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Detection of telomeric DNA circles in human ALT cells using rolling circle amplification Tkac, Jan 2009

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DETECTION OF TELOMERIC DNA CIRCLES IN HUMAN ALT CELLS USING ROLLING CIRCLE AMPLIFICATION  by  Jan Tká B.Sc., The University of Waterloo, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in The Faculty of Graduate Studies (Medical Genetics) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  July 2009 © Jan Tkáë, 2009  Abstract  Telomeres, the nucleoprotein structures at the ends of linear chromosomes, maintain genomic stability by protecting chromosome ends from fusion, degradation, and processing by the DNA double-strand break repair machinery. Telomere shortening, which occurs naturally in somatic cells during aging, leads to cellular senescence or apoptosis. In contrast, germline cells and cancer cells acquire unlimited replicative potential by activating a telomere lengthening mechanism, generally via reactivating the enzyme telomerase reverse transcriptase. To date, drug development targeting cellular immortalization in cancer has focused on telomerase inhibition. However, in a subset of tumours and in vitro-immortalized cell lines, telomeres are maintained by homologous recombination-mediated pathways, termed alternative lengthening of telomeres (ALT). ALT tumours are expected to be refractory to anti-telomerase therapies, so the ability to rapidly and reliably screen for ALT status in tumour-derived cells is essential for guiding therapeutic strategies that target cellular immortalization. One characteristic of ALT-mediated telomere maintenance is the presence of extrachromosomal telomeric repeat-containing DNA circles (t-circles), which provide an attractive target for detection in screening applications. Current methods oft-circle detection require considerable amounts of cells, making them unsuitable for analysis of limited clinical samples. We optimized a screen for ALT status based on a novel technique of rolling circle amplification (RCA) oft-circles from extrachromosomal DNA extracts of human ALT cells. We demonstrate that RCA requires a much lower number of cells than previously established t-circle detection methods, and screening many samples can be performed in parallel, making RCA suitable for analyzing clinical samples. T-circles were reproducibly detected in human immortalized ALT cell lines, but not in telomerase-utilizing cell lines. In addition, ectopic over-expression of telomerase in an ALT cell line does not appear to affect t-circle formation. This suggests that presence of active telomerase within a cell does not inhibit all telomeric recombination reactions. The potential for RCA as a tool to screen tumour samples for ALT activity and the link between telomerase and ALT-based telomere lengthening mechanisms are discussed.  II  Table of Contents  Abstract  .  Table of Contents  ii iii  List of Tables  v  List of Figures  vi  List of Abbreviations  vii  Acknowledgements  viii  Dedication  ix  Chapter 1: Introduction  1  1.1 Telomeres  1  1.1.1 Telomere structure  2  1.1.2 Telomere function and maintenance  4  1.2 Alternative lengthening of telomeres (ALT)  7  1.2.1 ALT is a recombination-mediated mechanism  8  1.2.2 Telomere length in ALT cells  9  1.2.3 ALT-associated promyelocytic leukemia nuclear bodies  10  1.2.4 Instability at minisatellite MS32  11  1.2.5 Telomeric circles (t-circles) and other telomeric extrachromosomal DNA (ecDNA) molecules  12  1.3 Proposed mechanisms oft-circle generation in ALT cells  13  1.4 Proposed mechanisms of telomere elongation mediated by t-circles  15  1.SALTincancer  16  1.6 Objectives and rationale  17  III  Chapter 2: Materials and methods  19  2.1 Cell culture  19  2.2 Extrachromosomal DNA isolation  19  2.3 Rolling circle amplification  20  2.4 Tn-gel detection of RCA reaction products  21  2.5 Telomere Repeat Amplification Protocol (TRAP)  22  2.6 Ectopic over-expression of telomerase in ALT cells  23  Chapter 3: Results  25  3.1 RCA amplifies telomeric sequence-containing circular plasmid DNA  25  3.2 RCA-based assay detects t-circles in the human ALT cell line GM847  30  3.3 Thio-C-primed RCA is a linear reaction which facilitates highly-sensitive detection oft-circles 33 3.4 RCA detects t-circles in human ALT cell lines but not in human telomerase utilizing cell lines  37  3.5 Telomerase reconstitution in ALT cells does not affect t-circle formation  40  Chapter 4: Discussion  47  4.1 RCA-based detection oft-circles is potentially more sensitive than the widely used 2D PFGE method 4.1.1 Detection of telomeric circles in single cells  47 49  4.2 The effects of telomerase activity on the ALT pathway  51  4.3 T-circles as by-products of overly long telomeres  54  4.4 General conclusions  56 57  References  iv  A  saiolTo-1 .ioj uoaios pasnq-yj>j org ut posn souij OJfl3)W U01431?O1 AuSSU  JVJJJ  rz aqn  seqej jo  Sfl  List of Figures  Figure 1.1  Telomeres form a protective cap sequestering the 3 ‘-G-rich overhang into a t-loop  3  Figure 1.2  Telomere length is under strict regulatory control  6  Figure 1.3  Potential mechanisms oft-circle formation in ALT cells  14  Figure 1.4  Potential mechanisms of telomere elongation mediated by t-circles  15  Figure 3.1  Rolling circle amplification  25  Figure 3.2  Amplification of plasmid DNA by RCA yields long, non-migrating ssDNA product  27  Figure 3.3  SSB improves the efficiency of RCA  29  Figure 3.4  Determination of optimal thio-C and SSB concentrations to detect t-circles in GM847 ALT cells  32  Figure 3.5  Linear versus exponential RCA reaction  34  Figure 3.6  Time-course RCA  35  Figure 3.7  Titration of cell number in the RCA reaction  36  Figure 3.8  RCA-based screen detects t-circles in human ALT cell lines  39  Figure 3.9  MSCV-based expression constructs containing telomerase subunits  41  Figure 3.10 Expression of telomerase components in VA-13 ALT cells  42  Figure 3.11 TRAP assay confirms reconstituted telomerase activity in VA-13 + hTERT + hTR cells  44  Figure 3.12 Telomerase reconstitution in VA-13 ALT cells does not affect t-circle formation  46  Figure 4.1  Telomere dynamics in VA- 13 ALT cells over-expressing ectopic telomerase, .53  vi  List of Abbreviations  ALT APB  Alternative lengthening of telomeres Alternative lengthening of telomeres-associated promyelocytic leukemia nuclear body CO-FISH Chromosome orientation fluorescence in situ hybridization D-Ioop Displacement loop dsDNA Double-stranded DNA Extrachromosomal DNA ecDNA EM Electron microscopy EtBr Ethidium bromide FACS Fluorescence-activated cell sorting Green fluorescent protein GFP Holliday junction HJ IRES Internal ribosome entry site LTR Long terminal repeat MSCV Murine stem cell virus mtDNA Mitochondrial DNA PD Population doubling 2D PFGE Two-dimensional pulsed-field gel electrophoresis PGK Phosphoglycerin kinase PML Promyelocytic leukemia protein POT1 Protection of telomeres 1 Q-FISH Quantitative fluorescence in situ hybridization Rad50151152 Radiation sensitive isolate 50/51/52 Repressor/activator protein 1 Rapi Rolling circle amplification RCA SCE Sister chromatid exchange SSB Single-stranded DNA binding protein ssDNA Single-stranded DNA SV4O Simian virus 40 T-circle Telomeric repeat-containing circular DNA molecule T-loop Telomere loop T-SCE Telomeric sister chromatid exchange TERT Telomerase reverse transcriptase catalytic subunit TIN2 TRF 1-interacting nuclear factor 2 TPP1 Tll’.12- and POT1-interacting protein 1 TR Telomerase RNA component TRAP Telomere repeat amplification protocol TRE? Terminal restriction fragment TRF1/2 Telomeric repeat binding factor 1/2 YFP Yellow fluorescent protein  VII  Acknowledgements  I would like to thank Dr. Peter Lansdorp for giving me the opportunity to be part of this truly exciting field of research and for all his advice. All members of the Lansdorp laboratory for providing scientific discussion, technical advice, and most importantly, a terrific work environment. Dr. Karel Riha (Gregor Mendel Institute, Vienna, Austria) for sharing his rolling circle amplification prQtocol. Dr. David Huntsman, Dr. Louis Lefebvre, and Dr. Ann Rose, for their insight and helpful discussion of this study. Dr. Ester Falconer and Dr. Mark Hills for critically reviewing this dissertation. This work was largely funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) Postgraduate Scholarship.  VIII  Dedication  ‘Tüto prácu venujem mofim roéicom, Ivanovi ‘Thátovi a R,uene ‘Tháéovej. a&ujem ‘Vdm zapomoc a fi(avne za 1ásu, kjorou ma neustá(e oôoliacujete. cDauje.m ‘Vdm, e som, kjo som.  ix  Chapter 1: Introduction  1.1 Telomeres Seminal experiments, performed more than 60 years ago by Hermann MUller and Barbara McClintock, provided initial glimpses into the biological importance of the structures that cap the ends of linear chromosomes. Muller, studying chromosomal rearrangements in Drosophila melanogaster, observed that deletions and inversions of chromosome ends are extremely rare [MUller, 1938]. He posited that chromosome stability requires specialized terminal structures, which he called “telomeres” [Zakian, 1989]. McClintock, studying chromosome dynamics in Zea mays, noted that broken chromosomes have a tendency to fuse with one another, whereas natural chromosome ends are stable and do not fuse with broken ends or with one another [McClintock, 1941]. These and numerous subsequent studies established that telomeres have important functions in protecting chromosome ends from fusion and degradation, thus stabilizing chromosomes to ensure faithful DNA replication of the entire “coding” genome and proper segregation of genetic material into daughter cells upon cell division. These functions are indispensable for cellular fidelity and survival in all organisms with linear genomes. In the following sections, the current understanding of telomere structure and function is summarized along with the molecular mechanisms used to maintain functional telomeres.  I  1.1.1 Telomere structure Telomeres are nucleoprotein complexes that cap the ends of linear chromosomes. The DNA sequence of vertebrate telomeres consists of tandem arrays of 5 ‘-(TTAGGG)-3’ repeats [de Lange et a!., 1990; Moyzis eta!., 1988]. The majority of the telomeric tract is doublestranded (dsDNA); however, telomeres terminate in a single-stranded (ssDNA) overhang of the 3’-G-rich strand, which is 130-210 nucleotides in length [Allshire et at., 1989; de Lange et al., 1990; Makarov et a!., 1997]. This 3 ‘-G-rich overhang is crucially important for telomere capping function and for protection of chromosomal integrity [Henderson et a!., 1987; Henderson and Blackburn, 1989]. The capping mechanism in mammalian cells relies on the folding of telomeric DNA and strand invasion of the 3 ‘-G-rich overhang into proximal duplex telomeric tracts, resulting in the formation of a lariat structure called the telomere ioop, or t-loop [Griffith et a!., 1999]. The t-loop sequesters the free end of the chromosome, preventing inappropriate DNA repair reactions and activation of DNA damage checkpoints. A simplified schematic oft-loop formation and stabilization by protein binding is shown in Figure 1.1.  T-loops are stabilized by several telomere-binding proteins. Six proteins  —  telomeric repeat  binding factor (TRF) 1, TRF2, protection of telomeres 1 (POT 1), TRF 1-interacting nuclear factor 2 (T1N2), repressor/activator protein 1 (Rap 1), and Tll’sT2 and POT 1-interacting protein 1 (TPP 1)  —  are constitutively localized to telomeres, although their exchange at  telomeric sites is very dynamic [Mattern et at., 2004]. Together, these proteins form a complex known as “shelterin” [de Lange, 2005]. TRF1 and TRF2 interact with telomeric dsDNA, whereas POT1 binds the ssDNA G-rich overhang (Figure 1.1A. Rapi, TPP1, and  2  TIN2 interact directly with the TRF1/TRF2 protein complexes [Aubert and Lansdorp, 2008]. TRF 1 and TRF2 can fold telomeric DNA, stimulating t-loop formation (Figure 1.1 B) [Amiard eta!., 2007; Bianchi et aL, 1997]. In addition, TRF2 stabilizes t-loops by promoting the formation and protecting from cleavage the Holliday junction (HJ)-like structure that occurs at the site of strand invasion of the 3 ‘-G-rich overhang into proximal duplex telomeric sequences (Figure l.1C) [Poulet eta!., 2009]. Strand invasion produces an internal ssDNA displacement ioop (D-loop), which is stabilized by binding of POll [de Lange, 2005].  3’-G-rich overhang  TTAGGG A  ILATCCC ‘‘  5•  TRFI  .  •TRF2  • POTI  Foldin  Strand invasion  T4oop  C Figure 1.1 Telomeres form a protective cap sequestering the 3’-G-rich overhang into a t-loop. (A) “Open” telomere conformation with an exposed ssDNA 3’-G-rich overhang. (B) TRF1 and TRF2 bend telomeric DNA upon binding. (C) “Closed” telomere conformation results from TRF2-stimulated strand invasion of the 3’-G-rich overhang into proximal telomeric regions, forming the t-loop. A ssDNA D-loop created at the site of strand invasion is stabilized by binding of POT 1. The arrowhead indicates the HJ-like structure formed at the site of strand invasion. 3  1.1.2 Telomere function and maintenance To form t-loops and protect chromosomes from degradation or fusion, telomeres must have a certain length. Telomere attrition results from several cellular processes and accompanies organismal aging in long-lived species [Aubert and Lansdorp, 20081. Small consistent losses occur due to the “end-replication problem”, whereby gradual shortening is observed after each replication of linear DNA molecules [Olovnikov, 1973]. The end-replication problem results from the inability of conventional DNA polymerases to replicate the 5’ end of linear chromosomes during lagging strand synthesis [Watson, 1972]. For human somatic cells in vitro, the end replication problem and post-replication exonucleolytic processing of the 5’-Crich strand result in a telomere length decline of 50-150 base pairs (bp) with each population doubling [Martens et al., 2000]. Tn addition, larger sporadic truncations may occur via stochastic events, such as failure to repair oxidative damage to telomeric DNA, failure to properly process higher-order structures of G-rich DNA, and homologous recombination reactions [Lansdorp, 2005]. Loss of telomeric DNA eventually leads to growth arrest accompanied by cellular senescence and apoptosis [Martens et a!., 2000]. Senescence is induced when shorter telomeres, which are likely unable to form stable t-loops and thus perform their protective functions, are recogiiized by the DNA repair machinery as DNA double-strand breaks [d’Adda di Fagagna et a!., 2003]. Since cancer cells require many cell divisions to form tumours, by limiting the cellular replicative capacity, telomere attrition serves as a tumour suppressor mechanism in long-lived multicellular organisms, including humans [Aubert and Lansdorp, 2008].  4  Germline cells and some somatic cells, such as B lymphocytes, must maintain their replicative capacity and therefore maintain telomere length throughout the life of the organism. In these cells, telomeres are continuously maintained by the enzyme telomerase, the ribonucleoprotein reverse transcriptase capable of de novo addition of telomeric repeats to the 3’ end of the telomere. First discovered in the ciliate protozoan Tetrahymena [Greider and Blackburn, 1985; Greider and Blackburn, 1987], telomerase has since been identified in eukaryotes as diverse as fungi, plants, and mammals, including humans [Aubert and Lansdorp, 2008]. The minimal telomerase enzyme consists of the telomerase reverse transcriptase (TERT) catalytic subunit, and the telomerase RNA component (TR), which folds into a secondary structure with an exposed telomere repeat template region. TERT catalyzes the addition of telomeric repeats to the 3 ‘-G-rich strand using the TR RNA subunit as template.  While telomerase activity is detectable in the germline and in some stem cells, most somatic cells have little or no detectable telomerase activity. Thus, cells of the soma undergo telomere shortening until the telomeres are too short to form a protective cap and become recognized as double-strand breaks by the DNA repair machinery, inducing a signaling cascade leading to cellular senescence [d’Adda di Fagagna et al., 2003]. However, some cells may continue to divide post-senescence with increasing genomic instability due to uncapped telomeres. These cells eventually reach a point called crisis, where the majority of cells die by apoptosis. Sporadically, a minority of cells in crisis acquires the ability to lengthen telomeres, becoming immortalized (gaining unlimited replicative potential). Somatic cellular immortalization in vivo leads to tumorigenic growth. In humans, immortalization occurs via  5  two general mechanisms. The majority of cancer cells express and utilize telomerase for  immortalization; however, about 10 % of cancers and up to 40 % of in vitro-irnmortalized cell lines utilize a poorly-understood, recombination-mediated pathway termed alternative lengthening of telomeres (ALT) [Bryan et al., 1995; Muntoni and Reddel, 2005; Mumane et aL, 1994]. An illustration of telomere length dynamics with increasing cell divisions is shown in Figure 1.2.  Germane cells (t&omerase-expressinqj  ALT  ]r  TelomeraseJ  C,.  Mechanisms of cell immortalization  Cell divisions  Figure 1.2 Telomere length is under strict regulatory control. Telomere length is constitutively maintained in cells of the germline by telomerase. In somatic cells, which do not express telomerase, telomere length declines at a predictable rate with increasing cell divisions, as well as via unpredictable stochastic telomere truncation events. When telomeres become too short to perform their chromosome protective functions, senescence ensues. Some cells continue to divide pots-senescence until reaching a point of unbearable genomic instability in crisis, where most cells undergo apoptosis. Sporadic survivors emerge out of crisis by activating a telomere maintenance mechanism, either telomerase or ALT (indicated in red). These immortalized cells can lead to cancer.  6  1.2 Alternative lengthening of telomeres (ALT) ALT is defined by the ability of a cell to maintain its telomeres and replicate indefinitely in the absence of detectable telomerase activity [Kim et al., 1994]. Human ALT cell lines have undetectable expression of the full length hTERT transcript [Kilian et al., 1997]. The definition of ALT is somewhat problematic, as it is based on the absence of a particular phenotypic marker (telomerase activity) and not on the presence of any specific phenotype. Thus, many studies since the first identification of human ALT cells [Bryan et al., 1995; Murnane et al., 1994], have focused on characterizing the ALT phenotype beyond its nonreliance on telomerase. This work has resulted in the identification of some common phenotypic features identified in subsets of ALT cells. These include telomeric recombination and sister chromatid exchange (SCE) events, a distinct telomere length profile, presence of ALT-associated promyelocytic leukemia (PML) nuclear bodies (APB5), instability at a specific DNA minisatellite (MS32), and presence of telomeric repeatcontaining extrachromosomal DNA (ecDNA) [Royle et al., 2008]. These characteristics have been observed in some, but not all human ALT cells. Due to this phenotypic heterogeneity associated with ALT, it is likely that multiple molecular pathways of telomere lengthening in a non-telomerase setting are in existence [Stewart, 2005]. However, the overriding theme of the commonalities between different ALT cells is that ALT constitutes recombination mediated telomere maintenance, potentially involving inter- and intramolecular homologous recombination reactions, gene conversion-like events, and templating of telomere extension by telomeric ecDNA molecules [Henson et at., 2002].  7  12.1 ALT is a recombination-mediated mechanism Recombination-mediated telomere elongation has been most studied in yeast, where it occurs in mutants that lack telomerase {Lendvay et al., 1996; Lundblad and Szostak, 1989; Lundblad and Blackburn, 1993; McEachern and Blackburn, 1996; Teng and Zakian, 1999]. In Saccharomyces cerevisiae, deletion of telomerase leads to gradual telomere shortening over 50-100 generations, eventually resulting in growth senescence [Lundblad and Blackburn, 1993]. Only a minority of cells is able to bypass this replicative block and can continue to be serially streaked. These survivors rely on homologous recombination mechanisms to maintain telomeres, as evidenced by their dependence on Radiation sensitive isolate 52 (Rad52), a recombinase protein that is required for a majority of mitotic recombination reactions [Lundblad and Blackburn, 1993; Teng and Zakian, 1999]. The unusual arrangement of chromosome ends in S. cerevisiae, with telomeric repeat arrays separated by 6-7 kb subtelomeric elements termed Y’, leads to the emergence of two main types of survivors. These employ distinct pathways of telomeric recombination. Type I survivors are Rad5 1-dependent, and are typified by amplification of the subtelomeric Y’ elements and very short tracts of telomeric repeats. Type II survivors are Rad5 0-dependent and lack the Y’ amplification, but instead display greatly amplified telomeric repeat DNA [Lundblad and Blackburn, 1993]. It has been proposed that S. cerevisiae type II recombination is mechanistically similar to the mammalian ALT pathway [Lustig, 2003].  Unambiguous evidence that the ALT mechanism in human cells relies on some form of telomere-telomere recombination was provided by experiments wherein a plasmid DNA tag that had been targeted to some telomeres within an ALT cell clone had been propagated to a  8  number of other telomeres over the course of several population doublings (PDs) [Dunham et aL, 2000]. There was a progressive increase in the number of tagged telomeres with increasing PDs. This phenomenon was not seen when the tag was inserted into the subtelorneric region in ALT cells, nor when inserted into the telomeres of telomerase utilizing cell lines. This suggests that increased recombination rates are restricted to the telomeres in ALT cells and that telomeric recombination does not occur in telomerase utilizing cells. In subsequent years, evidence for inter- and intra-telomeric recombination and gene conversion-like events at the telomeres in ALT cells has been accumulated [Muntoni et al., 2009; Varley et al., 2002].  In addition, telomeric sister chromatid exchange (T-SCE) events (recombination between sister chromatids on the same chromosome), which are extremely rare in mortal and telomerase-immortalized cells, occur with a high frequency in ALT cells [Londoflo-Vallejo et a!., 2004]. Similarly to inter-chromosomal telomeric recombination events described above, the increased frequency of SCEs is limited to the telomeres in ALT cells, because no correlation was found with SCE rates at non-telomeric chromosome sites, and T-SCEs were not observed in non-ALT, Bloom syndrome cells which have very high global SCE rates [Londoho-Vallejo et a!., 2004].  1.2.2 Telomere length in ALT cells ALT cells have a telomere length phenotype that is distinct from telomerase-expressing immortal cells. The telomere length in human telomerase-utilizing cell lines is relatively short and homogeneous, with a mean telomere length of less than 10 kb [Bryan eta!., 1995;  9  Park et at., 1998]. In contrast, human ALT cells have a very heterogeneous distribution of telomere lengths, ranging from a few hundred base pairs to over 50 kb, with a mean length of approximately 20 kb [Bryan et at., 1995; Jeyapalan et at., 2008]. Furthermore, telomere lengths are heterogeneous within individual ALT cells, as evidenced by telomere visualization using quantitative fluorescence in situ hybridization (Q-FISH) of telomere repeat probes on metaphase chromosomes in mouse and humans [Hande et at., 1999; Perrem et at., 2001]. Some chromosome ends have no detectable telomere sequence, whereas others within the same cell have very strong telomere signal. In cells that become immortalized in the absence of telomerase activity during in vitro culturing, the timing. of the immortalization event (overgrowth of a culture in crisis by immortalized cells) correlates with rapid telomere lengthening and generation of length heterogeneity that are characteristic of the ALT mechanism [Yeager et at., 1999].  1.2.3 ALT-associated promyelocytic leukemia nuclear bodies The majority of ALT cell lines analyzed to date contain subnuclear structures called ALTassociated promyelocytic leukemia (PML) nuclear bodies (APBs) [Yeager et at., 1999]. PML nuclear bodies are punctate proteinaceous structures bound to the nuclear matrix in the cells of most, but not all tissues [Henson et at., 2002; Stuurman et at., 1992]. Numerous proteins transiently localize to the PML bodies, which seem to be extremely functionally diverse [Bernardi and Pandolfi, 2007]. PML nuclear bodies have been implicated  iii  numerous  cellular processes. These include tumour suppression, cell cycle regulation, senescence, apoptosis, inflammatory responses, antigen presentation, protein refolding and degradation, and differentiation [Henson et at., 2002].  10  In ALT cells, about 5 % of nuclei contain PML bodies that co-localize with telomeric DNA and/or telomere-associated proteins such as TRF 1 and TRF2 [Yeager et al., 1999]. These ALT-specific structures are termed APBs. APB function is not well understood, but it has been suggested that they may serve as repositories for proteins required for the ALT pathway [Royle eta!., 2008]. In various studies, APBs were shown to contain numerous proteins known to function in DNA replication, recombination, and repair: RAD5 1, RAD52, RPA, MRE1 1, RAD5O, NBS1, BLM and WRN [Johnson eta!., 2001; Wu eta!., 2000; Wu et al., 2003; Yankiwski et a!., 2000; Yeager et al., 1999]. Cell cycle analysis has shown that large APBs are most abundant during the G2 phase of the cell cycle, which coincides with the timing of telomere elongation in ALT cells [Grobelny et a!., 2000] This data suggests that APBs may be the site for telomere elongation in ALT cells, however there is no direct evidence for this. Telomeric-repeat containing ecDNA molecules (discussed in detail in Section 1.2.5) also localize to APBs [Muntoni and Reddel, 2005]. Interestingly, short linear extrachromsomal fragments preferentially localize within APBs and it was proposed that such localization may allow the cell to evade the DNA damage response triggered by the unprotected DNA ends [Fasching et a!., 2007].  1.2.4 Instability at minisatellite MS32 Although T-SCEs are elevated in ALT cells, the frequency of SCE at non-telomeric sites is not increased relative to telomerase-utilizing immortal cells [Londoño-Vallejo et a!., 2004]. Likewise, the overall frequency of homologous recombination is similar in ALT and telomerase-utilizing cell lines [Bechter et a!., 2003]. Thus the recombination-mediated telomere maintenance in ALT cells does not appear to be accompanied by a global increase  11  in recombination frequency. In light of this information, it was surprising that a specific minisatellite (a tandem array of 10  —  100 bp repeats {Sudbery, 2002]), MS32 was found to be  highly unstable in a subset of ALT cell lines, but not in telomerase-utilizing or mortal cells [Jeyapalan et al., 2005]. MS32 instability was also found in a subset of ALT-utilizing sarcomas, indicating that MS32 instability occurs in vivo and is not simply a cell culture artifact. The MS32 minisatellite is located on the distal portion of chromosome 1 (1q42.3) and consists of a tandem array of 29 bp repeats [Royle et al., 2008]. It is relatively GC-rich  (65 %), which may be indicative of a common mutational mechanism with telomeric DNA. Six other minisatellites (MS31A, CEB1, MS205, B6.7, MS1, DXYS14) distributed on various chromosomes did not show increased instability in ALT cells [Jeyapalan et al., 2005; Royle et al., 2008]. The initiation of M532 instability coincides with activation of ALT, suggesting an overlap between the underlying mechanisms and possibly between the proteins involved [Royle et al., 2008]. Apart from these associations, it is currently unclear why this specific minisatellite is so highly mutable in ALT cells.  1.2.5 Telomeric circles (t-circles) and other telomeric extrachromosomal DNA (ecDNA) molecules ALT cells contain a significant amount of telomeric repeat-containing ecDNA which can be linear or circular [Cesare and Griffith, 2004; Ogino et a!., 1998; Tokutake et al., 1998; Wang  et a!., 2004; Yeager et a!., 1999]. These molecules are thought to arise by homologous recombination events between telomeric repeats. Recently, a detailed study of the structures of telomeric DNA molecules in ALT cells showed that the majority of telomeric ecDNA consists of dsDNA circles and ssDNA of the G-rich strand [Nabetani and Ishikawa, 2009].  12  The study further identified a novel form of telomeric ecDNA, termed “t-complex” DNA, which consists of highly branched molecules thought to represent complex homologous recombination intermediates. Circular telomeric ecDNA molecules (known as telomeric circles or t-circles) are particularly interesting in the context of ALT, because they could be produced by intramolecular recombination within the characteristic long telomeres and may potentially serve as templates for telomere elongation by a rolling circle amplification mechanism [Nataraj an and McEachem, 2002]. T-circles are characteristic of the yeast type II survivor recombination mechanism (see Section 1.2.1), which results in amplification of telomeric DNA [Nosek eta!., 2006]. Furthermore, t-circles have been observed in a variety of distantly-related organisms, including yeast, plants, amphibians, and mammals (reviewed by [Tomaska et a!., 2004]).  1.3 Proposed mechanisms oft-circle generation in ALT cells T-circles have been identified in a number of cell lines which maintain telomeres by the ALT mechanism [Cesare and Griffith, 2004; Wang eta!., 2004]. The generation oft-circles in ALT cells relies on homologous intramolecular recombination between telomeric repeats [Dunham et a!., 2000; Henson et aL, 2002]. Although the exact mechanism of this process has not been elucidated, several models have been proposed (Figure 1.3). Currently, the most popular model oft-circle formation is based on a process termed “t-loop excision”, which results from recombination-mediated resolution of the Holliday junction-like structure (Figure 1.1 C) formed at the site of strand invasion of duplex telomeric DNA by the single stranded 3’ G-rich overhang (Figure 1 .3A). This process has been described in yeast type II survivors and also in Rapi mutants, which undergo telomere rapid deletion events [Lustig,  13  2003]. In human cell lines, evidence oft-loop excision came from studies where expression of a mutant allele of TRF2 (TRF2B) in several cell types caused massive sudden telomere loss [Wang et a!., 20041. This telomere shortening was accompanied by the generation oftloop-sized t-circles in TRF2-expressing cells. In this study, t-circle formation was dependent on XRCC3, a Rad5 1-related protein associated with Holliday junction resolvase activity in vitro [Liu et a!., 2004]. In addition to t-loop excision, t-circles may also arise by intramolecular recombination between duplex telomeric DNA tracts (Figure 1 .3B) or by ligation of linear telomeric ecDNA fragments (Figure 1 .3C) [Tomaska et a!., 2004].  A  B  C  Figure 1.3 Potential mechanisms of t-circle formation in ALT cells. (A) T-loop excision. (B) Intramolecular homologous recombination. (C) Circularization of linear telomeric ecDNA. [Tomaska et a!., 2004]  14  1.4 Proposed mechanisms of telomere elongation mediated by t-circles Several mechanisms have likewise been proposed for telomere lengthening mediated by t circles. T-circles may serve as templates for telomere extension by a rolling circle amplification (RCA) mechanism, primed by the 3 ‘-G-rich overhang. The overhang could invade duplex t-circles or bind to C-rich ssDNA t-circles (Figure 1 .4A). Branch migration of the overhang would allow essentially unlimited elongation of the chromosomal telomere [Henson et al., 2002]. Linear telomeric ecDNA fragments could likewise be extended by RCA on a t-circle template (Figure 1 .4B). Subsequent recombination with telomeres or copying of the elongated linear ecDNA by a gene conversion-like mechanism could lengthen chromosomal telomeres. Intermolecular recombination mechanisms may also mediate direct integration oft-circles into duplex telomeric DNA (Figure 1 .4C).  A  B  C  Figure 1.4 Potential mechanisms of telomere elongation mediated by t-circles. (A) Rolling circle amplification using dsDNA or ssDNA t-circles as templates. (B) Rolling circle amplification of linear telomeric ecDNA. (C) Recombinational integration of a t-circle. [Tomaska et al., 2004]  15  1.5 ALT in cancer The vast majority of human cancers actively maintain telornere lengths, allowing the tumour cells unlimited replicative potential. Surveys across many types olcancer have revealed that approximately 90 % express telomerase and thereby maintain a stable and homogeneous telomere length [Kim et al., 1994]. The remaining 10 % of tumours activate the ALT pathway of telornere maintenance [Bryan et al., 1995]. However, the prevalence of ALT activation in specific cancer types, such as some tumours of mesenchymal origin appears to be much higher. For example, about 47 % of osteosarcomas, 25 % of liposarcornas, and 34 % of astrocytomas activate ALT [Costa et al., 2006; Henson et al., 2005; Johnson et al., 2005]. The reasons for this tissue specificity in the utilization of ALT are not yet understood, but it has been proposed that expression of the telornerase reverse transcriptase catalytic subunit (TERT) may he more heavily repressed in tissues of mesenchymal origin [Henson et al., 2002].  To date, therapies targeting cellular immortalization in cancer have focused on inhibition of telomerase. However, as discussed above, a significant number of cancers utilize ALT for telomere maintenance and resultant acquisition of limitless replicative capacity. These tumours are expected to be refractory to telomerase-targeting therapies. Indeed, it has been shown that introduction of a dominant-negative form of hTERT into the GM847 human ALT cell line did not affect telomere length nor cell growth; whereas introduction of the same dominant-negative hTERT mutant in telomerase-utilizing tumour cell lines caused telomere shortening and induced cell death [Hahn et al., 1999]. It has been further suggested that ALT may constitute an escape mechanism for telomerase-utilizing tumours treated with anti-  16  telomerase therapies, by selecting for ALT cells within a given tumour mass [Henson et at., 2002; Stewart, 2005]. Similarly, primary telomerase-utilizing turnours  may give rise to  ALT-  utilizing secondary tumours, as evidenced by observation of a patient with a telonierase positive glioblastorna multiforme who presented with an ALT-positive reculTent tumour [Hakin-Smith et at., 2003].  1.6 Objectives and rationale To guide treatment strategies that target cellular immortalization, it is essential to develop a robust method for determining ALT status in tumours and tumour-derived cell lines.  Whether t-circles are a by-product or active mediators of telomeric recombination processes, their presence in ALT cells makes them an attractive target for detection in screening applications. Currently, the most commonly used method oft-circle detection is neutralneutral 2-dimensional pulsed-field gel electrophoresis (2D PFGE). 2D PFGE is based on the electrophoretic separation of DNA molecules by size (i.e., molecular weight) in the first dimension and by molecular structure/topology (e.g., linear vs. circular, “relaxed” vs. supercoiled) in the second dimension. T-circles migrate through the gel in the second dimension (separation by molecular structure or shape) at a slower rate relative to linear telomeric DNA [Wang et at., 2004]. This method requires that digested genomic DNA from approximately 1.5-3.5 x 106 human diploid cells is loaded in a single 2D gel [Cesare and Griffith, 2004; Wang et at., 2004; Wang et al., 2004]. The requirement for these large samples precludes analysis of clinical tumour-derived cells, if these are low in number. An additional drawback of 2D PFGE is that only a single sample can be loaded in a 2D gel, so  17  evaluation of numerous samples requires several gels and additional controls for between-gel variability of hybridization efficiency and exposure differences between different X-ray films.  These problems may be overcome using a novel, rolling circle amplification (RCA)-based method oft-circle detection. (A detailed comparison of RCA and 2D PFGE t-circle detection methods is provided in Chapter 4: Discussion.) An RCA-based t-circle assay detected t circles in DNA extracts from Arabidopsis thaliana mutant cells which maintain telomeres by homologous recombination [Zellinger eta!., 2007]. Using telomeric sequence-specific primers and a highly-processive DNA polymerase from phage 429, t-circles were amplified by RCA and detected by denaturing agarose gel electrophoresis and subsequent probing.  Our primary goal was to adapt this RCA-based method for the detection oft-circles in human immortalized cell lines. Using RCA, t-circles may potentially be detected with much higher sensitivity and many samples may be analyzed in parallel on a single denaturing gel. These potential advantages of RCA over currently-used t-circle detection methods may allow for development of a reliable screen for ALT status with potential clinical applicability.  Our secondary goal was to examine the effects of telomerase on telomere maintenance by the ALT pathway, by determining whether reconstitution of telomerase activity in ALT cells affects t-circle generation.  18  Chapter 2: Materials and methods  2.1 Cell culture Cell lines HeLa, GM847, and SUSM-1, were grown in Dulbecco’s Modified Eagle Medium with 4.5 gIL D-glucose (STEMCELL Technologies, Vancouver, Canada), supplemented with 10 % fetal calf serum (FCS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 streptomycin. WT-38 VA-13 subline 2RA (VA-13) fibroblasts and PG-13 virus packaging cells were grown in Modified Eagle Medium-a (Invitrogen, Carlsbad, CA, U.S.A.) supplemented with 10 % FCS, 2 mlvi L-glutamine, 100 jiM MEM non-essential amino acid solution, 100 U/mE penicillin, and 100 pgImL streptomycin. HCT1 16 cells were grown in McCoy’s 5A Medium (Invitrogen, Carlsbad, CA, U.S.A.) supplemented with 10 % FCS, 2 mlvi L-glutamine, 100 U/mL penicillin, and 100 jig/mL streptomycin. All cells were incubated at 37 °C and 5 % CO 2 for expansion.  2.2 Extrachromosomal DNA isolation ecDNA was isolated using a modified Hirt extraction procedure [Hirt, 1967]. 2 x 106 cells were washed twice in phosphate-buffered saline (PBS), resuspended in 400 j.tL of lysis buffer (10 mM Tris-HC1, pH 7.5; 10 mM EDTA, pH 8.0; 0.6 % SDS) and incubated for 5 minutes at room temperature. 100 jiL of 5 M NaCl was added to each tube and mixed thoroughly by inverting the tube 4-5 times. The samples were incubated overnight at 4 °C, followed by centrifugation at 13,000 r.p.m. for 30 mm at 4 °C, in a tabletop centrifuge. The chromosomal DNA pellet was discarded and the ecDNA was extracted from the supernatant using phenol:chloroform:isoamyl alcohol (25:24:1, w/v). ecDNA was precipitated using 1 mL (2  19  volumes) of absolute isopropanol and 50 iL (0.1 volumes) of 3M sodium acetate (pH5.2), then washed in 500 jiL 70 % ethanol and air dried. ecDNA was resuspended in 65 tL of Tris-EDTA buffer (TE) containing 20 p.g!mL RNase A and incubated at 37 °C for 30 mm to digest any RNA in the sample. The isolated ecDNA samples were quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, U.S.A.) stored at  -  20 °C until use.  2.3 Rolling circle amplification The oligonucleotide used to prime the RCA reaction was telomeric sequence 2 imer (“thio C”: 5 ‘-CCCTAACCCTAACCCTAACsCsC-3, containing two thiophosphate linkages (denoted by “s”) between the three 3’ terminal nucleotides (University of Calgary Core DNA Services, DNA/RNA Synthesis Laboratory, Calgary, Canada).  In a 0.2 mL PCR tube, we combined 6.5 jiL of ecDNA extract (2 x i0 cell-equivalents), 2.5 jiL of 1 iM thio-C primer, and 1 jiL of 10 x annealing buffer (200 mM Tris-HC1, pH 7.5; 1 mM EDTA, pH 8.0; 0.2 M KC1). The mixture was covered with a drop of mineral oil, denatured at 95 °C for 3 mm, and then placed on ice for 15 mm to anneal the thio-C primer to complementary telomeric sequences in the ecDNA extract. The total RCA reaction volume was 20 jiL, containing the post-annealing DNA mixture, 0.2 mM dNTP mix, 33 mM Tris acetate (pH 7.9), 10 mM magnesium acetate, 66 mM potassium acetate, 0.1 % (v/v) Tween 20, 1 mM DT.T, 50 .tg/mL Escherichia coli single-stranded DNA binding protein (USB, Cleveland, OH, U.S.A.), and 7.5 U of F29 DNA polymerase (Fermentas, Burlington, Canada). RCA was carried out at 30 °C for 16 hr. c129 DNA polymerase was inactivated by  20  incubation at 65 °C for 10 mm. Only extracts from cell lines which contain t-circles provide appropriate templates for amplification by RCA under these conditions.  2.4 In-gel detection of RCA reaction products Half of the RCA reaction product (10 pL) was loaded onto a denaturing agarose gel (0.8 % agarose; 50 mM NaOH; 1 mM EDTA, pH 8.0) and separated by electrophoresis in alkaline running buffer (50 mM NaOH; 1 mM EDTA, pH 8.0) at approximately 2 V/cm for 16-18 hr. The gel was stained with SYBR Green II (Invitrogen, Carlsbad, CA, U.S.A.), which preferentially binds RNA and ssDNA for 40 mm at room temperature and imaged using Quantity One gel analysis software (Bio-Rad Laboratories, Hercules, CA, U.S.A.). The gel was baked at 60 °C for 45 mm under vacuum. The flattened gel was denatured (0.5 M NaOH, 1.5 M NaCl) for 20 mm and neutralized (0.5 M Tris-HC1, pH 7.5; 1.5 M NaC1) for 20 mm.  The gel was pre-hybridized in 6X SSC, 0.1 % SDS for 1 hr at 37 °C. The gel was probed by 5 oligonucleotide in 6 x SSC, 0.1 % SDS at hybridization of ‘yP-5’-end-labeled (TTAGGG) 32 37 °C for 16-18 hr. The probed gel was washed twice for 5 mm in 2 x SSC, 0.05 % SDS at room temperature, blotted on a piece of filter paper and air-dried. X-ray films (GE Healthcare, Little Chalfont, U.K.) were exposed to the probed gel for various amounts of time to ensure optimal resolution of DNA bands. The presence oft-circles in the original sample of ecDNA is inferred from the appearance of long, non-migrating single strands (RCA reaction products) of telomeric DNA on the probed gel. The gel bands resulting from  21  RCA were quantified using Bio-Rad Laboratories Quantity One gel analysis software (Bio Rad Laboratories, Hercules, CA, U.S.A.).  2.5 Telomere Repeat Amplification Protocol (TRAP) Cells were trypsinized, harvested in PBS and counted using a hemocytometer. Following centrifugation at 1,000 rpm for 5 mm in a tabletop centrifuge (Beckman model TJ-6), the sup ernatant was aspirated and the cells were resuspended in CHAPS lysis buffer (10 mM Tris-HC1, pH 7.5; 1 mM MgCl ; 1 mM EGTA; 0.1 mM benzamidine; 5 mM f 2 3 mercaptoethanol; 0.5 % w/v CHAPS; 10 % w/v glycerol) at a concentration of 1 x iü cells/200 jtL CHAPS buffer. The lysate was incubated on ice for 30 mm, then centrifuged at 12,000 rpm for 30 mill at 4 °C. The DNA-containing pellet was discarded and the proteincontaining sup ernatant was fractionated into aliquots of 1 x 1 ü cell-equivalents and stored at -80 °C until use.  Serial ten-fold dilutions of the lysate supematant (from 2,000 to 20 cell-equivalents) were prepared. Each TRAP reaction contained a total volume of 50 p.L and was made up according to Table 1 in a 0.2 mL PCR tube. The samples were incubated for 20 mm at room temperature for the telomerase extension reaction to proceed. The telomerase-extended products were then amplified using PCR in a thermocycler (MJ Research PTC-225; settings: 95 °C for 1 mm, 50 °C for 45 s, 72 °C for 1 mm; 30 cycles). The PCR products were loaded onto a 1 x Tris-borate-EDTA (TBE) polyacrylamide gel and separated by electrophoresis at 500 V for approximately 1 hr. The gel was stained with SYBR Green for 30 mm at room  22  temperature and imaged using Quantity One gel analysis software (Bio-Rad Laboratories, Hercules, CA, U.S.A.).  Table 2.1 TRAP assay reaction mixture. Reaction component primer U2 (5 ‘-ATCGCTTCTCGGCCTTTT-3’) primer TSU2 (5 ‘-AATCCGTCGAGCAGAGTTAAAA GGCCGAGAAGCGAT-3 t) primer ACX (5 ‘-GCGCGGCTTACCCTTACCCTTACCCTAACC-3) primer TS (5 ‘-AATCCGTCGAGCAGAGT 3’) dNTP mix (25 mM each dNTP) TRAP reaction buffer (1 OX) Taq Polymerase (Boehringer Marmheim) 0 2 DEPC-treated H  Volume (tL) 0.1 0.1 0.1 0.1 0.1 5.0 0.5 x = 50 lysate volume other components -  -  2.6 Ectopic over-expression of telomerase in ALT cells The hTERT-IRES-GFP and U3-hTR/PGK-YFP constructs used in this study were previously cloned into murine stem cell virus (MSCV)-based expression vectors and transduced into PG-13 murine virus packaging cells, in our laboratory. The vector-containing PG-13 cells were expanded at 37 °C and 5 % CO 2 in DMEM supplemented with 10 % FCS, 2 mM L glutamine, 100 U/mL penicillin, and 0.1 mglmL streptomycin. The packaged MSCV virus was released into the supematant by incubating PG- 13 cells in medium which is optimal for the VA-13 cells that were to be infected (MEM-a supplemented with 10 % FCS, 2 mM L glutamine, 100 jiM MEM non-essential amino acid solution, 100 U/mL penicillin, and 100 jig/mL streptomycin) at 32 °C, the optimal temperature for MSCV replication. After 48  23  t7  vsn ‘v ‘qEnbssJ ‘uoTsaiJ poqddy) wosCs uiwt ip  uisn uofl1?uTuIex oidoosoiiw Aq pu.uguoo osj  ATJ  aro uOTsiA1pu  SM uoTpnpsu1fl isns  -VA ponpsuiu jo sainijno aind Ajuiogjns unqo oi puuojid aM uoiindod jpo  uoisuidx  UTUAJUT  uop  ‘  pu urnos jo spunoi arqj  iuAsDvI) ut11os  Jo uorssaidx aoj pios  p  aIM satnjno ijj  Vsn ‘n..i ‘sajw[ uTpJuuJ  jo  ‘U0SUDJOTG  to-ouo3sa1onIJ Xq S1O3A ponpsui.i  sip  J1AS  .ioj ppudx uq ‘uoTpjui jo  punoa qo uwq inpu in uotwqnout q quuAo 1AO3I oi pMoJJ pu SHd UI TM SJJ3 1TJ p3npSuJ  OJM SflO  1M UOIoflpsU1 JO SUflOJ  ‘qi  UOIpflpSUJ J1IAOJ1J OUi JJA  J -yjS jo qwnu uogjns  ‘lcJM0JS  PA!P SIPO El-VA D LE  OD % S  qi atnsuo o puuojod UOtsiATp  I? IUUAO (j: j)  JJO Uuflp  Slfl330  rnpw pu iuuidns  ASN IJ1JM uoiqnouT q srn.rsuo tjoq .o ‘&TA-)T9d/U’-J-f1 ‘&TD-S[NI  -jjjq ijpi  pnpsun pui sjd IPM-9  UI UAOJ SAX  El-VA  ‘QUIT  •35fl filun 3 08pu ‘satod inn  qnong patjg ‘PSAJnJ SM uipn.udns  TP° LIV UUTrnJ {J  sonbij qw ç  in paIos  ‘uoipripoad snhIAJO sinoq  p  Chapter 3: Results  3.1 RCA amplifies telomeric sequence-containing circular plasmid DNA RCA is an isothermal (30 °C) DNA replication reaction, catalyzed by the highly-processive 4)29 DNA polymerase, the enzyme responsible for replicating the 18 kb genome of the bacteriophage 4)29 [Watabe eta!., 1982]. 4)29 DNA polymerase has efficient strand displacement activity, which allows it to generate ssDNA products longer than 100 kb using small DNA circles as templates [Blanco and Salas, 1996; Lizardi et a!., 1998]. Tn the current study, the reaction was primed with a C-rich telomeric sequence 2lmer oligonucleotide denoted “thio-C”, because it contains two thiophosphate linkages between the three 3’ terminal nucleotides (see Chapter 2: Materials and Methods for primer sequence). Thiophosphate linkages prevent nucleolytic degradation of the primer by 4)29 DNA polymerase, which possesses 3’ to 5’ exonuclease activity acting preferentially on singlestranded DNA [Garmendia eta!., 1992]. An illustration of the RCA reaction is shown in Figure 3.1.  Primer  -  Figure 3.1 Rolling circle amplification. A simplified illustration of RCA catalyzed by 4)29 polymerase, resulting in the synthesis of a long ssDNA strand which consists of tandem replicates of the circle sequence. 25  In order to develop an RCA-based protocol to detect t-circles in human ALT cell lines, it was first necessary to optimize a protocol to amplify known amounts of purified telomere repeatcontaining circular plasmid DNA in vitro. To this end, pBluescript II KS(+) plasmid vector containing an insert of 1.6 kb of telomeric repeat sequence (denoted “pTel 1.6”) was used to template the RCA reaction.  Following thio-C-primed RCA of the pTel 1.6 template, a long ssDNA product was detected, which did not migrate from the wells during agarose gel electrophoresis (Figure 3.2; lanes 26). As a positive control, the reaction was primed with random sequence-containing, thiophosphate-modified hexamers, which are expected to amplify input DNA regardless of its sequence (Figure 3.2B; lane 2). As a negative control, the reaction was carried out in the absence of the 29 DNA polymerase (all other reaction components were included). No long ssDNA product was detected in the well when the polymerase was excluded (Figure 3.2; lane 1).  To determine the concentration of primer molecules that would yield the most ssDNA product following RCA, a titration of the thio-C primer was performed. The experiment revealed that the yield of the RCA reaction varies with respect to the primer concentration, when the amount of the template is held constant (Figure 3.2; lanes 3-6). The optimal concentration of thio-C primer to amplify approximately 150 ng of pTel template was shown to be between 1-3 iM (Figure 3.2; lanes 4 and 5).  26  Lane #: I 029 p01: Primer:  23456 +  -  +  +  +  7  ÷  [Thio-C]  E 90 .  I—  (U  .  -  ft:  Cr)  a  db  1e 41b  —12 kb  EtBr  Figure 3.2 Amplification of plasmid DNA by RCA yields long, non-migrating ssDNA product. The circular plasmid pTel 1.6 was amplified using RCA primed with varying concentrations of the telomere-specific thio-C primer (lanes 3-6). The reaction yield of long ssDNA product (indicated by the arrow) was shown to be sensitive to the concentration of the thio-C primer in the reaction, with optimal yield seen at a concentration of 1-3 iiM (lanes 4 and 5). Lane 1, negative control: the reaction mixture subjected to RCA conditions in the absence of 29 polymerase. Lane 2, positive control: RCA primed by random hexamer oligonucleotides. The black arrowhead indicates the pTel 1.6 plasmid; the white arrowhead likely indicates a concatemer of the highly recombinogenic pTel 1.6 plasmid DNA. The nonmigrating DNA present in the 1 kb DNA size marker (lane 7, indicated by asterisk) results from association of smaller ladder fragments which contain sticky ends. This band can be resolved by denaturing gel electrophoresis (see Figure 3.10, which contains the same DNA ladder on a denaturing gel). EtBr, ethidium bromide.  27  To improve reaction efficiency and increase yield of the long ssDNA product, bacterial (K coli) single-stranded DNA binding protein (SSB) was added to the reaction mixture. Previous work has shown that SSB improves the efficiency and accuracy of RCA, most likely by preventing the re-annealing of the ssDNA reaction product to the circular template, minimizing non-specific DNA products that may result from primer dimers, and potentially minimizing secondary structure formation [Inoue eta!., 2006]. An illustration of SSB binding during RCA is shown in Figure 3.3A.  In this study, 150 ng of pTel was used to template the reaction and 1 .tM thio-C was used to prime the reaction. To determine the effects of SSB on RCA yield under these conditions, titres of SSB (concentration range 0  —  100 ig/mL) were added to each reaction (Figure  3.3B). The yield of the ssDNA reaction product was increased in the presence of SSB (Figure 3.3B; compare lanes 2 and 3). However, further increases of SSB concentration greater than 10 ig!mL did not appear to enhance the yield significantly (Figure 3.3B; lanes 3-6). This suggests that most of the ssDNA product is saturated with SSB binding at a concentration of 10 tg/mL and additional SSB molecules do not have available binding targets.  28  Lane#: 029po1:  I -  234567 +  +  +  +  +  [SSB]  -J  o  o  _J  _J  —  .-.  .  -J  .  U) -  -J  c’i  0O  ——  -10kb  B  EtBr  Figure 3.3 SSB improves the efficiency of RCA. (A) A schematic of E. coil SSB binding to the newly synthesized ssDNA as RCA proceeds. (B) The presence of SSB in during RCA increased the reaction product (lanes 2 and 3); however, increases of SSB concentration over 10 tg/mL did not further enhance RCA yield. Arrow indicates long ssDNA reaction product; the black arrowhead indicates the pTel 1.6 plasmid; the white arrowhead indicates concatenated plasmid DNA. EtBr, ethidium bromide.  29  3.2 RCA-based assay detects t-circles in the human ALT cell line GM847 To determine whether the RCA-based protocol will amplify t-circles in samples isolated from human cells, the protocol was applied to ecDNA extracts of the known, t-circle containing, human ALT cell line, GM847. GM847 is an SV4O-immortalized skin fibroblast [Henson et al., 2002] which has long, heterogeneous telomeres and undetectable telomerase activity [Bryan et a!., 1995]. The presence oft-circles in GM847 cells was previously demonstrated using 2D PFGE and electron microscopy (EM) [Cesare and Griffith, 2004; Wang et a!., 2004]. In the current study, GM847 ecDNA was isolated using a modified Hirt extraction procedure [Hirt, 1967]. Hirt extraction consists of cell lysis using SDS, DNA precipitation in the presence of high NaC1 concentration, and centrifugation to separate chromosomal DNA in the pellet from low molecular weight ecDNA in the supernatant (see Chapter 2: Materials and Methods for details). T-circles present in the GM847 ecDNA extract were subsequently taken through the thio-C-primed RCA procedure. The reaction product was separated by denaturing agarose gel electrophoresis, probed by in-gel hybridization of yP-5 ‘-end-labeled (TTAGGG) 32 5 oligonucleotide, and visualized by exposure of X-ray films.  In order to establish whether conditions optimized to amplify pTel plasmid were consistent with those required for amplification oft-circles in GM847 ecDNA, titrations of thio-C primer and E. coli S SB protein were performed. T-circles present in the ecDNA extract of 2 x 1 0 GM847 cells served as templates for each reaction. Titration of thio-C primer molecules revealed that the highest levels of ssDNA product were obtained at a primer concentration of 0.1-1 jiM (Figure 3.4A; lanes 4 and 5). Thio-C concentrations below 0.01 jiM were  30  insufficient to yield any detectable product (Figure 3.4A; lanes 1 and 2). On the opposite end of the spectrum, concentrations above 10 M seem to inhibit RCA extension by 29 DNA polymerase, presumably by saturating the t-circle templates with thio-C primer molecules (Figure 3 .4A; lane 7). This data indicates that RCA is highly sensitive to the relative concentrations of primer and template molecules in the reaction.  Titration of SSB showed a positive correlation between increasing SSB concentration in the RCA reaction mix and the yield of ssDNA produced by the reaction (Figure 3.4B). In contrast with the results obtained from amplification of pTel 1.6 plasmids, increasing the SSB concentration above 10 j.iglmL further increased the yield of ssDNA RCA product (compare Figures 3 .3B and 3 .4B). Several factors may be responsible for this discrepancy. First, the concentration oft-circles in ecDNA extracted from 2 x 1 0 GM847 cells may be significantly higher than the approximately 150 ng of pTel 1.6. An increased amount of template will yield a greater amount of ssDNA reaction product, providing more available binding sites for SSB. Second, larger circular templates would likewise yield more ssDNA product by providing more primer binding sites. If a significant proportion oft-circles in GM847 are larger than 1.6 kb (the length of telomeric DNA in a molecule of pTel 1.6), saturation of the product with SSB would occur at higher SSB concentrations. Finally, ecDNA extracts contain linear telomeric ecDNA molecules (much of which is G-rich ssDNA [Nabetani and Ishikawa, 2009]) and chromosomal telomeric DNA which may have sheared off the ends of chromosomes during the extraction procedure. Indeed, a significant amount of “contaminating” chromosomal DNA has been shown to remain in the supernatant (ecDNA) fraction following flirt extraction [Nab etani and Ishikawa, 2009]. Any telomeric DNA  31  molecules, and especially G-rich ssDNA, will compete with t-circles for thio-C primer binding. Addition of SSB to the RCA reaction may sequester appreciable amounts of non-t circle telomeric ssDNA, freeing up primer molecules for t-circle binding. However, at higher SSB concentrations, the yield of the reaction would be expected to plateau (as in the reaction with plasmid template, Figure 3 .3B) and eventually decrease as a significant proportion of the primer molecules themselves are bound by SSB.  Importantly, the optimization of thio-C and SSB reaction conditions showed that t-circle amplification products can be detected using RCA in the ALT cell line GM847. These reaction conditions may potentially be applied to screen other ALT cell lines for t-circles (see Section 3.4). A  B  [Thio-C]  E o  coo  0  [SSB]  —  o -  _j  _I  o 0  o 0  —  U) c’i  0 U)  -  Telomere probe  Figure 3.4 Determination of optimal thio-C and SSB concentrations to detect t-circles in GM847 ALT cells. (A) Titration of thio-C oligonucleotide primer reveals that optimal RCA yield was obtained using primer concentrations of 0.1-1 jiM (lanes 4 and 5) when the t circles in the ecDNA extract of 2 x 1 0 GM847 ALT cells were used as templates. (B) Titration of E. coli SSB shows that RCA-based t-circle detection in GM847 is enhanced by increasing the concentration of SSB. 32  3.3 Thio-C-primed RCA is a linear reaction which facilitates highly-sensitive detection of t-circles RCA primed by a single, sequence-specific oligonucleotide normally proceeds with linear reaction kinetics and produces long ssDNA amplification products. However, RCA may likewise be primed by multiple oligonucleotides, such as in the reaction shown in Figure 3.2, lane 2; which was primed by random-sequence hexamers. This type of reaction is known as “hyperbranched” or “multiple displacement” RCA [Demidov, 2005] and has been used to generate high-fidelity templates for DNA sequencing from low-abundance circular vectors [Nelson et a!., 2002]. As a result of annealing and priming on already produced ssDNA, random primed-RCA proceeds with exponential geometric kinetics and yields hyperbranched dsDNA products [Demidov, 2005]. An illustration depicting linear and exponential RCA reactions is shown in Figure 3.5. Thio-C-primed RCA uses a single, sequence-specific oligonucleotide which can only anneal to one strand of telomeric DNA, producing an ssDNA replicate of the complimentary strand. Thus the reaction was expected to proceed with linear kinetics.  33  A  Sequencespecific primer P29poI -  B Random primer  o Figure 3.5 Linear versus exponential RCA reaction. (A) Extension of a single, sequencespecific primer by RCA yields increasing amounts of ssDNA in a linear fashion, on a plot of reaction time versus reaction yield. (B) Extension of multiple random sequence primers by RCA yields increasing amounts of dsDNA in an exponential manner.  To address the reaction dynamics of thio-C-primed RCA, a time-course experiment was conducted (Figure 3.6). T-circles present in ecDNA isolated from 2 x 1 O GM847 ALT cells were used as template. Amplification time varied from 0 to 16 hours. Following denaturing agarose gel electrophoresis, probing with radioactively-labeled telomeric oligonucleotides, and exposure of an X-ray film, image analysis software was used to quantify the RCA reaction yield of long ssDNA (determined by the non-migrating band volume  =  band  intensity x band area). Band volume quantification confirmed that thio-C-primed RCA of GM847 t-circles proceeds with linear reaction kinetics (Figure 3.8B).  34  RCAtime:  4  2  1  0  8  16h  A 4000  3000 y= 2386x 3101  2000  R= 0.996  E 1000 0 C  £0  0 0  B  10  5  15  20  RA time (h)  GM847 ALT cells was amplified by t-circle-specific RCA for the indicated amounts of time. T-circles were detectable following 1 hour of RCA. (B) Thio-C-primed RCA oft-circle templates proceeds with linear reaction kinetics. Figure 3.6 Time-course RCA. (A) ecDNA from 2 x  To determine whether RCA-based detection oft-circles is more sensitive than the commonly-used 2D PFGE method which does not include any DNA amplification, titration of the ecDNA extract input was performed. T-circles present in the ecDNA extracts ranging from 5 x  to 4 x i0 GM847 cell-equivalents were used to template thio-C-primed RCA  (Figure 3.9). Amplification products from t-circles were detectable in the ecDNA extract of as few as 5 x 1  cells. Thus t-circles were detected with a much higher sensitivity than what  is possible using 2D PFGE, which commonly utilizes DNA extracted from at least 1.5 x 106 ALT cells [Cesare and Griffith, 2004; Wang et al., 2004; Wang et al., 2004].  35  RCA yield increased in a linear fashion relative to the input cell number until it reached a plateau at approximately 2 x 1 O cell-equivalents (Figure 3 .7B). This plateau likely represents the point at which no more primer molecules are available to bind the extra t circles in the sample. If this is the case, addition of higher amounts of thio-C primer to the RCA would allow the reaction to proceed linearly with higher input ecDNA. Alternately, the amount of SSB and/or ‘F29 DNA polymerase may likewise be limiting at higher t-circle template concentrations. Using the above-described reaction conditions, ecDNA extracts from 2 x 1 O cell-equivalents were used as the template for all subsequent experiments.  Cellequivalents.  0  0  ‘Z 0  c  0  0 C  0 C  —  8,000  Cc.ccd C —  N  •  —  6,000 C 4I  4,000  . 2,000 -U C  • I 0  I  0  A  Telomere probe  B  100.000 200.000 300,000 400,000  Number of cell-equivalents  Figure 3.7 Titration of cell number in the RCA reaction. (A) ecDNA from the indicated number of GM847 ALT cells was extracted and subjected to t-circle-specific RCA. (B) A plot of the number of cell-equivalents per ecDNA extract used in the RCA reaction versus the reaction yield of long ssDNA as quantified by the non-migrating band volume.  36  3.4 RCA detects t-circles in human ALT cell lines but not in human telomerase utilizing cell lines To evaluate whether the RCA-based t-circle detection method that was optimized for GM847 ALT cells is applicable for screening purposes, a panel of cell lines was evaluated using this technique. The ecDNA extracts of established ALT-utilizing and telomerase-utilizing human immortalized cell lines were subjected to thio-C-primed RCA. The characteristics of these cell lines are summarized in Table 3.1.  Table 3.1 Cell lines used in the RCA-based screen for t-circles.  Transformation Spontaneous Spontaneous SV4O Chemical SV4O  Cell line Cell type HeLa Epithelial, cervix HCT1 16 Epithelial, colon GM847 Fibroblast, skin SUSM-1 Fibroblast, liver VA-13 Fibroblast, lung * TMM, telomere maintenance mechanism  TMIVI* Telomerase Telomerase ALT ALT ALT  For controls within this study, RCA was again used to amplify pTel 1.6 plasmid DNA. The negative control reaction was carried out in the absence of 429 DNA polymerase (Figure 3.8; lanes 2 and 4), whereas the positive control reaction was carried out in the presence of the enzyme (Figure 3.8; lanes 3 and 5). As expected, the control experiment revealed that RCA only proceeded in the presence of cF29 DNA polymerase. However, probing of the gel with radioactively-labeled telomeric oligonucleotides revealed that telomeric DNA in the non-migraing ssDNA product was detected with extremely low efficiency (compare the relative amounts of pTel 1.6 plasmid and ssDNA RCA product as they appear on the ethidium bromide (EtBr)-stained gel and after probing, in lanes 3 and 5, respectively). The  37  low efficiency of probing for the long ssDNA product may potentially result from the presence of intervening plasmid vector sequences, or from an inability of the probe to hybridize to DNA which was essentially “clumped” within the well during overnight denaturing gel electrophoresis. This finding indicates that probing of the ssDNA RCA product may likewise be inefficient in amplified ecDNA extracts from cell lines.  Nevertheless, RCA reproduciblydetected t-circles in the ALT cell lines GM847, SUSM-1, and VA- 13, but did not detect any t-circles in the telomerase-utilizing cell lines HeLa and HCT 116 (Figure 3.8; lanes 7-11). The observations are in agreement with previous studies where both types of cell lines were analyzed by 2D PFGE and EM [Cesare and Griffith, 2004; Wang et al., 2004].  Interestingly, GM847 and SUSM- 1 consistently showed a higher level oft-circles than VA13 when the same number (2 x 1 O) of cell-equivalents was analyzed. This may be attributed to VA- 13 having lower number oft-circles. On the other hand, the result may be an artifact of the amplification-based method. It is feasible that the RCA reaction conditions optimized for t-circle detection in GM847 are suboptimal in amplifying t-circles in VA- 13 ecDNA extracts. For example, the RCA reaction may potentially proceed with a higher efficiency if a different number of cell-equivalents or thio-C primers were used.  38  Lane#: 029 pol:  1  2  3  4  5  -  +  -  +  cq  to  ‘-  ‘-  .q ‘-  4)  Template:  12kb  I—  .0.  ‘-  4)  I-  0.  4)  I-  0.  6  0.  8  9  10  11  +  +  +  +  +  t.-.  ‘;.  (0  I 4,0 I I -j  4)  I  7  ><  co 0  C,,  D Cl)  —  Telomere probe  SYBR Green II  Figure 3.8 RCA-based screen detects t-circles in human ALT cell lines. pTel 1.6 was used as a control template to ensure the RCA reaction proceeded in the presence of 129 polymerase (lanes 2 and 4), but not in its absence (lanes 3 and 5). T-circles were detected in the ALT cell lines GM847, SUSM- 1 and VA- 13, but not in the telomerase-utilizing cell lines HeLa and HCT1 16. “X” (lane 6) indicates that no sample was loaded. Arrow indicates long ssDNA reaction product; the black arrowhead indicates the pTel 1.6 plasmid; the white arrowhead indicates concatenated plasmid DNA.  39  3.5 Telomerase reconstitution in ALT cells does not affect t-circle formation Based on the existence of ALT-specific cellular phenotypes (e.g., telomeric recombination, APBs, MS32 instability, telomeric ecDNA) and the absence oft-circles in the telomerase expressing cell lines evaluated above, we postulated that telomere maintenance by telomerase and ALT are mutually exclusive in a given cell line. This appears to be the case in yeast [Teng and Zakian, 1999], but studies of human ALT cell lines have produced conflicting observations [Cerone eta?., 2001; Ford eta?., 2001; Grobelny eta?., 2001; Perrem eta?., 2001; Wen, 1998] (see Chapter 4: Discussion). To directly assess whether reconstitution of telomerase activity affects t-circle generation in ALT cells, the telomerase components hTERT and hTR were ectopically overexpressed in VA-13 ALT cells. hTERT, the reverse transcriptase subunit of telomerase, catalyzes the addition of telomeric repeats to the 3’ end of the telomere using the hTR RNA subunit as template.  VA- 13 cells were transduced by murine stem cell virus (MSCV)-derived expression vectors (previously cloned in our laboratory) using a retroviral infection protocol. Since VA- 13 cells grow relatively slowly and are only transduced during cellular replication, three rounds of MSCV infection were perfomLed to ensure sufficient transduction efficiency. The hTERT containing construct (called MSCV-hTERT-IRES-GFP) expresses green fluorescent protein (GFP) from an internal ribosome entry site (IRES). The hTR-containing construct (called U3-hTR!PGK-YFP) expresses hTR from a U3 promoter and yellow fluorescent protein (YFP) from a separate, phosphoglycerin kinase (PGK) promoter. GFP and YFP were included in the vectors to facilitate tracking of transgene expression in the transduced cells. Simplified maps of these constructs are shown in Figure 3.9.  40  Figure 3,9 MSCV-based expression constructs containing telomerase subunits. (A) MSCV-hTERT-IRES-GFP. (B) U3-hTRIPGK-YFP. Displayed are the cloned genes, fluorescent proteins, promoter regions and the 5’ and 3’ long terminal repeat (LTR) regions, which are used to integrate retroviral DNA into host genomes.  The transduced cells were examined under a fluorescence microscope for expression ofthe fluorescent proteins. GFP (peak emission wavelength  514 nm) and YFP (peak emission  wavelength = 535 nm) cannot be distinguished using our fluorescence microscope, which detects both using the same filter. Thus fluorescence-activated cell sorting (FACS) was used to confirm their expression in the cells doubly-transduced with both constructs. To obtain cultures of sufficient purity, transduced cells were sorted three times by FACS for the expression of GFP, YFP, or both. The resulting purity values at the time of ecDNA isolation were approximately 63 % for the VA-13 cells over-expressing hTERT, 92 % for cells over expressing hTR, and 62 % for cells over-expressing both hTERT and hTR. Representative microscope images and FACS purity plots are shown in Figure 3.10.  41  VA-I 3  VA-13 + hTERT  VA-13 + hTR  +  VA-I 3 hTERT+ hTR  I 0 C., 0  0 0  0 U-  a’  0  LL >-  .I. t  -x  ;a.  r. •1.  —I  a,  •  -  -  a’  _.a,  a’  GFP Figure 3.10 Expression of telomerase components in VA-13 ALT cells. Expression of the MSCV-based constructs MSCV-hTERT-IRES-GFP and U3-hTR/PGK-YFP in VA-13 ALT cells was confirmed by microscopy and FACS analysis. Representative images of cells under brightfield (top panels) and fluorescent (middle panels) illumination are shown. Note that the fluorescence images are pseudo-coloured, due to the fact that GFP and YFP emission cannot be discerned using our equipment. FACS analysis (purity plots shown in the bottom panels) confirmed that both constructs were expressed in the doubly-transduced VA-13 ALT cells. Even after three rounds of sorting, significant variation in the expression of the fluorescent proteins was observed. The size marker in the top left panel is 30 pm.  42  To determine whether expression of telomerase components lead to reconstitution of an active telomerase complex in doubly-transduced VA- 13, a telomere repeat amplification protocol (TRAP) assay was performed (Figure 3.11). The TRAP assay relies on endogenous telomerase within protein extracts from a sample of cells to add 6 bp telomeric repeats to a set of specially designed oligonucleotides (see Chapter 2: Materials and Methods for the sequences). Addition of telomeric repeats is visualized as a “ladder” when amplified by PCR and separated by polyacrylamide gel electrophoresis. A 50 bp PCR product results from oligonucleotide annealing in the absence of addition of any telomeric repeats and serves as an internal control band (indicated by an arrow in Figure 3.11). Tn this experiment, the leukemic lymphoblast cell line K-562, which is known to express active telomerase, was used as a positive control (Figure 3.11, lanes 3-6). Untransduced VA- 13 cells served as the negative control (Figure 3.11, lanes 7-10). 10-fold titrations of protein extracts of 2,000, 200 and 20 cell-equivalents of each cell type were included in the assay. Additional negative controls for all cell lines were provided by heat-inactivated (HI; 80 °C for 10 mm) protein extracts from 2,000 cell-equivalents (Figure 3.11, lanes 6,10,14).  The TRAP assay revealed minor laddering in the untransduced VA-13 samples (indicated by a black arrowhead in Figure 3.11), although this cell line was previously shown to lack any detectable telomerase activity [Ford et a!., 2001; Wen, 1998]. Additional low molecular weight bands were also observed in the extracts of 2,000 and 200 cells in K-562 and VA-13 cell lines (indicated by a white arrowhead in Figure 3.11). These observations likely represent PCR artifacts due to oligonucleotide mispriming and indicate that further optimization of TRAP reaction conditions is necessary.  43  Nevertheless, these preliminary results showed that the doubly-transduced hTERT- and hTR expressing VA- 13 cells had significant telomerase activity (note the ladders in Figure 3.11, lanes 11-13). Telomerase activity in these cells was higher than in the telomerase-utilizing K562 positive control cell line, presumably due to significant over-expression of the enzyme components. The reconstitution of telomerase activity in the ALT cell line VA- 13 facilitates direct analysis of the effects of the telomerase enzyme on t-circle formation.  Lane#: 1 Protein extract:  2  3  0 4.’  c  4  5  6  7  K-562 0 0  c’i —  8  9 10 11 12 13 14 15 VA-13 VA-13 + hTERT+hTR  0  c%4  CJ  0 0 0 C’4 0— C4  0 0 0  c..J  0 0 0 C1 0 00—  c•4c1J:  -330 bps  -  H .  -  200 100  -50  SYBR Green I  Figure 3.11 TRAP assay confirms reconstituted telomerase activity in VA-13 + hTERT hTR cells. The telomerase-utilizing K-562 cell line showed telomerase activity in protein extracts from 2,000 and 200, but not 20 cell-equivalents. The ALT cell line VA-13 did not show any telomerase activity in any of the three extract titrations. The doubly-transduced VA-13 + hTERT + hTR cells displayed laddering in all three extract titrations, including the extract of only 20 cell-equivalents, indicating that the over-expressed telomerase components reconstituted an active telomerase enzyme complex. “No extract”, negative control reaction which did not contain any protein extract but included all other TRAP components; HI, heatinactivated protein extract from the indicated cell lines. +  44  Based on the assumption that telomerase and ALT are mutually exclusive mechanisms for telomere maintenance, we hypothesized that t-circle formation would be inhibited in the presence of active telomerase. To address this possibility, the RCA-based t-circle assay was performed in VA-13, VA-13 over-expressing one of hTERT or hTR, and VA-13 overexpressing both hTERT and hTR, approximately 20 PDs after the final round of retroviral transduction. The 20 PD lag between transduction and t-circle detection was included to ensure that any t-circles already present in ALT cells at the time of telomerase reconstitution are diluted out via numerous cell divisions. Following RCA, no changes in t-circle levels were detected in the cells expressing active telomerase (VA- 13  +  hTERT  +  hTR) relative to  control cells (Figure 3.12). It is possible that small changes in the amount oft-circles detected following telomerase reconstitution were obscured in this assay due to suboptimal purity of the doubly-transduced VA-13 cells (approximately 62 % at the time of ecDNA isolation). Nevertheless, these results indicate that telomerase activity does not completely inhibit t-circle formation in VA- 13 ALT cells and suggests that telomeric recombination reactions can occur in the presence of telomerase.  45  Lane#: 029 p01:  I  23 +  4567 +  +  +  +  I -C +  ww I(0  (q  ‘-  -  4)  Template:  I-  0.  C)  .C + C)  -C +  1— +  4)  I  0.  — 12kb  —  7  Tel omere probe  Figure 3.12 Telomerase reconstitution in VA-13 ALT cells does not affect t-circle formation. RCA-based detection oft-circles did not detect significant changes in t-circle levels in VA- 13 ALT cells upon over-expression of active telomerase (compare lanes 4-7). Arrow indicates long ssDNA reaction product. (Note that the DNA size marker and the pTel containing control lanes in this figure are the same as those shown in Figure 3.10. The two experiments were performed in parallel and RCA reaction products were run on the same denaturing agarose gel.)  46  Chapter 4: Discussion  4.1 RCA-based detection of t-circles is potentially more sensitive than the  widely-used 2D PFGE method In the current study, RCA was used to amplify t-circles in ecDNA extracts of human ALT cell lines. The amplification conditions were modified from the initially-published method [Zellinger et al., 2007] by the use of ecDNA extracts instead of terminal restriction fragments (which contain chromosomal telomeres that are expected to sequester the thio-C primer) and by addition of E. coli SSB to the reaction mixture, which improved reaction efficiency. Using this protocol, t-circles were detected in ecDNA extracts of as few as 5 x 1 0 GM847 cellequivalents under optimal conditions (Figure 3.9). It is possible that t-circles may be detected from even lower amounts of cells using the RCA-based method. To address this, further cell number titration experiments are necessary, which will likely require further titration of thio C primer, titration of SSB protein, and longer exposure of X-ray films to visualize the minute non-migrating bands indicative of ssDNA amplification product.  Nevertheless, the ability to detect t-circles from as few as 5 x 1 0 ALT cells is significantly more sensitive than neutral-neutral 2D PFGE, which has been used for t-circle detection in the vast majority of publications to date. 2D PFGE requires that terminal restriction fragments (TRFs) from approximately 1.5-3.5 x 106 human diploid cells be loaded onto a single gel [Cesare and Griffith, 2004; Wang et al., 2004; Wang et al., 2004]. 2D PFGE is based on the electrophoretic separation of DNA molecules by size in the first dimension and by molecular structure/topology (e.g., linear vs. circular, “relaxed” vs. supercoiled) in the  47  second dimension. This method takes advantage of the fact that a linear molecule migrates through a gel matrix at a different rate than a non-linear molecule of equal mass [Bell and Byers, 1983; Oppenheim, 1981]. Thus t-circles migrate through the gel in the second dimension (separation by molecular structure or shape) at a slower rate relative to linear DNA restriction fragments [Wang et aL, 2004]. Increased sensitivity oft-circle detection was expected from the RCA-based method since it involves DNA amplification. However, in the absence of experiments to directly compare the RCA and 2D PFGE techniques in parallel, we cannot at this time make quantitative conclusions about the increased sensitivity provided by RCA. Titration experiments comparing input DNA required for t-circle detection by RCA and 2D PFGE in parallel are needed to address this question  RCA-based t-circle detection has other advantages relative to 2D PFGE. RCA amplifies t circles of any size, whereas t-circles smaller than approximately 2 kb are difficult or impossible to resolve on 2D gels [Nabetani and Ishikawa, 2009]. RCA can amplify t-circles as small as 40 nucleotides (Karel Riha, personal communication). Also, the ability of RCAbased detection to run multiple samples in a single gel streamlines t-circle detection of a collection of different samples. In contrast, only a single sample can be loaded on a 2D gel, so screens involving numerous samples require several gels and additional controls for the variability of hybridization efficiency between different gels and exposure differences between different X-ray films.  The disadvantages of RCA relative to 2D PFGE include lack of information about the molecular weight and structure (i.e., single- or double-stranded, relaxed or supercoiled) of t  48  circle molecules in the sample, and the variability introduced as a result of enzymatic amplification. RCA-based t-circle analysis is indirect; the DNA fragments that are detected on denaturing gels are long ssDNA amplicons oft-circle templates, but are not the t-circles themselves. As with any amplification-based methods of high sensitivity, there is an increased risk that contaminating factors may produce artifacts and false positives. In contrast, 2D PFGE detects t-circles directly. Thus 2D PFGE provides data on the t-circle size and structure, with the exception oft-circles smaller than approximately 2 kb, which cannot be resolved on 2D gels [Nabetani and Ishikawa, 2009]. Tn addition, the enzymatic amplification and indirect detection that serve as great advantages in the sensitivity of the RCA-based method may conversely pose some challenges to t-circle quantification. RCA yield is very sensitive to the relative concentrations of primer molecules, t-circle templates, and SSB in the RCA reaction. For example, the lower reaction yield from amplifying VA-13 compared to GM847 and SUSM-1 (Figure 3.10) may indicate suboptimal primer concentration relative to the t-circle templates specifically present in VA- 13 ecDNA extracts. Therefore, interpretations of RCA data must take into consideration that different reaction conditions may be necessary for optimal amplification of various samples.  4.1.1 Detection of telomeric circles in single cells All established t-circle detection methods require populations of cells. As a result, they cannot provide some desirable data about the molecular biology oft-circles in ALT cells. For example, the fraction of cells in a given ALT cell population that contains t-circles and the number oft-circles per cell are not known. It is unclear whether there is a relationship between t-circle size and abundance, and the lengths of chromosomal telomeres within  49  individual cells. Likewise, it is unknown whether t-circles are dynamically formed and degraded (or integrated into chromosomal telomeres by recombination) within given clones, or stably maintained through mitotic divisions. The development of in situ t-circle detection methods may provide these insights into t-circle biology, which are unattainable by currently-available methods.  One of the most attractive features of RCA is that it may provide the basis oft-circle detection within individual cell nuclei. Fluorescent in situ hybridization (FISH) of telomeric repeat-oligonucleotides can identify telomeric ecDNA [Ford et al., 2001; Hande et al., 20011 but does not tell us whether these molecules are t-circles, linear telomeric DNA, or the recently-identified t-complex DNA [Nabetani and Ishikawa, 2009]. Amplification by in situ RCA could potentially provide this distinction, since only t-circles (and not linear or t complex molecules) would be expected to template very long telomere repeat-containing ssDNA products.  Indeed, RCA has been utilized as a highly specific in situ signal amplification method for direct visualization of limited targets, such as point mutations in interphase nuclei [Zhong et al., 2001] and single antibody molecules bound to antigens [Schweitzer et al., 2000]. These experiments relied on the addition of exogenously-synthesized DNA circles to template RCA. The reactions were primed by DNA oligonucleotides consisting of two regions joined by a linker. One region was complementary to the circle templates and another region was specific for the point mutation [Zhong et al., 20011 or covalently linked to an antibody [Schweitzer et al., 2000].  50  We aimed to adapt our RCA protocol to detect t-circles directly in GM847 ALT cells, grown and fixed on glass slides. Preliminary experiments were unsuccessful, showing abundant non-specific binding of the fluorescent probe used to detect the ssDNA amplification product. Fluorescently-labeled RCA products indicative oft-circles were not observed (data not shown). Unlike previously-described in situ RCA methods [Schweitzer et aL, 2000; Zhong et al., 2001], which used exogenously-synthesized DNA circles to template the reaction, our method relies on endogenous t-circles to template the RCA. It is possible that in our experiment, t-circles were fixed to the glass slides or cross-linked to other molecules in a way that prevents primer annealing and/or polymerase extension around the entire molecule. We anticipate that further development of an RCA-based in situ t-circle detection method will require the optimization of several key experimental conditions, including fixation strategies, DNA denaturation and primer annealing, and (fluorescent) detection of the RCA extension product. Despite these technical challenges, the development of such a method remains desirable and should be pursued.  4.2 The effects of telomerase activity on the ALT pathway As discussed earlier, the effects of telomerase activity on telomere maintenance by ALT are not well understood. An important insight into the evolutionary origins oft-circles and ALT was gained by studies of yeast with linear mitochondrial DNA (mtDNA). The mitochondria of Candida parapsilosis contain abundant circular molecules derived from telomeric repeats, in addition to linear mtDNA genomes [Tomaska et al., 2000J. In a study which compared the mtDNA architecture of various C. parapsilosis subspecies, the presence oft-circles correlated with the occurrence of linear mitochondrial genomes [Rycovska et al., 2004]. The  51  authors proposed that recombination-based telomere lengthening involving ecDNA t-circles constitutes an archaic telomere maintenance mechanism which accompanied the evolution of linear genomes from their circular ancestors. Subsequently, this mechanism was replaced by the enzyme telomerase, which provides a more efficient way to synthesize telomeric arrays and thus ensure stability for the linear chromosomal form [Nosek et a!., 2006]. ALTutilization by mammalian tumour cells may thus be thought of as a reversion to an ancestral telomere maintenance mechanism in an aberrant physiological setting.  Consistent with the notion that telomerase has evolutionarily outcompeted recombination based pathways of telomere maintenance, recombination-mediated telomere elongation in S. cerevisiae telomerase-null survivors (see Section 1.2.1) is repressed by reconstitution of telomerase activity [Teng and Zakian, 1999]. On the other hand, reconstitution of telomerase in human ALT cells has yielded divergent observations in some studies [Cerone eta!., 2001; Ford et a!., 2001; Grobelny eta!., 2001; Perrem eta!., 2001; Wen, 1998]. lii the current study, ectopic over-expression of telomerase in the ALT cell line VA- 13 did not affect t circle levels detected using the RCA-based method, suggesting that telomerase activity does not affect telomere maintenance by ALT. Continued t-circle formation in the presence of telomerase shows that recombination reactions at the telomeres (at least those responsible for generating t-circles) may occur in telomerase-expressing cells. However, in the absence of telomere length analysis, it remains unclear whether t-circles contribute to elongation of chromosomal telomeres following telomerase reconstitution. Conversely, upon reconstitution, telomerase may be targeted to telomeres in a dominant manner and prevent the use oft-circles as templates for lengthening (Figure 4.1). The findings also indicate that t  52  circle formation is inhibited in human telomerase-utilizing cancer cells, such as HeLa and HCT116 (Figure 3.8), by means other than the enzyme itself. These cells may contain an unidentified inhibitory factor, or (more likely) possess a different telomere length, telomere topology, andlor telomeric chromatin relative to ALT cells.  T-circle formation 4  Tcirclemediated telornere etongation  +  Ii  Figure 4.1 Telomere dynamics in VA-13 ALT cells over-expressing ectopic telomerase. Upon reconstitution of telomerase activity, t-circles continue to be formed in the VA- 13 cell line. It remains unclear whether these t-circles contribute to telomere lengthening in the presence of telomerase, or whether they are inhibited from doing so by competition for the same molecular targets.  Several studies which evaluated other phenotypic characteristics of ALT concluded that telomerase and ALT can coexist and be concurrently active within a cell population [Cerone et a!., 2001; Grobelny eta!., 2001; Perrem eta!., 2001]. The long heterogeneous telomere length profile and the proportion of cells with APBs were shown to be unaffected by telomerase reconstitution [Cerone et aL, 2001; Grobelny et al., 2001; Perrem eta!., 2001]. Tn one study, telomere length heterogeneity continued to be generated after more than 100 PDs with telomerase activity [Perrem et a!., 2001].  53  In contrast, there is limited evidence that utilization of ALT for telomere maintenance may be affected by telomerase over-expression in some cases. One group reported a loss of long heterogeneous telomeres and a reduction in the fraction of cells containing APBs in two of nine late passage GM847 ALT cell clones ectopically over-expressing telomerase [Ford et a?., 20011. It is feasible that ALT-utilization may be lost as a stochastic event in some cells, which can continue to divide when exogenous telomerase is expressed. Alternately, ALT and telomerase may compete for access to the telomeres or other molecular targets and ALT may be repressed only in cases where a particularly high level of over-expressed telomerase subunits is present (Figure 4.1) [Henson et a?., 2002]. Further study of the molecular interactions between telomerase- and ALT-mediated telomere maintenance is needed to address these possibilities.  4.3 T-circles as by-products of overly long telomeres Recently, several phenotypic characteristics that were considered exclusive to the ALT mechanism were observed in non-ALT immortalized human cell lines with ectopically enhanced telomerase activity [Pickett et a?., 2009]. The long-term effects of exogenous hTR over-expression in the telomerase-utilizing tumour cell lines HeLa and HT1O8O resulted in increased telomerase activity, leading to long-term ( 300 PDs) telomere lengthening. Telomere lengths also became more heterogeneous with increasing PDs, resembling the telomere length profile in ALT cells. However, important hallmarks of telomere maintenance by ALT were not detected. The hTR-over-expressing cells did not show an increased rate of T-SCEs and were unable to propagate a telomere-inserted tag to other telomeres, as was previously demonstrated in ALT cells [Dunham eta?., 2000; Londoño-Vallejo et a?., 2004].  54  However, other features of ALT, such as the presence of APBs, telomeric ecDNA, and t circles were observed in these telomerase-utilizing cell lines. Thus these subnuclear structures likely represent by-products of overly long telomeres in ALT cells, rather than being --active mediators of telomere maintenance by ALT [Pickett et al., 2009]. The findings suggest that in addition to telomere lengthening mechanisms, cells also have the means to shorten overly long telomeres by t-loop excision. An important implication of this work is that recombination reactions at the telomeres are triggered when telomere lengths become generally dysregulated; both when telomeres are too short (initiating ALT-mediated lengthening) [Morrish and Greider, 2009] and when they are too long [Pickett et al., 2009].  Despite showing that t-circles are not restricted to the ALT pathway, these findings do not preclude the applicability of our RCA-based assay for t-circle detection in the context of categorizing human tumour samples based on their telomere maintenance mechanism. Human telomerase-utilizing cancer cells generally have short homogeneous telomere lengths, which would not be expected to generate t-circles at a high frequency. Conversely, the heterogeneity of telomere lengths in the majority of ALT-utilizing cancer cells means that a subset of telomeres reach a threshold length where t-loops would be excised to generate t circles and other forms of ecDNA.  55  4.4 General conclusions The major goal of this study was to develop an RCA-based assay for ALT status with the potential for screening tumour-derived samples from patients. RCA was optimized to amplify telomeric repeat-containing circular plasmid DNA as well as t-circles in ALT cell extracts. The technique allows indirect detection oft-circles from as few as 5 x 1 O cells with the potential of further scaling down cell numbers. Additionally, multiple RCA-amplified samples can be loaded onto a single denaturing gel, allowing the screening of numerous samples in parallel. Therefore, this method oft-circle screening for ALT status has potential use in the clinic to screen for this tumour immortalization mechanism and thereby guide appropriate treatment.  An additional goal of this study was to determine if reactivation of the telomerase-based telomere lengthening mechanism would affect ALT-based telomere maintenance. Phenotypic markers of ALT, including t-circles evaluated in the current study, are generally absent in telomerase-utilizing cancer cells. We found that ectopic telomerase over-expression did not affect t-circle formation in the ALT cell line VA- 13. This suggests that telomeric recombination reactions are not inhibited by telomerase, at least in this cell line. Further study is needed to determine the molecular interactions between telomerase and ALT pathways of telomere maintenance.  56  References  Allshire, R.C., Dempster, M. and Hastie, N.D. Human telomeres contain at least three types of G-rich repeat distributed non-randomly. 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