
The induction of p16INK4a protein expression did not affect in vitro growth of cells. In contrast, p53 protein in its wild-type conformation provoked a growth arrest accompanied by transactivation of p21Cip1 gene and accumulation of p21Cip1, bax and mdm-2 proteins. p53-induced growth arrest was due to a cell cycle arrest at the G1/S transition, probably mediated by p21Cip1 protein, which inhibits cyclin-dependent kinase 2/cyclin E complexes.


CONCLUSIONS
The lack of detectable pRb protein and resistance of cells to p16TNK4a strongly suggest that p53 is able to arrest the growth of HCC cells by a mechanism independent of "p53-retinoblastoma pathway". These findings are applicable to HCC with abberrations of both p53 and pRb genes, and may not represent the universal effects of p53 in hepatic cells.
Morel
A P
AP
INSERM U453, Centre Leon Berard, Lyon, France.
Unsal
K
K
Cagatay
T
T
Ponchel
F
F
Carr
B
B
Ozturk
M
M
D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology*; D002352:Carrier Proteins / Q000235:genetics*; D002455:Cell Division / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D016158:Genes, p53; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology*; D016160:Retinoblastoma Protein / Q000235:genetics*; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D016160:Retinoblastoma Protein