Telomere shortening and chromosomal instability in myelodysplastic syndromes

Telomere shortening and chromosomal instability are believed to play an important role in the development of myeloid neoplasia. So far, published data are only available on the average telomere length in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), but not on the telomere length of individual chromosomes. We used a new technique, telomere/centromere‐fluorescence in situ hybridization (T/C‐FISH), which combines fluorescence R‐banding and FISH using a probe against the telomere repeats to measure the telomere length of each chromosome arm in 78 patients with MDS. In line with the previous results, patients with MDS showed significantly shorter telomeres than those of healthy controls. Telomere lengths did not differ significantly between distinct morphological subtypes of MDS. However, there was a significant difference in telomere length between patients with an isolated monosomy 7 and patients with a normal karyotype (P < 0.05). Notably, patients with an isolated monosomy 7 showed significantly longer telomeres than patients with a normal karyotype in many chromosome arms, among them 7p and 7q. Neo‐telomeres were found in two patients with a complex karyotype, in one case at the fusion site of a dic(14;20). Normal and aberrant metaphases of the same patient did not differ in telomere length, thus indicating to telomere shortening as a basic mechanism affecting all hematopoietic cells in patients with MDS. In some MDS subtypes, like MDS with isolated monosomy 7, telomeres may be stabilized and even increase in length because of the activation of telomerase or alternative mechanisms. © 2009 Wiley‐Liss, Inc.     

Q‐FISH analysis of telomere and chromosome instability in the oesophagus with and without squamous cell carcinoma in situ

Chromosomal and genomic instability due to telomere dysfunction is known to play an important role in carcinogenesis. To study telomere dysfunction in the surrounding background epithelium of squamous cell carcinoma in situ (CIS) of the oesophagus, we measured telomere lengths of basal and parabasal cells of epithelia with and without CIS using quantitative fluorescence in situ hybridization (Q‐FISH) and our original software, Tissue Telo. Additionally, we assessed histological inflammation and the anaphase bridge index. In non‐cancerous epithelium, telomeres in basal cells were significantly longer than those in parabasal cells, whereas CIS showed a homogeneous telomere pattern in the basal and parabasal cells. Telomeres in basal and parabasal cells were significantly shorter in the background with CIS than in epithelium from age‐matched normal controls. Significant negative correlation was observed between the normalized telomere : centromere ratio (reflected telomere length) and the anaphase bridge index in non‐cancerous epithelia from both normal controls and the CIS background with no histological inflammation. These findings indicate that tissue stem cells may be located among basal cells, and that telomere length distribution in component cell types differs between CIS and non‐cancerous epithelium. We have demonstrated conclusively that oesophageal CIS arises from epithelium with short telomeres and chromosomal instability in the absence of histological inflammation. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Different TP53 mutations are associated with specific chromosomal rearrangements,telomere length changes,and remodeling of the nuclear architecture of telomeres

TP53 mutations are the most common mutations in human cancers, and TP53‐R175H and TP53‐R273H are the most frequent. The impact of these mutations on genomic instability after tumor initiation is still uncovered. To gain insight into this, we studied the effects of three specific TP53 mutants (TP53‐V143A, TP53‐R175H, and TP53‐R273H) on genomic instability using four isogenic lines of LoVo cells. Multicolor fluorescence in situ hybridization (FISH), three‐dimensional (3D) quantitative FISH (Q‐FISH) on interphase and Q‐FISH on metaphases were used to investigate genomic instability. We found that LoVo cells expressing mutant TP53‐R175H displayed the highest level of chromosomal instability among the LoVo cell lines. Furthermore, we observed that mutant TP53‐R175H and TP53‐V143A showed more alterations in their 3D nuclear architecture of telomeres than the mutant TP53‐R273H and the wild type. Moreover, we noted an association between some chromosomal abnormalities and telomere elongation in the mutant TP53‐R175H. Taken together, our results indicate that the mutation TP53‐R175H is more likely to cause higher levels of genomic instability than the other TP53 mutations. We proposed that the type of TP53 mutations and the genetic background of a cancer cell are major determinants of the TP53‐dependent genomic instability. © 2014 Wiley Periodicals, Inc.     

Genetic instability in inherited and sporadic leukemias

Genetic instability due to increased DNA damage and altered DNA repair is of central significance in the initiation and progression of inherited and sporadic human leukemias. Although very rare, some inherited DNA repair insufficiency syndromes (e.g., Fanconi anemia, Bloom's syndrome) have added substantially to our understanding of crucial mechanisms of leukemogenesis in recent years. Conversely, sporadic leukemias account for the main proportion of leukemias and here DNA damaging reactive oxygen species (ROS) play a central role. Although the exact mechanisms of increased ROS production remain largely unknown and no single pathway has been detected thus far, some oncogenic proteins (e.g., the activated tyrosine kinases BCR‐ABL1 and FLT3‐ITD) seem to play a key role in driving genetic instability by increased ROS generation which influences the disease course (e.g., blast crisis in chronic myeloid leukemia or relapse in FLT3‐ITD positive acute myeloid leukemia). Of course other mechanisms, which promote genetic instability in leukemia also exist. A newly emerging mechanism is the genome‐wide alteration of epigenetic marks (e.g., hypomethylation of histone H3K79), which promotes chromosomal instability. Taken together genetic instability plays a critical role both in inherited and sporadic leukemias and emerges as a common theme in both inherited and sporadic leukemias. Beyond its theoretical impact, the analysis of genetic instability may lead the way to the development of innovative therapy strategies. © 2010 Wiley‐Liss, Inc.     

Heterozygous mutations in the PALB2 hereditary breast cancer predisposition gene impact on the three‐dimensional nuclear organization of patient‐derived cell lines

PALB2/FANCN is a BRCA1‐ and BRCA2‐interacting Fanconi Anemia (FA) protein crucial for key BRCA2 genome caretaker functions. Heterozygous germline mutations in PALB2 predispose to breast cancer and biallelic mutations cause FA. FA proteins play a critical role in the telomere maintenance pathway, with telomeric shortening observed in FA cells. Less is known about telomere maintenance in the heterozygous state. Here, we investigate the roles of PALB2 heterozygous mutations in genomic instability, an important carcinogenesis precursor. Patient‐derived lymphoblastoid (LCL) and fibroblast (FCL) cell lines with monoallelic truncating PALB2 mutations were investigated using a combination of molecular imaging techniques including centromeric FISH, telomeric Q‐FISH and spectral karyotyping (SKY). Mitomycin C and Cisplatin sensitivity was assayed via cellular metabolism of WST‐1. The PALB2 c.229delT FCL showed increases in telomere counts associated with increased mean intensity compared with two wild‐type FCLs generated from first‐degree relatives (P =1.04E‐10 and P =9.68E‐15) and it showed evidence of chromosomal rearrangements. Significant differences in centromere distribution were observed in one of three PALB2 heterozygous FCLs analyzed when compared with PALB2 wild‐type, BRCA1 and BRCA2 heterozygous FCLs. No significant consistently increased sensitivity to Mitomycin C or Cisplatin was observed in LCLs. Our results are suggestive of an altered centromere distribution profile and a telomere instability phenotype. Together, these may indicate critical nuclear organization defects associated with the predisposition to transformation and early stage development of PALB2‐related cancers. © 2013 Wiley Periodicals, Inc.     

Chromosomal instability and telomere lengths of each chromosomal arm measured by Q-FISH in human fibroblast strains prior to replicative senescence

We monitored the telomere lengths and chromosomal instability characteristics of fibroblasts at different population doubling levels (PDLs) to gain further insight into the role of telomere shortening in chromosomal instability. We used 7 normal diploid human fibroblast strains (TIG-1, 3, 7, 103, 104, 112, and 114) and a quantitative fluorescence in situ hybridization method to measure telomere lengths of the p- and q-arms of individual chromosomes. We also enumerated morphologic signs of chromosomal instability, including fusion or loss of chromosomes, and anaphase bridges. In strains TIG-1, 3, 7, 103, and 114 at the late (phase 3) stage (≧40 PDLs), 29 (96.6%) of 30 fusions were associated with one or both of the chromosomal arms that bear significantly shorter telomeres in those populations. In TIG-1 at 62 PDL, 6 fusions were associated with Xq (n = 3), 21q (n = 3), and other (n = 6) chromosomes. Xq and 21q had significantly shorter telomeres, and anaphase bridges were often associated with chromosomes X and/or 21 (74.6%). Our results indicate that chromosomes having excessively shortened telomeres at late PDLs begin to show features of instability such as fusions and anaphase bridges.     

Measurement of telomere length in cells from pleural effusion: Asbestos exposure causes telomere shortening in pleural mesothelial cells

We estimated the telomere lengths of neoplastic and non‐neoplastic mesothelial cells and examined their correlation with asbestos exposure and the expression of markers of mesothelial malignancy. Cell blocks of pleural effusion obtained from 35 cases of non‐neoplastic disease (NN), 12 cases of malignant mesothelioma (MM) and 12 cases of carcinomatous effusion due to lung adenocarcinoma (LA) were examined. Fifteen of the 35 NN cases had pleural plaques (NNpp+) suggestive of asbestos exposure, and the other 20 cases had no pleural plaques (NNpp‐). Telomere length was measured using the tissue quantitative fluorescence in situ hybridization method, and expressed as normalized telomere‐to‐centromere ratio. NN cases had significantly longer telomeres than MM (P < 0.001) and LA (P < 0.001) cases. Telomeres in NNpp+ cases were slightly shorter than those of NNpp‐ cases (P = 0.047). MM and LA showed almost the same telomere length. NN cases with shorter telomeres tended to show aberrant expression of epithelial membrane antigen (EMA), CD146, glucose transporter 1 (GLUT1) and IGF‐II messenger RNA‐binding protein 3 (IMP3). These results suggest that telomere shortening and subsequent genetic instability play an important role in the development of MM. Measurement of telomere length of cells in pleural effusion might be helpful for earlier detection of MM.     

Localization of telomeres and telomere-associated proteins in telomerase-negative Saccharomyces cerevisiae

Cells lacking telomerase cannot maintain their telomeres and undergo a telomere erosion phase leading to senescence and crisis in which most cells become nonviable. On rare occasions survivors emerge from these cultures that maintain their telomeres in alternative ways. The movement of five marked telomeres in Saccharomyces cerevisiae was followed in wild-type cells and through erosion, senescence/crisis and eventual survival in telomerase-negative (est2::HYG) yeast cells. It was found that during erosion, movements of telomeres in est2::HYG cells were indistinguishable from wild-type telomere movements. At senescence/crisis, however, most cells were in G₂ arrest and the nucleus and telomeres traversed back and forth across the bud neck, presumably until cell death. Type I survivors, using subtelomeric Y′ amplification for telomere maintenance, continued to show this aberrant telomere movement. However, Type II survivors, maintaining telomeres by a sudden elongation of the telomere repeats, became indistinguishable from wild-type cells, consistent with growth properties of the two types of survivors. When telomere-associated proteins Sir2p, Sir3p and Rap1p were tagged, the same general trend was seen—Type I survivors retained the senescence/crisis state of protein localization, while Type II survivors were restored to wild type. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Squamous cell carcinomas of the esophagus arise from a telomere-shortened epithelial field

Critically shortened telomeres make chromosomes susceptible to the instability and widespread cytogenetic alterations that characterize most human cancers. We hypothesized that the very rapid cell proliferation observed in esophageal squamous cell carcinomas might accelerate telomere shortening and chromosomal instability associated with carcinogenesis. We used a number of telomere measurement techniques including quantitative fluorescence in situ hybridization (Q-FISH) to compare chromosomal aberrations and telomere lengths of individual chromosomes in esophageal squamous cell carcinomas (ESCCs) and nearby non-neoplastic esophageal epithelium (NNEE) cells. Our results showed that the mean telomere length in ESCC cells was significantly less than that in adjacent NNEE cells, and that telomeres in all NNEE cells were significantly shorter than those in normal esophageal epithelium (reported previously). In addition, there was no evidence linking telomere shortening of a particular chromosome to field cancerization in ESCC. However, a mechanistic link between telomere shortening and chromosomal instability was supported by a higher frequency of anaphase/telophase bridges and an increase in the frequency of aneuploidy. This study furthers our understanding of the mechanism by which telomere shortening and chromosomal instability lead to carcinogenesis and field cancerization in the esophagus.     

Telomere length and LINE1 methylation is associated with chromosomal aberrations in peripheral blood

The frequency of chromosomal aberrations in peripheral blood predicts a probable cancer risk. The individual telomere length and methylation of repetitive elements may be susceptibility factors for chromosomal aberrations. A cohort of healthy Norwegian men (N = 364) recruited during 1980–1999 were analyzed for chromosomal aberrations in phytohemagglutinin‐stimulated lymphocytes from peripheral blood. Chromosome‐type or chromatid‐type aberrations were scored. DNA was extracted from slides cytogenetically analyzed and relative average telomere length and methylation of LINE1 repeats were determined by quantitative polymerase chain reaction and bisulfite pyrosequencing, respectively. Information about individuals with malignant tumors (N = 49) diagnosed after chromosomal aberrations testing until end of 2008 was obtained and two matched controls per case were used in a nested case–control analysis. Shorter relative telomere length and higher methylation of LINE1 were associated with higher frequency of total chromosomal aberrations (β = ?0.76, P = 0.022; and β = 0.042, P = 0.048, respectively; age‐adjusted ordinal regression). The telomere length was stronger associated with chromosome‐type (β = ?1.00, P = 0.006) than with chromatid‐type aberrations (β = ?0.49, P = 0.115). The LINE1 methylation was stronger associated with chromatid‐type (β = 0.062, P = 0.003) than with chromosome‐type aberrations (β = 0.018, P = 0.41). Telomere length [individuals with short telomeres odds ratio (OR) = 0.87, 95% confidence interval (CI) 0.38–2.0], LINE1 (individuals with high methylation OR = 1.04, 95% CI 0.43–2.5) and chromosomal aberrations (individuals with high frequency OR = 1.6, 95% CI 0.63–3.9) at baseline did not predict cancer risk, but the conclusions were hampered by low statistical precision. The results suggest that shorter telomere length and higher LINE1 methylation in peripheral blood lymphocytes are predisposition factors for increased frequency of chromosomal aberrations. © 2012 Wiley Periodicals, Inc.     

Jumping translocations: Short telomeres or pathogenic TP53 variants as underlying mechanism in acute myeloid leukemia and myelodysplastic syndrome?

Chromosomal rearrangements involving one donor chromosome and two or more recipient chromosomes are called jumping translocations. To date only few cases of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) with jumping translocations have been described and the underlying mechanisms remain unclear. Here, we analyzed 11 AML and 5 MDS cases with jumping translocations. The cases were analyzed by karyotyping, FISH, telomere length measurement, and next‐generation sequencing with an AML/MDS gene panel. Cases with jumping translocations showed significantly (P < .01) shorter telomeres in comparison to healthy age‐matched controls. Additional neo‐telomeres were found in two cases. In total, eight cases showed recipient chromosomes with a breakpoint in the centromeric region all of them harboring a pathogenic variant in the TP53 gene (n = 6) and/or a loss of TP53 (n = 5). By contrast, no pathogenic variant or loss of TP53 was identified in the six cases showing recipient chromosomes with a breakpoint in the telomeric region. In conclusion, our results divide the cohort of AML and MDS cases with jumping translocations into two groups: the first group with a telomeric breakpoint of the recipient chromosome is characterized by short telomeres and a possibly telomere‐based mechanism of chromosomal instability formation. The second group with a centromeric breakpoint of the recipient chromosome is defined by mutation and/or loss of TP53. We, therefore, assume that both critically short telomeres as well as pathogenic variants of TP53 influence jumping translocation formation.     

The yeast VPS genes affect telomere length regulation

Eukaryotic cells invest a large proportion of their genome in maintaining telomere length homeostasis. Among the 173 non-essential yeast genes found to affect telomere length, a large proportion is involved in vacuolar traffic. When mutated, these vacuolar protein-sorting (VPS) genes lead to telomeres shorter than those observed in the wild type. Using genetic analysis, we characterized the pathway by which VPS15, VPS34, VPS22, VPS23 and VPS28 affect the telomeres. Our results indicate that these VPS genes affect telomere length through a single pathway and that this effect requires the activity of telomerase and the Ku heterodimer, but not the activity of Tel1p or Rif2p. We present models to explain the link between vacuolar traffic and telomere length homeostasis.     

Thiopurine‐induced mitotic catastrophe in Rad51d‐deficient mammalian cells

Thiopurines are part of a clinical regimen used for the treatment of autoimmune disorders and childhood acute lymphoblastic leukemia. However, despite these successes, there are also unintended consequences such as therapy‐induced cancer in long‐term survivors. Therefore, a better understanding of cellular responses to thiopurines will lead to improved and personalized treatment strategies. RAD51D is an important component of homologous recombination (HR), and our previous work established that mammalian cells defective for RAD51D are more sensitive to the thiopurine 6‐thioguanine (6TG) and have dramatically increased numbers of multinucleated cells and chromosome instability. 6TG is capable of being incorporated into telomeres, and interestingly, RAD51D contributes to telomere maintenance, although the precise function of RAD51D at the telomeres remains unclear. We sought here to investigate: (1) the activity of RAD51D at telomeres, (2) the contribution of RAD51D to protect against 6TG‐induced telomere damage, and (3) the fates of Rad51d‐deficient cells following 6TG treatment. These results demonstrate that RAD51D is required for maintaining the telomeric 3′ overhangs. As measured by γ‐H2AX induction and foci formation, 6TG induced DNA damage in Rad51d‐proficient and Rad51d‐deficient cells. However, the extent of γ‐H2AX telomere localization following 6TG treatment was higher in Rad51d‐deficient cells than in Rad51d‐proficient cells. Using live‐cell imaging of 6TG‐treated Rad51d‐deficient cells, two predominant forms of mitotic catastrophe were found to contribute to the formation of multinucleated cells, failed division and restitution. Collectively, these findings provide a unique window into the role of the RAD51D HR protein during thiopurine induction of mitotic catastrophe. Environ. Mol. Mutagen. 59:38–48, 2018. © 2017 Wiley Periodicals, Inc.     

Distinct telomere length regulation in premalignant cervical and endometrial lesions: implications for the roles of telomeres in uterine carcinogenesis

Mouse models show that progressive shortening of telomeres with ageing causes chromosomal instability, which can lead to the initiation of cancer. However, it is unclear what roles telomere shortening plays in human carcinogenesis. The present study has investigated the involvement of telomere dynamics in uterine carcinogenesis. Using telomere-FISH (telo-FISH) assays, telomere lengths in premalignant and malignant cervical and endometrial lesions were measured and compared with chromosomal arm loss or gain. Telo-FISH signals were visualized with Cy3-labelled telomere-specific probes and presented as telomere intensity (TI). Early-stage cervical intraepithelial neoplasias (CINs), especially CIN2, had significantly shorter telomeres than corresponding normal squamous epithelia (p = 0.019), together with increased rates of chromosomal arm loss/gain (p < 0.001). Cervical cancers had relatively short telomeres, but they also showed greater heterogeneity than other sampled tissues, including those with long telomeres. In contrast, there was no significant difference between the telomere length of normal endometrium and of endometrial hyperplasia and endometrial cancer. There was no significant difference in the rate of chromosomal arm loss/gain between normal endometrium and endometrial hyperplasia. These findings suggest that progressive shortening of telomeres occurs in CIN, in association with chromosomal instability, which may play critical roles in cervical carcinogenesis. In contrast, endometrial hyperplasias have relatively stable telomeres without widespread chromosome alteration, implying that endometrial carcinogenesis involves mechanisms distinct from those of cervical carcinogenesis, possibly including microsatellite instability.     

Three‐dimensional nuclear telomere architecture changes during endometrial carcinoma development

Endometrioid or type‐I endometrial carcinoma (EC) develops from hyperproliferative glandular pathologies. Inactivation of the tumor suppressor gene PTEN is frequently associated with type‐I EC. Using a previously characterized Pten heterozygous (Pten+/‐) mouse model, this study investigates the three‐dimensional (3D) telomere profiles during progression from hyperplastic lesions to EC to test the hypothesis that altered 3D telomere profiles can be detected prior to Pten loss in early hyperproliferative lesions. We used immunohistochemistry and 3D‐telomere fluorescent in‐situ hybridization to investigate Pten expression, telomere length and signal distribution, average number and spatial distribution of telomeres and formation of telomere aggregates in uterine glandular epithelial cells from wildtype and Pten+/‐ mice. Pten showed nuclear and cytoplasmic localization in WT, predominantly cytoplasmic staining in simple hyperplasia (SH) and was markedly reduced in atypical hyperplasia (AH). Telomere length in glandular epithelial cells does not shorten with age. The average number of telomeres per nucleus was not different in WT and Pten+/‐ mice indicating the lack of substantial numeric chromosome aberrations during EC development. We observed telomere aggregates in lesions of AH and EC. SH lesions in Pten+/‐ mice differed from normal glandular epithelium by an increased relative number of shorter telomeres and by a telomere signal distribution indicative of a heterogeneous cell population. Our study revealed that alterations in the nuclear 3D telomere architecture are present in early proliferative lesions of mouse uterine tissues indicative of EC development. The changes in telomere length distribution and nuclear signal distribution precede the loss of Pten. © 2013 Wiley Periodicals, Inc.     

Elevated telomere-telomere recombination in WRN-deficient, telomere dysfunctional cells promotes escape from senescence and engagement of the ALT pathway

Werner Syndrome (WS) is characterized by premature aging, genomic instability, and cancer. The combined impact of WRN helicase deficiency and limiting telomere reserves is central to disease pathogenesis. Here, we report that cells doubly deficient for telomerase and WRN helicase show chromosomal aberrations and elevated recombination rates between telomeres of sister chromatids. Somatic reconstitution of WRN function, but not a WRN helicase-deficient mutant, abolished telomere sister chromatid exchange (T-SCE), indicating that WRN normally represses T-SCEs. Elevated T-SCE was associated with greater immortalization potential and resultant tumors maintained telomeres via the alternative lengthening of telomere (ALT) pathway. We propose that the increased incidence of chromosomal instability and cancer in WS relates in part to aberrant recombinations between sister chromatids at telomeres, which facilitates the activation of ALT and engenders cancer-relevant chromosomal aberrations and tumor formation.     

Analysis of alternative lengthening of telomere markers in BRCA1 defective cells

Telomeres are specialized structures responsible for the chromosome end protection. Previous studies have revealed that defective BRCA1 may lead to elevated telomere fusions and accelerated telomere shortening. In addition, BRCA1 associates with promyelocytic leukemia (PML) bodies in alternative lengthening of telomeres (ALTs) positive cells. We report here elevated recombination rates at telomeres in cells from human BRCA1 mutation carriers and in mouse embryonic stem cells lacking both copies of functional Brca1. An increased recombination rate at telomeres is one of the signs of ALT. To investigate this possibility further we employed the C‐circle assay that identifies ALT unequivocally. Our results revealed elevated levels of ALT activity in Brca1 defective mouse cells. Similar results were obtained when the same cells were assayed for the presence of another ALT marker, namely the frequency of PML bodies. These results suggest that BRCA1 may act as a repressor of ALT. © 2016 The Authors Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc.