ANALYSIS OF GENETIC ALTERATIONS DURING HEPATOCARCINOGENESIS

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Mutations in the putative p53 tumor suppressor gene were analyzed in a series of 9 aflatoxin B1 (AFLB1) induced monkey tumors, 6 AFLB1 transformed rat liver cell lines, as well as a group of 19 human hepatocellular carcinomas (HCC) obtained from Qidong, China, and 17 HCCs induced by the food mutagen, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in cynomolgus monkeys. Among the AFLB1 induced monkey tumors only a single point mutation at codon 175 (G to T transversion) was identified in only one HCC by sequencing analysis of the four conserved domains (II to V) in the p53 gene. In 3/17 of the IQ induced monkey tumors the same G to T transversion was detected at codons 159, 175, and 248. Sequence analysis of amplified cDNA by RT-PCR of p53 mRNA from the six AFLB1 transformed rat liver cell lines showed the same point mutation, a transition from G to A in codon 173 (CGC to CAC) which results in a change of the encoded amino acid from arginine to histidine. No wild- type alleles of the p53 gene were observed in any of the AFLB1 cell lines by genomic DNA analysis. The half life of the mutant p53 protein in the AFLB1 transformants was greater than 3 hours versus 30 minutes for normal p53 protein. Analysis of 19 HCCs from Qidong using PCR-SSCP as well as RFLP analysis revealed that 10/19 HCCs (53%) showed p53 mutation. In two tumors single mutations occurred in exons 5 and 6, while in 8 HCCs a G to T mutation at the third position of codon 249 in exon 7 was observed. Loss of heterozygosity (LOH) of the p53 and retinoblastoma (RB) genes was detected in 7/9 (78%) and 3/6 (50%) of informative cases, respectively. No LOH of adenomatis polyposis coli locus was observed in 10 informative cases. LOH of chromosomes 16q and 16p was detected in 10/18 (56%) and 6/13 (46%) informative cases, respectively. These data suggest that the selective mutation and frequent LOH of the p53 play an important role in the development of HCCs from Qidong and at least three additional tumor suppressor genes might be involved in the human hepatocarcinogenesis.