Hepatocellular carcinoma (HCC) is a prevalent cancer in sub-Saharan Africa and eastern Asia. Hepatitis B virus and aflatoxins are risk factors for HCC, but the molecular mechanism of human hepatocellular carcinogenesis is largely unknown. Abnormalities in the structure and expression of the tumour-suppressor gene p53 are frequent in HCC cell lines, and allelic losses from chromosome 17p have been found in HCCs from China and Japan. Here we report on allelic deletions from chromosome 17p and mutations of the p53 gene found in 50% of primary HCCs from southern Africa. Four of five mutations detected were G----T substitutions, with clustering at codon 249. This mutation specificity could reflect exposure to a specific carcinogen, one candidate being aflatoxin B1 (ref. 7), a food contaminant in Africa, which is both a mutagen that induces G to T substitution and a liver-specific carcinogen.

Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae.

Cell cycle regulation is critical for maintenance of genome integrity. A prominent factor that guarantees genomic stability of cells is p53 (ref. 1). The P53 gene encodes a transcription factor that has a role as a tumour suppressor. Identification of p53-target genes should provide greater insight into the molecular mechanisms that mediate the tumour suppressor activities of p53. The rodent Pc3/Tis21 gene was initially described as an immediate early gene induced by tumour promoters and growth factors in PC12 and Swiss 3T3 cells. It is expressed in a variety of cell and tissue types and encodes a remarkably labile protein. Pc3/Tis21 has a strong sequence similarity to the human antiproliferative BTG1 gene cloned from a chromosomal translocation of a B-cell chronic lymphocytic leukaemia. This similarity led us to speculate that BTG1 and the putative human homologue of Pc3/Tis21 (named BTG2) were members of a new family of genes involved in growth control and/or differentiation. This hypothesis was recently strengthened by the identification of a new antiproliferative protein, named TOB, which shares sequence similarity with BTG1 and PC3/TIS21 (ref. 7). Here, we cloned and localized the human BTG2 gene. We show that BTG2 expression is induced through a p53-dependent mechanism and that BTG2 function may be relevant to cell cycle control and cellular response to DNA damage.

There is little information regarding the molecular mechanisms of hepatocarcinogenesis. We studied the p53 gene at the DNA, RNA, and protein level in seven human hepatocellular carcinoma (HCC)-derived cell lines; six of seven showed p53 abnormalities. By Southern blotting, the p53 gene was found to be partially deleted in Hep 3B and rearranged in SK-HEP-1 cells. Mahlavu cells showed a slower migration on SDS/polyacrylamide gels suggesting it was an abnormal protein. In' Huh7 cells, p53 protein had a prolonged half-life leading to its accumulation in the nuclei; increased levels of p53 protein were also found by immunoblotting. The p53 gene and its expression appeared to be unaltered in the hepatoblastoma-derived Hep G2 cell line. We found that the loss of p53 expression did not occur as a late in vitro event in the FOCUS cell line because p53 protein was also nondetectable at an early passage. We conclude that the loss of p53 expression or the presence of abnormal forms of the protein are frequently associated with HCC cell line-. These observations suggest that alterations in p53 may be important events in the transformation of hepatocytes to the malignant phenotype.Human hepatocellular carcinoma (HCC) is one of the most frequent tumors worldwide. Although HCC is relatively rare in North America and Europe, it ranks as one ofthe four most prevalent malignant diseases of adults in China, Taiwan, Korea, and sub-Saharan Africa (1). Infection with hepatitis B virus (HBV) as well as alcoholic cirrhosis and other factors associated'with chronic inflammatory and hepatic regenerative changes are found to be important risk factors for hepatocarcinogenesis (1-3). However, the molecular mechanisms involved in malignant transformation of hepatocytes remain largely unknown'. Because of strong epidemiological evidence showing a close correlation between the prevalence of hepatitis B surface antigen chronic carriers and the incidence of HCC, the role of HBV in hepatocarcinogenesis has been extensively studied (4). However, the HBV genome is unlikely to carry oncogenic sequences, and there is little evidence to suggest that integration of viral DNA into hepatocyte DNA activates cellular protooncogenes (3). Indeed, no specific association between activation of any known oncogene and HCC has yet been found.The role of recessive oncogenes in HCC has not been studied in detail. T'ang et al. (5) Immunoblotting. Cell and tissue extracts were prepared as described (24) and samples adjusted to'equal protein concentrations (100-200 ,ug) were separated by SDS/PAGE, electrotransferred to nitrocellulose paper, and analyzed by using the 1251-labeled Pabl22 antibody (25).Indirect Immunofluorescence. Cells grown on cover slips under standard culture conditions were used for indirect immunostaining with monoclonal antibody (mAb) Pabl22 by using a described protocol with minor modifications (26). Briefly, cells were fixed in 4% (vol/vol) formaldehyde and incubated with 1% bovine serum albumin (BSA)/10 mM phosphate/140 mM NaCl, pH 7.2...

Senescence induction could be used as an effective treatment for hepatocellular carcinoma (HCC). However, major senescence inducers (p53 and p16 Ink4a) are frequently inactivated in these cancers. We tested whether transforming growth factor-b (TGF-b) could serve as a potential senescence inducer in HCC. First, we screened for HCC cell lines with intact TGF-b signaling that leads to small mothers against decapentaplegic (Smad)-targeted gene activation. Five cell lines met this condition, and all of them displayed a strong senescence response to TGF-b1 (1-5 ng/mL) treatment. Upon treatment, c-myc was down-regulated, p21Cip1 and p15 Ink4bwere up-regulated, and cells were arrested at G 1 . The expression of p16 Ink4a was not induced, and the senescence response was independent of p53 status. A short exposure of less than 1 minute was sufficient for a robust senescence response. Forced expression of p21Cip1 and p15Ink4b recapitulated TGF-b1 effects. Senescence response was associated with reduced nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) induction and intracellular reactive oxygen species (ROS) accumulation. The treatment of cells with the ROS scavenger N-acetyl-L-cysteine, or silencing of the NOX4 gene, rescued p21 Cip1 and p15 Ink4b accumulation as well as the growth arrest in response to TGF-b. Human HCC tumors raised in immunodeficient mice also displayed TGF-b1-induced senescence. More importantly, peritumoral injection of TGFb1 (2 ng) at 4-day intervals reduced tumor growth by more than 75%. In contrast, the deletion of TGF-b receptor 2 abolished in vitro senescence response and greatly accelerated in vivo tumor growth. Conclusion: TGF-b induces p53-independent and p16 Ink4a -independent, but Nox4-dependent, p21 Cip1-dependent, p15 Ink4b-dependent, and ROS-dependent senescence arrest in well-differentiated HCC cells. Moreover, TGF-b-induced senescence in vivo is associated with a strong antitumor response against HCC. (HEPATOLOGY 2010;52:966-974)