Telomerase Catalytic Subunit mRNA Research Articles

Expression of Cellular and Extracellular TERRA, TERC and TERT in Hepatocellular Carcinoma.

Non-coding RNAs are transcribed from telomeres and the telomeric repeat-containing RNAs (TERRA) are implicated in telomere homeostasis and in cancer. In this study, we aimed to assess in hepatocellular carcinoma (HCC) the cellular and extracellular expression of TERRA, the telomerase RNA subunit (TERC) and the telomerase catalytic subunit (TERT). We determined by qPCR the expression level of TERRA 1_2_10_13q, TERRA 15q, TERRA XpYp, TERC and of TERT mRNA in HCC tissues and in the plasma of HCC patients. Further, we profiled the same transcripts in the HCC cell lines, HA22T/VGH and SKHep1C3, and in the extracellular vesicles (EVs) derived from their secretomes. We found that the expression of TERRA and TERT mRNA was significantly deregulated in HCC, being TERRA downregulated and TERT mRNA upregulated in HCC tissues vs. the peritumoral (PT) ones, and the receiver operating characteristic (ROC) curve analyses revealed a significant ability in discriminating HCC from PT tissue. Further, the determinations of circulating TERRA and TERC showed higher amounts of these transcripts in the plasma of HCC patients vs. controls and ROC analyses gave significant results. The expression characterization of the cultured HCC cells showed their ability to produce and secrete TERRA and TERC into the EVs; the ability to produce TERT mRNA that was not detectable in the EVs; and the ability to respond to sorafenib treatment increasing TERRA expression. Our results highlight that: (i) both cellular and extracellular expressions of TERRA and TERC are dysregulated in HCC as well as the cellular expression of TERT mRNA and (ii) the combined detection of TERRA and TERC in plasma may represent a promising approach for non-invasive diagnostic molecular indicators of HCC.

Apoptotic endonuclease EndoG induces alternative splicing of telomerase catalytic subunit hTERT and death of tumor cells

Telomerase activity is known to be regulated by alternative splicing of its catalytic subunit hTERT (human Telomerase Reverse Transcriptase) mRNA. Induction of non-active spliced hTERT leads to inhibition of telomerase activity. However, very little is known about the mechanism of hTERT mRNA alternative splicing. The aim of this study was to determine the role of apoptotic endonuclease EndoG in alternative splicing of hTERT and telomerase activity. Strong correlation was found between expression of EndoG and hTERT splice-variants in 12 colon cancer cell lines. Overexpression of EndoG in СаСо-2 cells downregulated the expression of active full-length hTERT variant and upregulated non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, dramatically shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. These data indicated the participation of EndoG in alternative splicing of mRNA of telomerase catalytic subunit, regulation of telomerase activity and cell fate.

PinX1 regulation of telomerase activity and apoptosis in nasopharyngeal carcinoma cells.

BackgroundHuman interacting protein X1 (PinX1) has been identified as a critical telomerase inhibitor and proposed to be a putative tumor suppressor gene. Loss of PinX1 has been found in a large variety of malignancies, however, its function in inhibiting telomerase activity of tumor cells is not well documented. Here we show that PinX1 is essential for down-regulation telomerase activity of nasopharyngeal carcinoma.MethodsExpression vectors of human PinX1 (pEGFP-C3-PinX1) and its small interfering RNA (PinX1-FAM-siRNA) were constructed and transfected into NPC. Their effects on mRNA of telomerase catalytic subunit (hTERT), telomerase activity, cell proliferation, cell migration, wound healing, cell cycles and apoptosis were examined using semi-quantitative RT-PCR, stretch PCR, MTT assay, Transwell, scratch assay and flow cytometry, respectively.ResultsTransfection of pEGFP-C3-PinX1 and PinX1-FAM-siRNA increased and reduced PinX1 mRNA by 1.6-fold and 70%, respectively. Over-expression of PinX1 decreased hTERT mRNA by 21%, reduced telomerase activity, inhibited cell growth, migration and wound healing ability, arrested cells in G0/G1 phase, and increased apoptotic index. In contrast, down-regulation of PinX1 did not alter the above characteristics.ConclusionsPinX1 may play important roles in NPC proliferation, migration and apoptosis and has application potential in tumor-targeted gene therapy.

Expression profiles of proliferative and antiapoptotic genes in sporadic and colitis‐related mouse colon cancer models

Elevated levels of survivin, telomerase catalytic subunit (TERT), integrin-linked kinase (ILK), cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS) and the regulatory factors c-MYB and Tcf-4 are often found in human cancers including colorectal cancer (CRC) and have been implicated in the development and progression of tumorigenesis. The aim of this study was to determine the expression of these genes in mouse models of sporadic and colitis-associated CRC. To address these issues, we used qRT-PCR approach to determine changes in gene expression patterns of neoplastic cells (high-grade dysplasia/intramucosal carcinoma) and surrounding normal epithelial cells in A/J and ICR mouse strains using laser microdissection. Both strains were injected with azoxymethane and ICR mice were also given drinking water that contained 2% dextran sodium sulphate. In both sporadic (A/J mice) and colitis-associated (ICR mice) models of CRC, the levels of TERT mRNA, COX-2 mRNA and Tcf-4 mRNA were higher in neoplastic cells than in surrounding normal epithelial cells. In contrast, survivin mRNA was upregulated only in neoplastic cells from A/J mice and ILK mRNA was upregulated only in neoplastic cells from ICR mice. However, the expression of iNOS mRNA was similar in normal and neoplastic cells in both models and c-MYB mRNA was actually downregulated in neoplastic cells compared with normal cells in both models. These findings suggest that the genetic background and/or the molecular mechanisms of tumorigenesis associated with genotoxic insults and colonic inflammation influence the gene expression of mTERT, COX-2, Tcf-4, c-MYB, ILK and survivin in colon epithelial neoplasia.

Sulfhydryl Angiotensin-Converting Enzyme Inhibitor Promotes Endothelial Cell Survival through Nitric-Oxide Synthase, Fibroblast Growth Factor-2, and Telomerase Cross-Talk

The protective effect exerted by angiotensin-converting enzyme inhibitors (ACEI) in cardiovascular diseases caused by endothelial injury and aging has been attributed to the restoration of endothelial cell functions. Recently, we demonstrated a central role of the fibroblast growth factor-2 (FGF-2)/FGF receptor-1 system in mediating the acquisition of an angiogenic phenotype in coronary microvascular endothelium exposed to ACEI. Here, we report on the rescuing effect of ACEI on impaired endothelium and the intracellular signaling mechanisms that lead endothelial cells to enter apoptosis and to senesce. Conditions mimicking pathological cell damage (serum deprivation) lead to endothelial apoptosis as evidenced by increased caspase-3 activity. ACEI enhanced cell survival through activation of prosurvival and antiaging signals involving Akt phosphorylation, endothelial nitric-oxide synthase (eNOS) expression and activation, FGF-2 and telomerase catalytic subunit (TERT) up-regulation, and delayed senescence. In microvascular endothelial cells exposed to ACEI, Akt/eNOS pathway-dependent FGF-2 was necessary for gene transcription of TERT. These protective effects were particularly evident for sulfhydryl-containing ACEI (zofenoprilat), which were reported to exhibit potent antioxidant effects. In conclusion, ACEI with antioxidant properties up-regulate eNOS, FGF-2, and TERT mRNA, which favor endothelial cell survival and prolong their lifespan, thus restoring endothelial cell functions after vascular damage. These effects could explain the beneficial effects of these drugs in various cardiovascular diseases associated with endothelial injury and aging.

Differential expression of human telomerase catalytic subunit mRNA by In situ hybridization in pheochromocytomas

In pheochromocytomas, it is very difficult to predict malignant potential by conventional histology or immunohistochemical and molecular markers. We investigated the expression of human telomerase catalytic component (hTERT) mRNA, hTERT protein, Ki-67 antigen, and p27kip1 in pheochromocytomas (27 benign, 7 suspected malignant, and 7 malignant), and evaluated the possibility of expressions of these proteins, and hTERT mRNA serve as diagnostic markers for predicting the biological behavior of these tumors. All tumors showed the classical histology and typical immunohistochemical pattern. By in situ hybridization, hTERT mRNA was expressed in 5/7 malignant tumors (defined as the presence of metastasis and/or extensive local invasion) as compared with 3/27 benign tumors. We examined the hTERT by immunohistochemistry to confirm the mRNA. hTERT mRNA expression was correlated with hTERT protein expression. All benign tumors exhibited no immunopositivity or <1% of cells stained for Ki-67 antigen. Six out of seven malignant tumors have shown either hTERT mRNA expression or Ki-67 immunoreactivity. While no statistical difference in p27kip1 expressions was observed among benign, malignant, and suspected malignant tumors, there was a statistical difference between the normal adrenal medulla samples and tumors (p < 0.001). Thus, hTERT mRNA detection by in situ hybridization, hTERT expression, and Ki-67 antigen expression are all useful tools for differentiating malignant from benign pheochromocytomas.

Spontaneous Immortalization of Human Epidermal Cells with Naturally Elevated Telomerase

This work explores spontaneous immortalization in keratinocytes, derived from two skin samples, that display naturally elevated telomerase activity. Serially passaged with 3T3 feeder layer support, the keratinocytes were examined for colony-forming ability, telomerase activity, telomere length, and finally gene expression using Affymetrix DNA microarrays. The cells initially exhibited normal karyotypes and low colony-forming efficiencies typical of normal epidermal cells, but after 40 passages (approximately 400 generations) colony-forming ability increased markedly, yielding immortalized lines exhibiting a small number of chromosomal aberrations and functionally normal p53. An improved protocol for analysis of microarray data permitted detection of 707 transcriptional changes accompanying immortalization including reduced p16(INK4A) mRNA. Telomerase activity was clearly elevated in cells even at low passage from both samples, and telomerase catalytic subunit mRNA was greatly elevated in those with elevated colony-forming ability. The data raise the possibility of an unusual natural phenotype in which aberrant telomerase regulation extends keratinocyte lifespan until rare variants evade senescence. In addition to revealing a potential tumor-prone syndrome, the findings emphasize the desirability of carefully minimizing the degree or timing of elevated expression of telomerase used to immortalize cells for therapeutic purposes.

The Role of Human Telomerase Catalytic Subunit mRNA Expression in Cervical Dysplasias

Telomerase activity and human telomerase reverse transcriptase (hTERT) mRNA expression were investigated in cervical specimens and were correlated with cytologic findings and the presence of human papilloma virus (HPV) infection. Telomerase activity was evaluated by the telomeric repeat protocol assay and hTERT mRNA expression was evaluated by reverse transcriptase polymerase chain reaction (PCR). HPV DNA was detected by PCR, as well as restriction endonuclease digestion. HPV DNA was detected in all 82 specimens with abnormal cytologic findings and in 4 of 34 normal samples. Low-grade squamous intraepithelial lesions (LGSILs) were present in 74 of 82 specimens (90.2%) and high-grade squamous intraepithelial lesions (HGSILs) were present in 8 of 82 (9.75%) specimens. Seven of the eight HGSIL (87.5%) and 26 of 74 LGSIL (35.1%) specimens were hTERT positive, whereas all normal specimens were hTERT mRNA negative. Telomerase activity was detected in 21 of 74 (28.4%) LGSIL/atypical squamous epithelial cells of undetermined significance (ASCUS) and in five of eight (62.5%) HGSIL samples. A correlation was observed among telomerase activity, hTERT mRNA expression, and high-risk HPV infection in HGSIL samples (P < 0.001). High-risk HPV infection assessment showed 75% sensitivity and 72.2% specificity for HGSILs. Telomerase activity assessment in cervical smears showed sensitivity and negative predictive value (NPV) for HGSILs 62.5% and 96.7%, whereas specificity and positive predictive value (PPV) were 80.5% and 19.2%, respectively. hTERT mRNA expression assessment showed 87.5% sensitivity and 98.7% NPV for HGSILs, whereas specificity and PPV were 76% and 21.2%, respectively. Based on the above-described telomerase assessment values, it is suggested that the telomerase system might not be an appropriate diagnostic marker for cytology, given that the final evaluation must rely on a combination of all available test assessment data, clinical diagnosis, as well as the follow-up of all LGSIL samples that were positive for telomerase activation.

Telomerase catalytic subunit in laryngeal carcinogenesis—an immunohistochemical study

We recently demonstrated that (1) telomerase catalytic subunit messenger RNA (mRNA) relative quantities increase progressively with the degree of laryngeal epithelial abnormalities and that (2) telomerase catalytic subunit gene re-expression represents an early event in laryngeal carcinogenesis. The aim of the study was to determine whether telomerase catalytic protein immunohistochemisty reflects telomerase catalytic subunit gene expression in different grades of laryngeal epithelial abnormalities and squamous cell carcinomas of the larynx. Telomerase catalytic protein was analysed immunohistochemically in 106 laryngeal epithelial tissue samples: 10 normal epithelia, 15 squamous cell hyperplasias, 14 basal/parabasal cell hyperplasias, 10 atypical hyperplasias, eight intraepithelial carcinomas and 49 squamous cell carcinomas. At least 200 nuclei of each lesion were quantified per slide and the number of positive signals per nucleus was expressed as a telomerase catalytic protein index. The mean telomerase catalytic protein index increased progressively with the degree of laryngeal epithelial abnormalities: from 0.17 in normal epithelia, 0.44 in squamous cell hyperplasia, 0.54 in basal/parabasal cell hyperplasia, 0.91 in atypical hyperplasia, 1.05 in intraepithelial carcinoma to 0.96 in squamous cell carcinomas. Statistical analysis revealed three different groups of laryngeal epithelial changes according to the number of telomerase catalytic protein signals per nucleus: (1) normal epithelium, (2) regenerative epithelium (squamous cell hyperplasia, basal/parabasal cell hyperplasia), and (3) atypical hyperplasia, intraepithelial carcinoma and squamous cell carcinoma (P<0.0033). Telomerase catalytic protein immunohistochemistry parallels well with telomerase catalytic subunit mRNA relative quantities in laryngeal carcinogenesis. In normal and regenerative laryngeal epithelia, telomerase catalytic protein is present in occasional basal/parabasal nuclei, becomes undetectable with maturation or differentiation of epithelial cells, and reflects the regenerative capacity of squamous epithelium. Nevertheless, several telomerase catalytic protein signals in the majority of nuclei in precancerous lesions, intraepithelial carcinomas and squamous cell carcinomas, are consistent with telomerase catalytic subunit gene re-expression, an early event in laryngeal carcinogenesis.

Effect of daidzein on cell growth, cell cycle, and telomerase activity of human cervical cancer in vitro.

Phytoestrogens are some plant compounds exhibiting estrogen-like activities. However, some studies have shown that they also affect the growth of some nonhormone-dependent diseases. In this study, daidzein--one of the most common phytoestrogens--was used to investigate its effects on human cervical cancer cells HeLa in vitro. First, the cell growth was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Then, the distributions of cell cycle and apoptosis were analyzed with the help of flow cytometry. Finally, the telomerase activity was detected by using real-time quantitative reverse transcription-polymerase chain reaction. The results showed that at the concentrations from 6.25 to 100 micro mol/l, daidzein inhibited the growth of HeLa cells. Flow cytometric analysis showed that cancer cells were arrested at G(0)/G(1) or G(2)/M phase with daidzein. The inductive effects of apoptosis were more obviously observed in low-concentration groups. After HeLa cells were treated with daidzein, the expression of human telomerase catalytic subunit mRNA decreased. These meant that daidzein affected human nonhormone-dependent cervical cancer cells in several ways, including cell growth, cell cycle, and telomerase activity in vitro.

Inhibition of Telomerase Activity and Secretion of Prostate Specific Antigen by Silibinin in Prostate Cancer Cells

The androgen sensitive prostate cancer cell line LNCaP is strongly positive for dihydrotestosterone (DHT) dependent telomerase activity, which is an important factor in cellular immortality and carcinogenesis. In this study we determined the potential of silibinin as an anticancer drug that down-regulates telomerase activity and prostate specific antigen (PSA) together with the co-activator of the androgen receptor prostate epithelium specific Ets transcription factor. LNCaP cells were treated with various concentrations of silibinin in the presence or absence of 5alpha-DHT. We used real-time reverse transcriptase-polymerase chain reaction to quantify mRNA expression of PSA, prostate epithelium specific Ets transcription factor and the catalytic subunit of telomerase vs the housekeeping gene porphobilinogen deaminase with gene specific, dual labeled fluorescence probes. PSA secretion from LNCaP cells in conditioned medium was measured with an Elecsys System 2010 (Roche Diagnostics, Mannheim, Germany) and telomerase activity in extracts from LNCaP cells was measured with a TRAP (telomeric repeat amplification protocol) assay. Silibinin down-regulated PSA mRNA expression and PSA secretion in conditioned medium. Simultaneous stimulation with silibinin and 10(-8) M DHT also resulted in PSA down-regulation, whereas DHT alone increased PSA secretion. Telomerase catalytic subunit mRNA decreased significantly after silibinin stimulation. Telomerase activity was down-regulated by silibinin and stimulated by DHT. The 2 agents in combination resulted in telomerase down-regulation. The down-regulation of PSA by silibinin and its counteraction on DHT effects indicate that this compound can interact with the expression of genes that are regulated through the androgen receptor. Silibinin can also inhibit the telomerase activity that mediates cell immortality and carcinogenesis. The 2 effects underline the possible therapeutic use of silibinin as an antiproliferative agent in intervention for prostate cancer.

Absence of telomerase activity and telemorase catalytic subunit mRNA in melanocyte cultures.

The classic model of activation of telomerase, for which activity has been found in most cancers including cutaneous malignant melanoma (CMM), dictates that enzyme activity is generated by pathological reactivation of telomerase in telomerase-negative somatic cells. However, recent data demonstrated physiological up-regulation in some normal cell types when established as proliferating cultures, indicating that, in some cancer types, telomerase is expressed by the process of up-regulation in telomerase-competent precursor cells. In this study, cultures of epidermal melanocytes, progenitor cells of CMM, were established and harvested in the logarithmic phase of growth. Telomerase activity was looked for using a non-isotopic variant of the telomeric repeat amplification protocol, and transcript expression of the hTERT gene, the rate-limiting catalytic telomerase subunit, was investigated by the reverse transcription polymerase chain reaction. Neither telomerase activity nor hTERT mRNA could be detected in proliferating melanocyte cultures. Our in vitro data argue against the model of telomerase as a common biomarker of cell proliferation. The results further suggest that telomerase is tightly controlled in normal melanocytes, and that telomerase is reactivated rather than up-regulated in melanocytic precursors during melanoma initiation or progression. © 2000 Cancer Research Campaign

Levels of telomerase catalytic subunit mRNA as a predictor of potential malignancy.

This study investigated the relationship between the levels of human telomerase reverse transcriptase (hTERT) mRNA and that of telomerase activity in hepatocellular carcinoma (HCC). A significant correlation between hTERT mRNA expression and telomerase activity by transfecting the gene encoding hTERT into telomerase-negative human fibroblast cells has clearly been demonstrated. However, the relationship between levels of telomerase activity and that of hTERT mRNA has yet to be elucidated. In this study, the levels of hTERT mRNA were analyzed in 24 HCC patients by real-time PCR. And the intensity of telomerase activity was analyzed by fluorescence-based TRAP method. The difference of hTERT mRNA level was highly significant between tumor tissues and non-cancerous liver tissues. And there were significant correlations between the levels of hTERT mRNA and that of telomerase activity (r=0.751) in tumor tissues. We observed a strong correlation between levels of hTERT mRNA and that of telomerase activity in HCC. Our results suggest that the levels of hTERT mRNA would be useful in genetic diagnostic tests, instead of telomerase activity, to screen at-risk patients of HCC in human liver tissues.

Genetic diagnostic test of hepatocellular carcinoma by telomerase catalytic subunit mRNA.

This study investigated the relationship between telomerase activity and telomere length and between telomerase reverse transcriptase (hTERT) mRNA and telomere length. Both cancerous and non-cancerous tissues were studied in individuals with hepatic carcinoma. In this study, the telomere length in HCC livers had a wide range, no clear significant correlation was found between hTERT mRNA and telomere length. Telomerase activity was more strongly correlated with hTERT mRNA than with telomere length. The correlation between hTERT mRNA and telomerase activity shown here indicates that hTERT mRNA has potential for cancer diagnosis.