FAS AND MUTANT ESTROGEN-RECEPTOR CHIMERIC GENE - A NOVEL SUICIDE VECTOR FOR TAMOXIFEN-INDUCIBLE APOPTOSIS

Several cancer gene therapy strategies involve suicide genes to kill t he neoplasm, or to regulate effector cells such as lymphocytes, We hav e developed an inducible apoptosis system with a Fas-estrogen receptor fusion protein (MfasER) for rapid elimination of transduced cells. In the present study, we further improved this molecular switch for estr ogen-inducible apoptosis to overcome concerns with the wild-type estro gen receptor and its natural ligand, 17 beta-estradiol (E-2). The liga nd-binding domain of MfasER was replaced with that of a mutant estroge n receptor which is unable to bind estrogen yet retains affinity for a synthetic ligand, 4-hydroxytamoxifen (Tm), The resultant fusion prote in (MfasTmR) and MfasER were expressed in L929 cells for examination o f their ligand specificities. Tm induced apoptosis in MfasTmR-expressi ng cells (L929MfasTmR) at 10(-8) M or higher concentrations, but induc ed no apoptosis in MfasER-expressing cells (L929MfasER) at up to 10(-6 ) M, On the other hand, E-2 induced apoptosis in L929MfasER at concent rations as low as 10(-10)-10(-9) M, while it did so partially in L929M fasTmR at concentrations greater than 10(-7) M, Thus, L929MfasTmR cell s were highly susceptible to Tm, but refractory to E-2, with 100-1,000 times more tolerance than L929MfasER, These results suggest that the MfasTmR/Tm system would induce apoptosis in the target cells more safe ly in vitro, working independently of endogenous estrogen.