THE ROLE OF PROMYELOCYTIC LEUKEMIA (PML) AND SMALL UBIQUITIN–LIKE MODIFIER (SUMO) PROTEINS IN THE ALTERNATIVE LENGTHENING OF TELOMERES YONG WEI YAN JACKLYN (B.SC (HONS), NATIONAL UNIVERSITY OF SINGAPORE) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY YONG LOO LIN SCHOOL OF MEDICINE, NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS I would like to express my most heartfelt gratitude to both Assoc. Professor M. Prakash Hande and Dr Martin Lee for the opportunity to work under their supervision. They have been selfless in imparting their knowledge and wisdom unto me and I sincerely thank both of them for their guidance. I would also like to express my gratitude and appreciation for their encouragement for my participation in various international scientific conferences. The exposure and experience gained at these conferences were invaluable. I would like to thank my friends in both Genome Stability and Nuclear Receptor laboratory. I extend my heartfelt gratitude to Mdm Wang Yaju and Mr Khaw Aik Kia, who have taught me various experimental techniques and imparted their laboratory knowledge unto me. In particular, I would also like to thank Dr Grace Low, Miss Diana Hay, Ms Asha, Mr Khaw Aik Kia and Mr Resham Gurung for their company, support and encouragement for the past years. I have gained a lot of insight from our discussions which have extended beyond science. I would also like to thank the members from both laboratories who have provided help and feedback with regard to my project. I would also like to extend my appreciation towards all staff members and their respective laboratories in the Department of Physiology for generously sharing the research equipments and materials. Special thanks towards the administrative staff, especially Ms Asha Das for her help whenever needed. In addition, my heartfelt gratitude towards my examiners for undertaking this thesis examination. Finally, I would like to thank my family members, particularly my spouse. A big ‘Thank You’ to him for being so very understanding about my commitment towards my project and for his tolerance when weekends had to be spent in the laboratory. I would also like to express my gratitude towards him for his expert help in the formatting of this thesis. My gratitude also to my mother, who has been extremely encouraging for me undertaking graduate studies and for grooming me into who I am today. My utmost appreciation to all of my family members for just loving me for who I am.   i LIST OF SCIENTIFIC CONFERENCES Yong JWY, Lee MB, and Hande MP. The coiled-coil domain of the promyelocytic leukemia protein is required for the formation of Alternative Lengthening of Telomeres-associated nuclear bodies. Telomeres and Telomerase Meeting, Cold Spring Harbor Laboratories Meeting. April-May 2009. Long Island, New York, USA. Yong JWY, Lee MB, and Hande MP. The role of the promyelocytic leukemia protein in the Alternative Lengthening of Telomeres. 100th Annual Meeting of the American Association for Cancer Research. April 2009. Denver, Colorado, USA. Yong JWY, Hande MP, and Lee MB. SUMO-mediated regulation of p53 in cancer cells exhibiting Alternative Lengthening of Telomeres. Centennial Conference of the American Association for Cancer Research. November 2007. Singapore, Singapore.   ii TABLE OF CONTENTS ACKNOWLEDGEMENTS I LIST OF SCIENTIFIC CONFERENCES II TABLE OF CONTENTS III LIST OF TABLES XI LIST OF FIGURES XIII LIST OF ILLUSTRATIONS XVII ABBREVIATIONS XVIII SUMMARY XXIII 1CHAPTER INTRODUCTION 1.1 POST-TRANSLATIONAL MODIFICATIONS 1.1.1 SMALL UBIQUITIN-LIKE MODIFIER (SUMO) 1.1.2 SUMOYLATION 1.1.3 REGULATION OF SUMO CONJUGATION 1.1.4 BIOLOGICAL FUNCTIONS OF SUMOYLATION 1.2 PROMYELOCYTIC LEUKEMIA (PML) 1.2.1 RING FINGER MOTIF 10 1.2.2 B-BOXES 10 1.2.3 COILED-COIL DOMAIN 11 1.2.4 PML NUCLEAR BODIES 11   iii 1.3 CANCER 12 1.4 HALLMARKS OF CANCER 13 1.5 TELOMERES 15 1.5.1 STRUCTURE OF TELOMERES 15 1.5.2 FUNCTIONS OF TELOMERES 16 1.5.3 TELOMERE MAINTENANCE MECHANISMS 17 1.5.4 TELOMERASE 18 1.6 ALTERNATIVE LENGTHENING OF TELOMERES 20 1.6.1 HALLMARKS OF ALT 20 1.6.2 MECHANISMS OF ALT 26 1.6.3 ALT AS A POSSIBLE CONSEQUENCE OF TELOMERE DYSFUNCTION 30 1.6.4 ALT AND TELOMERASE 31 1.6.5 EXISTENCE OF ALT REPRESSOR GENES 32 1.6.6 GENES POTENTIALLY INVOLVED IN ALT 34 1.6.7 ALT IN HUMAN CANCER 35 1.6.8 ALT AND PROGNOSIS 36 1.6.9 ALT AND CANCER THERAPY 37 2CHAPTER OBJECTIVES 39 3CHAPTER MATERIALS AND METHODS 42 3.1 CELL CULTURES 42 3.1.1 CELL LINES AND CULTURE CONDITIONS 42 3.1.2 PASSAGING CELLS 43 3.1.3 STORING CELLS 43 3.2 DETERMINATION OF NUCLEI ACID CONCENTRATION 43   iv 3.3 PREPARATION OF CACL2 COMPETENT E.COLI CELLS 44 3.4 PLASMIDS AMPLIFICATION 45 3.4.1 TRANSFORMATION 45 3.4.2 QIAGEN MINIPREP KIT 45 3.4.3 QIAGEN HISPEED PLASMID KIT 46 3.5 PLASMID CONSTRUCTS 46 3.5.1 ADDITION OF HA TAG TO SENP1 46 3.5.2 PCR MUTAGENESIS TO GENERATE PML KR MUTANTS 48 3.5.3 SUB-CLONING OF HA-PML TO PCI NEO VECTOR 49 3.5.4 ADDITION OF FLAG TAG TO PML C/C- 50 3.6 RESTRICTION ENDONUCLEASE DIGESTION OF DNA 51 3.7 AGAROSE GEL ELECTROPHORESIS 52 3.8 PURIFICATION OF DNA FROM AGAROSE GEL 52 3.9 DNA LIGATION 52 3.10 DNA FAST PREP 53 3.11 AUTOMATED DNA SEQUENCING 53 3.12 TRANSIENT TRANSFECTION 54 3.13 DETERMINATION OF GENETICIN DOSAGE 55 3.14 GENERATION OF STABLY OVER-EXPRESSING CELL CLONES (STABLE TRANSFECTION)   55 3.15 CELL CYCLE SYNCHRONIZATION 56 3.16 PREPARATION OF WHOLE CELL EXTRACTS 56 3.17 DETERMINATION OF PROTEIN CONCENTRATION BY BRADFORD METHOD 57 3.18 WESTERN BLOT ANALYSIS 58 3.18.1 SEPARATION OF PROTEINS BY POLYACRYLAMIDE GEL ELECTROPHORESIS 58   v 3.18.2 PROTEIN TRANSFER 58 3.18.3 WESTERN BLOTTING 59 3.19 IMMUNOPRECIPITATION 61 3.20 IMMUNOFLUORESCENCE 61 3.21 CONFOCAL MICROSCOPY ANALYSIS 62 3.22 CRYSTAL VIOLET CELL VIABILITY ASSAY 62 3.23 CELL CYCLE ANALYSIS BY PROPIDIUM IODIDE STAINING 63 3.24 COLONY FORMATION ASSAY 64 3.25 TERMINAL RESTRICTION FRAGMENT ANALYSIS 64 3.25.1 PREPARATION OF DNA FROM CELLS 65 3.25.2 RESTRICTION ENDONUCLEASE DIGESTION OF DNA 65 3.25.3 DNA SEPARATION BY AGAROSE GEL ELECTROPHORESIS 66 3.25.4 SOUTHERN BLOTTING 66 3.26 METAPHASE CHROMOSOMES PREPARATION 68 3.27 FLUORESCENCE IN SITU HYBRIDIZATION 68 3.28 TELOMERASE ACTIVITY ASSAY (TRAP) 69 3.29 BIOINFORMATICS AND BIOSTATISTICS 70 4CHAPTER RESULTS 4.1 SUMOYLATION OF P53 71 71 4.1.1 DIFFERENT GLOBAL SUMO-1 AND SUMO-2 CONJUGATION PATTERNS IN ALT 71 AND NON-ALT CANCER CELL LINES 4.1.2 SUMO-P53 IS DETECTED IN JFCF-6/T.1R CELLS AND NOT IN MCF7 CELLS 74 4.1.3 PIASY IS THE MOST STABLY OVER-EXPRESSED MEMBER AMONG THE PIAS FAMILY   79 vi 4.1.4 OVER-EXPRESSION OF P53 IN JFCF-6/T.1R CELLS BARELY AFFECTS SUMOYLATED P53 LEVELS 79 4.1.5 STABILITY OF OVER-EXPRESSED P53 IN MCF7 CELLS IS AFFECTED BY SUMO 80 AND PIAS 4.1.6 PIAS AFFECTS THE STABILITY OF OVER-EXPRESSED P53 81 4.1.7 SUMO1 AND PIAS STABILIZES OVER-EXPRESSED P53 FURTHER 82 4.2 EFFECTS OF SUMO-P53 84 4.2.1 SUMO-P53 AND CELL VIABILITY 84 4.2.2 SUMO-P53 AND CELL CYCLE PROGRESSION 87 4.3 FACTORS THAT AFFECT SUMOYLATION 89 4.3.1 ARSENITE REDUCES GLOBAL SUMOYLATION AS WELL THAT OF P53 IN ALT 89 CELLS 4.3.2 PROPORTION OF SUMO-P53 IN JFCF-6/T.1R CELLS VARIES IN DIFFERENT PHASES OF THE CELL CYCLE 4.4 PML IN CANCER 92 101 4.4.1 LYSINE160 IS IMPORTANT FOR SUMOYLATION OF PML AND THE COILED-COIL 101 DOMAIN IS REQUIRED FOR SUMOYLATION 4.4.2 TRANSIENTLY TRANSFECTED PML KR MUTANTS CONTINUE TO FORM APBS 105 BUT NOT THE COILED-COIL DOMAIN DELETION MUTANT 4.4.3 TRANSIENT OVER-EXPRESSION OF PML AND PML C/C- ENHANCES THE VIABILITY OF ALT CELLS 112 4.4.4 TRANSIENT OVER-EXPRESSION OF PML AND PML C/C- INCREASES THE 115 POPULATION OF ALT CELLS IN G2/M PHASE OF THE CELL CYCLE 4.4.5 U2OS AND MCF7 CLONES OF STABLY OVER-EXPRESSED PML AND PML C/C- WERE GENERATED 120 4.4.6 STABLY OVER-EXPRESSED PML C/C- DOES NOT FORM APBS IN ALT CELLS 122   vii 4.4.7 WILD-TYPE PML AND PML C/C- ALT CLONES HAVE A SLOWER POPULATION 129 DOUBLING RATE 4.4.8 WILD-TYPE PML INHIBITS THE CLONOGENICITY OF U2OS CELLS 132 4.4.9 HIGHER PROPORTION OF CELLS IN SUB-G1 AND G2/M PHASE OF THE CELL 135 CYCLE IN U2OS PML CLONES 4.4.10 WILD-TYPE PML INCREASES TELOMERE LENGTH SLIGHTLY WHILE PML C/C143 REDUCES TELOMERE LENGTH IN U2OS CELLS 4.4.11 TELOMERE LENGTHENING AND ACCUMULATION OF MCF7 CLONES EXHIBITING ALT-LIKE TELOMERE PHENOTYPE 149 4.4.12 MCF7 PML STC10 CLONE HAS A MUCH LOWER TELOMERASE ACTIVITY 151 4.4.13 MCF7 CLONES DISPLAYING ALT-LIKE PHENOTYPES ARE MORE SENSITIVE TO 153 DOXORUBICIN 5CHAPTER DISCUSSION 156 5.1 SUMOYLATION OF P53 156 5.1.1 DIFFERENT GLOBAL SUMO-1 AND SUMO-2 CONJUGATION PATTERNS IN ALT 156 AND NON-ALT CELL LINES 5.1.2 SUMO-P53 IS DETECTED IN JFCF-6/T.1R AND NOT IN MCF7 CELLS 156 5.1.3 PIASY IS THE MOST STABLY OVER-EXPRESSED MEMBER AMONG THE PIAS 158 FAMILY 5.1.4 OVER-EXPRESSION OF P53 IN JFCF-6/T.1R CELLS BARELY AFFECTS SUMOYLATED P53 LEVELS 159 5.1.5 STABILITY OF OVER-EXPRESSED P53 IN MCF7 CELLS IS AFFECTED BY SUMO 159 AND PIAS 5.1.6 PIAS AFFECTS THE STABILITY OF OVER-EXPRESSED P53 160 5.1.7 SUMO1 AND PIAS STABILIZES OVER-EXPRESSED P53 FURTHER 160 5.2 EFFECTS OF SUMO-P53 161 5.2.1 SUMO-P53 AND CELL VIABILITY 161   viii 5.2.2 SUMO-P53 AND CELL CYCLE PROGRESSION 162 5.3 FACTORS THAT AFFECT SUMOYLATION 164 5.3.1 ARSENITE REDUCES GLOBAL SUMOYLATION AS WELL THAT OF P53 IN ALT 164 CELLS 5.3.2 PROPORTION OF SUMO-P53 IN JFCF-6/T.1R CELLS VARIES IN DIFFERENT 166 PHASES OF THE CELL CYCLE 5.4 PML IN CANCER 167 5.4.1 LYSINE160 IS IMPORTANT FOR SUMOYLATION OF PML AND THE COILED-COIL 167 DOMAIN IS REQUIRED FOR SUMOYLATION 5.4.2 TRANSIENTLY TRANSFECTED PML KR MUTANTS CONTINUE TO FORM APBS 168 BUT NOT THE COILED-COIL DOMAIN DELETION MUTANT 5.4.3 TRANSIENTLY OVER-EXPRESSION OF PML AND PML C/C- ENHANCES THE 170 VIABILITY OF ALT CELLS 5.4.4 TRANSIENT OVER-EXPRESSION OF PML AND PML C/C- INCREASES THE 170 POPULATION OF ALT CELLS IN G2/M PHASE OF THE CELL CYCLE 5.4.5 U2OS AND MCF7 CLONES OF STABLY OVER-EXPRESSED PML AND PML C/C- WERE GENERATED 171 5.4.6 STABLY OVER-EXPRESSED PML C/C- DOES NOT FORM APBS IN ALT CELLS 172   5.4.7 WILD-TYPE PML AND PML C/C- ALT CLONES HAVE A SLOWER POPULATION 173 DOUBLING RATE 5.4.8 WILD-TYPE PML INHIBITS THE CLONOGENICITY OF U2OS CELLS   174 5.4.9 HIGHER PROPORTION OF CELLS IN SUB-G1 AND G2/M PHASE OF THE CELL 175 CYCLE IN U2OS PML CLONES 5.4.10 WILD-TYPE PML INCREASES TELOMERE LENGTH SLIGHTLY WHILE PML C/C177 REDUCES TELOMERE LENGTH IN U2OS CELLS   ix Garcia M, J. A., Ward EM, Center MM, Hao Y, Siegel RL, Thun MJ. (2007) Global Cancer Facts & Figures 2007. Atlanta, GA: American Cancer Society. Gartel, A. L., C. Feliciano & A. L. Tyner (2003) A new method for determining the status of p53 in tumor cell lines of different origin. Oncol Res, 13, 405-8. Geiss-Friedlander, R. & F. Melchior (2007) Concepts in sumoylation: a decade on. Nat Rev Mol Cell Biol, 8, 947-56. Gonzalo, S., I. Jaco, M. F. Fraga, T. Chen, E. Li, M. Esteller & M. A. Blasco (2006) DNA methyltransferases control telomere length and telomere recombination in mammalian cells. Nat Cell Biol, 8, 416-24. Gottifredi, V. & C. Prives (2001) P53 and PML: new partners in tumor suppression. Trends Cell Biol, 11, 184-7. Griffith, J. D., L. Comeau, S. Rosenfield, R. M. Stansel, A. Bianchi, H. Moss & T. de Lange (1999) Mammalian telomeres end in a large duplex loop. Cell, 97, 503-14. Grobelny, J. V., A. K. Godwin & D. Broccoli (2000) ALT-associated PML bodies are present in viable cells and are enriched in cells in the G(2)/M phase of the cell cycle. J Cell Sci, 113 Pt 24, 4577-85. Grobelny, J. V., M. Kulp-McEliece & D. Broccoli (2001) Effects of reconstitution of telomerase activity on telomere maintenance by the alternative lengthening of telomeres (ALT) pathway. Hum Mol Genet, 10, 1953-61. Grudic, A., A. Jul-Larsen, S. J. Haring, M. S. Wold, P. E. Lonning, R. Bjerkvig & S. O. Boe (2007) Replication protein A prevents accumulation of singlestranded telomeric DNA in cells that use alternative lengthening of telomeres. Nucleic Acids Res, 35, 7267-78. Guiducci, C., M. A. Cerone & S. Bacchetti (2001) Expression of mutant telomerase in immortal telomerase-negative human cells results in cell cycle deregulation, nuclear and chromosomal abnormalities and rapid loss of viability. Oncogene, 20, 714-25. Hahn, W. C., S. A. Stewart, M. W. Brooks, S. G. York, E. Eaton, A. Kurachi, R. L. Beijersbergen, J. H. Knoll, M. Meyerson & R. A. Weinberg (1999) Inhibition of telomerase limits the growth of human cancer cells. Nat Med, 5, 1164-70. Hakin-Smith, V., D. A. Jellinek, D. Levy, T. Carroll, M. Teo, W. R. Timperley, M. J. McKay, R. R. Reddel & J. A. Royds (2003) Alternative lengthening of   195 telomeres and survival in patients with glioblastoma multiforme. Lancet, 361, 836-8. Hanahan, D. & R. A. Weinberg (2000) The hallmarks of cancer. Cell, 100, 5770. Hande, M. P., E. Samper, P. Lansdorp & M. A. Blasco (1999) Telomere length dynamics and chromosomal instability in cells derived from telomerase null mice. J Cell Biol, 144, 589-601. Harley, C. B., A. B. Futcher & C. W. Greider (1990) Telomeres shorten during ageing of human fibroblasts. Nature, 345, 458-60. Heideker, J., J. J. Perry & M. N. Boddy (2009) Genome stability roles of SUMO-targeted ubiquitin ligases. DNA Repair (Amst), 8, 517-24. Henson, J. D., J. A. Hannay, S. W. McCarthy, J. A. Royds, T. R. Yeager, R. A. Robinson, S. B. Wharton, D. A. Jellinek, S. M. Arbuckle, J. Yoo, B. G. Robinson, D. L. Learoyd, P. D. Stalley, S. F. Bonar, D. Yu, R. E. Pollock & R. R. Reddel (2005) A robust assay for alternative lengthening of telomeres in tumors shows the significance of alternative lengthening of telomeres in sarcomas and astrocytomas. Clin Cancer Res, 11, 217-25. Henson, J. D., A. A. Neumann, T. R. Yeager & R. R. Reddel (2002) Alternative lengthening of telomeres in mammalian cells. Oncogene, 21, 598610. Heun, P. (2007) SUMOrganization of the nucleus. Curr Opin Cell Biol, 19, 350-5. Hoare, S. F., L. A. Bryce, G. B. Wisman, S. Burns, J. J. Going, A. G. van der Zee & W. N. Keith (2001) Lack of telomerase RNA gene hTERC expression in alternative lengthening of telomeres cells is associated with methylation of the hTERC promoter. Cancer Res, 61, 27-32. Hock, L. C. (2002) An overview of the cancer control programme in Singapore. Jpn J Clin Oncol, 32 Suppl, S62-5. Hodges, M., C. Tissot, K. Howe, D. Grimwade & P. S. Freemont (1998) Structure, organization, and dynamics of promyelocytic leukemia protein nuclear bodies. Am J Hum Genet, 63, 297-304. Hofmann, T. G. & H. Will (2003) Body language: the function of PML nuclear bodies in apoptosis regulation. Cell Death Differ, 10, 1290-9.   196 Hunter, T. & H. Sun (2008) Crosstalk between the SUMO and ubiquitin pathways. Ernst Schering Found Symp Proc, 1-16. Ishov, A. M., A. G. Sotnikov, D. Negorev, O. V. Vladimirova, N. Neff, T. Kamitani, E. T. Yeh, J. F. Strauss, 3rd & G. G. Maul (1999) PML is critical for ND10 formation and recruits the PML-interacting protein daxx to this nuclear structure when modified by SUMO-1. J Cell Biol, 147, 221-34. Jegou, T., I. Chung, G. Heuvelman, M. Wachsmuth, S. M. Gorisch, K. M. Greulich-Bode, P. Boukamp, P. Lichter & K. Rippe (2009) Dynamics of telomeres and promyelocytic leukemia nuclear bodies in a telomerasenegative human cell line. Mol Biol Cell, 20, 2070-82. Jensen, K., C. Shiels & P. S. Freemont (2001) PML protein isoforms and the RBCC/TRIM motif. Oncogene, 20, 7223-33. Jeyapalan, J. N., A. Mendez-Bermudez, N. Zaffaroni, Y. E. Dubrova & N. J. Royle (2008) Evidence for alternative lengthening of telomeres in liposarcomas in the absence of ALT-associated PML bodies. Int J Cancer, 122, 2414-21. Jeyapalan, J. N., H. Varley, J. L. Foxon, R. E. Pollock, A. J. Jeffreys, J. D. Henson, R. R. Reddel & N. J. Royle (2005) Activation of the ALT pathway for telomere maintenance can affect other sequences in the human genome. Hum Mol Genet, 14, 1785-94. Jiang, W. Q., Z. H. Zhong, J. D. Henson, A. A. Neumann, A. C. Chang & R. R. Reddel (2005) Suppression of alternative lengthening of telomeres by Sp100mediated sequestration of the MRE11/RAD50/NBS1 complex. Mol Cell Biol, 25, 2708-21. Jiang, W. Q., Z. H. Zhong, J. D. Henson & R. R. Reddel (2007) Identification of candidate alternative lengthening of telomeres genes by methionine restriction and RNA interference. Oncogene, 26, 4635-47. Jiang, W. Q., Z. H. Zhong, A. Nguyen, J. D. Henson, C. D. Toouli, A. W. Braithwaite & R. R. Reddel (2009) Induction of alternative lengthening of telomeres-associated PML bodies by p53/p21 requires HP1 proteins. J Cell Biol, 185, 797-810. Jiang, X. R., G. Jimenez, E. Chang, M. Frolkis, B. Kusler, M. Sage, M. Beeche, A. G. Bodnar, G. M. Wahl, T. D. Tlsty & C. P. Chiu (1999) Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat Genet, 21, 111-4. Johnson, E. S. (2004) Protein modification by SUMO. Annu Rev Biochem, 73, 355-82.   197 Johnson, F. B., R. A. Marciniak, M. McVey, S. A. Stewart, W. C. Hahn & L. Guarente (2001) The Saccharomyces cerevisiae WRN homolog Sgs1p participates in telomere maintenance in cells lacking telomerase. EMBO J, 20, 905-13. Johnson, J. E. & D. Broccoli (2007) Telomere maintenance in sarcomas. Curr Opin Oncol, 19, 377-82. Kamitani, T., K. Kito, H. P. Nguyen, H. Wada, T. Fukuda-Kamitani & E. T. Yeh (1998) Identification of three major sentrinization sites in PML. J Biol Chem, 273, 26675-82. Kang, H., E. T. Kim, H. R. Lee, J. J. Park, Y. Y. Go, C. Y. Choi & J. H. Ahn (2006) Inhibition of SUMO-independent PML oligomerization by the human cytomegalovirus IE1 protein. J Gen Virol, 87, 2181-90. Karlseder, J., A. Smogorzewska & T. de Lange (2002) Senescence induced by altered telomere state, not telomere loss. Science, 295, 2446-9. Katoh, M., M. Kameyama, H. Kugoh, M. Shimizu & M. Oshimura (1998) A repressor function for telomerase activity in telomerase-negative immortal cells. Mol Carcinog, 21, 17-25. Kilian, A., D. D. Bowtell, H. E. Abud, G. R. Hime, D. J. Venter, P. K. Keese, E. L. Duncan, R. R. Reddel & R. A. Jefferson (1997) Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types. Hum Mol Genet, 6, 2011-9. Kim, J. H., G. E. Lee, J. C. Kim, J. H. Lee & I. K. Chung (2002) A novel telomere elongation in an adriamycin-resistant stomach cancer cell line with decreased telomerase activity. Mol Cells, 13, 228-36. Kim, K. I. & S. H. Baek (2009) Small ubiquitin-like modifiers in cellular malignancy and metastasis. Int Rev Cell Mol Biol, 273, 265-311. Kim, Y. E., D. Y. Kim, J. M. Lee, S. T. Kim, T. H. Han & J. H. Ahn (2005) Requirement of the coiled-coil domain of PML-RARalpha oncoprotein for localization, sumoylation, and inhibition of monocyte differentiation. Biochem Biophys Res Commun, 330, 746-54. Komata, T., Y. Kondo, T. Kanzawa, S. Hirohata, S. Koga, H. Sumiyoshi, S. M. Srinivasula, B. P. Barna, I. M. Germano, M. Takakura, M. Inoue, E. S. Alnemri, J. W. Shay, S. Kyo & S. Kondo (2001) Treatment of malignant glioma cells with the transfer of constitutively active caspase-6 using the human telomerase catalytic subunit (human telomerase reverse transcriptase) gene promoter. Cancer Res, 61, 5796-802.   198 Komata, T., Y. Kondo, T. Kanzawa, H. Ito, S. Hirohata, S. Koga, H. Sumiyoshi, M. Takakura, M. Inoue, B. P. Barna, I. M. Germano, S. Kyo & S. Kondo (2002) Caspase-8 gene therapy using the human telomerase reverse transcriptase promoter for malignant glioma cells. Hum Gene Ther, 13, 101525. Kotaja, N., U. Karvonen, O. A. Janne & J. J. Palvimo (2002) PIAS proteins modulate transcription factors by functioning as SUMO-1 ligases. Mol Cell Biol, 22, 5222-34. Kumakura, S., T. W. Tsutsui, J. Yagisawa, J. C. Barrett & T. Tsutsui (2005) Reversible conversion of immortal human cells from telomerase-positive to telomerase-negative cells. Cancer Res, 65, 2778-86. Kumata, M., M. Shimizu, M. Oshimura, M. Uchida & T. Tsutsui (2002) Induction of cellular senescence in a telomerase negative human immortal fibroblast cell line, LCS-AF.1-3, by human chromosome 6. Int J Oncol, 21, 851-6. Kwek, S. S., J. Derry, A. L. Tyner, Z. Shen & A. V. Gudkov (2001) Functional analysis and intracellular localization of p53 modified by SUMO-1. Oncogene, 20, 2587-99. Le, S., J. K. Moore, J. E. Haber & C. W. Greider (1999) RAD50 and RAD51 define two pathways that collaborate to maintain telomeres in the absence of telomerase. Genetics, 152, 143-52. Lombard, D. B., K. F. Chua, R. Mostoslavsky, S. Franco, M. Gostissa & F. W. Alt (2005) DNA repair, genome stability, and aging. Cell, 120, 497-512. Londono-Vallejo, J. A., H. Der-Sarkissian, L. Cazes, S. Bacchetti & R. R. Reddel (2004) Alternative lengthening of telomeres is characterized by high rates of telomeric exchange. Cancer Res, 64, 2324-7. Lukyanova, N. Y., N. V. Rusetskya, N. A. Tregubova & V. F. Chekhun (2009) Molecular profile and cell cycle in MCF-7 cells resistant to cisplatin and doxorubicin. Exp Oncol, 31, 87-91. Lundblad, V. & E. H. Blackburn (1993) An alternative pathway for yeast telomere maintenance rescues est1- senescence. Cell, 73, 347-60. Maeda, D., M. Seki, F. Onoda, D. Branzei, Y. Kawabe & T. Enomoto (2004) Ubc9 is required for damage-tolerance and damage-induced interchromosomal homologous recombination in S. cerevisiae. DNA Repair (Amst), 3, 335-41.   199 Makarov, V. L., Y. Hirose & J. P. Langmore (1997) Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening. Cell, 88, 657-66. Marciniak, R. A., D. Cavazos, R. Montellano, Q. Chen, L. Guarente & F. B. Johnson (2005) A novel telomere structure in a human alternative lengthening of telomeres cell line. Cancer Res, 65, 2730-7. Maringele, L. & D. Lydall (2004) EXO1 plays a role in generating type I and type II survivors in budding yeast. Genetics, 166, 1641-9. Maringele, L. & D. Lydall (2004) Telomerase- and recombination-independent immortalization of budding yeast. Genes Dev, 18, 2663-75. Mathieu, N., L. Pirzio, M. A. Freulet-Marriere, C. Desmaze & L. Sabatier (2004) Telomeres and chromosomal instability. Cell Mol Life Sci, 61, 641-56. Matsuo, T., J. W. Shay, W. E. Wright, E. Hiyama, S. Shimose, T. Kubo, T. Sugita, Y. Yasunaga & M. Ochi (2009) Telomere-maintenance mechanisms in soft-tissue malignant fibrous histiocytomas. J Bone Joint Surg Am, 91, 92837. Matunis, M. J. (2002) On the road to repair: PCNA encounters SUMO and ubiquitin modifications. Mol Cell, 10, 441-2. Matunis, M. J., X. D. Zhang & N. A. Ellis (2006) SUMO: the glue that binds. Dev Cell, 11, 596-7. McCracken, M., M. Olsen, M. S. Chen, Jr., A. Jemal, M. Thun, V. Cokkinides, D. Deapen & E. Ward (2007) Cancer incidence, mortality, and associated risk factors among Asian Americans of Chinese, Filipino, Vietnamese, Korean, and Japanese ethnicities. CA Cancer J Clin, 57, 190-205. Melchior, F. (2000) SUMO--nonclassical ubiquitin. Annu Rev Cell Dev Biol, 16, 591-626. Melchior, F. & L. Hengst (2002) SUMO-1 and p53. Cell Cycle, 1, 245-9. Misri, S., S. Pandita, R. Kumar & T. K. Pandita (2008) Telomeres, histone code, and DNA damage response. Cytogenet Genome Res, 122, 297-307. Miyauchi, Y., S. Yogosawa, R. Honda, T. Nishida & H. Yasuda (2002) Sumoylation of Mdm2 by protein inhibitor of activated STAT (PIAS) and RanBP2 enzymes. J Biol Chem, 277, 50131-6.   200 Molenaar, C., K. Wiesmeijer, N. P. Verwoerd, S. Khazen, R. Eils, H. J. Tanke & R. W. Dirks (2003) Visualizing telomere dynamics in living mammalian cells using PNA probes. EMBO J, 22, 6631-41. Montgomery, E., P. Argani, J. L. Hicks, A. M. DeMarzo & A. K. Meeker (2004) Telomere lengths of translocation-associated and nontranslocation-associated sarcomas differ dramatically. Am J Pathol, 164, 1523-9. Morales, C. P., S. E. Holt, M. Ouellette, K. J. Kaur, Y. Yan, K. S. Wilson, M. A. White, W. E. Wright & J. W. Shay (1999) Absence of cancer-associated changes in human fibroblasts immortalized with telomerase. Nat Genet, 21, 115-8. Muller, S., M. Berger, F. Lehembre, J. S. Seeler, Y. Haupt & A. Dejean (2000) c-Jun and p53 activity is modulated by SUMO-1 modification. J Biol Chem, 275, 13321-9. Muller, S., A. Ledl & D. Schmidt (2004) SUMO: a regulator of gene expression and genome integrity. Oncogene, 23, 1998-2008. Muntoni, A. & R. R. Reddel (2005) The first molecular details of ALT in human tumor cells. Hum Mol Genet, 14 Spec No. 2, R191-6. Murnane, J. P., L. Sabatier, B. A. Marder & W. F. Morgan (1994) Telomere dynamics in an immortal human cell line. EMBO J, 13, 4953-62. Murphy, L. C., B. Peng, A. Lewis, J. R. Davie, E. Leygue, A. Kemp, K. Ung, M. Vendetti & R. Shiu (2005) Inducible upregulation of oestrogen receptorbeta1 affects oestrogen and tamoxifen responsiveness in MCF7 human breast cancer cells. J Mol Endocrinol, 34, 553-66. Nabetani, A. & F. Ishikawa (2009) Unusual telomeric DNAs in human telomerase-negative immortalized cells. Mol Cell Biol, 29, 703-13. Nabetani, A., O. Yokoyama & F. Ishikawa (2004) Localization of hRad9, hHus1, hRad1, and hRad17 and caffeine-sensitive DNA replication at the alternative lengthening of telomeres-associated promyelocytic leukemia body. J Biol Chem, 279, 25849-57. Naka, K., K. Ikeda & N. Motoyama (2002) Recruitment of NBS1 into PML oncogenic domains via interaction with SP100 protein. Biochem Biophys Res Commun, 299, 863-71. Nakabayashi, K., T. Ogata, M. Fujii, H. Tahara, T. Ide, R. Wadhwa, S. C. Kaul, Y. Mitsui & D. Ayusawa (1997) Decrease in amplified telomeric sequences and induction of senescence markers by introduction of human chromosome or its segments in SUSM-1. Exp Cell Res, 235, 345-53.   201 Nakamura, T. M., J. P. Cooper & T. R. Cech (1998) Two modes of survival of fission yeast without telomerase. Science, 282, 493-6. Natarajan, S. & M. J. McEachern (2002) Recombinational telomere elongation promoted by DNA circles. Mol Cell Biol, 22, 4512-21. Niida, H., Y. Shinkai, M. P. Hande, T. Matsumoto, S. Takehara, M. Tachibana, M. Oshimura, P. M. Lansdorp & Y. Furuichi (2000) Telomere maintenance in telomerase-deficient mouse embryonic stem cells: characterization of an amplified telomeric DNA. Mol Cell Biol, 20, 4115-27. Nittis, T., L. Guittat & S. A. Stewart (2008) Alternative lengthening of telomeres (ALT) and chromatin: is there a connection? Biochimie, 90, 5-12. Nobert, G. S., M. M. Kraak & S. Crawford (2006) Estrogen dependent growth inhibitory effects of tamoxifen but not genistein in solid tumors derived from estrogen receptor positive (ER+) primary breast carcinoma MCF7: single agent and novel combined treatment approaches. Bull Cancer, 93, E59-66. Ogino, H., K. Nakabayashi, M. Suzuki, E. Takahashi, M. Fujii, T. Suzuki & D. Ayusawa (1998) Release of telomeric DNA from chromosomes in immortal human cells lacking telomerase activity. Biochem Biophys Res Commun, 248, 223-7. Opitz, O. G., Y. Suliman, W. C. Hahn, H. Harada, H. E. Blum & A. K. Rustgi (2001) Cyclin D1 overexpression and p53 inactivation immortalize primary oral keratinocytes by a telomerase-independent mechanism. J Clin Invest, 108, 725-32. Opresko, P. L., M. Otterlei, J. Graakjaer, P. Bruheim, L. Dawut, S. Kolvraa, A. May, M. M. Seidman & V. A. Bohr (2004) The Werner syndrome helicase and exonuclease cooperate to resolve telomeric D loops in a manner regulated by TRF1 and TRF2. Mol Cell, 14, 763-74. Owerbach, D., E. M. McKay, E. T. Yeh, K. H. Gabbay & K. M. Bohren (2005) A proline-90 residue unique to SUMO-4 prevents maturation and sumoylation. Biochem Biophys Res Commun, 337, 517-20. Palvimo, J. J. (2007) PIAS proteins as regulators of small ubiquitin-related modifier (SUMO) modifications and transcription. Biochem Soc Trans, 35, 1405-8. Perrem, K., T. M. Bryan, A. Englezou, T. Hackl, E. L. Moy & R. R. Reddel (1999) Repression of an alternative mechanism for lengthening of telomeres in somatic cell hybrids. Oncogene, 18, 3383-90.   202 Perrem, K., L. M. Colgin, A. A. Neumann, T. R. Yeager & R. R. Reddel (2001) Coexistence of alternative lengthening of telomeres and telomerase in hTERT-transfected GM847 cells. Mol Cell Biol, 21, 3862-75. Pickett, H. A., A. J. Cesare, R. L. Johnston, A. A. Neumann & R. R. Reddel (2009) Control of telomere length by a trimming mechanism that involves generation of t-circles. EMBO J, 28, 799-809. Pinsky, B. A. & S. Biggins (2002) Top-SUMO wrestles centromeric cohesion. Dev Cell, 3, 4-6. Plevova, P., J. Bouchal, M. Fiuraskova, M. Papezova, A. Krepelova, R. Curik, L. Foretova, M. Navratilova, J. Zapletalova, T. Posolda & Z. Kolar (2007) PML and TRF2 protein expression in hereditary and sporadic colon cancer. Neoplasma, 54, 269-77. Potts, P. R. & H. Yu (2007) The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins. Nat Struct Mol Biol, 14, 581-90. Quimby, B. B., V. Yong-Gonzalez, T. Anan, A. V. Strunnikov & M. Dasso (2006) The promyelocytic leukemia protein stimulates SUMO conjugation in yeast. Oncogene, 25, 2999-3005. Razak, Z. R., R. J. Varkonyi, M. Kulp-McEliece, C. Caslini, J. R. Testa, M. E. Murphy & D. Broccoli (2004) p53 differentially inhibits cell growth depending on the mechanism of telomere maintenance. Mol Cell Biol, 24, 5967-77. Reddel, R. R. (2000) The role of senescence and immortalization in carcinogenesis. Carcinogenesis, 21, 477-84. Reddel, R. R. (2001) An alternative lifestyle for immortalized oral keratinocytes. J Clin Invest, 108, 665-7. Reddel, R. R. (2003) Alternative lengthening of telomeres, telomerase, and cancer. Cancer Lett, 194, 155-62. Reddel, R. R., T. M. Bryan, L. M. Colgin, K. T. Perrem & T. R. Yeager (2001) Alternative lengthening of telomeres in human cells. Radiat Res, 155, 194200. Reddel, R. R., T. M. Bryan & J. P. Murnane (1997) Immortalized cells with no detectable telomerase activity. A review. Biochemistry (Mosc), 62, 1254-62. Reineke, E. L. & H. Y. Kao (2009) Targeting promyelocytic leukemia protein: a means to regulating PML nuclear bodies. Int J Biol Sci, 5, 366-76.   203 Reymond, A., G. Meroni, A. Fantozzi, G. Merla, S. Cairo, L. Luzi, D. Riganelli, E. Zanaria, S. Messali, S. Cainarca, A. Guffanti, S. Minucci, P. G. Pelicci & A. Ballabio (2001) The tripartite motif family identifies cell compartments. EMBO J, 20, 2140-51. Rizki, A. & V. Lundblad (2001) Defects in mismatch repair promote telomerase-independent proliferation. Nature, 411, 713-6. Royle, N. J., J. Foxon, J. N. Jeyapalan, A. Mendez-Bermudez, C. L. Novo, J. Williams & V. E. Cotton (2008) Telomere length maintenance--an ALTernative mechanism. Cytogenet Genome Res, 122, 281-91. Royle, N. J., A. Mendez-Bermudez, A. Gravani, C. Novo, J. Foxon, J. Williams, V. Cotton & A. Hidalgo (2009) The role of recombination in telomere length maintenance. Biochem Soc Trans, 37, 589-95. Ruckova, E., J. Friml, P. Prochazkova Schrumpfova & J. Fajkus (2008) Role of alternative telomere lengthening unmasked in telomerase knock-out mutant plants. Plant Mol Biol, 66, 637-46. Ruggero, D., Z. G. Wang & P. P. Pandolfi (2000) The puzzling multiple lives of PML and its role in the genesis of cancer. Bioessays, 22, 827-35. Rytinki, M. M., S. Kaikkonen, P. Pehkonen, T. Jaaskelainen & J. J. Palvimo (2009) PIAS proteins: pleiotropic interactors associated with SUMO. Cell Mol Life Sci, 66, 3029-41. Saitoh, H. & J. Hinchey (2000) Functional heterogeneity of small ubiquitinrelated protein modifiers SUMO-1 versus SUMO-2/3. J Biol Chem, 275, 62528. Salomoni, P. (2009) Stemming out of a new PML era? Cell Death Differ, 16, 1083-92. Sanders, R. P., R. Drissi, C. A. Billups, N. C. Daw, M. B. Valentine & J. S. Dome (2004) Telomerase expression predicts unfavorable outcome in osteosarcoma. J Clin Oncol, 22, 3790-7. Sankala, H. M., N. C. Hait, S. W. Paugh, D. Shida, S. Lepine, L. W. Elmore, P. Dent, S. Milstien & S. Spiegel (2007) Involvement of sphingosine kinase in p53-independent induction of p21 by the chemotherapeutic drug doxorubicin. Cancer Res, 67, 10466-74. Satyanarayana, A., M. P. Manns & K. L. Rudolph (2004) Telomeres, telomerase and cancer: an endless search to target the ends. Cell Cycle, 3, 1138-50.   204 Scheel, C. & C. Poremba (2002) Telomere lengthening in telomerasenegative cells: the ends are coming together. Virchows Arch, 440, 573-82. Scheel, C., K. L. Schaefer, A. Jauch, M. Keller, D. Wai, C. Brinkschmidt, F. van Valen, W. Boecker, B. Dockhorn-Dworniczak & C. Poremba (2001) Alternative lengthening of telomeres is associated with chromosomal instability in osteosarcomas. Oncogene, 20, 3835-44. Schmidt, D. & S. Muller (2002) Members of the PIAS family act as SUMO ligases for c-Jun and p53 and repress p53 activity. Proc Natl Acad Sci U S A, 99, 2872-7. Seeler, J. S. & A. Dejean (1999) The PML nuclear bodies: actors or extras? Curr Opin Genet Dev, 9, 362-7. Seeler, J. S. & A. Dejean (2001) SUMO: of branched proteins and nuclear bodies. Oncogene, 20, 7243-9. Seger, Y. R., M. Garcia-Cao, S. Piccinin, C. L. Cunsolo, C. Doglioni, M. A. Blasco, G. J. Hannon & R. Maestro (2002) Transformation of normal human cells in the absence of telomerase activation. Cancer Cell, 2, 401-13. Shammas, M. A., H. Koley, R. C. Bertheau, P. Neri, M. Fulciniti, P. Tassone, S. Blotta, A. Protopopov, C. Mitsiades, R. B. Batchu, K. C. Anderson, A. Chin, S. Gryaznov & N. C. Munshi (2008) Telomerase inhibitor GRN163L inhibits myeloma cell growth in vitro and in vivo. Leukemia, 22, 1410-8. Sharrocks, A. D. (2006) PIAS proteins and transcriptional regulation--more than just SUMO E3 ligases? Genes Dev, 20, 754-8. Shay, J. W. & S. Bacchetti (1997) A survey of telomerase activity in human cancer. Eur J Cancer, 33, 787-91. Shay, J. W. & W. E. Wright (2000) Hayflick, his limit, and cellular ageing. Nat Rev Mol Cell Biol, 1, 72-6. Shen, T. H., H. K. Lin, P. P. Scaglioni, T. M. Yung & P. P. Pandolfi (2006) The mechanisms of PML-nuclear body formation. Mol Cell, 24, 331-9. Shigeeda, N., M. Uchida, J. C. Barrett & T. Tsutsui (2003) Candidate chromosomal regions for genes involved in activation of alternative lengthening of telomeres in human immortal cell lines. Exp Gerontol, 38, 64151. Siddiqa, A., D. A. Cavazos & R. A. Marciniak (2006) Targeting telomerase. Rejuvenation Res, 9, 378-90.   205 Singh, B., K. R. Cook, L. Vincent, C. S. Hall, J. A. Berry, A. S. Multani & A. Lucci (2008) Cyclooxygenase-2 induces genomic instability, BCL2 expression, doxorubicin resistance, and altered cancer-initiating cell phenotype in MCF7 breast cancer cells. J Surg Res, 147, 240-6. Smogorzewska, A., B. van Steensel, A. Bianchi, S. Oelmann, M. R. Schaefer, G. Schnapp & T. de Lange (2000) Control of human telomere length by TRF1 and TRF2. Mol Cell Biol, 20, 1659-68. Stagno D'Alcontres, M., A. Mendez-Bermudez, J. L. Foxon, N. J. Royle & P. Salomoni (2007) Lack of TRF2 in ALT cells causes PML-dependent p53 activation and loss of telomeric DNA. J Cell Biol, 179, 855-67. Stavropoulos, D. J., P. S. Bradshaw, X. Li, I. Pasic, K. Truong, M. Ikura, M. Ungrin & M. S. Meyn (2002) The Bloom syndrome helicase BLM interacts with TRF2 in ALT cells and promotes telomeric DNA synthesis. Hum Mol Genet, 11, 3135-44. Stehmeier, P. & S. Muller (2009) Regulation of p53 family members by the ubiquitin-like SUMO system. DNA Repair (Amst), 8, 491-8. Stewart, S. A., W. C. Hahn, B. F. O'Connor, E. N. Banner, A. S. Lundberg, P. Modha, H. Mizuno, M. W. Brooks, M. Fleming, D. B. Zimonjic, N. C. Popescu & R. A. Weinberg (2002) Telomerase contributes to tumorigenesis by a telomere length-independent mechanism. Proc Natl Acad Sci U S A, 99, 12606-11. Strahl, C. & E. H. Blackburn (1996) Effects of reverse transcriptase inhibitors on telomere length and telomerase activity in two immortalized human cell lines. Mol Cell Biol, 16, 53-65. Sun, B., M. Chen, C. L. Hawks & P. J. Hornsby (2005) Immortal ALT+ human cells not require telomerase reverse transcriptase for malignant transformation. Cancer Res, 65, 6512-5. Synowiec, E., D. Ksiazek, J. Blasiak & K. Wozniak (2008) [Role of SUMO modification in the maintenance of genome stability]. Postepy Biochem, 54, 234-41. Takahashi, Y., V. Yong-Gonzalez, Y. Kikuchi & A. Strunnikov (2006) SIZ1/SIZ2 control of chromosome transmission fidelity is mediated by the sumoylation of topoisomerase II. Genetics, 172, 783-94. Takemura, M., K. Sugimura, K. Okumura, S. Limsirichaikul, M. Suzuki, Y. Yamada & S. Yoshida (2008) Hyper-phosphorylated retinoblastoma protein suppresses telomere elongation. Biosci Biotechnol Biochem, 72, 630-5.   206 Tanaka, K., J. Nishide, K. Okazaki, H. Kato, O. Niwa, T. Nakagawa, H. Matsuda, M. Kawamukai & Y. Murakami (1999) Characterization of a fission yeast SUMO-1 homologue, pmt3p, required for multiple nuclear events, including the control of telomere length and chromosome segregation. Mol Cell Biol, 19, 8660-72. Tarsounas, M., P. Munoz, A. Claas, P. G. Smiraldo, D. L. Pittman, M. A. Blasco & S. C. West (2004) Telomere maintenance requires the RAD51D recombination/repair protein. Cell, 117, 337-47. Temime-Smaali, N., L. Guittat, T. Wenner, E. Bayart, C. Douarre, D. Gomez, M. J. Giraud-Panis, A. Londono-Vallejo, E. Gilson, M. Amor-Gueret & J. F. Riou (2008) Topoisomerase IIIalpha is required for normal proliferation and telomere stability in alternative lengthening of telomeres. EMBO J, 27, 151324. Teng, S. C., J. Chang, B. McCowan & V. A. Zakian (2000) Telomeraseindependent lengthening of yeast telomeres occurs by an abrupt Rad50pdependent, Rif-inhibited recombinational process. Mol Cell, 6, 947-52. Teng, S. C. & V. A. Zakian (1999) Telomere-telomere recombination is an efficient bypass pathway for telomere maintenance in Saccharomyces cerevisiae. Mol Cell Biol, 19, 8083-93. Tilman, G., A. Loriot, A. Van Beneden, N. Arnoult, J. A. Londono-Vallejo, C. De Smet & A. Decottignies (2009) Subtelomeric DNA hypomethylation is not required for telomeric sister chromatid exchanges in ALT cells. Oncogene, 28, 1682-93. Tokutake, Y., T. Matsumoto, T. Watanabe, S. Maeda, H. Tahara, S. Sakamoto, H. Niida, M. Sugimoto, T. Ide & Y. Furuichi (1998) Extrachromosomal telomere repeat DNA in telomerase-negative immortalized cell lines. Biochem Biophys Res Commun, 247, 765-72. Tomaska, L., M. J. McEachern & J. Nosek (2004) Alternatives to telomerase: keeping linear chromosomes via telomeric circles. FEBS Lett, 567, 142-6. Tomaska, L., J. Nosek, J. Kramara & J. D. Griffith (2009) Telomeric circles: universal players in telomere maintenance? Nat Struct Mol Biol, 16, 1010-5. Torres-Rosell, J., I. Sunjevaric, G. De Piccoli, M. Sacher, N. Eckert-Boulet, R. Reid, S. Jentsch, R. Rothstein, L. Aragon & M. Lisby (2007) The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus. Nat Cell Biol, 9, 923-31. Tsutsui, T., S. Kumakura, Y. Tamura, T. W. Tsutsui, M. Sekiguchi, T. Higuchi & J. C. Barrett (2003) Immortal, telomerase-negative cell lines derived from a   207 Li-Fraumeni syndrome patient exhibit telomere length variability and chromosomal and minisatellite instabilities. Carcinogenesis, 24, 953-65. Ulaner, G. A., A. R. Hoffman, J. Otero, H. Y. Huang, Z. Zhao, M. Mazumdar, R. Gorlick, P. Meyers, J. H. Healey & M. Ladanyi (2004) Divergent patterns of telomere maintenance mechanisms among human sarcomas: sharply contrasting prevalence of the alternative lengthening of telomeres mechanism in Ewing's sarcomas and osteosarcomas. Genes Chromosomes Cancer, 41, 155-62. Ulaner, G. A., H. Y. Huang, J. Otero, Z. Zhao, L. Ben-Porat, J. M. Satagopan, R. Gorlick, P. Meyers, J. H. Healey, A. G. Huvos, A. R. Hoffman & M. Ladanyi (2003) Absence of a telomere maintenance mechanism as a favorable prognostic factor in patients with osteosarcoma. Cancer Res, 63, 1759-63. Ulrich, H. D. (2009) The SUMO system: an overview. Methods Mol Biol, 497, 3-16. Ulrich, H. D., S. Vogel & A. A. Davies (2005) SUMO keeps a check on recombination during DNA replication. Cell Cycle, 4, 1699-702. Varley, H., H. A. Pickett, J. L. Foxon, R. R. Reddel & N. J. Royle (2002) Molecular characterization of inter-telomere and intra-telomere mutations in human ALT cells. Nat Genet, 30, 301-5. Verdun, R. E. & J. Karlseder (2007) Replication and protection of telomeres. Nature, 447, 924-31. Vidal-Cardenas, S. L. & C. W. Greider (2009) Comparing effects of mTR and mTERT deletion on gene expression and DNA damage response: a critical examination of telomere length maintenance-independent roles of telomerase. Nucleic Acids Res. Wang, R. C., A. Smogorzewska & T. de Lange (2004) Homologous recombination generates T-loop-sized deletions at human telomeres. Cell, 119, 355-68. Watson, I. R. & M. S. Irwin (2006) Ubiquitin and ubiquitin-like modifications of the p53 family. Neoplasia, 8, 655-66. Watts, F. Z. (2006) Sumoylation of PCNA: Wrestling with recombination at stalled replication forks. DNA Repair (Amst), 5, 399-403. Weidtkamp-Peters, S., T. Lenser, D. Negorev, N. Gerstner, T. G. Hofmann, G. Schwanitz, C. Hoischen, G. Maul, P. Dittrich & P. Hemmerich (2008) Dynamics of component exchange at PML nuclear bodies. J Cell Sci, 121, 2731-43.   208 Wen, J., Y. S. Cong & S. Bacchetti (1998) Reconstitution of wild-type or mutant telomerase activity in telomerase-negative immortal human cells. Hum Mol Genet, 7, 1137-41. Westin, E. R., E. Chavez, K. M. Lee, F. A. Gourronc, S. Riley, P. M. Lansdorp, F. D. Goldman & A. J. Klingelhutz (2007) Telomere restoration and extension of proliferative lifespan in dyskeratosis congenita fibroblasts. Aging Cell, 6, 383-94. Wilson, V. G. & D. Rangasamy (2001) Intracellular targeting of proteins by sumoylation. Exp Cell Res, 271, 57-65. Wilson, V. G. & G. Rosas-Acosta (2005) Wrestling with SUMO in a new arena. Sci STKE, 2005, pe32. World Health Organization [Internet]. Available from: http://www.who.int/en/ Wu, G., X. Jiang, W. H. Lee & P. L. Chen (2003) Assembly of functional ALTassociated promyelocytic leukemia bodies requires Nijmegen Breakage Syndrome 1. Cancer Res, 63, 2589-95. Wu, G., W. H. Lee & P. L. Chen (2000) NBS1 and TRF1 colocalize at promyelocytic leukemia bodies during late S/G2 phases in immortalized telomerase-negative cells. Implication of NBS1 in alternative lengthening of telomeres. J Biol Chem, 275, 30618-22. Yang, S. H. & A. D. Sharrocks (2004) SUMO promotes HDAC-mediated transcriptional repression. Mol Cell, 13, 611-7. Yasumoto, S., C. Kunimura, K. Kikuchi, H. Tahara, H. Ohji, H. Yamamoto, T. Ide & T. Utakoji (1996) Telomerase activity in normal human epithelial cells. Oncogene, 13, 433-9. Yates, K. E., G. A. Korbel, M. Shtutman, I. B. Roninson & D. DiMaio (2008) Repression of the SUMO-specific protease Senp1 induces p53-dependent premature senescence in normal human fibroblasts. Aging Cell, 7, 609-21. Yeager, T. R., A. A. Neumann, A. Englezou, L. I. Huschtscha, J. R. Noble & R. R. Reddel (1999) Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body. Cancer Res, 59, 4175-9. Yeh, E. T. (2009) SUMOylation and De-SUMOylation: wrestling with life's processes. J Biol Chem, 284, 8223-7. Zeng, S., T. Xiang, T. K. Pandita, I. Gonzalez-Suarez, S. Gonzalo, C. C. Harris & Q. Yang (2009) Telomere recombination requires the MUS81 endonuclease. Nat Cell Biol, 11, 616-23.   209 Zhang, X., V. Mar, W. Zhou, L. Harrington & M. O. Robinson (1999) Telomere shortening and apoptosis in telomerase-inhibited human tumor cells. Genes Dev, 13, 2388-99. Zhong, Z. H., W. Q. Jiang, A. J. Cesare, A. A. Neumann, R. Wadhwa & R. R. Reddel (2007) Disruption of telomere maintenance by depletion of the MRE11/RAD50/NBS1 complex in cells that use alternative lengthening of telomeres. J Biol Chem, 282, 29314-22. Zhou, L., D. Zheng, M. Wang & Y. S. Cong (2009) Telomerase reverse transcriptase activates the expression of vascular endothelial growth factor independent of telomerase activity. Biochem Biophys Res Commun, 386, 739-43. Zhu, X. D., B. Kuster, M. Mann, J. H. Petrini & T. de Lange (2000) Cell-cycleregulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres. Nat Genet, 25, 347-52.   210 [...]... groups, to proteins Post-translational modifications may also involve structural changes of a protein, including the formation of disulphide bridges between cysteine amino acids and proteolytic cleavage (of a peptide bond) Post-translational modifications of a protein may also involve the attachment of other proteins or peptides, such as ubiquitin and the small ubiquitin- like modifier (SUMO) Subsets of a... suppressor in APL and in other cancers; PML has also been implicated in the repression of gene expression and the promotion of both intrinsic and extrinsic apoptotic pathway (Jensen, Shiels and Freemont 2001, Ruggero, Wang and Pandolfi 2000) There are three cysteine-rich zinc-binding domains in PML, a RING-finger domain and two B-boxes (B1 and B2) Together with a predicted α-helical coiled-coil domain, these... localization signal in exon 6 is not present in all PML isoforms and this results in both nuclear and cytoplasmic isoforms of PML Thus while the RBCC motif is conserved in all PML isoforms, the isoforms differ in their C-terminal regions   9 1.2.1 RING finger motif The RING finger motif is a cysteine-rich zinc binding domain The conserved RING structural elements consists of two zinc atoms bound via... of proteins may be modified post-translationally according to the cellular conditions and microenvironment as part of the cellular response and regulatory processes 1.1.1 Small Ubiquitin- like Modifier (SUMO) The Small Ubiquitin- like Modifier (SUMO) protein is a 10-11 KDa polypeptide that has a strong structural homology to ubiquitin (Melchior 2000, Ulrich 2009, Johnson 2004) However, SUMO has distinct... (protein inhibitor of activated STAT) family is the most prominent among the SP-RING proteins In general, the E3 ligases appear to play a part in conferring substrate selectivity to the SUMO conjugation process 1.1.2.3.1 PIAS PIAS (protein inhibitor of activated STAT) proteins were named after their ability to interact and inhibit STAT proteins (Palvimo 2007) In mammals, four genes encode the PIAS proteins; ... family members and this suggests that it is an important determinant of the overall motif and its function (Reymond et al 2001) The B-boxes, B1 and B2, are two distinct cysteine-rich motifs adjacent to the RING domain Both bind zinc but differed in terms of the number and spacing of conserved Cys and His ligands (Borden et al 1996) While substitution of the conserved zinc ligands in B1 and/ or B2 disrupted... arrangement of Cys and His ligands Mutations of these ligands in PML disrupted its nuclear body formation and led to loss of its growth suppression and apoptosis abilities (Jensen et al 2001) The requirement of an intact RING finger for PML nuclear body formation could be due to specific proteinprotein interactions that are mediated by the RING motif In addition, the RING finger motif in PML specifically interacts... Besides being involved in the cleavage of the SUMO precursor to the mature form, SENPs are also involved in the deconjugation of SUMO from target proteins and in the processing of SUMO polymers The C-terminal hydrolase activity of SENP converts the SUMO precursor to its mature form The removal of SUMO from target proteins is accomplished through the isopeptidase activity of SENP and this occurs in a single... xxi TIN2 – TRF1-Interacting Partner TMM – Telomere Maintenance Mechanism TPG – Total Product Generated TPP1 – POT1 -and- TIN2 binding protein TRAP – Telomeric Repeat Amplification Protocol TRF – Terminal Restriction Fragment TRF1 – Telomeric Repeat Binding Factor 1 TRF2 – Telomeric Repeat Binding Factor 2 UV – Ultra-violet WRN – Werner Syndrome protein   xxii SUMMARY The Alternative Lengthening of Telomeres. .. fidelity of chromosome transmission (Takahashi et al 2006) and the SUMOylation of RAD52 to the regulation of recombination events at the ribosomal gene locus (Torres-Rosell et al 2007)   8 1.2 Promyelocytic Leukemia (PML) The promyelocytic leukemia (PML) protein is a growth and tumor suppressor that is inactivated in acute promyelocytic leukemia (APL) through the fusion of the PML gene with the retinoic . THE ROLE OF PROMYELOCYTIC LEUKEMIA (PML) AND SMALL UBIQUITIN LIKE MODIFIER (SUMO) PROTEINS IN THE ALTERNATIVE LENGTHENING OF TELOMERES YONG WEI YAN JACKLYN. MB, and Hande MP. The coiled-coil domain of the promyelocytic leukemia protein is required for the formation of Alternative Lengthening of Telomeres- associated nuclear bodies. Telomeres and. Telomerase Meeting, Cold Spring Harbor Laboratories Meeting. April-May 2009. Long Island, New York, USA. Yong JWY, Lee MB, and Hande MP. The role of the promyelocytic leukemia protein in the Alternative