Telomerase Activity Expression Research Articles

Alternative pathways for the extension of cellular life span: inactivation of p53/pRb and expression of telomerase.

Telomere shortening may be one of several factors that contribute to the onset of senescence in human cells. The p53 and pRb pathways are involved in the regulation of cell cycle progression from G1 into S phase and inactivation of these pathways leads to extension of life span. Short dysfunctional telomeres may be perceived as damaged DNA and may activate these pathways, leading to prolonged arrest in G1, typical of cells in senescence. Inactivation of the p53 and pRb pathways, however, does not lead to cell immortalization. Cells that overcome senescence and have an extended life span continue to lose telomeric DNA and subsequently enter a second phase of growth arrest termed 'crisis'. Forced expression of telomerase in human cells leads to the elongation of telomeres and immortalization. The development of human cancer is frequently associated with the inactivation of the pRb and p53 pathways, attesting to the importance of senescence in restricting the tumor-forming ability of human cells. Cancer cells must also maintain telomere length and, in the majority of cases, this is associated with expression of telomerase activity.

Analysis of Telomerase Activity in Ovarian Cystadenomas, Low-Malignant-Potential Tumors, and Invasive Carcinomas

Objective. Inappropriate telomerase expression has been reported to be associated with the development and/or progression of malignancies. Therefore, the purpose of the study was to determine and evaluate the levels of telomerase activity in normal ovary, cystadenomas, low-malignant-potential tumors, and carcinomas of the ovary.Methods. In the present study, telomerase activity was examined in frozen tissue specimens of normal ovary (n = 6), ovarian cystadenomas (n = 13), ovarian low-malignant-potential (LMP) tumors (n = 12), and ovarian invasive carcinomas (n = 81). Clinicopathological information including age at diagnosis, histological grade, FIGO stage, presence of distant metastasis at diagnosis, and residual disease was available for all patients with ovarian carcinomas (n = 81). Telomerase activity was assessed by the telomeric repeat amplification protocol (TRAP). Arbitrary values were assigned to processivity of telomerase activities based on the molecular weights of the telomeric repeat DNA ladders, and were graded as “negative,” “moderate” (≤99 bp), or “high” (>100 bp) activities. The specificity of telomerase activity was determined by the pretreatment of telomerase-positive control or tumor samples with RNase that led to the abolition of the activity. In addition, to determine the possibility of false negativity due to the presence of telomerase inhibitors, TRAP assay was performed on each of the telomerase-negative specimens by mixing them individually with the telomerase-positive control.Results. Telomerase activity in the progression of ovarian carcinogenesis was evaluated. In comparison with normal ovary/cystadenoma (32%), a much higher frequency of the moderate activity was observed in LMP tumors (67%) or invasive carcinomas (57%), suggesting a close association between the latter two categories. The results reflect a subpopulation of telomerase-positive LMP tumor cells with the potential to develop invasive carcinomas. None of the specimens of the benign or LMP tumors exhibited high activity. In contrast, 18% of ovarian invasive carcinomas showed high telomerase activity (P = 0.013, Fisher exact test) and further 57%, moderate activity (75% in all). A statistically significant difference was observed in the expression of telomerase activity between normal ovary/benign cystadenomas and ovarian invasive carcinomas (P = 0.001, χ2 test).Conclusions. The study showed a high prevalence of telomerase activity in LMP tumors or invasive carcinomas, the high levels of telomerase activity being associated exclusively with the invasive ovarian carcinomas. Therefore, the levels of processivity of telomerase activity and evidence of its statistically significant association with ovarian carcinoma suggest its role in the progression of ovarian carcinogenesis.

Telomerase-independent senescence of human immortal cells induced by microcell-mediated chromosome transfer.

Maintenance of telomeres, commonly through expression of telomerase activity, is necessary but may not be sufficient for human cells to escape from the cellular senescence program and become immortal. We report here that human tumor cells could undergo cellular senescence in the presence of telomerase activity when a specific normal human chromosome was introduced via microcell-mediated chromosome transfer. The cell models studied include SiHa (uterine cervical carcinoma cells expressing E6 and E7 oncoproteins of human papillomavirus type 16) with a transferred chromosome 2, CC1 (choriocarcinoma cells expressing an amino-terminally truncated p53 protein) with a transferred chromosome 7, and JTC-32 (bladder carcinoma cells) with a transferred chromosome 11. The microcell hybrids with the indicated chromosomes ceased to divide after five to 10 population doublings and showed senescence-associated beta-galactosidase activity but still expressed the genes encoding three components of human telomerase, consistent with the retention of telomerase activity. These results are evidence for barriers to human cell immortalization, which involve activation of unidentified senescence-inducing genes that function independently of inactivation of telomerase.

Progressive Increase in Telomerase Activity From Benign Melanocytic Conditions to Malignant Melanoma

The expression of telomerase activity and the in situ localization of the human telomerase RNA component (hTR) in melanocytic skin lesions was evaluated in specimens from sixty-three patients. Specimens of melanocytic nevi, primary melanomas and subcutaneous metastases of melanoma were obtained from fifty-eight patients, whereas metastasized lymph nodes were obtained from five patients. Telomerase activity was determined in these specimens by using a Polymerase Chain Reaction—based assay (TRAP). High relative mean telomerase activity levels were detected in metastatic melanoma (subcutaneous metastasess = 54.5, lymph node metastasess = 56.5). Much lower levels were detected in primary melanomas, which increased with advancing levels of tumor cell penetration (Clark II = 0.02, Clark III = 1.1, and Clark IV = 1.9). Twenty-six formalin-fixed, paraffin-embedded melanocytic lesions were sectioned and analyzed for telomerase RNA with a radioactive in situ hybridization assay. In situ hybridization studies with a probe to the template RNA component of telomerase confirmed that expression was almost exclusively confined to tumor cells and not infiltrating lymphocytes. These results indicate that levels of telomerase activity and telomerase RNA in melanocytic lesions correlate well with clinical stage and could potentially assist in the diagnosis of borderline lesions.

Tuberculin purified protein derivative up-regulates the telomerase activity of peripheral blood mononuclear cells from patients with pulmonary tuberculosis

Telomerase activity was detectable in cells of tuberculous pleural effusions at high percentage. To investigate the possible role of telomerase in the immune function, we examined the proliferating state and the expression of telomerase activity in peripheral blood mononuclear cells (PBMC) from 13 patients with pulmonary tuberculosis (TB) and 13 healthy volunteers in response to tuberculin purified protein derivative (PPD) challenge. Exposure of cells to phytohemagglutinin (PHA) significantly promoted PBMC proliferation during a 6 day-period in both TB patient and healthy volunteer groups. PPD treatment also significantly promoted PBMC proliferation during a 6 day-period in TB patient group, but had no significant effect in healthy volunteer group. During the same period, telomerase activity was detected in every PHA- and PPD-treated samples of the TB patient group. However, the telomerase activity was not detected in PPD-treated samples from healthy donors and all the untreated samples. Our results indicate that the telomerase activity in PBMC could be induced by PPD stimulation in TB patients. Telomerase activity may thus play a permissive role in cell division and clonal expansion of the immune cells in response to TB.

Correlation of clinical features and telomerase activity in human gliomas.

Telomerase is a ribonucleoprotein containing an RNA template that synthesizes telomeric DNA. The expression of telomerase activity is concomitant with the attainment of immortality in tumor tissues and cells. In this report, we analyzed telomerase activity in 39 human gliomas with different histological, and in 10 meningiomas, 3 neurinomas, and 2 normal brain tissues by using a polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay. Telomerase activity was detectable in almost all of the gliomas (36 of 39), but not in any of the meningiomas, neurinomas, or normal brain tissues. In addition, we also analyzed the level of telomerase activity in the 36 gliomas with positive telomerase activity. The relative telomerase activity of the glioma showed a clear association with the pathological grade of glioma; i.e., most of the tumors with high telomerase activity were pathologically of high grade. And also the relative level of telomerase activity could be correlated with the survival time of the patients. These results suggest that the level of telomerase activity in brain tumors is a diagnostic marker indicating the prognosis of the patient as well as the malignant potential of the tumor.

Expression of Telomerase Activity in Human Endometrium Is Localized to Epithelial Glandular Cells and Regulated in a Menstrual Phase-Dependent Manner Correlated with Cell Proliferation

Telomerase activity is observed in most malignant tumors and germ cells, whereas normal somatic cells usually do not express it. Human endometrium is composed of glandular and stromal components and exhibits dramatic changes in proliferative activity during the menstrual cycle, which is exquisitely regulated by estrogen function. We previously reported that normal human endometrium expresses telomerase activity. However, it remains unclear which of the above components are the major sources of telomerase activity and how levels of telomerase activity are regulated over the menstrual cycle. Quantitative analysis of telomerase activity revealed that it changes dramatically over the course of the menstrual cycle and is strictly regulated in a menstrual-phase-dependent manner. Maximal activity equivalent to that in endometrial cancer was present in late proliferative phase, and minimal activity in late secretory phase. Postmenopausal endometrium and endometrium treated with anti-estrogen drugs exhibited decreased telomerase activity. Testing isolated epithelial glandular cells and stromal cells, we found that telomerase activity was localized to epithelial glandular cells. In situ RNA hybridization analysis also revealed epithelial-specific expression of human telomerase RNA. In vitro analysis of cultured epithelial cells demonstrated that telomerase activity is correlated with epithelial proliferation but not affected by estrogen treatment. These findings suggest that expression of telomerase activity is specific to epithelial cells and linked to cell proliferative status. The involvement of estrogen in telomerase regulation remains to be elucidated.

Elevated Levels of Telomerase Activity in Malignant Pheochromocytoma

BACKGROUND Although malignant pheochromocytoma is a life-threatening illness, there is no excretory profile that is predictive of malignancy. Reliable prediction of malignant behavior on the basis of histopathology is also notoriously difficult. Although DNA ploidy can be a helpful indicator, aneuploidy per se is not considered to be a specific marker of malignancy. Mutations in p53 have been reported to occur frequently in cases of benign pheochromocytoma. Thus, molecular analysis of this protein is not a useful diagnostic tool. The authors analyzed telomerase activity in benign and malignant pheochromocytomas and assessed its utility as a prognostic marker. METHODS Telomerase activity was estimated in 16 benign pheochromocytomas, 3 malignant pheochromocytomas, and 16 normal adrenal medullae by the fluorescence-based telomeric repeat amplification protocol. The telomerase activities of each group of samples were compared. RESULTS No telomerase activity was detected in any of the normal adrenal medullae or the benign pheochromocytomas. By contrast, all of the three malignant pheochromocytomas had elevated telomerase activity. The telomerase activity in each tumor was determined to be 87.6 units, 71.2 units, and 180.3 units, respectively. CONCLUSIONS The expression of telomerase activity in pheochromocytoma clearly suggests the malignant behavior of the component cells. Analysis of telomerase activity appears to be a powerful tool for the diagnosis of malignant pheochromocytoma. Cancer 1998;82:176-9. © 1998 American Cancer Society.

Telomerase activity as an indicator of potentially malignant adrenal tumors

Telomerase is an enzyme that adds repeated telomere sequences to the ends of chromosome arms. It helps maintain both the length of telomere and infinite cell proliferation. In recent years, telomerase activity has been considered an important characteristic that differentiates between normal and cancerous cells. Because the authors often encountered difficulties in distinguishing between benign and malignant adrenal tumors, they investigated whether the expression of telomerase activity could distinguish potentially malignant adrenal tumors. The authors examined telomerase activity in 48 samples of adrenal tumor tissue and 27 samples of adjacent normal adrenal tissue. All samples were obtained from 48 patients who underwent surgery at Hamamatsu University Hospital in Hamamatsu, Japan. Based on the clinical and postoperative pathologic examinations, 45 samples were diagnosed as benign and 3 were diagnosed as malignant. Telomerase activity was examined using a telomerase repeat amplification protocol (TRAP) assay. Of the 48 adrenal tumor samples, 7 (14.6%) had telomerase activity. All adjacent normal adrenal tissues were negative for telomerase activity. Of the telomerase positive samples, two were clinically known adrenocortical carcinoma, and another was metastatic adrenal tumor from lung carcinoma. Four other telomerase positive samples were diagnosed as benign after clinical and initial pathologic examinations. However, two of the patients from whom these samples were taken developed metastatic lesions after adrenalectomy. A telomerase assay of adrenal tumors may help predict their malignant potential.

Telomerase activity as an indicator of potentially malignant adrenal tumors

BACKGROUND Telomerase is an enzyme that adds repeated telomere sequences to the ends of chromosome arms. It helps maintain both the length of telomere and infinite cell proliferation. In recent years, telomerase activity has been considered an important characteristic that differentiates between normal and cancerous cells. Because the authors often encountered difficulties in distinguishing between benign and malignant adrenal tumors, they investigated whether the expression of telomerase activity could distinguish potentially malignant adrenal tumors. METHODS The authors examined telomerase activity in 48 samples of adrenal tumor tissue and 27 samples of adjacent normal adrenal tissue. All samples were obtained from 48 patients who underwent surgery at Hamamatsu University Hospital in Hamamatsu, Japan. Based on the clinical and postoperative pathologic examinations, 45 samples were diagnosed as benign and 3 were diagnosed as malignant. Telomerase activity was examined using a telomerase repeat amplification protocol (TRAP) assay. RESULTS Of the 48 adrenal tumor samples, 7 (14.6%) had telomerase activity. All adjacent normal adrenal tissues were negative for telomerase activity. Of the telomerase positive samples, two were clinically known adrenocortical carcinoma, and another was metastatic adrenal tumor from lung carcinoma. Four other telomerase positive samples were diagnosed as benign after clinical and initial pathologic examinations. However, two of the patients from whom these samples were taken developed metastatic lesions after adrenalectomy. CONCLUSIONS A telomerase assay of adrenal tumors may help predict their malignant potential. Cancer 1998;83:772-776. © 1998 American Cancer Society.

Telomerase activity in lung cancer cell lines and tissues

Using TRAP assay, we studied the activity of the telomerase in the lung cancer cell lines, and lung cancer and normal tissues in which expression appears to be related to the immortality of cancer cells. All the human lung cancer cell lines and the majority of human lung cancer tissues (78%) expressed telomerase activity, but this was undetectable in normal human lung tissues. Positivity for telomerase activity in lung cancer cell lines was higher than in lung cancer tissues; this result implies the expression of telomerase activity may play a crucial role in the development or progression of lung cancer, and also suggests that improved method of detection may lead to the higher positivity for telomerase activity in primary lung cancer tissues. To determine whether there is a definite causal relationship between telomerase and cancer, and to develop new anti-cancer agents which inhibit telomerase, further study is needed.

Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase.

The expression of telomerase, the enzyme that synthesizes telomeric DNA de novo, is suppressed in normal somatic human cells but is reactivated during tumorigenesis. This reactivation appears to arrest the normal loss of telomeric DNA incurred as human cells divide. Since continual loss of telomeric DNA is predicted to eventually limit cell proliferation, activation of telomerase in cancer cells may represent an important step in the acquisition of the cell immortalization which occurs during tumor progression. The telomerase holoenzyme is composed of both RNA and protein subunits. In humans, mRNA expression of hTERT (hEST2), the candidate telomerase catalytic subunit gene, appears to parallel the levels of telomerase enzyme activity, suggesting that induction of hTERT is necessary and perhaps sufficient for expression of telomerase activity in tumor cells. To test this model directly, we ectopically expressed an epitope-tagged version of hTERT in telomerase-negative cells and show that telomerase activity was induced to levels comparable to those seen in immortal telomerase-positive cells and that the expressed hTERT protein was physically associated with the cellular telomerase activity. We conclude that synthesis of the hTERT telomerase subunit represents the rate-limiting determinant of telomerase activity in these cells and that this protein, once expressed, becomes part of the functional telomerase holoenzyme.

Elevated levels of telomerase activity in malignant pheochromocytoma

Although malignant pheochromocytoma is a life-threatening illness, there is no excretory profile that is predictive of malignancy. Reliable prediction of malignant behavior on the basis of histopathology is also notoriously difficult. Although DNA ploidy can be a helpful indicator, aneuploidy per se is not considered to be a specific marker of malignancy. Mutations in p53 have been reported to occur frequently in cases of benign pheochromocytoma. Thus, molecular analysis of this protein is not a useful diagnostic tool. The authors analyzed telomerase activity in benign and malignant pheochromocytomas and assessed its utility as a prognostic marker. Telomerase activity was estimated in 16 benign pheochromocytomas, 3 malignant pheochromocytomas, and 16 normal adrenal medullae by the fluorescence-based telomeric repeat amplification protocol. The telomerase activities of each group of samples were compared. No telomerase activity was detected in any of the normal adrenal medullae or the benign pheochromocytomas. By contrast, all of the three malignant pheochromocytomas had elevated telomerase activity. The telomerase activity in each tumor was determined to be 87.6 units, 71.2 units, and 180.3 units, respectively. The expression of telomerase activity in pheochromocytoma clearly suggests the malignant behavior of the component cells. Analysis of telomerase activity appears to be a powerful tool for the diagnosis of malignant pheochromocytoma.

Expression of Telomerase Activity in Human Chorion

Telomerase activation is required for cellular immortalization and is found in most malignant tumors. Normal somatic cells are generally telomerase-negative, except for stem cells in renewing tissues. During pregnancy, human trophoblast continues to proliferate and acts as proliferating stem cells for the development of chorion and the formation of placenta. In the present study, a total of 105 chorions from placentas at various weeks of gestation were examined for telomerase activity using the telomeric repeat amplification protocol (TRAP) assay. Twenty-five of 33 (76%) normal early chorions at 5 to 9 weeks gestation were telomerase-positive. Chorions from early spontaneous abortions also exhibited telomerase activity but at a low level. In contrast, only 2 (4%) late chorions at 34 to 41 weeks gestation expressed telomerase activity. Significant telomerase activity was observed in trophoblast cell fractions of chorion, demonstrating trophoblast to be the source of the activity. Expression of human telomerase catalytic subunit (hTRT) was observed in early chorions, but not in late placenta, and there was a close correlation between telomerase activity and hTRT expression. In contrast, expression of human telomerase RNA component (hTR) was observed in both early and late chorions and was not liked to telomerase activity. These findings suggest that telomerase activity in chorion is critically regulated over the course of gestation, associated with hTRT expression. The findings of the present study also appear to support the emerging concept that normal somatic cells with stem cell-like characteristics can express telomerase activity.

Gradual phenotypic conversion associated with immortalization of cultured human mammary epithelial cells.

Examination of the process of immortal transformation in early passages of two human mammary epithelial cell (HMEC) lines suggests the involvement of an epigenetic step. These lines, 184A1 and 184B5, arose after in vitro exposure of finite lifespan 184 HMEC to a chemical carcinogen, and both are clonally derived. Although early-passage mass cultures of 184A1 and 184B5 maintained continuous slow growth, most individual cells lost proliferative ability. Uniform good growth did not occur until 20-30 passages after the lines first appeared. Early-passage cultures expressed little or no telomerase activity and telomeres continued to shorten with increasing passage. Telomerase activity was first detected when the telomeres became critically short, and activity levels gradually increased thereafter. Early-passage cultures had little or no ability to maintain growth in transforming growth factor-beta (TGFbeta); however, both mass cultures and clonal isolates showed a very gradual increase in the number of cells displaying progressively increased ability to maintain growth in TGFbeta. A strong correlation between capacity to maintain growth in the presence of TGFbeta and expression of telomerase activity was observed. We have used the term "conversion" to describe this process of gradual acquisition of increased growth capacity in the absence or presence of TGFbeta and reactivation of telomerase. We speculate that the development of extremely short telomeres may result in gradual, epigenetic-based changes in gene expression. Understanding the underlying mechanisms of HMEC conversion in vitro may provide new insight into the process of carcinogenic progression in vivo and offer novel modes for therapeutic intervention.

Inhibition of Telomerase Activity by PKC Inhibitors in Human Nasopharyngeal Cancer Cells in Culture

Telomerase is a specialized ribonucleoprotein polymerase that adds hexanucleotides (TTAGGG) onto human chromosomal ends. The expression of telomerase activity has been associated with cell immortalization and the malignant phenotype in most cancers. How the telomerase activity is regulated in cancer cells is presently not known. In this work, the effects of cell cycle blockers, DNA damaging agents, TopII inhibitors and proteins kinase inhibitors on the telomerase activity were examined in cultured nasopharyngeal carcinoma cells NPC-076. Agents which interfere with tubulin assembly (Taxol and vinblastine) and agents which arrest cells at S phase (methotrexate and 5-fluorouracil) did not inhibit telomerase activity of treated cells. Agents which damage DNA (cisplatin, methyl methanesulfonate, and UV radiation) and TopII inhibitors (etoposide and daunorubicin) also did not inhibit telomerase activity of treated cells. Among the protein kinase inhibitors examined, no significant inhibition of telomerase activity was observed with cells treated with quercetin, H-89, or herbimycin A. On the other hand, two protein kinase C (PKC) inhibitors (bisindolylmaleimide I and H-7) were found to produce a big inhibition of telomerase activity in treated cells. Staurosporine produced a moderate inhibition, and sphingosine had a small inhibitory effect. The inhibition of telomerase activity by PKC inhibitors appears to be specific since the treated cells were mostly viable (i.e., greater than 75%) and still retained significant levels of protein synthesis capability. These results implicate that protein kinase C is involved in the regulation of telomerase activityin vivo.

Differential expression of telomerase activity in human cervical cancer and cervical intraepithelial neoplasia lesions.

Telomeres are tandem arrays of repeated DNA sequences located at the ends of eukaryotic chromosomes, and are synthesized by the enzyme telomerase. Loss of telomeric DNA may play an important role in the development of human cancers. However, very little is known about the status of telomerase during human cervical cancer development. Telomerase activity was measured by telomere repeat amplification protocol (TRAP) assay in 24 cervical cancers, one carcinoma in situ (CIS), and 20 cervical intraepithelial neoplasia (CIN) lesions. Adjacent nontumor cervical tissue from the same 24 cervical cancer patients and normal cervical tissues from 11 control individuals also were examined for the presence of telomerase activity. Twenty two of the 24 (91.7%) cervical cancer specimens and the single CIS tissue were strongly positive for telomerase activity. Relatively weak but distinctive telomerase activity also was detectable in one of four CIN-I (25%), two of eight CIN-II (25%), and two of eight CIN-III (25%), respectively. However, telomerase activity was not found in the 24 corresponding nontumor cervical tissues from the same cervical cancer patients and the 11 normal cervical tissues from control individuals. The majority of cervical cancers contain strong telomerase activity. Significant proportions of noncancerous CIN tissues also contain telomerase activity, although weaker than that in cervical cancer. It seems that there is a progressive increase of telomerase activity in association with an increased degree of cervical malignancy. These results seem to suggest that the expression of telomerase may play a crucial role in cervical cancer carcinogenesis.

Telomeres and telomerase in normal and malignant haematopoietic cells

The normal haematopoietic system harbours telomerase-competent cells with a capacity to upregulate the activity to notable levels in a telomere length-independent manner. Strong telomerase activity is found in progenitor stem cells and activated lymphocytes in vitro as well as in vivo, indicating that cells with high growth requirements can readily upregulate telomerase. Despite detection of telomerase activity, a gradual telomere erosion occurs in stem cells and lymphocytes, with significantly shortened telomeres at higher ages, a phenomenon that might be of importance for developing immunosenescence and exhausted haematopoiesis. In malignant haematopoietic disorders telomerase activity is a general finding with large differences in activity levels. The strongest telomerase expression has been shown in acute leukaemias and non-Hodgkin's lymphomas, especially high grade cases. There are indications that the level of activity might parallel tumour progression and be of prognostic relevance, but studies of larger patient materials are needed. An association between the cell cycle and telomerase activity exists, especially for normal haematopoietic cells, and induction of a differentiation programme in immortalised cell lines downregulates telomerase activity. The expression of telomerase activity seems to be regulated at different levels, since for immature bone marrow cells the level of activity seemed to parallel better the phenotype than the proliferation state. The frequent expression of telomerase in leukaemias and lymphomas makes these disorders interesting targets for future anti-telomerase therapy.

Regulated expression of telomerase activity in human T lymphocyte development and activation.

Telomerase, a ribonucleoprotein that is capable of synthesizing telomeric repeats, is expressed in germline and malignant cells, and is absent in most normal human somatic cells. The selective expression of telomerase has thus been proposed to be a basis for the immortality of the germline and of malignant cells. In the present study, telomerase activity was analyzed in normal human T lymphocytes. It was found that telomerase is expressed at a high level in thymocyte subpopulations, at an intermediate level in tonsil T lymphocytes, and at a low to undetectable level in peripheral blood T lymphocytes. Moreover, telomerase activity is highly inducible in peripheral T lymphocytes by activation through CD3 with or without CD28 costimulation, or by stimulation with phorbol myristate acetate (PMA)/ionomycin. The induction of telomerase by anti-CD3 plus anti-CD28 (anti-CD3/CD28) stimulation required RNA and protein synthesis, and was blocked by herbimycin A, an inhibitor of S pi protein tyrosine kinases. The immunosuppressive drug cyclosporin A selectively inhibited telomerase induction by PMA/ionomycin and by anti-CD3, but not by anti-CD3/CD28. Although telomerase activity in peripheral T lymphocytes was activation dependent and correlated with cell proliferation, it was not cell cycle phase restricted. These results indicate that the expression of telomerase in normal human T lymphocytes is both developmentally regulated and activation induced. Telomerase may thus play a permissive role in T cell development and in determining the capacity of lymphoid cells for cell division and clonal expansion.

Differential expression of telomerase activity in hematopoietic progenitors from adult human bone marrow.

The loss of telomeric DNA may serve as a mitotic clock which signals cell senescence and exit from cell cycle. Telomerase, and enzyme which synthesizes telomeric repeats de novo, is required to maintain telomere lengths. In humans, significant telomerase activity has been found in cells with essentially unlimited replicative potential such as reproductive cells in ovaries and testes, immortal cell lines and cancer tissues, but not in most normal somatic cells or tissues. We have now examined telomerase expression in subpopulations of hematopoietic cells from adult human bone marrow using a sensitive polymerase chain reaction-based telomeric repeat amplification protocol. Telomerase activity was found at low levels in the highly enriched primitive hematopoietic cells (CD34+CD71loCD45RAlo) and was increased transiently when these cells were cultured in the presence of a mixture of cytokines. In contrast, the early progenitors (CD34+CD71+) expressed telomerase activity at a higher level which was subsequently downregulated in response to cytokines. Telomerase activity remained low in the more mature CD34-cells upon exposure to cytokines. Taken together, our results suggest that telomerase is expressed at a basal level in all hematopoietic cell populations examined, is induced in a primitive subset of hematopoietic progenitor cells and is downregulated upon further proliferation and differentiation of these cells. We have previously observed telomere shortening in cytokine-stimulated primitive hematopoietic cells. The low and transient activation of telomerase activity described here thus appears insufficient to maintain telomere lengths in cultured hematopoietic cells.