Hepato1og.v 2000; 33: 254-265 Printed in Denmark Ail rights reserved Mwrksgaard Copenhagen Journal of Hepatology ISSN 0168-8278 ‘I ~53 but not ~16~~~ induces growth arrest in retinoblastomadeficient hepatocelhilar carcinoma cells Anne Pierre Morel”, Kezban Unsal**, Tolga Cagatay2, Frederique Ponchel’, Brian Carr” and Mehmet Ozturk’,2 NSERM U453, Centre Leon Berm-d, Lyon, France, ‘Department zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey, and 3Pittsburgh Transplantution Institute, University zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONML of Pittsburgh, Pittsburgh, PA, USA Background/Aim: Both ~16’~~” and p53 proteins are negative regulators of the cell cycle. In human hepato- cellular carcinomas @ICC), the loss of function of ~53, retinoblastoma @Rb) and ~16’~~~~ genes by dif- ferent mechanisms has been largely documented, but their hepatocellular effects are poorly known. We compared the growth-inhibitory effects of p16rNK4” and p53 proteins in Hep3B cell line-derived clones. Met/z&: Cells were transfected with inducible ~16’~~~” and p53 expression vectors, and stable clones were analyzed for transgene expression by Western blotting and immunoperoxidase staining. Ef- fects 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 p21 ‘iPI transcripts and by Western blotting of p2P1, mdm-2, bax, cyclin-dependent kinase 2 and cyclin E proteins. The pRb protein was studied by Western blotting and immunoprecipitation assays. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHG H EPATOCELLULAR carcinoma (HCC), which is etio- logically associated to hepatitis B virus (HBV), hepatitis C virus (HCV) and aflatoxins, is one of the most frequent cancers world-wide (1). Genetic studies have revealed so far that a dozen genes are altered in these cancers. These genetic alterations (mostly so- matic) are related to at least four different pathways, including DNA damage response (~53 gene), cell cycle regulation (retinoblastoma, p 1 61NK4a and cyclin D genes), TGF-P (M6P/IGF2R, SMAD2 and SMAD4 * A. P M. and K. U. contributed equally to this work. Received 4 October: revised 17 December; accepted 24 December 1999 Correspondence: Mehmet Ozturk, Department of Mol- ecular Biology and Genetics, Bilkent University, 06533 Bilkent Ankara, Turkey. Tel: 90 3 12 266 50 81. Fax: 90 312 266 50 97. e-mail: ozturk@fen.bilkent.edu.tr Results: The induction of ~16’~~’ 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 p21Cipr gene and accumulation of p21ciP’, bax and mdm-2 proteins. p53-induced growth arrest was due to a cell cycle arrest at the GUS transition, probably mediated by p21ciP’ protein, which inhibits cyclin-de- pendent kinase 2/cyclin E complexes. Conclusions: The lack of detectable pRb protein and resistance of cells to p16rNK4”strongly suggest that p53 is able to arrest the growth of HCC cells by a mechanism independent of “p53-retinoblastoma path- way”. These findings are applicable to HCC with ab- berrations of both p53 and pRb genes, and may not represent the universal effects of p53 in hepatic cells. Key words: Cell cycle arrest; Cyclin E; Hepatoma; p16rNK4”; p21ciP’; ~53; Retinoblastoma. genes) and wnt (B-catenin and APC genes) signaling pathways (2). p53 was the first tumor suppressor gene found to be mutated in HCC (3). Many reports now indicate that the p53 gene, which is located at chromosome 17p, is somatically mutated in about 30% of HCCs worldwide (2). Both the frequency and the type of p.53 mutations are different depending on the geographical location and suspected etiology of these tumors. An HCC-spe- cific codon 249 mutation (AGG+AGT leading to p53- 249Ser), suspected to be induced by aflatoxins, was found in most HCCs from geographical areas with a high incidence of HCC and a high risk of exposure to aflatoxins (4,5). The frequency of all p53 mutations in HCC varies between 15% in Europe and 42% in China (2). On the other hand, physical and functional interac- tion between p53 protein and HBx viral antigen has 254