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UBC Theses and Dissertations

Combination chemotherapy with telomerase inhibitors and genotoxic compounds against breast and colorectal… Tamakawa, Raina Ayako 2010

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COMBINATION CHEMOTHERAPY WITH TELOMERASE INHIBITORS AND GENOTOXIC COMPOUNDS AGAINST BREAST AND COLORECTAL CANCERS  by Raina Ayako Tamakawa B.Sc., Pacific University, 2006  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in  THE FACULTY OF GRADUATE STUDIES (Pharmaceutical Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  October 2010 Raina Ayako Tamakawa, 2010  ABSTRACT Telomerase is the specialized reverse transcriptase responsible for the de novo synthesis of telomeric repeats at chromosome ends.  Telomerase plays  important roles in tumor development and is responsible for the indefinite growth phenotype in cancer. Telomerase over-expression is found in more than 85% of human tumors surveyed. In contrast, normal somatic cells have low or undetectable telomerase expression, making the enzyme an appealing target for the development of anticancer therapy. However, there is a significant time lag between the start of telomerase inhibition therapy and growth inhibition effects, restricting the use of telomerase inhibitors in clinical applications. In addition to telomere maintenance, telomerase participates in cellular recovery processes following genotoxic insults. Genetic suppression of the human telomerase catalytic subunit, telomerase reverse transcriptase (hTERT), diminishes cellular DNA repair capability following double-stranded DNA damage induction, suggesting that the enzyme is involved in the regulation of DNA repair response. I hypothesize that transient telomerase inhibition at the time of genotoxic stimulus will increase cytotoxicity in tumor cells.  My studies showed that short-term  telomerase inhibition potentiates the cytotoxic effects of DNA damage inducing agents in MCF-7 breast cancer and HT29 colorectal cancer cells, in a cell-cycle dependent and DNA damage mechanism-specific manner. Additionally, I found that the Ataxia Telangiectasia Mutated kinase may interact with telomerase dependent DNA damage repair pathways to further augment cancer cell death.  This study  provides new mechanistic insight into the roles of telomerase function in cancer cell ii  survival and impetus to design new telomerase-based clinical therapies against breast and colorectal cancers.  iii  TABLE OF CONTENTS ABSTRACT.............................................................................................................................. ii TABLE OF CONTENTS......................................................................................................... iv LIST OF TABLES .................................................................................................................. vii LIST OF FIGURES .............................................................................................................. viii LIST OF ABBREVIATIONS................................................................................................... x ACKNOWLEDGEMENTS ...................................................................................................xiii INTRODUCTION .................................................................................................................... 1 Telomeres .......................................................................................................................................1 Telomere structure............................................................................................................................................... 1 Telomere function ................................................................................................................................................. 6 Telomerase .....................................................................................................................................8 Telomerase structure .......................................................................................................................................... 8 TER expression and domain structure.................................................................................................... 11 H/ACAs .................................................................................................................................................................... 12 Staufen and L22 ................................................................................................................................................. 14 SmB/SmD3 .......................................................................................................................................................... 15  TERT synthesis/localization......................................................................................................................... 15 Hsp90 and p23 ................................................................................................................................................... 19 14-3-3 .................................................................................................................................................................... 19 Nucleolin .............................................................................................................................................................. 20 PINX1..................................................................................................................................................................... 20  TERT post-translational modifications ................................................................................................... 21 TER-TERT assembly ......................................................................................................................................... 22  iv  Pontin and reptin .............................................................................................................................................. 22 TCAB ...................................................................................................................................................................... 23  Targeting telomerase holoenzyme to telomere ................................................................................. 23 hEST1A.................................................................................................................................................................. 25 hnRNPs ................................................................................................................................................................. 25  Telomerase catalytic cycle....................................................................................................... 26 Alternative lengthening of telomeres................................................................................... 29 Telomerase and cancer ............................................................................................................ 29 NON-telomere maintenance roles of telomerase .............................................................. 31 Telomerase inhibition as a clinical therapy ....................................................................... 34 Small molecule inhibitor of telomerase .................................................................................................. 35 Other clinical strategies targeting telomeres or telomerase ....................................................... 37 Time lag between the start of therapy to the observation of cytotoxicity: the achilles heel of telomerase-based chemotherapies ........................................................................................... 39 Anti-cancer chemotherapeutic agents ................................................................................. 40 Current DNA damaging chemotherapeutic agents ........................................................................... 40 Telomerase inhibition in anti-cancer chemotherapies .................................................................. 42 GRN163L: specific inhibitor of telomerase ........................................................................................... 45  HYPOTHESIS........................................................................................................................ 48 Goals of present study............................................................................................................... 50 Specific aim 1 ........................................................................................................................................................ 50 Specific aim 2 ........................................................................................................................................................ 50 Specific aim 3 ........................................................................................................................................................ 50  METHODS ............................................................................................................................. 51 RESULTS ............................................................................................................................... 58  v  Testing GRN163L for effective telomerase inhibition ..................................................... 58 Telomerase inhibition increases the cytotoxicity of cell cycle specific genotoxic compounds................................................................................................................................... 60 Cytotoxicity of etoposide is further increased with telomerase inhibition and parallel ATM inhibition. ........................................................................................................... 80  DISCUSSION ......................................................................................................................... 92 Insights into the mechanism of telomerase’s function in DNA repair ......................... 92 The effects of telomerase inhibition effects on chromatin structure .......................... 94 The relationship between ATM kinase and telomerase in homologous recombination repair during S/G2 phase of the cell cycle ............................................. 95 The future of telomerase-based clinical therapy in human cancer ............................. 97  REFERENCES..................................................................................................................... 100 APPENDIX ......................................................................................................................... 120 Methods ...................................................................................................................................... 122 Results ......................................................................................................................................... 124 Discussion .................................................................................................................................. 127  APPENDIX REFERENCES................................................................................................ 129  vi  LIST OF TABLES Table 1: Cell line properties…………………………………………………………………………….....57  vii  LIST OF FIGURES Figure 1: Human telomeres………………………………………………………………………………….3 Figure 2: Telomere shortening and cancer initiation………….…………………….……………9 Figure 3: Human TER and TERT organization....…………………...……………………………..13 Figure 4: Telomere repeat synthesis…………………………………………………………………..27 Figure 5: Hypothesis………………………………………………………………………………………....48 Figure 6: Effect of GRN163L on telomerase activity in MCF-7, HT29 and LS180 cells…………………………………………………………………..…………………….60 Figure 7: Cytotoxicity of etoposide in MCF-7 cells is increased by the addition of telomerase inhibitor GRN163L……………………………………..……62 Figure 8: Cytotoxicity of irinotecan in MCF-7 cells is increased by the addition of telomerase inhibitor GRN163L……………..……………………………64 Figure 9: Lack of potentiation of cytotoxicity in MCF-7 cells between telomerase inhibition by GRN163L and non cell cycle specific bleomycin.……………………………………………………………………………66 Figure 10: Lack of potentiation of cytotoxicity in MCF-7 cells between telomerase inhibition by GRN163L and non cell cycle specific oxaliplatin.……………………………………………………………………………68 Figure 11: Cytotoxicity of etoposide in MCF-7 cells is increased by the addition of telomerase inhibitor GRN163L……………………………………..……71 Figure 12: Cytotoxicity of irinotecan in MCF-7 cells is increased by the addition of telomerase inhibitor GRN163L……………..……………………………73 Figure 13: Lack of potentiation of cytotoxicity in MCF-7 cells between telomerase inhibition by GRN163L and non cell cycle specific bleomycin.……………………………………………………………………………75 Figure 14: Lack of potentiation of cytotoxicity in MCF-7 cells between telomerase inhibition by GRN163L and non cell cycle specific oxaliplatin.……………………………………………………………………………77 Figure 15: Etoposide cytotoxicity was further augmented by parallel viii  ATM inhibition in MCF-7 cells…………..……….………………………………….……81 Figure 16: ATM inhibition did not increase the cytotoxicity in HT29 cells treated with etoposide and 10 M GRN163………………………………………...83 Figure 17: Etoposide cytotoxicity was further augmented by parallel ATM inhibition in LS180 cells…………..……….…………………………….…………86 Figure 18: Immunostaining for MCF-7 and HT29 cells treated with etoposide or in combination with 10 M Ku55933.………………..……………88 Figure 19: p53 western blot analysis for MCF-7 and LS180 cells treated with etoposide or in combination with 10 M Ku55933.………….90 Figure A1: Schematic of the order of addition of GRN163L in MCF-7 cells breast cancer cells…………………………….………………………………..………….122 Figure A2: Inhibition of telomerase 24 hours prior to the induction DNA damage resulted in an optimal potentiation of etoposide toxicity…………………………………………………………………………….124  ix  LIST OF ABBREVIATIONS 3’ UTR: 3’ untranslated region Akt/PKB: protein kinase B ALT: Alternative lengthening of telomeres ATM: Ataxia telangiectasia mutated AZT: 3’-azido-2’, 3’-dideoxythymidine BIO: biogenesis bp: base pair CAB box: cajal body specific localization signal CBs: cajal bodies CFU: colony forming unit assay CR4: conserved region 4 CR5: conserved region 5 d-loop: displacement loop DAT: dissociates activities of telomerase ddG: dideoxyguanosine DMEM: Dulbecco’s modified Eagle’s medium DN: dominant negative dNTPs: deoxynucleotide triphosphates HLA: human leukocyte antigen hTERT: human telomerase reverse transcriptase IFD: insertion in fingers domain IR: ionizing radiation x  LD50: lethal dose, 50% MHC: major histone complex MT-hTER: mutant-template human telomerase RNA NES: nuclear export signal NF-Y: nuclear factor Y nt: nucleotide Pol II: polymerase II POT1: protection of telomeres 1 Rap1: repressor/activator protein 1 Rb: retinoblastoma RID1: RNA interacting domain 1 RID1: RNA interacting domain 2 RNP: ribonucleoprotein RT: reverse transcriptase t-loop: telomeric loop TCAB1: telomerase cajal body protein 1 TEN: TERT essential N-terminal TER: telomerase RNA TERT: telomerase reverse transcriptase TIN2: TRF1 and TRF2 interacting protein 2 TPP1: formally known as PTOP, PIP1 or TINT1 TRAP: telomere repeat amplification protocol TRBD: telomerase RNA binding domain  xi  TRF1: telomere repeat binding factor 1 TRF2: telomere repeat binding factor 2  xii  ACKNOWLEDGEMENTS This thesis would not have been possible without the help and guidance of several individuals throughout the preparation and completion of this project. First and foremost, I would like to sincerely thank my advisor Dr. Judy Wong for her patient guidance and continuous support through the course of this project. My gratitude also goes out to the rest of the Wong lab, especially Dr. Helen Fleisig and Xi Lei Zeng, for their assistance and support. I would also like to thank my committee members, Dr. Kish Wasan, Dr. Wayne Riggs, Dr. David Grierson, Dr. Marc Levine and Dr. Mary Ensom for their valuable input and guidance. Many thanks goes to the Geron Corporation for the generous supply of GRN163L, as well as the assistance of Robert Tressler. I would also like to thank the Center for Drug Development and Research for use of their TyphoonTM Imager, as well as the BioImaging Facility for use of their Zeiss Pascal Excite laser scanning confocal microscope. Funding for this project was provided by the Canadian Cancer Society Research Institute and Michael Smith Foundation for Health Research. Lastly, I would like to thank my family and friends. Without your continuous encouragement and support, I would have not made it this far. I truly appreciate each and every one of you.  xiii  INTRODUCTION Telomeres Telomere structure At the ends of most eukaryotic chromosomes are highly conserved, tandem DNA repeats. These highly repetitive sequences are associated with their specific binding proteins, and together, these chromosome end structures are known as telomeres. Telomeres cap chromosome ends and protect them from non-specific nuclease digestion, as well as prevent them from being recognized as doublestranded DNA breaks. In the absence of telomeres, erroneous DNA repair will lead to chromosomal end-to-end fusions and genetic recombination (de Lange 2002). In all vertebrate chromosomes, telomeres are made up of the G-rich hexanucleotide sequence (TTAGGG)n (Moyzis, Buckingham et al. 1988). The length of these repeats varies between species, ranging from ~300-600bp in yeasts (Chan and Tye 1983), to ~150kb in mice (Kipling and Cooke 1990). Human telomeres measure ~5-15 kb in length (de Lange, Shiue et al. 1990; Harley, Futcher et al. 1990). These repeats run 5’-3’, terminating in a single-stranded 3’ overhang of the G-rich strand (Makarov, Hirose et al. 1997; McElligott and Wellinger 1997). The length of this overhang is also species specific, measuring ~50-100 nucleotides in length in mouse and human telomeres (Greider 1999). Mammalian telomeres were previously thought to be linear.  However,  electron microscopy analysis of psoralene cross-linked telomeric DNA from human and mouse were visualized to end as large duplex loops (Griffith, Comeau et al. 1999). Double-stranded DNA fold back onto themselves to form a lariat structure 1  termed the telomeric loop.  This allows for the G-rich 3’ overhang to invade the  duplex section of telomeric repeats, forcing the formation of a single stranded DNA displacement loop (Klobutcher, Swanton et al. 1981) (Fig 1a). The resulting higher order chromatin structure is distinct from damaged DNA and thus serves to differentiate the normal chromosomal terminus, preventing them from being recognized as double strand breaks. This differentiation mechanism is crucial in preventing the initiation of DNA damage checkpoint responses (Greider 1999). A 6-member protein complex, termed shelterin, associates with the telomere ends in a sequence specific manner. The shelterin complex facilitates the formation of the telomeric loop to protect chromosome ends from DNA damage surveillance mechanism, as well as functionally maintain telomere length. The shelterin complex is comprised of 6 distinct proteins: telomere repeat binding factors 1 and 2 (TRF1 and TRF2), protection of telomeres 1 (POT1), TRF1- and TRF2-interacting nuclear protein 2 (TIN2), repressor/activator protein 1 (Rap1), and TPP1 (formally known as PTOP, PIP1, or TINT1) (de Lange 2005; Palm and de Lange 2008). TRF1 and 2 are sequence specific telomeric DNA binding proteins that recruit the other four proteins to the telomeres (Palm and de Lange 2008). Both TRF1 and TRF2 contain a C-terminal SANT/Myb-type DNA binding domain that binds to the 5’-TTAGGG-3’ sequence in duplex DNA, making the entire shelterin  2  a)  b)  Figure 1. Human telomeres. a) Telomere repeats at chromosome ends fold back to form a lariat structure (t-loop). The 3’ telomeric DNA overhang invades the double stranded DNA region of telomeric repeats to form a displacement-loop (d-loop). b) Shelterin protein complex aids in t-loop formation and stabilization: TRF1 and TRF2 interact with double stranded telomeric repeats, recruiting the other four shelterin proteins, POT1, TIN2, TPP1, and Rap1, to the telomere end. TIN2 links TRF1 to TRF2, contributing to the stabilization of these proteins on the telomere. POT1, which has strong specificity for single-stranded telomeric repeats and its heterodimeric partner TPP1, associate with TRF1 and TRF2 through a bridge formed by TIN2. Rap1 is recruited by TRF2, forming a TRF2-Rap complex. Figure courtesy of Judy Wong and Helen Fleisig.  3  complex highly specific for telomeric repeats (Bianchi, Stansel et al. 1999; Court, Chapman et al. 2005) (Fig 1b). TRF1 is a homodimeric protein that aids in telomeric loop formation and stabilization (Shay 1999). Its binding to arrays of telomeric repeats induced shallow bends and resulted in the formation of DNA loops, demonstrating the protein’s architectural role on telomeres (Bianchi, Stansel et al. 1999). This protein has also been shown to affect telomere length. Over-expression of TRF1 resulted in telomere shortening while expression of a dominant negative TRF1 mutant, lacking the Mybtype domain, caused telomere lengthening which indicate a negative correlation between TRF1 function and telomere length (van Steensel and de Lange 1997; Smogorzewska, van Steensel et al. 2000).  Accumulations of TRF1 and TRF2 at  telomere ends were shown to correlate with telomere length (van Steensel and de Lange 1997; Smogorzewska, van Steensel et al. 2000). This led to a protein counting theory of telomere length regulation, which proposed that a feedback mechanism mediated by protein interactions with TRF1 is responsible for steady-state telomere length maintenance (van Steensel and de Lange 1997). Like TRF1, TRF2 also binds to double-stranded telomeric DNA as a homodimer (Broccoli, Smogorzewska et al. 1997) and plays a role in telomeric loop assembly. In contrast to TRF1, TRF2 is believed to bind near the loop-tail junction where it stabilizes the G-rich single-stranded telomeric overhang at the displacement loop by facilitating strand invasion and prevents the single-strand sequence from being recognized as a DNA break (Griffith, Comeau et al. 1999; Karlseder, Broccoli et al. 1999). In corroboration to this model, electron microscopy 4  of a model telomere DNA, containing ~2kb of telomeric repeats at the end of a linearized DNA plasmid and terminating in a 3’ single-stranded overhang, revealed the location specific binding of TRF2 at the telomeric loop junction (Stansel, de Lange et al. 2001). Like TRF1, TRF2 also serves as a negative regulator of telomere length.  TRF2 over-expression resulted in shortened telomeres and induced  senescence in telomerase negative cells (Karlseder, Smogorzewska et al. 2002) POT1 is the most highly conserved component of shelterin and has a strong specificity for single-stranded 5’-(T)TAGGGTTAG-3’ sites (Lei, Podell et al. 2004; Loayza, Parsons et al. 2004). This protein’s accumulation at the chromosome ends is believed to regulate telomerase activity by relaying telomere length information from the double stranded region of the telomeric loop to the singlestranded region through its interaction with TRF1 (Loayza and De Lange 2003). Studies have also demonstrated POT1 to play a positive role in telomere length maintenance as ectopic expression of POT1 resulted in an increase in telomeric DNA (Colgin, Baran et al. 2003; Armbruster, Linardic et al. 2004). TPP1, the heterodimeric partner of POT1 (Wang, Podell et al. 2007; Xin, Liu et al. 2007), enhances POT1 affinity for single-stranded telomeric DNA (Xin, Liu et al. 2007). Majority of the POT1-TPP1 complexes are associated with TRF1 and TRF2 through a bridge formed by TIN2, which function to stabilize the interactions between those proteins (O'Connor, Safari et al. 2006). In addition to the protection of telomere ends, the TPP1-POT1 complex also serves as a regulator of telomere length maintenance. Through its oligonucleotide and oligosaccharide-binding fold, TPP1 has been suggested to regulate telomerase activity and the enzyme’s access to  5  single-stranded telomeric DNA, both negatively and positively in a context dependent manner (Xin, Liu et al. 2007). TIN2 co-localizes with TRF1 on metaphase chromosomes (Kim, Kaminker et al. 1999). TIN2 forms bridges that join POT1 to TRF1 and TRF2 and also TRF1 to TRF2, contributing to the stabilization of these proteins on the telomeres (Liu, Safari et al. 2004; Ye and de Lange 2004). The binding of TRF1 to TIN2 leads to the compaction of telomeric DNA and telomeric loop stabilization. Both events limit the accessibility of telomerase to telomere ends and thereby functioning as a negative regulator of telomere length (Kim, Kaminker et al. 1999). Rap1 is recruited to the telomere by protein interaction with TRF2, forming a TRF2-Rap1 complex (Li, Oestreich et al. 2000).  Rap1 affects telomere length  homeostasis through its interactions with telomere length regulator proteins Rif1 and Rif2. Like the other shelterin proteins, Rap1 is a negative regulator of telomere length. Over-expression of Rap1 led to telomere shortening while expression of dominant negative mutants resulted in the gain of telomere length (Li and de Lange 2003).  Telomere function In addition to structurally protecting the ends of chromosomes, telomeres also serve as a solution to the end-replication problem, where DNA polymerases fail to completely copy chromosomes to the very end. The end replication problem leads to a loss of approximately 50-100bp of DNA terminal sequence every cell cycle. The placement of telomeres at the ends of chromosome allows them to buffer  6  gene coding sequence from being eroded (Olovnikov 1973; Harley, Futcher et al. 1990). Instead, 50-100bp of telomeric DNA is lost after a single round of cell replication. Telomeric DNA loss is cumulative, resulting in the continual depletion of these tandem DNA repeats, as cell division continues.  With continual  proliferation, telomeres will eventually reach a critical short length. At this point, genome surveillance mechanisms will trigger replicative senescence, an irreversible cellular growth arrest state where cells can no longer divide into daughter progeny but remain metabolically active (Wright and Shay 1992; de Lange 2002; Smogorzewska and de Lange 2002; Takai, Smogorzewska et al. 2003). This short telomere checkpoint serves as a “mitotic clock”, counting down the number of cell divisions in each cell lineage. Incidentally, this process can be viewed as a tumor suppressive mechanism: by limiting the number of cell divisions that can occur in a particular cell lineage, one can reduce the accumulation of deleterious mutations that precede cellular transformation (Fleisig and Wong 2007). In rare cases, some somatic cells are able to bypass this short telomere checkpoint by the inactivation of the genome surveillance mechanisms mediated by the tumor suppressor genes p53 and the retinoblastoma protein (Rb). Further cell divisions in p53/Rb-inactivated cells continue to deplete telomeric DNA, leading to the loss of telomeric protein binding and the disruption of the telomeric loop structure (Rajaraman, Choi et al. 2007). Uncapped telomeres are recognized by cellular repair mechanisms as damaged DNA, resulting in cells attempting to repair this damage. Erroneous repair leads to chromosome end fusions and rampant  7  genomic instability. When this happens, a second checkpoint termed ‘crisis’ (Wright and Shay 1992) will be activated and cells are triggered to undergo apoptosis. However, a rare cell (~1 in 10 million) can reactivate a specialized cellular reverse transcriptase, termed telomerase, which is capable of adding telomeric repeats to the chromosome end (Wright and Shay 1992). Telomerase expression allows cells to replace loss telomeric repeats and prevent further genomic instability. In these cases of forced reactivation of telomerase enzyme expression, constitutive telomerase activity confers the unlimited proliferative capacity required for the formation of a malignant tumor cell (Fig 2).  Telomerase Telomerase structure The human telomere terminal transferase enzyme, more commonly referred to as telomerase, is a ribonucleoprotein (RNP) responsible for the de novo synthesis of telomere repeats. This unique reverse transcriptase extends chromosome ends by utilizing an integral RNA subunit as template, to synthesize the TTAGGG telomeric DNA repeats. The core components of this enzyme complex, which were first characterized in Tetrahymena thermophila (Greider and Blackburn 1985), consist of the telomerase reverse transcriptase catalytic subunit (TERT) and the telomerase RNA (TER), which contains the template sequence for telomere synthesis. In the human enzyme, chaperone proteins such as the H/ACA proteins  8  Figure 2. Telomere shortening and cancer initiation. With each cell division, approximately 50-100bp of telomeric DNA is lost from chromosome ends. With continual proliferation, telomeres will eventually reach a critical short length and are triggered to enter replicative senescence. Inactivation of genome surveillance mechanisms mediated by tumor suppressor genes p53 and Rb allow continual cell divisions, further depleting telomeric DNA leading to rampant genomic instability and the induction of apoptosis. A rare cell (~1 in 10 million) can be forced to reactivate telomerase, allowing the cell to replace lost telomeric repeats, prevent further genomic instability and confer the unlimited proliferative capacity required for the formation of a malignant tumor.  9  dyskerin, Nop10 and Nhp2 are also found to associate with the core enzyme complex. Other proteins transiently associate with the core enzyme complex, and they play important roles in the regulation of the catalytic activity, enzyme stability, the cellular localization and intracellular trafficking of the enzyme (Mitchell, Cheng et al. 1999; Dragon, Pogacic et al. 2000; Wang and Meier 2004). TER and TERT were identified as the catalytic core of this complex by virtue of their ability to form a complex and elongate telomeres in vitro, in the absence of other protein factors (Weinrish et al., 1997). However, in vivo, telomerase employs an intricate biogenesis pathway involving specific factors for enzyme assembly, trafficking and localization of the holoenzyme complex.  TER transcription is  ubiquitous in all human cells. The stability of TER is dependent on biogenesis proteins factors Shq1 and NAF-1 mediated complex formation with the chaperone H/ACA proteins (dyskerin, Nhp2 and Nop10). TER association with the H/ACA complex results in the formation of a stable but inactive telomerase RNP. Assembly of this inactive telomerase RNP with TERT is required for catalytic activity (Harrington 2003). Telomerase enzyme assembly is cell cycle and subcellular localization dependent (Wong, Kusdra et al. 2002). Numerous biogenesis factors, including staufen, L22, SmB/SmD3, PinX1, 14-3-3, nucleolin, pontin, reptin, Hsp90, p23, and telomerase Cajal body protein 1 (TCAB1) have all been demonstrated to play important roles in TER/TERT localization and enzyme assembly.  Finally,  following the formation of a functional telomerase enzyme, additional trafficking  10  factors, such as TCAB, hEST1A, and hnRNPs, are required for proper transport of the active enzyme to chromosome ends.  TER expression and domain structure TER is a non-coding RNA that serves as a template for TERT-dependent addition of telomeric repeats. Ubiquitously expressed, human TER is synthesized by RNA polymerase II (pol II) and processed into a mature 451-nucleotide (nt) product with a 5’ trimethyl cap and lacks a polyadenosine tail at its 3’ end (Feng, Funk et al. 1995; Zaug, Linger et al. 1996). TER contains a 341-nt pol II-type promoter region upstream of the transcription start site (Zhao, Hoare et al. 1998). Nuclear factor-Y (NF-Y), Sp1 and Sp3 are essential regulators of TER promoter function. NF-Y is composed of three subunits, NF-Y A, B, and C, and is a heterotrimeric transcriptional activator that binds to the CAAT region of the TER promoter (Maity and de Crombrugghe 1998). Inhibition of NF-Y activity has been shown to diminish TER promoter activity (Zhao, Glasspool et al. 2000). Sp1 and 3 are members of a family of mammalian Kruppel-like transcription factors that bind to GC-rich sequences and have been shown to be involved in cell cycle regulation, chromatin modeling, and maintenance of methylation-free islands (Lania, Majello et al. 1997). Both have been demonstrated to bind to the Sp binding regions of the TER promoter with Sp1 activating and Sp3 repressing gene expression (Zhao, Glasspool et al. 2000). Primary and secondary structure elements of TER contain many motifs that are essential for telomerase activity as well as cellular accumulation of mature TER (Fig 3a). The 11-nt telomeric repeat template sequence is contained within the 5’  11  portion of TER in the pseudoknot domain (nt 1-209). Deletional mutant containing only the pseudoknot domain together with the Conserved Region 4 (CR4) and CR5 domain (nt 241-330), can reconstitute telomerase activity in vitro, indicating the importance of these domains to the enzyme’s catalytic activity (Tesmer, Ford et al. 1999). The H/ACA motif (nt 275-451) is essential for TER association with the chaperone H/ACA protein complex.  Association with the H/ACA proteins is  essential for cellular accumulation and 3’end processing of TER. The 3’terminal hairpin domain (CR7; nt 408-422) contains a Cajal body specific localization signal (CAB box), necessary for the accumulation of TER to the Cajal bodies (CBs), as well as a biogenesis box (BIO box), which is necessary for in vivo accumulation of TER (Chen, Blasco et al. 2000; Fu and Collins 2003).  H/ACAs The H/ACA proteins dyskerin, Nop10 and Nhp2 form the core trimer that acts as chaperone to promote the in vivo accumulation of TER. The binding of these proteins with TER immediately following transcription is essential for its cellular accumulation, processing and stability (Dez, Henras et al. 2001). In contrast to other protein factors described in the later sections, H/ACA proteins associate with TER throughout the enzyme’s lifespan and are considered stable components of the telomerase holoenzyme, as illustrated by affinity purification experiments (Cohen, Graham et al. 2007).  12  a)  b)  Figure 3. Human TER and TERT organization. a) Secondary structure of hTER. The 451-nt RNA includes the 11-nt template region in addition to conserved regions: pseudoknot domain (nt 1-209), CR4/CR5 (nt 214-330), CR7 3’terminal hairpin domain, which contains the CAB box and BIO box, and H/ACA domain (275-441). b) Functional organization of hTERT protein. The reverse transcriptase (RT) domain is flanked by an N-terminal domain which is subdivided into an RNA binding domain (TRBD/RID2) and a TERT essential N-terminal (TEN/RID1) domain. The seven universally conserved RT motifs are illustrated as purple boxes. Figure courtesy of Judy Wong and Helen Fleisig.  13  Following TER transcription, the chaperone H/ACA protein complex is loaded onto TER by the biogenesis protein factors SHQ1 and NAF1. SHQ1 is believed to function as a chaperone protein, binding to and stabilizing newly synthesized dyskerin until its association with NOP10 and NHP2 (Grozdanov, Roy et al. 2009). On the other hand, NAF1 first associates with TER, and mediates the loading of the trimer H/ACA complex (Wang and Meier 2004). This NAF1 containing complex is then shuttled to the nucleolus, where NAF1 is replaced by GAR1 resulting in the formation of mature, but inactive telomerase ribonucleoprotein (Darzacq, Kittur et al. 2006). H/ACA proteins bind to the TER H/ACA motif (nt 275-441) (Mitchell, Cheng et al. 1999). Mutations in the H/ACA motif in TER, as well as in the members of the H/ACA core trimer complex (dyskerin, Nhp2 and Nop10), are associated with genetic diseases with the common etiology of telomerase deficiencies and overlapping clinical presentations of pre-mature tissue aging phenotypes (Heiss, Knight et al. 1998; Mitchell, Cheng et al. 1999; Vulliamy, Marrone et al. 2001; Collins and Mitchell 2002; Wong and Collins 2003)  Staufen and L22 RNA binding proteins, staufen and L22, have been shown to independently associate with hTER in vivo and are involved in hTER processing, localization and telomerase assembly (Le, Sternglanz et al. 2000). Staufen shuttles between the cytoplasm and nucleus (Miki, Takano et al. 2005) and both proteins have been shown to localize to the nucleoli (Toczyski, Matera et al. 1994; Le, Sternglanz et al.  14  2000), where hTER is also shown to accumulate (Mitchell, Cheng et al. 1999; Narayanan, Lukowiak et al. 1999; Speckmann, Narayanan et al. 1999). Because TER processing and telomerase enzyme assembly both take place in the nucleolus (Pederson 1998; Olson, Dundr et al. 2000), it is conceivable that Staufen and L22 are involved in hTER processing and telomerase assembly.  SmB/SmD3 Sm-fold proteins SmB and SmD3 have been shown to associate with TER and are involved in its subcellular localization to Cajal bodies. SmB and SmD3 both interact with the CAB box sequence of TER, located in the CR7 domain, through an extended C-terminal tail modified with symmetric dimethyl-arginine. Deletion of this modified C-terminal sequence disrupted their association with TER (Fu and Collins 2007). However, it is not known whether this association is mediated through direct interactions between Sm proteins and TER or through the interactions with a tether protein. Mutations in TER’s CAB box resulted in a significant decrease in SmB and SmD3 association and a loss of CB localization (Goldberg, Sargent et al. 2004; Jady, Bertrand et al. 2004; Fu and Collins 2006). However, these mutations are not reported to affect telomere length maintenance in cell culture models.  TERT synthesis/localization Catalytic activation of the telomerase complex requires the transcriptional activation of hTERT.  The hTERT gene, located on chromosome 5p15.33, is  15  comprised of 16 exons and encompasses more than 37kb (Cong, Wen et al. 1999; Wick, Zubov et al. 1999). The GC-rich promoter region is located 1100bp upstream from the ATG start codon (Horikawa, Cable et al. 1999; Wick, Zubov et al. 1999). This region lacks both TATA and CAAT boxes (Cong, Wen et al. 1999) and was found hypermethylated in somatic cells, correlated with its transcriptional inactive state. The TERT promoter contains numerous c-myc, as well as other oncogenic transcription factor, such as c-Jun and c-fos binding sites, which have been demonstrated to mediate hTERT transcriptional activation in transformed cells (Wu, Grandori et al. 1999). Transcription of the TERT locus produces a full length TERT-mRNA as well as a variety of alternative spliced forms. TERT alternative splicing is believed to regulate the levels of functional telomerase, in a development stage specific manner (Yi, White et al. 2000). Following protein translation of the full length 125 kDa polypeptide (Meyerson, Counter et al. 1997; Yang, Chang et al. 1999), TERT associates with chaperones Hsp90 and p23, and is transported to the nucleus via its nuclear localization signal, where it is assembled with the TERH/ACA complex to form the fully functional telomerase enzyme (Holt, Aisner et al. 1999). TERT contains a central reverse transcriptase (RT) domain that is flanked by a N-terminal region and a C-terminal domain. The TERT N-terminal region is further subdivided into two domains: an RNA binding domain (TRBD) and a TERT essential N-terminal (TEN) domain. These two N-terminal regions are separated by a large non-conserved linker (Kelleher, Teixeira et al. 2002) (Fig 3b).  16  The RT domain contains the seven universally conserved RT motifs (1, 2, A, B’, C, D, and E) (Xiong and Eickbush 1990). Compared to viral RTs, telomerase RT domain contains substantially higher number of amino acid residues intervening between the A and B’ motifs (~100 amino acids as compared to 20 amino acids) (Lingner, Hughes et al. 1997; Nakamura, Morin et al. 1997), suggesting that this specific “finger” region, termed the insertion in fingers domain (IFD), may harbor telomerase specific properties (Nugent and Lundblad 1998). An invariant trio of aspartic acids (found in motifs A and C), was shown to directly involve in catalysis, as mutations of these residues result in abolished catalytic activity in vitro and in vivo (Harrington, Zhou et al. 1997; Lingner, Hughes et al. 1997; Weinrich, Pruzan et al. 1997; Beattie, Zhou et al. 1998; Nakayama, Tahara et al. 1998). Mutations of other amino acid residues in any of the conserved RT motifs were also found to reduce or eliminate telomerase reverse transcriptase activity (Lingner, Hughes et al. 1997; Weinrich, Pruzan et al. 1997; Nakayama, Tahara et al. 1998). The high affinity RNA binding domain (TRBD), also known as the RNA interacting domain 2 (RID 2), contains telomerase specific motifs CP, QFP, and T, also referred to as domains II, III, and IV respectively (Bryan, Goodrich et al. 2000; Xia, Peng et al. 2000; Bosoy, Peng et al. 2003).  These motifs mediate TER  recognition and have a relatively high binding affinity to structured RNA stem loop, interacting with the CR4/CR5 domain of TER (Lai, Mitchell et al. 2001). This domain plays a role in promoting stable enzyme assembly, as mutations in these motifs result in severe defects in TER-TERT association (Moriarty, Huard et al. 2002).  17  The TERT essential N-terminal (TEN) domain or RNA interacting domain 1 (RID 1), contains the non-conserved extreme N-terminus motif (Lue 2004) and moderately conserved GQ motif (also referred to as domain I) (Kelleher, Teixeira et al. 2002; Moriarty, Huard et al. 2002). The GQ motif is further divided into domains IA and IB, separated by a DAT (dissociates activities of telomerase) domain (Armbruster, Banik et al. 2001).  The TEN domain interacts with the TER  pseudoknot-template domain (Lai, Mitchell et al. 2001), but is not considered a major TER binding surface as mutations in this region only result in modest reductions of TER-TERT association (Moriarty, Huard et al. 2002). This region also displays high single-stranded telomeric DNA binding affinity, implicating an important role in substrate recognition and primer binding.  Accordingly,  mutagenesis experiments showed that the TEN domain is essential for product alignment and enzyme processivity (Armbruster, Banik et al. 2001; Lai, Mitchell et al. 2001; Lee, Wong et al. 2003; Moriarty, Ward et al. 2005). The smaller, less-conserved C-terminal domain (TEC or CDAT) plays several roles in telomerase function: it contributes to telomerase catalytic activity (Bachand and Autexier 2001; Lai, Mitchell et al. 2001), regulates the cellular localization of the enzyme, and plays a role in the polymerase processivity (Hossain, Singh et al. 2002; Huard, Moriarty et al. 2003). However, this domain is not essential for RNA binding as mutations in this region were not found to impair TER-TERT association (Huard, Moriarty et al. 2003).  18  Hsp90 and p23 Molecular chaperone proteins p23 and Hsp90 were identified as key factors in the assembly and functionality of the telomerase holoenzyme. Both were found to associate with hTERT and aid in its nuclear localization and import. They were also demonstrated to be required for the assembly of active telomerase enzyme both in vitro and in vivo, as inhibition of either chaperone protein disrupts telomerase assembly leading to a reduction in enzyme activity (Holt, Aisner et al. 1999). However, their exact role in the catalytic activity of telomerase remains unknown.  14-3-3 The nuclear retention of TERT is dependent on its association with the 14-33 proteins, a protein family involved in intracellular trafficking/targeting, cell cycle regulation, cytoskeleton structure and transcription (Aitken 2006). hTERT and 143-3 interact via their respective C-termini. This interaction is required for the nuclear accumulation of hTERT, as dominant negative 14-3-3 or hTERT-3A, a hTERT mutant unable to bind 14-3-3, both resulted in the redistribution of hTERT to the cytoplasm (Seimiya, Sawada et al. 2000). 14-3-3 proteins promote the nuclear retention of hTERT by masking the nuclear export signal (NES)-like motif in the Cterminal region of hTERT. Binding of 14-3-3 inhibits the binding of CRM1/exportin 1 to TERT NES, resulting in the nuclear accumulation of the reverse transcriptase.  19  Nucleolin Nucleolin is another important protein factor in the nuclear localization of hTERT.  This major nuclear phosphorprotein binds to hTERT through its RNA  binding domain 4 and the carboxyl terminal RGG domain. RNA binding domain 1 may also be involved in the nucleolar localization of telomerase holoenzyme through its interactions with hTER.  Biochemical experiments showed that the  binding of hTERT with the nucleolin-4R fragment, which lacks a nucleolar localization signal, resulted in the mislocalization of hTERT in the cytoplasm, thereby implicating this protein in the nuclear localization of hTERT (Khurts, Masutomi et al. 2004).  PINX1 PINX1, a PIN2/TRF1 interacting protein, is involved in TERT nucleolar localization and has also been characterized as an inhibitor of telomerase activity and a negative regulator of telomere length.  Inhibition of endogenous PINX1  resulted in an increase in telomerase activity, whereas over-expression of PINX1 decreases telomerase activity and shortens telomeres (Zhou and Lu 2001). PINX1 was found to bind directly with hTERT at its RNA binding domain and indirectly associate with TER through TERT (Banik and Counter 2004). Inhibitory effect of PINX1 on human telomerase involves its interaction with an assembled hTERT-TER complex, in contrast to yeast PINX1p, which regulates telomerase activity by forming an inactive telomerase complex that lacks TER.  20  TERT post-translational modifications Telomerase activity is regulated via post-translational modifications of TERT. The E3 ubiquitin ligase MKRN1 was shown to have a negative role on telomere length homeostasis.  MKRN1 is responsible for the ubiquitination of hTERT,  targeting hTERT for protease degradation. Over-expression of MKRN1 results in the decrease of telomerase activity and subsequently in the shortening of telomere length (Kim, Park et al. 2005). Several studies have demonstrated that the phosphorylation of hTERT is required for catalytic activity of the enzyme (Li, Zhao et al. 1998; Kang, Kwon et al. 1999; Breitschopf, Zeiher et al. 2001; Haendeler, Hoffmann et al. 2003). Protein kinase B (Akt) and protein kinase C have both been shown to interact with and phosphorylate TERT in vitro and in vivo (Li, Zhao et al. 1998; Kang, Kwon et al. 1999; Breitschopf, Zeiher et al. 2001), resulting in the increase in telomerase activity. Conversely, protein phosphatase 2A inhibits telomerase activity via the dephosphorylation of TERT directly (Li, Zhao et al. 1997; Li, Zhao et al. 1998) or indirectly, through the dephosphorylation and inhibition of Akt. c-Abl protein tyrosine kinase associates with TERT and mediates TERT phosphorylation in vitro and in vivo. In contrast to the activation models above, cAbl phosphorylation of TERT resulted in the inhibition of telomerase activity, making this kinase a negative regulator of TERT (Kharbanda, Kumar et al. 2000).  21  TER-TERT assembly Association of the stable TER-H/ACA RNP complex with TERT is required for the production of a functional telomerase enzyme in vivo. Assembly of telomerase occurs primarily in the nucleolus, although Cajal bodies are also implicated as the assembly sites. In the following sections, the major biogenesis factors responsible for the TERT-TER assembly process will be discussed: Pontin and reptin Pontin and reptin, members of the AAA+ family of DNA helicases (Huber, Menard et al. 2008), play pivotal roles in telomerase assembly. These helicases are found to bind to dyskerin and play a role in the formation of the TER-dyskerin complex. Subsequently, these helicases bind to endogenous hTERT and mediate the assembly with TER-dyskerin complex to form the catalytically active telomerase enzyme (Venteicher, Meng et al. 2008). The formation of the TERT-pontin-reptin complex is regulated by cell cycle stages, with the highest level of complex formation occurring during S-phase, providing evidence for another level of cell cycle dependent regulation of TERT. However, because the purified TERT-pontin-reptin complex exhibited low catalytic activity, it is believed that this complex may represent an intermediate of the assembly process, which require further modifications, or association with additional factors, to form the mature form of the enzyme.  22  TCAB Nucleoplasmic Cajal bodies (CBs) have been suggested as one of the sites for telomerase assembly. The novel RNA binding protein TCAB1 was shown to be required for telomerase localization to these sites. Knockdown of TCAB using retroviral shRNA and RNA interference resulted in a significant reduction in the percentage of cells with TER staining in CBs by microscopic analysis (Venteicher, Abreu et al. 2009), indicating its role in CBs localization of telomerase. TCAB1 was found to associate with TER by binding specifically to the CAB-box sequence (CR7 motif).  This interaction occurs after the assembly of the TER-TERT-dyskerin  complex (Venteicher, Abreu et al. 2009), suggesting that TCAB acts as a CBs targeting or retention factor and could be involved in additional assembly steps following TERT-TER holoenzyme formation. Inhibition of TCAB1 by shRNA also reduced the amount of TER at telomeres during S phase of the cell cycle, resulting in telomere shortening. These data suggested that TCAB1 may play a role in controlling the access of telomerase complex to telomeres, representing an additional level of enzyme activity regulation (Venteicher, Abreu et al. 2009).  Targeting telomerase holoenzyme to telomere Newly assembled, catalytically active telomerase enzyme must travel to and associate with the limited number of telomere ends for its proper function.  As  illustrated with the earlier discussion on TCAB, the Cajal Bodies were suggested as sites where the delivery of the active enzyme to the telomeres occurs (Zhu,  23  Tomlinson et al. 2004; Tomlinson, Ziegler et al. 2006; Cristofari, Adolf et al. 2007). TER is found localized at CBs in cancer cells throughout the cell cycle (Jady, Bertrand et al. 2004; Zhu, Tomlinson et al. 2004). Mutations in the CAB box motif decreases the accumulation of TER in CBs as well as the frequency of hTER association with telomeres, resulting in shorter telomere length (Jady, Bertrand et al. 2004; Cristofari, Adolf et al. 2007). However, the same mutations, when tested in primary human cells, did not show substantial effects on telomere length maintenance (Errington, Fu et al. 2008). These results indicate TER localization to CBs may be an important regulatory factor in telomere length homeostasis, especially in cells with high proliferative demands and constitutive telomerase activity requirements. The presence of hTERT was also found to be necessary for the localization and accumulation of hTER in CBs as well as trafficking of telomerase to telomeres during S phase of the cell cycle (Zhu, Tomlinson et al. 2004; Jady, Richard et al. 2006; Tomlinson, Abreu et al. 2008). However, outside of S phase, hTERT resides in subnuclear foci, termed TERT foci (Tomlinson, Ziegler et al. 2006), indicating that these two components are not transported to CBs as a assembled complex. Inhibition of hTERT resulted in a decrease of hTER colocalized with CBs and telomeres without affecting the levels of hTER in cells. Additionally, expression of hTERT in telomerase negative cells resulted in the accumulation of hTER at both sites (Tomlinson, Abreu et al. 2008). These observations again suggest that CB localization of telomerase is connected to enzyme biogenesis and catalytic activity in transformed cells.  24  hEST1A hEST1A has also been suggested to play an important role in telomere maintenance in a manner similar to its yeast homologue Est1p.  Yeast Est1p  interacts with TER and the yeast telomere binding protein Cdc13, thereby bringing telomerase to the proximity of the telomeres (Pennock, Buckley et al. 2001; Evans and Lundblad 2002). A high-affinity TER binding domain has been identified in hEST1A, as well as a domain in hTERT that is responsible for the association of this protein with TERT in vitro (Redon, Reichenbach et al. 2007). hEST1A exhibits direct telomeric DNA binding activity and may mediate the telomere-telomerase interaction, but the exact sequence of binding and targeting events remained largely unknown.  hnRNPs There are also several lines of evidence that support the involvement of hnRNPs in the localization of telomerase to telomere ends for the de novo synthesis of telomere repeats.  Several in vitro studies have demonstrated that hnRNPs  A1/UP1, A2, A3, C1/C2, and D bind to hTER and single-stranded telomeric DNA, thereby suggesting their roles in the association between telomeres with the telomerase holoenzyme. In parallel, hnRNP A1/UP1 is found at telomere ends in vivo and is suggested to stimulate telomerase activity through the disruption of Gquadruplex structures formed during translocation of the telomerase enzyme during telomere synthesis (Zhang, Yang et al. 2006).  25  Telomerase catalytic cycle TERT directs the addition of deoxynuclotide triphosphates (dNTPs) to the ends of the G-rich strand of the chromosome by copying the last six nucleotide of the 11-nt telomere repeat template sequence of TER (Shippen-Lentz and Blackburn, 1990, Blackburn, 1992). This activity results in the de novo synthesis of a single, 6nt repeat. Because the TER RNA template is quite short, to generate multiple repeats within a single catalytic event, telomerase holoenzyme undergoes multiple rounds of transient dissociation from the DNA substrate, to reposition the enzymesubstrate complex. Telomerase relies on its unique ability to transiently move away from the active site after the addition of a single 6-nt repeat, translocate towards the 3’end of the newly synthesized chromosome and mediate the realignment of the new chromosome end with the TER RNA template, in order to continue subsequent rounds of multiple telomeric repeat addition (Fig 4). The ability of the enzyme to carry out these two movement behaviors is referred to as nucleotide addition processivity and repeat addition processivity, respectively (Collins 1999; Peng, Mian et al. 2001). Nucleotide addition processivity is a common characteristic of all reverse transcriptases; however, repeat addition processivity is unique to the telomerase enzyme (Lue 2004).  26  Figure 4. Telomere repeat synthesis. Due to its short RNA template sequence, telomerase relies on two movement behaviors to add multiple 6-nuleotide (nt) telomeric sequences to chromosome ends. Addition of each 6-nt repeat to the 3’ end of the template is followed by telomerase translocation. This mediates realignment of the chromosome end from the 5’ end to the 3’ end of the template to enable subsequent rounds of repeat addition. Telomerase’s ability to carry out these two movement behaviors is termed nucleotide addition processivity and repeat addition processivity, respectively.  27  The 11-nt template region of TER can be subdivided into a 5-nt alignment domain and the 6-nt elongation domain for polymerization (Hamilton, Pitts et al. 1997; Drosopoulos, Direnzo et al. 2005). The alignment domain, located 3’ to the elongation domain, repositions the newly translocated product through basepairing interactions and plays a pivotal role in mediating repeat addition processivity (Lue 2004). Following the addition of each telomeric repeat, the enzyme may either disassociate from the chromosome end, stay bound without continuing elongation, or translocate and continue additional cycles of repeat addition (Fulton and Blackburn 1998; Lue 2004). Translocation of the enzyme requires the DNA substrate to remain bound to telomerase. This interaction is mediated through an “anchor site” within the N-terminal domain of TERT (Moriarty, Ward et al. 2005; Wyatt, Lobb et al. 2007). Several domains in both hTERT protein and TER have been demonstrated to play roles in the enzyme’s processivity. The TEN/RID1 domain, which contains the GQ motif and non-conserved “N-region”, mediate repeat addition processivity through its interaction with the hTER pseudoknot template domain. Deletion of some conserved residues or the entire domain from hTERT resulted in defective processivity (Moriarty, Marie-Egyptienne et al. 2004). The T motif was also shown to affect repeat addition processivity. Mutations in the conserved FYXTE telomerase signature sequence within this motif resulted in an increase in repeat addition rates, suggesting this motif might have a restrictive role in this type of processivity (Drosopoulos and Prasad). Mutations in the C-terminal domain resulted in impaired  28  nucleotide addition processivity and also influenced repeat addition processivity, indicating this region to be important for both (Huard, Moriarty et al. 2003).  Alternative lengthening of telomeres Telomere maintenance can also be achieved by a process called alternative lengthening of telomeres (ALT) (Reddel, Bryan et al. 2001; Henson, Neumann et al. 2002).  ALT was initially discovered by Bryan and colleagues in 1995 when  telomere elongation was observed in immortal human cells without detectable telomerase activity (Bryan, Englezou et al. 1995). In yeast, this process involves either rolling circle recombination or strand exchange recombination mechanisms. ALT is believed to occur by similar processes in humans (Cerone, Londono-Vallejo et al. 2001), as it requires the activity of many homologous recombination protein factors including Rad50, MRE11, and NBS1 (Bechter, Zou et al. 2003; Jiang, Zhong et al. 2005). Although detected in human cells, ALT is not considered to be the normal physiological process for the maintenance of telomeres in humans. It has only been observed in a small number of human tumors (carcinoma and osteocarcinoma) and some transformed cell types in culture (mainly fibroblasts) (Bryan, Englezou et al. 1995). Long-term telomerase inhibition could potentially select tumor cells for ALT activation, but such phenomenon has not been observed.  Telomerase and cancer Most normal human somatic cells do not express detectable levels of telomerase activity as TERT expression is rapidly down-regulated following  29  embryonic development (Wright, Piatyszek et al. 1996). In some human cell types, such as germline cells and stem cells, where there is a high demand for proliferation, the TERT gene is activated to allow for transient expression of the enzyme. In contrast, more than 85% of human tumors surveyed harbor robust telomerase activity (Shay and Bacchetti, 1997). In almost all cases, the transcriptional upregulation of hTERT is responsible for the increase in ectopic telomerase activity in tumor cells (Kim, Piatyszek et al. 1994), while a small percent (less than 5-10%) exhibit the recombination-based mechanism for telomere maintenance, ALT. This indicates that telomere maintenance by telomerase is required and preferred over ALT for the continual proliferation and immortal phenotype of cancer cells. Indeed, proof of concept experiments showed that the inhibition of this enzyme in human cancer cells resulted in telomere-induced crisis and apoptosis in cell culture models (Hahn, Stewart et al. 1999; Zhang, Mar et al. 1999) Telomerase expression is not considered oncogenic, as it alone does not lead to the development of cancer (Harley, 2002). Additionally, it has been shown that hTERT expression alone is not sufficient for the immortalization of human mammary epithelial cells, keratinocytes (Kiyono et al., 1998), prostate epithelial cells (Berger et al., 2004), or airway epithelial cells (Lundberg et al., 2002). Cooperation between TERT and other oncogenic factors are essential for the transformed phenotype (Hahn, Counter et al. 1999) Paradoxically, early neoplastic lesions typically have undetectable or low telomerase activity as compared to advanced malignant lesions that over-express the enzyme (Chadeneau, Hay et al. 1995; Engelhardt, Drullinsky et al. 1997). This  30  suggests that initiation of tumor development may require the absence of telomerase activity.  Indeed, data from tumor cytogenetic studies have  demonstrated that telomere length from precancerous lesions are much shorter than in normal tissues (de Lange, Shiue et al. 1990; Miura, Horikawa et al. 1997; Rudolph, Millard et al. 2001).  Several studies have reported critically short  telomeres as a common early feature of many human cancers, such as colon (Engelhardt, Drullinsky et al. 1997), lung (Lantuejoul, Soria et al. 2005), breast (Meeker, Hicks et al. 2004), pancreatic (van Heek, Meeker et al. 2002), and prostate (Meeker, Hicks et al. 2002). The telomere dysfunction model of carcinogenesis suggested that rampant genomic instability following the uncapping of dysfunctional, short telomeres contributes to the eventual development of aneuploidy, a genetic signature of cellular transformation and carcinogenesis (Wright and Shay 1992; Gisselsson, Jonson et al. 2001). Telomere dysfunction is thus recognized as a late event in the process of cancer initiation. After which, telomerase activity has to be induced to prevent further genomic instability that hinders cancer growth, and provides a mechanism for the indefinite proliferation and immortality phenotype in malignant tumors (DePinho 2000).  NON-telomere maintenance roles of telomerase Besides its role in telomere maintenance, there is growing evidence pointing to telomerase’s additional role in the cancer biology. If telomere maintenance is the sole requirement for telomerase expression in cancer, then cells with the ALT mechanism should be able to substitute for telomerase in tumorigenesis. This was  31  proven not to be the case: expression of the oncogenic H-Ras allele in the immortal human fibroblast ALT cell line GM847 did not result in malignant transformation when injected into nude mice. In contrast, the co-expression of hTERT in these cells imparted a tumorigenic phenotype (Stewart et al., 2002).  This tumorigenic  phenotype was also observed with the introduction of a mutant hTERT, hTERT HA, which retains its enzymatic activity in vitro but is incapable of maintaining telomere length in vivo. Thus, the ALT recombination mechanism is concluded not to be able to replace telomerase in the process of cellular transformation, implicating an additional role of hTERT, independent of telomere length maintenance. Direct evidence for the presence of a non-telomere length related telomerase function was demonstrated using non-transformed, human fibroblasts. Normal, diploid human fibroblasts over-expressing hTERT were found to be more resistant to apoptosis and necrosis induced by DNA damage, but equally susceptible to the cytotoxic effects of oxidative agents as normal fibroblasts without TERT expression (Gorbunova, Seluanov et al. 2002). This suggested that telomerase is involved in enhancing cellular survival following genotoxic stress. In a separate study, hTERT was shown to affect genomic stability and DNA repair functions. Higher mRNA levels of several DNA repair and chromatin modifying genes, as well as better double-stranded break repair kinetics, were observed in human foreskin fibroblast cells expressing hTERT as compared to cells lacking ectopic expression of the enzyme (Sharma, Gupta et al. 2003). Importantly, these effects occurred rapidly before any significant telomere lengthening was observed. A transcriptome study done by Smith and colleagues in 2005 demonstrated that the ectopic expression of  32  telomerase in human mammary epithelial cells reduced the need for exogenous mitogens for cellular proliferation, correlating with the telomerase dependent induction of gene expression that promote cell growth and survival. This latter study provided evidence for a role of telomerase in cellular proliferation by affecting the expression profiles of growth and survival-related genes. In 2005, William Hahn’s group provided direct evidence implicating telomerase’s role as a regulator of the DNA damage response pathway.  By  suppressing endogenous hTERT expression in diploid human fibroblasts using either an hTERT-coding sequence specific shRNA or an hTERT 3’untranslated region-specific shRNA (hTERT 3’UTR shRNA), they demonstrated that hTERT participates in DNA damage responses and chromatin maintenance in a manner that is separate from its role in telomere length maintenance.  Following ionizing  radiation (IR), irinotecan, or etoposide treatment, phosphorylation of H2AX and the ataxia telangiectasia mutated (ATM) protein was greatly impaired as compared to control cells expressing normal levels of TERT.  As a direct consequence, the  phosphorylation of BRCA1 tumor suppressor proteins was not observed and protein levels of p53 were not up-regulated. These results indicate impaired DNA damage responses in cells lacking hTERT. Telomerase knock-down cells also exhibited increased sensitivity to IR as shown by the decreased relative survival in clonogenic growth assays.  When wildtype recombinant hTERT was introduced into cells  expressing the hTERT 3’UTR shRNA, which does not target these recombinant copies of hTERT, the cells ability to respond to DNA damage was restored.  33  In addition, the introduction of several recombinant hTERT mutants (NDAT92, N-DAT122, and C-DAT1127), which maintained catalytic activity in vitro but were unable to elongate telomeres in vivo, into cells whose endogenous hTERT was suppressed via hTERT 3’UTR-specific shRNA, restored the cells’ ability to phosphorylate H2AX as well as stabilize p53 levels following IR. The catalytically inactive DN-TERT mutant failed to restore these DNA damage responses (Masutomi, Possemato et al. 2005). These results indicate that the effect of hTERT on DNA damage response pathways is dependent on its catalytic competency, yet independent of its telomere synthesis activity. The molecular mechanism of how TERT perform this role to promote DNA damage survival remains unclear, but is suggested to be associated with TERT’s chromatin remodeling activities.  Telomerase inhibition as a clinical therapy Telomerase reactivation and over-expression is universal in all human cancers. Breast cancer is the most common malignancy among women in the United States and is the second leading cause of cancer death after lung cancer (American Cancer Society statistics). Telomerase activity is detected in approximately 75-95% of primary invasive breast cancers (Umbricht, Sherman et al. 1999) and is 6.9-fold higher than that of normal human mammary epithelial cells (Hiyama, Gollahon et al. 1996; Ramachandran, Fonseca et al. 2002). Similarly, colorectal cancer is one of the most common forms of cancer and is the second leading cause of cancer deaths in the western world and the fourth worldwide (World Health Organization statistics). Tumors of this type of cancer also exhibit high levels of telomerase activity (93%)  34  and have been correlated with rapid disease progression and poor prognosis in clinical studies (Malaska, Kunicka et al. 2004; Neal, Garcea et al. 2006). Telomerase is constitutively over-expressed in over 85% of all human cancers (Shay and Bacchetti 1997). Together with the apparent lack of TERT expression in normal somatic cells made telomerase an ideal target for anticancer therapies. Early proof-of-principle experiments demonstrated that the expression of a dominant negative form of hTERT completely inhibited telomerase activity and substantially reduced telomere length in several cancer models. The resulting telomere dysfunctions led to the formation of dicentric chromosomes and other types of chromosome fusions, resulting in the loss of cellular viability and apoptosis. This inhibition of hTERT was demonstrated to limit tumorigenicity of mouse xenograph models of cancer (Hahn, Stewart et al. 1999).  Small molecule inhibitor of telomerase Telomerase inhibition leads to the loss of cancer cell viability (Hahn, Stewart et al. 1999). These findings prompted the active development and investigation of anti-cancer therapies targeting telomerase and telomeres.  Since HIV reverse  transcriptase and telomerase reverse transcriptase shared many structural and mechanistic similarities, it is conceivable that HIV reverse transcriptase inhibitors could exhibit inhibition activity against telomerase. Accordingly, the effects of two HIV retroviral reverse transcriptase inhibitors were measured on telomerase activity and telomere length dynamics. Dideoxyguanosine (ddG) and AZT (3’-azido2’,3’-dideoxythymidine) both demonstrated inhibition of cellular telomerase activity  35  and repeated exposure resulted in telomere shortening. Surprisingly, a decrease in cellular growth rate and telomere length-dependent senescence was not observed with prolonged passaging of cells in the presence of these agents (Strahl and Blackburn 1996).  However, it remained possible that under extreme selective  pressure, some cancer cells could develop resistance mechanisms to counter the effect of these anti-metabolite classes of agents by altering the metabolic activation pathways of these agents, or by increasing the cellular elimination mechanisms. The effects of drug therapy targeting the protein stability of TERT were investigated with the agent geldanamycin.  Geldanamycin is a benzoquinone  ansamycin antibiotic and inhibits the binding of cofactor ATP and partner p23 to the molecular chaperone Hsp90 (Grenert, Sullivan et al. 1997; Prodromou, Roe et al. 1997). Geldanamycin was demonstrated to block the assembly of active telomerase both in vitro and in vivo (Holt, Aisner et al. 1999). However, as HSP90 and p23 form chaperone complexes that have integral roles in different biological processes, geldanamycin mediated inhibition of HSP90 function lacks specificity for the telomerase pathway. Other small molecules targeting telomerase and telomere functions include agents that stabilize G-quadruplex structures. The 3’ telomeric DNA overhang is guanine rich and can form secondary structures, known as G-quadruplexes, stable 4stranded DNA structures made up of G-rich sequences where the guanine residues form square arrangements. G-quadruplexes were demonstrated to have important functions in yeast telomeres (Liu, Lee et al. 1995). Several different g-quadruplex inhibitors have been shown to disrupt the binding of telomere-associated proteins,  36  inhibit telomerase activity and induce apoptosis in vitro (Sun, Thompson et al. 1997; Perry, Gowan et al. 1998; Perry, Gowan et al. 1999; Perry, Read et al. 1999; Gowan, Heald et al. 2001). Unfortunately, a major problem with these compounds is their inability to penetrate the cell membrane and a proper delivery protocol for these types of drugs has yet to be developed.  Other clinical strategies targeting telomeres or telomerase Based on the selective activation of the TERT promoter in cancer cells, several groups reported the use of recombinant DNA vectors, with the TERT promoter driving the expression of various transgenes, delivering cytotoxic/suicidal activities in a cancer cell specific manner (Abdul-Ghani, Ohana et al. 2000; Gu, Kagawa et al. 2000; Koga, Hirohata et al. 2000; Boyd, Mairs et al. 2001; Koga, Hirohata et al. 2001; Koga, Kondo et al. 2001; Komata, Koga et al. 2001; Komata, Kondo et al. 2001; Majumdar, Hughes et al. 2001; Plumb, Bilsland et al. 2001). While these proof-of-principle experiments provided the framework for a cancer specific targeting strategy, more work is still needed for the development, delivery and clinical validity of cancer gene therapy. Other studies have looked at the expression of mutant-template human telomerase RNA (MT-hTER), in telomerase positive cells. MT-hTERs assemble with endogenous TERT and the recombinant enzyme then erroneously adds DNA repeats with mutant sequence to chromosome ends. A few copies of mutant DNA repeats are enough to disrupt the binding of telomeric proteins. The resulting compromised  37  telomere structure leads to the loss in cellular viability and the increase in apoptosis (Guiducci, Cerone et al. 2001; Kim, Rivera et al. 2001; Li, Rosenberg et al. 2004). Telomerase is also tested as a novel target for cancer immunotherapy.  In  telomerase-positive cancers, hTERT peptides are presented as epitopes on the tumor cell surface by the major histocompatibility complex (MHC) class I pathway. TERT antigen presentation was demonstrated to produce cytotoxic T lymphocyte responses (Vonderheide, Hahn et al. 1999; Minev, Hipp et al. 2000; Nair, Heiser et al. 2000). Two first-generation vaccines have been developed: GRNVAC1 and GV1001. Telomerase cancer vaccine, GRNVAC1, uses an ex vivo process where mature dendritic cells are isolated from the patient’s blood and transfected with hTERT mRNA. These cells are then returned to the body where they stimulate the production of CD4+ and CD8+ T-cells specific for hTERT (Su, Dannull et al. 2005). GV1001 is a peptide vaccine derived from the active functional domain of telomerase.  GV1001 binds multiple human leukocyte antigen (HLA) class II  molecules and harbors putative HLA class I epitopes, and also illicit CD4+ and CD8+ T-cell responses specific for hTERT (Bernhardt, Gjertsen et al. 2006). Both vaccines were test successful in phase I/II clinical trials for efficacy in producing telomerase specific CD4+ and CD8+ T-lymphocytes (Su, Dannull et al. 2005; Bernhardt, Gjertsen et al. 2006). GV1001 is currently in two phase III clinical trials for the treatments of pancreatic cancer while GRNVAC1 is being investigated in a phase II clinical trial in patients with acute myeloid leukemia (Shay and Keith 2008).  38  Time lag between the start of therapy to the observation of cytotoxicity: the achilles heel of telomerase-based chemotherapies Despite the demonstrations of several successful strategies targeting telomeres and telomerase in cancer cells, their usefulness in the clinics has been marred by several deficiencies.  The timeline of inducing cyototoxicity by  telomerase inhibition relies completely on the kinetics of telomere shortening to a critically short length. As telomere length decreases at a rate of 50-100bp per cell division, this process can be quite long, and tumor specific. This time lag can range from weeks to months of continual telomerase inhibition therapy.  However,  prolonged inhibition of the telomerase enzyme could affect normal human cells that also dependent on transient telomerase activity for their functionality (Fleisig and Wong 2007). In these cases, telomere erosion in off-target cells from telomerase inhibition therapy could precipitate adverse treatment effects in these normal cell types. Premature telomere shortening translate to the accelerated rate of tissue aging. If these cells were allowed to divide beyond the short telomere check point, due to the inactivation of tumor suppressive mechanism, new rounds of genomic instability cycles could trigger the development of secondary tumors. This paradox, in addition to the lack of proper delivery method for genetic-based inhibition of TERT function, argues that telomerase inhibition on its own is not efficacious as an anti-cancer therapy.  39  Anti-cancer chemotherapeutic agents  Current DNA damaging chemotherapeutic agents Currently, cancer is treated using chemotherapy, radiation therapy and/or surgery. Many of the chemotherapeutic agents induced DNA damages, such as topoisomerase inhibitors, alkylating agents, and anti-tumor antibiotics, and can be divided into cell cycle specific and non cell cycle specific agents. Etoposide, a topoisomerase II inhibitor, and irinotecan, a topoisomerase I inhibitor, are examples of cell cycle specific DNA damaging agents. Etoposide has been found to be anti-neoplastic and very effective against cells in late S/G2 phases of the cell cycle (Stahelin 1973). It induces double-stranded DNA breaks through its inhibitory effects on the scission-reunion reaction of mammalian topoisomerase II (van Maanen, Retel et al. 1988).  Irinotecan is a semi-synthetic derivative of  camptothecin, a plant alkaloid from Camptotheca acuminata (Armand, Ducreux et al. 1995). Irinotecan is a prodrug that is activated by cellular carboxylesterase into its active form, 7-ethyl-lo-hydroxy-camptothecin (SN-38), which acts as a potent topoisomerase I inhibitor and causes single-stranded DNA breaks and the arrest of DNA replication (Armand, Ducreux et al. 1995).  However, in cases where these  unrepaired ends are allowed to pass through S phase of the cell cycle, the passage of the replication fork along these lesions convert the single-stranded nicks into double-stranded DNA breaks.  Accordingly, cytotoxicity of irinotecan is at a  maximum during S-phase of the cell cycle (Zhang, D'Arpa et al. 1990; Armand, Ducreux et al. 1995). Notably, irinotecan is indicated to be the first-line therapy in  40  advanced colorectal cancers in combination with 5-fluorouracil (FU). Bleomycin, an anti-tumor antibiotic, oxaliplatin and cyclophosphamide, both alkylating agents, are examples of non cell cycle specific DNA damaging agents. Bleomycin functions as a DNA-cleaving glycopeptide produced by the bacteria Streptomyces verticillus (Suzuki, Nagai et al. 1970). It binds to DNA, forms a DNAbleomycin-Fe (II) complex, and catalyzes the reduction of molecular oxygen, resulting in the formation of free radicals, which are responsible for creating singleand double-stranded DNA breaks as well as inhibiting DNA synthesis. Oxaliplatin, a third generation diaminocyclohexane platinum chemotherapy drug, is commonly used in the treatment of metastatic colorectal cancer. Its platinum component forms both intrastrand and interstrand cross-links with DNA, resulting in the inhibition of DNA replication and RNA transcription, thereby causing direct DNA strand breaks (Raymond, Faivre et al. 1998). Even though the mechanism of action is similar between oxaliplatin and the previous generations of platinum compounds, the mode of DNA repair in response to the lesions generation by oxaliplatin is notably different from that of the other platinum compounds, as illustrated by their non-overlapping drug resistant mechanisms (Raymond, Faivre et al. 2002). Cyclophosphamide is a nitrogen mustard alkylating agent that crosslinks DNA, thereby preventing DNA replication. Cyclophosphamide is a prodrug and must be activated by the cytochrome P450 mixed-function oxidase system. It is converted to its active metabolite 4-hydroxycyclophosphamide which is then broken down into two cytotoxic metabolites, phosphoramide mustard and acrolein (Struck, Kirk et al. 1975; Hales 1982). Cyclophosphamide is most commonly used in combination with  41  methotrexate and flurouracil for the treatment of leukemia, breast and ovarian cancers (D'Incalci, Bolis et al. 1979; Bonadonna, Valagussa et al. 1995).  Telomerase inhibition in anti-cancer chemotherapies Telomerase inhibition has been demonstrated to increase the sensitivity to chemotherapeutic agents by overwhelming the DNA repair mechanism, with the creation of unprotected chromosome ends. For example, telomere dysfunction in late generation TERC-/- mice, lacking the mouse telomerase RNA gene, resulted in decreased cellular survival after exposure to IR (Wong, Chang et al. 2000). At the cellular level, the rate of apoptosis in gastrointestinal crypt cells and primary thymocytes was higher in telomerase deficient mice as compared to control. Additionally, mouse embryonic fibroblasts showed a much lower dose-dependent clonogenic survival. These TER-/- cells also displayed delayed DNA break repair kinetics, as well as persistent chromosomal breaks, complex chromosomal aberrations and massive fragmentation. Reduction of telomerase activity also resulted in increased cell sensitivity to topoisomerase inhibitors. The MCF-7 breast cancer cell line and HBL-100 immortal breast cell line expressing an anti-TERT ribozyme, which cleaves human telomerase mRNA, resulted in inhibition of telomerase activity, decreased telomere length and induced apoptosis. Additionally, an increased sensitivity to the topoisomerase II inhibitor doxorubicin was also observed in these cell lines. Interestingly, when exogenous hTERT was introduced into telomerase-negative human fibroblasts, there was a decrease in the sensitivity of these cell lines to doxorubicin, as well as  42  two other topoisomerase inhibitors: mitoxantrone and etoposide (Ludwig, Saretzki et al. 2001). Telomerase inhibition via the ectopic expression of dominant negativehTERT (DN-hTERT) in human cancer cells resulted in telomere shortening, growth arrest and apoptosis (Hahn, Stewart et al. 1999; Zhang, Mar et al. 1999). Expression of recombinant DN-TERT in BCR-ABL positive leukemia cells completely inhibited endogenous telomerase activity and resulted in an increase in apoptosis following treatment with the tyrosine kinase inhibitor imatinib (Tauchi, Nakajima et al. 2002). Telomerase inhibition was also demonstrated to increase telomerase positive pharynx Fadu tumor cell’s sensitivity to paclitaxel, which causes telomere erosion (Multani, Li et al. 1999). Telomerase inhibition was achieved using either antisense hTER, which blocks the template for telomere synthesis, or 3’-azido3’deoxythymidine (AZT), a nucleoside analog reverse transcriptase inhibitor. AZT was also shown to increase the in vivo effects of paclitaxel. The combination of AZT and paclitaxel resulted in decreased tumor size, increased apoptosis and prolonged survival in FaDu xenograft tumor mice models. This telomerase inhibition induced cytotoxic effect was not observed in telomerase negative human osteocarcinoma Saos-2 cells, indicating that the increase in sensitivity to paclitaxel was due to telomerase inhibition (Mo, Gan et al. 2003). Knockdown of telomerase activity in human cells can also be achieved via retroviral transfer of siRNA targeting TERT. These telomerase knockdown cells displayed increased sensitivity to IR and chemotherapeutic agents etoposide, bleomycin and doxorubicin (Nakamura, Masutomi et al. 2005). In addition, the  43  combination therapy using the hTERT siRNA increased the apoptotic effect of cisplatin, a platinum-based chemotherapeutic agent, on the hepatocellular cell line SMMC7721 in vitro and also greatly reduced SMMC7721 and HepG2 tumor growth in the mouse xenograft model as compared to cisplatin monotherapy (Guo, Wang et al. 2008). The results from these combination studies provided genetic and biological evidence  linking  telomere  dysfunction  and  increased  sensitivity  to  chemotherapeutic agents, making telomerase inhibition an effective therapeutic option for many different types of cancers. However, telomerase inhibition was achieved using gene knockout, RNA interference or dominant-negative TERT expression strategies. In addition to the fact that these methods employ a longer time course to achieve complete telomerase inhibition, many of these strategies have yet to be proven to be clinically useful, due to the lack of a proper delivery protocol. Many of these studies concluded that the observed increase in sensitivity of cancer cells to cytotoxic agents was telomere length dependent. Telomere shortening caused by the continuous inhibition of telomerase resulted in the disruption of telomere homeostasis and the activation of short telomere checkpoint, suggesting that longer term of telomerase inhibition will lead to greater clinical effect. However, prolonged inhibition of the enzyme may affect normal human cell types that also require telomerase activity for growth and proliferation (Fleisig and Wong 2007). In 2005, Ward and Autexier reported the effects of telomerase inhibition on drug resistant leukemia and breast cancer cells by the non-nucleosidic small  44  molecule inhibitor BIBR1532, a propriety formulation from Boehringer Ingelheim (Damm, Hemmann et al. 2001). This drug impairs telomere elongation by affecting telomerase translocation or promoting the disassociation of the enzyme from the telomere end (Pascolo, Wenz et al. 2002).  They observed an increase in  chemotherapy sensitivity when drug resistant leukemia and breast cancer cells were concurrently treated with BIBR1532. However, because continuous BIBR1532 treatment was found to decrease the proliferative capacity of these cells as the number of population doublings increased and progressively sensitized cells to chemotherapeutic agents, the effects of BIBR1532 treatment were also determined to be telomere length dependent (Ward and Autexier 2005).  GRN163L: specific inhibitor of telomerase Oligonucleotide-based inhibitors of telomerase designed to target the TER template may provide a more promising telomerase-based, anti-tumor therapy. Pharmacokinetic studies of phosphorothioate oligonucleotides show that they do not cross the blood-brain-barrier, are well absorbed and rapidly distributed. They also demonstrate low levels of toxicity during clinical trials (Geary, Yu et al. 2001). DNA oligonucleotides complementary to hTER have been demonstrated to inhibit telomerase activity (Feng, Funk et al. 1995), but are readily hydrolyzed by nucleases, decreasing their stability in cells. Studies then began looking at using oligonucleotide motifs known to be nuclease resistant. Peptide nucleic acids (PNA) were reported to inhibit telomerase activity with higher specificity and affinity than phosphorothioate oligomers (Norton, Piatyszek et al. 1996). PNAs targeting hTERT  45  were also reported to reduce cell growth and induce apoptosis in human prostate cancer cells (Folini, Brambilla et al. 2005). Unfortunately, although strategies for the delivery of these PNAs in cultured cells have been reported (Hamilton, Simmons et al. 1999; Villa, Folini et al. 2000), cellular uptake is inefficient. The human prostate carcinoma cell line DU145 treated with a 2’-O-methoxyethyl oligonucleotide, which acts as a potent telomerase inhibitor by binding to the hTER template, showed an increase in sensitivity to cisplatin or carboplatin. Long-term, sustained telomerase inhibition (55 or 65 days) prior to treatment resulted in an increase in the DNA damaging agents’ cytotoxicity, however, short-term inhibition (30 or 45 days) showed no effects, leading to the conclusion that telomere shortening is required for any observable synergistic effect (Chen, Koeneman et al. 2003). GRN163L is a 13-base, lipid modified N3’-P5’ thiophosphoramidate oligomer, complementary to the template region of hTER. GRN163L binds with high affinity to telomerase (Akiyama, Hideshima et al. 2003; Asai, Oshima et al. 2003) and has been demonstrated to effectively inhibit the enzyme, resulting in telomere length shortening and subsequent growth arrest. The 5’-lipid palmitoyl domain facilitates cellular and tissue penetration, as well as makes it more acid resistant than other anti-telomerase oligonucleotides, thereby increasing the cellular uptake and bioavailability of the drug (Herbert, Gellert et al. 2005). GRN163L shows antitumor effects in several human cancers, including breast, liver, lung, and multiple myeloma, both in vitro and in vivo (Dikmen, Gellert et al. 2005; Djojosubroto, Chin et al. 2005; Herbert, Gellert et al. 2005; Gellert, Dikmen et al. 2006; Hochreiter, Xiao et al. 2006). This drug is currently undergoing clinical trials in patients with chronic  46  lymphocytic leukemia, multiple myeloma, solid tumor malignancies, locally recurrent or metastatic breast cancer and advanced or metastatic non small cell lung cancer (Brower 2010).  47  HYPOTHESIS We hypothesize that telomerase plays a positive role in DNA damage protection and/or repair.  Transient inhibition of telomerase at the time of genotoxic stress  will potentiate the cytotoxicity of DNA damaging agents in a telomere length independent manner (Fig 5).  48  Figure 5. Hypothesis: Telomerase plays a positive role in DNA damage protection and/or repair. Inhibition of telomerase at the time of genotoxic stress will potentiate the cytotoxicity of DNA damaging agents in a telomere length independent manner.  49  Goals of present study Specific aim 1 Determine the appropriate dose and treatment timeframe for telomerase inhibitor GRN163L required for the effective inhibition of telomerase in human carcinoma cell lines MCF-7, HT29 and LS180.  Specific aim 2 Evaluate the effects of concurrent telomerase inhibition on the cytotoxicity of DNA damaging agents etoposide, irinotecan, bleomycin and oxaliplatin on MCF-7 and HT29 cell lines.  Specific aim 3 Investigate the relationship between homologous recombination DNA repair regulator ATM kinase and telomerase inhibition in determining the cytotoxic effects of DNA damaging agent etoposide.  50  METHODS Cell culture: The human breast adenocarcinoma cell line MCF-7 and human colorectal carcinoma cell lines HT29 and LS180 (ATTC, Manassas, VA) were cultured in T-75 flasks at a starting density of 1  106 cells in Dulbecco’s modified Eagle’s  medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and 100U each of penicillin and streptomycin. Cells were maintained at 37 C and in the presence of 5% CO2. Media was renewed 2 or 3 times per week and cells were split upon reaching 80-90% confluency.  GRN163L preparation: GRN163L (Geron Corporation, Menlo Park, CA) was prepared as a 5mM stock solution in PBS. 60% of the calculated volume of PBS was used to dissolve the GRN163L powder and vortexed for 15 minutes in a 15ml conical tube.  The oligonucleotide concentration was measured using UV-  spectrophotometer absorbance at A260nm and GRN163L concentration was calculated as mg/ml, using the supplied formula (ODdilution)/23.7.  Due to  solubility issues, the stock solution was re-suspended and oligonucleotide concentration measured before every use to ensure an accurate estimate of the stock concentration.  GRN163L treatment: To determine the dose of telomerase inhibitor needed for the effective inhibition of telomerase activity, MCF-7, HT29 or LS180 cells were plated in six-well plates at a starting density of 3 105 cells/well. 24 hours after seeding,  51  cells were treated with 1 ml of a 2-fold dilution series (0.1-32 μM) of GRN163L in DMEM and incubated for 24 hours. Treated cells were harvested using trypsinEDTA, transferred into 1.5ml eppendorf tubes and spun down for 5 min at 1500 rpm.  Whole cell lysate preparation: Harvested cells were washed twice with ice cold PBS and centrifuged for 1 minute at 5000 rpm at 4 C. Cell lysate was prepared in 50 l of cell hypotonic cell lysis buffer (20mM Hepes, 2mM MgCl 2, 0.2mM EGTA, 1.0mM DTT, 0.1mM PMSF, 10% glycerol). Cell suspensions were then snap frozen in liquid nitrogen for 30 seconds, thawed in a 37 C water bath and vortexed well. This freeze/thaw procedure was repeated three times. 5.0M NaCl was then added to the lysate in two parts to a final concentration of 400mM and left on ice for 15 minutes for soluble nuclear protein extraction.  Lysates were cleared by  centrifugation at 13,200 rpm for 15 minutes and supernatant (whole cell lysate fraction) was saved in aliquots for subsequent analysis.  Bradford protein assay: protein concentrations of whole cell lysate fractions were measured using the Bradford protein assay (Bio-Rad Laboratories Inc., Hercules, CA). The dye reagent was prepared by diluting one part Dye Reagent Concentrate with 4 parts deionized water. A protein standard series was prepared using a 2-fold dilution series of Bovine Serum Albumin (0.0625-0.5 mg/ml). Whole cell lysate samples were prepared by diluting 2 l of each sample with 98 l of lysis buffer. 10 l of each standard and diluted samples were pipetted into separate wells in a 96-well  52  plate. 200 l of the diluted dye reagent was added to each well. The plate was incubated at room temperature for 5 minutes and absorbance was measured at 595nm.  Telomerase repeat amplification protocol (TRAP): TRAP assay was carried out using 0.5, 0.2, 0.08, 0.032 g (2.5-fold dilution series) of whole cell lysate as the source of telomerase. Samples were incubated with the forward primer M2 (5’AATCCGTCGAGCAGAGTT-3’), and telomerase mediated addition of the 6-nt telomeric repeats to the 3’-end of the primer were carried out at 30 C for 1 hour, in the presence of 2ng/ l M2 primer, 1x PCR buffer (20mM Tris-Cl (pH = 8.0), 15mM MgCl2, 680mM KCL, 0.5% Tween 20, 10mM EDTA), and 50 M dNTPs. Reactions were heat-inactivated at 95 C for 30 seconds. The primer extension products were then amplified by PCR using 1.25U Pfu DNA polymerase and 0.1 g of a Cy5fluorophor-labeled reverse primer CX3 (CY5-5’-CCGCGCCCTAACCCTAACCT-3’) in 1x PCR buffer with 10-7 copies of ITAS internal control. The resulting products were analyzed by gel electrophoresis using 10% nondenaturing polyacrylamide gel (10% bis:acryl (19:1), 0.5x TBE, 0.1% APS, 0.1% TEMED) and visualized using the TyphoonTM scanner fitted with the appropriate laser and filters (Center for Drug Research and Development, UBC).  Combination treatments: Telomerase inhibition: MCF-7 or HT29 cells were plated in 6-well plates at a starting density of 3 105 cells/well. For combination treatment, 24 hours after seeding, 1ml 53  of 10µM of GRN163L in DMEM was added to each well and the cells were incubated for 24 hours. 48 hours following seeding, all cells were treated with a 2-fold dilution series of etoposide (Sigma, Saint Louis, MO) (MCF7: 0.025-12.8µM; HT29: 0.05-12.8 µM; in 0.15% DMSO vehicle control), bleomycin (Calbiochem, San Diego, CA) (MCF7: 0.01-10.24µg/ml; HT29: 0.02-20.48µg/ml), irinotecan (Sigma) (MCF-7: 0.1102.4µM; HT29: 0.1-102.4 µM) or oxaliplatin (Tocris Bioscience, Bristol, UK) (MCF7: 0.05-12.8µM; HT29: 0.1-102.4 µM) in the presence or absence of 10µM GRN163L. 24 hours following the addition of DNA damaging agents, cells were harvested using trypsin-EDTA and collected for the colony forming unit assays and western blot analysis.  ATM inhibition: MCF-7, HT29 or LS180 cells were divided into four treatment groups: etoposide alone, etoposide + 10 M GRN163L, etoposide + 10 M Ku55933, and etoposide with 10 M GRN163L + 10 M Ku55933. described above.  Cells were plated as  For combination treatment with telomerase inhibition, 24h  following seeding, cells were treated with 10 M GRN163L. 48h following seeding, all cells were treated with a 2-fold dilution series of etoposide (Sigma) (MCF7: 0.025-12.8µM; HT29: 0.05-12.8µM; LS180: 0.05-12.8µM; 0.15% DMSO vehicle control) in the presence or absence of 10µM GRN163L and/or 10µM of ATM inhibitor Ku55933 (Sun, Luo et al. ; Hickson, Zhao et al. 2004; Pereg, Shkedy et al. 2005) (Calbiochem, San Diego, CA). 24 hours after the initiation of the cocktail combination treatment, cells were harvested using trypsin-EDTA and collected for the colony forming unit assays, immunostaining and western blot analysis.  54  Colony forming unit (CFU) assay: Following combination treatments, MCF-7, HT29 or LS180 cells were harvested using trypsin EDTA, transferred into 1.5ml eppendorf tubes and spun down for 5 min at 1500 rpm. Cells were then re-suspended in 0.9 ml of media consisting of 0.4% agar, 20% 2x DMEM, 45% 1x DMEM and10% FBS at a density of 3 104 cells/ml and poured over a 2 ml base layer of media consisting of 0.53% agar, 26.7% 2x DMEM, 30% 1x DMEM and 10% FBS and allowed to solidify. Suspended cells were incubated at 37°C and 5% CO2 for 2 weeks and CFUs were quantified by counting colonies >50 microns in diameter. The number of CFUs formed were compared to the control (no DNA damaging agents) cells for the series and reported as a percent loss of CFUs. For example, cells receiving GRN163L and DNA damaging agent etoposide, in varying doses, will be normalized to cells receiving GRN163L alone. The dose response relationships and LD 50 values were compared between cells treated with DNA damage alone and cells treated with the combination therapy.  Each treatment group was done in duplicate and each  experiment was repeated at least 3 times. Dose-response and statistical analyses were performed using GraphPad Prism software.  Statistical analysis: Dose response curves and LD50 values were generated using the GraphPad Prism software.  Cells treated with DNA damaging agent in  combination with 10 M GRN163L and/or 10 M Ku55933 were normalized to the control group for the series (these are defined as cells receiving 10 M GRN163L/10 M Ku55933/10 M GRN163L + 10 M Ku55933, respectively, but  55  were not incubated with any DNA damaging agent). LD50 values from different treatment groups were compared using the unpaired t-test with the GraphPad Prism software. Two-tailed p values  0.05 were determined to be statistically  significant.  Western blot:  Following combination treatment, MCF-7 or HT29 cells were  harvested using trypsin EDTA and whole cell lysate was prepared as described above. 40 g of whole cell lysate was separated by SDS-PAGE electrophoresis on a 10% gel (separating gel: 10% acryl, 0.13% bis, 0.375M Tris (pH= 8.8), 0.1% SDS; stacking gel: 5.3% acryl, 0.14% bis, 0.125M Tris (pH= 6.8), 0.1% SDS) and transferred onto polyvinylidene fluoride membranes using the Hoeffer Western Transfer system, for one hour at 500mA. Each PVDF blot was stained with 2% Ponceau S dye to evaluate the effectiveness of the gel transfer.  The PVDF  membranes were blocked using 4% BSA in 1xPBS, supplemented with 0.04% NP-40 for one hour at 4 C, then incubated with polyclonal antibodies against p53 (Cell Signaling Technology, Inc, Boston, MA) (1:1000 dilution in 4% BSA in PBS with 0.04% NP-40) overnight at 4 C. Following incubation, the PVDF membranes were washed twice in PBS with 0.04% NP-40 and once with PBS. The membranes were probed with a peroxidase-conjugated AffiniPure donkey anti-rabbit IgG secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) (1:10000 dilution in 4% BSA in PBS with 0.04% NP-40). The membranes were washed as previously  described  and  detection  was  performed  using  enhanced  56  chemiluminescence (AmershamTM ECL Plus Western Blotting Detection System, GE Healthcare UK Ltd, Buckinghamshire, UK) against Kodak X-ray film.  Immunostaining: Following combination treatment, MCF-7 and HT29 cells growing on glass coverslips were fixed with 3.7% paraformaldehyde for 15 minutes. Cells were washed and solubilized with PBS-t (PBS + 0.1% triton X-100 (Sigma)) 3 times for one minute each. Cells were then incubated in PBS-t containing 3% normal goat serum (NGS) (Vector Laboratories Inc., Burlingame, CA) at room temperature for one hour. Following 3 one minute washes with PBS-t, cells were incubated in rabbit polyclonal H2AX (Ser139) antibody (Millipore, Billerica, MA) (1:400 dilution in PBS-t + 3% NGS) at 4 C overnight. Cells were washed 3 times with PBS-t for one minute each, then incubated in Texas Red (Sigma) (1:1500 dilution in PBS + 3% NGS) for one hour at room temperature, covered with foil to minimize light exposure. Cells were washed 3 times with PBS for one minute each and DNA was counterstained with YO-PRO-1 iodide (Invitrogen) (1:2000 in PBS + 3% NGS) at room temperature for 15 minutes. After washing the cells 3 times with PBS for one minute each, coverslips were mounted onto glass microscope slides using Fluoromount-G (SouthernBiotech Associates, Birmingham, AL).  Immunostained  cells were visualized using a Zeiss Pascal Excite laser scanning confocal microscope fitted with appropriate lasers and filters (BioImaging Facility, UBC).  57  RESULTS Testing GRN163L for effective telomerase inhibition We used MCF-7 (breast cancer), LS180 and HT29 (colorectal cancer) cells in our cellular study. MCF-7 cells are human mammary epithelial adenocarcinoma cells, while both HT29 and LS180 cells are human colorectal epithelial adenocarcinoma cells, and all have high telomerase activity. To our knowledge, our study represents the first characterization of telomerase inhibition combination therapy in colorectal cancers. The cytogenetic and other molecular characteristics of these cell lines are listed (Table 1).  Cell line  Karyotype  p53 status  Microsatellite stability  MCF-7  Hypertriploid-  Wildtype  Stable  hypertetraploid HT29  Hypertetraploid  R273H  Stable  LS180  Diploid  Wildtype  Unstable  Table1. Cell line properties  Telomerase inhibition was achieved using GRN163L, a competitive telomerase antagonist. In order to determine the effective dose of GRN163L on telomerase activity inhibition in MCF-7, HT29 and LS180 cells, each cell line was treated with a two-fold dilution series of GRN163L in media (0.25-32µM) for 24h at 37 C. We observed that GRN163L stock solution concentration decreased in 4 C storage. We 58  suspected that the GRN163L drug molecule might have precipitated out of solution overtime, leading to the changes in soluble drug concentration. To compensate for this, stock solution was vigorously vortexed for at least 15 minutes before each use, and drug concentration was verified using spectrophotometric absorbance analysis. The dose of GRN163L needed for effective (>100x) inhibition of telomerase activity for each cell line was determined using the telomere repeat amplification protocol (TRAP) assay. This in vitro telomerase activity assay is divided into two parts. First, active telomerase from sample whole cell extracts is allowed to elongate a singlestranded oligo primer, with the addition of the signature 6-nt telomeric repeats to the 3’-end of this primer. The primer extension products are single stranded and vary in length depending on the numbers of telomeric repeats added during the synthesis reaction. In the second half of the protocol, the primer extension products are amplified by PCR. These double-stranded TRAP products are then separated by polyacrylamide gel electrophoresis. Telomerase products appear as a ladder, where each step of the ladder is separated from the previous product by 6-bp in length, corresponding to the quantum addition of telomeric DNA products in units of 6nt repeats. The dose required for sufficient inhibition of telomerase activity was determined to be at least 8µM for MCF-7 (Fig 6a), HT29 (Fig 6b) cells and LS180 cells (Fig 6c). Comparison of telomerase activity of cell extracts treated with 8µM to control profiles shows an ~125-fold decrease in activity.  We determined a 24h  treatment with 10µM of GRN163L should be sufficient for effective (>100x) telomerase inhibition, at the time of the introduction of DNA damaging agents.  59  Telomerase inhibition increases the cytotoxicity of cell cycle specific genotoxic compounds The effects of concurrent telomerase inhibition on the cytotoxicity of DNA damaging agents was determined with MCF-7 breast cancer and HT29 colorectal cancer cells. With the CFU assay, changes in cell viability, cell proliferation and DNA damage repair potential were studied.  Augmentation in treatment effects  (cytotoxicity) due to the addition of GRN163L was measured by graphical/statistical means. Cells were treated with chemotherapeutic agents that induce DNA damage through different mechanisms of action and target different phases of the cell cycle. Etoposide and irinotecan are cell cycle specific agents, while bleomycin and oxaliplatin are non cell cycle specific chemotherapeutic agents. To determine the effects of telomerase inhibition on the cytotoxicity of these genotoxic compounds, MCF-7 cells were treated with a two-fold serial dilution of etoposide (0.025-12.8µM; solubilized in 0.15% DMSO and the same concentration of DMSO was added as a vehicle control), bleomycin (0.01-10.24 µg/ml in PBS), irinotecan (0.1-102.4µM in PBS), or oxaliplatin (0.05-12.8µM in PBS) for 24h in the presence or absence of 10µM GRN163L (previously determined optimal GRN163L concentration required for effective inhibition (>100x) of telomerase activity). GRN163L was introduced 24h prior to the addition of genotoxic compounds, and cells were incubated for an additional 24h following the initiation of DNA damage. Cells were harvested by trypsinization, counted with a Coulter Cell Counter  60  Figure 6. Effect of GRN163L on telomerase activity in a) MCF-7, b) HT29, and c) LS180 cells. Cells were plated in 6-well plates and treated with a two-fold serial dilution of GRN163L (0.2532μM). We determined a 24h treatment with 10µM of GRN163L to be sufficient for effective (>100x) telomerase inhibition for all cell lines.  61  and seeded for CFU assays. CFU trays were allowed to recover for two weeks under standard tissue culture conditions.  Following this recovery period, the CFU  numbers were determined for each treatment, and the percent loss of CFU was calculated and compared to control cells.  Dose response relationships were  estimated using the graphical program GraphPad Prism.  The presence of  telomerase inhibition increased the cytotoxic effects of S/G2 phase specific double strand DNA damaging agent etoposide, with the combination treatment leading to a greater loss of MCF-7’s anchorage-independent growth. LD50 for etoposide alone was 0.38 to 0.14  0.07µM, while the addition of 10µM GRN163L reduced the LD 50 2.8-fold 0.02µM (p<0.005) (Fig 7). The shift in the dose response curve argues that  the addition of GRN163L potentiates the effect of etoposide treatment. In addition, the parallel dose response curves indicate that the cytotoxic mechanism of etoposide is conserved.  Increased cytotoxicity by the addition of telomerase  inhibitor to the S phase specific genotoxic agent irinotecan was also observed. The LD50 for irinotecan alone was 4.42 therapy was 2.08  0.97µM, while the LD50 for the combination  0.67µM (p=0.02), a 2.1-fold decrease (Fig 8). In contrast,  concurrent telomerase inhibition did not increase the cytotoxicity in MCF-7 cells treated with non cell cycle specific genotoxic agents bleomycin (bleomycin alone LD50= 0.13  0.003µg/ml; combination LD50 = 0.13  oxaliplatin (oxaliplatin alone LD50= 0.38  0.03µg/ml; p=0.90) (Fig 9) or  0.04µM; combination LD50 = 0.39  0.07µM; p=0.77) (Fig 10). TRAP assays were performed on all corresponding treatment groups to ensure that 10 M of GRN163L inhibited telomerase activity as compared to cells receiving DNA damaging agent alone. 62  Figure 7. Cytotoxicity of etoposide in MCF-7 cells is increased by the addition of telomerase inhibitor GRN163L. a) Dose response curves showing growth inhibition effect of etoposide treatments, as a single agent or in combination with 10μM GRN163L in MCF-7 breast cancer cells. Experiments were repeated three times and each dose response assay was done in duplicates. b) Mean LD50 values for MCF-7 cells treated with etoposide alone (E alone) or in combination with 10μM GRN163L (E + G). Statistical significance was determined using the unpaired t-test. Two-tailed p-values of 0.05 were considered as statistically significant. Graphs were generated by the GraphPad Prism software. Error bars denote SD.  63  Figure 7 continued. PAGE of TRAP products showing the telomerase activity profile for MCF7 cells treated with etoposide alone or in combination with 10 M GRN163L. c) Etoposide treatment does not apparently affect steady state telomerase activity. d) GRN163L treatment effectively inhibited telomerase activity (>100x). Fluorescent gel scanning was performed with the Typhoon imager.  64  Figure 8. Cytotoxicity of irinotecan in MCF-7 cells is increased by the addition of telomerase inhibitor GRN163L. a) Dose response curves showing growth inhibition effect of irinotecan treatments, as a single agent or in combination with 10μM GRN163L on MCF-7 breast cancer cells. Experiments were repeated three times and each dose response assay was done in duplicates. b) Mean LD50 values for MCF-7 cells treated with irinotecan alone (I alone) or in combination with 10μM GRN163L (I + G). Statistical significance was determined using the unpaired t-test. Two-tailed p-values of 0.05 were considered as statistically significant. Graphs were generated by the GraphPad Prism software. Error bars denote SD.  65  Figure 8 continued. PAGE of TRAP products showing the telomerase activity profile for MCF7 cells treated with irinotecan alone or in combination with 10 M GRN163L. c) Irinotecan treatment does not apparently affect steady state telomerase activity. d) GRN163L treatment effectively inhibited telomerase activity (>100x). Fluorescent gel scanning was performed with the Typhoon imager.  66  Figure 9. Lack of potentiation of cytotoxicity in MCF-7 cells between telomerase inhibition by GRN163L and non cell cycle specific bleomycin. a) Dose response curves showing growth inhibition effect of bleomycin treatments, as a single agent or in combination with 10μM GRN163L on MCF-7 breast cancer cells. Experiments were repeated three times and each dose response assay was done in duplicates. b) Mean LD50 values for MCF-7 cells treated with bleomycin alone (B alone) or in combination with 10μM GRN163L (B + G). Statistical significance was determined using the unpaired t-test. Two-tailed p-values of 0.05 were considered as statistically significant. Graphs were generated by the GraphPad Prism software. Error bars denote SD.  67  Figure 9 continued. PAGE of TRAP products showing the telomerase activity profile for MCF7 cells treated with bleomycin alone or in combination with 10 M GRN163L. c) Bleomycin treatment does not apparently affect steady state telomerase activity. d) GRN163L treatment effectively inhibited telomerase activity (>100x). Fluorescent gel scanning was performed with the Typhoon imager.  68  Figure 10. Lack of potentiation of cytotoxicity between telomerase inhibition by GRN163L and non cell cycle specific oxaliplatin. a) Dose response curves showing growth inhibition effect of oxaliplatin treatments, as a single agent or in combination with 10μM GRN163L on MCF-7 breast cancer cells. Experiments were repeated three times and each dose response assay was done in duplicates. b) Mean LD50 values for MCF-7 cells treated with oxaliplatin alone (O alone) or in combination with 10μM GRN163L (O + G). Statistical significance was determined using the unpaired t-test. Two-tailed p-values of 0.05 were considered as statistically significant. Graphs were generated by the GraphPad Prism software. Error bars denote SD.  69  Figure 10 continued. PAGE of TRAP products showing the telomerase activity profile for MCF-7 cells treated with oxaliplatin alone or in combination with 10 M GRN163L. c) Oxaliplatin treatment does not apparently affect steady state telomerase activity. d) GRN163L treatment effectively inhibited telomerase activity (>100x). Fluorescent gel scanning was performed with the Typhoon imager.  70  We repeated the same experiments with the colorectal cancer HT29 cells. Cells were treated with a two-fold serial dilution of etoposide (0.05-12.8µM; solubilized in 0.15% DMSO and the same concentration of DMSO was added as a vehicle control), bleomycin (0.02-10.24 µg/ml in PBS), irinotecan (0.1-102.4µM in PBS), or oxaliplatin (0.1-102.4µM in PBS) for 24h in the presence or absence of 10µM GRN163L. Similar to the results observed with the MCF-7 breast cancer cells, a statistically significant increase in cytotoxicity was observed with HT29 cells treated with cell cycle specific agents etoposide and irinotecan in combination with telomerase inhibition, when compare to the treatments with the genotoxic agents alone. The LD50 for etoposide as a single agent therapy was 2.4  0.41µM while the  LD50 for etoposide in combination with telomerase inhibition was reduced 2.5-fold to 0.96 0.24µM (p<0.01) (Fig 11). The LD50 for irinotecan as a single agent therapy was 4.28  1.10µM while the LD50 for irinotecan in combination with telomerase  inhibition was reduced 1.9-fold to 2.28  0.39µM (p=0.02) (Fig 12). In contrast,  analysis of graphical data, as well as statistical determination, of the cytotoxicity in cells treated with bleomycin in the presence of 10 M GRN163L showed no changes in the loss of viability compared with cells treated with bleomycin alone (bleomycin alone LD50= 0.63  0.15µg/ml; combination LD50 = 0.51  0.07µg/ml; p=0.3) (Fig  13). Lastly, similar to the case in MCF-7, telomerase inhibition in HT29 did not result in an increase in the cytotoxicity of oxaliplatin (oxaliplatin alone LD50 = 1.85 0.23µM; combination LD50 = 2.12 0.39µM; p>=0.30) (Fig 14). The increase in cytotoxicity with the addition of telomerase inhibition was only observed with cell cycle specific DNA damaging agents. Addition of GRN163L 71  Figure 11. Cytotoxicity of etoposide is increased by the addition of telomerase inhibitor GRN163L. a) Dose response curves showing growth inhibition effect of etoposide treatments, as a single agent or in combination with 10μM GRN163L on HT29 colorectal cancer cells. Experiments were repeated three times and each dose response assay was done in duplicates. b) Mean LD50 values for HT29 cells treated with etoposide alone (E alone) or in combination with 10μM GRN163L (E + G). Statistical significance was determined using the unpaired t-test. Two-tailed p-values of 0.05 were considered as statistically significant. Graphs were generated by the GraphPad Prism software. Error bars denote SD.  72  Figure 11 continued. PAGE of TRAP products showing the telomerase activity profile for HT29 cells treated with etoposide alone or in combination with 10 M GRN163L. c) Etoposide treatment does not apparently affect steady state telomerase activity. d) GRN163L treatment effectively inhibited telomerase activity (>100x). Fluorescent gel scanning was performed with the Typhoon imager.  73  Figure 12. Cytotoxicity of irinotecan is increased by the addition of telomerase inhibitor GRN163L. a) Dose response curves showing growth inhibition effect of irinotecan treatments, as a single agent or in combination with 10μM GRN163L on HT29 colorectal cancer cells. Experiments were repeated three times and each dose response assay was done in duplicates. b) Mean LD50 values for HT29 cells treated with irinotecan alone (I alone) or in combination with 10μM GRN163L (I + G). Statistical significance was determined using the unpaired t-test. Two-tailed p-values of 0.05 were considered as statistically significant. Graphs were generated by the GraphPad Prism software. Error bars denote SD.  74  Figure 12 continued. PAGE of TRAP products showing the telomerase activity profile for HT29 cells treated with irinotecan alone or in combination with 10 M GRN163L. c) Irinotecan treatment does not apparently affect steady state telomerase activity. d) GRN163L treatment effectively inhibited telomerase activity (>100x). Fluorescent gel scanning was performed with the Typhoon imager.  75  Figure 13. Lack of potentiation of cytotoxicity between telomerase inhibition by GRN163L and non cell cycle specific bleomycin a) Dose response curves showing growth inhibition effect of bleomycin treatments, as a single agent or in combination with 10μM GRN163L on HT29 colorectal cancer cells. Experiments were repeated three times and each dose response assay was done in duplicates. b) Mean LD50 values for HT29 cells treated with bleomycin alone (B alone) or in combination with 10μM GRN163L (B + G). Statistical significance was determined using the unpaired t-test. Two-tailed p-values of 0.05 were considered as statistically significant. Graphs were generated by the GraphPad Prism software. Error bars denote SD.  76  Figure 13 continued. PAGE of TRAP products showing the telomerase activity profile for HT29 cells treated with bleomycin alone or in combination with 10 M GRN163L. c) Bleomycin treatment does not apparently affect steady state telomerase activity. d) GRN163L treatment effectively inhibited telomerase activity (>100x). Fluorescent gel scanning was performed with the Typhoon imager.  77  Figure 14. Lack of potentiation of cytotoxicity between telomerase inhibition by GRN163L and non cell cycle specific oxaliplatin a) Dose response curves showing growth inhibition effect of oxaliplatin treatments, as a single agent or in combination with 10μM GRN163L on HT29 colorectal cancer cells. Experiments were repeated four times and each dose response assay was done in duplicates. b) Mean LD50 values for HT29 cells treated with oxaliplatin alone (O alone) or in combination with 10μM GRN163L (O + G). Statistical significance was determined using the unpaired t-test. Two-tailed p-values of 0.05 were considered as statistically significant. Graphs were generated by the GraphPad Prism software. Error bars denote SD.  78  Figure 14 continued. PAGE of TRAP products showing the telomerase activity profile for HT29 cells treated with oxaliplatin alone or in combination with 10 M GRN163L. a) Oxaliplatin treatment does not apparently affect steady state telomerase activity. b) GRN163L treatment effectively inhibited telomerase activity (>100x). Fluorescent gel scanning was performed with the Typhoon imager.  79  to non cell cycle specific DNA damaging agents did not result in an increase in their cytotoxic effects. Our results suggest that the potential protective functions of telomerase occur specifically during the S and G2 phases of the cell cycle.  Cytotoxicity of etoposide is further increased with telomerase inhibition and parallel ATM inhibition. We next tested the effect of GRN163L telomerase inhibition on the S/G2 phase specific DNA damaging agent etoposide cytotoxicity while concurrently inhibiting ataxia telangiectasia mutated (ATM) kinase. Our data indicated telomerase activity protects against double-stranded DNA breaks during this phase of the cell cycle and the ATM-mediated homologous recombination repair mechanism is the dominant form of repair during this period. This serine/threonine-specific protein kinase is a key player in the cell’s response to double-stranded DNA breaks, specifically homologous recombination. ATM phosphorylates a number of tumor suppressor proteins, such as p53, NBS1, BRCA1, Chk2 and H2AX, in response to doublestranded DNA breaks, leading to the activation of DNA repair, cell cycle arrest or apoptosis. Following 24h of telomerase inhibition with 10µM of GRN163L, MCF-7 and HT29 were treated with 10µM Ku55933, a small molecule ATM inhibitor that binds to the ATP active site of ATM (Hickson, Zhao et al. 2004), in combination with the same 2-fold dilution series of etoposide for each cell line as described above for an additional 24h. The increased cytotoxicity observed due to telomerase inhibition in combination with etoposide treatment was further augmented by the addition of  80  10µM Ku55933 in MCF-7 cells (LD50: etoposide alone = 0.36 GRN163L = 0.19 0.03µM; w/10µM Ku55933 = 0.12 10µM Ku55933 = 0.08  0.08µM; w/10µM  0.03µM; w/10µM GRN163L +  0.01µM; p values for comparison between these treatments  are listed with the figures) (Fig 15).  TRAP assays were performed on the  corresponding treatment groups to ensure that 10 M GRN163L inhibited telomerase activity. In contrast, 10 M Ku55933 treatment did not display any effect. We repeated the same experiment with the colorectal cancer cells HT29. HT29 cells were treated with a 2-fold dilution series of etoposide ranging from 0.0512.8µM (solubilized in 0.15% DMSO; same concentration of DMSO was added as a vehicle control) in the presence of 10µM GRN163L and/or KU55933. The triple agents combination experiments illustrated the same super shift of the doseresponse curves (LD50: etoposide alone = 1.95 with 10µM GRN163L = 0.66 Ku55933 = 0.35  1.02µM; etoposide in combination  0.32µM; etoposide in combination with 10µM  0.02µM; etoposide in combination with 10µM GRN163L and  10µM Ku55933 = 0.32  0.04µM; p values for comparison between these treatments  are listed with the figures) (Fig 16). However, the comparison between etoposide cytotoxicity in cells receiving parallel KU55933 treatments did not reach statistical significance when compared to cells receiving both GRN163L and KU55933 treatments. TRAP assays were performed to monitor telomerase inhibition by GRN163L. In parallel, HT29 cells were analyzed for H2AX foci formation, and ATM inhibition by KU55933 was confirmed. We performed western analysis for p53 stabilization in this cell model; however, HT29 harbors a dominant negative version 81  Treatment Etoposide alone Etoposide + 10 M GRN163L Etoposide + 10 M Ku55933 Etoposide + 10 M GRN163L + 10 M Ku55933 MCF-7 p values E E+G E+K E+G+K  LD50 0.36 0.19 0.12 0.08  0.08 0.03 0.03 0.01  E  E+G  E+K  E+G+K  -<0.01 <0.001 <0.001  <0.01 --<0.01  <0.001 --<0.05  <0.001 <0.01 <0.05 --  M M M M  Figure 15. Etoposide cytotoxicity was further augmented by parallel ATM and telomerase inhibition in MCF-7 cells. a) Dose response curves showing growth inhibition effect of etoposide treatments, as a single agent or in combination with 10μM GRN163L and or 10μM Ku55933 on MCF-7 breast cancer cells. Experiments were repeated four times and each dose response assay was done in duplicates. b) Mean LD50 values for MCF-7 cells treated with etoposide alone (E alone), in combination with 10μM GRN163L (E + G), in combination with 10μM Ku55933 (E + K), or in combination with both 10μM GRN163L and 10μM Ku55933 (E + G + K). LD50 and p values are listed in the tables below. Statistical significance was determined using the unpaired t-test. Twotailed p-values of 0.05 were considered as statistically significant. Graphs were generated by the GraphPad Prism software. Error bars denote SD.  82  Figure 15 continued. PAGE of TRAP products showing the telomerase activity profile for MCF-7 cells treated with etoposide alone or in combination with 10 M GRN163L and or 10 M Ku55933. c) Etoposide treatment does not apparently affect steady state telomerase activity. d) GRN163L treatment effectively inhibited telomerase activity (>100x). e) Ku55933 treatment does not apparently affect steady-state telomerase activity. f) GRN163L treatment still effectively inhibited telomerase activity in combination with Ku55933 treatment. Fluorescent gel scanning was performed with the Typhoon imager.  83  Treatment Etoposide alone Etoposide + 10 M GRN163L Etoposide + 10 M Ku55933 Etoposide + 10 M GRN163L + 10 M Ku55933 HT29 p values E E+G E+K E+G+K  1.95 0.66 0.35 0.32  E  E+G  E+K  E+G+K  -<0.05 <0.01 <0.01  <0.05 --<0.05  <0.01 -->0.05  <0.01 <0.05 >0.05 --  LD50 1.02 0.32 0.02 0.04  M M M M  Figure 16. ATM inhibition did not increase cytotoxicity in HT29 cells treated with etoposide and GRN163L. a) Dose response curves showing growth inhibition effect of etoposide treatments, as a single agent or in combination with 10μM GRN163L and or 10μM Ku55933 on HT29 colorectal cancer cells. Experiments were repeated five times and each dose response assay was done in duplicates. b) Mean LD50 values for HT29 cells treated with etoposide alone (E alone), in combination with 10μM GRN163L (E + G), in combination with 10μM Ku55933 (E + K), or in combination with both 10μM GRN163L and 10μM Ku55933 (E + G + K). LD50 and p values are listed in the tables below. Statistical significance was determined using the unpaired t-test. Twotailed p-values of 0.05 were considered as statistically significant. Graphs were generated by the GraphPad Prism software. Error bars denote SD.  84  Figure 16 continued. PAGE of TRAP products showing the telomerase activity profile for LS180 cells treated with etoposide alone or in combination with 10 M GRN163L and or 10 M Ku55933. c) Etoposide treatment does not apparently affect steady state telomerase activity. d) GRN163L treatment effectively inhibited telomerase activity (>100x). e) Ku55933 treatment does not apparently affect steady-state telomerase activity. f) GRN163L treatment still effectively inhibited telomerase activity in combination with Ku55933 treatment. Fluorescent gel scanning was performed with the Typhoon imager.  85  of p53 which appears to play a role in the carcinogenesis process. We observed an over-expression of p53 signal in resting (without DNA damage) HT29 cells and concluded that quantification of this protein signal will not reflect the regulation by ATM kinase. In contrast to MCF-7 breast cancer cells, HT29 colorectal cancer cells exhibit a lack of potentiation by GRN163L when ATM function is inhibited. To differentiate whether the lack of GRN163L potentiation is a function of HT29’s properties, or a common observation in colorectal cancer models, we tested the same combination treatments with another colorectal cancer line LS180. LS180 cells displayed regular p53 regulation and high resting telomerase activity. LS180 cells were treated with 10µM GRN163L and/or KU55933, together with a 2-fold dilution series of etoposide ranging from 0.05-12.8µM (solubilized in 0.15% DMSO and the same concentration of DMSO was added as a vehicle control). Our data showed that, unlike HT29 cells but in agreement with MCF-7 breast cancer cells, pairwise comparisons of all treatment groups showed significant differences (LD50: etoposide alone = 0.90 0.03µM; etoposide in combination with10µM GRN163L = 0.37 in combination with 10µM Ku55933 = 0.52 with 10µM GRN163L and 10µM Ku55933 = 0.25  0.05µM; etoposide  0.04µM; etoposide in combination 0.03µM; p values for comparison  between these treatments are listed with the figures) (Fig 17). Again, TRAP activity profiles were determined in these cells to confirm the effectiveness of GRN163L inhibition of telomerase. In parallel, H2AX foci cytostaining (Fig 18) and p53 western signal analysis (Fig 19) both confirmed the suppression of ATM kinase activity by the actions of KU55933.  86  Treatment Etoposide alone Etoposide + 10 M GRN163L Etoposide + 10 M Ku55933 Etoposide + 10 M GRN163L + 10 M Ku55933 LS180 p values E E+G E+K E+G+K  0.90 0.37 0.52 0.25  LD50 0.03 0.05 0.04 0.03  E  E+G  E+K  E+G+K  -<0.005 <0.005 <0.005  <0.005 --<0.01  <0.005 --<0.05  <0.005 <0.01 <0.05 --  M M M M  Figure 17. Etoposide cytotoxicity was further augmented by parallel ATM and telomerase inhibition in LS180 cells. a) Dose response curves showing growth inhibition effect of etoposide treatments, as a single agent or in combination with 10μM GRN163L and or 10μM Ku55933 on MCF-7 breast cancer cells. Experiments were repeated three times and each dose response assay was done in duplicates. b) Mean LD50 values for MCF-7 cells treated with etoposide alone (E alone), in combination with 10μM GRN163L (E + G), in combination with 10μM Ku55933 (E + K), or in combination with both 10μM GRN163L and 10μM Ku55933 (E + G + K). LD50 and p values are listed in the tables below. Statistical significance was determined using the unpaired t-test. Twotailed p-values of 0.05 were considered as statistically significant. Graphs were generated by the GraphPad Prism software. Error bars denote SD.  87  Figure 17 continued. PAGE of TRAP products showing the telomerase activity profile for LS180 cells treated with etoposide alone or in combination with 10 M GRN163L and or 10 M Ku55933. c) Etoposide treatment does not apparently affect steady-state telomerase activity. d) GRN163L treatment effectively inhibited telomerase activity (>100x). e) Ku55933 treatment does not apparently affect steady state telomerase activity. f) GRN163L treatment still effectively inhibited telomerase activity in combination with Ku55933 treatment. Fluorescent gel scanning was performed with the Typhoon imager.  88  Figure 18. Immunostaining for MCF-7 cells treated with etoposide alone or in combination with 10 M Ku55933. Cells were treated with antibodies specific for H2AX foci (red) and the nucleus was identified with YO-PRO-1 iodide (green). H2AX foci (red) form in response to etoposide treatment. Treatment with 10 M Ku55933 reduced the number of nuclear H2AX foci.  89  Figure 18 continued. Immunostaining for MCF-7 cells treated with etoposide alone or in combination with 10 M Ku55933. Cells were treated with antibodies specific for H2AX foci (red) and the nucleus was identified with YO-PRO-1 iodide (green). H2AX foci (red) form in response to etoposide treatment. Treatment with 10 M Ku55933 reduced the number of nuclear H2AX foci.  90  a)  b)  Figure 19. p53 expression is decreased in combination studies. a) MCF-7 and b) LS180 cells treated with etoposide in combination with 10 M Ku55933 expressed lower levels of p53 as compared to cells treated with etoposide alone.  91  DISCUSSION Telomerase activity is responsible for the de novo synthesis of telomeric repeats, maintaining chromosome end structure and genome integrity. Continuous telomerase expression in tumor cells is necessary for the immortal phenotype associated with cancer growth. My data implicate that the high telomerase activity observed in tumor cells as necessary for the survival of cancer cells, in a manner independent of its role in telomere length maintenance. My data also present the first characterization of the mechanism of this telomerase protective function against cellular stress and provides the impetus for further preclinical research on combining telomerase inhibitor and genotoxic chemicals in breast and colorectal cancer therapies.  Insights into the mechanism of telomerase’s function in DNA repair Previous data demonstrated that telomerase is involved in regulating DNA damage response by mediating cellular repair capacity (Masutomi, Possemato et al. 2005).  Based on this earlier observation, we hypothesized that combining  telomerase inhibition with genotoxic chemotherapeutic treatment should result in a more effective anti-cancer therapy than the genotoxic agent alone. In this study, the specific telomerase inhibitor GRN163L, a 13-base oligomer complimentary to the template sequence of TER, was used for the inhibition of telomerase activity. Unlike genetic methods for telomerase inhibition, the use of a small molecule inhibitor allowed for a tight control on the timing of telomerase inhibition. In addition, treating cells with GRN163L for a short time frame (less than 92  48 hours) allowed me to presume that any potentiation in cytotoxicity of the DNA damaging agents by GRN163L are likely telomere length independent. This is of particular importance, from a basic science point of view, in the validation of the non-canonical mode of telomerase actions. Although previous combination studies demonstrated synergistic effects of GRN163L in combination with ionizing radiation (Gomez-Millan, Goldblatt et al. 2007) treatments, the conclusion drawn pointed to a telomere-length dependent mechanism. Enhanced radiation sensitivity by GRN163L application was observed following long-term (42 days) drug treatment, with no observed differences in short-term (2 and 9 days) and intermediate inhibition (20 days) (Gomez-Millan, Goldblatt et al. 2007). Accordingly, this synergistic effect was attributed to the generation of critically short telomeres following long-term telomerase inhibition. My data revealed an important distinction from the conclusions obtained with these earlier combination studies.  I argue that in these earlier studies,  telomerase inhibition has an apparent additive mode of action with the genotoxic compounds. In contrast, when telomere length is unlikely to be affected, such as in the short inhibition time course followed with my experimental scheme, a clear, mechanism-based pattern of potential GRN163L partners emerges:  while  telomerase inhibition had no apparent potentiation effect on the cytotoxicities of the non-cell cycle specific genotoxic agents bleomycin and oxaliplatin, the combination of GRN163L with cell cycle specific DNA damaging agents etoposide and irinotecan led to marked increase in their respective cytotoxic effects. To my knowledge, this is the first demonstration linking telomerase actions with a specific DNA repair  93  mechanism. My data demonstrated that telomerase is involved in DNA repair occurring in the S/G2 phase of the cell cycle. Based on this functional specificity of telomerase action, GRN163L potentiation of genotoxicity is only observed with those chemotherapeutic compounds with the correct (S/G2 specific) mode of action.  The effects of telomerase inhibition effects on chromatin structure A previous study showed that GRN163L acts synergistically with paclitaxel (Goldblatt, Gentry et al. 2009), a chemotherapeutic agent that stabilizes microtubules and inhibits cellular proliferation (Jordan, Wendell et al. 1996; Rowinsky 1997). In this study, even though GRN163L treatment was carried out for 5 days, the synergistic mechanism with paclitaxel was determined to be telomere length independent. It was suggested that GRN163L induced telomerase inhibition affects cytoskeleton architecture, thereby resulting in changes in actin filament organization and focal adhesion formation. This model is extrapolated with data from a previous report suggesting telomerase’s involvement in DNA chromatin structural maintenance (Masutomi, Possemato et al. 2005). While my data cannot support or dispute this notion, given that my experiments were designed to examine possible DNA damage response mechanisms of telomerase rather than its function in chromatin maintenance.  However, it is unlikely that telomerase, a nuclear  enzyme, will have direct effects on cytoplasmic polymer protein organization. The colony forming unit (CFU) assay was picked as a functional readout, based on its ability to reveal cytotoxicity data beyond that of immediate cell death. Upon the induction of DNA damage, three different responses could be elicited: 1)  94  damaged cells can engage in programmed or non-programmed cell death, which is quantitatively measured by many cell based colorimetric cell viability assays such as the MTT and the trypan blue exclusion assays; 2) cells could be permanently arrested from re-entering the cell cycle, in a process known as stress-induced senescence. Growth arrested cells are no longer able to give rise to progeny, but yet remain metabolically active, thereby counted as viable by the colorimetric assays; 3) finally, cells could temporally be arrested in the various phases of the cell cycle awaiting the completion of DNA repair, regardless of fidelity (either faithfully restored to the correct nucleotide sequence after repair, or introduced erroneous sequence/mutation by imprecise repair mechanism); these populations of cells are also regarded as viable by the colorimetric cell viability assays. In contrast, only cells that are temporarily arrested and able to return to regular growth, within the two-week incubation/recovery period will be counted as viable in the CFU assay. It remained possible that choosing the more stringent CFU assay as the functional readout may have augmented my ability to uncover the protective effect of telomerase, specifically for DNA damage during the S/G2 phase of the cell cycle, in a telomere length independent manner.  The relationship between ATM kinase and telomerase in homologous recombination repair during S/G2 phase of the cell cycle The homologous recombination DNA repair mechanism is prominent during the S/G2 phase of the cell cycle, where the damaged chromosome is repaired using the paired sister chromatid. This repair mechanism is controlled by the master  95  regulator ATM kinase (Morrison, Sonoda et al. 2000), which auto-phosphorylates upon the induction of DNA damage, as well as phosphorylates several key proteins involved in DNA repair, damage checkpoints, and apoptosis pathways, such as p53, NBS1, BRCA1, and CHK2. Inhibition of ATM by the small molecule inhibitor Ku55933 resulted in an additional increase of the cytotoxicity of the DNA damaging agent etoposide in combination with GRN163L in the breast cancer cell line MCF-7, as well as colorectal cell lines HT29 and LS180. The potentiation of the cytotoxicity of etoposide when both ATM and telomerase are inhibited suggests that these two enzymes could work in an independent, parallel mechanism. Alternatively, it is highly possible that ATM and telomerase work in the same pathway, yet each enzyme is involved in several other non-overlapping mechanisms in response to the induction of DNA damages. When comparing the addition of GRN163L inhibition to ATM-inhibited cells, I came to a different conclusion, based on the identity of the cancer cells. The addition of GRN163L further increased the cytotoxicity of etoposide in ATM-inhibited LS180 and MCF-7 cells, but this difference was insignificant in HT29 colorectal cancer cells. Since both LS180 and HT29 are colorectal cancer cell types, it was ruled out that this lack of additional cytotoxicity is based on the cancer type. When I compared the two colorectal cancer cell lines’ characteristics, it was found that while LS180 cells are microsatellite unstable and thereby defective in mismatch DNA repair capacity (de la Chapelle 2003), HT29 cells are microsatellite stable. On the other hand, HT29 cells carry a dominant negative mutant form of p53 (R237H), an important downstream effector of the ATM kinase functions, while LS180 cells carry the  96  wildtype version of the protein. The R237H p53 mutation found in the central DNA binding domain of the protein abolished the transcription factor’s DNA binding affinity. Since p53 works as a tetramer, the presence of one non-DNA-binding molecule is enough to poison the complex, rendering any cells harboring even a single mutant allele null for p53 function (Cho, Gorina et al. 1994; Rolley, Butcher et al. 1995). This mutation is described as a “hot spot” mutant as it is one of the most common p53 mutations in human cancers (Hollstein, Rice et al. 1994). Given the intimate functional dependency between ATM kinase and p53, I was inclined to model my data using the p53 interaction hypothesis. Based on this model, the present data suggest that the telomerase enzyme may have a regulatory role in p53 maintenance/activation. The lack of a functional p53 pathway in HT29 cells results in the lack of GRN163L addition effects on etoposide cytotoxicity in the presence of ATM inhibition. This is an interesting observation, and the potential for further investigation using biochemical or genetic means is warranted.  The future of telomerase-based clinical therapy in human cancer Previous combination studies have demonstrated that inhibiting telomerase leads to an increase of cellular sensitivity to chemotherapeutic agents. However, in these reports telomerase inhibition was achieved using gene knockout, RNA interference or dominant negative strategies which employ a long time course to achieve complete telomerase inhibition, leading to the conclusion that the cytotoxic synergism is telomere length dependent. My data demonstrated the presence of a telomere length independent DNA damage protective/repair function by  97  telomerase. Using the small molecule oligomer telomerase inhibitor GRN163L to precisely control the timing of exposure, I concluded that this effect is specific to the S/G2 phase of the cell cycle. This is an exciting finding, both in the perspective of telomerase mechanism discovery, as well as from the cancer therapeutic perspective. Given that small molecule inhibitors of telomerase are in various phases of the clinical development pipeline (Harley 2008; Shay and Keith 2008), the potential to design new chemotherapy, based on the transient inhibition of telomerase function, while inducing DNA damages in a S-G2 cell cycle phase specific phase manner, should produce effective new treatment strategies against breast, colorectal and other cancer types. In contrast to etoposide, which is only active at the S/G2 phase of the cell cycle, GRN163L-dependent potentiation was reduced in cells treated with another cell cycle specific agent, irinotecan. Inhibition of topoisomerase I by irinotecan could create single-strand breaks in a non cell-cycle specific manner, by virtue of the enzyme’s involvement in transcription complexes.  However, cytotoxicity of  irinotecan peaked at the S phase of the cell cycle, when single stranded DNA breaks were converted into double-strand breaks as they went through DNA replication. Thus, irinotecan can induce toxicity with the creation of single-strand breaks in a non cell cycle specific manner, but cytotoxicity related to the generation of doublestrand DNA breaks are produced in a cell cycle (S/G2) specific manner.  As  telomerase protection against DNA damage is only effective during the S/G2 phase of the cell cycle, the reduction in the potentiation effect by GRN163L may reflect irinotecan’s promiscuous mechanisms during different phases of the cell cycle. To  98  extrapolate from this observation, the lack of potentiation by GRN163L, observed with cell cycle non-specific agents bleomycin and oxaliplatin, may simply reflect the small overall percentage of cells passing through S/G2 phase at the time when DNA damage was induced. Based on the conclusion from my data, a significant increase in cytotoxicity of S/G2 specific DNA damaging agents in the presence of telomerase inhibition, is due to the removal of telomerase mediated DNA damage protection/repair; one could design rational, new chemotherapy combinations that exploit this fact. In addition to the inclusion of S/G2 specific DNA damaging agents (for which the topoisomerase II inhibitors are the only drug class that fits this bill), cell cycle restricted agents that specifically arrest cancer cells at the S/G2 junction could be included in a designer cocktail therapy. By including compounds that force an increase population of cells to arrest at the S/G2 phase, one could maximize the effects of the other components of this cocktail: telomerase inhibition by small molecule inhibitors, in combination with any genotoxic compounds. 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"Genomic organization and promoter characterization of the gene encoding the human telomerase reverse transcriptase (hTERT)." Gene 232(1): 97-106. Wong, J. M. and K. Collins (2003). "Telomere maintenance and disease." Lancet 362(9388): 983-8. Wong, J. M., L. Kusdra and K. Collins (2002). "Subnuclear shuttling of human telomerase induced by transformation and DNA damage." Nat Cell Biol 4(9): 731-6. Wong, K. K., S. Chang, S. R. Weiler, S. Ganesan, J. Chaudhuri, C. Zhu, S. E. Artandi, K. L. Rudolph, G. J. Gottlieb, L. Chin, F. W. Alt and R. A. DePinho (2000). "Telomere dysfunction impairs DNA repair and enhances sensitivity to ionizing radiation." Nat Genet 26(1): 85-8. Wright, W. E., M. A. Piatyszek, W. E. Rainey, W. Byrd and J. W. Shay (1996). "Telomerase activity in human germline and embryonic tissues and cells." Dev Genet 18(2): 173-9. Wright, W. E. and J. W. Shay (1992). "Telomere positional effects and the regulation of cellular senescence." Trends Genet 8(6): 193-7. Wright, W. 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"Origin and evolution of retroelements based upon their reverse transcriptase sequences." EMBO J 9(10): 3353-62. Yang, J., E. Chang, A. M. Cherry, C. D. Bangs, Y. Oei, A. Bodnar, A. Bronstein, C. P. Chiu and G. S. Herron (1999). "Human endothelial cell life extension by telomerase expression." J Biol Chem 274(37): 26141-8. Ye, J. Z. and T. de Lange (2004). "TIN2 is a tankyrase 1 PARP modulator in the TRF1 telomere length control complex." Nat Genet 36(6): 618-23. Yi, X., D. M. White, D. L. Aisner, J. A. Baur, W. E. Wright and J. W. Shay (2000). "An alternate splicing variant of the human telomerase catalytic subunit inhibits telomerase activity." Neoplasia 2(5): 433-40. Zaug, A. J., J. Linger and T. R. Cech (1996). "Method for determining RNA 3' ends and application to human telomerase RNA." Nucleic Acids Res 24(3): 532-3. Zhang, X., V. Mar, W. Zhou, L. Harrington and M. O. Robinson (1999). "Telomere shortening and apoptosis in telomerase-inhibited human tumor cells." Genes Dev 13(18): 2388-99. Zhang, Z., X. Yang, Y. Zhang, B. Zeng, S. Wang, T. Zhu, R. B. Roden, Y. Chen and R. Yang (2006). "Delivery of telomerase reverse transcriptase small interfering RNA in complex with positively charged single-walled carbon nanotubes suppresses tumor growth." Clin Cancer Res 12(16): 4933-9. Zhao, J. Q., R. M. Glasspool, S. F. Hoare, A. Bilsland, I. Szatmari and W. N. Keith (2000). "Activation of telomerase rna gene promoter activity by NF-Y, Sp1, and the retinoblastoma protein and repression by Sp3." Neoplasia 2(6): 531-9. Zhao, J. Q., S. F. Hoare, R. McFarlane, S. Muir, E. K. Parkinson, D. M. Black and W. N. Keith (1998). "Cloning and characterization of human and mouse telomerase RNA gene promoter sequences." Oncogene 16(10): 1345-50. Zhou, X. Z. and K. P. Lu (2001). "The Pin2/TRF1-interacting protein PinX1 is a potent telomerase inhibitor." Cell 107(3): 347-59. Zhu, Y., R. L. Tomlinson, A. A. Lukowiak, R. M. Terns and M. P. Terns (2004). "Telomerase RNA accumulates in Cajal bodies in human cancer cells." Mol Biol Cell 15(1): 81-90.  119  APPENDIX Telomerase has been suggested to participate in biological pathways that are important for cellular survival following genotoxic stress, independently of its role in telomere length maintenance (Kondo, Kondo et al. 1998; Fu, Begley et al. 1999; Ren, Xia et al. 2001; Gorbunova, Seluanov et al. 2002; Stewart, Hahn et al. 2002; Sharma, Gupta et al. 2003; Zhang, Chan et al. 2003).  Direct evidence has  demonstrated telomerase as a regulator of chromatin architecture and DNA repair in response to genotoxic stimuli (Masutomi, Possemato et al. 2005). However, the exact mechanism of how telomerase might achieve this novel function is not fully understood.  Aside from increased DNA repair, telomerase may participate in  several other possible cellular responses to DNA damaging events. Telomerase’s roles in DNA damage recognition, the promotion of cell survival by inhibiting apoptosis or cellular senescence, and the increase in the cellular tolerance to DNA damage by inducing adaptation (the restoration of normal cell division in site of persistent DNA damage signals).  These previous studies achieved telomerase  inhibition with RNA interference, gene knockout models, and dominant negative strategies, where telomerase inhibition was established before the onset of DNA damage initiation, making it difficult to determine the timeline of the enzyme’s involvement. The availability of small molecule inhibitor of telomerase provided us with the impetus to revisit this question. Using the telomerase specific GRN163L, one can initiate telomerase inhibition rather uniformly in a population of cells, in a timely manner, without the intervening genetic selection process. We hypothesized that 120  introduction of GRN163L at different time points (i.e. 24 hours before, in parallel or 4.5 hours after DNA damage induction by S/G2 phase specific etoposide), should allow us to glean on the requirements for telomerase activity in DNA damage survival pathways. The use of a small molecule inhibitor would allow us to control of the timing of telomerase inhibition and further probe the requirements for telomerase activity.  Because telomerase will be inhibited continuously once  GRN163L is added, due to its low Km, the different time points of addition will allow us to eliminate the enzyme’s involvement in the various mechanisms. We reasoned that if telomerase inhibition potentiates the cytotoxicity of DNA damaging agents when GRN163L is introduced after the genotoxic stimuli, then this would indicate that telomerase might potentially involve in late DNA damage response events, such as participation in cell recovery, survival or the adaptation process. If an increase in cytotoxicity is observed when GRN163L is added in parallel with etoposide, then this will suggest possible telomerase participation in DNA repair and damage survival. Lastly, if applying GRN163L 24 hours prior to the induction of DNA damage is necessary for the potentiation of cytotoxicity effects, then one would argue that telomerase activity is possible at play during the early events in DNA damage recognition, but we cannot rule out that continual telomerase inhibition during repair and/or damage survival could also be involved in the potentiation mechanism.  121  Methods  MCF-7 cells were plated in 6-well plates at a starting density of 3 105 cells/well. Telomerase inhibition was induced at different time points: 1) 24 hours after seeding, 1ml of 10µM of GRN163L in DMEM was added to each well and the cells were treated for 24 hours. Following treatment, the cells were then treated with 1ml of a 2-fold dilution series of etoposide (0.025-12.8µM in 0.15% DMSO vehicle control). 2) 48 hours after seeding, 1ml of 10µM of GRN163L in DMEM was added to each well in combination with 1ml of a 2-fold dilution series of etoposide (0.025-12.8µM in 0.15% DMSO vehicle control). 3) 48 hours after seeding, 1ml of a 2-fold dilution series of etoposide (0.025-12.8µM in 0.15% DMSO vehicle control) was added. 4.5 hours following etoposide treatment 1ml of 10µM of GRN163L in DMEM was added to each well. 24 hours following etoposide treatment, all cells were harvested using trypsin-EDTA and collected for colony forming unit assays and TRAP assays to ensure effective inhibition of telomerase activity (Appendix figure 1).  122  Figure A1. Schematic of the order of addition of GRN163L experiments in MCF-7 breast cancer cells. Telomerase inhibition by GRN163L was initiated at different time points: 24 hours prior to, concurrently with or 4.5 hours following the addition of varying dose of etoposide. 24 hours following the beginning of etoposide treatment, cells were collected for CFU assay and other biochemical analysis (TRAP and protein lysate collection).  123  Results  Treatment with GRN163L 24 hours prior to induction of DNA damage by etoposide resulted in a 1.7-fold reduction in LD50 value as compared to cells treated with etoposide alone (LD50: etoposide alone = 0.45 0.04µM; combination = 0.26 0.05µM; p=0.001). The addition of GRN163L in parallel with or 4.5 hours following etoposide increased cytotoxicity to a lesser extent, both producing an LD50 value of 0.37 0.04µM (p=0.02 and 0.03, respectively) (Appendix figure 2).  124  Treatment Etoposide alone Etoposide + 10 M GRN163L 24h before Etoposide + 10 M GRN163L Etoposide + 10 M GRN163L 4.5h after LS180 p values E E+G 24h before E+G E+G 4.5h after  E alone -<0.001 <0.05 <0.05  E+G 24h before <0.001 -<0.01 >0.05  0.45 0.26 0.37 0.37 E+G <0.05 <0.01 ->0.05  LD50 0.04 0.05 0.02 0.03  M M M M  E+G 4.5h after <0.05 >0.05 >0.05 --  Figure A2. Inhibition of telomerase 24 hours prior to the induction of DNA damage resulted in an optimal potentiation of etoposide cytotoxicity. a) Dose response curves showing growth inhibition effect of etoposide treatments, as a single agent or in combination with 10μM GRN163L introduced at different time points relative to the addition of the DNA damaging agent, on MCF-7 breast cancer cells. Experiments were repeated four times and each dose response assay was done in duplicates. b) Mean LD50 values for MCF-7 cells treated with etoposide alone (E alone) or in combination with 10μM GRN163L 24 hours before etoposide addition (E + G (24h before), concurrently with etoposide (E + G) or 4.5 hours following etoposide addition (E + G (4.5h after). LD50 and p values are listed in the tables below. Two-tailed p-values of 0.05 were considered as statistically significant. Graphs were generated by the GraphPad Prism software. Error bars denote SD.  125  Figure A2 continued. PAGE of TRAP products showing the telomerase activity profile for MCF-7 cells treated with etoposide alone or in combination with 10 M GRN163L. GRN163L treatment inhibited telomerase activity when added 24 hours before, concurrently, or 4.5 hours following the addition of etoposide, when cells are harvested 24 hours after the addition of etoposide (i.e. 48, 24 and 19.5 hours following the addition of GRN163L). Etoposide treatment does not apparently affect steady state telomerase activity. Fluorescent gel scanning was performed with the Typhoon imager.  126  Discussion  Introduction of telomerase inhibitor GRN163L at different time points in respect to the induction of DNA damage by etoposide allowed us to glean the possible mechanism of telomerase action in the DNA damage response pathway. Our results indicate that the timing of telomerase inhibition by GRN163L affects the potentiation of cytotoxicity of etoposide. Inhibition of telomerase 24 hours prior to the induction of DNA damage resulted in the optimal potentiation of etoposide cytotoxicity. These results suggest that telomerase is involved early in the DNA damage response pathway, either in DNA damage recognition and/or initial DNA repair events. Inhibition of telomerase concurrently with or 4.5 hours after the induction of DNA damage by the S/G2 phase specific genotoxic agent etoposide resulted in suboptimal potentiation of its cytotoxicity as compared to cells treated with GRN163L 24 hours prior to the initiation of genotoxicity. We reasoned that the addition of etoposide to a non-synchronized population of cells may provide an explanation for these results. At the time of etoposide addition, only the proportion of the cells in the S/G2 phases of the cell cycle will be immediately affected. In the remaining populations of cells, etoposide actions were delayed, due to the lag time required for these cells in reaching S/G2 phase of the cell cycle. This time lag allowed for GRN163L to initiate effective telomerase inhibition, and may be sufficient to allow for the observed potentiation of etoposide cytotoxicity in these subpopulation of cells.  127  Our results indicate that effective telomerase inhibition in MCF-7 cells at the time of genotoxic stimuli is necessary for the optimal potentiation of the cytotoxicity of etoposide. This finding also suggests that telomerase is involved in DNA damage recognition and early damage repair events.  Further experiments need to be  performed to determine the detailed molecular mechanism behind this novel noncanonical function of telomerase.  128  APPENDIX REFERENCES Fu, W., J. G. Begley, M. W. Killen and M. P. Mattson (1999). "Anti-apoptotic role of telomerase in pheochromocytoma cells." J Biol Chem 274(11): 7264-71. Gorbunova, V., A. Seluanov and O. M. Pereira-Smith (2002). "Expression of human telomerase (hTERT) does not prevent stress-induced senescence in normal human fibroblasts but protects the cells from stress-induced apoptosis and necrosis." J Biol Chem 277(41): 38540-9. Kondo, Y., S. Kondo, Y. Tanaka, T. Haqqi, B. P. Barna and J. K. Cowell (1998). "Inhibition of telomerase increases the susceptibility of human malignant glioblastoma cells to cisplatin-induced apoptosis." Oncogene 16(17): 2243-8. Ren, J. G., H. L. Xia, Y. M. Tian, T. Just, G. P. Cai and Y. R. Dai (2001). "Expression of telomerase inhibits hydroxyl radical-induced apoptosis in normal telomerase negative human lung fibroblasts." FEBS Lett 488(3): 133-8. Sharma, G. G., A. Gupta, H. Wang, H. Scherthan, S. Dhar, V. Gandhi, G. Iliakis, J. W. Shay, C. S. Young and T. K. Pandita (2003). "hTERT associates with human telomeres and enhances genomic stability and DNA repair." Oncogene 22(1): 131-46. 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 and R. A. Weinberg (2002). "Telomerase contributes to tumorigenesis by a telomere length-independent mechanism." Proc Natl Acad Sci U S A 99(20): 12606-11. Zhang, P., S. L. Chan, W. Fu, M. Mendoza and M. P. Mattson (2003). "TERT suppresses apoptotis at a premitochondrial step by a mechanism requiring reverse transcriptase activity and 14-3-3 protein-binding ability." FASEB J 17(6): 767-9.  129  

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