GENETIC ALTERATIONS AT THE SPLICE JUNCTION OF P53 GENE IN HUMAN HEPATOCELLULAR-CARCINOMA

The tumor-suppressor gene p53 may transactivate the transcription of g enes that down-regulate cellular growth-related genes and may become o ncogenic as a result of the production of mutant proteins or the loss of its protein expression. This study reports that alterations of the highly conserved consensus intervening sequences at the splice junctio ns may lead to the inactivation of the p53 gene. Analyses with the com bined polymerase chain reaction and single-strand conformational polym orphism and direct DNA sequencing of DNAs amplified by means of asymme tric polymerase chain reaction demonstrated sequence alterations at th e splice junctions of introns 5 and 7 in four human hepatocellular car cinomas, with a single base substitution at the splice junction in thr ee and a 10-bp deletion starting from the dinucleotide AG of the accep tor site of intron 5 in the fourth. Restriction fragment length polymo rphism analysis disclosed allele loss in all three informative cases. The p53 mRNA concentrations were remarkably reduced or undetectable in two hepatocellular carcinomas, whereas the two tumors (cases 2 and 3) that had single base changes at the acceptor site of intron 7 had bot h normal and abnormally sized p53 mRNAs. Immunocytochemistry failed to detect the wild-type and mutant p53 proteins in all four tumors. West ern-blot analysis disclosed an abnormal, larger p53 protein of 55 kD i n the tumor of case 3. These findings suggest that the inactivation of p53 gene caused by the genetic alterations at the splice junction may occur more often than perceived and plays an important role in human hepatocarcinogenesis because of the inactivation of the p53 gene by wa y of the loss of the protein or production of an abnormal protein.