p 53 - and ATM - Dependent Apoptosis Induced by Telomeres Lacking TRF 2
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چکیده
al., Nature 383, 99 (1996); H. Kawasaki et al., ibid. 393, 284 (1998). 5. P. H. Stern, in Vitamin D, D. Feldman, F. H. Glorieux, J. W. Pike, Eds. (Academic Press, New York, 1997), pp. 343–345; A. Takeshita et al., J. Biol. Chem. 273, 14738 (1998). 6. T. Yoshizawa et al., Nature Genet. 16, 391 (1997). 7. S. Kato et al., Science 270, 1491 (1995); K. Takeyama et al., ibid. 277, 1827 (1997). 8. K. Ebihara et al., Mol. Cell. Biol. 16, 3393 (1996). 9. S. Kato et al., ibid. 15, 5858 (1995). 10. COS-1 cells were maintained in Dulbecco’s modified Eagle’s medium without phenol red, supplemented with fetal bovine serum (5%) treated with dextran-coated charcoal. The cells were transfected at 40 to 50% confluency in 10-cm petri dishes with a total of 20 mg of the indicated plasmids using calcium phosphate. All assays were done in the presence of 3 mg of pCH110 (Pharmacia), a b-galactosidase expression vector, as an internal control to normalize for variations in transfection efficiency. Cognate ligands were added to the medium 1 hour after transfection and at each exchange of medium. After 24-hour incubation with the calcium phosphate–precipitated DNA, the cells were washed with fresh medium and incubated for an additional 24 hours. Cell extracts were prepared by freezing and thawing and were assayed for CAT activity after normalization for b-galactosidase activity as described (9). 11. The mammalian expression vector pcDNA3 (Invitrogen) was used for the expressions of Smad and SRC-1 proteins. Constitutively active and catalytically inactive forms of TbR-I, BMPR-IA, and BMPR-IB were as described (12). Full-length VDR and VDR mutants were inserted into the mammalian expression vector pSG5 (pSG5-VDR). DEF domains of VDR were inserted into the pM vector (Clontech) [GAL4-VDR(DEF)] and full-length Smad2 and Smad3 were inserted into pVP (Clontech) ( VP16-Smad2 and VP16-Smad3). 12. T. Imamura et al., Nature 389, 622 (1997). 13. J. Yanagisawa et al., unpublished data. 14. J. Massague, Cell 85, 947 (1996); C.-H. Heldin, K. Miyazono, P. ten Dijke, Nature 390, 465 (1997); J. R. Howe et al., Science 280, 1086 (1998). 15. M. Kretzschmar et al., Genes Dev. 11, 984 (1997); A. Suzuki et al., Dev. Biol. 184, 402 (1997); J. M. Graff, A. Bansal, D. A. Melton, Cell 85, 479 (1996). 16. P. A. Hoodless et al., Cell 85, 489 (1996); F. Liu et al., Nature 381, 620 (1996). 17. M. Macias-Silva et al., Cell 87, 1215 (1996); A. Nakao et al., EMBO J. 16, 5353 (1997); J. C. Baker and R. M. Harland, Genes Dev. 10, 1880 (1996). 18. G. Lagna et al., Nature 383, 832 (1996); Y. Zhang et al., ibid., p. 168. 19. X. Chen, M. J. Rubock, M. Whitman, ibid., p. 691; J. Kim et al., ibid. 388, 304 (1997); S. Dennler et al., EMBO J. 17, 3091 (1998); L. Zawel et al., Mol. Cell 1, 611 (1998). 20. Y. Zhang, T. Musci, R. Derynck, Curr. Biol. 7, 270 (1997). 21. GAL4 DNA-binding domain fusions were generated in pM, and VP16 fusions within pVP16. Interactions were tested in COS-1 cells. Activation of the CAT reporter– bearing GAL4-binding elements (17Mx5) was assayed in the presence or absence of 1,25(OH)2D3. 22. COS-1 cells were transfected with the indicated plasmids, lysed in TNE buffer [10 mM Tris-HCl (pH 7.8), 1% NP-40, 0.15 M NaCl, 1 mM EDTA], and immunoprecipitated with monoclonal antibody to FLAG (IBI; Eastman Kodak). Interacting proteins were separated by 8% SDS–polyacrylamide gel electrophoresis (SDSPAGE), transferred onto polyvinylidene difluoride membranes (Bio-Rad), and then detected with antibody to VDR and antibody to rabbit immunoglobulin G conjugated with alkaline phosphatase (Promega). 23. GST-fused proteins were expressed in Escherichia coli and purified on glutathione-Sepharose beads (Pharmacia). The beads were incubated with [35S]methionine-labeled proteins. Bound proteins were eluted and analyzed by SDS-PAGE. 24. Proteins were translated in vitro in the presence of [35S]methionine with the reticulocyte lysate system (Promega). 25. D. M. Heery et al., Nature 387, 733 (1997); H. Masuyama et al., Mol. Endocrinol. 11, 1507 (1997); C. H. Jin et al., ibid. 10, 945 (1996). 26. Cells were harvested and washed twice with ice-cold phosphate-buffered saline. Centrifuged cells were resuspended in 4 ml ice-cold lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% (v/v) NP-40] and incubated on ice for 15 min, then centrifuged again for 5 min at 500g. The sedimented nuclear fraction was resuspended in TNE buffer [10 mM tris-HCl (pH 7.8), 1% NP-40, 0.15 M NaCl, 1 mM EDTA], incubated for 30 min on ice, and centrifuged. The supernatant was used as nuclear extract for the experiments. 27. T. Spencer et al., Nature 389, 194 (1997); T. Yao et al., Proc. Natl. Acad. Sci. U.S.A. 93, 10626 (1997). 28. J. J. Voegel et al., EMBO J. 17, 507 (1998). 29. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. X indicates any residue. 30. We thank P. Chambon for critical reading of the manuscript and for providing nuclear receptor expression vectors, H. Gronemeyer for providing TIF2 expression vectors, R. H. Goodman for the CBP cDNA, and S. Hanazawa and A. Takeshita for helpful discussion.
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