Telomerase expression in respiratory epithelium during the multistage pathogenesis of lung carcinomas.

To investigate the role of telomerase in the multistage pathogenesis of lung cancer, we examined 205 fresh and archival tissue samples obtained from 40 patients, 34 of whom had invasive lung carcinoma, 5 with carcinoma in situ (CIS) without invasion, and 1 without lung carcinoma. We analyzed samples for telomerase enzyme activity using the semiquantitative PCR-based telomeric repeat amplification protocol assay (131 samples) or by a radioactive in situ hybridization method for expression of the RNA component of human telomerase (hTR; 74 samples). A subset of samples was assayed by both methods, and the correlation was excellent (30 of 36; 83%). With the exception of a carcinoid tumor and a necrotic squamous cell carcinoma, all tumor cells were moderate to strongly positive for both hTR and telomerase activity, except for foci of keratinization in squamous cell carcinomas. Telomerase positivity, with weak enzyme activity and/or low hTR expression, was present in basal epithelial cells of large bronchi, both histologically normal (26%) and hyperplastic (71%), and in 23% of peripheral lung samples (in epithelium of small bronchi and bronchioles or lymphoid aggregates). More advanced epithelial changes (metaplasia, dysplasia, and CIS) were associated with telomerase dysregulation. Dysregulation in preneoplasia was manifested in three ways: almost all such lesions expressed hTR, although enzyme activity levels were several-fold lower than in the corresponding invasive tumors; cells throughout these multilayered processes expressed hTR; and intense, focal up-regulation of hTR occurred in CIS foci in the vicinity of invasive cancers. Alveolar cells and areas of atypical adenomatous hyperplasia (possible precursor lesions for peripheral adenocarcinomas) were negative. Our studies demonstrate that dysregulation of telomerase occurs early in the multistage pathogenesis of bronchogenic lung carcinomas and that intense focal localized hTR expression in CIS may indicate imminent invasion.

Telomerase activity in ordinary meningiomas predicts poor outcome.

Telomerase, the enzyme that stabilizes telomere length, is reactivated with almost all cancer types, and it may be necessary for unlimited cell proliferation. Assessment of malignancy in ordinary meningiomas is inconclusive because no clear-cut correlation exists between aggressive clinical behavior and histological features or karyotypic abnormalities. We analyzed telomerase activity in 52 cases of meningioma by using the highly sensitive telomeric repeat amplification protocol and then compared clinical behavior in telomerase-positive and -negative ordinary meningiomas. Twenty-six of the 52 tumors (50%) had detectable telomerase activity. Twenty-one of the 22 neoplasms classified as malignant or atypical (95%) had detectable telomerase activity, and these tumors generally had a poor outcome. Interestingly, 5 of 30 ordinary (morphologically benign) meningiomas (17%) also showed detectable telomerase activity. Of the 5 patients with telomerase-positive ordinary meningiomas, 3 had rapid regrowth of the tumor despite gross total resection. The remaining 2 patients also had other primary malignancies. We observed a highly significant correlation in ordinary meningiomas between the presence of telomerase activity and a poor prognosis for the patient (P = .0002). Telomerase activity in benign meningiomas is clinically relevant because the presence of the enzyme suggests that these benign-appearing tumors may contain a population of immortal cells. The detection of telomerase activity may help to identify benign meningiomas that would be more likely to continue to grow and to recur clinically if surgical resection were incomplete.

Telomerase activity and in situ telomerase RNA expression in malignant and non-malignant lymph nodes.

AIMS/BACKGROUND: Telomerase, an enzyme associated with cellular immortality, is expressed by most malignant tumours, but is inactive in normal somatic cells except for male germ cells and proliferating stem cells. Thus, the measurement of telomerase activity in tissue samples may provide useful diagnostic and prognostic information. The aim of this study was to determine whether telomerase expression is useful for the detection of occult malignant cells in lymph nodes. METHODS: Telomerase activity was compared with histological findings in 123 surgically removed lymph nodes submitted for routine or frozen section diagnosis. Telomerase activity was measured using a modified, semi-quantitative PCR-based telomeric repeat amplification protocol (TRAP). The assay was adapted for single 5 microns OCT embedded cryostat sections. In either fresh tissues or cryostat sections, normalised activity was linear when compared with protein concentration. Furthermore, using an in situ hybridisation method, the human telomerase RNA (hTR) component was measured in a subset of negative and positive nodes. RESULTS: Most (96%) of the 97 histologically negative nodes expressed low levels of activity (mean value of positive samples = 3.0 units/microgram protein) which may be derived from activated lymphocytes that express telomerase activity. All 26 malignant nodes (17 metastases, nine lymphomas) expressed telomerase (mean value = 17.8 units/microgram protein). The rank order levels between the two groups differed significantly (p = 0.0002). In situ results showed clearly that the hTR was expressed relatively highly in metastatic cancer cells and at lower levels in germinal centres of secondary follicles. CONCLUSIONS: Although expression of telomerase by activated lymphocytes may limit its usefulness, measurement of enzyme activity combined with detection of hTR using in situ hybridisation may assist in the histopathological diagnosis of lymph nodes.

Chromosome end-to-end associations and telomerase activity during cancer progression in human cells after treatment with alpha-particles simulating radon progeny.

Chromosome end-to-end associations seen at metaphase involve telomeres and are commonly observed in cells derived from individuals with ataxia telangiectasia and most types of human tumors. The associations may arise because of short telomeres and/or alterations of chromatin structure. There is a growing consensus that telomere length is stabilized by the activity of telomerase in immortal cells; however, it is not clear why some immortal cells display chromosome end-to-end associations. In the present study we evaluated chromosome end-to-end associations, telomere length and telomerase activity with the tumorigenic status of human bronchial epithelial cells immortalized with human papillomavirus. Oncogenic transformation was initiated using radon simulated alpha-particles and cells evaluated as primary, secondary and metastatic transformants. The fewest chromosome end associations and lowest telomerase activity were observed in the parental immortalized cells. However, increased levels of telomerase activity were detected in alpha-particle survivors while robust telomerase activity was seen in the tumorigenic cell lines. The tumorigenic cells that were telomerase positive and had the highest frequency of cells with chromosome end-to-end associations were also metastatic. No correlation was found between telomere length and the different stages of carcinogenicity.

Telomerase activity in plant extracts.

Telomeres of most eucaryotes terminate in long stretches of short, guanine-rich repeats. Telomerase, a specialized enzyme with reverse transcriptase-like activity, has been shown to synthesize these repeats in many lower eucaryotes and several animal species. Although a sequence (TTTAGGG)n that matches the eucaryotic consensus sequence Tx(A)Gy is present in several plant species, the activity and expression patterns of plant telomerase have not been reported. Here we document the presence of telomerase activity in plant tissues using a modification of the human Telomeric Repeat Amplification Protocol (TRAP) assay. Telomerase activity was detected in barley embryo, anther and carpel tissues and in immature seeds of Arabidopsis thaliana, but not in barley leaf tissue.

Telomerase activity in human renal cell carcinoma.

Telomeres have a vital role in maintaining chromosome stability and are essential for long term viability. Since the very ends of linear chromosomes cannot replicate, telomeres shorten in normal somatic cells eventually resulting in growth inhibition. However, most immortal cell lines maintain stable telomeres indicating that mechanisms exist to compensate for the end replication problem. Telomerase activity, leading to synthesis of telomeric DNA repeats, has been proposed to be an important step in the immortalization process of tumor cells. In the present study, 56 renal cell carcinomas were tested for telomerase activity using the sensitive TRAP (telomeric repeat amplification protocol). Forty of the analysed tumors (71%) were positive for telomerase activity, whereas none of the 56 corresponding normal kidney samples showed telomerase activity. All telomerase negative tumors had a reduction in mean telomere restriction fragment (TRF) length and a decrease in total telomere repeat hybridization signal, though cases were observed with an increase in peak TRF lengths. No obvious association between the presence of telomerase activity and clinicopathological parameters (histopathologic grade, DNA-ploidy, stage and clinical outcome) was found. The high frequency of detection of telomerase activity in the renal cell carcinomas indicates that this enzyme is likely to be an important factor involved in the evolution of this tumor type.

Inhibition of human telomerase activity by peptide nucleic acids.

We report the inhibition of human telomerase activity by peptide nucleic acids (PNAs). PNAs recognize the RNA component of human telomerase (hTR) and inhibit activity of the enzyme with IC50 values in the picomolar to nanomolar range. Inhibition depends on targeting exact functional boundaries of the hTR template and is 10- to 50-fold more efficient than inhibition by analogous phosphorothioate (PS) oligomers. In contrast to high selectivity of inhibition by PNAs, PS oligomers inhibit telomerase in a non-sequence-selective fashion. These results demonstrate that PNAs can control the enzymatic activity of ribonucleoproteins and possess important advantages relative to PS oligomers in both the affinity and the specificity of their recognition. These observations should facilitate the development of effective inhibitors of telomerase activity and affinity probes of telomerase structure.

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.

Experimental elongation of telomeres extends the lifespan of immortal x normal cell hybrids.

Hybrids between immortal cells that express telomerase and normal cells that lack telomerase have a limited lifespan. We demonstrate that telomerase is repressed in such hybrids. Treatment of immortal human cell lines with certain oligonucleotides resulted in telomere elongation. We took advantage of this observation to test the hypothesis that elongation of telomeres would extend the lifespan of cells in culture. An immortal human cell line was treated with an oligonucleotide to lengthen its telomeres and then was fused with mortal cells. The lifespan of these hybrid cells was longer than that of the hybrids in which telomeres had not been elongated. These observations provide the first direct evidence supporting the hypothesis that telomere length determines proliferative capacity of human cells.

Detection of telomerase activity in malignant and nonmalignant skin conditions.

Telomeres are the end regions of linear chromosomes, and in normal somatic cells the lengths of telomeres shorten with successive cell divisions. Telomerase, a ribonucleoprotein enzyme, maintains the length of telomeres in immortal and germline cells. Although present in human fetal tissues, shortly after birth telomerase activity is not detectable except in germline cells, hematopoietic cells, and most human primary tumors. In the present study we show telomerase activity to be present in 73 of 77 basal cell carcinomas, 15 of 18 nonmetastatic cutaneous squamous cell carcinomas, and 6 of 7 cutaneous melanomas, contrasting with extremely low levels detected in sun-protected skin. Sun-damaged skin, psoriatic lesional skin, and skin from lesions of poison ivy dermatitis, however, have increased levels of telomerase activity compared to sun-protected skin, although less than that detected in tumor tissue. Because telomerase activity can be found in inflammatory lesions of the skin, this indicates that telomerase activity does not always correlate with the malignant phenotype. In addition, we show that telomerase activity is localized to the epidermis of newborn foreskin, which suggests that telomerase is expressed constitutively by cells in the epidermis. Finding higher levels of telomerase activity in sun-exposed skin compared to nonexposed skin suggests that environmental factors may modulate telomerase activity.

Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues.

Telomerase activity has been detected in many human immortal cells lines and in tumor tissues, whereas it is generally absent from primary cell strains and from many tumor adjacent tissue samples. With the recently cloned human telomerase RNA (hTR), we used Northern analysis to follow the levels of hTR in primary, precrisis, and immortalized cells. It was surprising that the amount of hTR was high in cell strains that lacked telomerase activity, and the levels did not parallel the increases in telomerase activity, which accompanies immortalization. In addition, although the hTR levels were somewhat higher in tumor samples compared to nontumor tissues, the level of hTR in a variety of different human tumors did not predict the level of telomerase activity in the tumor. Thus, whereas hTR was detected in all samples that have telomerase activity, the presence of the RNA was not a good predictor of the presence or amount of telomerase activity.

Telomerase activity in human breast tumors.

BACKGROUND: The activity of the ribonucleoprotein enzyme telomerase is not detected in normal somatic cells; thus, with each cell division, the ends of chromosomes consisting of the telomeric repeats TTAGGG progressively erode. The current model gaining support is that telomerase activity in germline and immortal cells maintains telomere length and thus compensates for the "end-replication problem." PURPOSE: Our objective was to determine when telomerase activity is reactivated in the progression to malignant breast cancer and if knowledge of telomerase activity may be an indicator for the diagnosis and potential treatment of breast cancer. METHODS: Using a polymerase chain reaction-based telomerase activity assay, we examined telomerase activity in 140 breast cancer specimens (from 140 patients), four phyllodes tumors (from four patients), 38 noncancerous lesions (20 fibroadenomas, 17 fibrocystic diseases, one gynecomastia; from 38 patients), and 55 adjacent noncancerous mammary tissues (from 55 of the 140 breast cancer patients). In addition, 33 fine-needle-aspirated breast samples (from 33 patients) were analyzed. RESULTS: Among surgically resected samples, telomerase activity was detected in 130 (93%) of 140 breast cancers. Telomerase activity was detected in 68% of stage I primary breast cancers, in 73% of cancers smaller than 20 mm, and in 81% of axillary lymph node-negative cancers. Moreover, the activity was detected in more than 95% of advanced stage tumors but in only two (4%) of 55 adjacent noncancerous tissues. While telomerase activity was not detected in any of 17 specimens of fibrocystic disease, surprisingly low levels of telomerase activity were detected in nine (45%) of 20 fibroadenomas. Among samples obtained by fine-needle aspiration, 14 (100%) of 14 patients whose fine-needle-aspirated specimen contained telomerase activity and who subsequently underwent surgery were confirmed to have breast cancer. Multivariate analysis of 125 specimens from patients for whom data were available on age at surgery, stage of disease, tumor size, lymph node status tumor histology, and menopausal status indicated that stage classification exhibited the strongest association with telomerase activity (for stage I versus stages II-IV: odds ratio = 1.0 versus 73.4; 95% confidence interval = 2.0-959.0; P = .02). CONCLUSION: Telomerase activity was detected in more than 95% of advanced stage breast cancers. It was absent in 19%-32% of less advanced cancers. Since a determination of any association between telomerase activity and patient survival is not possible at the present time, it remains to be determined whether lack of telomerase activity predicts for favorable outcome.

Telomerase activity: a prevalent marker of malignant human prostate tissue.

We urgently need biochemical markers to detect the malignant nature and pathological states of the human prostate. We report that telomerase activity is associated with prostate cancer but absent in the benign disease and normal gland. Telomerase is, therefore, a potential diagnostic marker for prostate cancer. Twenty-five human prostates resected at the time of radical prostatectomy were dissected to obtain matched adjacent areas of normal, central zone benign prostatic hyperplasia (BPH), and pathologically confirmed cancer tissue. These matched tissue samples were assayed for telomerase activity using a sensitive PCR technique. None of the normal tissues exhibited telomerase activity. In contrast, 21 of the 25 (84%) cancers were strongly positive. At the time of prostatectomy, four lymph nodes were positive for metastases and all were strongly positive for telomerase activity. In adjacent BPH tissues taken from the cancerous prostates, only 3 of the 25 tissues (12%) were weakly positive. Telomerase activity was not detected in ten BPH samples recovered from patients who underwent open surgery solely for BPH. All five available cell lines of human prostate cancer (DU145, LNCaP, PC3, PPC1, and TSU) were strongly positive. Short telomere lengths have been observed in several human cancers. We also measured the telomere lengths in 27 matched samples of normal, BPH, and cancer tissue taken from nine radical prostatectomies. The telomeres from cancer tissue were significantly and consistently shorter than either the adjacent normal or adjacent BPH tissues. Our results indicate that telomerase activity, as well as telomere lengths, may be markers for distinguishing prostate cancer from normal and benign prostate tissues.

Telomerase activity in human germline and embryonic tissues and cells.

Telomerase is a ribonucleoprotein that synthesizes telomere repeats onto chromosome ends and is involved in maintaining telomere length in germline tissues and in immortal and cancer cells. In the present study, the temporal regulation of expression of telomerase activity was examined in human germline and somatic tissues and cells during development. Telomerase activity was detected in fetal, newborn, and adult testes and ovaries, but not in mature spermatozoa or oocytes. Blastocysts expressed high levels of telomerase activity as did most human somatic tissues at 16-20 weeks of development with the exception of human brain tissue. This activity could no longer be detected in the somatic tissues examined from the neonatal period onward. Neither placenta nor cultured fetal amniocytes contained detectable telomerase activity. Fetal tissues explanted into primary cell culture showed a dramatic decline in telomerase activity which became undetectable after the first passage in vitro. Elucidation of the regulatory pathways involved in the repression of telomerase activity during development may lead to the ability to manipulate telomerase levels and explore the consequences both for cellular aging and for the survival of cancer cells.

Telomerase activity in human brain tumours.

Malignant gliomas are invasive into surrounding brain and are refractory to therapy. Telomerase stabilises telomere length and may immortalise cells to allow unlimited proliferation. Our analysis of telomerase activity in 90 human gliomas showed that 19 of 19 oligodendrogliomas and 38 of 51 glioblastoma multiformes have detectable telomerase activity. The absence of telomerase activity in anaplastic astrocytomas (2/20 positive) and in one-quarter (13/51) of the glioblastomas suggests that these tumours follow different pathways of neoplastic progression. Thus we have found that a distinct subgroup of brain tumour consists of transformed yet pre-immortal cells.

Activation of telomerase in human lymphocytes and hematopoietic progenitor cells.

This is the first report describing up-regulation of telomerase activity in human normal cells. Telomerase, a ribonucleoprotein enzyme, has been thought to be involved in maintaining telomere length stability in germline and most cancer cells, but not in normal cells. However, in the present study, we demonstrate that telomerase activity is detectable at low levels in normal human T and B cells, increases by in vitro mitogenic stimulation, increases in hematopoietic progenitor cells upon their proliferation and differentiation, and decreases with aging. Understanding the regulation of telomerase activity in normal cells may provide important insights not only into the mechanisms of normal cellular senescence but also into the mechanisms of telomerase activity deregulation as part of cancer development.

Telomere shortening is associated with cell division in vitro and in vivo.

In humans, the amount of terminal (TTAGGG)n, telomeric DNA decreases during aging of various somatic cell types in vitro and in vivo. While the factors accounting for telomere shortening have not been thoroughly established, the inability of the DNA replication machinery to completely copy chromosomal termini (the "end replication problem") and the absence in somatic cells of telomerase, the enzyme that synthesizes telomeric DNA de novo, is a likely mechanism. One prediction of this hypothesis is that telomere shortening should be dependent on cell division. Thus we analyzed telomere length in actively dividing and quiescent cells in vitro and in vivo. In circular outgrowths of cultured human diploid fibroblasts (HDF), cells at the outer periphery had a significantly lower mean terminal restriction fragment (TRF) length (P = 0.011) and telomeric signal intensity (P = 0.024) than cells at the center. Also, the rate of telomere shortening over time for HDFs held quiescent was not statistically significant (m = -12 bp/day, P = 0.16) while that for serially passaged cells was significant (m = -34 bp/day, P = 0.017). To examine the rate of telomere shortening for quiescent cells in vivo, we measured mean TRF length in brain tissue from adult donors ranging in age from 32-75 years. No significant decrease was observed as a function of donor age (P = 0.087), in contrast to the shortening of telomere length that occurs during in vivo aging of mitotically active cells (P = 0.0001). These observations show that telomere shortening is largely, if not entirely, dependent on cell division and support the end replication problem as a mechanism for this process and the use of telomere length as a biomarker for replicative capacity.

Telomerase activity in gastric cancer.

Although many genetic alterations have been reported in gastric cancer, it is not known whether all gastric tumors are capable of indefinite proliferative potential, e.g., immortality. The expression of telomerase and stabilization of telomeres are concomitant with the attainment of immortality in tumor cells; thus, the measurement of telomerase activity in clinically obtained tumor samples may provide important information useful both as a diagnostic marker to detect immortal cancer cells in clinical materials and as a prognostic indicator of patient outcome. Telomerase activity was analyzed in 66 primary gastric cancers with the use of a PCR-based assay. The majority of tumors (85%) displayed telomerase activity, but telomerase was undetectable in 10 tumors (15%), 8 of which were early stage tumors. Most of the tumors with telomerase activity were large and of advanced stages, including metastases. Survival rate of patients of tumors with detectable telomerase activity was significantly shorter than that of those without telomerase activity. Alterations of telomere length (reduced/elongated terminal restriction fragments) were detected in 14 of 66 (21%) gastric cancers, and all 14 had telomerase activity. Cellular DNA contents revealed that all 22 aneuploid tumors had detectable telomerase activity. The present results indicate that telomerase activation may be required as a critical step in the multigenetic process of tumorigenesis, and that telomerase is frequently but not always activated as a late event in gastric cancer progression.

Telomerase activity in small-cell and non-small-cell lung cancers.

BACKGROUND: Telomerase is an enzyme that adds hexameric TTAGGG nucleotide repeats onto the ends of vertebrate chromosomal DNAs (i.e., telomeres) to compensate for losses that occur with each round of DNA replication. Somatic cells do not have telomerase activity and stop dividing when the telomeric ends of at least some chromosomes have been shortened to a critical length. It has been suggested that immortalized cells (including some, but probably not all, cancer cells) continue to proliferate indefinitely because they express telomerase. PURPOSE: To investigate whether expression of telomerase is a prerequisite for the development of naturally occurring human cancers, we assayed the levels of telomerase activity in specimens of human lung tumor and adjacent normal tissue. METHODS: Using a polymerase chain reaction-based assay, we examined telomerase activity in 136 primary lung cancer tissues and 68 adjacent noncancerous tissues obtained by surgical resection. We also studied telomerase activity in four primary and 23 metastatic lesions obtained through biopsy, (two patients) or autopsy (10 patients). Relative telomerase activity levels were estimated by serial dilutions of extracts prepared from the specimens. Telomerase activity was also assayed in extracts of cells present in pleural fluids from three patients with adenocarcinoma of the lung. RESULTS: Among surgically resected samples, telomerase activity was detected in 109 (80.1%) of 136 primary lung cancer tissues and in three (4.4%) of 68 normal adjacent tissues. All 11 surgically resected specimens of primary small-cell lung cancer (from 11 patients) revealed high levels of telomerase activity, whereas the activity ranged from undetectable to high levels in the 125 surgically resected specimens of primary non-small-cell lung cancer tissue (from 125 patients). Generally, high levels of telomerase activity were observed in metastatic lesions and tumors with altered telomere length. A few primary and, surprisingly, some metastatic tumors did not appear to have detectable telomerase activity. Telomerase activity was, however, detected in cells present in all tested pleural fluids obtained (from three patients with adenocarcinoma of the lung). CONCLUSION: The subset of non-small-cell lung cancers that exhibits only low or undetectable levels of telomerase activity may contain primarily mortal cancer cells. Cancers that exhibit high levels of telomerase activity, such as all of the small-cell lung cancers examined in this study, are likely to consist mainly of immortal cells. IMPLICATIONS: Telomerase activity may be useful both as a diagnostic marker to detect the existence of immortal lung cancer cells in clinical materials and as a target for therapeutic intervention.