Investigation Of The Relationship Between Point Mutation Of Ha-Ras Oncogene And Biologic Behaviors Of Bladder Cancer

Though bladder cancer is one of the most common malignancies, little is known about its mechanism of carrcinogenesis and progression. Identification of Ha-ras onco-gene and subsequent profound investigations were shown to be very promising for the study of the molecular mechanism. Many data have suggested that activated Ha-ras oncogene is relevant to the occurrence and progression of bladder cancer. However, the methods for detecting point mutation and investigating the dominat mechanism of Ha-ras oncogene activation have not been widely adopted sensitivity, because of their low and the repoeted incidence of Ha-ras gene mutations is less than 30%. As a result, there have been few detailed investigations employing these methods. In this study, we investigate point mutations at codon 12 of Ha - ras gene from paraffin - embedded spescimens of human bladder cancer using the more recently devised polymerase chain reaction (PCR), together with assays using restriction fragment length polymorphisms (RFLP). In addition, the relationship between the incidence of Ha-ras gene mutations at codon 12 and the clinical stage and pathologic grading of bladder cancer is also stud ied. The aim is to evaluate the possible roles of the point mutations at codon 12 of Haras gene in the occurrence and progression of bladder cancer on the gene level and find whether the identification of the mutated ras gene correlates with the cilinicao-pathological parameters.Genomic DNA was first extracted from 24 paraffin-embedded bladder cancer specimens and plasmid DNA of pUC EJ6. 6 and genomic DNA of normal bladder tissue were prepared as positive and negative control, respectively. Then, PCR primers targeting the 1st exon of Ha-ras gene were designed and synthetized according to the published Ha -ras gene sequence. The first exon covering codon 12, a hot point for mutation, of the Ha-ras gene was amplified by PCR, from plasmid, normal bladder tissue and bladder cancer, and the amplified fragments were recovered and subjected to second round of PCR. The products of the second PCR were digested with Hpa â…¡ or MspI, the restriction enzymes specific for codon 12 mutated Ha-ras gene, or with Sau3A as a contrrol for PCR and digestion reaction.The findings were as follows :(1) DNA samples of plasmid, paraffin-embedded specimens and fresh tissue could...