RESUMO
Sequence alterations in the exon 1 region of the rat c-Ki-ras gene were studied in DNA isolated from aflatoxin B1 (AFB1)-induced rat liver carcinomas and precursor lesions appearing 56 weeks after administration of the carcinogen. To detect the mutations with high sensitivity, DNA samples were analyzed by using polymerase chain reaction (PCR) amplification in conjunction with allele-specific oligonucleotide (ASO) hybridization together with a modified PCR-G+C clamp-denaturing gradient gel electrophoresis (DGGE) method. Mutations in the Ki-ras gene were present in all adenomas and carcinomas examined. The predominant mutation observed was a G.C-to-A.T base transition in codon 12 (GGT to GAT). Also present, but at low frequency, was a G.C-to-T.A base transversion in the same codon (GGT to TGT). In addition, 20% of the samples contained a G.C-to-T.A transversion in the second base position of codon 12 (GGT to GTT), a mutation not previously observed in AFB1-induced rat liver tumors. These results confirm and extend our previous findings that Ki-ras mutation is a prevalent event in hepato-cellular carcinogenesis induced in Fischer 344 rats by AFB1. The modified DGGE method described is applicable to the screening of multiple mutations in neoplastic lesions with high fidelity and sensitivity.
Assuntos
Genes ras , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenoma/genética , Aflatoxina B1 , Animais , Sequência de Bases , Eletroforese em Gel de Ágar , Regulação Neoplásica da Expressão Gênica , Dados de Sequência Molecular , Mutação , Desnaturação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Reação em Cadeia da Polimerase , Lesões Pré-Cancerosas/genética , RatosRESUMO
The TPR-MET oncogenic rearrangement was originally observed in an in vitro transformed human osteosarcoma cell line. Recently, we detected the expression of this rearrangement at very low levels in several cell lines derived from human tumors of nonhematopoietic origin using a highly sensitive method based on polymerase chain reaction amplification of the transcript. We report here the results of analysis of TPR-MET expression in cell lines derived from human gastric tumors and 22 biopsy samples of human gastric mucosa showing cancer or precursor lesions. The rearranged RNA was expressed in all four cell lines as well as in biopsy samples from 12 of the 22 patients. Overexpression of TPR-MET RNA in superficial gastritis lesions with hyperplasia of glandular neck cells suggests the possible involvement of this oncogene at an early stage of gastric tumorigenesis. Analysis of gastric biopsy samples for RAS gene mutations showed base substitutions occurring in the codon 12 region of Ki- and Ha-RAS genes in four cases, including two precursor lesions.
Assuntos
Rearranjo Gênico , Oncogenes , Lesões Pré-Cancerosas/genética , Neoplasias Gástricas/genética , Sequência de Bases , Biópsia , Linhagem Celular , Códon , Mucosa Gástrica/patologia , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Osteossarcoma/genética , Reação em Cadeia da Polimerase , Polimorfismo Genético , Lesões Pré-Cancerosas/patologia , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Mapeamento por Restrição , Neoplasias Gástricas/patologiaRESUMO
Activation of the MET protooncogene by a rearrangement involving the fusion of TPR and MET specific gene sequences has been observed in a human osteosarcoma cell line (HOS) treated in vitro with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). No information has been available about the possible occurrence of this rearrangement in human tumors. To facilitate rapid screening of human cell lines and tumor samples for this specific gene rearrangement, we developed a sensitive detection method based on polymerase chain reaction (PCR) amplification of TPR-MET mRNA. cDNA was generated from cellular transcripts by using one of the PCR primers, which was then used as a template for PCR amplification of a 205-base-pair region carrying the breakpoint. An end-labeled internal probe was hybridized in solution to an aliquot of the PCR product for detecting amplification. Cells could be directly screened by the assay without prior isolation of RNA. A 205-base-pair DNA fragment characteristic of the TPR-MET rearrangement was detected in cell lines previously known to contain this altered sequence. The rearrangement was also detected at very low levels in the parental (nontransformed) cell line, HOS TE-85. A preliminary survey of cell lines derived from a variety of human tumors indicates that TPR-MET rearrangement occurred and was expressed at very low frequencies by cells from 7 of 14 tumors of nonhematopoietic origin.
