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 determi ned in 7 human hepatocellular carcinoma (HCC) cell lines. Two 20-base oligomers complementary to bases 872-891 of human p53 cDNA with a sing le nucleotide difference in the third position of codon 249 were end-l abelled with biotin-conjugated dATP using terminal deoxynucleotidyltra nsferase (TdT). The hybridized oligomer was visually detected in situ using streptavidin-alkaline phosphatase (AP) conjugate and AP substrat e. 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 co don 249 wild-type p53. The transcripts of the p53 gene were undetectab le in Hep3B cells (derived from an American patient). Hybridizations o f the codon 249 specific oligomers were specific to the p53 transcript s, since the cells that expressed p53 gene homogeneously were stained in the cytoplasm only by differential hybridization with a codon 249 s pecific oligomer; moreover, hybridization with a labelled oligomer non -complementary to the p53 cDNA showed nuclear stainings. Thus, detecti on of the codon 249 mutant p53 mRNA by differential in situ hybridizat ion is a specific method for studying the mutation-specific expression of the p53 gene in liver cancers at the cellular level, while simulta neously visualizing the cell morphology. The results also support the notion that the p53 gene codon 249 mutation may have etiological impli cations involving HCC from various geographic areas.