THE ROLE OF TIS1 AND TIS21 IN TRANSFORMATION OF JB6 CELLS

Abstract

The 12-0-tetradecanoylphorbol-13-acetate (TPA) inducible sequence genes are primary response genes induced in Swiss 3T3 cells by TPA (Lim, et al., 1987). Previous studies showed that a subset of TIS genes, TIS21 and TIS1, were preferentially induced by TPA and also by epidermal growth factor in promotion resistant (P-) murine epidermal JB6 cells. Their protein levels were also greater in TPA-treated promotion resistant (P-) cells than promotion sensitive (P+) cells (Cmarik, et al., 1994). Based on these observations, it was proposed that the high level of TIS1 and TIS21 proteins in P- cells could be a factor that inhibits tumor promoterinduced transformation. The strategy used to test this hypothesis was to cause repression of TIS1 or TIS21 protein synthesis by constructing TIS21 or TIS1 antisense RNA expression plasmids. TIS21 cDNA was subcloned in the pcDNA3 vector and TIS1 in the pCEP4 vector. Antisense orientation was confirmed by restriction digestion and sequencing. These plasmids were transfected into P- cells subsequently grown in selective medium containing G418 medium for TIS21 transfectants and hygromycin for TIS1 transfectants. Greater than 25 independent clones of TIS21 or TIS1 were obtained. They were then analyzed for introduced TIS21 or TIS1 antisense RNA expression by northern analysis. The endogenous TIS21 or TIS1 mRNA was expressed, whereas the antisense RNA expression was not observed. Protein extracts from some TIS21 or TIS1 transfectants were subjected to western analysis. There was no decrease in the TPA-induced level of TIS21 or TIS1 proteins in antisense transfectants compared with that of the vector controls as would be observed with functional antisense. Thus, if any antisense RNAs were expressed, they did not inhibit the synthesis of their respective proteins. Southern analysis was carried out to determine if TIS1 or TIS21 antisense DNA is present in the cell. Of six TIS21 transfectants tested, none showed the presence of the TI521 antisense DNA, whereas two out of six TIS1 transfectants, pET1j and pET1p appeared to contain the TIS1 antisense DNA. These observations reflect that most of the clones obtained do not have the introduced gene and the few that do have the gene did not express the antisense RNA. Probably during clonal selection the cells lost the antisense DNA and most of the clones obtained are the ones without the antisense DNA. Because no clones with functional antisense that effectively blocked TIS1 or TIS21 protein induction in the P- cells were isolated, the hypothesis that the high level of TIS1 or TIS21 proteins in the P- cells can inhibit tumor promoter-induced transformation could not be validated.