Telomere length and telomerase activity predict survival in patients with B cell chronic lymphocytic leukemia

The telomere-telomerase hypothesis states that the vast majority of human tumors have a prolonged replicative life span throughout expressing telomerase, which compensates the cell division-associated loss of telomere DNA. The use of telomere length and telomerase expression as new biological markers in cancer patients requires their correlation with disease prognosis. We, therefore, correlated the mean telomere length based on a telomere restriction fragment assay and the activity of telomerase measured with a telomeric repeat amplification protocol with clinical data and overall survival in 58 patients with B cell chronic lymphocytic leukemia (B-CLL). Telomere length showed a highly inverse correlation to telomerase activity. Patients with telomeres below 6.0 kb were associated with high telomerase activity, whereas patients with a telomere length >6.0 kb generally showed low enzyme activity (P <0.001). Patients in Binet A exhibited significantly longer telomeres and had less telomerase activity than did patients in Binet B or Binet C, where significantly shorter telomeres and higher telomerase activity were observed (P=0.031). Short telomere length and high telomerase activity were significantly associated with a shorter median survival (P=0.02 and P <0.001), and telomerase activity was the most significant prognostic factor for overall survival in B-CLL (P <0.001). Our data provide evidence that telomere length, as well telomerase activity, exerts a strong impact on the survival of B-CLL patients and that telomerase activity can be used as a new prognostic marker in this disease.     

Sensitivity of the N-methyl-D-aspartate receptor channel to butyrophenones is dependent on the epsilon2 subunit

Li-Fraumeni Syndrome (LFS) is characterized by heterozygous germline mutations in the p53 gene. Accompanied by genomic instability and loss or mutation of the remaining wild type p53 allele, a low frequency of spontaneous immortalization in LFS fibroblasts occurs. It is believed that the loss of p53 wild type function contributes to immortalization of these LFS fibroblasts, but it is not clear if this is sufficient. Because stabilization of telomere length is also thought to be a necessary step in immortalization, telomerase activity, expression of the telomerase RNA component (hTR) and telomere length were anlaysed at various passages during the spontaneous immortalization of LFS skin fibroblasts. One LFS strain which immortalized, MDAH087 (087), had no detectable telomerase activity whereas another LFS strain which immortalized, MDAH041 (041), had detectable telomerase activity. In preimmortal cells from both strains, hTR was not detected by in situ hybridization. Immortal 087 cells remained negative for hTR, while immortal 041 cells demonstrated strong hTR in situ hybridization signals. 087 cells had long and heterogenous telomeres whereas telomeres of 041 cells had short, stable telomere lengths. Tumorigenicity studies in nude mice with ras-transformed 087 and 041 cells resulted in both cell lines giving rise to tumors and retaining telomerase status. Overall these results suggest that strain specificity may be important in telomerase re-activation and that both abrogation of p53 function and a mechanism to maintain telomeres are necessary for immortalization.     

Telomere, telomerase and cytogenetic changes in myelodysplastic syndromes

Myelodysplastic syndrome (MDS) is a heterogenous but clonal disorder characterized by cytopenia and dysplastic features. Telomere length in MDS vary but some of them show shortened telomeres. Telomerase activity in MDS also vary but about 60% of them show slightly elevated telomerase activity. According to the disease progression of MDS, MDS patients categorize into 3 groups, i.e., (1) normal telomere length before and after disease progression, (2) short telomere length before and after progression, and (3) shortened telomere with disease progression. Telomerase change with disease progression is not obscure, indicating impairment of telomere dynamics in MDS. These observations may indicate that some MDS show telomerase upregulation possible due to telomere shortening, while the another pathway without telomerase upregulation associated with complex chromosome changes may link to the pathogenesis of MDS.     

Multiple pathways to cellular senescence: role of telomerase repressors

Telomeres progressively shorten with age in somatic cells in culture and in vivo because DNA replication results in the loss of sequences at the 5' ends of double-stranded DNA. Whereas somatic cells do not express the enzyme, telomerase, which adds repeated telomere sequences to chromosome ends, telomerase activity is detected in immortalised and tumour cells in vitro and in primary tumour tissues. This represents an important difference between normal cells and cancer cells, suggesting that telomere shortening causes cellular senescence. Hybrids between immortal cells and normal cells senesce, indicating that immortal cells have lost, mutated or inactivated genes that are required for the programme of senescence in normal cells. Genes involved in the senescence programme have been mapped to over ten different genetic loci using microcell fusion to introduce human chromosomes and restore the senescence programme. Multiple pathways of cellular senescence have also been demonstrated by chromosome transfer, indicating that the functions of the mapped senescence genes are probably different. One possibility is that one or more of these senescence genes may suppress telomerase activity in immortal cells, resulting in telomere shortening and cellular senescence. To test this hypothesis, telomerase activity and the length of terminal restriction fragments (TRFs) have been examined in microcell hybrids. Re-introduction of a normal chromosome 3 into the renal cell carcinoma cell line RCC23, which has the short arm of chromosome 3, restored cellular senescence. The loss of indefinite growth potential was associated with the loss of telomerase activity and shortening of telomeres in the RCC cells containing the introduced 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 the parental RCC23. Thus, restoration of cellular senescence by chromosome 3 is associated with repression of telomerase function in RCC cells. This evidence suggests that telomerase suppression is one of several pathways involved in immortalisation.     

Cell cycle-regulated generation of single-stranded G-rich DNA in the absence of telomerase

Current models of telomere replication predict that due to the properties of the polymerases implicated in semiconservative replication of linear DNA, the two daughter molecules have one end that is blunt and one end with a short 3' overhang. Telomerase is thought to extend the short 3' overhang to produce long single-stranded overhangs. Recently, such overhangs, or TG1-3 tails, were shown to occur on both telomeres of replicated linear plasmids in yeast. Moreover, indirect evidence suggested that the TG1-3 tails also occurred in a yeast strain lacking telomerase. We report herein a novel in-gel hybridization technique to probe telomeres for single-stranded DNA. Using this method, it is shown directly that in yeast strains lacking the TLC1 gene encoding the yeast telomerase RNA, TG1-3 single-stranded DNA was generated on chromosomal and plasmid telomeres. The single-stranded DNA only appeared in S phase and was sensitive to digestion with a single-strand-specific exonuclease. These data demonstrate that during replication of telomeres, TG1-3 tails can be generated in a way that is independent of telomerase-mediated strand elongation. In wild-type strains, these TG1-3 tails could subsequently serve as substrates for telomerase and telomere binding proteins on all telomeres.     

Telomerase and telomere length in the development and progression of premalignant lesions to colorectal cancer

Telomerase and telomere length are increasingly studied as prognostic markers in malignancy. Telomerase is also known to be expressed in certain nonmalignant cells, although generally at low levels. We investigated telomerase activity and telomere length in premalignant, malignant, inflammatory, and normal colon specimens to determine whether significant differences exist and whether telomerase may serve as a marker for early- or late-stage colorectal cancer. Telomerase activity was evaluated in 130 frozen specimens from human colon cancer (n = 50), adjacent normal colon tissue (n = 50), colon polyps (n = 20), and colitis (n = 10) using a modified telomeric repeat amplification protocol assay, and telomere length was assessed by terminal restriction fragment analysis. High to moderate levels of telomerase activity were detected in 90% of colorectal tumors. Weakly positive activity was detected in 10%. None of the normal tissues exhibited telomerase activity. In polyps and colitis, telomerase activity was found in 60% (12 of 20) and 40% (4 of 10), respectively. Telomerase activity in both nonmalignant lesions was 25- to 54-fold lower than that detected in colon cancer (P < 0.001). We found a positive correlation between tumor cell infiltration determined in cryostat sections and telomerase activity (r = 0.886; P > 0.0001). Late-stage tumors (Dukes C + D) demonstrated increased telomerase activity compared to early-stage tumors (Dukes A + B). Telomere restriction fragments in colon tumors had peak values of 4.8 +/- 1 kbp that were significantly and consistently shorter than those of the adjacent normal tissues (7.54 +/- 1.3 kbp), polyps (7.5 +/- 0.7 kbp), and colitis specimens (7.7 +/- 0.5kbp; P < 0.0001). Telomeres were 0.6 kbp longer in tumors with high telomerase activity and in late-stage cancers (Dukes C + D) compared to those in tumors with low telomerase activity and in early-stage cancers (Dukes A + B). Our data demonstrate that telomerase in colon cancer was commonly acquired, and activity was higher than that in polyps and colitis. However, weak telomerase activity was detected in premalignant and inflammatory lesions. Telomeres in colon cancer were considerably shorter, an indication of extensive cell proliferation and population divisions, whereas adjacent normal colon specimens, polyps, and colitis had comparable telomere lengths. Our results indicate that increased telomerase activity occurs in colon cancer cells that have undergone extensive telomere shortening relative to surrounding normal tissues and in which telomerase-induced stabilization of telomeres may be critical for the continued proliferation of the malignant clone. The link between telomerase activity and stage suggests that telomerase is up-regulated as a function of increased tumor cell invasion, tumor progression, and metastatic potential in colon cancer.     

Telomerase activity and telomere lengths in various cell lines: changes of telomerase activity can be another method for chemosensitivity evaluation

For the cancer cells which have overcome the second mitotic clock (M2), activated telomerase is essential and used as another marker of immortality. Many trials had been initiated to target telomerase, which is known to be specific to tumors. To determine the best in vitro cell system for testing the efficacy of telomerase inhibitors, we evaluated the telomerase activity of various cancer cell lines and measured their telomere lengths. We also treated some cancer cell lines with adriamycin and measured the changes of telomerase activity. Telomerase activity was evaluated in various cell lines with the TRAP (telomeric repeat amplification protocol) assay. Telomerase activity was calculated and translated into arbitrary units by computer-assisted densitometry with the control of telomerase activity in the 293 control cell line. Also, terminal restriction fragment lengths were measured using Southern blotting. We also measured telomerase activity and telomere lengths in 11 benign breast tumor tissues and 19 paired stomach cancer and normal tissues. Cancer cell lines treated with adriamycin we evaluated for changes of telomerase activity and the cell proliferation by MTT assay and dye exclusion test. Telomerase activity of cell lines was 95.3 24.1 unit with a range of 27.6-129.6 unit, while the telomere lengths of those cell lines were variable from 5.0 to 10.4 kbp with a median of 6 kbp. In 11 cancer cell lines which were not yet firmly established, we could not detect any telomerase activity. Low telomerase activity was detected in only 2 benign tumor tissues of breast with a median telomere length of 8.8 (7-10.5) kbp. Among paired 19 gastric cancer and normal tissues, only 7 cancer tissues showed weak telomerase activity. After adriamycin treatment, telomerase activity in YCC-S-1, YCC-S-3, MCF-7 and MCF-7/ADR was decreased in accordance with the changes of the cell numbers. Telomerase is specific to cancer tissues and is expressed differently from organ to organ. Telomerase activity by TRAP assay could be used as a chemosensitivity assay.     

Sexual differences in branched chain amino acid metabolism into fatty acids and cholesterol in Harderian gland of golden hamster

PURPOSE: To investigate the telomere hypothesis of cellular aging as the mechanism for cell cycle arrest in normal human corneal endothelium. METHODS: The corneal endothelium and epithelium from 21 human corneas from 13 donors 5 weeks to 84 years of age were dissected and frozen at -70 degrees C. Purified DNA, digested with the restriction enzyme, HinfI, was run on 0.7% agarose gels, probed with radiolabeled (AATCCC)4, and exposed to a phosphor screen. The length of the terminal restriction fragment (TRF) was determined by densitometry. RESULTS: The cells of the corneal endothelium had TRF lengths ranging from 11.0 to 14.0 kbp (mean, 12.2 +/- 0.9). Corneal epithelial specimens showed TRF lengths that were always less than (mean, 10.4 +/- 1.0; range 9.0-12.0) the corresponding endothelial TRF lengths. Human corneal endothelial cells, transformed with human papillomavirus type 16 oncogenes E6 and E7, showed decreasing TRF lengths from 11 kbp at population doubling level (PDL) 15 to 9.5 kbp at PDL 73. Neither the endothelial and epithelial cells from human donors nor the transformed pre-immortalized human endothelial cells showed evidence of telomerase activity. CONCLUSIONS: Human corneal endothelial cells have long telomeres throughout life. Their limited replicative ability does not appear to result from critically short telomere lengths.     

Diagnostic significance of telomerase activity and telomere length in endoscopic sample of colorectal cancer

Telomeres are located on both ends of individual chromosomes in eukaryotes. It has been reported that telomerase activity and telomere reduction can be detected in most human cancers. We examined telomerase activity and telomere length in colorectal cancer tissues obtained by colonoscopy. Telomerase activity was examined by the TRAP (telomeric repeat amplification protocol) assay and was detected in 21 of 26 (81%) primary colorectal carcinoma tissues. Two of 9 (22%) colorectal polyp were telomerase positive. Telomere length was analyzed by Southern blotting and there was reduction in telomere lengths in 12 of 15 (80%) primary colorectal carcinoma and 3 of 6 colorectal polyp, compared to the corresponding normal colonic mucosa. Therefore, telomerase activity and telomere length may serve as an useful tool for preoperative cancer diagnosis.     

Community hospitals and general practice: extended attachments for medical students

Telomerase activation has been shown to be an almost universal property of malignant tumors evoking its role in the immortalisation process. We used the recently described sensitive and rapid detection assay called telomeric repeat amplification protocol (TRAP) to detect telomerase activity in neoplastic and non neoplastic tissues. Human telomerase is a ribonucleoprotein which functions as a telomere terminal transferase by adding multiple repeats of the TTAGGG hexamer at the 3'-OH ends of either telomeres or oligonucleotide specifically designed for the TRAP assay. Whenever present, telomerase activity is revealed on acrylamide gel by a six nucleotide ladder of extension products. Detection of telomerase activity may be evaluated for differentiating neoplastic from non neoplastic tissues. In addition, quantitation of telomerase activity may serve as prognostic marker for malignant tumors.     

Processing of telomeric DNA ends requires the passage of a replication fork

During telomere replication in yeast, chromosome ends acquire a long single-stranded extension of the strand making the 3' end. Previous work showed that these 3' tails are generated late in S-phase, when conventional replication is virtually complete. In addition, the extensions were also observed in cells that lacked telomerase. Therefore, a model was proposed that predicted an activity that recessed the 5' ends at yeast telomeres after conventional replication was complete. Here, we demonstrate that this processing activity is dependent on the passage of a replication fork through yeast telomeres. A non-replicating linear plasmid with telomeres at each end does not acquire single-stranded extensions, while an identical construct containing an origin of replication does. Thus, the processing activity could be associated with the enzymes at the replication fork itself, or the passage of the fork through the telomeric sequences allows a transient access for the activity to the telomeres. We therefore propose that there is a mechanistic link between the conventional replication machinery and telomere maintenance.     

Rap1 protein regulates telomere turnover in yeast

Telomere length is maintained through a dynamic balance between addition and loss of the terminal telomeric DNA. Normal telomere length regulation requires telomerase as well as a telomeric protein-DNA complex. Previous work has provided evidence that in the budding yeasts Kluyveromyces lactis and Saccharomyces cerevisiae, the telomeric double-stranded DNA binding protein Rap1p negatively regulates telomere length, in part by nucleating, by its C-terminal tail, a higher-order DNA binding protein complex that presumably limits access of telomerase to the chromosome end. Here we show that in K. lactis, truncating the Rap1p C-terminal tail (Rap1p-DeltaC mutant) accelerates telomeric repeat turnover in the distal region of the telomere. In addition, combining the rap1-DeltaC mutation with a telomerase template mutation (ter1-kpn), which directs the addition of mutated telomeric DNA repeats to telomeres, synergistically caused an immediate loss of telomere length regulation. Capping of the unregulated telomeres of these double mutants with functionally wild-type repeats restored telomere length control. We propose that the rate of terminal telomere turnover is controlled by Rap1p specifically through its interactions with the most distal telomeric repeats.     

Telomere length regulation in mice is linked to a novel chromosome locus

Little is known about the mechanisms that regulate species-specific telomere length, particularly in mammalian species. The genetic regulation of telomere length was therefore investigated by using two inter-fertile species of mice, which differ in their telomere length. Mus musculus (telomere length >25 kb) and Mus spretus (telomere length 5-15 kb) were used to generate F1 crosses and reciprocal backcrosses, which were then analyzed for regulation of telomere length. This analysis indicated that a dominant and trans-acting mechanism exists capable of extensive elongation of telomeres in somatic cells after fusion of parental germline cells with discrepant telomere lengths. A genome wide screen of interspecific crosses, using M. spretus as the recurrent parent, identified a 5-centimorgan region on distal chromosome 2 that predominantly controls the observed species-specific telomere length regulation. This locus is distinct from candidate genes encoding known telomere-binding proteins or telomerase components. These results demonstrate that an unidentified gene(s) mapped to distal chromosome 2 regulates telomere length in the mouse.     

Aging and cancer: are telomeres and telomerase the connection?

There is substantial evidence for the progressive loss of the telomeric ends of chromosomes during aging, both in cell culture and in vivo. The loss of telomeres may eventually induce antiproliferative signals that result in cellular senescence. A hypothesis gaining prominence is that the activation of telomerase, a ribonucleoprotein enzyme that is important in maintaining telomere length stability, is necessary for the sustained growth of most tumors. The interrelationships between telomere shortening and aging, and how activation of telomerase may be necessary for cells to become immortal and malignant, are reviewed here.     

Possible mechanisms for the regulation of telomere length

Since DNA polymerases can only synthesise a new DNA strand in the 5'-3' direction and need a primer that provides a free 3' OH end, the cellular replication machinery is unable to duplicate the 3' ends of linear chromosomes unless special mechanisms are operative. While the telomeres seem to shorten continuously in human somatic cells because of the "end replication" problem, it appears that telomere length is maintained in cancer cells, the germ line and unicellular organisms like yeast and Tetrahymena by a mechanism involving the enzyme telomerase, which elongates the 3' ends of telomeres. However, telomerase must be part of a more complicated mechanism to ensure that there is no net gain or loss of telomeric ends. Here we describe a simple theoretical model that can explain several experimental findings. The simulations show that (i) the proposed mechanism is able to maintain telomeres at a constant length, (ii) this length constancy is independent of the initial telomere length, (iii) mutations of the telomeric sequence lead to an elongation of telomeres, (iv) inhibition of telomerase causes telomeric shortening, and (v) it reproduces and explains the experimental result that the addition of oligonucleotides to the culture medium leads to an increase of telomere length.     

Telomerase activity of sarcoma cell lines and fibroblasts is independent of p53 status

Telomerase activity is necessary for the stabilization of telomeres, which function to overcome cellular senescence and are linked to unlimited cell proliferation. Activation of telomerase is characteristic of immortalized cell lines and most tumors. The p53 gene has been implicated as a crucial barrier to unlimited cell proliferation, and its absence has been shown to allow direct immortalization of cells by certain oncogenes. The p53 gene may have an additional function of signaling cell growth arrest in response to telomere shortening, which occurs with repeated cellular divisions and ultimately threatens chromosomal stability. This prompted us to consider whether the enzyme telomerase, responsible for adding new telomeres to chromosomal ends, may be affected by the p53 status of normal and malignant cells. We investigated whether a relationship between telomerase and p53 could be demonstrated in a human sarcoma cell line containing a missense p53 mutation and several stable transfectants that express the wild-type p53 gene or a temperature-sensitive mutant of p53. All cell lines had readily detectable telomerase activity regardless of p53 status. In addition, murine fibroblast cell strains established from tissues of p53+/+ and p53-/- (p53 knockout) mice expressed telomerase regardless of the p53 status of their tissue of origin. Levels of telomerase subunit mRNA (hEST2) were comparable among cell lines and tissues with different p53 status. These results imply that p53 status is not associated with telomerase activity per se and that activation of telomerase can occur either in cells completely devoid of p53 or in cells that have functional p53.     

Telomerase activity in human acute myelogenous leukemia: inhibition of telomerase activity by differentiation-inducing agents

The terminal regions of human chromosomes, the telomeres, shorten with each cell division in most normal somatic cells. Telomerase, a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends, is activated in germline cells and almost all tumor cells. Telomerase activity maintains the stability of telomere length, resulting in indefinite cellular proliferation (immortality). In the present study, telomerase activity was analyzed in leukemic mononuclear blood cells obtained from 56 patients with acute myelogenous leukemia (AML) with known cytogenetic alterations. Heterogenous levels of telomerase activity were observed and generally correlated with cytogenetic status. Patients with 11q abnormalities and -5/-7 (unfavorable cytogenetics) tended to have high telomerase activity compared with cells obtained from AML patients with other types of cytogenetics. Additional studies with a larger cohort of patients will determine whether these differences are statistically significant. Chemotherapy agents that result in differentiation of leukemic cells also resulted in inhibition of telomerase activity. Knowledge of telomerase activity in patients with AML, before and throughout therapy, may have clinical utility for following disease progression and may predict early cancer relapse.