Differential in situ hybridization for determination of mutational specific expression of the p53 gene in human hepatoma cell lines.
The codon 249 mutation specific expression of the p53 gene was determined in 7 human hepatocellular carcinoma (HCC) cell lines. Two 20-base oligomers complementary to bases 872-891 of human p53 cDNA with a single nucleotide difference in the third position of codon 249 were end-labelled with biotin-conjugated dATP using terminal deoxynucleotidyltransferase (TdT). The hybridized oligomer was visually detected in situ using streptavidin-alkaline phosphatase (AP) conjugate and AP substrate. Expression of the codon 249 mutant p53 was steady in PLC/PRF/5 and Mahlavu cells (derived from African patients), while Huh4, Huh6, Huh7 and HCC-M cells (derived from Japanese patients) expressed only the codon 249 wild-type p53. The transcripts of the p53 gene were undetectable in Hep3B cells (derived from an American patient). Hybridizations of the codon 249 specific oligomers were specific to the p53 transcripts, since the cells that expressed p53 gene homogeneously were stained in the cytoplasm only by differential hybridization with a codon 249 specific oligomer; moreover, hybridization with a labelled oligomer non-complementary to the p53 cDNA showed nuclear stainings. Thus, detection of the codon 249 mutant p53 mRNA by differential in situ hybridization is a specific method for studying the mutation-specific expression of the p53 gene in liver cancers at the cellular level, while simultaneously visualizing the cell morphology. The results also support the notion that the p53 gene codon 249 mutation may have etiological implications involving HCC from various geographic areas.