Fas and mutant estrogen receptor chimeric gene: a novel suicide vector for tamoxifen-inducible apoptosis.

摘要

Several cancer gene therapy strategies involve suicide genes to kill the neoplasm, or to regulate effector cells such as lymphocytes. We have 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 estrogen-inducible apoptosis to overcome concerns with the wild-type estrogen receptor and its natural ligand, 17beta-estradiol (E2). The ligand-binding domain of MfasER was replaced with that of a mutant estrogen receptor which is unable to bind estrogen yet retains affinity for a synthetic ligand, 4-hydroxytamoxifen (Tm). The resultant fusion protein (MfasTmR) and MfasER were expressed in L929 cells for examination of their ligand specificities. Tm induced apoptosis in MfasTmR-expressing cells (L929MfasTmR) at 10(-8) M or higher concentrations, but induced no apoptosis in MfasER-expressing cells (L929MfasER) at up to 10(-6) M. On the other hand, E2 induced apoptosis in L929MfasER at concentrations as low as 10(-10)-10(-9) M, while it did so partially in L929MfasTmR at concentrations greater than 10(-7) M. Thus, L929MfasTmR cells were highly susceptible to Tm, but refractory to E2, 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 safely in vivo, working independently of endogenous estrogen.