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 and should be a useful tool for studying controlled activation of wild-type p53 function in a variety of experimental cell systems. The protein product of this gene, p53ERTM, is expressed in cells constitutively but is not functional unless associated with tamoxifen or 4-hydroxytamoxifen. p53ERTM was introduced into p53-deficient mouse embryo fibroblasts (MEFs) expressing the E1A and T24 H-ras oncogenes. Activation of p53 in these transformed cells by the addition of tamoxifen or 4-hydroxytamoxifen resulted in apoptosis. In addition to engaging the apoptotic machinery, the tamoxifen-activated fusion protein exhibited other functions characteristic of wild-type p53, such as induction of WAF1 and MDM2 gene expression and activation of the p53-dependent spindle checkpoint in cells treated with nocodazole. Activation of p53ERTM expressed in p53-positive MEFs coexpressing E1A and ras had, at most, only a small cytotoxic effect. When three cell lines of transformed p53+/+ fibroblasts not expressing p53ERTM were tested for sensitivity to the DNA-damaging drug doxorubicin, the p53+/+ clones displayed either comparable sensitivity, or at most an increase in drug sensitivity of less than fourfold, as compared to several p53-/- cell lines. Our data show that restoration of wild-type p53 activity is sufficient to trigger apoptosis in p53-/- MEFs transformed with E1A and T24 H-ras and suggest that rare propagable clones of p53-normal MEFs expressing the E1A and T24 H-ras oncogenes have suffered compensatory alterations that compromise the ability to undergo p53-dependent apoptosis.
Vater
C A
CA
Apoptosis Technology, Inc, Cambridge, Massachusetts 02139-4239, USA.
Bartle
L M
LM
Dionne
C A
CA
Littlewood
T D
TD
Goldmacher
V S
VS
D017500:Adenovirus E1A Proteins / Q000235:genetics*; D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D002461:Cell Line, Transformed; D004622:Embryo, Mammalian / Q000166:cytology; D004965:Estrogen Antagonists / Q000494:pharmacology; D005347:Fibroblasts; D005786:Gene Expression Regulation; D011905:Genes, ras; D051379:Mice; D011960:Receptors, Estrogen / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D017500:Adenovirus E1A Proteins; D004965:Estrogen Antagonists; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D013629:Tamoxifen; C016601:afimoxifene