The p53 tumor suppressor gene is most noted for its frequent mutations in human cancers. The wild-type p53 protein is well characterized as a sequence specific transcriptional factor that regulates cell cycle arrest, apoptosis, and genome stability through its target genes. Loss of p53 function has strongly correlated with neoplastic transformation, however, emerging evidence indicates that the "gain of function" of mutant p53 also contributes significantly to the acquisition of malignant phenotypes. To understand the effect of mutant p53 in gene expressions related to cell transformation, a p53^val135 gene was transduced into Rat 6 embryo fibroblasts through a retroviral vector. The established p53^val135-overexpressing cells exhibited high levels of spontaneous transformants in long-te...[ Read more ]

The p53 tumor suppressor gene is most noted for its frequent mutations in human cancers. The wild-type p53 protein is well characterized as a sequence specific transcriptional factor that regulates cell cycle arrest, apoptosis, and genome stability through its target genes. Loss of p53 function has strongly correlated with neoplastic transformation, however, emerging evidence indicates that the "gain of function" of mutant p53 also contributes significantly to the acquisition of malignant phenotypes. To understand the effect of mutant p53 in gene expressions related to cell transformation, a p53^val135 gene was transduced into Rat 6 embryo fibroblasts through a retroviral vector. The established p53^val135-overexpressing cells exhibited high levels of spontaneous transformants in long-term cultures. Clonal transformed cell lines were derived from individual spontaneous transformants.

In order to identify genes that were altered in the course of cell transformation induced by the mutant p53^val135, the gene expression patterns of the parental p53^val135-overexpressors R6#13-8 and its derived spontaneous transformant T2 were analyzed by the mRNA differential display. This study reports the identification of three differentially expressed genes. Two genes, one identified as a rat homolog of Cyr61 and the other as a nonmuscle myosin heavy chain-B (r-nmMHC-B), were down-regulated in T2 cells. The third gene, a rat frizzled related protein (rFRP) was expressed in p53^val135-overexpressors, but not in the parental Rat 6 cells. Further characterization was done on both r-nmMHC-B and rFRP. The full-length r-nmMHC-B cDNA, driven by an ecdysone-inducible promoter, was introduced into T2 cells lacking the expression of the endogenous nmMHC-B gene. The induced clonal transfectants displayed a flatter morphology, exhibited slower growth rates, and reached to lower saturation densities compared with the parental transformed cells. In soft agar assay, induced nmMHC-B transfectants formed fewer and substantially smaller colonies than the parental cells. It was also remarkably effective in suppressing the tumorigenicity of the T2 cells when tested in nude mice. Thus, our findings suggest that nmMHC-B may act as tumor suppressor gene. Another gene, rFRP, whose expression was reportedly down regulated in a broad range of cancers and suggested to be a candidate cancer gene. The rFRP cDNA, shown in our study to be specifically expressed in ovary, was isolated and sequenced. To further understand the derepression of rFRP observed in mutant p53^val135-overexpressors, the promoter region of the rFRP was cloned and analyzed by transient reporter assays. Our results identified a 60-bp fragment which is located 136 bp upstream to the transcriptional starting site, and may be responsible for the basal promoter activity. Furthermore, analysis of the promoter region revealed few cis-acting elements which may account for the ovary-specific expression of rFRP observed in our study.

This current study of the cloning and functional analysis of both the r-nmMHC- B and rFRP has provided important clues to understanding the molecular basis of cell transformation and etiology related to p53 tumor suppressor gene.