臺灣博碩士論文加值系統

四股螺旋結構 (G-quadruplex) 是一非典型B form DNA結構，在生理上具重要意義，其參與端粒、DNA複製和轉錄上的調控。我們實驗室和中研院原分所張大釗老師合作，合成能穩定G-quadruplex的小分子藥物BMVC4。已知BMVC4對於非小細胞肺癌細胞H1299處理後，會使H1299端粒酶活性下降、端粒縮短、老化、DNA damage response和DNA strand breaks，然而，藉由免疫螢光染色實驗，我們發現DNA斷裂的區域不只出現在端粒，也出現在染色體其他區域，所以我們推測BMVC4所造成的影響不只在端粒和端粒酶上，可能和染色體其他區域出現的G-quadruplex的穩定有關。WRN和BLM屬於RecQ helicase family，其已被證實對於G-quadruplex、replication fork和Holliday junction的結構有特異性、有偏好性的去解開這些結構；此外，其在DNA repair、DNA replication和telomere maintenance也是重要的調控因子。所以，我們想知道，BMVC4穩定G-quadruplex後所造成細胞的DNA damage是否和WRN和BLM有關。首先，我們將WRN和BLM表現抑制後，不管是否處理BMVC4，pATM和total ATM的表現都下降，接著當WRN和BLM表現恢復時，pATM和total ATM的表現也跟著恢復，推測WRN和BLM在BMVC4所造成的DNA damage response中可能是必須的，而且其會互相調控，缺一不可。我們也發現，當WRN和BLM表現量下降，不管是否處理BMVC4的情況下，都會使H1299生長遲緩，接著想知道是否會影響細胞老化，藉由SA-β-galactosidase活性測定，發現BMVC4的處理與否，WRN和BLM的表現一但受到抑制，就會使H1299產生老化的現象。因此，WRN和BLM不僅在BMVC4造成的DNA damage response中扮演重要調控者，其也在細胞生長和細胞老化上有重要影響。

G-quadruplex is a non-canonical B form DNA structure, it plays an important role in the sense of physiology. For example, G-quadruplex participates in the regulation of telomere, DNA replication and transcriptional regulation. BMVC4 is a novel G-quadruplex stabilizer. It was synthesized through the collaboration with Dr. Chang, Academic Sinica. We found that BMVC4 could not only inhibit telomerase activity in H1299, non-small cell lung cancer, but also shorten telomere length, eventually lead to cellular senescence. BMVC4 also caused DNA damage response and DNA strand breaks. After the measurement of immunofluorescence, we found only 20% DNA damage response that arose from telomere. Therefore, the occurrence of DNA damage response maybe come from the other site of the chromosomes. WRN and BLM belong to RecQ helicase family. It is well established that WRN and BLM have capability in preferentially binding to G-quadruplex, replication fork and holliday junction structure. Both of them have high efficiency in resolving these special structures. They play a vital role in DNA repair, DNA replication and telomere maintenance. Because WRN and BLM could resolving G-quadruplex structure, we test if there is a correlation between WRN or BLM and the stabilization of G-quadruplex by BMVC4. We knocked down the expression of WRN and BLM respectively and found that the expression of pATM and total ATM was in concert with the expression of WRN or BLM. It is possible that WRN or BLM may function as the upstream signal of DNA damage response, which caused by the effect of BMVC4. That is, WRN or BLM might be required for eliciting the DNA damage response. We also found that WRN or BLM depletion caused H1299 cells to stop proliferating after a short period, no matter the treatment of BMVC4 or not. In addition, through SA-β-gal analysis, we found that the depletion of WRN or BLM induced cellular senescent phenotype. In conclusion, WRN and BLM are not simply the pivotal regulator of DNA damage response caused by BMVC4, but likewise have deeply influence in cell growth and cellular senescence.

口試委員會審定書．．．．．．．．．．．．．．．．．．．．．．．．1
致謝詞．．．．．．．．．．．．．．．．．．．．．．．．．．．．．2
中文摘要．．．．．．．．．．．．．．．．．．．．．．．．．．．．5
英文摘要．．．．．．．．．．．．．．．．．．．．．．．．．．．．6
目錄．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．8
附圖目錄．．．．．．．．．．．．．．．．．．．．．．．．．．．．9
縮寫表．．．．．．．．．．．．．．．．．．．．．．．．．．．．．11
一、緒論．．．．．．．．．．．．．．．．．．．．．．．．．．．．13
二、實驗材料與方法．．．．．．．．．．．．．．．．．．．．．．．22
三、實驗結果．．．．．．．．．．．．．．．．．．．．．．．．．．29
四、討論．．．．．．．．．．．．．．．．．．．．．．．．．．．．39
五、參考文獻．．．．．．．．．．．．．．．．．．．．．．．．．．42
六、附圖．．．．．．．．．．．．．．．．．．．．．．．．．．．．52

Aggarwal, M. and R. M. Brosh, Jr. (2009). "Hitting the bull''s eye: novel directed cancer therapy through helicase-targeted synthetic lethality." J Cell Biochem 106(5): 758-763.

Aggarwal, M., et al. (2010). "Delineation of WRN helicase function with EXO1 in the replicational stress response." DNA Repair (Amst) 9(7): 765-776.

Aggarwal, M., et al. (2011). "Inhibition of helicase activity by a small molecule impairs Werner syndrome helicase (WRN) function in the cellular response to DNA damage or replication stress." Proc Natl Acad Sci U S A 108(4): 1525-1530.

Arnoult, N., et al. (2009). "Human POT1 is required for efficient telomere C-rich strand replication in the absence of WRN." Genes Dev 23(24): 2915-2924.

Balasubramanian, S., et al. (2011). "Targeting G-quadruplexes in gene promoters: a novel anticancer strategy?" Nat Rev Drug Discov 10(4): 261-275.

Barefield, C. and J. Karlseder (2012). "The BLM helicase contributes to telomere maintenance through processing of late-replicating intermediate structures." Nucleic Acids Res 40(15): 7358-7367.

Bischof, O., et al. (2001). "Regulation and localization of the Bloom syndrome protein in response to DNA damage." J Cell Biol 153(2): 367-380.

Bloom, D. (1954). "Congenital telangiectatic erythema resembling lupus erythematosus in dwarfs; probably a syndrome entity." AMA Am J Dis Child 88(6): 754-758.

Bochman, M. L., et al. (2012). "DNA secondary structures: stability and function of G-quadruplex structures." Nat Rev Genet 13(11): 770-780.

Bryant, H. E. and T. Helleday (2006). "Inhibition of poly (ADP-ribose) polymerase activates ATM which is required for subsequent homologous recombination repair." Nucleic Acids Res 34(6): 1685-1691.

Bryant, H. E., et al. (2005). "Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase." Nature 434(7035): 913-917.

Bugaut, A. and S. Balasubramanian (2008). "A sequence-independent study of the influence of short loop lengths on the stability and topology of intramolecular DNA G-quadruplexes." Biochemistry 47(2): 689-697.

Burge, S., et al. (2006). "Quadruplex DNA: sequence, topology and structure." Nucleic Acids Res 34(19): 5402-5415.

Buseman, C. M., et al. (2012). "Is telomerase a viable target in cancer?" Mutat Res 730(1-2): 90-97.

Capra, J. A., et al. (2010). "G-quadruplex DNA sequences are evolutionarily conserved and associated with distinct genomic features in Saccharomyces cerevisiae." PLoS Comput Biol 6(7): e1000861.

Cesare, A. J. and R. R. Reddel (2010). "Alternative lengthening of telomeres: models, mechanisms and implications." Nat Rev Genet 11(5): 319-330.

Cheng, W. H., et al. (2008). "WRN is required for ATM activation and the S-phase checkpoint in response to interstrand cross-link-induced DNA double-strand breaks." Mol Biol Cell 19(9): 3923-3933.

Cheng, W. H., et al. (2005). "Werner syndrome protein associates with gamma H2AX in a manner that depends upon Nbs1." FEBS Lett 579(6): 1350-1356.

Cheng, W. H., et al. (2004). "Linkage between Werner syndrome protein and the Mre11 complex via Nbs1." J Biol Chem 279(20): 21169-21176.

Cogoi, S. and L. E. Xodo (2006). "G-quadruplex formation within the promoter of the KRAS proto-oncogene and its effect on transcription." Nucleic Acids Res 34(9): 2536-2549.

Collins, K. and J. R. Mitchell (2002). "Telomerase in the human organism." Oncogene 21(4): 564-579.

Cooper, M. P., et al. (2000). "Ku complex interacts with and stimulates the Werner protein." Genes Dev 14(8): 907-912.

Crabbe, L., et al. (2004). "Defective telomere lagging strand synthesis in cells lacking WRN helicase activity." Science 306(5703): 1951-1953.

Croteau, D. L., et al. (2014). "Human RecQ helicases in DNA repair, recombination, and replication." Annu Rev Biochem 83: 519-552.

Curtin, N. J. (2012). "DNA repair dysregulation from cancer driver to therapeutic target." Nat Rev Cancer 12(12): 801-817.

De Cian, A., et al. (2007). "Reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action." Proc Natl Acad Sci U S A 104(44): 17347-17352.

Dunham, M. A., et al. (2000). "Telomere maintenance by recombination in human cells." Nat Genet 26(4): 447-450.

Eddy, J. and N. Maizels (2006). "Gene function correlates with potential for G4 DNA formation in the human genome." Nucleic Acids Res 34(14): 3887-3896.

Ellis, N. A., et al. (1995). "The Bloom''s syndrome gene product is homologous to RecQ helicases." Cell 83(4): 655-666.

Ellis, N. A., et al. (1995). "Somatic intragenic recombination within the mutated locus BLM can correct the high sister-chromatid exchange phenotype of Bloom syndrome cells." Am J Hum Genet 57(5): 1019-1027.

Farmer, H., et al. (2005). "Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy." Nature 434(7035): 917-921.

Fernando, H., et al. (2006). "A conserved quadruplex motif located in a transcription activation site of the human c-kit oncogene." Biochemistry 45(25): 7854-7860.

Gellert, M., et al. (1962). "Helix formation by guanylic acid." Proc Natl Acad Sci U S A 48: 2013-2018.

German, J. (1993). "Bloom syndrome: a mendelian prototype of somatic mutational disease." Medicine (Baltimore) 72(6): 393-406.

German, J. (1997). "Bloom''s syndrome. XX. The first 100 cancers." Cancer Genet Cytogenet 93(1): 100-106.

Goldstein, S. (1990). "Replicative senescence: the human fibroblast comes of age." Science 249(4973): 1129-1133.

Gray, M. D., et al. (1997). "The Werner syndrome protein is a DNA helicase." Nat Genet 17(1): 100-103.

Guedin, A., et al. (2010). "How long is too long? Effects of loop size on G-quadruplex stability." Nucleic Acids Res 38(21): 7858-7868.

Hand, R. and J. German (1975). "A retarded rate of DNA chain growth in Bloom''s syndrome." Proc Natl Acad Sci U S A 72(2): 758-762.

Hardin, C. C., et al. (2000). "Thermodynamic and kinetic characterization of the dissociation and assembly of quadruplex nucleic acids." Biopolymers 56(3): 147-194.

Hazel, P., et al. (2006). "Predictive modelling of topology and loop variations in dimeric DNA quadruplex structures." Nucleic Acids Res 34(7): 2117-2127.

Hsu, Y. H. and J. J. Lin (2005). "Telomere and telomerase as targets for anti-cancer and regeneration therapies." Acta Pharmacol Sin 26(5): 513-518.

Huang, F. C., et al. (2008). "G-quadruplex stabilizer 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide induces accelerated senescence and inhibits tumorigenic properties in cancer cells." Mol Cancer Res 6(6): 955-964.

Huang, F. C., et al. (2012). "Induction of senescence in cancer cells by the G-quadruplex stabilizer, BMVC4, is independent of its telomerase inhibitory activity." Br J Pharmacol 167(2): 393-406.

Huang, S., et al. (1998). "The premature ageing syndrome protein, WRN, is a 3''-->5'' exonuclease." Nat Genet 20(2): 114-116.

Huber, M. D., et al. (2006). "A conserved G4 DNA binding domain in RecQ family helicases." J Mol Biol 358(4): 1071-1080.

Huppert, J. L. and S. Balasubramanian (2005). "Prevalence of quadruplexes in the human genome." Nucleic Acids Res 33(9): 2908-2916.

Huppert, J. L. and S. Balasubramanian (2007). "G-quadruplexes in promoters throughout the human genome." Nucleic Acids Res 35(2): 406-413.

Jaskelioff, M., et al. (2011). "Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice." Nature 469(7328): 102-106.

Kamath-Loeb, A. S., et al. (1998). "Werner syndrome protein. II. Characterization of the integral 3'' --> 5'' DNA exonuclease." J Biol Chem 273(51): 34145-34150.

Karmakar, P., et al. (2006). "BLM is an early responder to DNA double-strand breaks." Biochem Biophys Res Commun 348(1): 62-69.

Kim, N. W., et al. (1994). "Specific association of human telomerase activity with immortal cells and cancer." Science 266(5193): 2011-2015.

Lan, L., et al. (2005). "Accumulation of Werner protein at DNA double-strand breaks in human cells." J Cell Sci 118(Pt 18): 4153-4162.

Lee, J. H. and T. T. Paull (2005). "ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex." Science 308(5721): 551-554.

Levy, M. Z., et al. (1992). "Telomere end-replication problem and cell aging." J Mol Biol 225(4): 951-960.

Li, Q., et al. (2012). "Searching drug-like anti-cancer compound(s) based on G-quadruplex ligands." Curr Pharm Des 18(14): 1973-1983.

Lillard-Wetherell, K., et al. (2004). "Association and regulation of the BLM helicase by the telomere proteins TRF1 and TRF2." Hum Mol Genet 13(17): 1919-1932.

London, T. B., et al. (2008). "FANCJ is a structure-specific DNA helicase associated with the maintenance of genomic G/C tracts." J Biol Chem 283(52): 36132-36139.

Machwe, A., et al. (2011). "The Werner and Bloom syndrome proteins help resolve replication blockage by converting (regressed) holliday junctions to functional replication forks." Biochemistry 50(32): 6774-6788.

Machwe, A., et al. (2006). "The Werner and Bloom syndrome proteins catalyze regression of a model replication fork." Biochemistry 45(47): 13939-13946.

Machwe, A., et al. (2004). "TRF2 recruits the Werner syndrome (WRN) exonuclease for processing of telomeric DNA." Oncogene 23(1): 149-156.

Makarov, V. L., et al. (1997). "Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening." Cell 88(5): 657-666.

Mao, F. J., et al. (2010). "The human WRN and BLM RecQ helicases differentially regulate cell proliferation and survival after chemotherapeutic DNA damage." Cancer Res 70(16): 6548-6555.

Mohaghegh, P., et al. (2001). "The Bloom''s and Werner''s syndrome proteins are DNA structure-specific helicases." Nucleic Acids Res 29(13): 2843-2849.

Moser, R., et al. (2012). "MYC-driven tumorigenesis is inhibited by WRN syndrome gene deficiency." Mol Cancer Res 10(4): 535-545.

Muller, S., et al. (2012). "Pyridostatin analogues promote telomere dysfunction and long-term growth inhibition in human cancer cells." Org Biomol Chem 10(32): 6537-6546.

Murai, J., et al. (2012). "Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors." Cancer Res 72(21): 5588-5599.

Naasani, I., et al. (1998). "Telomerase inhibition, telomere shortening, and senescence of cancer cells by tea catechins." Biochem Biophys Res Commun 249(2): 391-396.

Nguyen, G. H., et al. (2013). "A small molecule inhibitor of the BLM helicase modulates chromosome stability in human cells." Chem Biol 20(1): 55-62.

Nimonkar, A. V., et al. (2011). "BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair." Genes Dev 25(4): 350-362.

Nimonkar, A. V., et al. (2008). "Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair." Proc Natl Acad Sci U S A 105(44): 16906-16911.

O''Sullivan, R. J. and J. Karlseder (2010). "Telomeres: protecting chromosomes against genome instability." Nat Rev Mol Cell Biol 11(3): 171-181.

Opresko, P. L., et al. (2005). "POT1 stimulates RecQ helicases WRN and BLM to unwind telomeric DNA substrates." J Biol Chem 280(37): 32069-32080.

Opresko, P. L., et al. (2002). "Telomere-binding protein TRF2 binds to and stimulates the Werner and Bloom syndrome helicases." J Biol Chem 277(43): 41110-41119.

Paeschke, K., et al. (2010). "Telomeres: structures in need of unwinding." FEBS Lett 584(17): 3760-3772.

Paeschke, K., et al. (2005). "Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo." Nat Struct Mol Biol 12(10): 847-854.

Pascolo, E., et al. (2002). "Mechanism of human telomerase inhibition by BIBR1532, a synthetic, non-nucleosidic drug candidate." J Biol Chem 277(18): 15566-15572.

Postberg, J., et al. (2012). "A telomerase-associated RecQ protein-like helicase resolves telomeric G-quadruplex structures during replication." Gene 497(2): 147-154.

Qin, Y. and L. H. Hurley (2008). "Structures, folding patterns, and functions of intramolecular DNA G-quadruplexes found in eukaryotic promoter regions." Biochimie 90(8): 1149-1171.

Ralf, C., et al. (2006). "The Bloom''s syndrome helicase can promote the regression of a model replication fork." J Biol Chem 281(32): 22839-22846.

Reddel, R. R. (2003). "Alternative lengthening of telomeres, telomerase, and cancer." Cancer Lett 194(2): 155-162.

Reddy, S., et al. (2010). "Processing of human telomeres by the Werner syndrome protein." Cell Cycle 9(16): 3137-3138.

Rezler, E. M., et al. (2005). "Telomestatin and diseleno sapphyrin bind selectively to two different forms of the human telomeric G-quadruplex structure." J Am Chem Soc 127(26): 9439-9447.

Riou, J. F., et al. (2002). "Cell senescence and telomere shortening induced by a new series of specific G-quadruplex DNA ligands." Proc Natl Acad Sci U S A 99(5): 2672-2677.

Rizzo, A., et al. (2009). "Stabilization of quadruplex DNA perturbs telomere replication leading to the activation of an ATR-dependent ATM signaling pathway." Nucleic Acids Res 37(16): 5353-5364.

Rodriguez, R., et al. (2008). "A novel small molecule that alters shelterin integrity and triggers a DNA-damage response at telomeres." J Am Chem Soc 130(47): 15758-15759.

Salvati, E., et al. (2007). "Telomere damage induced by the G-quadruplex ligand RHPS4 has an antitumor effect." J Clin Invest 117(11): 3236-3247.

Samanta, S. and P. Karmakar (2012). "Recruitment of HRDC domain of WRN and BLM to the sites of DNA damage induced by mitomycin C and methyl methanesulfonate." Cell Biol Int 36(10): 873-881.

Sanders, C. M. (2010). "Human Pif1 helicase is a G-quadruplex DNA-binding protein with G-quadruplex DNA-unwinding activity." Biochem J 430(1): 119-128.

Shammas, M. A., et al. (2004). "Telomerase inhibition and cell growth arrest after telomestatin treatment in multiple myeloma." Clin Cancer Res 10(2): 770-776.

Shay, J. W. and S. Bacchetti (1997). "A survey of telomerase activity in human cancer." Eur J Cancer 33(5): 787-791.

Sidorova, J. M., et al. (2013). "Distinct functions of human RECQ helicases WRN and BLM in replication fork recovery and progression after hydroxyurea-induced stalling." DNA Repair (Amst) 12(2): 128-139.

Sidorova, J. M., et al. (2008). "The RecQ helicase WRN is required for normal replication fork progression after DNA damage or replication fork arrest." Cell Cycle 7(6): 796-807.

Stewart, S. A. and R. A. Weinberg (2006). "Telomeres: cancer to human aging." Annu Rev Cell Dev Biol 22: 531-557.

Stiff, T., et al. (2006). "ATR-dependent phosphorylation and activation of ATM in response to UV treatment or replication fork stalling." EMBO J 25(24): 5775-5782.

Sun, H., et al. (1998). "The Bloom''s syndrome helicase unwinds G4 DNA." J Biol Chem 273(42): 27587-27592.

Tauchi, T., et al. (2006). "Telomerase inhibition with a novel G-quadruplex-interactive agent, telomestatin: in vitro and in vivo studies in acute leukemia." Oncogene 25(42): 5719-5725.

Tejera, A. M., et al. (2001). "Chronic in vitro exposure to 3''-azido-2'', 3''-dideoxythymidine induces senescence and apoptosis and reduces tumorigenicity of metastatic mouse mammary tumor cells." Breast Cancer Res Treat 65(2): 93-99.

Todd, A. K., et al. (2007). "Sequence occurrence and structural uniqueness of a G-quadruplex in the human c-kit promoter." Nucleic Acids Res 35(17): 5799-5808.

Wang, Q., et al. (2011). "G-quadruplex formation at the 3'' end of telomere DNA inhibits its extension by telomerase, polymerase and unwinding by helicase." Nucleic Acids Res 39(14): 6229-6237.

Williamson, J. R., et al. (1989). "Monovalent cation-induced structure of telomeric DNA: the G-quartet model." Cell 59(5): 871-880.

Yang, D. and L. H. Hurley (2006). "Structure of the biologically relevant G-quadruplex in the c-MYC promoter." Nucleosides Nucleotides Nucleic Acids 25(8): 951-968.

Yu, C. E., et al. (1996). "Positional cloning of the Werner''s syndrome gene." Science 272(5259): 258-262.

Zahler, A. M., et al. (1991). "Inhibition of telomerase by G-quartet DNA structures." Nature 350(6320): 718-720.

Zakian, V. A. (1995). "Telomeres: beginning to understand the end." Science 270(5242): 1601-1607.