INDUCTION OF APOPTOSIS BY TAMOXIFEN-ACTIVATION OF A P53-ESTROGEN RECEPTOR FUSION PROTEIN EXPRESSED IN E1A AND T24 H-RAS TRANSFORMED P53(- -) MOUSE EMBRYO FIBROBLASTS/

A fusion gene consisting of wild-type p53 linked to a modified ligand binding domain of the murine estrogen receptor has been constructed an d should be a useful tool for studying controlled activation of wild-t ype p53 function in a variety of experimental cell systems, The protei n product of this gene, p53ER(TM), is expressed in cells constitutivel y but is not functional unless associated with tamoxifen or 4-hydroxyt amoxifen, p53ER(TM) was introduced into p53-deficient mouse embryo fib roblasts (MEFs) expressing the EIA and T24 H-ras oncogenes, Activation of p53 in these transformed cells by the addition of tamoxifen or 4-h ydroxytamoxifen resulted in apoptosis, In addition to engaging the apo ptotic machinery, the tamoxifen-activated fusion protein exhibited oth er functions characteristic of wild-type p53, such as induction of WAF 1 and MDM2 gene expression and activation of the p53-dependent spindle checkpoint in cells treated with nocodazole. Activation of p53ER(TM) expressed in p53-positive MEFs coexpressing E1A and ras had, at most, only a small cytotoxic effect, When three cell Lines of transformed p5 3(+/+) fibroblasts not expressing p53ER(TM) were tested for sensitivit y to the DNA-damaging drug doxorubicin, the p53(+/+) clones displayed either comparable sensitivity, or at most an increase in drug sensitiv ity of less than fourfold, as compared to several p53(-/-) cell lines, Our data show that restoration of wild-type p53 activity is sufficien t to trigger apoptosis in p53(-/-) MEFs transformed with EIA and T24 H -ras and suggest that rare propagable clones of p53-normal MEPs expres sing the EIA and T24 H-ras oncogenes have suffered compensatory altera tions that compromise the ability to undergo p53-dependent apoptosis.