Doctor of Philosophy / Activation of a telomere maintenance mechanism is a vital step in the development of most cancers and provides a target for the selective killing of cancer cells. Cancers can use either telomerase or Alternative Lengthening of Telomeres (ALT) to maintain their telomeres and inhibition of either telomere maintenance mechanism can cause cancer cells to undergo senescence or apoptosis. Although telomerase inhibitors are undergoing clinical trials, on commencing this study very little was known about the role of ALT in cancer, what proteins were involved in its mechanism and regulation and how it could be targeted clinically. The primary aim of this thesis was to develop an assay for ALT suitable for examining archived tumour specimens and to begin using it to examine the prevalence and clinical significance of ALT in cancer. This assay and gene expression analysis was also used to identify genes that are involved in or associated with the activation of the ALT mechanism, to contribute towards the overall goal of an ALT cancer therapy. The ALT mechanism involves recombination mediated replication and ALT cells have a marked increase in a range of recombinational events specifically at their telomeres. Presumably, as a consequence of this the telomere lengths of ALT cells are very heterogeneous and on average long. This can be detected by terminal restriction fragment (TRF) Southern analysis, which has been used previously as the definitive test for ALT activity. However, TRF analysis requires intact genomic DNA and is unsuitable for tumour specimens which are commonly archived by paraffin embedding. Another hallmark of ALT is ALT-associated PML bodies (APBs) which are the subset of PML bodies that contain telomeric DNA. Work done in this study to consolidate APBs as a hallmark of ALT, combined with published data, showed 29/31 ALT[+], 3/31 telomerase[+] and 0/10 mortal cell lines/strains are APB[+]. The three APB[+]/telomerase[+] cell lines identified here had an order of magnitude lower frequency of APB[+] nuclei than the ALT[+] cell lines. APBs may be functionally linked to the ALT mechanism and contain the recombination proteins that are thought to be involved in the ALT mechanism. This study, in collaboration with Dr W-Q Jiang, strengthened this functional link by demonstrating that loss of ALT activity (as determined by TRF analysis) coincided with the disruption of APBs. The detection of APBs was developed into a robust assay for ALT in archived tumour specimens using a technique of combined immunofluorescence and telomere fluorescence in situ hybridisation. It was demonstrated that the APB assay concurred exactly with the standard assay for ALT (TRF analysis) in 60 tumours for which TRF analysis gave unequivocal results. The APB assay may be a more appropriate technique in the case of tumour specimen heterogeneity, which may explain why the APB assay was able to give definitive results when TRF analysis was equivocal. We demonstrated that intratumoral heterogeneity for ALT does exist and this could explain why about 3% of tumours in this study were APB[+] but with more than a ten-fold reduction in the frequency of APB[+] nuclei. This study also made the novel discovery of single stranded C-rich telomeric DNA inside APBs which potentially could be used to make the APB assay more suitable for routine pathology laboratory use. The APB assay was used to show that ALT is a significant concern for oncology. ALT was utilised in approximately one quarter of glioblastoma multiforme (GBM), one third of soft tissue sarcomas (STS) including three quarters of malignant fibrous histiocytomas (MFH), half of osteosarcomas and one tenth of non-small cell lung carcinomas (NSCLC). Furthermore, the patients with these ALT[+] tumours had poor survival; median survivals were 2 years for ALT[+] GBM, 4 years for ALT[+] STS including 3.5 years for ALT[+] MFH and 5 years for ALT[+] osteosarcoma. ALT[+] STS and osteosarcomas were also just as aggressive as their ALT[-] counterparts in terms of grade and patient outcome. ALT status was not found to be associated with response to chemotherapy in osteosarcomas or survival in STS. ALT was however, less prevalent in metastatic STS. The APB assay was a prognostic indicator for GBM and was correlated with three fold increased median survival in GBM (although this survival was still poor). ALT was more common in lower grade astrocytomas (88% ALT[+]) than GBM (24% ALT[+]) and ALT[+] GBM had an identical median age at diagnosis to that reported for secondary GBM. It is discussed that these data indicate that ALT was indirectly associated with secondary GBM and is possibly an early event in its progression from lower grade astrocytoma. This is relevant because secondary GBM have distinct genetic alterations that may facilitate activation of the ALT mechanism. Putative repressors of ALT could explain why this study found that ALT varied among the different STS subtypes. ALT was common in MFH (77%), leiomyosarcoma (62%) and liposarcoma (33%) but rare in rhabdomyosarcoma (6%) and synovial sarcoma (9%). ALT was not found in colorectal carcinoma (0/31) or thyroid papillary carcinoma (0/17) which have a high prevalence of telomerase activity and a reduced need for a telomere maintenance mechanism (low cell turnover), respectively. A yeast model of ALT predicts that one of the five human RecQ helicases may be required for ALT. Using the APB assay to test for the presence of ALT in tumours from patients with known mutations in either WRN or RECQL4 it was demonstrated that neither of these RecQ helicases is essential for ALT. Although p53 and mismatch repair (MMR) proteins have been suggested to be possible repressors of ALT, there was no apparent increase in the frequency of ALT in tumours from patients with a germline mutation in p53 codon 273 or in colorectal carcinomas that had microsatellite instability and thus MMR deficiency. Also contrary to being a repressor of ALT but consistent with its ability to interact with a protein involved in the ALT mechanism, the MMR protein MLH1, was demonstrated to be present in the APBs of an ALT[+] cell line. To further test for genes that may be involved in the ALT mechanism or associated with its activation, RNA microarray was used to compare the gene expression of 12 ALT[+] with 12 matched telomerase[+] cell lines; 240 genes were identified that were significantly differentially expressed (p<0.005) between the ALT[+] and telomerase[+] cell lines. Only DRG2 and SFNX4 were significantly differentially expressed after adjusting for the estimated false positive rate. Overall, DRG2, MGMT and SATB1 were identified as most likely to be relevant to the ALT[+] tumours and Western analysis indicated that DRG2 and MGMT levels were down-regulated after activation of ALT and up-regulated after activation of telomerase, whereas SATB1 protein levels appeared to be up-regulated after immortalisation but to a higher degree with activation of ALT compared to telomerase. Since lack of MGMT is known to be a determinant of temozolomide sensitivity in GBM, the possibility that ALT and the APB assay could be used to predict temozolomide sensitivity is discussed. The microarray data was consistent with MGMT expression being suppressed by EGF (p < 0.05), indicating that caution may be needed with combining EGFR inhibitors with temozolomide in ALT cancers. One ALT[+] cell line which did not express MGMT had TTAA sequence in its telomeres. This could possibly have resulted from mutations due to lack of MGMT expression and a possible role for MGMT in the ALT mechanism is discussed. Further analysis of the microarray data identified two groups of co-regulated genes (p < 5x10-5): CEBPA, TACC2, SFXN4, HNRPK and MGMT, and SIGIRR, LEF1, NSBP1 and SATB1. Two thirds of differentially expressed genes were down-regulated in ALT. Chromosomes 10 and 15 had a bias towards genes with lower expression in ALT while chromosomes 1, 4, 14 and X had a bias towards genes with higher expression levels in ALT. This work has developed a robust assay for ALT in tumour specimens which was then used to show the significance of ALT in sarcomas, astrocytomas and NSCLC. It has also identified genes that could possibly be molecular targets for the treatment of ALT[+] cancers.

Telomeres are specialised structures, consisting of TTAGGG DNA repeats and binding proteins, that cap the ends of human chromosomes and maintain chromosome integrity. It has been shown that telomeres shorten with each round of cell division in most normal human somatic cells. It has become generally accepted that this shortening is due, in part, to the inability of DNA polymerases to replicate the extreme ends of chromosomes which is a phenomenon known as the �end replication problem�. An intriguing hypothesis that has emerged from these observations is that critically shortened telomeres trigger growth arrest and senescence. This is regarded as a key determining factor in the limited lifespan of normal cells in culture and is commonly known as the �Telomere Hypothesis of Senescence�. In support of this hypothesis it has been demonstrated that immortalised human cells, that have an unlimited lifespan in culture, maintain stable telomere lengths and do not undergo progressive telomere shortening. In most cases this is due to the ribonucleoprotein enzyme telomerase, the activation of which is as a key step in the immortalisation process. Telomerase compensates for sequential telomere shortening by utilising an RNA template to catalyse the addition of repeat sequences by reverse transcription. It is absent from most normal tissue but is present in the germline and is presumably downregulated during development. Significantly, analysis of human tumour cells has shown that a majority also have active telomerase, which supports the importance of immortalisation in tumourigenesis. Previous work in this laboratory has shown that, although the majority of in vitro immortalised cells and tumour cells that have been studied maintain telomeres by reactivation of telomerase, a proportion do not have detectable telomerase activity. These telomerase-negative cells still maintain telomeres, however, and this is via a mechanism(s) yet to be fully elucidated known as Alternative Lengthening of Telomeres (ALT). ALT is characterised, in addition to lack of telomerase activity, by extreme telomere length heterogeneity with telomere lengths ranging from over 50 kilobases (kb) of DNA to almost undetectable. This phenotype is evident, by Southern analysis and fluorescent in situ hybridisation (FISH), in all ALT cells. Alternative mechanisms of telomere maintenance, via retrotransposition and recombination, had already been characterised in lower eukaryotes. It has been shown in this laboratory that ALT cell lines and tumours contain a novel type of PML body, referred to as ALT-associated PML bodies (APBs). APBs have been found in all of the ALT cell lines so far tested and also in archival tumour sections, and contain a number of factors which co-localise. These include PML, TTAGGG repeats, TRF 1 & TRF 2 telomere binding proteins and proteins involved in homologous recombination: RAD51 & RAD52. More recently, it has been shown that the RAD50/Mre11/Nbs1 complex, which is involved in cell cycle checkpoint control and repair of DNA damage, is also present in APBs. The presence of these RAD proteins in APBs is of great interest as a recombination between telomeres has been proposed as the central mechanism by which ALT lengthens telomeres. Studies in yeast have identified such a mechanism and it was proposed that a similar process occurred in human immortal cells that utilise ALT. It has now been shown by this laboratory that a recombination mechanism is indeed evident at the telomeres of ALT cells. To date all in vitro immortalised cell lines and most tumour cell types that have been studied have a telomere maintenance mechanism either via telomerase or ALT. Targeting telomerase has become a major focus of anti-cancer research as inhibitors have the potential to treat a wide variety of different tumour types. An understanding of ALT and its regulation is likely to be important in such therapeutic strategies, as selective pressure due to telomerase inhibition may result in ALT revertants within the tumour mass. Development of inhibitors of both telomerase and ALT may therefore be required when targeting telomere maintenance. The main focus of this thesis is the understanding of ALT repression in the SV40 immortalised skin fibroblast cell line GM847, as a means to further understanding the mechanism of ALT. The data presented provide new insights into the repression of ALT and also the relationship between telomerase and ALT which is important for our understanding of telomere maintenance in human cancer. Hybrids formed by fusion of normal cells and ALT cells underwent rapid telomere loss followed by senescence, indicating that normal cells contain factors that repress ALT. This strongly suggests that ALT is recessive and is activated in part by loss or mutation of repressors. Similar experiments were performed with ALT cells and telomerasepositive cells, and the resulting hybrids were all telomerase-positive and ALT repressed. It is possible that the same negative regulators are involved as additional data show that telomerase does not act as an ALT inhibitor. Exogenous expression of telomerase in ALT cells did not repress ALT, but both mechanisms co-existed in these transfected cells. This result provides a further argument for targeting both ALT and telomerase in any future treatments of tumours as it demonstrates in principle that these mechanisms are not mutually exclusive. A serendipitous finding was that a dominant-negative telomerase catalytic subunit caused telomere shortening in ALT cells, had not been reported elsewhere, and indeed was in contrast to previous findings. This provided further evidence for a link between telomerase and ALT as it suggested that there were essential components that were common to both pathways. As a further means to understanding ALT repression, a series of experiments was performed to determine the chromosomal localisation of ALT repressor(s) by microcell mediated chromosome transfer. This was done to facilitate the eventual isolation of repressors. A repressor of ALT in the chemically immortalised fibroblast cell line SUSM-1, had been reported to be localised to chromosome 7. This result could not be repeated in the GM847 cell line, but ALT repression was evident in GM847 cells upon transfer of chromosome 6. Another important question regarding the nature of ALT is the structure and sequence of the long heterogeneous telomeres generated by ALT specific recombination, which is the focus of the final series of data that is presented. ALT telomere length heterogeneity was detected under denaturing conditions, ruling out the possibility that it was an artefact of electrophoresis conditions due to novel secondary structure. Although the hybridisation signal intensity of TTAGGG increases at the onset of immortalisation in ALT cells, it had been demonstrated by restriction digests that degenerate repeats did exist at the telomeres of some ALT cell lines. Sequences containing telomere repeats were cloned from the ALT cell line WI38 VA13/2RA (SV40 immortalised fibroblasts) and these were found to be interspersed with a number of other sequence fragments. The significance of these sequences in relation to the mechanism of ALT is discussed.

Telomeres are specialised structures, consisting of TTAGGG DNA repeats and binding proteins, that cap the ends of human chromosomes and maintain chromosome integrity. It has been shown that telomeres shorten with each round of cell division in most normal human somatic cells. It has become generally accepted that this shortening is due, in part, to the inability of DNA polymerases to replicate the extreme ends of chromosomes which is a phenomenon known as the �end replication problem�. An intriguing hypothesis that has emerged from these observations is that critically shortened telomeres trigger growth arrest and senescence. This is regarded as a key determining factor in the limited lifespan of normal cells in culture and is commonly known as the �Telomere Hypothesis of Senescence�. In support of this hypothesis it has been demonstrated that immortalised human cells, that have an unlimited lifespan in culture, maintain stable telomere lengths and do not undergo progressive telomere shortening. In most cases this is due to the ribonucleoprotein enzyme telomerase, the activation of which is as a key step in the immortalisation process. Telomerase compensates for sequential telomere shortening by utilising an RNA template to catalyse the addition of repeat sequences by reverse transcription. It is absent from most normal tissue but is present in the germline and is presumably downregulated during development. Significantly, analysis of human tumour cells has shown that a majority also have active telomerase, which supports the importance of immortalisation in tumourigenesis. Previous work in this laboratory has shown that, although the majority of in vitro immortalised cells and tumour cells that have been studied maintain telomeres by reactivation of telomerase, a proportion do not have detectable telomerase activity. These telomerase-negative cells still maintain telomeres, however, and this is via a mechanism(s) yet to be fully elucidated known as Alternative Lengthening of Telomeres (ALT). ALT is characterised, in addition to lack of telomerase activity, by extreme telomere length heterogeneity with telomere lengths ranging from over 50 kilobases (kb) of DNA to almost undetectable. This phenotype is evident, by Southern analysis and fluorescent in situ hybridisation (FISH), in all ALT cells. Alternative mechanisms of telomere maintenance, via retrotransposition and recombination, had already been characterised in lower eukaryotes. It has been shown in this laboratory that ALT cell lines and tumours contain a novel type of PML body, referred to as ALT-associated PML bodies (APBs). APBs have been found in all of the ALT cell lines so far tested and also in archival tumour sections, and contain a number of factors which co-localise. These include PML, TTAGGG repeats, TRF 1 & TRF 2 telomere binding proteins and proteins involved in homologous recombination: RAD51 & RAD52. More recently, it has been shown that the RAD50/Mre11/Nbs1 complex, which is involved in cell cycle checkpoint control and repair of DNA damage, is also present in APBs. The presence of these RAD proteins in APBs is of great interest as a recombination between telomeres has been proposed as the central mechanism by which ALT lengthens telomeres. Studies in yeast have identified such a mechanism and it was proposed that a similar process occurred in human immortal cells that utilise ALT. It has now been shown by this laboratory that a recombination mechanism is indeed evident at the telomeres of ALT cells. To date all in vitro immortalised cell lines and most tumour cell types that have been studied have a telomere maintenance mechanism either via telomerase or ALT. Targeting telomerase has become a major focus of anti-cancer research as inhibitors have the potential to treat a wide variety of different tumour types. An understanding of ALT and its regulation is likely to be important in such therapeutic strategies, as selective pressure due to telomerase inhibition may result in ALT revertants within the tumour mass. Development of inhibitors of both telomerase and ALT may therefore be required when targeting telomere maintenance. The main focus of this thesis is the understanding of ALT repression in the SV40 immortalised skin fibroblast cell line GM847, as a means to further understanding the mechanism of ALT. The data presented provide new insights into the repression of ALT and also the relationship between telomerase and ALT which is important for our understanding of telomere maintenance in human cancer. Hybrids formed by fusion of normal cells and ALT cells underwent rapid telomere loss followed by senescence, indicating that normal cells contain factors that repress ALT. This strongly suggests that ALT is recessive and is activated in part by loss or mutation of repressors. Similar experiments were performed with ALT cells and telomerasepositive cells, and the resulting hybrids were all telomerase-positive and ALT repressed. It is possible that the same negative regulators are involved as additional data show that telomerase does not act as an ALT inhibitor. Exogenous expression of telomerase in ALT cells did not repress ALT, but both mechanisms co-existed in these transfected cells. This result provides a further argument for targeting both ALT and telomerase in any future treatments of tumours as it demonstrates in principle that these mechanisms are not mutually exclusive. A serendipitous finding was that a dominant-negative telomerase catalytic subunit caused telomere shortening in ALT cells, had not been reported elsewhere, and indeed was in contrast to previous findings. This provided further evidence for a link between telomerase and ALT as it suggested that there were essential components that were common to both pathways. As a further means to understanding ALT repression, a series of experiments was performed to determine the chromosomal localisation of ALT repressor(s) by microcell mediated chromosome transfer. This was done to facilitate the eventual isolation of repressors. A repressor of ALT in the chemically immortalised fibroblast cell line SUSM-1, had been reported to be localised to chromosome 7. This result could not be repeated in the GM847 cell line, but ALT repression was evident in GM847 cells upon transfer of chromosome 6. Another important question regarding the nature of ALT is the structure and sequence of the long heterogeneous telomeres generated by ALT specific recombination, which is the focus of the final series of data that is presented. ALT telomere length heterogeneity was detected under denaturing conditions, ruling out the possibility that it was an artefact of electrophoresis conditions due to novel secondary structure. Although the hybridisation signal intensity of TTAGGG increases at the onset of immortalisation in ALT cells, it had been demonstrated by restriction digests that degenerate repeats did exist at the telomeres of some ALT cell lines. Sequences containing telomere repeats were cloned from the ALT cell line WI38 VA13/2RA (SV40 immortalised fibroblasts) and these were found to be interspersed with a number of other sequence fragments. The significance of these sequences in relation to the mechanism of ALT is discussed.

Doctor of Philosophy / Activation of a telomere maintenance mechanism is a vital step in the development of most cancers and provides a target for the selective killing of cancer cells. Cancers can use either telomerase or Alternative Lengthening of Telomeres (ALT) to maintain their telomeres and inhibition of either telomere maintenance mechanism can cause cancer cells to undergo senescence or apoptosis. Although telomerase inhibitors are undergoing clinical trials, on commencing this study very little was known about the role of ALT in cancer, what proteins were involved in its mechanism and regulation and how it could be targeted clinically. The primary aim of this thesis was to develop an assay for ALT suitable for examining archived tumour specimens and to begin using it to examine the prevalence and clinical significance of ALT in cancer. This assay and gene expression analysis was also used to identify genes that are involved in or associated with the activation of the ALT mechanism, to contribute towards the overall goal of an ALT cancer therapy. The ALT mechanism involves recombination mediated replication and ALT cells have a marked increase in a range of recombinational events specifically at their telomeres. Presumably, as a consequence of this the telomere lengths of ALT cells are very heterogeneous and on average long. This can be detected by terminal restriction fragment (TRF) Southern analysis, which has been used previously as the definitive test for ALT activity. However, TRF analysis requires intact genomic DNA and is unsuitable for tumour specimens which are commonly archived by paraffin embedding. Another hallmark of ALT is ALT-associated PML bodies (APBs) which are the subset of PML bodies that contain telomeric DNA. Work done in this study to consolidate APBs as a hallmark of ALT, combined with published data, showed 29/31 ALT[+], 3/31 telomerase[+] and 0/10 mortal cell lines/strains are APB[+]. The three APB[+]/telomerase[+] cell lines identified here had an order of magnitude lower frequency of APB[+] nuclei than the ALT[+] cell lines. APBs may be functionally linked to the ALT mechanism and contain the recombination proteins that are thought to be involved in the ALT mechanism. This study, in collaboration with Dr W-Q Jiang, strengthened this functional link by demonstrating that loss of ALT activity (as determined by TRF analysis) coincided with the disruption of APBs. The detection of APBs was developed into a robust assay for ALT in archived tumour specimens using a technique of combined immunofluorescence and telomere fluorescence in situ hybridisation. It was demonstrated that the APB assay concurred exactly with the standard assay for ALT (TRF analysis) in 60 tumours for which TRF analysis gave unequivocal results. The APB assay may be a more appropriate technique in the case of tumour specimen heterogeneity, which may explain why the APB assay was able to give definitive results when TRF analysis was equivocal. We demonstrated that intratumoral heterogeneity for ALT does exist and this could explain why about 3% of tumours in this study were APB[+] but with more than a ten-fold reduction in the frequency of APB[+] nuclei. This study also made the novel discovery of single stranded C-rich telomeric DNA inside APBs which potentially could be used to make the APB assay more suitable for routine pathology laboratory use. The APB assay was used to show that ALT is a significant concern for oncology. ALT was utilised in approximately one quarter of glioblastoma multiforme (GBM), one third of soft tissue sarcomas (STS) including three quarters of malignant fibrous histiocytomas (MFH), half of osteosarcomas and one tenth of non-small cell lung carcinomas (NSCLC). Furthermore, the patients with these ALT[+] tumours had poor survival; median survivals were 2 years for ALT[+] GBM, 4 years for ALT[+] STS including 3.5 years for ALT[+] MFH and 5 years for ALT[+] osteosarcoma. ALT[+] STS and osteosarcomas were also just as aggressive as their ALT[-] counterparts in terms of grade and patient outcome. ALT status was not found to be associated with response to chemotherapy in osteosarcomas or survival in STS. ALT was however, less prevalent in metastatic STS. The APB assay was a prognostic indicator for GBM and was correlated with three fold increased median survival in GBM (although this survival was still poor). ALT was more common in lower grade astrocytomas (88% ALT[+]) than GBM (24% ALT[+]) and ALT[+] GBM had an identical median age at diagnosis to that reported for secondary GBM. It is discussed that these data indicate that ALT was indirectly associated with secondary GBM and is possibly an early event in its progression from lower grade astrocytoma. This is relevant because secondary GBM have distinct genetic alterations that may facilitate activation of the ALT mechanism. Putative repressors of ALT could explain why this study found that ALT varied among the different STS subtypes. ALT was common in MFH (77%), leiomyosarcoma (62%) and liposarcoma (33%) but rare in rhabdomyosarcoma (6%) and synovial sarcoma (9%). ALT was not found in colorectal carcinoma (0/31) or thyroid papillary carcinoma (0/17) which have a high prevalence of telomerase activity and a reduced need for a telomere maintenance mechanism (low cell turnover), respectively. A yeast model of ALT predicts that one of the five human RecQ helicases may be required for ALT. Using the APB assay to test for the presence of ALT in tumours from patients with known mutations in either WRN or RECQL4 it was demonstrated that neither of these RecQ helicases is essential for ALT. Although p53 and mismatch repair (MMR) proteins have been suggested to be possible repressors of ALT, there was no apparent increase in the frequency of ALT in tumours from patients with a germline mutation in p53 codon 273 or in colorectal carcinomas that had microsatellite instability and thus MMR deficiency. Also contrary to being a repressor of ALT but consistent with its ability to interact with a protein involved in the ALT mechanism, the MMR protein MLH1, was demonstrated to be present in the APBs of an ALT[+] cell line. To further test for genes that may be involved in the ALT mechanism or associated with its activation, RNA microarray was used to compare the gene expression of 12 ALT[+] with 12 matched telomerase[+] cell lines; 240 genes were identified that were significantly differentially expressed (p<0.005) between the ALT[+] and telomerase[+] cell lines. Only DRG2 and SFNX4 were significantly differentially expressed after adjusting for the estimated false positive rate. Overall, DRG2, MGMT and SATB1 were identified as most likely to be relevant to the ALT[+] tumours and Western analysis indicated that DRG2 and MGMT levels were down-regulated after activation of ALT and up-regulated after activation of telomerase, whereas SATB1 protein levels appeared to be up-regulated after immortalisation but to a higher degree with activation of ALT compared to telomerase. Since lack of MGMT is known to be a determinant of temozolomide sensitivity in GBM, the possibility that ALT and the APB assay could be used to predict temozolomide sensitivity is discussed. The microarray data was consistent with MGMT expression being suppressed by EGF (p < 0.05), indicating that caution may be needed with combining EGFR inhibitors with temozolomide in ALT cancers. One ALT[+] cell line which did not express MGMT had TTAA sequence in its telomeres. This could possibly have resulted from mutations due to lack of MGMT expression and a possible role for MGMT in the ALT mechanism is discussed. Further analysis of the microarray data identified two groups of co-regulated genes (p < 5x10-5): CEBPA, TACC2, SFXN4, HNRPK and MGMT, and SIGIRR, LEF1, NSBP1 and SATB1. Two thirds of differentially expressed genes were down-regulated in ALT. Chromosomes 10 and 15 had a bias towards genes with lower expression in ALT while chromosomes 1, 4, 14 and X had a bias towards genes with higher expression levels in ALT. This work has developed a robust assay for ALT in tumour specimens which was then used to show the significance of ALT in sarcomas, astrocytomas and NSCLC. It has also identified genes that could possibly be molecular targets for the treatment of ALT[+] cancers.

Telomeres are repetitive DNA sequences found at the ends of eukaryotic chromosomes that help maintain genome stability. Telomeres shorten every time a cell divides, eventually inducing replicative senescence. To gain replicative immortality cancer cells establish mechanisms to maintain telomere length over many cell divisions. Around 10% of cancers do this using a recombination-based pathway called the Alternative Lengthening of Telomeres (ALT). ALT resembles a specific type of homology-directed repair called break-induced replication (BIR). Through this body of work, we aimed to better understand both the genetics underlying ALT positive cancers and the mechanistic basis of ALT. ALT positive cancers frequently carry loss of function mutations in the genes for ATRX/DAXX, which function to regulate heterochromatin. Recently, we identified a novel chromosomal fusion event in ALT positive osteosarcoma causing defects in DAXX function. Additionally, we identified several osteosarcoma tumors with wild-type ATRX/DAXX that had abnormalities in SLX4IP or SMARCAL1, proteins recently shown to regulate the ALT pathway. These data suggest that a more thorough understanding of the ALT mechanism may reveal additional factors that are defective in ALT positive tumors. Building on this, we aimed to further define the mechanism of ALT by investigating the DNA translocase RAD54 in the ALT pathway. During BIR, a broken DNA strand invades a homologous template, forming a structure called a displacement loop (D-loop) where a strand of template DNA is displaced to allow base pairing between the broken DNA strand and the homologous template. The D-loop recruits DNA polymerases, leading to extension and repair of the broken DNA strand. RAD54 is known to regulate both the formation and resolution of D-loops. In this work, we found that RAD54 promotes elongation at ALT telomeres by mediating branch migration and dissolution of the D-loop. D-loops formed at ALT telomeres must be resolved before mitosis to prevent the formation of ultra-fine anaphase bridges. These data demonstrate that by mediating D-loop migration RAD54 plays an important role in both promoting telomere elongation and maintaining genome stability in ALT cells. Together this body of work represents advances in defining both the genetic and mechanistic basis of ALT. / 2021-01-30T00:00:00Z

Des mutations dans ATRX, une protéine de remodelage de la chromatine, ont été associées, dans plusieurs études cliniques, avec la voie télomérase-indépendante de maintenance des télomères (voie ALT) dans plusieurs types de cancer. Grâce à des expériences d’immunoprécipitation de chromatine (ChIP), nous avons montré qu’ATRX était localisée au niveau subtélomérique de cellules tumorales humaines en culture. Nous avons également montré, par ChIP, que l’inactivation génétique d’ATRX provoquait une diminution des quantités de cohésine/SMC1 présentes dans les régions subtélomériques. L’inactivation d’ATRX a conduit en outre à une diminution des quantités de TERRA, transcrits non codants de l’ADN télomérique. Nos données suggèrent qu’ATRX pourrait établir des interactions fonctionnelles avec la cohésine au niveau de la chromatine subtelomérique afin de contrôler les niveaux de TERRA et que l’un ou l’autre de ces évènements pourrait avoir un rapport avec la voie ALT. / Mutations in ATRX, a chromatin remodeling protein, have been found, in several clinical studies, associated with the telomerase-independent ALT pathway of telomere maintenance in several types of cancer. Using chromatin immunoprecipitation (ChIP), we have shown that ATRX localized to subtelomeric regions of human tumor cells in culture. Cohesin has recently been shown to be part of telomeric chromatin. Here, using ChIP, we showed that genetic inactivation of ATRX provoked a diminution in the amount of cohesin in subtelomeric regions of telomerase-positive glioma cells. Moreover, inactivation of ATRX also led to a diminution in the amount of TERRAs, non-coding RNAs resulting from transcription of telomeric DNA. Our data suggest that ATRX might establish functional interactions with cohesin on subtelomeric chromatin in order to control TERRA levels and that one or the other or both of these events might be important for ALT mechanisms.

In the absence of telomerase activity, a subset of cancerous and immortalized cells maintain telomere length by means of a poorly understood mechanism, termed alternative lengthening of telomeres (ALT). Many details of telomere maintenance in ALT positive cells remain unclear, but significant evidence implicates a homologous recombination mechanism. ALT specific nuclear structures, known as ALT-associated promyelocytic leukemia nuclear bodies (APBs), are thought to serve as the site of telomere extension. Using electron spectroscopic imaging we have demonstrated that APBs contain substantial amounts of nucleic acid sequestered within the bodies. In contrast, promyelocytic leukemia nuclear bodies in non-ALT cell lines contain no significant nucleic acid. We show that the nucleic acid found in APBs is not RNA and provide evidence that it is in fact telomeric repeat DNA. This evidence supports a role for APBs to sequester extrachromosomal telomeric DNA in order to suppress the activation of DNA repair.

In the absence of telomerase activity, a subset of cancerous and immortalized cells maintain telomere length by means of a poorly understood mechanism, termed alternative lengthening of telomeres (ALT). Many details of telomere maintenance in ALT positive cells remain unclear, but significant evidence implicates a homologous recombination mechanism. ALT specific nuclear structures, known as ALT-associated promyelocytic leukemia nuclear bodies (APBs), are thought to serve as the site of telomere extension. Using electron spectroscopic imaging we have demonstrated that APBs contain substantial amounts of nucleic acid sequestered within the bodies. In contrast, promyelocytic leukemia nuclear bodies in non-ALT cell lines contain no significant nucleic acid. We show that the nucleic acid found in APBs is not RNA and provide evidence that it is in fact telomeric repeat DNA. This evidence supports a role for APBs to sequester extrachromosomal telomeric DNA in order to suppress the activation of DNA repair.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, caused by a GAA repeat expansion mutation within intron 1 of the FXN gene, resulting in reduced level of frataxin protein. Normal individuals have 5 to 40 GAA repeat sequences, whereas affected individuals have approximately 70 to more than 1000 GAA triplets. Frataxin is a mitochondrial protein involved in iron-sulphur cluster and heme biosynthesis. The reduction in frataxin expression leads to oxidative stress, mitochondrial iron accumulation and consequential cell death with the primary sites of neurons of the dorsal root ganglia and the dentate nucleus of the cerebellum. FRDA, which is the most common inherited ataxia, affecting 1:50,000 Caucasians, is characterised by neurodegeneration, cardiomyopathy, diabetes mellitus and skeletal deformities. To investigate FRDA molecular disease mechanisms and therapy, several human FXN YAC transgenic mouse models have been established: Y47R, containing normal-sized (GAA)9 repeats; YG8R and YG22R, which initially contained expanded GAA repeats of 90-190 units and 190 units, respectively, but which have subsequently been bred to now contain expanded GAA repeats of 120-220 units and 170-260 units, respectively, and YG8sR (YG8R with a small GAA band) that was recently generated from YG8R breeding. To determine the FXN transgene copy number in the enhanced GAA repeat expansion-based FRDA mouse lines, a TaqMan qPCR assay was developed. The results demonstrated that the YG22R and Y47R lines had a single copy of the FXN transgene while the YG8R line had two copies. The YG8s lines showed less than one copy of the target gene, suggesting potential deletion of the FXN gene. Single integration sites of all transgenes were confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. However, in the YG8s line, at least 25% of the YG8s cells had no signals, while the remaining cells showed one signal corresponding to the transgenic FXN gene. In addition, the analysis of FXN exons in YG8s rescue mice by PCR confirmed the presence of all FXN exons in these lines, suggesting the incidence of somatic mosaicism in these lines. Extended functional analysis was carried out on these mice from 4 to 12 months of age. Coordination ability of YG8R, YG8sR and YG22R ‘FRDA-like’ mice, together with Y47R and C57BL6/J wild-type control mice, was assessed using accelerating rotarod analysis. The results indicated a progressive decrease in the motor coordination of YG8R, YG22R and YG8sR mice compared to Y47R or C57BL6/J controls. Locomotor activity was also assessed using an open field beam-breaker apparatus followed by four additional functional analyses including beam-walk, hang wire, grip strength and foot print tests. The results indicated significant functional deficits in the FRDA mouse models. Glucose and insulin tolerance tests were also conducted in the FRDA mouse models, indicating glucose intolerance and insulin hypersensitivity in the aforementioned lines. To investigate the correlation between the FRDA-like pathological phenotype and frataxin deficiency in the FRDA mouse models, frataxin mRNA and protein levels as well as somatic GAA repeat instability were examined. The results indicated that somatic GAA repeats increased in the cerebellum and brain of YG22R, YG8R and YG8sR mice, together with significantly reduced levels of FXN mRNA and protein in the liver of YG8R and YG22R compared to Y47R. However, YG8sR lines showed a significant decrease in FXN mRNA in all of the examined tissues compared to Y47R human FXN and C57BL6/J mouse Fxn mRNA. Protein expression levels were also considerably reduced in all the tissues of YG8sR mice compared to Y47R. Subsequently, the telomere length of human and mouse FRDA and control fibroblasts was assessed using qPCR and Q-FISH. The results indicated that the FRDA cells had chromosomes with relatively longer telomeric repeats in comparison to the controls. FRDA cells were screened for expression of telomerase activity using the TRAP assay and a quantitative assay for hTERT mRNA expression using TaqMan qRT-PCR. The results indicated that telomerase activity was not present in the FRDA cells. To investigate whether FRDA cells maintained their telomeres by ALT associated PML bodies (APBs), co-localisation of PML bodies with telomeres was assessed in these cells using combined immunofluorescence to PML and Q-FISH for telomere detection. The results demonstrated that the FRDA cells had significantly higher co-localised PML foci with telomeric DNA compared to the normal cells. Moreover, telomere sister chromatid exchange (T-SCE) frequencies were analysed in the human FRDA cell lines using chromosome orientation FISH (CO-FISH). The results indicated a significant increase in T-SCE levels of the FRDA cell lines relative to the controls. Furthermore, growth curve and population doubling analysis of the human FRDA and control fibroblasts was carried out. The results showed that the FRDA fibroblast cell cultures underwent growth arrest with higher cumulative population doubling compared to the controls. Though, further analysis of telomere length at different passage numbers revealed that the FRDA cells lost telomeres faster than the controls. Finally, the telomere dysfunction-induced foci (TIF) assay was performed to detect DNA damage in the human FRDA fibroblast cells using an antibody against DNA damage marker γ-H2AX and a synthetic PNA probe for telomeres. The frequency of γ-H2AX foci was significantly higher in the FRDA cells compared to the controls. Similarly, the FRDA cells had greater frequencies of TIFs in comparison to the controls, suggesting induced telomere dysfunction in the FRDA cells.

Telomeres are nucleoprotein complexes located at the end of chromosomes. They have an essential role in protecting chromosome ends. Telomerase or ALT (alternative lengthening of telomeres) mechanisms maintain telomeres by compensating natural telomeric loss. We have set up a flow-FISH method and using mouse lymphoma cell lines we identified unexpectedly the presence of subpopulations of cells with different telomere lengths. Subpopulations of cells with different telomere lengths were also observed in a human ALT and non-ALT cell line. Differences in telomere length between subpopulations of cells were significant and we term this phenomenon TELEFLUCS (TElomere LEngth FLUctuations in Cell Subpopulations). By applying flow-FISH we could successfully measure telomere lengths during replicative senescence in human primary fibroblasts with different genetic defects that confer sensitivity to ionising radiation (IR). The results from this study, based on flow-FISH and Southern hybridisation measurements, revealed an accelerated rate of telomere shortening in radiosensitive fibroblasts. We also observed accelerated telomere shortening in murine BRCA1 deficient cells, another defect conferring radiosensitivity, in comparison with a BRCA1 proficient cell line. We transiently depleted BRCA1 by siRNAs in two human mammary epithelial cell lines but could not find changes in telomere length in comparison with control cells. Cytological evidence of telomere dysfunction was observed in all radiosensitive cell lines. These results suggest that mechanisms that confer sensitivity to IR may be linked with mechanisms that cause telomere dysfunction. Furthermore, we have been able to show that human ALT positive cell lines show dysfunctional telomeres as detected by either the presence of DSBs at their telomeres or cytogenetic analysis and usually cells with dysfunctional telomeres are sensitive to IR. Finally, we assessed hTERT mRNA splicing variants and telomerase activity in brain tumours, which exhibit considerable chromosome instability suggesting that DNA repair mechanisms may be impaired. We demonstrated that high levels of hTERT mRNAs and telomerase activity correlate with proliferation rate. The presence of hTERT splice variants did not strictly correlate with absence of telomerase activity but hTERT spliced transcripts were observed in some telomerase negative brain tumours suggesting that hTERT splicing may contribute to activation of ALT mechanisms.