Expression Of Human Telomerase RNA Research Articles

La Protein Required for Internal Ribosome Entry Site–Directed Translation Is a Potential Therapeutic Target for Hepatitis C Virus Replication

Translation of the hepatitis C virus (HCV) is mediated by an internal ribosome entry site (IRES). Here, we analyzed the functional relevance of La protein for replication of HCV using an infectious HCV clone, JFH-1. A single-nucleotide mutation from A to U was introduced at the 338th nucleotide in the stem-loop domain IV structure of HCV IRES, which stabilized stem-loop IV and abolished translation and replication of JFH-1 almost completely. During JFH-1 replication, translation initiation factors required for HCV IRES activity, including La protein, polypyrimidine tract binding protein (PTB), PSMA7, and PCBP2, were significantly induced in Huh-7.5 cells. Interestingly, JFH-1 infection increased telomerase activity and induced the expression of human telomerase RNA (hTR) in Huh-7.5 cells. In 37 tissue specimens from patients with chronic hepatitis C, La protein significantly correlated with the representative essential telomerase components hTR, p23, and HSP90 (P<.001). Recombinant adenovirus that expressed short-hairpin RNA against La protein successfully suppressed the levels of La protein and core protein of JFH-1 to 30% of that in the control cells. HCV infection might be strongly related to telomerase activity in the liver through La protein induction. Inhibition of La protein substantially repressed JFH-1 replication; therefore, La protein is a potential therapeutic target for HCV.

Effect of Helicobacter pylori eradication on oncogenes and cell proliferation

Helicobacter pylori , the main cause of chronic gastritis, is a class 1 gastric carcinogen. However, it remains unclear whether H. pylori affects molecular alterations in chronic gastritis. Thus, this study was designed to investigate the effect of H. pylori eradication on the expression of human telomerase RNA (hTR), human telomerase reverse transcriptase (hTERT), c-myc and proliferation nuclear cell antigen (PCNA) in H. pylori associated chronic gastritis. hTR was determined by in situ hybridization, hTERT, c-myc and PCNA were detected by immunohistochemistry using stomach tissues obtained from 39 H. pylori-infected and 21 H. pylori-negative patients with chronic gastritis before and after H. pylori eradication therapy or treatment for symptom relief only. Levels of hTR, hTERT, c-myc and PCNA were significantly higher in H. pylori-infected mucosa (51.3%, 53.8%, 53.8% and 16.8 +/- 5.8, respectively) when compared to H. pylori-negative mucosa before therapy (19.0%, 23.8%, 28.6%, 8.8 +/- 3.4, respectively; P < 0.05 in all cases). In patients with successful eradication of H. pylori the levels of hTR, hTERT, c-myc and PCNA (55.5%, 59.3%, 59.3%, 16.8 +/- 5.8, respectively) were significantly reduced after the therapy (22.2%, 22.2%, 14.8%, 7.0 +/- 5.0, respectively; P < 0.05 in all cases). In the H. pylori failed eradication and H. pylori-negative groups, there was no statistical difference in all four measurements. H. pylori infection may induce the overexpression of hTR, hTERT, c-myc and stimulate cell proliferation. Eradication of H. pylori may reverse the aberrant expression of these oncoproteins and thus correct the abnormal cell proliferation.

Detection of human telomerase RNA in the tumour‐surrounding mucosa of bladder carcinomas as a marker for premalignant transformation

To investigate tumour tissue and non-malignant tumour-surrounding bladder mucosa (NTSBM) for expression of human telomerase RNA (hTR) as a possible marker for premalignant transformation of urothelial cells. From 67 patients with superficial transitional cell carcinoma (TCC), sections with representative tumour tissue and NTSBM were selected for evaluation. Sections with moderate or severe dysplasia were omitted from evaluation. hTR expression was detected with in situ hybridization using a 35S-UTP-labelled riboprobe, and analysed semiquantitatively by counting the hybridization signals. In 45 of the 67 patients hTR expression was moderate or strong in tumour tissue, and in 20 hTR expression was moderate or strong in NTSBM. Moderate or strong hTR expression was detected in the NTSBM from 19 of 60 patients with pTa/pT1 tumours. Of the 56 patients who were treated conservatively, eight had tumour recurrence, of whom five had moderate or strong hTR expression in the TSBM, compared with only 14 of 48 patients without tumour recurrence. Detecting hTR expression and the morphological distribution of hTR hybridization signals in NTSBM by in situ hybridization might indicate premalignant alterations involved in tumour recurrence.

The study on the correlation between telomerase and histopathologic features of retinoblastoma

To investigate the correlation between the expression of human telomerase RNA (hTR), human telomerase reverse transcriptase (hTRT) and the proliferation and differentiation of retinoblastoma (RB). In 37 RB specimens, the expression of hTR and hTRT was investigated with the methods of in situ hybridization (ISH). Immunohistochemical method was performed to detect the expression of proliferating cell nuclear antigen (PCNA) in RB. All data were analyzed statistically with SPSS. Of 37 RB specimens, positive expression of hTR and hTRT was 83.8% and 89.2%, respectively, while negative results were observed in 2 normal retina tissues. The expressions of hTR and hTRT showed a close correlation to the proliferation and differentiation of RB. Furthermore, there was a close correlation among the expression of hTR and hTRT. These results suggest that higher expression of hTRT and hTR may be used as new parameters for evaluating the degree of malignancy of RB. The close correlation between the expression of hTR and hTRT and proliferation and differentiation degrees of RB indicats that the expression of telomerase can influence the proliferation and differentiation of RB. Because the telomerase is presented in the Rb, therefore, modulating the telomerase activity may be used as a novel treatment for the RB.

Telomerase activity in human hepatocellular carcinoma: parallel correlation with human telomerase reverse transcriptase (hTERT) mRNA isoform expression but not with cell cycle modulators or c-Myc expression

Background: To explore the possible regulatory mechanisms of telomerase, we examined the telomerase activity (TA), expression of human telomerase RNA (hTR), human telomerase reverse transcriptase (hTERT) mRNA isoforms and cell cycle modulators in human hepatocellular carcinoma (HCC) cell lines (J5, J7) and a normal human immortalized hepatic epithelial cell line (Chang-liver). Methods: The cell lines were chemically synchronized in either G1, G1/S, G2/M or M phases. TA was measured by polymerase chain reaction (PCR)-based telomerase repeat amplification protocol assay. The hTR and hTERT mRNA levels were analyzed by reverse transcriptase–polymerase chain reaction. Western blotting and immunocytochemistry were used to assay the cell cycle modulators. Results: The TA of J5, J7 and Chang-liver cell lines tested was highest in M phase. The expression level of hTERT mRNA associated with the highest TA detected in the M phase of HCC cell lines. Chang-liver expressed markedly less TA and hTERT mRNA than J5 or J7. The elevated TA and expression of hTERT mRNA isoforms in M phase of HCC cell lines did not significantly correlate with that of the cell cycle modulators and c-Myc. Conclusions: The results implicate that regulation of TA is related to hTERT mRNA isoform expression, and that regulation is different between the cell immortalization and tumorigenesis.

Telomerase Can Inhibit the Recombination-based Pathway of Telomere Maintenance in Human Cells

Telomere length can be maintained by telomerase or by a recombination-based pathway. Because individual telomeres in cells using the recombination-based pathway of telomere maintenance appear to periodically become extremely short, cells using this pathway to maintain telomeres may be faced with a continuous state of crisis. We expressed telomerase in a human cell line that uses the recombination-based pathway of telomere maintenance to test whether telomerase would prevent telomeres from becoming critically short and examine the effects that this might have on the recombination-based pathway of telomere maintenance. In these cells, telomerase maintains the length of the shortest telomeres. In some cases, the long heterogeneous telomeres are completely lost, and the cells now permanently contain short telomeres after only 40 population doublings. This corresponds to a telomere reduction rate of 500 base pairs/population doubling, a rate that is much faster than expected for normal telomere shortening but is consistent with the rapid telomere deletion events observed in cells using the recombination-based pathway of telomere maintenance (Murnane, J. P., Sabatier, L., Marder, B. A., and Morgan, W. F. (1994) EMBO J. 13, 4953-4962). We also observed reductions in the fraction of cells containing alternative lengthening of telomere-associated promyelocytic leukemia bodies and extrachromosomal telomere repeats; however, no alterations in the rate of sister chromatid exchange were observed. Our results demonstrate that human cells using the recombination-based pathway of telomere maintenance retain factors required for telomerase to maintain telomeres and that once the telomerase-based pathway of telomere length regulation is engaged, recombination-based elongation of telomeres can be functionally inhibited.

Telomerase activity and expression of telomerase subunits in the testicular tissue of infertile patients

Objective: Determination of telomerase activity and the expression of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in the testicular tissue of patients with infertility arising from various causes. Design: Prospective observational study. Setting: A university hospital. Patient(s): Thirty-three patients with azoospermia arising from various causes. There were 12 testicular biopsy specimens from patients with Sertoli cell–only syndrome, 9 from patients with maturation arrest, and 12 from patients with obstructive azoospermia and normal histologic findings. Intervention(s): Thirty-three testicular biopsies. Main Outcome Measure(s): Correlation of histologic findings at testicular biopsy with telomerase activity, hTERT, and hTR. Result(s): All 12 biopsy specimens from patients with obstructive azoospermia were positive for telomerase activity, hTR, and hTERT. Biopsy specimens from the 9 patients with maturation arrest were positive for telomerase activity in 8 cases, hTR in 9 cases, and hTERT in 5 cases. None of the patients with Sertoli cell–only syndrome showed either telomerase activity or hTERT, but all of them showed hTR. Conclusion(s): Telomerase activity and evidence of hTERT in testicular tissue are highly sensitive and highly specific markers of gametogenesis, which could gain in importance as part of the fertility workup before microinjection procedures.

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

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

In Situ Hybridization Analysis of the Expression of Human Telomerase RNA in Normal and Pathologic Conditions of the Skin

Human telomerase RNA (hTER) expression in skin was examined by in situ hybridization analysis. All newborn foreskins examined (n = 5) expressed hTER in epidermal basal cells at moderate levels. Telomerase RNA was not detectable in most adult specimens from sun protected areas (six of seven), whereas all samples obtained from sun exposed areas (n = 8) showed moderate hTER signals in epidermal basal cells. Telomerase RNA was also detected at moderate to strong levels in basal cells of psoriasis, contact dermatitis, and the proliferative cells of the anagen hair bulb. Basal cell carcinoma samples (14 of 15) had moderate to high hTER expression throughout the entire tumor, whereas squamous cell carcinomas (seven of eight) showed variable intensities of hTER expression but only in the cells at the periphery of tumor nests. All melanomas examined (n = 5) had moderate hTER expression in all tumor cells. The hTER signal intensities in skin tumors did not correlate with the age or sex of the donors, the clinical history of the lesions, or the histologic subtypes. To address whether hTER expression correlated with the proliferative state, sequential sections were stained with anti-Ki-67 antibody, a proliferation marker. In newborn foreskins, squamous cell carcinomas, and basal cell carcinomas, the distributions of hTER and Ki-67 were similar but not always identical. Telomerase RNA was more abundant than Ki-67 in the basal and suprabasal layer of newborn foreskins, suggesting that hTER expression is present both in actively cycling and in resting cells.

Expression of telomerase RNA component correlates with the MIB-1 proliferation index in ependymomas.

Although there is general agreement that certain morphologic subtypes of ependymoma are benign, the biologic behavior of other ependymal neoplasms is poorly understood and not clearly related to conventional histopathologic criteria. The absence of universally accepted standards has prompted the search for more objective biologic markers. Telomerase is an RNA-containing enzyme associated with immortality in proliferating stem cells and many tumors. We investigated the proliferative activity of 26 ependymomas as determined by MIB-1 immunolabeling and compared the results with the in situ expression of human telomerase RNA (hTR) and WHO tumor grade. The study included 9 WHO grade I ependymomas (6 subependymomas and 3 myxopapillary ependymomas), 13 WHO grade II ependymomas, and 4 anaplastic (WHO grade III) ependymomas. The proliferation index (PI) and telomerase RNA expression were significantly increased in grade III ependymomas (p < 0.0001 for PI and p = 0.0015 for hTR). In these tumors, the PI and hTR expression were highly correlated (p = 0.0001). Of note, a single case designated grade II showed both increased proliferative activity and the highest hTR expression detected in this series of ependymal neoplasms. Our results suggest that the PI and hTR expression may be important biologic markers, independent of other histopathologic criteria of tumor grade. Future studies examining the correlation of MIB-1 cell kinetics and hTR expression with clinical parameters in selected ependymoma subtypes are needed to determine the prognostic relevance of these markers.

Expression of human telomerase RNA is an early event of stomach carcinogenesis.

Expression of human telomerase RNA (hTR) and telomerase activity in gastric cancer and corresponding non‐cancerous mucosa were studied. Telomerase activity was detected in 23 (88%) of 26 carcinoma tissues. Although all tumor specimens and non‐cancerous mucosa expressed various levels of hTR, 21 (81%) of 26 cases expressed hTR at a higher level in the tumor than that in the corresponding mucosa. All 8 gastric carcinoma cell lines also expressed hTR at high levels. Nine (35%) of 26 non‐cancerous mucosa showed telomerase activity and all of them contained intestinal metaplasia. The incidence of telomerase‐positive mucosa in grade 2 intestinal metaplasia was significantly higher than that in grade 0 or grade 1 intestinal metaplasia, whereas hTR overexpression was found in grade 0 or grade 1 intestinal metaplasia as well as grade 2 intestinal metaplasia. The degree of Heticobacter pylori infection increased in parallel with the level of hTR expression and telomerase positivity. These results overall suggest that Helicobacter pylori infection may he a strong trigger for hTR overexpression in intestinal metaplasia, and this may lead to telomerase reactivation.