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Japanese Journal of Cancer Research : Gann logoLink to Japanese Journal of Cancer Research : Gann
. 1998 Jul;89(7):741–747. doi: 10.1111/j.1349-7006.1998.tb03279.x

Fas and Mutant Estrogen Receptor Chimeric Gene: A Novel Suicide Vector for Tamoxifen‐inducible Apoptosis

Hiroshi Kodaira 1,✉, Akihiro Kume 1, Yoji Ogasawara 1, Masashi Urabe 1, Kiyoshi Kitano 3, Akira Kakizuka 4, Keiya Ozawa 1,2,✉
PMCID: PMC5921882  PMID: 9738981

Abstract

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 Fasestrogen 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, 17β‐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 10‐8M or higher concentrations, but induced no apoptosis in MfasER‐expressing cells (L929MfasER) at up to 10‐6M. On the other hand, E2 induced apoptosis in L929MfasER at concentrations as low as 10‐10–10‐9M, while it did so partially in L929MfasTmR at concentrations greater than 10‐7M. 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.

Keywords: key words, Fas, Mutant estrogen receptor, Tamoxifen, Inducible apoptosis, Gene therapy

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REFERENCES

  • 1. ) Moolten , F. L.Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy . Cancer Res. , 46 , 5276 – 5281 ( 1986. ). [PubMed] [Google Scholar]
  • 2. ) Obaru , K. , Fujii , S. , Matsushita , S. , Shimada , T. and Takatsuki , K.Gene therapy for adult T cell leukemia using human immunodeficiency virus vector carrying the thymidine kinase gene of herpes simplex virus type 1 . Hum. Gene Ther. , 7 , 2203 – 2208 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 3. ) Franken , M. , Estabrooks , A. , Cavacini , L. , Sherburne , B. , Wang , F. and Scadden , D. T.Epstein‐Barr virus‐driven gene therapy for EBV‐related lymphomas . Nat. Med. , 2 , 1379 – 1382 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 4. ) Bordignon , C. , Bonini , C. , Verzeletti , S. , Nobili , N. , Maggioni , D. , Traversari , C. , Giavazzi , R. , Servida , P. , Zappone , E. , Benazzi , E. , Bernardi , M. , Porta , F. , Ferrari , G. , Mavilio , F. , Rossini , S. , Blaese , R. M. and Candotti , F.Transfer of the HSV‐tk gene into donor peripheral blood lymphocytes for in vivo modulation of donor anti‐tumor immunity after allogeneic bone marrow transplantation . Hum. Gene Ther. , 6 , 813 – 819 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 5. ) Bonini , C. , Ferrari , G. , Verzeletti , S. , Servida , P. , Zappone , E. , Ruggieri , L. , Ponzoni , M. , Rossini , S. , Mavilio , F. , Traversari , C. and Bordignon , C.HSV‐TK gene transfer into donor lymphocytes for control of allogeneic graft‐versus‐leukemia . Science , 276 , 1719 – 1724 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 6. ) Aoki , Y. , Tani , K. , Takahashi , K. , Fukushima , M. , Ozawa , K. and Asano , S.Regulation of recombinant human granulocyte colony‐stimulation factor production using herpes simplex virus 1 thymidine kinase gene . Biochem. Biophys. Res. Commun. , 200 , 1245 – 1251 ( 1994. ). [DOI] [PubMed] [Google Scholar]
  • 7. ) Borrelli , E. , Heyman , R. , Hsi , M. and Evans , R. M.Targeting of an inducible toxic phenotype in animal cells . Proc. Natl. Acad. Sci. USA , 85 , 7572 – 7576 ( 1988. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. ) Heyman , R. A. , Borrelli , E. , Lesley , J. , Anderson , D. , Richman , D. D. , Baird , S. M. , Hyman , R. and Evans , R. M.Thymidine kinase obliteration: creation of transgenic mice with controlled immune deficiency . Proc. Natl. Acad. Sci. USA , 86 , 2698 – 2702 ( 1989. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. ) Lupton , S. D. , Brunton , L. L. , Kalberg , V. A. and Overell , R. W.Dominant positive and negative selection using a hygromycin phosphotransferase‐thymidine kinase fusion gene . Mol. Cell. Biol. , 11 , 3374 – 3378 ( 1991. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. ) Anderson , W. F.Gene therapy for cancer . Hum. Gene Ther. , 5 , 1 – 2 ( 1994. ). [DOI] [PubMed] [Google Scholar]
  • 11. ) Takebayashi , H. , Oida , H. , Fujisawa , K. , Yamaguchi , M. , Hikida , T. , Fukumoto , M. , Narumiya , S. and Kakizuka , A.Hormone‐induced apoptosis by Fas‐nuclear receptor fusion proteins: novel biological tools for controlling apoptosis in vivo . Cancer Res. , 56 , 4164 – 4170 ( 1996. ). [PubMed] [Google Scholar]
  • 12. ) Kokubun , M. , Kume , A. , Urabe , M. , Mano , H. , Okubo , M. , Kasukawa , R. , Kakizuka , A. and Ozawa , K.Apoptosis‐mediated regulation of recombinant human granulocyte colony‐stimulating factor production by genetically engineered fibroblasts . Gene Ther. , 5 , ( 1998. ), in press . [DOI] [PubMed] [Google Scholar]
  • 13. ) Picard , D.Steroid‐binding domains for regulating the functions of heterologous proteins in cis . Trends Cell Biol. , 3 , 278 – 280 ( 1993. ). [DOI] [PubMed] [Google Scholar]
  • 14. ) Mattioni , T. , Louvion , J.‐F. and Picard , D.Regulation of protein activities by fusion to steroid binding domains . Methods Cell Biol. , 43 , 335 – 352 ( 1994. ). [DOI] [PubMed] [Google Scholar]
  • 15. ) Berthois , Y. , Katzenellenbogen , J. A. and Katzenellenbogen , B. S.Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen‐responsive cells in culture . Proc. Natl. Acad. Sci. USA , 83 , 2496 – 2500 ( 1986. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. ) Osborne , C. K. , Hobbs , K. and Clark , G. M.Effect of estrogens and antiestrogens on growth of human breast cancer cells in athymic nude mice . Cancer Res. , 45 , 584 – 590 ( 1985. ). [PubMed] [Google Scholar]
  • 17. ) Danielian , P. S. , White , R. , Hoare , S. A. , Fawell , S. E. and Parker , M. G.Identification of residues in the estrogen receptor that confer differential sensitivity to estrogen and hydroxytamoxifen . Mol. Endocrinol. , 7 , 232 – 240 ( 1993. ). [DOI] [PubMed] [Google Scholar]
  • 18. ) Littlewood , T. D. , Hancock , D. C. , Danielian , P. S. , Parker , M. G. and Evan , G. I.A modified oestrogen receptor ligand‐binding domain as an improved switch for the regulation of heterologous proteins . Nucleic Acids Res. , 23 , 1686 – 1690 ( 1995. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. ) Morgan , R. A. , Couture , L. , Elroy‐Stein , O. , Ragheb , J. , Moss , B. and Anderson , W. F.Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and applications to human gene therapy . Nucleic Acids Res. , 20 , 1293 – 1299 ( 1992. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. ) Urabe , M. , Hasumi , Y. , Ogasawara , Y. , Matsushita , T. , Kamoshita , N. , Nomoto , A. , Colosi , P. , Kurtzman , G. J. , Tobita , K. and Ozawa , K.A novel dicistronic AAV vector using a short IRES segment derived from hepatitis C virus genome . Gene , 200 , 157 – 162 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 21. ) Gorman , C. , Padmanabhan , R. and Howard , B. H.High efficiency DNA‐mediated transformation of primate cells . Science , 221 , 551 – 553 ( 1983. ). [DOI] [PubMed] [Google Scholar]
  • 22. ) Onishi , M. , Kinoshita , S. , Morikawa , Y. , Shibuya , A. , Phillips , J. , Lanier , L. L. , Gorman , D. M. , Nolan , G. P. , Miyajima , A. and Kitamura , T.Applications of retrovirus‐mediated expression cloning . Exp. Hematol. , 24 , 324 – 329 ( 1996. ). [PubMed] [Google Scholar]
  • 23. ) Sanford , K. K. , Hobbs , G. L. and Earle , W. R.The tumor‐producing capacity of strain L mouse cells after 10 years in vitro . Cancer Res. , 15 , 162 – 166 ( 1956. ). [PubMed] [Google Scholar]
  • 24. ) Itoh , N. , Yonehara , S. , Ishii , A. , Yonehara , M. , Mizushima , S. , Sameshima , M. , Hase , A. , Seto , Y. and Nagata , S.The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis . Cell , 66 , 233 – 243 ( 1991. ). [DOI] [PubMed] [Google Scholar]
  • 25. ) Watanabe‐Fukunaga , R. , Brannan , C. I. , Itoh , N. , Yonehara , S. , Copeland , N. G. , Jenkins , N. A. and Nagata , S.The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen . J. Immunol. , 148 , 1274 – 1279 ( 1992. ). [PubMed] [Google Scholar]
  • 26. ) Itoh , N. and Nagata , S.A novel protein domain required for apoptosis . J. Biol. Chem. , 268 , 10932 – 10937 ( 1993. ). [PubMed] [Google Scholar]
  • 27. ) Ushiroyama , T. , Yoshikawa , M. , Saeki , M. , Okuda , K. and Sugimoto , O.Hypergonadotropinemia with estradiol secretion in peri‐ and postmenopausal period . Acta Obstet. Gynecol. Scand. , 68 , 139 – 143 ( 1989. ). [DOI] [PubMed] [Google Scholar]
  • 28. ) Ushiroyama , T. and Sugimoto , O.Endocrine function of the peri‐ and postmenopausal ovary . Horm. Res. , 44 , 64 – 68 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 29. ) Chinnaiyan , A. M. , O'Rourke , K. , Tewari , M. and Dixit , V. M.FADD, a novel death domain‐containing protein, interacts with the death domain of Fas and initiates apoptosis . Cell , 81 , 505 – 512 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 30. ) Boldin , M. P. , Varfolomeev , E. E. , Pancer , Z. , Mett , I. L. , Camonis , J. H. and Wallach , D.A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain . J. Biol. Chem. , 270 , 7795 – 7798 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 31. ) Hsu , H. , Xiong , J. and Goeddel , D. V.The TNF receptor 1‐associated protein TRADD signals cell death and NF‐KB activation . Cell , 81 , 495 – 504 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 32. ) Stanger , B. Z. , Leder , P. , Lee , T.‐H. , Kim , E. and Seed , B.RIP: a novel protein containing a death domain that interacts with Fas/APO‐1 (CD95) in yeast and causes cell death . Cell , 81 , 513 – 523 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 33. ) Ting , A. T. , Pimentel‐Muinos , F. X. and Seed , B.RIP mediates tumor necrosis factor receptor 1 activation of NF‐KB but not Fas/APO‐1‐initiated apoptosis . EMBO J. , 15 , 6189 – 6196 ( 1996. ). [PMC free article] [PubMed] [Google Scholar]
  • 34. ) Pan , G. , O'Rourke , K. , Chinnaiyan , A. M. , Gentz , R. , Ebner , R. , Ni , J. and Dixit , V. M.The receptor for the cytotoxic ligand TRAIL . Science , 276 , 111 – 113 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 35. ) Pan , G. , Ni , J. , Wei , Y.‐F. , Yu , G.‐I. , Gentz , R. and Dixit , V. M.An antagonist decoy receptor and a death domain‐containing receptor for TRAIL . Science , 277 , 815 – 818 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 36. ) Sheridan , J. P. , Marsters , S. A. , Pitti , R. M. , Gurney , A. , Skubatch , M. , Baldwin , D. , Ramakrishnan , L. , Gray , C. L. , Baker , K. , Wood , W. I. , Goddard , A. D. , Godowski , P. and Ashkenazi , A.Control of TRAIL‐induced apoptosis by a family of signaling and decoy receptors . Science , 277 , 818 – 821 ( 1997. ). [DOI] [PubMed] [Google Scholar]

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