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 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.
Kodaira
H
H
Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi.
Kume
A
A
Ogasawara
Y
Y
Urabe
M
M
Kitano
K
K
Kakizuka
A
A
Ozawa
K
K
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology*; D015316:Genetic Therapy; D051379:Mice; D009154:Mutation; D011960:Receptors, Estrogen / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology; D019014:fas Receptor / Q000235:genetics*
D004965:Estrogen Antagonists; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D019014:fas Receptor; D013629:Tamoxifen; C016601:afimoxifene; D004958:Estradiol