Telomerase activity in human ovarian carcinoma.

Telomeres fulfill the dual function of protecting eukaryotic chromosomes from illegitimate recombination and degradation and may aid in chromosome attachment to the nuclear membrane. We have previously shown that telomerase, the enzyme which synthesizes telomeric DNA, is not detected in normal somatic cells and that telomeres shorten with replicative age. In cells immortalized in vitro, activation of telomerase apparently stabilizes telomere length, preventing a critical destabilization of chromosomes, and cell proliferation continues even when telomeres are short. In vivo, telomeres of most tumors are shorter than telomeres of control tissues, suggesting an analogous role for the enzyme. To assess the relevance of telomerase and telomere stability in the development and progression of tumors, we have measured enzyme activity and telomere length in metastatic cells of epithelial ovarian carcinoma. We report that extremely short telomeres are maintained in these cells and that tumor cells, but not isogenic nonmalignant cells, express telomerase. Our findings suggest that progression of malignancy is ultimately dependent upon activation of telomerase and that telomerase inhibitors may be effective antitumor drugs.     

Developmental and tissue-specific regulation of mouse telomerase and telomere length.

Telomere shortening and telomerase activation in human somatic cells have been implicated in cell immortalization and cellular senescence. To further study the role of telomerase in immortalization, we assayed telomere length and telomerase activity in primary mouse fibroblasts, in spontaneously immortalized cell clones, and in mouse tissues. In the primary cell cultures, telomere length decreased with increased cell doublings and telomerase activity was not detected. In contrast, in spontaneously immortalized clones, telomeres were maintained at a stable length and telomerase activity was present. To determine if telomere shortening occurs in vivo, we assayed for telomerase and telomere length in tissues from mice of different ages. Telomere length was similar among different tissues within a newborn mouse, whereas telomere length differed between tissues in an adult mouse. These findings suggest that there is tissue-specific regulation of mouse telomerase during development and aging in vivo. In contrast to human tissues, most mouse tissues had active telomerase. The presence of telomerase in these tissues may reflect the ease of immortalization of primary mouse cells relative to human cells in culture.     

Telomere lengthening and telomerase activation during human B cell differentiation

The function of the immune system is highly dependent on cellular differentiation and clonal expansion of antigen-specific lymphocytes. However, little is known about mechanisms that may have evolved to protect replicative potential in actively dividing lymphocytes during immune differentiation and response. Here we report an analysis of telomere length and telomerase expression, factors implicated in the regulation of cellular replicative lifespan, in human B cell subsets. In contrast to previous observations, in which telomere shortening and concomitant loss of replicative potential occur in the process of somatic cell differentiation and cell division, it was found that germinal center (GC) B cells, a compartment characterized by extensive clonal expansion and selection, had significantly longer telomeric restriction fragments than those of precursor naive B cells. Furthermore, it was found that telomerase, a telomere-synthesizing enzyme, is expressed at high levels in GC B cells (at least 128-fold higher than those of naive and memory B cells), correlating with the long telomeres in this subset of B cells. Finally, both naive and memory B cells were capable of up-regulating telomerase activity in vitro in response to activation signals through the B cell antigen receptor in the presence of CD40 engagement and/or interleukin 4. These observations suggest that a novel process of telomere lengthening, possibly mediated by telomerase, functions in actively dividing GC B lymphocytes and may play a critical role in humoral immune response by maintaining the replicative potential of GC and descendant memory B cells.     

Barley telomeres shorten during differentiation but grow in callus culture

Eukaryotic chromosomes terminate with long stretches of short, guanine-rich repeats. These repeats are added de novo by a specialized enzyme, telomerase. In humans telomeres shorten during differentiation, presumably due to the absence of telomerase activity in somatic cells. This phenomenon forms the basis for several models of telomere role in cellular senescence. Barley (Hordeum vulgare L.) telomeres consist of thousands of TTTAGGG repeats, closely resembling other higher eukaryotes. In vivo differentiation and aging resulted in reduction of terminal restriction fragment length paralleled by a decrease of telomere repeat number. Dedifferentiation in callus culture resulted in an increase of the terminal restriction fragment length and in the number of telomere repeats. Long-term callus cultures had very long telomeres. Absolute telomere lengths were genotype dependent, but the relative changes due to differentiation, dedifferentiation, and long-term callus culture were consistent among genotypes. A model is presented to describe the potential role of the telomere length in regulation of a cell's mitotic activity and senescence.     

Analysis of telomere length and telomerase activity in tree species of various life-spans,and with age in the bristlecone pine <Emphasis Type="BoldItalic">Pinus longaeva</Emphasis>

Normal somatic cells have a finite replicative capacity. With each cell division, telomeres (the physical ends of linear chromosomes) progressively shorten until they reach a critical length, at which point the cells enter replicative senescence. Some cells maintain telomere length by the action of the telomerase enzyme. The bristlecone pine, Pinus longaeva, is the oldest known living eukaryotic organism, with the oldest on record turning 4770 years old in 2005. To determine what changes occur, if any, in telomere length and telomerase activity with age, and what roles, if any, telomere length and telomerase activity may play in contributing to the increased life-span and longevity of P. longaeva with age, as well as in other tree species of various life-spans, we undertook a detailed investigation of telomere length and telomerase activity in such trees. The results from this study support the hypothesis that both increased telomere length and telomerase activity may directly/indirectly contribute to the increased life-span and longevity evident in long-lived pine trees (2000–5000 year life-spans) compared to medium-lived (400–500 year life-span) and short-lived (100–200 year life-span) pine trees, as well as in P. longaeva with age.     

Telomere maintenance and telomerase activity are differentially regulated in asexual and sexual worms

In most sexually reproducing animals, replication and maintenance of telomeres occurs in the germ line and during early development in embryogenesis through the use of telomerase. Somatic cells generally do not maintain telomere sequences, and these cells become senescent in adults as telomeres shorten to a critical length. Some animals reproduce clonally and must therefore require adult somatic mechanisms for maintaining their chromosome ends. Here we study the telomere biology of planarian flatworms with apparently limitless regenerative capacity fueled by a population of highly proliferative adult stem cells. We show that somatic telomere maintenance is different in asexual and sexual animals. Asexual animals maintain telomere length somatically during reproduction by fission or when regeneration is induced by amputation, whereas sexual animals only achieve telomere elongation through sexual reproduction. We demonstrate that this difference is reflected in the expression and alternate splicing of the protein subunit of the telomerase enzyme. Asexual adult planarian stem cells appear to maintain telomere length over evolutionary timescales without passage through a germ-line stage. The adaptations we observe demonstrate indefinite somatic telomerase activity in proliferating stem cells during regeneration or reproduction by fission, and establish planarians as a pertinent model for studying telomere structure, function, and maintenance.     

Telomerase: regulation, function and transformation

Work from several laboratories over the past decade indicates that the acquisition of constitutive telomerase expression is a critical step during the malignant transformation of human cells. Normal human cells transiently express low levels of telomerase, the ribonucleoprotein responsible for extending and maintaining telomeres, and exhibit telomere shortening after extended passage, whereas most cancers exhibit constitutive telomerase expression and maintain telomeres at stable lengths. These observations establish a direct connection between immortalization and stabilization of telomere structure. However, recent work suggests that telomerase also contributes to cancer development beyond its role in maintaining stable telomere lengths. In this review, we summarize recent observations that support the concept that telomerase plays multiple roles in facilitating human cell transformation.     

Depletion of Ku70/80 reduces the levels of extrachromosomal telomeric circles and inhibits proliferation of ALT cells

In normal cells, telomeres shorten each time a cell divides ultimately resulting in cell senescence. In contrast, cancer cells counteract the loss of telomeric DNA either by inducing the expression of telomerase or by activating the alternative lengthening of telomeres (ALT) pathway. ALT cells are characterized by heterogeneous telomeres and the presence of extrachromosomal circular double-stranded DNA molecules containing telomeric repeat sequences. These telomeric circles (t-circles) are though to be generated through a recombination process and utilized as templates for telomere elongation by rolling-circle-replication, although their precise mechanism of formation and role in telomere maintenance and cell proliferation is largely unknown. Here we show that shRNA-mediated knockdown of the Ku70/80 heterodimer, a factor with functions at both pathological and natural DNA ends, inhibits ALT cell growth and results in a significant decrease in the levels of t-circles without affecting overall telomere length. These findings demonstrate that non homology-based processes contribute to the maintenance of t-circles and proliferation of ALT cells.     

Telomeres and telomerase in ageing and cancer

Telomeres lie at the ends of human chromosomes and contain long tandem repeats of a simple nucleotide sequence. Because DNA replication cannot proceed to the very end of chromosomes, copies of these repeats are lost at each cell division. If the telomeres shorten below a critical length, the cells will eventually die as a result of genomic instability. Aging cells usually avoid death by entering senescence before the critical telomere length is reached. Malignantly transformed, immortal cells overcome senescence but they must still avoid the final, critical shortening of telomeres to survive. In the vast majority of cases, tumor cells achieve this by activating the telomerase enzyme, a ribonucleoprotein complex which repairs the end of chromosomes and prevents telomere shortening. Normal mortal cells do not normally express telomerase, although some stem cell populations which must regenerate thought the life span of the organism, retain enzyme activity. Cellular senescence can be overcome by inducing telomerase expression in mortal cells, firmly establishing the role of telomere length in the senescence signaling pathway. In tumor cells, the evidence of a role for telomerase in immortality is still largely correlative, with 80–90% of tumors expressing telomerase activity. To establish whether telomerase activity is important in maintaining the malignant phenotype, attempts have been made to inactivate it in tumor cells, using a variety of approaches, where there is evidence that disrupting telomerase function can result in the induction of apoptosis. The background and implications of these observations is discussed.     

Regulation of murine telomere length via Rtel

Normal somatic cells have a finite replicative capacity. With each cell division, telomeres (the physical ends of linear chromosomes) progressively shorten until they reach a critical length, at which point the cells enter replicative senescence. Some cells can maintain telomere length by the action of the telomerase enzyme. A recent article described in detail the organization and function of Rtel, a murine gene encoding a DNA helicase-like protein. The Rtel protein was found to be essential for genomic stability and embryonic development and survival, and is proposed to be a dominant regulator of murine telomere length.     

Telomere length predicts replicative capacity of human fibroblasts.

When human fibroblasts from different donors are grown in vitro, only a small fraction of the variation in their finite replicative capacity is explained by the chronological age of the donor. Because we had previously shown that telomeres, the terminal guanine-rich sequences of chromosomes, shorten throughout the life-span of cultured cells, we wished to determine whether variation in initial telomere length would account for the unexplained variation in replicative capacity. Analysis of cells from 31 donors (aged 0-93 yr) indicated relatively weak correlations between proliferative ability and donor age (m = -0.2 doubling per yr; r = -0.42; P = 0.02) and between telomeric DNA and donor age (m = -15 base pairs per yr; r = -0.43; P = 0.02). However, there was a striking correlation, valid over the entire age range of the donors, between replicative capacity and initial telomere length (m = 10 doublings per kilobase pair; r = 0.76; P = 0.004), indicating that cell strains with shorter telomeres underwent significantly fewer doublings than those with longer telomeres. These observations suggest that telomere length is a biomarker of somatic cell aging in humans and are consistent with a causal role for telomere loss in this process. We also found that fibroblasts from Hutchinson-Gilford progeria donors had short telomeres, consistent with their reduced division potential in vitro. In contrast, telomeres from sperm DNA did not decrease with age of the donor, suggesting that a mechanism for maintaining telomere length, such as telomerase expression, may be active in germ-line tissue.     

Telomerase activity in normal leukocytes and in hematologic malignancies

Telomeres are essential for function and stability of eukaryotic chromosomes. In the absence of telomerase, the enzyme that synthesizes telomeric DNA, telomeres shorten with cell division, a process thought to contribute to cell senescence and the proliferative crisis of transformed cells. We reported telomere stabilization concomitant with detection of telomerase activity in cells immortalized in vitro and in ovarian carcinoma cells, and suggested that telomerase is essential for unlimited cell proliferation. We have now examined the temporal pattern of telomerase expression in selected hematologic malignancies. We found that, unlike other somatic tissues, peripheral, cord blood, and bone marrow leukocytes from normal donors expressed low levels of telomerase activity. In leukocytes from chronic lymphocytic leukemia (CLL) patients, activity was lower than in controls in early disease, and comparable with controls in late disease. Relative to bone marrow, telomerase activity was enhanced in myelodysplastic syndrome (MDS) and more significantly so in acute myeloid leukemia (AML). Regardless of telomerase levels, telomeres shortened with progression of the diseases. Our results suggest that early CLL and MDS cells lack an efficient mechanism of telomere maintenance and that telomerase is activated late in the progression of these cancers, presumably when critical telomere loss generates selective pressure for cell immortality.     

Abrupt disruption of capping and a single source for recombinationally elongated telomeres in Kluyveromyces lactis

Eukaryotic cells, including some human cancers, that lack telomerase can sometimes maintain telomeres by using recombination. It was recently proposed that recombinational telomere elongation (RTE) in a telomerase-deletion mutant of the yeast Kluyveromyces lactis occurs through a roll-and-spread mechanism as described in our previous work. According to this model, a tiny circle of telomeric DNA is copied by a rolling-circle mechanism to generate one long telomere, the sequence of which is then spread to all other telomeres by gene-conversion events. In support of this model, we demonstrate here that RTE in K. lactis occurs by amplification of a sequence originating from a single telomere. When a mutationally tagged telomere is of normal length, its sequence is spread to all other telomeres at a frequency (≈10%) consistent with random selection among the 12 telomeres in the cell. However, when the mutationally tagged telomere is considerably longer than other telomeres, cellular senescence is partially suppressed, and the sequence of the tagged telomere is spread to all other telomeres in >90% of cells. Strikingly, the transition between a state resistant to recombination and a state capable of initiating recombination is abrupt, typically occurring when telomeres are ≈3–4 repeats long. Last, we show that mutant repeats that are defective at regulating telomerase are also defective at regulating telomere length during RTE.     

Analysis of telomere length and telomerase activity in tree species of various lifespans, and with age in the bristlecone pine Pinus longaeva

Normal somatic cells have a finite replicative capacity, and with each cell division telomeres progressively shorten, unless the telomerase enzyme is present. The bristlecone pine, Pinus longaeva, is the oldest known living eukaryotic organism, with the oldest on record turning 4770 years old in 2005. The results from our study of telomere length and telomerase activity in samples (needle, root, core) from P. longaeva with age, and in other tree species of various lifespans, support the hypothesis that both increased telomere length and telomerase activity may contribute to the increased lifespan and longevity evident in long-lived pine trees (i.e., 2000- to 5000-year lifespan) compared with medium-lived (400- to 500-year lifespan) and short-lived (100- to 200-year lifespan) pine trees, as well as in P. longaeva with age.     

Telomerase regulation and telomere dynamics in germinal centers

Telomere length maintenance, usually executed by telomerase, is a prerequisite for an extended or infinite division potential. Nevertheless most telomerase positive normal cells exhibit telomere shortening. This study details the telomerase expression and telomere dynamics in purified tonsil B cell subsets during the germinal center (GC) reaction. Significant telomere lengthening was observed as naive B cells matured to centroblasts and when centroblasts matured further to centrocytes, resulting in an increase in telomere length of about 4 kbp determined by Southern blotting. Immunopurified cell populations were also studied by fluorescence in situ hybridization and flow cytometry (flow-FISH) confirming that the GC B cells exhibited lengthened telomeres. These data were further verified in unpurified tonsil cells by combining flow-FISH and immunophenotyping using selected surface markers. Centroblasts expressed high levels of telomerase activity, which was increased in centrocytes, whereas resting naive, activated naive and memory B cells were telomerase activity negative. Expression levels of the catalytic subunit (hTERT) RNA paralleled the telomerase activity levels. The unique telomere elongation in GC B cells permits extensive proliferation during the GC reaction and provides the memory cells with a substantial increase in division potential. Understanding the telomere biology of GC cells is important in defining requirements for telomere elongation in vivo, with implications for the normal immune system as well as for lymphomas, and could provide insights into how the division potential of cells can be manipulated in vitro.     

Telomere and telomerase in the initial stage of immortalization of esophageal epithelial cell

AIM: To search for the biomarker of cellular immortalization, the telomere length, telomerase activity and its subunits in cultured epithelial cells of human fetal esophagus in the process of immortalization. METHODS: The transgenic cell line of human fetal esophageal epithelium (SHEE) was established with E(6)E(7) genes of human papillomavirus (HPV) type 18 in our laboratory. Morphological phenotype of cultured SHEE cells from the 6th to 30th passages, was examined by phase contrast microscopy, the telomere length was assayed by Southern blot method, and the activity of telomerase was analyzed by telomeric repeat amplification protocol (TRAP). Expressions of subunits of telomerase, hTR and hTERT, were assessed by RT-PCR. DNA content in cell cycle was detected by flow cytometry. The cell apoptosis was examined by electron microscopy (EM) and TUNEL label. RESULTS: SHEE cells from the 6th to 10th passages showed cellular proliferation with a good differentiation. From the 12th to the 16th passages, many senescent and apoptotic cells appeared, and the telomere length sharply shortened from 23kb to 17kb without expression of hTERT and telomerase activity. At the 20th passage, SHEE cells overcame the senescence and apoptosis and restored their proliferative activity with expression of telomerase and hTERT at low levels, but the telomere length shortened continuously to the lowest of 3kb. After the 30th passage cells proliferation was restored by increment of cells at S and G2M phase in the cell cycle and telomerase activity expressed at high levels and with maintenance of telomere length. CONCLUSION: At the early stage of SHEE cells, telomeres are shortened without expression of telomerase and hTERT causing cellular senescence and cell death. From the 20th to the 30th passages, the activation of telomerase and maintenance of telomere length show a progressive process for immortalization of esophageal epithelial cells. The expression of telomerase may constitute a biomarker for detection of immortalization of cells.     

Telomere length, telomeric proteins and genomic instability during the multistep carcinogenic process

Telomeres form specialized structures at the ends of eukaryotic chromosomes, preventing them from being wrongly recognized as DNA damage. The human telomere DNA sequence is a tandem repetition of the sequence TTAGGG. In normal cells, the DNA replication machinery is unable to completely duplicate the telomeric DNA; thus, telomeres are shortened after every cell division. Having reached a critical length, telomeres may be recognized as double strand break DNA lesions, and cells eventually enter senescence. Carcinogenesis is a multistep process involving multiple mutations and chromosomal aberrations. One of the most prevalent aberrations in pre-cancerous lesions is telomere shortening and telomerase activation. We discuss the role and homeostasis of telomeres in normal cells and their implication in the early steps of carcinogenesis. We also discuss various techniques used, and their limitations, in the study of telomeres and genome instability and their role in carcinogenesis and related genomic modifications.