Telomeres and telomerase in hematologic neoplasia

Normal hematopoietic cells express telomerase activity, however the presence of telomerase does not necessarily imply stable and thus unchanging telomere length. Gradual telomere loss with aging and rapid cycling of hematopoietic stem cells might contribute to immunosenescence, exhausted hematopoiesis, and increased likelihood of malignant transformation. In leukemias and lymphomas, telomere length may reflect the cellular proliferative history, prior to immortalization. The level of telomerase activity is generally influenced by the fraction of cells in the proliferative pool. Shortened telomeres and high telomerase activity almost always correlates with disease severity in hematologic neoplasias such as relapsed leukemia and high-grade lymphomas, indicating that measurement of telomere length and telomerase activity might be useful to monitor disease condition. Since the mode of action of telomerase inhibitors may require telomeric shortening before induction of apoptosis, anti-telomerase therapy might be helpful for adjuvant therapy following conventional chemotherapy, in vitro purging of neoplastic cells in stem cell transplantation, and treating minimal residual disease. Some promising areas of tissue engineering include rejuvenation of hematopoietic stem cells for improving stem cell transplants or enhancing general immunity for older patients.     

Telomerase activity in pleural malignant mesotheliomas

New treatments are needed for malignant pleural mesothelioma (MPM), which currently has a poor prognosis. Cellular immortalisation, one of the hallmarks of cancer, depends on the activity of a telomere length maintenance mechanism (TMM) - either telomerase or alternative lengthening of telomeres (ALT). The TMMs are widely regarded as potential targets for cancer therapies and telomerase inhibitors have entered clinical trials. The aim of this study was to determine what proportion of MPMs use ALT and/or telomerase. Forty-three MPMs from 42 patients were examined for telomerase and ALT activity. Telomerase activity was detected by immunoaffinity purification followed by the telomere repeat amplification protocol (TRAP), and ALT activity was determined by the C-circle assay and by assessing telomere lengths using terminal restriction fragment analyses. We found that 43 of 43 MPMs were telomerase-positive[+] and ALT-negative[−]. Therefore, to investigate whether pleural mesothelial cells are unusually susceptible to activation of telomerase, we examined activation of the TMMs in an in vitro model of cellular immortalisation, in which normal pleural mesothelial cells were transduced with simian virus 40 (SV40) oncogenes. We found that normal mesothelial cells were TMM-negative, and that expression of the SV40 oncogenes did not directly activate telomerase or ALT. Immortalisation, which in this experimental system results from additional genetic changes that have not yet been identified, was accompanied by activation of either TMM. Therefore, pleural mesothelial cells are capable of activating either TMM in vitro, and the observation that 100% of MPMs were telomerase[+] suggests that there are factors in vivo that select for telomerase activity during oncogenesis of this tumour type. We conclude that MPM is a tumour that could be considered for anti-telomerase therapy.     

Mutated telomeres sensitize tumor cells to anticancer drugs independently of telomere shortening and mechanisms of telomere maintenance

Telomerase is a ribonucleoprotein complex that maintains the stability of chromosome ends and regulates replicative potential. Telomerase is upregulated in over 85% of human tumors, but not in adjacent normal tissues and represents a promising target for anticancer therapy. Most telomerase-based therapies rely on the inhibition of telomerase activity and require extensive telomere shortening before inducing any antiproliferative effect. Disturbances of telomere structure rather than length may be more effective in inducing cell death. Telomerase RNA subunits (hTRs) with mutations in the template region reconstitute active holoenzymes that incorporate mutated telomeric sequences. Here, we analysed the feasibility of an anticancer approach based on the combination of telomere destabilization and conventional chemotherapeutic drugs. We show that a mutant template hTR dictates the synthesis of mutated telomeric repeats in telomerase-positive cancer cells, without significantly affecting their viability and proliferative ability. Nevertheless, the mutant hTR increased sensitivity to anticancer drugs in cells with different initial telomere lengths and mechanisms of telomere maintenance and without requiring overall telomere shortening. This report is the first to show that interfering with telomere structure maintenance in a telomerase-dependent manner may be used to increase the susceptibility of tumor cells to anticancer drugs and may lead to the development of a general therapy for the treatment of human cancers.     

Telomeres and telomerase in cancer stem cells

Alterations in telomere dynamics both suppress and facilitate malignant transformation by regulating genomic stability and cell lifespan. Checkpoints induced by telomere dysfunction play a major role in tumour suppression, whereas telomere shortening contributes to the initiation of cancer by inducing chromosomal instability. Since stem cells are exposed to various tumourigenic agents and stresses throughout their lifetime, the ageing stem cell is a major target of malignant transformation. This review summarises our knowledge of telomere length and telomerase activity in stem cells during ageing and carcinogenesis.     

Telomeres and Telomerase in Normal and Malignant Haematologic Cells

Telomeres, the ends of eukaryotic chromosomes, are structural and functional units composed of proteins and repetitive DNA sequences. Telomeres protect the ends of chromosomes from DNA loss caused by incomplete replication of 3' ends. The obligatory loss of terminal sequence with each cell division leads to telomere shortening, and is counteracted in germline cells by an enzymatic activity termed telomerase that resynthesizes telomeric DNA de novo. Telomere length and telomerase activity have been measured by several groups in both normal and malignant blood and marrow cells. Telomere length decreases with age in normal blood and bone marrow, despite the presence of a detectable telomerase activity. In most hematologic malignancies telomere length is short and telomerase activity is enhanced, compatible with the late activation of the enzyme in tumour development. The implications of these findings for tumour pathogenesis, diagnosis, and treatment are discussed.     

Relation between telomerase activity, hTERT and telomere length for intracranial tumours

Human linear chromosomes are capped by specialized DNA-protein structures called telomeres. The present study analysed the telomerase activity, hTERT protein and telomere length in meningiomas and gliomas in relation to their WHO grading. Fifty-three freshly dissected tumour biopsies were analysed for telomerase activity, hTERT protein expression and telomere length. Telomerase activity was examined in 41 of the 53 biopsies. Telomerase activity was detected in 3 of 35 (8.6%) screened meningiomas (1 benign, 1 atypical and 1 malignant meningioma). For hTERT expression, 56.4% of meningiomas were positive with a mean labelling index (hTERT LI) of 31.3% (SD=26.5) for the hTERT positive meningiomas. The mean telomere length for meningiomas was 6.983 kb (SD=1.969). For gliomas, no active telomerase was detected in 2 low-grade astrocytomas, whereas three of the four screened glioblastomas were positive for telomerase activity. The only hTERT protein positive astrocytoma had a mean labelling index of 9.0%. On the other hand, the hTERT LI for glioblastomas was 53.6% (SD=28.0). The two low-grade astrocytomas had a telomere length of 14.310 and 9.236 kb. The anaplastic astrocytoma had a telomere length of 4.903 kb and the glioblastomas 5.767 kb (SD=2.042). The normal meningeal and neuronal tissue is negative for telomerase activity and hTERT. The length was +/-10.000 kb. These results indicate that telomere shortening may be a critical step in pathogenesis of atypical and malignant meningiomas and gliomas. Critical telomere shortening in vitro was shown to activate telomerase.     

Recent advances in telomere biology: implications for human cancer

PURPOSE OF REVIEW: Research into the basic biology of telomeres continues to reveal details relevant to fundamental aspects of human cancer. The goal of this review is to highlight discoveries made within the last year, with emphasis on their relevance to cancer prevention, diagnosis, prognostics, and treatment. RECENT FINDINGS: Increasing evidence indicates that dysfunctional telomeres likely play a causal role in the process of malignant transformation, in at least a fraction of human cancers, by initiating chromosomal instability. Telomeres form protective capping structures composed of telomeric DNA complexed with a multitude of associated proteins, the loss of which can have profound effects on telomeric stability. Critical telomeric shortening can lead to telomere "uncapping" and may occur at the earliest recognizable stages of malignant transformation in epithelial tissues. The widespread activation of the telomere synthesizing enzyme telomerase in human cancers not only confers unlimited replicative potential but also prevents intolerable levels of chromosomal instability. Several details regarding telomere structure and telomerase regulation have recently been elucidated, providing new targets for therapeutic exploitation. Various therapeutic strategies aimed at either telomerase or its telomeric substrate are showing promise and may synergize with established anti-cancer agents. Further support for anti-telomerase approaches comes from recent studies indicating that telomerase may possess additional functions, beyond telomere maintenance, that support the growth and survival of tumor cells. SUMMARY: Substantial progress has been made in understanding the complex relationships that exist between telomeres and cancer. However, important issues, such as transient activation of telomerase in normal cells and the potential for tumor cell immortalization via telomerase independent means, remain to be clarified.     

Telomerase activity in B-cell non-Hodgkin lymphomas is regulated by hTERT transcription and correlated with telomere-binding protein expression but uncoupled from proliferation

Telomere maintenance is a prerequisite for immortalisation, and in most malignant cells is carried out by telomerase, an enzyme that synthesis new telomeric repeats on the chromosome ends. In normal or reactive tissues with a high regenerative capacity, telomerase is regulated according to the telomere loss that occurs during proliferation. To evaluate the interaction of proliferation and telomerase activity in malignant lymphomas, we quantified telomerase expression in different non-Hodgkin lymphomas in comparison to normal or reactive lymph nodes. Surprisingly, the activity levels were the same in most of the lymphomas analysed as compared to reactive lymph nodes. Significantly higher activity was detected only in Burkitt's lymphoma. Telomerase activity correlated well with hTERT and c-myc expression, but was independent of proliferation. To evaluate interactions of telomere-binding protein expression on telomerase expression in non-Hodgkin lymphoma, the mRNA levels of TRF1, TRF2, tankyrase and hPif1 were assessed by real-time RT-PCR. We demonstrate here that the magnitude of telomerase upregulation does not necessarily reflect the requirement of telomere compensation caused by proliferation. Telomerase regulation in non-Hodgkin lymphomas is therefore uncoupled from proliferative stimuli found in reactive lymphoid tissue. We suggest that the upregulation of specific telomere-binding proteins like TRF2 may contribute to telomere maintenance in malignant lymphoma.     

Comparison of Telomere Length and Telomerase Activationbetween Breast Fibroadenoma and Infiltrating DuctalCarcinoma in Malaysian Women

A study was initiated to explore possible differences in handling telomere attrition in the most commonmalignant and benign tumours of the breast in Malaysian women. Infiltrating ductal carcinoma (IDC) andfibroadenoma (FA) represented the malignant and benign prototypes respectively. 29 IDC, 28 FA and 22 benignnon-lesional control (BNL) breast tissue samples were analysed for telomerase activation using a Telomerase PCRELISA kit (Boehringer Mannheim). In addition, 23 IDC, 12 FA and 14 BNL were subjected to telomere lengthdetermination with a TeloTAGGG Telomere Length Assay Kit (Roche Diagnostic GmbH, Germany), followingdigestion of genomic DNA by frequently cutting restriction enzymes RsaI and HinfI. Mean telomerase activityin IDC (A450nm=0.3338), but not FA (A450nm=0.0003) was significantly raised (p<0.05) compared with BNL(A450nm=0.0031). Similarly IDC (1.2 kb), but not FA (2.2 kb), showed significant telomere shortening (p<0.05)relative to BNL (2.9 kb). The findings imply that telomere attrition and telomerase activation differ betweenmalignant and benign tumours of the breast and may be important for targeted therapy.     

Correlations of telomere length, telomerase activity, and telomeric-repeat binding factor 1 expression in colorectal carcinoma

BACKGROUND: Telomere maintenance has been proposed as an essential step for tumor cell immortalization. The objectives of the current study were to investigate the mechanisms implicated in telomere length in colorectal carcinoma (CRC) and to evaluate the prognostic impact of telomere status. METHODS: Ninety-one colorectal carcinoma samples that were obtained from patients who underwent surgery were analyzed to investigate the factors related to telomere function. The authors studied telomerase activity, terminal restriction fragment (TRF) length, and telomeric-repeat binding factor (TRF1) expression and analyzed the prognostic implications of those factors. RESULTS: Most tumors (81.3%) displayed telomerase activity. Overall, telomeres in CRC specimens were significantly shorter compared with telomeres in normal, adjacent specimens (P=0.02). Moreover, tumors that demonstrated shortened telomeres displayed higher TRF1 levels than tumors without telomere shortening. In relation to patient prognosis, a significantly poor clinical course was observed in the group of patients who had tumors with longer telomeres (P=0.02), and this finding emerged as an independent prognostic factor in a Cox proportional hazards model (P=0.04; relative risk, 6.48). Among patients with tumors classified as telomerase-positive, telomere length ratios (the ratio of tumor tissues to normal tissues)相似文献   

Repression of an alternative mechanism for lengthening of telomeres in somatic cell hybrids.

Some immortalized cell lines maintain their telomeres in the absence of detectable telomerase activity by an alternative (ALT) mechanism. To study how telomere maintenance is controlled in ALT cells, we have fused an ALT cell line GM847 (SV40 immortalized human skin fibroblasts) with normal fibroblasts or with telomerase positive immortal human cell lines and have examined their proliferative potential and telomere dynamics. The telomeres in ALT cells are characteristically very heterogeneous in length, ranging from very short to very long. The ALT x normal hybrids underwent a rapid reduction in telomeric DNA and entered a senescence-like state. Immortal segregants rapidly reverted to the ALT telomere phenotype. Fusion of ALT cells to telomerase-positive immortal cells in the same immortalization complementation group resulted in hybrids that appeared immortal and also exhibited repression of the ALT telomere phenotype. In these hybrids, which were all telomerase-positive, we observed an initial rapid loss of most long telomeres, followed either by gradual loss of the remaining long telomeres at a rate similar to the rate of telomere shortening in normal telomerase-negative cells, or by maintenance of shortened telomeres. These data indicate the existence of a mechanism of rapid telomere deletion in human cells. They also demonstrate that normal cells and at least some telomerase-positive immortal cells contain repressors of the ALT telomere phenotype.     

Telomere lengths and telomerase activity in dog tissues: a potential model system to study human telomere and telomerase biology

Studies on telomere and telomerase biology are fundamental to the understanding of aging and age-related diseases such as cancer. However, human studies have been hindered by differences in telomere biology between humans and the classical murine animal model system. In this paper, we describe basic studies of telomere length and telomerase activity in canine normal and neoplastic tissues and propose the dog as an alternative model system. Briefly, telomere lengths were measured in normal canine peripheral blood mononuclear cells (PBMCs), a range of normal canine tissues, and in a panel of naturally occurring soft tissue tumours by terminal restriction fragment (TRF) analysis. Further, telomerase activity was measured in canine cell lines and multiple canine tissues using a combined polymerase chain reaction/enzyme-linked immunosorbent assay method. TRF analysis in canine PBMCs and tissues demonstrated mean TRF lengths to range between 12 and 23 kbp with heterogeneity in telomere lengths being observed in a range of normal somatic tissues. In soft tissue sarcomas, two subgroups were identified with mean TRFs of 22.2 and 18.2 kbp. Telomerase activity in canine tissue was present in tumour tissue and testis with little or no activity in normal somatic tissues. These results suggest that the dog telomere biology is similar to that in humans and may represent an alternative model system for studying telomere biology and telomerase-targeted anticancer therapies.     

Telomere maintenance and dysfunction predict recurrence in paediatric ependymoma

We have recently described the enzymatic subunit of telomerase (hTERT) as an important prognostic marker for paediatric ependymoma. Because of the lack of good, representative pre-clinical models for ependymoma, we took advantage of our large cohort of ependymoma patients, some with multiple recurrences, to investigate telomere biology in these tumours. Our cohort consisted of 133 ependymomas from 83 paediatric patients and included 31 patients with recurrences. Clinical outcome was measured as overall survival, progression-free survival and response to therapy. In all 133 tumours, hTERT expression correlated with proliferative markers, including MIB-1 index (P<0.0001) and mitotic index (P=0.005), as well as overall tumour grade (P=0.001), but not with other markers of anaplasia. There was no correlation between telomere length and hTERT expression or survival. Surprisingly, prior radiation or chemotherapy neither induced sustained DNA damage nor affected telomere maintenance in recurrent tumours. There was an inverse correlation between hTERT expression and telomere dysfunction as measured by gamma H2AX expression (P=0.016). Combining gamma H2AX and hTERT expressions could segregate tumours into three different survival groups (log rank, P<0.0001) such that those patients whose tumours expressed hTERT and showed no evidence of DNA damage had the worst outcome. This study emphasises the importance of telomere biology as a prognostic tool and telomerase inhibition as a therapeutic target for paediatric ependymoma. Furthermore, we have demonstrated that analysing tumours as they progress in vivo is a viable approach to studying tumour biology in humans.     

Telomerase inhibition induces acute ATM-dependent growth arrest in human astrocytomas

The purpose of the study was to examine the degree of hTERT, the catalytic subunit of telomerase, expression in paediatric high-grade astrocytoma and to explore the potential of telomerase inhibition as a therapy for these tumours. hTERT was expressed at high levels in 36 of 44 paediatric astrocytomas. Telomerase inhibition induced acute DNA damage and ATM-pathway-dependent G2/M cell cycle arrest in astrocytomas in vitro, both occurring prior to telomere shortening itself. Our data suggest that telomerase inhibition could be a useful adjuvant therapy for high-grade astrocytomas, potentially inducing tumour growth arrest following short-term treatment.     

Apoptosis related to telomere instability and cell cycle alterations in human glioma cells treated by new highly selective G-quadruplex ligands

Telomerase represents a relevant target for cancer therapy. Molecules able to stabilize the G-quadruplex (G4), a structure adopted by the 3'-overhang of telomeres, are thought to inhibit telomerase by blocking its access to telomeres. We investigated the cellular effects of four new 2,6-pyridine-dicarboxamide derivatives displaying strong selectivity for G4 structures and strong inhibition of telomerase in in vitro assays. These compounds inhibited cell proliferation at very low concentrations and then induced a massive apoptosis within a few days in a dose-dependent manner in cultures of three telomerase-positive glioma cell lines, T98G, CB193 and U118-MG. They had also antiproliferative effects in SAOS-2, a cell line in which telomere maintenance involves an alternative lengthening of telomeres (ALT) mechanism. We show that apoptosis was preceded by multiple alterations of the cell cycle: activation of S-phase checkpoints, dramatic increase of metaphase duration and cytokinesis defects. These effects were not associated with telomere shortening, but they were directly related to telomere instability involving telomere end fusion and anaphase bridge formation. Pyridine-based G-quadruplex ligands are therefore promising agents for the treatment of various tumors including malignant gliomas.     

Telomerase activity in melanoma and non-melanoma skin cancer.

Telomeres are specialized structures consisting of repeat arrays of TTAGGGn located at the ends of chromosomes. They are essential for chromosome stability and, in the majority of normal somatic cells, telomeres shorten with each cell division. Most immortalized cell lines and tumours reactivate telomerase to stabilize the shortening chromosomes. Telomerase activation is regarded as a central step in carcinogenesis and, here, we demonstrate telomerase activation in premalignant skin lesions and also in all forms of skin cancer. Telomerase activation in normal skin was a rare event, and among 16 samples of normal skin (one with a history of chronic sun exposure) 12.5% (2 out of 16) exhibited telomerase activity. One out of 16 (6.25%) benign proliferative lesions, including viral and seborrhoeic wart samples, had telomerase activity. In premalignant actinic keratoses and Bowen's disease, 42% (11 out of 26) of samples exhibited telomerase activity. In the basal cell carcinoma and cutaneous malignant melanoma (CMM) lesions, telomerase was activated in 77% (10 out of 13) and 69% (22 out of 32) respectively. However, only 25% (3 out of 12) of squamous cell carcinomas (SCC) had telomerase activity. With the exception of one SCC sample, telomerase activity in a positive control cell line derived from a fibrosarcoma (HT1080) was not inhibited when mixed with the telomerase-negative SCC or CMM extracts, indicating that, overall, Taq polymerase and telomerase inhibitors were not responsible for the negative results. Mean telomere hybridizing restriction fragment (TRF) analysis was performed in a number of telomerase-positive and -negative samples and, although a broad range of TRF sizes ranging from 3.6 to 17 kb was observed, a relationship between telomerase status and TRF size was not found.     

Telomerase activity and Bcl-2 expression in human breast cancer.

AIMS: Telomerase is a ribonucleoprotein that synthesizes telomeres and plays an important role in cellular immortalization. Bcl-2 gene encodes for a mitochondrial protein thought to prevent apoptosis of normal cells. We previously reported telomerase activity in 74% of human invasive breast cancers and detected a significant association between telomerase activity and prognostic parameters such as nodal status, tumour size and cellular proliferation. We hypothesized that telomerase reactivation in human breast cancer was associated with increased immunohistochemical expression of Bcl-2. METHODS: Bcl-2 immunohistochemical expression was determined in 25 infiltrating breast carcinomas with known telomerase activity (17 telomerase-positive and 8 telomerase-negative). The percentage of strongly and moderately stained tumour cells for Bcl-2 was determined by a breast pathologist who was blinded to telomerase data. Fisher's exact test was used to examine the association between telomerase activity and Bcl-2 expression. RESULTS: The median percentage of strongly stained tumour cells was 50% for telomerase-positive tumours (range, 0--100%) and 45% for telomerase-negative tumours (range, 0--100%). Twelve (70%) of 17 telomerase-positive tumours expressed strong or moderate Bcl-2 staining in >50% of tumour cells compared with six (75%) of eight telomerase-negative tumours (P=1.0). CONCLUSION: Telomerase reactivation seems to be independent of Bcl-2 protein expression in human breast cancer.     

Telomerase inhibition, telomere shortening, cell growth suppression and induction of apoptosis by telomestatin in childhood neuroblastoma cells

Neuroblastoma is a tumour derived from primitive cells of the sympathetic nervous system and is the most common extracranial solid tumour in childhood. Unfavourable tumours are characterised not only by structural changes, including 1p deletion and amplification of the MYCN proto-oncogene, but also by high telomerase activity. Telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. In this study, we examined telomestatin, a G-quadruplex interactive agent, for its ability to inhibit telomere maintenance of neuroblastoma cells. Telomere length was determined by the terminal restriction fragment method, telomerase activity was measured by a quantitative telomeric repeat amplification protocol, and the expression of human telomerase by quantitative real-time polymerase chain reaction (RT-PCR). Short-term treatment with telomestatin resulted in dose-dependent cytotoxicity and induction of apoptosis. Long-term treatment with telomestatin at non-cytotoxic, but still telomerase activity-inhibiting, concentrations resulted in telomere shortening, growth arrest and induction of apoptosis. These results suggest that the effect of telomestatin is dose-dependent and at least 2-fold. Prolonged low-dose treatment with telomestatin limits the cellular lifespan of NB cells through disruption of telomere maintenance.