Detection of p53 gene mutations in rat hepatocellular carcinoma cell lines by denaturing gradient gel electrophoresis.
Structural alterations of the p53 gene were investigated in chemically induced rat hepatocellular carcinomas (HCCs), hyperplastic hepatic nodules (HPNs), and cell lines derived from rat neoplastic and normal liver cells. The mutations were detected by GC-clamped denaturing gradient gel electrophoresis using DNA that had been amplified from p53 mRNA by the reverse transcriptase-polymerase chain reaction. This method enabled us to find single-base changes within the p53 gene without using radioisotopes. The presence of mutations was subsequently confirmed by DNA sequencing. No mutations were detected in six primary HCCs and 12 HPNs induced by the Solt and Farber regimen (Nature 236: 701-703, 1976), suggesting that p53 gene mutations do not play a major role in rat hepatic carcinogenesis. However, five of seven HCC cell lines and one of two cell lines derived from normal liver cells had the mutated p53 gene and had lost the normal p53 gene. Five cell lines had a G-->T transversion at various codons, whereas one line had a 21-base deletion in exon 5. Therefore, we conclude that p53 gene mutations may occur in vitro during establishment of the cell lines or may be derived from very small populations within the primary tumors.
Fukuda
I
I
Department of Pathology, Asahikawa Medical College, Japan.
Ogawa
K
K
D000236:Adenoma / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002273:Carcinogens / Q000633:toxicity; D002460:Cell Line; D003062:Codon / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D016158:Genes, p53; D008099:Liver / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D051381:Rats; D014407:Tumor Cells, Cultured
D002273:Carcinogens; D003062:Codon; D004273:DNA, Neoplasm; D009838:Oligodeoxyribonucleotides