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

PURPOSE: 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. PATIENTS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.     

Telomerase activity and its clinicopathological significance in gastric cancer

In order to assess the role of telomerase in development of malignant gastric cancer, we measured the telomerase activity in gastric cancer tissues and normal tissues obtained from 95 patients by employing recently developed sensitive PCR (polymerase chain reaction)-based telomerase assay (telomeric repeat amplification protocol, TRAP). We also investigated how telomerase activity related to other clinicopathological findings including DNA ploidy and K-RAS gene point mutation. The telomerase activity was present in 85 of the 95 gastric cancer tissues, whereas we detected no telomerase activity in any normal tissue. The incidence of telomerase activity in gastric cancer tissues was not correlated to age, sex, tumour stage, histological grade, DNA ploidy or K-RAS mutation. Disease-free or overall survival of patients having tumours with detectable telomerase activity was not significantly different from that of those without telomerase activity. These findings suggest that telomerase may play a key role in the establishment and progression of the gastric cancer and further studies will be needed to elucidate the biological role of telomerase in gastric cancer.     

Epidural anaesthesia for caesarean section in an achondroplastic dwarf

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA on chromosomal ends. Telomerase activation has been seen in many immortal cell lines and cancers. Telomerase activity was analyzed in prostate carcinoma; in coexistent prostatic intraepithelial neoplasia (PIN), benign prostatic hyperplasia (BPH), atrophy and normal tissue; and in benign prostate glands. Telomerase activity was detected in 80 of 87 (92%) prostate cancers. Forty-one matched samples (from a total of 32 cases) were available for comparative analysis. The presence of telomerase activity in adjacent PIN, BPH, and normal tissue was correlated with telomerase activity in the malignant epithelium. In these adjacent tissues, telomerase activity was found in 11 of 15 (73%) PINs, 13 of 26 (50%) BPHs, and 1 of 6 (16%) atrophy and 4 of 11 (36%) normal tissues. In contrast to the BPH tissue from cancer-bearing glands, all 16 BPH specimens from patients only diagnosed with BPH were telomerase activity negative. In cancer samples, there was no correlation between telomerase activity and Gleason grade or preoperation prostate-specific antigen level. Our data indicate that telomerase activity is present in most prostate cancers. The high rate of telomerase activity in the benign-appearing areas of these glands may be attributed either to the presence of occult cancer cells or to early molecular alterations of cancer that were histologically inapparent.     

Telomerase activity in the normal and neoplastic rat mammary gland

The 1-methyl-1-nitrosourea-induced rat mammary tumor model system is well studied, reproducible, and widely used. We have investigated whether these tumors possess higher telomerase activity than normal mammary tissue. Using the telomeric repeat amplification protocol assay, we found significantly higher telomerase activity in 36 mammary carcinomas than in 72 mammary glands of virgin rats. The level of telomerase activity in virgin rats was unaffected by strain, age, stage of the estrous cycle, or ovariectomy. However, mammary glands obtained from pregnant rats exhibited telomerase activity comparable to that found in the tumors, possibly reflecting the high epithelial content of these tissues. Indeed, isolated epithelial cells from virgin and pregnant mammary glands and from carcinomas had similar telomerase activities. Thus, telomerase activity is constitutive in the rat mammary epithelium and is not a unique characteristic of malignant transformation in this tissue. These results underscore the importance of attributing biochemical properties to specific cell types in a tissue, a situation not paralleled in the interpretation of data from in vitro models.     

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

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

Telomerase activity in the differentiation of benign and malignant adrenal tumors

BACKGROUND: Telomerase is an RNA-dependent DNA polymerase that extends the ends of chromosomes by synthesizing the 6 oligonucleotide repeat TTAGGG and thus serves as a marker for cellular immortality. Although absent in most adult somatic tissues, telomerase activity is present in stem cells and is reactivated in nearly all primary human malignancies. In this study we sought to determine whether tumors of the adrenal glands contain telomerase activity and whether telomerase activity can be used to differentiate benign and malignant tumors of the adrenal glands. METHODS: Tissue was obtained from 23 specimens at adrenalectomy. Adjacent normal adrenal tissue was obtained for control. All specimens were rapidly frozen and stored at -80 degrees C until assay. Telomerase activity was determined by the telomeric repeat amplification protocol (TRAP). RESULTS: Telomerase activity was present in 5 of 23 (22%) of the adrenal tumors. All 3 malignant tumors were strongly TRAP positive. There was a single cortical adenoma that had very weak telomerase activity. The single TRAP-positive tumor of the adrenal medulla was a ganglioneuroma. CONCLUSIONS: Benign adrenal tumors infrequently contain telomerase activity, whereas telomerase reactivation appears to be common in malignant tumors of the adrenal glands. These data suggest that determination of telomerase activity may offer a novel way to facilitate the differentiation of benign and malignant adrenal tumors.     

In situ mRNA hybridization technique for analysis of human telomerase RNA in gastric precancerous and cancerous lesions

Telomerase, the ribonucleoprotein enzyme that elongates telomerase, is repressed in normal somatic cells but is reactivated during tumor progression. The purpose of this study was to investigate the localization of human telomerase RNA (hTR) expression in human gastric precancerous and cancerous lesions by using in situ mRNA hybridization (ISH) with avidin-biotin staining. We also examined telomerase activity in these lesions by using hybridization protection assay connected with a telomeric repeat amplification protocol (TRAP/HPA). Analyzed tissue samples were as follows; 132 cases of chronic atrophic gastritis without intestinal metaplasia, 115 incomplete-type intestinal metaplasias, 40 complete-type intestinal metaplasias, 23 hyperplastic polyps, 23 tubular adenomas and 26 adenocarcinomas. In ISH analysis, high levels of hTR expression were observed preferentially in the nuclei at the single-cell level. hTR-expressing cells in carcinomas and adenomas were significantly more frequent than those of the other lesions (P < 0.001). The expression pattern of hTR in carcinoma and adenoma tissues was heterogeneous and similar intratumor heterogeneity was detected in Ki-67 immunoreactivity. Infiltrating lymphocytes in tissue also exhibited high levels of hTR expression. In TRAP/HPA analysis, carcinomas had significantly more frequent positivity for telomerase activity and a higher level of telomerase activity than the other lesions (P < 0.05). However, the amount of telomerase activity did not parallel the expression level of hTR. Our data suggest that hTR expression increases in the early stages of stomach carcinogenesis and that sufficient synthesis of hTR is a prerequisite for telomerase reactivation in tumorigenesis.     

Decrease of telomere length in thyroid adenomas without telomerase activity

In somatic cells, telomeres shorten with population doubling, thus limiting their capacity to divide. Telomerase, which synthesizes telomeric repeats, can compensate for such shortening. Telomerase activity is known to be absent from most somatic differentiated cells but is present in germline cells, immortal cell lines, or a large majority of malignant tumors. Autonomous thyroid adenomas are benign tumors composed of highly differentiated cells characterized by TSH-independent function and growth. Telomere length and telomerase activity were measured in autonomous and hypofunctioning adenomas and their surrounding tissues. A significant decrease of 3.8+/-1.0 kilobases (kb) was observed in the length of the terminal restriction fragments (TRF) in 12 autonomous adenomas (8.6+/-1.1 kb), compared with the TRF length of their surrounding tissues (12.4+/-1.6 kb). The same kind of decrease, 3.5+/-1.2 kb, was also observed in 16 hypofunctioning adenomas (12.3+/-1.7 kb in surrounding tissue and 8.8+/-1.6 kb in the adenomas). No telomerase activity was detected either in the 12 autonomous adenomas studied or in most of the quiescent tissues (10 of 12). Most of the hypofunctioning adenomas tested (15 of 16) did not display telomerase activity. These results suggest that the cells have undergone a higher number of cell divisions in the adenomas than in the surrounding tissue. Moreover, there is a larger spread of the TRF length distribution in autonomous adenomas than in the collateral tissue. This could reflect the heterogeneity in proliferation status of the cells in the nodule, some of which have reached the end of their life span, whereas others are still proliferating (but with no malignant potential for the autonomous adenomas). In conclusion, benign adenomas exhibit a shorter and more variable telomere length than the normal collateral quiescent tissue, with no telomerase activity to compensate this loss in telomere length.     

Telomerase activity in benign and malignant thyroid diseases

BACKGROUND: Telomerase, an enzyme associated with cellular immortality, is expressed by most malignant cells and is inactive in most normal somatic cells, with the excitation of proliferative stem cells, male germ cells, and activated lymphocytes. The measurement of telomerase activity in clinically obtained tissue samples may provide useful information as both a diagnostic and prognostic marker. In this study, we sought to determine whether telomerase activity might prove helpful in the assessment of benign and malignant thyroid tumors. METHODS: A modified, semiquantitative polymerase chain reaction-based telomeric repeat amplification protocol assay was used for detection of telomerase activity in 59 samples obtained at thyroidectomy, including 15 thyroid cancers, 22 benign thyroid diseases, and 22 adjacent normal thyroid tissues. RESULTS: Four of 13 differentiated thyroid carcinomas (30%) and 2 of 2 medullary carcinomas (100%) expressed telomerase activity. Unexpectedly, we also detected activity in 3 of 22 (14%) adjacent normal thyroid tissues and 6 of 22 (28%) benign thyroid diseases. Pathologic review of the telomerase-positive benign specimens revealed that many contained extensive lymphoid infiltrates with germinal centers (six of nine, 67%), as did two of four telomerase-positive papillary carcinomas. CONCLUSIONS: In contradistinction to other epithelial carcinomas, telomerase does not appear to be frequently reactivated in differentiated thyroid carcinomas.     

Increased telomerase activities in human pancreatic duct adenocarcinomas

Telomerase is a key enzyme with regard to immortalization of cancer cells and increased activity has been demonstrated in various human malignant neoplasms. Since little is known of its role in pancreatic cancers, we investigated changes in telomerase activity in human pancreatic duct adenocarcinomas and compared the frequency of increased telomerase activity with the presence of K-ras gene mutations. The samples were obtained from 38 pancreatic duct adenocarcinomas and 7 tumor surrounding tissues at surgical resection. Telomerase activity was examined by telomeric repeat amplification protocol assay and terminal restriction fragment (TRF) length was examined by Southern analysis. K-ras mutation was examined by means of polymerase chain reaction-single strand conformation polymorphism analysis. Among 38 pancreatic carcinomas, 32 (84%) exhibited increased telomerase activities with no apparent relation to the histological type of tumor, tumor size, regional lymphnode involvement and distant metastasis or clinical stage. In tissue surrounding the tumor, telomerase activity was not detected. TRF length tended to be reduced in pancreatic carcinomas. Mutations of K-ras gene were found in 24 out of the 38 (63%) cases. Among the 38 cases, 14 showed increased telomerase activity without K-ras mutation and 4 cases showed K-ras mutation without telomerase activity. These results suggest that increased telomerase activity might be a sensitive genetic diagnostic marker and could be a target for future therapy of pancreatic duct carcinomas.     

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

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

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

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

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

Telomerase inhibitor

Human normal somatic cells and tissues have undetectable or very weak telomerase activity and shorten telomere size at each cell division resulting in a limited proliferative life span. Human germ tissues and most tumor tissues have telomerase activity and maintain telomere size during cell proliferation resulting in unlimited growth. Telomerase is expected to be a new target for cancer chemotherapy. Cultured tumor cells are shown to die after losing telomerase activity.     

Telomerase activity in human bladder cancer

Telomerase can synthesize telomeric DNA repeats onto chromosome ends. Telomere length and telomerase activity have recently been implicated in the control of the proliferative capacity of normal and malignant cells. The expression of telomerase activity is concomitant with the attainment of immortality in tumor tissues and cells. Thus, enzyme activity may indicate a prevalent or even ubiquitous tumor producer. In this report, telomerase activity was analyzed in 40 human bladder cancers, 7 normal tissues, and 2 bladder epithelia with dysplasia using a PCR-based telomeric repeat amplification protocol assay. Telomerase activity was detected in almost all bladder tumors (97.5%); only one sample, which was in an early stage, did not express telomerase activity. None of the normal tissues displayed telomerase activity. One of the two bladder epithelia with dysplasia expressed low telomerase activity. The expression of telomerase activity has a clear association with the pathological grade and clinical stage. Most of the tumors with high telomerase activity were in an advanced grade and had deep invasion. Thus, telomerase activity might be suggested to represent an additional required event in the multigenetic process of tumorigenesis in human bladder cancer.