Progression through the cell cycle is controlled by the induction of cyclins and activation of cognate cyclindependent kinases. The human hepatitis B virus-X (HBV-X) protein functions in gene expression alterations, 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) p21 wa¯/cipl , increased binding of p21 wa¯/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 p21 wa¯/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 p21 wa¯/cipl inhibitors, particularly in the HBV-X infected hepatoma which lacks functional p53. Oncogene (2000) 19, 3384 ± 3394.

A¯atoxin B1 (AFB1) induced mutation of the p53 gene at codon 249 (p53mt249) is critical during the formation of hepatocellular carcinoma (HCC) following hepatitis B virus (HBV) infection. p53mt249 markedly increases insulin-like growth factor II (IGF-II) transcription largely from promoter 4, accumulating the fetal form of IGF-II. Modulation of the transcription factor binding to IGF-II P4 by wild-type p53 and p53mt249 was identi®ed. Wild-type p53 inhibited binding of transcription factors Sp1 and TBP on the P4 promoter, while p53mt249 enhanced the formation of transcriptional complexes through enhanced DNA-protein (Sp1 or TBP) and protein ± protein (Sp1 and TBP) interactions. p53mt249 stimulates transcription factor Sp1 phosphorylation which might be a cause of increased transcription factor binding on the P4 promoter while wild-type p53 does not. Transfection of hepatocytes with p53mt249 impaired induction of apoptosis by the HBV-X protein and TNF-a. Therefore, the blocking of apoptosis through enhanced production of IGF-II should provide a favorable opportunity for the selection of transformed hepatocytes. These results explain the molecular basis for the genesis of HCC by p53mt249 which was found to be induced by a potent mutagen, AFB1. Oncogene (2000) 19, 3717 ± 3726.

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The possibility that hepatitis C virus core gene product (HCV-core) acts as a transactivator in insulin-like growth factor II (IGF-II) gene transcription was tested. HCV-core protein increases endogenous IGF-II expression from promoter 4 (P4) of the IGF-II gene through two cis-acting elements: Sp1 and Egr1 binding sites. Sp1 and Egr1 both bind to IGF-II P4 and functionally cooperate in mediating the maximal activity of IGF-II P4. HCV-core protein induced the binding of Sp1 and Egr1 on its binding sites on IGF-II P4. In addition, Sp1 and Egr1 were stimulated to phosphorylate by HCV-core, and its DNA binding activity was up-regulated upon HCV-core transfection. Transfection with HCV-core in HepG2 cells stimulated the membrane translocation of protein kinase C (PKC) and the treatment of HCV-core transfected cells with calphostin C, a PKC inhibitor, blocked induction of Sp1 and Egr1 DNA binding activity, and eventually transcriptional transactivations of the IGF-II gene. Increasing the DNA binding activity of the phosphorylated form of Sp1 and Egr1 might be an important mechanism for regulating IGF-II gene expression and for promoting cell division during hepatic carcinogenesis. These results indicate that HCV-core functions as a positive regulator of IGF-II transcription through the PKC pathway and that Sp1 and Egr1 are direct targets of the transcriptional regulation of the IGF-II gene which plays an important role in hepatitis C virus pathogenesis during the formation of hepatocellular carcinoma (HCC).

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