Relation between telomerase activity, hTERT and telomere length for intracranial tumours

Human linear chromosomes are capped by specialized DNA-protein structures called telomeres. The present study analysed the telomerase activity, hTERT protein and telomere length in meningiomas and gliomas in relation to their WHO grading. Fifty-three freshly dissected tumour biopsies were analysed for telomerase activity, hTERT protein expression and telomere length. Telomerase activity was examined in 41 of the 53 biopsies. Telomerase activity was detected in 3 of 35 (8.6%) screened meningiomas (1 benign, 1 atypical and 1 malignant meningioma). For hTERT expression, 56.4% of meningiomas were positive with a mean labelling index (hTERT LI) of 31.3% (SD=26.5) for the hTERT positive meningiomas. The mean telomere length for meningiomas was 6.983 kb (SD=1.969). For gliomas, no active telomerase was detected in 2 low-grade astrocytomas, whereas three of the four screened glioblastomas were positive for telomerase activity. The only hTERT protein positive astrocytoma had a mean labelling index of 9.0%. On the other hand, the hTERT LI for glioblastomas was 53.6% (SD=28.0). The two low-grade astrocytomas had a telomere length of 14.310 and 9.236 kb. The anaplastic astrocytoma had a telomere length of 4.903 kb and the glioblastomas 5.767 kb (SD=2.042). The normal meningeal and neuronal tissue is negative for telomerase activity and hTERT. The length was +/-10.000 kb. These results indicate that telomere shortening may be a critical step in pathogenesis of atypical and malignant meningiomas and gliomas. Critical telomere shortening in vitro was shown to activate telomerase.     

Telomerase activity in germ cell cancers and mature teratomas.

BACKGROUND: An inverse relationship has been reported between the presence of telomerase enzymatic activity and the induction of differentiation in human tumor cell lines. Male germ cell tumors represent an attractive clinical model to assess this relationship further because high telomerase activity is present in normal germ cell progenitors and in embryonal carcinomas that can differentiate into mature teratomas. To investigate how telomerase activity and the differentiation state of germ cell tumors are related, telomerase activities and telomere lengths were measured in benign testicular tissues, germ cell cancers, and mature or immature teratomas. METHODS: By use of a modified telomeric repeat amplification protocol (TRAP) assay, telomerase activity was measured in four specimens of benign testicular tissue, in 27 germ cell cancers, in seven mature teratomas, and in one immature teratoma. Telomere lengths were measured in all specimens by restriction digestion of genomic DNA and Southern blot hybridization analysis. Associations between telomerase activity and tissue histopathology were assessed with two-sided Fisher's exact tests. RESULTS: Telomerase activity was detected in all examined germ cell cancers and in the benign testicular tissue specimens. In marked contrast, telomerase activity was not detected in any mature teratoma (P<.0001). Very long telomeres were detected in some mature teratomas, consistent with telomerase repression as a late event in teratoma formation. The immature teratoma, with malignant transformation, had high telomerase activity. CONCLUSION: Telomerase is active in germ cell cancers and repressed in mature teratomas. The absence of telomerase activity may contribute to the limited proliferative capacity of mature teratomas. These findings support the existence of an inverse relationship between telomerase activity and the differentiation state of clinical germ cell tumors.     

Telomerase activity and telomere length in lymphocytes from patients with cutaneous T-cell lymphoma.

BACKGROUND: Telomeres shorten with successive cell divisions in normal somatic cells. Telomerase is a ribonucleoprotein enzyme associated with cellular proliferation and plays an important role in maintaining the stability of chromosomes and the length of DNA telomeres. Telomerase activity has been detected in tissues from many human tumors, but is not present in the majority of normal tissues. Thus, measurement of telomerase activity and telomere length may contribute to understanding the mechanism of tumorigenesis and provide useful diagnostic or prognostic information. The aim of this study was to investigate the telomerase activity and telomere length from patients with cutaneous T-cell lymphoma (CTCL). METHODS: Eighteen skin-homing T-cell lines were established from skin biopsies and 10 peripheral blood mononuclear cells (PBMC) were isolated from patients with various stages of CTCL together with 22 PBMC from healthy donors. For each sample an identical amount of cellular protein was measured quantitatively for telomerase activity using the telomerase polymerase chain reaction-enzyme-linked immunosorbent assay based on the telomeric repeat amplification protocol method. Telomere length was assayed using a commercial kit. RESULTS: Eight of ten PBMC and 16 of 18 skin-homing T-cell lines from patients with CTCL showed moderate to strong telomerase activity. Freshly obtained PBMC from healthy donors showed weak levels of telomerase activity. A shorter telomere length was found in cell lines and PBMC from patients with CTCL compared with healthy controls. Four skin-homing T-cell lines going into growth crisis showed sharply reduced telomerase activity. CONCLUSIONS: The results of the current study indicate that both skin-homing T-cells and PBMC from CTCL have high telomerase activity and short telomere length. These changes are similar to the changes observed in the majority of malignant cells including other types of T-cell lymphoma. It is interesting to note that even in the very early stages of CTCL such as parapsoriasis (which is a clinically benign disease) the changes already are present, indicating that a significantly high level of telomerase activity frequently occurs in CTCL and may be an important event in tumorigenesis. Telomerase activity and telomere length are useful markers for CTCL risk assessment.     

Telomerase activity in oral and maxillofacial tumors

Telomerase activity was measured in biopsy specimens as well as surgically resected tissues of 39 oral squamous cell carcinomas (SCCs), 22 oral leukoplakias, 13 normal oral mucosas, 12 malignant salivary gland tumors, 10 benign salivary tumors and five normal salivary gland tissues adjacent to tumors using a polymerase chain reaction (PCR)-based telomerase assay. Telomerase activity was detectable in 38.5% (5/13) of normal oral mucosa samples, 54.5% (12/22) of leukoplakia samples, 82.1% (32/39) of oral SCC samples, 83.3% (10/12) of malignant salivary gland tumor samples and 0% (0/10, 0/5) of benign salivary gland tumor and normal salivary gland samples. High-level enzyme activities were seen in 20% (3/15) of mild dysplasia specimens, 50% (2/4) of moderate-severe dysplasia specimens, 48.7% (19/39) of oral SCC tissue specimens, and no high activity was seen in the normal mucosa and hyperkeratosis specimens (P for trend, <0.001). We also analyzed the proliferative activity of dysplastic leukoplakia, oral SCC, and salivary gland tumor specimens using Ki-67 immunohistochemistry. The Ki-67 labeling indices (LI) were significantly higher in dysplastic leukoplakia and oral SCC with high telomerase activity than in dysplastic leukoplakias and oral SCC with low and negative telomerase activity (P<0.01 and P<0.05). These results indicate that telomerase activity has some correlation with the progression of multistep oral carcinogenesis with the cellular proliferation, and also indicate that telomerase may be a specific marker used to distinguish malignant salivary gland tumors from their benign counterparts.     

Implication of telomere length as a proliferation-associated marker in schwannomas

BACKGROUND AND OBJECTIVES: Some schwannomas in the central nervous system may demonstrate relatively aggressive behavior in pathological findings and clinical course. We evaluate the diagnostic values of telomerase activity and telomere length in the clinicopathological behavior of schwannomas. METHODS: Thirty surgical specimens from intracranial and intraspinal schwannomas were analyzed by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) for telomerase activity and terminal restriction fragments (TRFs) using Southern blot for telomere length. Proliferative indices were also studied. RESULTS: Telomerase activity could not be detected in all schwannomas. Elongated telomere length (mean 17,101 +/- 259 bp) was found in four specimens (13.3%). Three of these four were found to have mitotic figures, high vascularity, cellularity, and pleomorphism in the pathological findings. The proliferative indices (35) showed correlative high values. One patient died of this disease, and one was found to have recurrence at follow-up evaluation. Those that displayed benign histopathological pictures showed relatively short telomere length (8,866 +/- 271 base pairs) and low proliferative indices (21). These is a significant difference between these two groups (P = 0.001). CONCLUSIONS: Elongation of telomere length in schwannomas appears to predict aggressive clinicopathological behavior.     

Telomere length and telomerase activity in carcinogenesis of the stomach

Telomerase activity is generally absent in primary cell cultures and normal tissues. Telomerase is known to be induced upon immortalization or malignant transformation of human cells. In the present study, we analyzed both telomere length and telomerase activity in biopsy samples from mucosa undergoing metaplasia, adenoma and cancer of the stomach. We attempted to estimate the correlation between telomerase activity and telomere length in these tissues. Telomerase activity was estimated using the telomeric repeat amplification protocol and telomere length by Southern blot analysis. Extracts were defined as telomerase-negative when the signals were less intense than those for 10(2) KATO-III cells (positive control). We detected telomerase activity in 15%, 45% and 89% of the examined cases of intestinal metaplasia, adenoma and gastric cancer respectively. However, telomere length in the gastric mucosa became reduced as the mucosa underwent metaplasia and developed into adenoma. Gastric cancers showed a broad range of telomere length among cases. However, gastric adenomas showed the shortest telomere length. These results suggest that telomerase is expressed during the early phase (intestinal metaplasia through adenoma) of gastric carcinogenesis, although the activity at that stage is not high enough to fully restore the reduced telomeric DNA.      

Telomerase activity as a marker of breast carcinoma in fine-needle aspirated samples

BACKGROUND: Telomerase activity in breast fine-needle aspiration (FNA) samples may have diagnostic utility. The purpose of this study was to compare in FNA samples of breast tumor the diagnostic accuracy as correlated with histologic final diagnosis. METHODS: Fine-needle aspiration samples were obtained from 617 patients with palpable breast tumors. Slide preparation and cytology were performed according to a uniform approach. Extracts derived from 10(3) cells from the residual cells in the syringe were used for the telomeric repeat amplification protocol (TRAP) assay. Of the original 617 patients, 220 underwent open biopsy or surgery, and 93 cancers and 127 patients' benign diseases were diagnosed by histologic examination. RESULTS: All 62 tumors that were diagnosed as "malignant" or "probably malignant" by FNA cytology were cancerous, and 50 cases (81%) showed detectable telomerase activity. Among 17 "atypical" or "indeterminate" cases, all 10 tumors with detectable telomerase activity subsequently were diagnosed as breast carcinoma whereas 6 of 7 tumors without telomerase activity were diagnosed as benign. Among the 141 "benign" or "unsatisfactory" samples, 12 of 21 cases with detectable telomerase activity subsequently were diagnosed as cancer. CONCLUSIONS: The diagnostic accuracy of telomerase activity in FNA samples is considered to be equivalent or slightly higher to that of cytology (86% vs. 70%). Detection of telomerase activity should be considered an alert for false-negative results of FNA cytology and may be useful as a diagnostic marker for breast malignancy, especially in samples cytologically undetermined to be malignant. Cancer (Cancer Cytopathol) Copyright 2000 American Cancer Society.     

High telomerase activity is an independent prognostic indicator of poor outcome in colorectal cancer.

Telomerase activity and altered telomere length have been extensively studied in many kinds of malignant tumors for clinical diagnostic and/or prognostic utilities. In the present study, we investigated telomerase activity and telomere length in colorectal cancers and noncancerous colonic mucosa specimens in 100 patients between 1991 and 1996. To determine whether the level of telomerase activity or telomere length is a prognostic indicator of patient outcome, we followed these patients more than 3 years after surgery. Among 100 primary colorectal cancer specimens, 96 specimens had telomerase activity. Because noncancerous mucosa has some detectable telomerase activity, we divided the levels of telomerase activity into three categories: high (>50-fold more than that in noncancerous mucosa); moderate (10- to 50-fold); and low (<10-fold) levels. Among 100 cancer tissues, 28 showed moderate telomerase activity and 44 showed high telomerase activity. The frequency of tumors with moderate or high telomerase activity showed no significant relationship with any clinicopathological factors. The prognosis of the patients with high telomerase activity was significantly worse than that for patients with moderate and low telomerase activity (P < 0.01). Among the 87 patients with curative surgery, disease-free survival rate of those with high telomerase activity was also significantly poorer (P < 0.01). These results indicate that a high level of telomerase activity may be an independent prognosis-predicting factor in the patients with colorectal cancer.     

端粒酶活性与子宫内膜癌相关性研究

目的　研究正常子宫内膜组织、不典型增生过长、不同临床分期、组织学分级子宫内膜癌组织中端粒酶活性变化。方法　采用以PCR为基础的端粒酶重复扩增方法及非变性PAGE银染定性法检测不同组织中端粒酶活性。结果　端粒酶活性在子宫内膜癌组织中显著增高 ,在正常子宫内膜组织及不典型增生过长中亦有表达 ,而子宫内膜癌不同临床分期及组织学分级间则无显著差异 (P >0 0 5 )。结论　端粒酶的激活是诱发子宫内膜癌的重要因素 ,但其进展及恶性程度则与肿瘤的进展无显著相关。     

Telomerase activity and telomere length in different areas of renal cell carcinoma

Telomerase activity and telomere length were analyzed in a total of 59 surgically removed primary renal cell carcinoma (RCC). The study includes tissue from the centre of the tumor, several different peripheral tumor areas, metastases and secondary tumors. None of the normal renal cortex tissues used as control exhibited telomerase activity. In contrast, telomerase activity was detected in 55 out df 59 (=93%) tested primary RCC. There was no case with intratumoral heterogeneity concerning the telomerase activity status. All metastases and secondary tumors were telomerase-positive. In the four telomerase deficient tumors all measured telomeric repeat fragments were shortened in comparison to the normal tissue. As these patients exhibit no metastases or secondary tumors a less malignant variant of RCC is supposed. There was no correlation between telomerase activity and specific histopathological subtypes of RCC or specific chromosomal aberrations. As telomerase activity is not associated with advanced stages of tumors it may be an important early event in the development of RCC. Thus, telomerase activity may be a prevalent marker for early and late stages of all subtypes of RCC.     

Differential telomerase activity, expression of the telomerase catalytic sub-unit and telomerase-RNA in ovarian tumors.

Telomerase activity has been found in a variety of malignant tumors but only rarely in benign tumors or normal tissues. In this study, we investigated telomerase activation in 37 ovarian tumors, including benign, borderline and malignant neoplasms. Telomerase activity was detected using the telomeric repeat amplification protocol (TRAP) in 13/16 ovarian carcinomas, 9/10 borderline tumors and 3/11 cystadenomas/fibromas. mRNA expression of the putative human telomerase catalytic sub-unit gene (hTERT) was detected by RT-PCR in 14/15 ovarian carcinomas, 8/10 borderline tumors and 4/11 cystadenomas/fibromas. In situ hybridization was performed to evaluate telomerase-RNA (hTR) expression in the corresponding paraffin-embedded tumors. Variable expression levels of hTR were found over neoplastic tumor cells. The highest levels of hTR expression were found predominantly in ovarian carcinomas. Although the amount of telomerase activity varied, significantly high levels of telomerase activity were found predominantly in ovarian carcinomas. hTERT mRNA expression was closely associated with telomerase activity. These findings suggest that up-regulation of hTERT and hTR is important for telomerase activation during malignant-tumor progression. Telomerase activation might therefore be a valuable diagnostic parameter that could help to identify potentially progressive lesions. However, the diagnostic and therapeutic implications of telomerase activation need to be clarified in clinical trials. Int. J. Cancer (Pred. Oncol.) 84:426-431, 1999.     

Analysis of telomerase activity and telomere length in bone and soft tissue tumors

Telomerase activation is prevalent in most epithelial tumors, and may be a critical step in cellular immortalization and carcinogenesis. However, telomerase activity in tumors of mesenchymal origin is not well understood. In the present study, we examined telomerase activity in clinical samples from osteosarcoma and soft tissue sarcoma and representative sarcoma cell lines (HOS, OST and Saos2), using the telomeric repeat amplification protocol (TRAP) assay. The cell lines HOS and OST were telomerase-positive, but Saos2 cells lacked telomerase activity and hTERT mRNA expression. Treatment of Saos2 cells with the demethylating agent 5-aza-2'-deoxy-cytidine, alone or together with the histone deacetylase inhibitor tricostatin A, did not induce hTERT mRNA expression. Twenty-six of the 83 sarcoma samples (31.3%) were telomerase-positive [bone sarcoma, 15 of 42 samples (35.7%); soft tissue sarcoma, 11 of 41 samples (26.8%)], whereas neither benign tumors nor normal bone tissue expressed telomerase activity. There was no significant correlation between histological type, tumor staging and telomerase activity. However, patients with telomerase-positive tumors had significantly shorter survival than those with telomerase-negative tumors. There was heterogeneity in telomere length (range, 6-18 kb) among the tumors examined, but there was no significant difference in length between telomerase-positive and -negative tumors. Thus, these mesenchymal tumors comprise heterologous groups, some positive and some negative for telomerase, with long and short telomeres, suggesting multiple carcinogenesis pathways. The present results indicate that telomerase activation is not prevalent in mesenchymal tumors and is not a critical determinant of telomere length, but it may be a prognostic indicator of mesenchymal tumors.     

Telomerase activity in oral cancer

Telomerase activity can be detected in most human cancers. This is consistent with the telomere hypothesis, which predicts upregulation of telomerase expression after a number of mitotic divisions to prevent the progressive and catastrophic loss of telomeres. However, telomerase has not been fully analyzed in oral cancers. In this report, telomerase activity was analyzed in 31 human oral malignant tumors, 11 leukoplakias, three pleomorphic adenomas, and 40 samples taken from normal tissues of the oral cavity, using a polymerase chain reaction (PCR)-based telomeric repeat amplification protocol assay. Telomerase activity was detected in most oral cancers [squamous cell carcinoma (SCC), non-Hodgkin's lymphoma, adenoid cystic carcinomas, mucoepidermoid carcinoma, osteosarcoma, acinic cell carcinoma, rhabdomyosarcoma]. None of the normal tissues or pleomorphic adenomas displayed telomerase activity. In leukoplakia, telomerase activity was seen in moderate or severe dysplastic tissue and carcinoma in situ. Mild dysplasia did not reveal telomerase activity. In SCC, there was no clear association between relative telomerase activity and grade or stage. These results suggest that detection of telomerase activity in oral tissues could be used to differentiate malignant from benign or normal tissues.     

Telomere shortening and the clinicopathologic characteristics of human colorectal carcinomas.

BACKGROUND: It has been reported that shortening of telomeres and strong activation of telomerase occur frequently in colorectal carcinomas. In the current study, the authors examined the correlations between the telomere length of colorectal carcinomas and their clinicopathologic characteristics as well as the activity of telomerase to clarify whether telomere length might represent the biologic behavior of tumors and the mode of tumor development. METHODS: Telomere length was examined by Southern blot analysis in 61 invasive colorectal carcinomas and corresponding normal mucosas. Telomerase activity was assayed by the telomeric repeat amplification protocol with minor modifications. RESULTS: Shortening of the telomere was detected in 38 (62.3%) and elongation in 3 (4. 9%) of the 61 carcinomas. The telomere shortening occurred more frequently in nonulcerating polypoid carcinomas than in ulcerating carcinomas (P = 0.0373) and also occurred more frequently in ascending colon carcinomas than in sigmoid colon or rectal carcinomas (P = 0.0259 and P = 0.0407, respectively). However, no significant correlation was found between the activity of telomerase and the length of telomere. CONCLUSIONS: The results of this study indicate that telomere length may represent the biologic behavior of individual tumors and possibly the mode of development of colorectal carcinomas.     

Prognostic significance of telomerase activity in soft tissue sarcomas.

Only few reports on the prognostic significance of telomerase activity in human cancer exist. To find a new prognostic marker in soft tissue tumors, we investigated 60 soft tissue sarcomas of different histology and six benign tumors for telomerase activity. Telomerase activity was measured by using the non-radioactive PCR-based TRAP-assay. PCR products were analyzed on an automated fluorescence sequencer. Tumors of grade-II and grade-III histology showed a significantly poorer prognosis. Both disease-free (p<0.03) and the overall survival (p<0.02) were reduced in the highly malignant sarcoma patients. We found telomerase activity in 38.3% of the cases, there being a correlation with a more aggressive behavior of soft tissue sarcomas. Telomerase activity correlated with the grade of malignancy (p=0.04), but not with sex (p=0.64) or age (p=0. 48) of the patients. The total survival was significantly reduced in patients with telomerase-positive sarcomas (p=0.04). Both of the patients having grade I tumors with telomerase activity died of disease, whereas 10 of 11 patients with telomerase-negative grade I tumors are still alive. Only one of the benign tumors showed telomerase activity. We suggest that telomerase activity is a potential prognostic factor in malignant soft tissue tumors. Despite the histological heterogeneity of soft tissue tumors, single entities should be assessed for telomerase activity.     

Absence of telomerase activity in malignant bone tumors and soft-tissue sarcomas

Purpose: Telomerase activity appears to play a crucial role in the development of many tumors. More than 80% of all malignant human tumors show an increased telomerase activity. However, conflicting results have been reported about telomerase activity in sarcomas. The aim of the study was to obtain more information about telomerase activity in sarcomas based on a large number of cases.Methods: Telomerase activity was measured in 69 different tumor samples (33 malignant bone tumors and 36 soft tissue sarcomas). Tumor samples were obtained intraoperatively and frozen immediately in liquid nitrogen. Telomerase activity was detected by the telomeric repeat amplification assay (TRAP-assay).Results: Only 7% of the samples showed telomerase activity. No correlation between staging and telomerase activity could be observed.Discussion: The fact that only five out of 69 examined tumor samples showed a telomerase activity provides experimental evidence that in sarcomas the reactivation of telomerase may play a subordinate role. Our results suggest that alternative mechanisms for cell immortalization, yet to be determined, seem to be involved in the development and/or maintenance of soft-tissue sarcomas and malignant bone tumors.     

Telomeric lengths and telomerase activity in liposarcomas

To assess the role of telomerase in the development of liposarcomas, we measured telomerase activity in 36 malignant and seven benign lipomatous neoplasias from 34 patients. A sensitive polymerase chain reaction-based telomerase assay (the telomeric repeat amplification protocol) was applied. Shortening or elongation of telomeric repeat fragment lengths, as measured by using hybridization with a telomere-specific oligonucleotide probe, was correlated with the presence of telomerase activity. The latter was demonstrable in 69% of malignant tumors. Benign tumors can be distinguished from malignant neoplasias on the basis of telomerase activity. However, telomerase expression seems to be characteristic of poorly differentiated liposarcomas. Myxoid/round cell liposarcomas exhibited a higher telomerase activity level than the classical low-grade variants. Telomerase activity was not correlated with age at the time of diagnosis or with sex. In most cases, telomerase-positive tumors showed higher proliferation indices than did neoplasias lacking telomerase. All eight recurrences expressed telomerase activity, reflecting a close association of telomerase with the biological behavior of liposarcomas. Our findings suggest that telomerase may play a key role in the establishment and progression of malignant lipomatous tumors.