Telomerase activity in human intestine

In human somatic cells without the activity of telomerase, the ends of chromosomes consisting of the telomeric repeats TTAGGG progressively erode with each cell division. In germline and immortal cells telomerase activity maintains telomere length and thus compensates for the 'end-replication problem'. Progressive telomere shortening and reactivation of telomerase activity have been considered to be one of the key mechanisms in cellular senescence and immortalization. It has been shown that while most somatic cells do not have detectable telomerase activity, almost all cancers do have telomerase activity. Thus, detection of telomerase activity may have utility in the early diagnosis of cancer and may be a new target for therapeutic intervention. However, there is recent evidence that some cells of renewal tissues, such as hematopoietic cells and basal cells of the epidermis, have detectable telomerase activity. In the present study, we report detectable telomerase activity in normal human intestinal mucosa. This activity is localized to the lower third of each crypt and may be derived from intestinal stem cells. Since intestinal telomeric repeats are shorter in adults when compared to children, the telomerase activity in the intestine is insufficient to maintain telomere length but may be sufficient to provide extended proliferative capacity for such renewal tissues.     

A repressor function for telomerase activity in telomerase-negative immortal cells

Human telomerase, a ribonucleoprotein that adds TTAGGG repeats onto telomeres and compensates for their shortening, is repressed in most normal human somatic cells. Human somatic cells are considered to have a limited proliferation capacity because of the telomere shortening. Although immortalization of somatic cells is often associated with telomerase reactivation, there are some immortal cells in which telomerase activity is undetectable. In these cells, telomeres may be maintained by an unknown mechanism other than telomerase reactivation. To examine the genetic regulation of telomerase activity, we constructed hybrids between immortal cells with (HepG2) and without (KMST6) telomerase activity. These two cell lines had relatively short and long telomeres, respectively. The hybrid cells continued to proliferate without detectable telomerase activity even after 100 population doublings. Telomerase-positive subpopulations occasionally appeared after serial passages. Southern blot analysis revealed that the hybrids had long terminal restriction fragments similar to that of KMST6, regardless of telomerase activity, and fluorescence in situ hybridization with a telomeric probe showed high-intensity hybridization signals on telomeres, indicating relatively long telomeric repeats. These results suggest that the telomerase-negative immortal cells contain a gene or genes functioning as a telomerase repressor and maintain telomere length by a dominant mechanism other than telomerase reactivation. Mol. Carcinog. 21:17–25, 1998. © 1998 Wiley-Liss, Inc.     

Loss of telomeric DNA during aging may predispose cells to cancer (review)

In normal human somatic cells, gradual shortening of telomeres may activate the complex cascade of molecular events known as cellular senescence. Experimental evidence from our laboratory suggests that cellular mortality is regulated by two separate mechanisms that we have termed mortality stage 1 (M1) and mortality stage 2 (M2). In mammary epithelial cells, the M1 mechanism involves de-regulation of p53 whereas in fibroblasts both the retinoblastoma (Rb) and p53 gene products are implicated. Cells that overcome the function of these antiproliferative proteins (M1 controls) continue to divide until a second entirely independent mechanism, M2 is induced. As somatic cells age they gradually lose telomeric sequences at the termini of their chromosomes, a process that continues during the extended lifespan period between M1 and M2. Immortal and cancer cells, as well as cells that maintain telomere length (e.g. germ cells), express telomerase, a ribonucleoprotein which maintains (stabilizes) telomere length by synthesizing TTAGGG repeats. Because normal human somatic cells and cells prior to M2 do not express telomerase, we propose that the M2 mechanism involves either the direct or indirect induction of telomerase activity. In order for cells to overcome senescence and become immortal, they must first escape the checkpoints that limit the proliferative capacity of normal cells, the MI and M2 controls (a very rare event). However, the probability of immortalization and that of tumorigenesis increases with age and we propose telomere shortening and reactivation of telomerase are important components in these processes. Once immortal, cells can then follow many pathways that result in the acquisition and progression of cancer.     

Inhibition of telomerase activity correlates with a decrease in the RNA component of telomerase during differentiation

In most normal somatic cells the telomeres of human chromosomes shorten with each cell division because of low expression or lack of telomerase activity. Telomerase, a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends, is reactivated or upregulated in tumor cells and maintains the stability of telomere length. We previously showed that treatment of HL60 leukemia cells with differentiation-inducing agents resulted in inhibition of telomerase activity. In the present study, we found that the decrease in telomerase activity did not temporally correlate with the expression of a differentiation marker, CD11b, on the cell surface. Mixing of protein extracts from telomerase-negative differentiated HL60 cells with those from parental HL60 cells did not result in inhibition of telomerase activity, suggesting that a diffusible cellular telomerase inhibitor was not produced in the differentiated cells. However, a decrease in telomerase activity correlated with a selective decrease of telomerase RNA expression, a decrease in the levels of total cellular RNA, and an increase in cells at G(0) phase.     

Telomere dynamics, end-to-end fusions and telomerase activation during the human fibroblast immortalization process.

Loss of telomeric repeats during cell proliferation could play a role in senescence. It has been generally assumed that activation of telomerase prevents further telomere shortening and is essential for cell immortalization. In this study, we performed a detailed cytogenetic and molecular characterization of four SV40 transformed human fibroblastic cell lines by regularly monitoring the size distribution of terminal restriction fragments, telomerase activity and the associated chromosomal instability throughout immortalization. The mean TRF lengths progressively decreased in pre-crisis cells during the lifespan of the cultures. At crisis, telomeres reached a critical size, different among the cell lines, contributing to the peak of dicentric chromosomes, which resulted mostly from telomeric associations. We observed a direct correlation between short telomere length at crisis and chromosomal instability. In two immortal cell lines, although telomerase was detected, mean telomere length still continued to decrease whereas the number of dicentric chromosomes associated was stabilized. Thus telomerase could protect specifically telomeres which have reached a critical size against end-to-end dicentrics, while long telomeres continue to decrease, although at a slower rate as before crisis. This suggests a balance between elongation by telomerase and telomere shortening, towards a stabilized 'optimal' length.     

Telomeres and telomerase in chronic myeloid leukaemia: impact for pathogenesis,disease progression and targeted therapy

Telomeres are specialized structures localized at the end of human chromosomes. Due to the end replication problem, each cell division results in a loss of telomeric repeats in normal somatic cells. In germ line and stem cells, the multicomponent enzyme telomerase maintains the length of telomere repeats. However, elevated telomerase activity has also been reported in the majority of solid tumours as well as in acute and chronic leukaemia. Chronic myeloid leukaemia (CML) serves as a model disease to study telomere biology in clonal myeloproliferative disorders. In CML, telomere shortening correlates with disease stage, duration of chronic phase (CP), prognosis measured by the Hasford risk score and the response to disease‐modifying therapeutics such as the tyrosine kinase inhibitor Imatinib. In addition, telomerase activity (TA) is already increased in CP CML and further upregulated with disease progression to accelerated phase and blast crisis (BC). Furthermore, a correlation of TA with increased genetic instability as well as a shorter survival of the patients has been reported. Here, we review the current state of knowledge of the role of telomere and telomerase biology in CML and discuss the possible impact of novel treatment approaches. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

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.     

Restoration of the Cellular Senescence Program and Repression of Telomerase by Human Chromosome 3

Telomeres, at the end of chromosomes, shorten with each cell division, resulting in cellular senescence. Tumor cells, unlike normal somatic cells, express a telomerase that maintains the telomere length. Deletion of a gene(s) on chromosome 3 is common in human renal cell carcinoma (RCC) and reintroduction of a normal chromosome 3 into an RCC immortal cell line restored the program of cellular senescence. The loss of indefinite growth potential was associated with the loss of telomerase activity and shortening of telomeres in the RCC cells with a normal chromosome 3. However, microcell hybrids that escaped from senescence and microcell hybrids with an introduced chromosome 7 or 11 maintained telomere lengths and telomerase activity similar to those of the parental RCC23. Thus, restoration of the cellular senescence program by chromosome 3 is associated with repression of telomerase function in RCC cells.     

Impaired telomere regulation mechanism by TRF1 (telomere-binding protein), but not TRF2 expression, in acute leukemia cells

Telomere regulation is suggested to be an important mechanism in cellular proliferation and cellular senescence not only in normal diploid cells but also in neoplastic cells, including human leukemia cells. We studied the possible correlation among telomere length, telomerase (a ribonuclear protein that synthesizes the telemeres de novo) activity, hTERT (a catalytic subunit of telomerase) expression, and TRF1 and TRF2 (telomere DNA binding proteins) expression in human acute leukemia cells. The hTERT expression level was strongly associated with telomerase activity (P=0.0001), indicating that the expression level of the catalytic subunit (hTERT) regulates telomerase activity in human acute leukemia cells. TRF1 expression, which is believed to control telomere length, was significantly elevated in patients with acute lymphoblastic leukemia (ALL) (P=0.0232) compared to those in acute myeloid leukemia (AML); TRF1 expression tended to be higher in patients without telomere shortening (P=0.077) and in those with hTERT expression (P=0.055). This indicates that TRF1 may act to monitor telomere length under the condition of up-regulated telomerase activity in some neoplastic cells. In contrast, TRF2 expression in acute leukemia did not show any correlation with telomere parameters in this study. Although the precise regulation mechanism of telomere length is still uncertain, these results may suggest that regulation of telomere length is partially associated with TRF1 expression, whereas dysfunction of TRF1 expression may be speculated in a subset of acute leukemia.     

Telomerase promotes efficient cell cycle kinetics and confers growth advantage to telomerase-negative transformed human cells

Constitutive telomerase activity maintains telomere length and confers immortal phenotypes to human cancers. The prevalence of telomerase, rather than a homologous recombination-based mechanism, in telomere length maintenance suggests that telomerase also has auxiliary roles in tumorigenesis. Here, we investigate growth advantages provided by the telomerase enzyme in oncogene-transformed human cells that do not require telomerase activity for telomere length control. Our data suggest that in oncogene-transformed cells, telomerase activity accelerates cell growth kinetics in a cell cycle phase-specific manner and promotes anchorage-independent growth. Coculture experiments demonstrated that this growth advantage conferred by telomerase activity is not due to increased cellular cross-talk. Growth advantages provided by telomerase required all functional aspects of the enzyme. Dissociation-of-activity-in-telomerase mutants and other functionally defective versions of telomerase were unable to promote oncogene-transformed cell growth, suggesting that canonical telomerase activities may be involved. We conclude that telomerase provides advantages to oncogene-transformed human cells, thereby supporting the development of telomerase-based anticancer chemotherapies targeting these growth-promoting effects.     

Simultaneous targeting of telomeres and telomerase as a cancer therapeutic approach

Telomeres, which are important for maintaining chromosome integrity and functions, shorten with each cell division. Telomerase, responsible for telomere synthesis, is expressed in approximately 90% of human tumor cells but seldom in normal somatic cells. This study evaluated the hypothesis that simultaneous shortening of telomeres and inhibition of telomerase results in synergistic and tumor-selective cytotoxicity. In telomerase-positive human pharynx FaDu tumor cells, paclitaxel caused telomere erosion (first detected at 1 h) and apoptosis. Expression of antisense to the RNA component of human telomerase (hTR) inhibited telomerase activity, shortened telomere length, reduced cell growth rate, and resulted in a significant higher sensitivity to paclitaxel. Another telomerase inhibitor, 3'-azido-3'-deoxythymidine (AZT), at a concentration that produced little or no cell detachment or apoptosis, inhibited the telomerase activity and enhanced the paclitaxel-induced cell detachment and apoptosis. AZT also enhanced the activity of paclitaxel in mice bearing well-established s.c. FaDu xenograft tumors (i.e., reduced residual tumor size, enhanced apoptotic cell fraction, and prolonged survival time), without enhancing host toxicity. In contrast, AZT did not enhance the paclitaxel activity in the telomerase-negative osteosarcoma Saos-2 cells nor in FaDu cells where telomerase was already suppressed by antisense hTR, confirming that the AZT effect in parent FaDu cells is mediated through telomerase inhibition. These results demonstrate that combined use of agents targeting both telomere and telomerase yielded synergistic activity selective for tumors that depend on telomerase for telomere maintenance.