INFLUENCE OF EXOGENOUS RAS AND P53 ON P-GLYCOPROTEIN FUNCTION IN IMMORTALIZED RODENT FIBROBLASTS

The ability of ras oncogenes and mutant p53 to activate reporter gene expression from human and rodent mdr1 gene promoters was described, al though functional significance of this finding was unclear. We analyze d the influence of various forms of recombinant human ras and p53 on t he mdr1 gene expression and P-glycoprotein (Pgp) function in rodent im mortalized fibroblasts. The ras genes, in addition to activation of ex ogenous human mdr1 gene promoter, caused an increase in (i) expression of endogenous mdrl mRNA, (ii) Pgp activity as determined by flow cyto metry analysis of Rhodamine 123 exclusion, and (iii) resistance of cel ls to the cytotoxic action of colchicine and some other drugs. To eluc idate whether the same signalling pathway is responsible for multidrug resistance induced by various oncogenes and protein kinase C (PKC), w e tested the effects of v-mos and the PKC agonist 12-O-tetradecanoylph orbol-13-acetate. Similarly to cells transformed by ras, a Rat1 sublin e transformed by the v-mos oncogene was characterized by decreased dru g sensitivity. On the contrary, Rat1 cells treated with the protein ki nase C agonist 12-O-tetradecanoylphorbol-13-acetate showed neither inc reased mdr1 mRNA expression nor stimulation of Pgp function. Introduct ion by retrovirus-mediated gene transfer of wild-type p53 into Rat1 ce lls or into murine p53-deficient 10(1) and 10(3) cells did not change the Pgp function significantly, whereas in Rat1 cells transformed by a ctivated N-ras or v-mos, expression of wild-type p53 caused partial re version of oncogene-induced drug resistance. In agreement with previou sly published results, we observed that in a transient transfection as say a majority of p53 mutants stimulated reporter gene expression from an exogenous mdrl promoter in murine cells. However, in Rat1, 10(1) a nd 10(3) cell sublines, which permanently express various p53s as a re sult of retroviral transfer, we found neither increase in mdr1 mRNA ex pression and activation of the mdr1 gene promoter, nor increased drug resistance. Moreover, in ras- and mos-transformed Rat1 cells some p53 mutants (His-273, Trp-248), similarly to wild-type p53, caused a sligh t decrease in resistance to colchicine. On the other hand, Tyr-141 p53 decreased drug resistance of Rat1 and Rat/mos but not Rat/ras cells. Weak effects of physiological concentrations of p53 proteins on mdr1 g ene expression and Pgp function as well as differential influence of v arious p53 mutants on multidrug resistance in various cell contexts al low us to suppose that p53 may influence mdrl gene expression through other transcription factors, rather than by its direct interaction wit h the mdr1 gene.