[Cloning of cancer-related genes of Rat6 fibroblasts by using an improved differential display method]

The p53 gene is the most frequently mutated gene identified so far in human cancers. When a mutant p53(135)-val gene was allowed to be over-expressed in Rat6(R6) cells, a high incidence of spontaneous transformation was observed in long-term culture of this cell line(R6 # 13-8). To identify genes involved in cell transformation, parental p53 over-expressing cell, R6 # 13-8, and its spontaneous transformant T2, were analyzed by an improved mRNA differential display technique, which was reproducible, simpler, and was able to clone cDNA longer than 500 bp, and was with less false positives. When 33 10-mer or 12-mer single primers with arbitrary but defined sequence were used for PCR, over 90 discrete cDNAs were obtained from R6 # 13-8 and T2 cells. Three differentially expressed cDNAs were identified, one of them is highly expressed in T2 cells, while the other two, 0.8 kb and 0.9 kb long, are highly expressed in R6 # 13-8 cells. The latter were cloned and confirmed by Northern hybridization. Both cloned fragment were not homologous with any published sequence. Our results suggest that the activation and inactivation of genes are involved in the process of the spontaneous transformation from R6 # 13-8 to T2.