TERT mRNA Research Articles

TERT promoter mutations and gene amplification: promoting TERT expression in Merkel cell carcinoma.

Telomerase activation through the induction of its catalytic component TERT is essential in carcinogenesis. The regulatory mechanism and clinical significance underlying cancer-specific TERT expression have been extensively investigated in various human malignancies, but little is known about these in Merkel cell carcinoma (MCC), an aggressive neuroendocrine skin tumor. Here we addressed these issues by determining TERT promoter mutations, gene amplification, mRNA expression and association with clinical variables in MCC. TERT mRNA was expressed in 6/6 MCC cell lines and 41 of 43 tumors derived from 35 MCC patients. Telomerase activity was detectable in all 6 cell lines and 11 tumors analyzed. TERT promoter mutations were identified in 1/6 cell lines and 4/35 (11.4%) MCC cases. The mutation exhibited UV signature and occurred in sun-exposed areas. Increased TERT gene copy numbers were observed in 1/6 cell lines and 11/14 (79%) tumors, and highly correlated with its mRNA expression (r = 0.7419, P = 0.0024). Shorter overall survival was significantly associated with higher TERT mRNA levels in MCC patients (P = 0.032). Collectively, TERT expression and telomerase activity is widespread in MCC, and may be attributable to TERT promoter mutations and gene amplification. Higher TERT expression predicts poor patient outcomes.

P04.24 * MUTATIONS OF THE TERT PROMOTER CORRELATE WITH ENHANCED TELOMERASE ACTIVATION AND PREDICT A WORSE PROGNOSIS IN PRIMARY GLIOBLASTOMA PATIENTS

The stabilization of telomeres by upregulation of telomerase is compulsive for indefinite proliferation and cell immortalization therefore representing a main feature of malignant solid tumors, including gliomas. However, the mechanisms responsible for cancer-associated telomerase activation are not completely understood. Recently, defined mutations in the TERT promoter were identified in a variety of tumors, most frequent in primary glioblastomas (GBM). Presence of the mutations was associated with increased TERT expression. In the present study GBM derived tumor tissue from 126 patients operated at the Wagner Jauregg Hospital were screened for TERT promoter mutations. Subsequently the collected data were correlated with telomere associated parameters (telomerase activity, TERT mRNA expression, telomere lengths), the glioma biomarkers MGMT promoter methylation and IDH1 mutation as well as clinical parameters including patient survival. Using direct sequencing, the TERT promoter mutations (C228T, C250T) were found in 73% of tumors with predominance of C228T (72% of the mutated cases). Thirty-four (27%) samples contained none of the investigated TERT promoter mutations while mutations at both sites occurred in none of the investigated GBM cases. TERT promoter mutations were accompanied by a significant upregulation of telomerase activity (p = 0.0005) and TERT mRNA expression (p = 0.0004). Accordingly, telomere lengths of TERT promoter mutated tumors were significantly shorter compared to the TERT promoter wild-type subgroup (p = 0.001). Moreover, Kaplan-Meier survival analyses revealed a significantly shorter overall survival for GBM patients harbouring mutant tumors (p < 0.0001). In the multivariate Cox regression analysis TERT promoter mutation was found to have independent prognostic power (p = 0.049) but reached elevated significance in the interaction with age (p = 0.007). Accordingly, the prognostic quality of TERT promoter mutations was confined to the subgroup of patients aged younger than 65 years and completely absent in the older patient cohort. Significantly enhanced TERT mRNA expression and reduced telomere lengths in the aged patients were only observed in the subgroup lacking TERT promoter mutations. Summarizing, these results suggest that the negative prognostic value of TERT promoter mutation is restricted to patients of younger age further corroborating age-associated differences in the way of glioblastoma-associated telomere stabilization.

The circadian rhythm controls telomeres and telomerase activity

Circadian clocks are fundamental machinery in organisms ranging from archaea to humans. Disruption of the circadian system is associated with premature aging in mice, but the molecular basis underlying this phenomenon is still unclear. In this study, we found that telomerase activity exhibits endogenous circadian rhythmicity in humans and mice. Human and mouse TERT mRNA expression oscillates with circadian rhythms and are under the control of CLOCK–BMAL1 heterodimers. CLOCK deficiency in mice causes loss of rhythmic telomerase activities, TERT mRNA oscillation, and shortened telomere length. Physicians with regular work schedules have circadian oscillation of telomerase activity while emergency physicians working in shifts lose the circadian rhythms of telomerase activity. These findings identify the circadian rhythm as a mechanism underlying telomere and telomerase activity control that serve as interconnections between circadian systems and aging.

Human telomerase reverse transcriptase messenger RNA (TERT mRNA) as a tumour marker for early detection of hepatocellular carcinoma

Background and study aimsHepatocellular carcinoma (HCC) is the fifth most common malignancy in the world. Although tumour markers such as α-foetoprotein (AFP) are widely used and important for HCC detection in clinical scenes, they still do not provide a satisfactory solution to detect HCC at the early stage. The aim of our study was to illustrate the significance of serum human telomerase reverse transcriptase messenger RNA (hTERT mRNA) as a novel biomarker for early detection of HCC. Patients and methodsThirty-five patients with HCC, 15 patients with liver cirrhosis, and 10 healthy subjects were sex and age matched. History taking, full physical examination, and laboratory investigations including liver function tests, hepatitis markers, AFP, and quantification of serum human telomerase reverse transcriptase mRNA (hTERT mRNA) using real-time reverse transcription polymerase chain reaction (RT-PCR) were conducted. Ultrasonography (US), triphasic computed tomography (CT), and liver biopsy were carried out. ResultsThe hTERT was above the cutoff point (>144copies/ml) in 27 HCC patients with a sensitivity of 77.14% and a specificity of 100% and with a positive predictive value (PPV) of 100% and a negative predictive value (NPV) of 65.2%. AFP was above the cutoff point (>50ng/ml) in 23 HCC patients with a sensitivity of 65.71% and a specificity of 96% and with a PPV of 96.3% and an NPV of 53.8%.Our study also showed a statistically significant relationship between the size of the tumour in HCC patients and both AFP and hTERT, and hTERT appears to be more correlated with the size of the tumour than AFP. There is no direct correlation between hTERT or AFP and the number of focal lesions with p value >0.05. ConclusionSerum hTERT mRNA is more sensitive and specific than AFP in the early detection of HCC and its level correlates with the size of the tumour.

Simultaneous induction of Tri-antigen specific cytotoxic T lymphocytes(CTLs) using DCs transferred with WT1, survivin and TERT mRNA for adoptive T cell therapy

T cell to tumor cell ratios, increasing anti-tumor activity and improving patient survival. We therefore incorporated the OX40 and CD28 co-stimulatory endodomains into our GD2-CAR construct in the hope they would enhance in vivo expansion and persistence of CAR T-cells in patients with relapsed/refractory neuroblastoma. Methods: This is a Phase I, dose-escalating, safety trial administering 3 generation GD2-CAR T cells to patients with relapsed/refractory neuroblastoma. Results: We have manufactured 4 autologous 3 generation GD2-CAR T cell products (patient ages: 4 e 23 years). Starting with a median of 20x10 PBMCs, we obtained a median of 450x10 CAR T cells (range 240x10 to 480x10, 18-fold expansion) after 10 3 days of culture. The transduction efficiency was 79 5% by FACS. Phenotypically, the T cell lines were 27 11% CD4 and 67 10% CD8, with 8 2% central memory (CD45ROCD62LCCR7), and 12 10% naive (CD45RACCR7) cells in the final products. Cr release assays showed the T cell lines produced 70 7% and 55 8% lysis of GD2 targets (LAN-1 and CHLA255, respectively) at a 20:1 effector:target ratio. One patient has been treated, so far without significant toxicity. Clinical and immunological results are pending. Conclusion: Generation of autolgous 3 generation CAR T cells for the treatment of high-risk neuroblastoma appears feasible and the safety and antitumor activity of the approach are now being assessed.

Depletion of Sirtuin 1 (SIRT1) Leads to Epigenetic Modifications of Telomerase (TERT) Gene in Hepatocellular Carcinoma Cells

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase that is implicated in plethora of biological processes, including metabolism, aging, stress response, and tumorigenesis. Telomerase (TERT) is essential for telomere maintenance. Activation of TERT is considered a crucial step in tumorigenesis, and therefore it is a potential therapeutic target against cancer. We have recently found that SIRT1 expression is highly elevated in hepatocellular carcinoma, and the depletion of SIRT1 leads to substantial reduction in TERT mRNA and protein expression. However, the underlying molecular mechanism of SIRT1-dependent TERT expression remains uncharacterized. Here, we elucidated if SIRT1 regulates TERT expression via transcriptional, epigenetic and post-transcriptional mechanisms. We report that depletion of SIRT1 does not lead to significant change in transcriptional activity and CpG methylation patterns of the TERT promoter, nor does it affect mRNA stability or 3′-UTR regulation of TERT. Intriguingly, depletion of SIRT1 is associated with substantial induction of acetylated histone H3-K9 and reduction of trimethyl H3-K9 at the TERT gene, which are known to be associated with gene activation. Our data revealed that SIRT1 regulates histone acetylation and methylation at the TERT promoter. We postulated that SIRT1 may regulate TERT expression via long-range interaction, or via yet unidentified histone modifications.

TERT promoter mutations rather than methylation are the main mechanism for TERT upregulation in adult gliomas

observed TERT upregulation in some tumors without mutations in the hotspots. A small subset of tumors had neither TERT nor ATRX mutations [9]. Recently, it has been reported that DNA hypermethylation of the TERT promoter is a common finding in pediatric brain tumors and associated with TERT upregulation [2]. Hypermethylation of the TERT CpG island has been linked to increased expression levels in other cancers [2, 4, 5]. We, therefore, studied the association between TERT methylation and TERT mRNA levels to investigate the possibility that DNA methylation serves as an alternative mechanism for TERT upregulation in adult gliomas. Eighty-eight adult gliomas samples with known TERT promoter mutation status suitable for mRNA expression analysis, 43 of which had mutations, were examined in this study. TERT mRNA expression of 88 tumors including 48 primary glioblastomas that have previously been investigated was analyzed as described [1]. The methylation status of three regions within the CpG island (Regions 1–3; Fig. 1a), including the region methylated in pediatric tumors (Region 1) and the region that contains the two mutation hotspots (Region 3), was assessed by pyrosequencing of the PCR products amplified from bisulfitemodified genomic DNA. The methylation status was represented either as the mean methylation levels of all CpGs in each region or as a dichotomous variable (hypermethylated or unmethylated) at each region using the cut-off value of 15 % according to Castelo-Branco et al. [2]. More detailed information is available in Supplementary Materials and Methods. There was no significant difference in the mean methylation levels or the frequency of hypermethylated tumors in any of the regions between tumors of the different histological subtypes (astrocytic tumor, oligodendroglial tumor and glioblastoma), or between those with and without Telomere lengthening (TL) is mandatory for infinite proliferation of many cancer cells. This is generally achieved either by telomerase activation or in some cases by telomerase-independent alternative lengthening of telomeres (ALT) [3]. Recently, recurrent mutations at two hotspots termed C228T and C250T in the promoter region of TERT, a catalytic subunit of telomerase, have been reported in various types of cancers [1, 6, 7, 9, 12]. These mutations result in upregulation of TERT expression [1, 7], which is required for telomerase activation [11]. TERT promoter mutations are particularly common in adult gliomas [1, 9]. It is also known that a subset of astrocytomas harbors mutations of ATRX, which could lead to ALT [10]. We have previously shown that glioblastomas with TERT mutation had TERT mRNA upregulation [1]. We have also

Frequency of TERT promoter mutations in human cancers

Reactivation of telomerase has been implicated in human tumorigenesis, but the underlying mechanisms remain poorly understood. Here we report the presence of recurrent somatic mutations in the TERT promoter in cancers of the central nervous system (43%), bladder (59%), thyroid (follicular cell-derived, 10%) and skin (melanoma, 29%). In thyroid cancers, the presence of TERT promoter mutations (when occurring together with BRAF mutations) is significantly associated with higher TERT mRNA expression, and in glioblastoma we find a trend for increased telomerase expression in cases harbouring TERT promoter mutations. Both in thyroid cancers and glioblastoma, TERT promoter mutations are significantly associated with older age of the patients. Our results show that TERT promoter mutations are relatively frequent in specific types of human cancers, where they lead to enhanced expression of telomerase.

Telomerase reverse transcriptase promotes the proliferation of human laryngeal carcinoma cells through activation of the activator protein 1

TERT is the main functional unit of telomerase, which maintains telomere length and chromosome structure stability. TERT has been shown to act as a key factor in various biological processes, such as cell proliferation, via uncharacterized mechanisms. We transfected HEp-2 laryngeal carcinoma cells with a TERT overexpressing adenovirus (Ad-TERT) and TERT shRNA silencing adenovirus (Ad-sh-TERT), and examined the effect on TERT and the AP-1 transcription factor subunits c-Fos and c-Jun using RT-PCR and western blot analysis. TERT mRNA expression was quantified using RT-PCR in 24 human laryngeal carcinoma samples, and TERT protein co-expression with AP-1 was investigated in a human laryngeal carcinoma tissue microarray using quantum-dot based immunofluorescence. The effect of specific ERK and p38 inhibitors on ERK, p38, c-Jun and c-Fos phosphorylation was investigated in TERT-overexpressing HEp-2 cells. TERT overexpression led to increased TERT, c-Jun and c-Fos mRNA and protein expression and increased cell proliferation, while TERT silencing had the opposite effects. TERT mRNA expression levels were positively correlated with c-Fos and c-Jun mRNA in human laryngeal carcinoma tissue. TERT and AP-1 protein were expressed at high levels and positively correlated in laryngeal carcinoma tissues. Treatment of TERT-overexpressing HEp-2 cells with specific p38 and ERK inhibitors indicated that TERT modulates the expression and phosphorylation of the AP-1 subunits c-Jun and c-Fos through the p38 and ERK signaling pathways. In conclusion, the results of this study indicate that TERT is capable of promoting cell proliferation via activation of the AP-1 subunits, c-Jun and c-Fos, in laryngeal carcinoma cells.

Dyskerin Overexpression in Human Hepatocellular Carcinoma Is Associated with Advanced Clinical Stage and Poor Patient Prognosis

BackgroundDyskerin (encoded by the DKC1 gene) is an essential nucleolar protein involved in cell proliferation, where it is required for the pseudo-uridylation of ribosomal RNA (rRNA) molecules and the stabilization of the telomerase RNA component. Dyskerin expression has been reported to predict poor survival in some cancer patients. The aim of the present study was to analyze the expression of dyskerin in hepatocellular carcinoma (HCC) and to determine its correlation with clinicopathologic features, including the survival of patients with HCC.Methodology/Principal FindingsDyskerin protein expression was detected by immunohistochemistry in paraffin sections of 252 HCC cases and 80 noncancerous liver tissues. The correlation was analyzed between dyskerin expression levels and clinicopathologic variables and prognosis. Dyskerin protein was significantly overexpressed in HCC tissues when compared to noncancerous liver tissue. Dyskerin overexpression was positively correlated with the hepatitis B surface antigen status, serum alpha-fetoprotein, and advanced clinical stage in HCC patients. A survival analysis indicated that HCC patients with higher dyskerin expression had a significantly shorter overall survival and 5-year survival time when compared to those with low expression. A multivariate analysis suggested that dyskerin overexpression was an independent factor for prognosis (hazard risk, 2.912; P = 0.007). Expression of DKC1 mRNA was measured by quantitative RT-PCR in 80 HCC and 50 non-cancerous tissues. The relationship between DKC1, TERT, MKI67, and MYC mRNA expression in HCC tissues was also evaluated. DKC1 mRNA was significantly overexpressed in HCC tissues and showed a significant correlation with MKI67 and MYC mRNA but a weak correlation with TERT mRNA.Conclusions/SignificanceDyskerin overexpression in HCC patients was correlated with MYC and MKI67 expression and showed a possible involvement in the tumorigenic process. Dyskerin overexpression may be an unfavorable prognostic factor in patients with HCC.

Genistein induces growth arrest and suppresses telomerase activity in brain tumor cells

Genistein, a soy isoflavone, has been reported to exhibit multiple effects, such as inducing cell cycle arrest, triggering apoptosis, inhibiting the activation of NF(K) B and inactivating several signaling cascades in human cancer cells. In vivo studies demonstrating antiangiogenesis and antimetastatic effects of genistein have also been reported. Here, we demonstrate that genistein inhibits the growth of glioblastoma multiforme and medulloblastoma cells with different TP53 mutations and radio-responses by arresting the cells at G2/M phase of the cell cycle. The cell cycle arrest was found to be independent of DNA damage and such an arrest was sustainable for at least 10 days even after drug removal. Annexin V staining revealed absence of apoptotic or necrotic cell populations after genistein treatment. This supports the observation that genistein induces insignificant DNA damage and indicates that the cell cycle arrest triggered does not lead to cell death. Gene and protein expression studies reveal similar changes in the same pathways following treatment in the cell types tested. Genistein was also able to inhibit telomerase activity resulting in telomere shortening. Thus, we demonstrate, for the first time, that genistein induces growth arrest in association with telomerase inhibition in brain tumor cells via the suppression of TR- and TERT mRNA. By elucidating the mechanisms of anticancer effects after genistein treatment in brain tumor cells, there will be a premise for the incorporation of genistein dietary sources to complement radiotherapy in brain tumor patients.

Differential TERT promoter methylation and response to 5‐aza‐2′‐deoxycytidine in acute myeloid leukemia cell lines: TERT expression, telomerase activity, telomere length, and cell death

The catalytic subunit of human telomerase (TERT) is highly expressed in cancer cells, and correlates with complex cytogenetics and disease severity in acute myeloid leukemia (AML). The TERT promoter is situated within a large CpG island, suggesting that expression is methylation-sensitive. Studies suggest a correlation between hypermethylation and TERT overexpression. We investigated the relationship between TERT promoter methylation and expression and telomerase activity in human leukemia and lymphoma cell lines. DAC-induced demethylation and cell death were observed in all three cell lines, as well as telomere shortening in HL-60 cells. DAC treatment reduced TERT expression and telomerase activity in OCI/AML3 and HL-60 cells, but not in U937 cells. Control U937 cells expressed lower levels of TERT mRNA, carried a highly methylated TERT core promoter, and proved more resistant to DAC-induced repression of TERT expression and cell death. AML patients had significantly lower methylation levels at several CpGs than "well elderly" individuals. This study, the first to investigate the relationship between TERT methylation and telomerase activity in leukemia cells, demonstrated a differential methylation pattern and response to DAC in three AML cell lines. We suggest that, although DAC treatment reduces TERT expression and telomerase activity, this is unlikely to occur via direct demethylation of the TERT promoter. However, further investigations on the regions spanning CpGs 7-12 and 14-16 may reveal valuable information regarding transcriptional regulation of TERT.

Posttranscriptional regulation of telomerase reverse transcriptase in Human T cells

Telomerase, a reverse transcriptase consisting of a catalytic subunit (TERT) and a RNA template (TERC), plays an essential role in maintaining the length of telomeres and cellular replicative lifespan. TERT mRNA is present in resting human T cells but with undetectable level of telomerase activity. It is unclear whether the lack of telomerase activity is a result of non‐functional Alternate Splicing Products (ASP) of TERT mRNA and/or missing proper post‐translational modifications of TERT protein. It was hypothesized that missing proper post‐translational modifications of TERT protein lead to the lack of telomerase activity. We analyzed two common TERT splicing forms in resting and activated human T cell subsets using single cell RT‐PCR method. We found that 1) 39% of the resting T cells expressed TERT full‐length alone, 34% expressed both full‐length and ASP and 27% expressed only ASP; 2) activation decreased expression of full‐length TERT and increased expression of ASP; and 3) all live and dead T cells after activation had ASP. These findings show that resting T cells express mostly full‐length TERT whereas activated T cells express mostly ASP. Further studies will be needed to elucidate this seemingly paradoxical finding. The findings of this study may lead to pharmaceutical interventions to slow or stop the aging process.

Tolemerase Reverse Transcriptase Gene Expression as a Tumor Marker for Hepatocellular Carcinoma

Problem statement: Hepatocellular carcinoma will emerge as a major fo rm of malignancy in the coming decades. The continuing high incidenc e of hepatocellular carcinoma, suggests that this disease will continue to represent a global health problem far into the future. Different genes encode for the various components of the human telomerase complex. These components include the human Telomerase RNA Component (hTERC) and the Telomerase Catalytic Subunit (hTERT). Correlation between Telomerase Reverse Transcriptase (hTERT) expression and telomerase activity has been reported in cancer patients. This work aimed to cla rify the significance of human Telomerase Reverse Transcriptase (hTERT mRNA) as a potential molecular tumor marker for Hepatocellular Carcinoma (HCC). Approach: The current study included 25 patients of hepatoce llular carcinoma (HCC), 30 patients with liver cirrhosis and 25 age and sex matched individuals with normal laboratory and Image findings as a control group. h TERT mRNA was measured in plasma by Real time PCR in all patients samples in comparison with normal healthy controls. Results: The expression of hTERT mRNA by relative unit was 129.10 ±27.6 with range (67.72-69.6) Vs 5245.87 ±2382.48 (2053-12232.90) Vs 92782.76 ±16158 (61783.25-118596.47) for control Vs cirrhosis Vs HCC group respectively. The hTERT expression was significantly with 699 and 33 fold increase in HCC and cirrhosis groups correspondingl y when compared to that of controls p<0.05. Conclusion: It was suggested that this procedure was highly di scriminating between healthy subjects and cancer patients and strongly support t he idea that a valuable diagnostic test for cancer might be developed using this genetic marker in pla sma. However it needs to be combined with other markers in future studies to be more specific for liver cancer.

Combinational RNAi gene therapy of hepatocellular carcinoma by targeting human EGFR and TERT

Both human telomerase reverse transcriptase (hTERT) and epidermal growth factor receptor (hEGFR) are ideal targets for RNA interference (RNAi)-based gene therapy of hepatocellular carcinoma. Two shRNA expression plasmids pU6-shTERT and pU6-shEGFR targeting hTERT and hEGFR, respectively, were separately formulated as pegylated immuno-lipopolyplexes, a novel non-viral gene delivery system. In vitro studies showed that when pU6-shTERT and pU6-shEGFR were combined and applied to SMMC-7721 cells, there was a significant additive effect on cytotoxicity as well as cell apoptosis, compared to pU6-shTERT or pU6-shEGFR alone, with a cell viability of 50.9 ± 7.4%, 79.2 ± 3.6% and 77.1 ± 3.6%, respectively, and with a cell apoptotic rate of 44.8 ± 0.9%, 25.1 ± 0.4% and 29.5 ± 0.8%, respectively. In vivo study in SMMC-7721 xenograft tumor model demonstrated that intravenous administration of PILP-formulated pU6-shTERT and pU6-shEGFR caused an additive effect on tumor growth inhibition, compared to pU6-shTERT or pU6-shEGFR alone, with a tumor growth inhibition rate of 74.0%, 36.3% and 46.1%, respectively, which is consistent with the downregulated EGFR and TERT mRNA expression. The results suggest that combinational RNAi gene therapy of hepatocellular carcinoma by targeting human EGFR and TERT with pegylated immuno-lipopolyplexes is a new and good strategy.

Expression of TERT in precancerous gastric lesions compared to gastric cancer

The objective of this study was to determine the levels of TERT mRNA and TERT protein expression in stomach precancerous lesions such as intestinal metaplasia (IM) and gastric ulcer (GU) and compare them to gastric cancer (GC). Real-time PCR was performed to detect TERT mRNA expression levels in 35 biopsies of IM, 30 of GU, and 22 of GC and their respective normal mucosas. TERT protein was detected by immunohistochemistry in 68 samples, 34 of IM, 23 of GU, and 11 of GC. Increased TERT mRNA expression levels were observed in a significant number of cases, i.e., 46% of IM, 50% of GU, and 79% of GC. The relative mean level of TERT mRNA after normalization with the β-actin reference gene and comparison with the respective adjacent normal mucosa was slightly increased in the IM and GU groups, 2.008 ± 2.605 and 2.730 ± 4.120, respectively, but high TERT mRNA expression was observed in the GC group (17.271 ± 33.852). However, there were no statistically significant differences between the three groups. TERT protein-positive immunostaining was observed in 38% of IM, 39% of GU, and 55% of GC. No association of TERT mRNA and protein expression with Helicobacter pylori infection or other clinicopathological variables was demonstrable, except for the incomplete type vs the complete type of IM. This study confirms previous data of the high expression of both TERT mRNA and protein in gastric cancer and also demonstrates this type of changed expression in IM and GU, thus suggesting that TERT expression may be deregulated in precursor lesions that participate in the early stages of gastric carcinogenesis.

Effects of cadmium on telomerase activity, expressions of TERT, c-myc and P53, and apoptosis of rat hepatocytes

This study investigated the effect of cadmium on the telomerase activity, the expression of TERT, c-myc and p53 and the apoptosis of rat hepatocytes. The rats were administrated 5, 10 and 20 μmol/kg cadmium chloride intraperitoneally and sacrificed 48 h after the initial treatment. The telomerase activity of the rat hepatocytes was measured by the telomeric repeat amplification protocol (TRAP), and apoptosis was detected by flow cytometry. The mRNA expressions of TERT, c-myc and p53 were measured by reverse transcription-polymerase chain reaction (RT-PCR). C-myc and P53 proteins were determined by immunochemistry. The results showed that cadmium chloride increased the hepatocellular telomerase activity in a dose-dependant manner and induced the apoptosis of hepatocytes significantly. The value of relative coefficient between the telomerase activity and the apoptosis rate was 0.9398. RT-PCR revealed that specific bands corresponding to the TERT mRNA, c-myc mRNA, and p53 mRNA were displayed at 185, 342 and 538 bp respectively. Cadmium chloride could substantially increase the mRNA expressions of TERT, c-myc and p53 in rat hepatocytes, as compared with control. Moreover, cadmium chloride at the doses of 5, 10 and 20 μmol/kg could increase the content of P53 protein in rat hepatocytes obviously, but only that at the doses of 10 and 20 μmol/kg substantially promoted the c-myc protein level in rat hepatocytes. Our study herein suggested that cadmium may contribute to the carcinogenesis by activating telomerase, and overexpressing the mRNAs of TERT, c-myc and p53, and causing apoptosis of normal cells.

Leg Amputation Accelerates Senescence of Rat Lumbar Intervertebral Discs

Several senescence biomarkers were observed to investigate cell senescence in degenerative intervertebral lumbar discs of foreleg-amputated rats. To determine if cell senescence is accelerated in degenerative intervertebral lumbar disc cells in an upright-rat model. Cellular senescence was accelerated in human and sand rat degenerative intervertebral disc (IVD) cells. Repeated use of upright posture by rats contributed to degenerative disc changes. No convincing evidence of cell senescence was observed in the lumbar disc of the foreleg amputated rat. The forelimbs of 20 rats were amputated at 1 month of age such that they maintained an upright stance; rats were housed in custom-made cages. Nonamputated rats, also 1 month of age, were kept in regular cages and served as a control group. The lumbar IVDs were harvested from rats in 2 groups, at 5 or 9 months following amputation. Senescence-associated-β-galactosidase-positive staining was used to detect cell senescence. p16INK4a and p27KIP were assessed by immunohistochemistry analysis. Total RNA isolated from these samples was used to measure the gene expression of p16INK4a, RB, cyclin D1, CDK4, PTEN, p27KIP, p19ARF, p21, TERT, and RAGE by real-time polymerase chain reaction assay. The highest levels of SA-β-GAL activity were detected in 9-month amputated rats. Quantitative immunohistochemical analysis showed that there were highest rates of p16INK4a and p27KIP protein expression in the cartilage endplate and anulus fibrosus of 9-month amputated rats. The mRNA levels of p16INK4a, RB, PTEN, p27KIP, p19ARF, and RAGE were upregulated. The increased cyclin D1 mRNA level was statistically significant only at the ninth month following amputation; CDK4 and TERT mRNA levels were downregulated to a similar extent at both points compared with nonamputated controls. mRNA expression of p21 was significantly downregulated. Accelerated cell senescence was associated with forelimb amputation that causes abnormal loading in rat lumbar IVDs.

RNAi screen for telomerase reverse transcriptase transcriptional regulators identifies HIF1alpha as critical for telomerase function in murine embryonic stem cells.

In various types of stem cells, including embryonic stem (ES) cells and hematopoietic stem cells, telomerase functions to ensure long-term self-renewal capacity via maintenance of telomere reserve. Expression of the catalytic component of telomerase, telomerase reverse transcriptase (Tert), which is essential for telomerase activity, is limiting in many types of cells and therefore plays an important role in establishing telomerase activity levels. However, the mechanisms regulating expression of Tert in cells, including stem cells, are presently poorly understood. In the present study, we sought to identify genes involved in the regulation of Tert expression in stem cells by performing a screen in murine ES (mES) cells using a shRNA expression library targeting murine transcriptional regulators. Of 18 candidate transcriptional regulators of Tert expression identified in this screen, only one candidate, hypoxia inducible factor 1 alpha (Hif1alpha), did not have a significant effect on mES cell morphology, survival, or growth rate. Direct shRNA-mediated knockdown of Hif1alpha expression confirmed that suppression of Hif1alpha levels was accompanied by a reduction in both Tert mRNA and telomerase activity levels. Furthermore, gradual telomere attrition was observed during extensive proliferation of Hif1alpha-targeted mES cells. Switching Hif1alpha-targeted mES cells to a hypoxic environment largely restored Hif1alpha levels, as well as Tert expression, telomerase activity levels, and telomere length. Together, these findings suggest a direct effect of Hif1alpha on telomerase regulation in mES cells, and imply that Hif1alpha may have a physiologically relevant role in maintenance of functional levels of telomerase in stem cells.

Effects of combined siRNA-TR and -TERT on telomerase activity and growth of bladder transitional cell cancer BIU-87 cells

The effects of combined RNA interference (RNAi) of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) genes on telomerase activity in a bladder cancer cell line (BIU-87 cells) were investigated by using gene chip technology in vitro with an attempt to evaluate the role of RNAi in the gene therapy of bladder transitional cell cancer (BTCC). Three TR-specific double-stranded small interfering RNAs (siRNAs) and three TERT-specific double-stranded siRNAs were designed to target different regions of TR and TERT mRNA. The phTR-siRNA, phTERT-siRNA, and the combination of both plasmids phTR+phTERT-siRNA were transfected into BIU-87 cells. The expression of hTR and hTERT mRNA was detected by quantitative fluorescent reverse transcription-polymerase chain reaction, and a telomeric repeat amplification protocol was applied to detect telomerase activity. Growth inhibition of BIU-87 cells was measured by MTT assay. Gene chip analysis was performed to evaluate the effects of the combined RNAi of hTR+hTERT genes on telomerase activity and growth of BIU-87 cells in vitro. The results showed that the expression of hTERT and hTR mRNA was inhibited by pRNAT-hTERT-III, pRNAT-hTR-III, and pRNAT-hTR-III+hTERT-III in BIU-87 cells. The inhibition efficiency of pRNAT-hTERT-III, pRNAT-hTR-III, pRNAT-hTERT-III+pRNAT-hTR-III was 67% for TERT mRNA, 41% for TR mRNA, 57% for TR mRNA and 70% for TERT mRNA in BIU-87 cells respectively. The growth of BIU-87 cells was inhibited and telomerase activity was considerably decreased, especially in the cells treated with combined RNAi-hTR and -hTERT. Gene chip analysis revealed that 21 genes were down-regulated (ATM, BAX, BCL2, BCL2L1, BIRC5, CD44, CTNNB1, E2F1, JUN, MCAM, MTA1, MYC, NFKB1, NFKBIA, NME4, PNN, PNN, SERPINE1, THBS1, TNFRSF1A, and UCC1). The results indicated that hTR-siRNA and hTERT-siRNA, especially their combination, siRNA hTR+hTERT, specifically and effectively suppressed the expression of both hTR and hTERT mRNA and telomerase activity. Molecular biological mechanism by which combined siRNA-TR and -TERT inhibited telomerase activity and growth of BIU-87 cells in vitro may involve the down-regulation of the 21 genes.