Telomerase-associated Protein Research Articles

Differential regulation of telomerase activity by six telomerase subunits.

Telomerase is a specialized reverse transcriptase responsible for synthesizing telomeric DNA at the ends of chromosomes. Six subunits composing the telomerase complex have been cloned: hTR (human telomerase RNA), TEP1 (telomerase-associated protein 1), hTERT (human telomerase reverse transcriptase), hsp90 (heat shock protein 90), p23, and dyskerin. In this study, we investigated the role of each the telomerase subunit on the activity of telomerase. Through down- or upregulation of telomerase, we found that only hTERT expression changed proportionally with the level of telomerase activity. The other components, TEP1, hTR, hsp90, p23, and dyskerin remained at high and unchanged levels throughout modulation. In vivo and in vitro experiments with antisense oligonucleotides against each telomerase component were also performed. Telomerase activity was decreased or abolished by antisense treatment. To correlate clinical sample status, four pairs of normal and malignant tissues from patients with oral cancer were examined. Except for the hTERT subunit, which showed differential expression in normal and cancer tissues, all other components were expressed in both normal and malignant tissues. We conclude that hTERT is a regulatable subunit, whereas the other components are expressed more constantly in cells. Although hTERT has a rate-limiting effect on enzyme activity, the other telomerase subunits (hTR, TEP1, hsp90, p23, dyskerin) participated in full enzyme activity. We hypothesize that once hTERT is expressed, all other telomerase subunits can be assembled to form a highly active holoenzyme.

Mechanism of Human Telomerase Inhibition by BIBR1532, a Synthetic, Non-nucleosidic Drug Candidate

Telomerase, a ribonucleoprotein acting as a reverse transcriptase, has been identified as a target for cancer drug discovery. The synthetic, non-nucleosidic compound, BIBR1532, is a potent and selective telomerase inhibitor capable of inducing senescence in human cancer cells (). In the present study, the mode of drug action was characterized. BIBR1532 inhibits the native and recombinant human telomerase, comprising the human telomerase reverse transcriptase and human telomerase RNA components, with similar potency primarily by interfering with the processivity of the enzyme. Enzyme-kinetic experiments show that BIBR1532 is a mixed-type non-competitive inhibitor and suggest a drug binding site distinct from the sites for deoxyribonucleotides and the DNA primer, respectively. Thus, BIBR1532 defines a novel class of telomerase inhibitor with mechanistic similarities to non-nucleosidic inhibitors of HIV1 reverse transcriptase.

Expression of MRE11 Complex (MRE11, RAD50, NBS1) and hRap1 and Its Relation with Telomere Regulation, Telomerase Activity in Human Gastric Carcinomas

The MRE11 complex (MRE11, RAD50, NBS1) are required for the repair of DNA double-strand breaks and have another important function in regulating telomere length. The silent information regulator (Sir) proteins required for telomere position effect also bind telomeres. hRap1 protein is a human ortholog of yeast Rap1 which regulates telomere length by interacting with TRF2 and is recruited to telomeres by TRF2. We examined the expression of the MRE11 complex (MRE11, RAD50, NBS1), Sir2 and hRAP1 in 20 gastric carcinomas by reverse transcription polymerase chain reaction and then analyzed the relation between telomerase activity and other telomerase components such as human telomerase reverse transcriptase (TERT), human telomerase RNA component (hTR), human telomerase-associated protein (TEP1), telomeric repeat binding factor 1 (TRF1), TRF2- and TRF1-interacting, ankyrin-related ADP-ribose polymerase (tankyrase) as well as TRF1-interacting nuclear protein 2 (TIN2). Of twenty gastric carcinomas examined, 13 (65%), 14 (70%), 16 (80%), 12 (60%) and 13 (65%) expressed MRE11, RAD50, NBS1, Sir2 and hRap1 at higher levels than corresponding nonneoplastic gastric mucosa, respectively. No obvious correlation was observed between MRE11 complex expression and telomerase activity or expression of TERT, hTR, TEP1, tankyrase and TIN2. Carcinomas with high TRF1 expression expressed significantly higher levels of MRE11 and RAD50 than those with low TRF1 expression (p < 0.05). On the other hand, carcinomas with high TRF2 expression expressed significantly higher levels of MRE11, NBS1 and hRap1 than those with low TRF2 expression (p < 0.05). These results suggest that gastric carcinomas with high TRF1 and TRF2 expression may need a large quantity of the MRE11 complex. Moreover, gastric carcinomas with high TRF1 expression may require a large quantity of hRap1.

Clinicopathologic significance of telomerase activity and hTERT mRNA expression in non-small cell lung cancer

Telomerase has been reported to be a novel diagnostic marker for malignant diseases and has been recently proven to be composed of three main components, hTR (human telomerase RNA component), TP1 (telomerase-associated protein 1) and hTERT (human telomerase reverse transcriptase), the last of which plays a key role in telomerase activation. In the present study, quantitative levels of telomerase activity and hTERT gene mRNA ( hTERT) expression were analyzed in cancerous and non-cancerous lung tissues of 62 lung cancer patients by telomeric repeat amplification protocol and reverse transcription-PCR, respectively. The telomerase expression levels of each group of tissue samples were compared with clinicopathologic variables. Telomerase activity and hTERT were detected in cancerous tissues (75.8 and 75.8%, respectively), while these parameters were not observed in any non-cancerous tissues. In quantitative assessment of telomerase expression, both telomerase activity and hTERT were significantly correlated with lymph node metastasis (N0 vs. N1+2, P<0.05). Telomerase activity also correlated with tumor cell differentiation and stage classification ( P<0.05), but did not correlate with other clinicopathologic variables. The disease-free survival in patients with lung cancer demonstrated that patients with hTERT-positive tumor survived for a significantly shorter period than those with hTERT-negative tumor ( P=0.0334). Since hTERT levels are correlated with N factor which represents the true aggressiveness of patients’ disease concerning the evaluation of clinical outcome, hTERT was found to be one of the important markers revealing biological malignant potentials for lung cancer.

Expression of telomeric repeat binding factor 1 and 2 and TRF1-interacting nuclear protein 2 in human gastric carcinomas

Telomeric repeat binding factor 1 (TRF1) and 2 (TRF2) may play key roles in the maintenance of telomere function. TRF1 negatively regulates telomere elongation, while TRF2 protects the chromosome ends by inhibiting end-to-end fusions. We examined the expression of TRF1 and TRF2 in 20 gastric carcinomas by reverse transcription polymerase chain reaction and then analyzed the relation with telomerase activity and other telomerase components such as human telomerase reverse transcriptase (TERT), human telomerase RNA component (hTR), human telomerase-associated protein (TEP1) and TRF1-interacting, ankyrin-related ADP-ribose polymerase (tankyrase) as well as TRF1-interacting nuclear protein 2 (TIN2). Of 20 gastric carcinomas examined, 10 (50%) and 12 (60%) expressed TRF1 and TRF2 at higher levels than did non-neoplastic mucosa, respectively. No obvious correlation was observed between TRF1 expression and telomerase activity or expression of TERT, hTR and TEP1. Carcinomas with high TRF1 expression expressed significantly higher levels of tankyrase and TIN2 than did those with low TRF2 expression (p<0.05). The telomerase activities and the levels of TERT, hTR and TEP1 showed tendency to be lower in tumors expressing TRF1 at low levels, although it was not significant. On the other hand, carcinomas with short telomere length (shorter than 2 Kbp) expressed significantly stronger telomerase activities and higher TRF1 expression (p<0.05) and tended to express TRF2 and TIN2 at higher levels than those with long telomere length. The results suggest that gastric carcinomas with short telomeres need high levels of telomerase activity and large quantity of TRFs and TIN2, whereas those with long telomeres do not require high levels of telomerase activity and telomere associated proteins.

Human telomerase RNA-protein interactions.

Telomere length is maintained in most eukaryotic cells by telomerase. The core components of this ribonucleoprotein (RNP) enzyme include a protein catalytic subunit, composed of motifs conserved among reverse transcriptases (RT), and an RNA subunit that contains a short template sequence essential for the synthesis of telomeric repeats. We developed an electrophoretic mobility shift assay using active telomerase partially purified from 293 cells and radiolabeled, in vitro-transcribed human telomerase RNA (hTR) to investigate the molecular interactions of the human telomerase RT (hTERT) and telomerase-associated proteins with hTR. A specific hTR-protein complex was identified and shown to contain hTERT and human Staufen by antibody supershift assays. Variants of hTR altered in distinct structural elements were analyzed for their ability to competitively inhibit complex formation. Human telomerase RNAs lacking the CR4-CR5 domain were poor inhibitors of hTR-protein complex formation, suggesting that the CR4-CR5 domain of hTR is a potential protein-binding site. Furthermore, alterations in the telomerase RNA pseudoknot's P3 helix, the CR7 domain, or the H/ACA box efficiently inhibited formation of the complex, indicating that these domains are dispensable for the assembly of a telomerase RNP in vitro. Potential telomerase-associated proteins that bind hTR were also identified using a UV cross-linking assay.

Telomerase reverse transcriptase and telomeric-repeat binding factor protein 1 as regulators of telomerase activity in pancreatic cancer cells.

Telomerase adds hexameric repeats of 5′-TTAGGG-3′ termed telomeres to ends of chromosomal DNA. This enzyme has been implicated in cellular immortalization and cellular senescence. Recently, a number of relevant genes have been cloned, including these encoding three major components of human telomerase: human telomerase RNA component (hTR), human telomerase reverse transcriptase (hTERT), and telomerase-associated protein-1 (TEP1). Also important are genes encoding human telomeric-repeat binding factor protein (TRF) 1 and 2. To clarify mechanisms regulating telomerase activity, we studied telomerase activity, the telomeric restriction fragment (TRF) length and gene expression of these telomerase components and the telomeric-repeat binding factor proteins in sequential observation following X-irradiation of cultured pancreatic cancer cells. We previously reported that PANC-1 cells are better able to tolerate thermal stress, antineoplastic drugs, and exposure to tumour necrosis factor than MIAPaCa-2 cells. MIAPaCa-2 and PANC-1 cells were exposed to X-irradiation, their telomerase activity was increased at 2 days and then decreased gradually. Of the three telomerase components, only hTERT mRNA expression showed parallel changes. TRF length was stable just before and after X-irradiation. Among binding factor proteins, TRF1 mRNA showed reciprocal changes possibly directed toward maintaining a stable telomere length. In this study, our results demonstrate that not only hTERT but also TRF1 are important regulator of telomerase activity. © 2001 Cancer Research Campaign http://www.bjcancer.com

Possible Regulation of Telomerase Activity by Transcription and Alternative Splicing of Telomerase Reverse Transcriptase in Human Melanoma

To investigate the regulatory mechanisms of telomerase activity in human melanoma cells, we assessed the enzyme's catalytic activity and the expression of the telomerase subunits, the human telomerase RNA, the human telomerase-associated protein, and the human telomerase reverse transcriptase, in 52 melanoma lesions. Eight normal skin specimens were also studied. Telomerase activity was detected in 84.6% of melanomas, whereas all skin specimens were telomerase negative. Human telomerase-associated protein mRNA and human telomerase RNA were constitutively expressed in all melanoma and skin specimens. Although at a variable level of expression, human telomerase reverse transcriptase mRNA was detected in all but one melanomas, whereas it was never present in skin samples. Reverse transcriptase-polymerase chain reaction experiments were performed using primers within the reverse transcriptase domain of human telomerase reverse transcriptase and revealed the presence of multiple alternatively spliced transcripts in melanoma specimens. Among the 44 telomerase-positive melanomas, one showed the full-length transcript alone whereas in all other specimens a full-length message was present with different combinations of alternatively spliced variants. In these tumors the expression of the full-length transcript was generally equal to or higher than that of the alternatively spliced variants. The ratio full-length transcript to alternatively spliced species ranged from 0.6 to 5.26, with a median value of 1.18. Among the seven telomerase-negative melanomas, one displayed the beta deletion transcript alone, whereas in the remaining six tumors weak expression of the full-length transcript and a more abundant level of alternatively spliced transcripts were found. In these cases human telomerase reverse transcriptase ratio ranged from 0.09 to 1.1, with a median value of 0.40. The results suggest that transcription and alternative splicing of human telomerase reverse transcriptase are regulatory mechanisms controlling telomerase activity in melanoma.

Telomerase activation and p53 mutations in urethane-induced A/J mouse lung tumor development.

The mouse telomerase holoenzyme, which synthesizes telomeric DNA de novo, is a ribonucleoprotein complex that includes the mouse telomerase RNA component (mTERC), mouse telomerase-associated protein (mTEP1) and mouse telomerase reverse transcriptase (mTERT). To determine the role of telomerase in urethane-induced lung tumorigenesis in A/J mice we examined telomerase activity and the expression of each telomerase subunit in 20 tumor samples, harvested at 16, 28, 40 and 50 weeks after urethane treatment. The telomeric repeat amplification protocol assay showed that statistically significant telomerase activation occurred both early and late in tumorigenesis. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed that mRNA expression levels of mTEP1 and mTERT were up-regulated during tumor progression, while mTERC expression was not significantly different between tumors and normal lung. We further examined mTEP1 protein expression in normal lung tissue and lung tumors; western blot analysis showed preferential expression of mTEP1 protein in lung tumors compared with normal lung and immunohistochemistry revealed that a majority of the adenoma cells were positively stained in the nucleus, whereas only a few of the adjacent normal alveolar cells were immunoreactive. In addition, we investigated DNAs of the 20 tumor samples by single strand conformation polymorphism and sequencing analyses to examine whether alterations of the p53 gene in exons 5-8 were associated with telomerase activity. Although we found one nonsense, two missense, two silent and one simultaneous double mutation at different codons in six late stage tumors, there was no apparent correlation between telomerase activity and p53 mutations. Collectively, these results suggest that mTEP1 as well as mTERT may be involved in the regulation of telomerase activity and that telomerase activation may contribute to lung tumorigenesis in A/J mice independently of p53 gene alterations.

Expression of telomerase-associated genes: reflection of telomerase activity in gastric cancer?

Telomerase activation is a characteristic of immortalized tumor cells but not of normal cells. Telomerase activity has been detected in approximately 85% of malignant tumors, and assaying for telomerase activity is thought to be useful for diagnosing cancer. Three telomerase-associated molecules [human telomerase RNA component (hTR), telomerase-associated protein (TEP1), and human telomerase reverse transcriptase (hTERT)] have been cloned. We semiquantitatively measured telomerase activity and the expression of these genes in cancerous and noncancerous regions of gastric cancer patients. We also investigated whether the expression of these genes correlated with telomerase activity. Telomerase activity in cancerous regions was significantly higher than in noncancerous regions, but there was no correlation between telomerase activity and the expression of these genes. Furthermore, no clear difference was observed between cancerous and noncancerous regions. These data indicate that the level of three telomerase-associated genes (i.e., hTR, TEP1 mRNA, hTERT mRNA), do not reflect telomerase activity, and the RNA levels of these genes are not useful markers for diagnosing gastric cancer.

Expression of telomerase reverse transcriptase mRNA and its quantitative analysis in human endometrial cancer.

Three major components of telomerase, i.e., human telomerase RNA (hTERC), telomerase-associated protein (TEP1), and the human telomerase reverse transcriptase (hTERT), have been identified. Among them, TERT expression is very closely related to telomerase activity. The purpose of this study was to evaluate the implications of TERT expression and telomerase activity in endometrial cancer. Fresh surgical specimens of 36 endometrial carcinomas (CA group) and 9 samples of postmenopausal endometrial tissue without malignancy (NP group) were obtained at operation in our hospital. These specimens were analyzed for telomerase activity and TERT expression by TRAP assay and RT-PCR, respectively, and the detection and quantitative analysis were made. The results for endometrial cancer were compared with those for normal endometrium and with the clinical data. In the CA group, TERT expression was detected in 35/36 subjects (97.2%), whereas in 1/9 subject (11.1%) from the NP group. Relative TERT mRNA expression was 0.50 in the CA group, and this was significantly higher compared with the level of 0.10 in the NP group (p<0.05). Telomerase activity was detected in 34/36 subjects (94.4%) from the CA group and in 3/9 subjects (33.3%) from the NP group (p<0.05), while the RTA was 30.9 and 0.2, respectively (p<0.05). There was a significant correlation between the relative TERT expression and RTA (n=45, R=0.413, p<0.05). RTA was significantly higher at an advanced surgical stage (FIGO II, III or IV) than at an early stage (FIGO 0 or I) (52.4 vs. 20.4, p<0.05), but other clinical factors showed no relationship with TERT and RTA values. The detection and quantitative analysis of telomerase activity and TERT expression is helpful for distinguishing malignant from normal endometrium when the patient is postmenopausal, even if the tumor is very small or of low malignancy.

Role of human telomerase reverse transcriptase and telomeric-repeat binding factor proteins 1 and 2 in human hematopoietic cells.

Telomerase, an enzyme that adds hexameric repeats of 5′‐TTAGGG‐3′, termed telomeres, to the ends of chromosomal DNA, has been implicated in cellular immortalization and cellular senescence. Recently several relevant genes have been cloned, including those encoding three major components of human telomerase: human telomerase RNA component (hTR), human telomerase reverse transcriptase (hTERT), and telomerase‐associated protein‐1 (TEP1). Also important are genes encoding human telomeric‐repeat binding factor proteins (TRF) 1 and 2. We compared 10 human malignant hematopoietic cell lines, 19 samples from patients with acute leukemia and normal granulocytes and monocytes to study telomerase activity and expression of these various genes using a reverse transcription‐polymerase chain reaction (RT‐PCR). In all 10 malignant cell lines with telomerase activity, hTR, hTERT mRNA, and TEP1 mRNA were expressed, while in normal monocytes and granulocytes without telomerase activity, expression of hTR, but not hTERT mRNA was detected. TEP1 mRNA was expressed in normal monocytes, but not granulocytes. Expression of TRF1 and TRF2 mRNAs was greater in the normal cells than in human malignant hematopoietic cell lines and in 16 samples of patients with acute leukemia. When differentiation of the malignant hematopoietic cell line HL‐60 was induced using tumor‐necrosis‐factor 471 and all‐trans retinoic acid (ATRA), telomerase activity decreased gradually during differentiation. Of the three telomerase components, only hTERT mRNA expression showed changes paralleling telomerase activity, becoming undetectable with differentiation. In contrast, initially low expression of TRF1 and TRF2 mRNAs increased during differentiation. Not only hTERT, but also TRF1 and TRF2 are important regulators of telomerase activity that represent potential targets for gene therapy against cancer.

Telomerase-Associated Protein TEP1 Is Not Essential for Telomerase Activity or Telomere Length Maintenance In Vivo

TEP1 is a mammalian telomerase-associated protein with similarity to the Tetrahymena telomerase protein p80. Like p80, TEP1 is associated with telomerase activity and the telomerase reverse transcriptase, and it specifically interacts with the telomerase RNA. To determine the role of mTep1 in telomerase function in vivo, we generated mouse embryonic stem (ES) cells and mice lacking mTep1. The mTep1-deficient (mTep1(-/-)) mice were viable and were bred for seven successive generations with no obvious phenotypic abnormalities. All murine tissues from mTep1(-/-) mice possessed a level of telomerase activity comparable to that in wild-type mice. In addition, analysis of several tissues that normally lack telomerase activity revealed no reactivation of telomerase activity in mTep1(-/-) mice. Telomere length, even in later generations of mTep1(-/-) mice, was equivalent to that in wild-type animals. ES cells deficient in mTep1 also showed no detectable alteration in telomerase activity or telomere length with increased passage in culture. Thus, mTep1 appears to be completely dispensable for telomerase function in vivo. Recently, TEP1 has been identified within a second ribonucleoprotein (RNP) complex, the vault particle. TEP1 can also specifically bind to a small RNA, vRNA, which is associated with the vault particle and is unrelated in sequence to mammalian telomerase RNA. These results reveal that TEP1 is an RNA binding protein that is not restricted to the telomerase complex and that TEP1 plays a redundant role in the assembly or localization of the telomerase RNP in vivo.

Lung resistance-related protein/major vault protein and vaults in multidrug-resistant cancer.

Tumor cells that are insensitive to anticancer drugs frequently have a multidrug-resistant (MDR) phenotype. Proteins that can be involved in this phenomenon are transport-associated proteins such as P-glycoprotein, multidrug-resistance protein 1, breast cancer resistance protein, and lung resistance-related protein (LRP). LRP was identified as the major vault protein (MVP), the main component of multimeric vault particles. With the recent identification of the two minor vault proteins as telomerase-associated protein (TEP1) and vault-poly (ADP-ribose) polymerase (VPARP), and with high-resolution three-dimensional imaging, the composition of vaults is almost unraveled. Although the first direct evidence for a causal relationship between LRP/MVP expression and drug resistance has been obtained, many functional aspects of vaults in normal physiology and in MDR still need to be clarified. The current clinical data on LRP/MVP detection indicate that LRP/MVP expression can be of high clinical value to predict the response to chemotherapy of several tumor types.

Telomerase activity in primary and secondary glioblastomas multiforme as a novel molecular tumor marker.

Telomerase activity is responsible for cell immortality. To examine the role of telomerase in the carcinogenesis of human glioblastomas multiforme (GBMs), the authors studied telomerase activity, telomerase component expression, and telomere lengths in 42 GBM samples. In all samples, EGFR and MDM2 amplifications and overexpressions were examined using Southern and Northern blot analyses. The p53 mutation was analyzed using polymerase chain reaction-single strand conformational polymorphism and by direct sequence analysis. Specimens of tissues were immunostained with p53, EGFR, and MDM2 antibodies. Allelic loss on chromosomes 17p and 10 was assessed by loss of heterozygosity (LOH) assays. Telomerase activity, expression of its components (human telomerase reverse transcriptase [hTERT], human telomerase RNA component [hTERC], and telomerase-associated protein [TEP1]), and telomere lengths were analyzed using the telomeric repeat amplification protocol (TRAP)-hybridization protection assay, reverse transcription-polymerase chain reaction, and Southern blot analysis. According to the results of assessments of EGFR and MDM2 amplifications, p53 mutation, LOHs in chromosomes 17p and 10, and the clinical course of the disease, the 42 samples were classified into 22 primary and 20 secondary glioblastomas. Twenty-six (61.9%) of all 42 samples demonstrated detectable telomerase activity during the TRAP assay. Secondary GBMs displayed significantly higher levels of telomerase activity and hTERT expression than primary GBMs. Tumors with a p53 gene mutation demonstrated significantly higher telomerase activity than those without a p53 mutation. Four samples with a codon 175 mutation demonstrated an exceptionally high amount of telomerase activity. In secondary GBMs, the increase in telomerase activity and the hTERT expression level correlated with the increased frequency of p53 mutations. There was no significant difference in telomere length between primary and secondary GBMs. These results suggest that telomerase activity and p53 mutations both play important roles in the multistep carcinogenesis of GBMs. Telomerase activity and hTERT expression may be considered as novel distinctive factors in human GBMs.

Polymerization defects within human telomerase are distinct from telomerase RNA and TEP1 binding.

The minimal, active core of human telomerase is postulated to contain two components, the telomerase RNA hTER and the telomerase reverse transcriptase hTERT. The reconstitution of human telomerase activity in vitro has facilitated the identification of sequences within the telomerase RNA and the RT motifs of hTERT that are essential for telomerase activity. However, the precise role of residues outside the RT domain of hTERT is unknown. Here we have delineated several regions within hTERT that are important for telomerase catalysis, primer use, and interaction with the telomerase RNA and the telomerase-associated protein TEP1. In particular, certain deletions of the amino and carboxy terminus of hTERT that retained an interaction with telomerase RNA and TEP1 were nonetheless completely inactive in vitro and in vivo. Furthermore, hTERT truncations lacking the amino terminus that were competent to bind the telomerase RNA were severely compromised for the ability to elongate telomeric and nontelomeric primers. These results suggest that the interaction of telomerase RNA with hTERT can be functionally uncoupled from polymerization, and that there are regions outside the RT domain of hTERT that are critical for telomerase activity and primer use. These results establish that the human telomerase RT possesses unique polymerization determinants that distinguish it from other RTs.

Expression of human telomerase subunit genes in primary lung cancer and its clinical significance

Background. Three major components of human telomerase, RNA component (hTERC), telomerase-associated protein (TEP1), and catalytic subunit (hTERT) have been cloned recently. The aim of this study was to examine the expression of these genes and to search for clinical usefulness. Methods. Expression of these genes was evaluated by reverse transcription-polymerase chain reaction in 92 human lung cancers and in 32 non-neoplastic lung tissues. In 15 patients, both telomerase activity by telomeric repeat amplification protocol assay and expression were evaluated. Results. hTERT expression was best associated with telomerase activity with a concordance of 77%. In 92 lung cancer tissues, hTERC, TEP1, and hTERT were expressed in 100%, 93%, and 89%, respectively. Whereas most adjacent non-neoplastic lung tissues expressed hTERC and TEP1 (94% and 100%, respectively), hTERT was detected in only 1 of 32 normal lungs. However, there was no relationship between hTERT expression and clinicopathologic features. Conclusions. hTERT expression can be a surrogate for telomerase activity that may serve as a novel biomarker of lung cancer with high specificity and sensitivity.

The Saccharomyces telomere-binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase α and the telomerase-associated Est1 protein

Saccharomyces telomeres consist of approximately 350 bp of C(1-3)A/TG(1-3) DNA. Most of this approximately 350 bp is replicated by standard, semiconservative DNA replication. After conventional replication, the C(1-3)A strand is degraded to generate a long single strand TG(1-3) tail that can serve as a substrate for telomerase. Cdc13p is a single strand TG(1-3) DNA-binding protein that localizes to telomeres in vivo. Genetic data suggest that the Cdc13p has multiple roles in telomere replication. We used two hybrid analysis to demonstrate that Cdc13p interacted with both the catalytic subunit of DNA polymerase alpha, Pol1p, and the telomerase RNA-associated protein, Est1p. The association of these proteins was confirmed by biochemical analysis using full-length or nearly full-length proteins. Point mutations in either CDC13 or POL1 that reduced the Cdc13p-Pol1p interaction resulted in telomerase mediated telomere lengthening. Over-expression of the carboxyl terminus of Est1p partially suppressed the temperature sensitive lethality of a cdc13-1 strain. We propose that Cdc13p's interaction with Est1p promotes TG(1-3) strand lengthening by telomerase and its interaction with Pol1p promotes C(1-3)A strand resynthesis by DNA polymerase alpha.

Ectopic expression of c-myc fails to overcome downregulation of telomerase activity induced by herbimycin A, but ectopic hTERT expression overcomes it.

Telomerase plays a key role in the maintenance of chromosomal stability in tumors, but the mechanism regulating telomerase activity is still unclear. Recent studies have suggested that c-myc may be vital for regulation of hTERT mRNA expression and telomerase activity. In this study, we investigated the changes of telomerase activity and telomerase-related genes induced by herbimycin A in K562 human chronic myelogeous leukemic cells. Telomerase activity showed a biphasic pattern in herbimycin A-treated K562 cells. Initially, the telomerase activity decreased along with the decline of cells in S and G2/M phases, but it recovered slightly at the end of treatment. Expression of mRNA for the telomerase catalytic subunit (hTERT) was decreased before the decline of telomerase activity, and increased slightly before the reactivation of telomerase activity. During herbimycin A treatment, both c-myc and cyclin D1 mRNA showed transient downregulation before the increase of G1 cells. Herbimycin A treatment caused the downregulation of both telomerase activity and hTERT mRNA in cyclin D1-transfected K562 cells, while telomerase activity was partially restored in c-Myc-transfected cells. In contrast, hTERT-transfected K562 cells maintained a high level of telomerase activity during herbimycin A treatment. Neither the template RNA component of telomerase (hTERC) nor telomerase-associated protein (TEP-1) were altered in any of the transfected K562 cells. These results indicate that telomerase activity is mainly regulated by hTERT, and that c-Myc protein is one of the positive regulators of hTERT in leukemic cells but is not enough to counteract the downregulation of telomerase activity by herbimycin A completely.

Identification of Two RNA-binding Proteins Associated with Human Telomerase RNA

Telomerase plays a crucial role in telomere maintenance in vivo. To understand telomerase regulation, we have been characterizing components of the enzyme. To date several components of the mammalian telomerase holoenzyme have been identified: the essential RNA component (human telomerase RNA [hTR]), the catalytic subunit human telomerase reverse transcriptase (hTERT), and telomerase-associated protein 1. Here we describe the identification of two new proteins that interact with hTR: hStau and L22. Antisera against both proteins immunoprecipitated hTR, hTERT, and telomerase activity from cell extracts, suggesting that the proteins are associated with telomerase. Both proteins localized to the nucleolus and cytoplasm. Although these proteins are associated with telomerase, we found no evidence of their association with each other or with telomerase-associated protein 1. Both hStau and L22 are more abundant than TERT. This, together with their localization, suggests that they may be associated with other ribonucleoprotein complexes in cells. We propose that these two hTR-associated proteins may play a role in hTR processing, telomerase assembly, or localization in vivo.