Hepatitis B virus-X protein upregulates the expression of p21wafl/cipl and prolongs G1-S transition via a p53 independent pathway in human hepatoma cells

Abstract

Progression through the cell cycle is controlled by the induction of cyclins and activation of cognate cyclin- dependent kinases. The human hepatitis B virus-X (HBV-X) protein functions in gene expression altera- tions, in the sensitization of cells to apoptotic killing and deregulates cell growth arrest in certain cancer cell types. We have pursued the mechanism of growth arrest in Hep3B cells, a p53-mutant human hepatocellular carcinoma (HCC) cell line. In stable or transient HBV-X transformed Hep3B cells, HBV-X increased protein and mRNA levels of the cyclin-dependent kinase inhibitor (CDKI) p21wa¯/cipl, increased binding of p21wa¯/cipl with cyclin-dependent kinase 2 (CDK2), markedly inhibited cyclin E and CDK2 associated phosphorylation of histone H1 and induced the activation of a p21 promoter reporter construct. By using p21 promoter deletion constructs, the HBV-X responsive element was mapped to a region between 71185 and 71482, relative to the transcription start site. Promoter mutation analysis indicated that the HBV-X responsive site coincides with the ets factor binding sites. These data indicate that in human hepatocellular carcinoma cells, HBV-X can circumvent the loss of p53 functions and induces critical downstream regulatory events leading to transcriptional activation of p21wa¯/cipl. As a consequence, there is an increased chance of acquisition of mutations which can enhance the genesis of hepatomas. Our results also emphasize the chemotherapeutic potential of p21wa¯/cipl inhibitors, particularly in the HBV-X infected hepatoma which lacks functional p53. Oncogene (2000) 19, 3384 ± 3394. Keywords: hepatitis B virus-X; p21wa¯/cipl; cyclin-depen- dent kinase inhibitor; hepatocellular carcinoma; p53