Detection of telomerase activity in breast masses by fine-needle aspiration

Background: Telomerase is an RNA-dependent DNA polymerase that compensates for the telomere shortening that occurs in its absence. Reactivation of telomerase is thought to be an important step in cellular immortalization, and recent studies have indicated that telomerase activity is often detected in primary human malignancies. The clinical implications of telomerase activity in human tumors are currently under investigation. Methods: Eighty-nine samples (46 FNAs and 43 gross tissue biopsies) from 44 patients with breast masses were analyzed prospectively for the presence of telomerase activity by a modification of the telomere repeat amplification protocol (TRAP). All samples were obtained directly from the excised mass at the time of specimen removal in the operating room. Results: Telomerase activity was detected in 17 of 19 (90%) FNA samples and 15 of 18 (83%) invasive breast cancer tissue biopsies. Telomerase was also detected in 9 of 16 (56%) FNAs and 8 of 15 (53%) tissue biopsies from 16 fibroadenomas. Other benign proliferative lesions (n=5) did not have detectable telomerase activity in either FNA or tissue specimens. FNA-TRAP results correlated with the gross tissue specimen TRAP results in 95% of all cases. Conclusion: The FNA-TRAP assay for telomerase detection is a highly sensitive and accurate method for the detection of telomerase activity in breast masses. Future application of these techniques should facilitate evaluation of telomerase as a tumor marker in the clinical management of breast and other solid malignancies. These authors contributed equally to this work.     

Telomerase activity in periampullary tumors correlates with aggressive malignancy

OBJECTIVE: To determine the presence of telomerase activity in a variety of periampullary malignancies and pancreatic diseases and quantify its activity to establish any association with the stage or aggressiveness of malignancy. SUMMARY BACKGROUND DATA: Progressive shortening of telomeres, repetitive DNA sequences at the ends of chromosomes, plays a role in cell senescence. Telomerase catalyzes conservation of telomeric repeats and may promote cell immortality and hence malignancy. It is absent in normal tissues but upregulated in more than 80% of cancers. METHODS: Fresh specimens of 62 periampullary tumors were snap-frozen in liquid nitrogen and adjacent tissue was formalin-fixed for histopathology. The telomerase repeat amplification protocol (TRAP) was used to obtain telomerase DNA products. These were separated with gel electrophoresis, stained with SYBR green, and quantified by densitometry. Findings were confirmed with a fluorometric TRAP assay in which fluorescent primers specific for telomerase were selectively amplified in its presence. RESULTS: Telomerase activity was upregulated in 26 of 33 periampullary malignancies (79%): 17 of 21 pancreatic adenocarcinomas (81%), 2 of 2 cholangiocarcinomas, 2 of 2 duodenal carcinomas, and 5 of 8 ampullary carcinomas (63%). Poorly differentiated periampullary tumors had significantly higher telomerase activity than well-differentiated tumors, and tumors larger than 2 cm had significantly higher telomerase activity than those 2 cm or smaller. Pancreatic ductal adenocarcinomas with lymph node metastases had significantly greater activity than node-negative cancers. Two of 11 intraductal papillary mucinous tumors were positive for telomerase activity, but only in foci of invasive carcinoma. Chronic pancreatitis (n = 7), serous cystadenomas (n = 5), benign mucinous cystic neoplasms (n = 4), neuroendocrine cancer (n = 1), and acinar cell carcinoma (n = 1) had no detectable telomerase activity. CONCLUSION: Telomerase activity is common in periampullary carcinomas. The magnitude of activity correlates with aggressiveness in pancreatic adenocarcinoma and may prove useful as a molecular index for biologic staging.     

Telomerase Activity in Giant Cell Tumors of Bone

Background A giant cell tumor of bone (GCT) is a histologically benign neoplasma that has an unpredictable pattern of biological aggressiveness. In the present study, we investigated whether there was a correlation between telomere length or the levels of telomerase activity and other clinical features of GCTs, for the possible use of these factors as parameters of aggressiveness or prognosis. Methods In 16 surgically resected GCTs specimens, telomere length was assessed by terminal restriction fragments by Southern blot analysis. Telomerase activity was measured by a semiquantitative polymerase chain reaction–based telomeric repeat amplification protocol assay. Results Telomere length reduction was observed in 69% of the GCT samples. The telomere lengths of tumors were significantly shorter than those of normal tissue (P = .008). The mean telomere length of grade 3 tumors was significantly shorter than those of grade 1 and 2 tumors (P = .038). Telomerase activity was detected in 81% of tumor samples. The level of telomerase activity in tumors with local recurrence was significantly higher than in tumors without local recurrence (P = .011). Conclusions These results suggest that telomere length correlates with roentgenographic grade as a result of the frequency of cell division, and high telomerase activity indicates the aggressiveness of GCTs.     

Telomerase activity in diagnosis of bladder cancer

OBJECTIVE: Telomerase is an enzyme that can reconstitute the ends of chromosomes after cell division and thus circumvent the damage that occurs in normal adult somatic cells during successive mitotic cycles. Immortal cells have short but stable chromosomes and increased telomerase activity. Transitional cell carcinoma (TCC) has only a few useful markers of diagnostic or prognostic importance. The objectives of this study were to determine whether there was a correlation between telomerase activities and the grade or stage of TCC and whether the activity of the enzyme could serve as a biochemical marker of this tumor. MATERIAL AND METHODS: Telomerase activity was determined by examining, using a polymerase chain reaction-based assay designed using the telomeric repeat amplification protocol (TRAP), urine cell pellets obtained from 42 bladder cancer patients, 18 patients with primary hematuria, 19 patients with benign urologic disease, 14 patients with urologic malignancies other than TCC and 20 healthy volunteers. RESULTS: Telomerase activity was found in 24/31 patients with bladder tumors (77.4% sensitivity) and in 5/77 patients without tumors (93.5% specificity). No correlation was found between telomerase activity and the grade or stage of the tumor. Although none of the urine cell pellets obtained from the 20 healthy volunteers demonstrated telomerase activity, positive telomerase activity was found in two subjects in the benign urologic disease group and in three subjects in the other urologic malignancy group. It was demonstrated that gross hematuria was the cause of false-negative results in six of the nine patients (66.7%). but washing the pellets four times and diluting them before the TRAP assay solved this problem. CONCLUSION: These results indicate that telomerase activity may be a promising marker for TCC but the technical aspects of the technique must be improved before it is used in routine clinical practice as a standard method. False-negative results obtained using gross hematuric urine should be carefully reevaluated and cell pellets should be washed again and diluted before analysis.     

FOCAL INTRATUMORAL HETEROGENEITY FOR TELOMERASE ACTIVITY IN HUMAN PROSTATE CANCER

PURPOSE: The value of telomerase activity as a marker in clinical decision-making is closely related to how representative the analysis of a small tumor sample is for the whole tumor. We therefore evaluated the intratumoral distribution pattern of telomerase activity in prostatic carcinomas. MATERIALS AND METHODS: From 50 prostate cancer patients treated with radical prostatectomy, telomerase activity was determined using the telomeric repeat amplification protocol (TRAP assay). Comparative analysis of at least two separate cancer areas from a single tumor was performed in 42 cases. RESULTS: Telomerase activation has been demonstrated in 90% of the prostatic carcinomas. Focal intratumoral heterogeneity was found in 38.1% of the tumors with at least two different areas examined. Telomerase positivity of all samples from one given tumor was detected in 50%, telomerase negativity of all samples in 11.9%. A heterogeneous telomerase activity pattern was more frequently detected in tumors with a Gleason score < or = 7 than in those with a Gleason score > 7. Furthermore, there was an increase in the proportion of homogeneously telomerase-positive tumors with increase in severity of the Gleason score. The differences reached statistical significance. Telomerase activity was also detected in non-cancerous prostatic tissue samples. CONCLUSIONS: Telomerase activation is nearly ubiquitous in prostatic carcinomas, although a heterogeneous telomerase activity pattern within tumors might produce a false-negative result in the telomerase activity assay. This limits the value of telomerase activity assays for diagnostic means. There is evidence for a shift from telomerase-negative prostate cancer tissue toward telomerase positivity during the progression process of prostate cancer. The relatively high proportion of telomerase-positive nonmalignant prostatic tissue samples argues against cancer-specificity of telomerase activation.     

Expression of telomerase activity in thymoma and thymiccarcinoma tissues; a clinicopathological study

BACKGROUND: Telomerase is a nucleoprotein complex that caps the physical termini of all eukaryotic chromosomes. It is suggested that telomerase play important roles in unlimited cell division acquisition of the malignant phenotype. We studied the relation of telomerase activity in thymoma and thymic carcinoma to the clinicopathological features of these lesions. METHODS: Tissue specimens were surgically resected from patients with thymoma and thymic carcinoma. Telomerase activity was evaluated according to a modified telomeric repeat amplification protocol (TRAP) assay. RESULTS: Telomerase activity was detected in all thymic epithelial tumors. The activity (mean +/- SD: unit/microgram.protein) in thymoma (n = 17) was significantly higher than that in thymic carcinoma (n = 7) [431.8 +/- 400.1 vs. 68.8 +/- 39.8: p < 0.01]. Telomerase activities in thymoma and thymic carcinoma were significantly higher than that in primary lung adenocarcinoma (33.5 +/- 39.2: n = 47), studied as control (p < 0.01). In patients with thymoma, telomerase activity did not correlate with tumor stage according to Masaoka classification (p = 0.776). In patients with thymic carcinoma, however, telomerase activity positively correlated with tumor stage (p = 0.02). In thymoma, telomerase activity positively correlated with the ratio of induced lymphocytes according to Rosai's classification (p = 0.045). CONCLUSION: In thymoma, telomerase activity reflects the presence of immature T-cell lymphocytes in tumor tissue rather than tumor stage or malignant phenotype. In thymic carcinoma, telomerase activity derived directly from cancer cells may relate to tumor stage.     

Telomerase activity in malignant and benign renal tumors

OBJECTIVES: An important characteristic of malignant cells is their unlimited replicative potential, their immortality. In conferring this immortality, the enzyme telomerase is believed to play a crucial role. The detection of telomerase activity provides new knowledge regarding the biologic growth behavior of tumors and offers new diagnostic and therapeutic tools. METHODS: In the present study the sensitive TRAP assay (telomeric repeat amplification protocol) was used to examine 44 malignant renal tumors and 8 benign tumors of the kidney and 52 specimens of normal renal tissue for telomerase activity. RESULTS: Telomerase activity was detected in 63% of tissue samples obtained from histologically confirmed renal cell carcinomas. In cases of renal cell carcinoma restricted to the kidney, telomerase activity was detected in 58%. In cases in which tumor growth has progressed beyond the limits of the organ, telomerase activity was found in 69%. This stage dependence, however, did not reach statistical significance. No correlation to tumor grading was observed. Telomerase activity was found less frequent in chromophobe renal cell carcinomas. Neither the 8 benign renal tumors (4 oncocytomas and 4 angiomyolipomas) nor the specimens of normal kidney showed any evidence of telomerase activity. CONCLUSIONS: The proportion of remarkable slow-growing renal cell carcinomas showing telomerase activity is less than in other malignancies and may correlate with biologic growth behavior. Possible explanations include the presence of an alternative pathway, called ALT (alternative lengthening of telomeres) and an association with the loss or presence of the telomerase suppressor on the short arm of chromosome 3. Prolonged follow-up will be of special interest to determine whether lack of telomerase activity predicts favorable outcome.     

Semi-quantitative analysis of telomerase activity of exfoliated cells in urine of patients with urothelial cancers: Causative factors affecting sensitivity and specificity

We previously reported that detection of telomerase activity in urinary exfoliated cells obtained from urothelial cancer patients by telomeric repeat amplification protocol (TRAP) assay is a more sensitive method of diagnosis than conventional urine cytologic examination, particularly in patients with grade 1 tumors. To establish this method as a noninvasive screening test for the diagnosis of urothelial cancers, we performed the semi-quantitative analysis of telomerase activity using Telomerase PCR ELISA?. Spontaneous voided urine samples were obtained from 65 urothelial and 58 non-urothelial cancer patients. When the mean + 2 standard deviation of telomerase activity in urine sediments of non-urothelial cancer patients was arbitrarily determined as a cut-off, the sensitivity of TRAP enzyme-linked immunosorbent assay (ELISA) and the conventional cytology were 57% and 35%, respectively. Detection rate was significantly higher in semi-quantitative TRAP assay than in conventional cytologic examination in grade 1 cancer patients (52% vs. 5%, p = 0.00195). False positives were detected in 5% of non-urothelial cancer patients without pyuria and in 11% of non-urothelial cancer patients with pyuria (p = 0.395). Telomerase activity was enhanced in some cases after phenol extraction or extracting epithelial cells by using Dynabeads of macroscopic hematuria and pyuria, indicating that hematuria and pyuria might contribute to false negatives. In conclusion, the TRAP-ELISA method is superior to the standard TRAP assay in quantitativeness and simplicity of the experimental procedure. Detection of telomerase activity in urine sediments is particularly useful for the diagnosis of low-grade tumors. However, telomerase activity in patients with high grade tumors often might be underestimated due to the excessive amount of exfoliated epithelia with necrotic tissues, hematuria, and pyuria.     

Telomere Length and Telomerase Activity in Bladder and Prostate Cancer Cell Lines

Background: Specific repeats of oligonucleotides at the ends of chromosomes (telomeres) are shortened by cell division in somatic cells and are thought to be related to aging. Immortal cells such as germ-line cells or cancer cells have demonstrated increased activity of the telomere-elongating enzyme (telomerase). The length of the telomeres of these cells is stable regardless of cell division. We examined the telomere length and telomerase activity in 3 bladder (JTC30, JTC32, and T24) and 2 prostate cancer (LNCaP and DU145) cell lines.
Methods: Telomere lengths were evaluated by Southern blot analysis with a oligonucleotide probe, (TTAGGG)_S and telomerase activities were detected with a polymerase chain reaction-based assay method.
Results: Telomerase activity was detected in all of the cell lines. Each cell line had a specific telomere length. In 2 bladder cancer cell lines (JTC30 and JTC32), the telomere length decreased with increased passage of the cells.
Conclusion: The presence of telomerase may be a biological character of bladder and prostate cancers as well as other malignancies, although it does not always compensate telomere shortening.     

Consistent decrease in telomere length in parathyroid tumors but alteration in telomerase activity limited to malignancies: preliminary report

Telomerase is known to be activated and telomere length altered in various types of malignant and benign tumors, but whether this is also the case for parathyroid lesions has hitherto been unclear. We therefore investigated telomerase activity and telomere length in 3 parathyroid metastatic cancers, 6 adenomas, 2 cases of parathyroid hyperplasia, and 16 samples of normal parathyroid tissue. Telomerase activity, assayed by the telomeric repeat amplification protocol, was detected in all of the parathyroid cancers (100%), in none of the 8 parathyroid benign lesions, and in only 1 of the 16 normal parathyroid samples (8.3%). Telomere length, determined by the terminal restriction fragment assay, was reduced in the tumor tissues with a mean telomere length of 8.23 +/- 0.86 kbp compared with the 12.61 +/- 0.81 kbp for the 16 age-matched subjects (p = 0.002). The results indicate that telomerase activity and telomere length may reflect the biologic behavior of individual parathyroid lesions.     

膀胱癌癌旁组织端粒酶活性检测的临床意义

目的　探讨膀胱癌癌旁组织端粒酶活性检测的临床意义。　方法　采用端粒重复序列扩增 (TRAP)法 ,检测 2 4例膀胱癌组织及癌旁组织中端粒酶活性表达。　结果　 2 4例癌旁组织中端粒酶活性表达阳性 10例 (42 % ) ,癌旁组织端粒酶活性表达与原发灶癌病理分级分期相关 ,并与癌复发相关。　结论　膀胱癌癌旁组织端粒酶活性检测可作为判断膀胱癌预后的指标之一     

Telomerase activity Simplification of assay and detection in bladder tumor and urinary exfoliated cells

Detection of telomerase activity can differentiate malignant from benign cells. However, the original telomeric repeat amplification protocol (TRAP) methods had a number of limitations including a radioisotope labeling [α(32)P] dCTP [α(32)P] dGTP system. We developed digoxigenin labeled CX primer to detect telomerase activity without using radioisotope and attempted to detect telomerase activity of bladder tumor and exfoliated cells in bladder cancer patients. Telomerase activity was detected in 5 (71%) of 7 patients diagnosed with grade 1, 31 (97%) of 32 grade 2, and 11 (100%) of 11 grade 3 bladder tumors. In urinary exfoliated cells, 32 (82%) of 39 grades 1 or 2 bladder tumors were positive for telomerase activity but 20 (51%) of 39 were positive for urinary cytology (P < 0.01). Ten (91%) of 11 of grade 3 tumors were positive for telomerase activity and 11 (100%) of 11 were positive urinary cytology. Three of 100 noncancerous patients were positive for telomerase activity. Sensitivity, specificity, and positive predictive value of telomerase activity assay in urinary exfoliated cells were 84%, 97%, and 93%, respectively. Telomerase activity may be a useful diagnostic marker to detect the existence of immortal cancer cells in the urine.     

端粒酶活性及其结构基因在人脑胶质瘤中的表达

目的 研究分析端粒酶活性及相关结构基因在不同级别脑胶质瘤中的表达,探讨端粒酶与脑胶质瘤的相关性及其临床意义。方法 采集40例脑胶质瘤手术切除标本、4例正常脑组织,通过半定量端粒重复序列扩增（TRAP）－银染方法检测端粒酶活性水平;通过半定量逆转录－聚合酶链反应（RT－PCR）检测端粒酶相关结构基因hTR、TP1、hTRT的 mRNA表达水平,结果 在40例胶质瘤标本的33例（82.5%)中检测出端粒酶活性,而在正常脑组织中无端粒酶活性的表达,不同级别脑胶质瘤之间端粒酶活性水平差异有显著性,脑胶质瘤粒酶活性水平与hTRT基因的表达呈显著正相关,而与TP1、hTR 的表达无显著相关。结论 端粒酶活性可以作为脑胶质瘤的恶性标记之一,hTRT基因是一个端粒酶的正调控结构基因,hTRT的表达与细胞永生化和恶性肿瘤形成过程中的端粒酶的激活机制有关,hTR基因是端粒酶活性必须的组分,但不影响端粒酶活性的高低。     

TRAP-Sybr Green I法检测结直肠癌端粒酶活性

目的应用新型核酸荧光染料Sybr Green Ⅰ检测端粒酶活性，并探讨该酶活性在结直肠癌临床诊断中的意义。方法用端粒重复扩增(TRAP)结合Sybr Green Ⅰ技术检测46例结直肠癌及19例癌旁组织中端粒酶的活性。结果Sybr Green Ⅰ能清晰显示端粒酶扩增后的条带。端粒酶活性在结直肠癌中阳性率为89．13％，在癌旁组织中未发现端粒酶活性。端粒酶活性与细胞分化程度、Dukes分期、有无淋巴结转移均无关。结论用Sybr Green Ⅰ检测端粒酶活性对于诊断结直肠癌具有重要的临床辅助价值。     

端粒酶hTERT基因反义核酸对TNF-α诱导前列腺癌PC3细胞凋亡的影响

目的:探讨hTERT基因的两端部分硫代修饰反义寡核苷酸(AS PS-ODN)抑制前列腺癌细胞PC3端粒酶活性后,肿瘤坏死因子α(TNF-α)对PC3凋亡的增敏作用。方法:采用端粒重复序列扩增法及TRAP-PCR-ELISA法检测前列腺癌细胞的端粒酶活性;采用MTT比色试验观察hTERTAS PS-ODN与TNF-α共同作用对前列腺癌细胞PC3生长活力的影响;倒置显微镜观察凋亡细胞的形态变化;通过流式细胞仪测定凋亡细胞的百分率。结果:hTERTAS PS-ODN作用于PC3细胞48h,其端粒酶活性下降,作用72h,其端粒酶活性受到抑制,与正义寡核苷酸组及空白对照组相比差异有显著性(P<0.05)。hTERTAS PS-ODN作用于PC3细胞后加入TNF-α作用48h,PC3细胞的抑制率与对照组、S PS-ODN组、AS PS-ODN组、TNF-α组及S PS-ODN+TNF-α组相比有统计学差异(P<0.01)。hTERTAS PS-ODN作用于PC3细胞后加入TNF-α作用48h,细胞出现典型的凋亡形态变化。hTERT ASPS-ODN作用于PC3细胞后加入4μg/ml TNF-α作用48h,凋亡细胞的百分率分别与对照组、S PS-ODN组、AS PS-ODN组、TNF-α组及S PS-ODN+TNF-α组相比差异有显著性(P<0.05)。结论:hTERTAS PS-ODN能降低前列腺癌细胞PC3端粒酶活性;hTERT基因反义核酸对TNF-α诱导前列腺癌细胞PC3凋亡有增敏作用。     

Androgen depletion activates telomerase in the prostate of the nonhuman primate, Macaca mulatta

BACKGROUND: The activity of telomerase, an enzyme that synthesizes telomeric repeats at the ends of chromosomes, is not detectable in normal human prostate. However, the majority of human prostate cancers exhibit telomerase activity. Since androgens play a major role in prostate tumorigenesis, we investigated the effect of androgen-depletion on the expression of telomerase activity in the prostate. METHODS: Adult male rhesus monkeys were either bilaterally castrated or subjected to sham surgery (n = 5 each). Approximately 6 weeks later, the animals were killed and the different regions of the prostate gland were removed and frozen immediately. Telomerase activity was assayed using the telomeric repeat amplification protocol. RESULTS: All five regions of the prostate from sham operated control animals failed to exhibit telomerase activity. In the castrated monkey, all regions of the prostate, except for the anterior lobe, expressed high levels of telomerase activity. CONCLUSIONS: Our results indicate that in monkeys, androgen-ablation leads to up-regulation of telomerase activity. The negative-regulation of telomerase activity by androgens is probably lost during prostate tumorigenesis.     

乳腺癌端粒长度与端粒酶活性变化的相关性研究

目的　检测乳腺癌及其癌旁组织中的端粒 (TLM )长度、端粒酶 (TLMA )活性的表达 ,探讨乳腺癌端粒长度与端粒酶活性的关系。方法　采用端粒酶重复扩增实验 (TRAP) 银染法检测端粒酶活性 ,以地高辛标记的Southern杂交的方法检测端粒长度。结果　端粒酶活性在乳腺癌TNM分期的进展中由Ⅰ期的 5 9.1%增高到Ⅳ期的 92 .9% ,而端粒长度在乳腺癌TNM分期的进展中不断缩短 ,由Ⅰ期的 (7.5 4± 0 .95 )kb缩短到Ⅳ期的 (5 .0 3± 0 .5 3 )kb。恶性乳腺肿瘤癌旁组织中的平均端粒长度皆位于正常水平。乳腺癌中端粒酶阳性表达组的端粒长度为 (4 .45± 1.3 9)kb显著短于端粒酶阴性组的 (5 .70± 1.2 3 )kb。结论　恶性乳腺肿瘤将端粒酶激活并没有绝对延长其染色体末端的端粒长度 ,而且 ,随着肿瘤的发展端粒片段还会缩短 ,并与端粒酶活性似乎存在着反向的关系。     

Detection of telomerase activity in prostatic fluid specimens

We report here the extended use of the telomeric repeat amplification protocol (TRAP) assay for the detection of telomerase activity in fresh prostatic fluid obtained from anesthetized patients. Telomerase activity was detected in pellet extract and/or supernatant fluid of specimens obtained from 25 of 30 prostate cancer (PCa) patients (83%), whereas no activity was similarly detectable in specimens taken from 8 of 9 patients (89%) without clinical evidence of PCa. The positive predictive value (PPV) of the TRAP assay for PCa in this pilot study was 96%. We found a strong correlation between telomerase activity in prostatic fluid specimens and serum prostate specific antigen (PSA) values. Telomerase activity was found in 84% of specimens from patients with PSA values >4 ng/ml, whereas in specimens from patients with PSA values 4. In prostatic fluid from PCa patients with Gleason scores of 相似文献