Alteration of p53 and p21 during hepatocarcinogenesis in tree shrews.

Aim: To investigate p53 mutation and p21 expression in hepatocarcinogenesis induced by hepatitis B virus (HBV) and aflatoxin B(1) (AFB(1)) in tree shrews, and to reveal the role of these genes in hepatocarcinogenesis.

Methods: Tree shrews were divided into four groups: group A, those infected with HBV and fed with AFB(1) (n = 39); group B, those infected with HBV alone (n = 28); group C, those fed with AFB(1) alone (n = 29); and group D, normal controls (n = 20). The tree shrews underwent liver biopsies once every 15 wk. Expression of p53 and p21 proteins and genes in the biopsies and tumor tissues of the experimental tree shrews was detected, respectively, by immunohistochemistry, and by Southern blotting and reverse transcription-polymerase chain reaction and sequencing.

Results: The incidence of hepatocellular carcinomas (HCC) was higher in group A (66.7%) than that in group B (3.57%) and C (30%). The time of HCC occurrence was also earlier in group A than that in group C (120.0+/-16.6 wk vs 153.3+/-5.8 wk, respectively, P<0.01). p53 protein was not detected by immunohistochemistry in all groups before the 75(th) wk of the experiment. At the 105(th) wk, the positive rates fo p53 were 78.6%, 60% and 71.4% in groups A, B and C, respectively, which were significantly higher than that in group D (10%) (all P<0.05). An abnormal band of p53 gene was observed in groups A and C. The mutation points of p53 gene in tree shrews with HCC were at codons 275, 78 and 13. The nucleotide sequence and amino acid sequence of tree shrew's wild-type p53 showed 91.7% and 93.4% homologies with those of human p53, respectively. The immunopositivity for p21 was found before HCC development. The incidence of HCC was significantly higher in tree shrews that were positive for p21 than those negative for p21 (80.0% vs 11.0%, P<0.001). The incidence of HCC in p21 positive animals in group A was significantly higher than those positive for p21 in group C (P<0.05).

Conclusion: A remarkable synergistic effect on HCC development exists between HBV and AFB(1). p53 mutation promotes the development of HCC. HBV and AFB(1) may synergistically induce p53 gene mutation, and stimulate ras gene expression. ras gene is activated at the earlier stage during hepatocarcinogenesis. p21 protein may be an early marker, and the alterations of p53 may be a late event in the development of HCC.