hTERT Gene Expression Research Articles

Reprogramming of replicative senescence in hepatocellular carcinoma-derived cells

Tumor cells have the capacity to proliferate indefinitely that is qualified as replicative immortality. This ability contrasts with the intrinsic control of the number of cell divisions in human somatic tissues by a mechanism called replicative senescence. Replicative immortality is acquired by inactivation of p53 and p16INK4a genes and reactivation of hTERT gene expression. It is unknown whether the cancer cell replicative immortality is reversible. Here, we show the spontaneous induction of replicative senescence in p53-and p16INK4a-deficient hepatocellular carcinoma cells. This phenomenon is characterized with hTERT repression, telomere shortening, senescence arrest, and tumor suppression. SIP1 gene (ZFHX1B) is partly responsible for replicative senescence, because short hairpin RNA-mediated SIP1 inactivation released hTERT repression and rescued clonal hepatocellular carcinoma cells from senescence arrest.

Das pharmakologische Potential von Phytoöstrogenen in der Therapie des Prostatakarzinoms

Phytoestrogenes are plant-derived compounds that have been shown to exert an antiproliferative potential on prostate cancer cells, although the exact mechanisms are still unclear. In prostate cancer cells proliferation is regulated by modulation of the IGF-1 receptor (IGF-R-1) by the androgen receptor (AR) and its co-activator prostate derived Ets factor (PDEF). Phytooestrogenes interact with these mechanisms as demonstrated exemplarily in the presented study with the isoflavone tectorigenin derived from Belamcanda chinensis. Cultured androgen-sensitive LNCaP prostate cancer cells were treated with tectorigenin of 100 microM for 24 hours. The mRNA-expression of AR, PSA, PDEF, hTERT, TIMP-3 and IGF-R-1 were quantified by real-time RT-PCR. Furthermore, the expression or activity of PSA, telomerase and IGF-R-1 was measured on the protein level. In addition, we investigated in nude mice the influence of a diet of extracts of Belamcanda chinensis on the growth of subcutaneously injected LNCaP cells versus a control group of animals fed with a soy-free diet. In cultured LNCaP cells treatment with tectorigenin resulted in a significant down-regulation of the gene expression of AR, PDEF, PSA, IGF-R-1 and hTERT. On the protein level PSA secretion and the activity of telomerase and IGF-R-1 expression was also decreased. The gene expression of TIMP-3 was distinctly up-regulated by tectorigenin. Nude mice fed with Belamcanda chinensis extract showed a significantly decreased incidence and tumor growth compared to controls. Tectorigenin shows an inhibition of the IGF-1-R modulated cell proliferation of PCa-Cells, due to modulation of the activity the co-activator PDEF independently from the AR. Furthermore, tectorigenin has pro-apoptotic effects and decreases tissue invasion by up-regulation of TIMP-3. Therefore, phytooestrogenes are an interesting option in the therapy of prostate especially advanced prostate cancer.

CTCF binds the proximal exonic region of hTERT and inhibits its transcription

The expression of the catalytic subunit (hTERT) represents the limiting factor for telomerase activity. Previously, we detected a transcriptional repressor effect of the proximal exonic region (first two exons) of the hTERT gene. To better understand the mechanism involved and to identify a potential repressor, we further characterized this region. The addition of the hTERT proximal exonic region downstream of the hTERT minimal promoter strongly reduced promoter transcriptional activity in all cells tested (tumor, normal and immortalized). This exonic region also significantly inhibited the transcriptional activity of the CMV and CDKN2A promoters, regardless of the cell type. Therefore, the repressor effect of hTERT exonic region is neither cell nor promoter-dependent. However, the distance between the promoter and the exonic region can modulate this repressor effect, suggesting that nucleosome positioning plays a role in transcriptional repression. We showed by electrophoretic mobility shift assay that CCCTC-binding factor (CTCF) binds to the proximal exonic region of hTERT. Chromatin immunoprecipitaion assays confirmed the binding of CTCF to this region. CTCF is bound to hTERT in cells in which hTERT is not expressed, but not in telomerase-positive ones. Moreover, the transcriptional downregulation of CTCF by RNA interference derepressed hTERT gene expression in normal telomerase-negative cells. Our results suggest that CTCF participates in key cellular mechanisms underlying immortality by regulating hTERT gene expression.

Expression of human telomerase reverse transcriptase gene and protein, and of estrogen and progesterone receptors, in breast tumors: Preliminary data from neo-adjuvant chemotherapy

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, is very closely associated with telomerase activity. Telomerase has been implicated in cellular immortalization and carcinogenesis. In situ detection of hTERT will aid in determining the localization of telomerase-positive cells. The aim of this study was to detect expression of hTERT mRNA, hTERT protein, estrogen receptor (ER) and progesterone receptor (PR) in paraffin-embedded breast tissue samples and to investigate the relationship between hTERT expression and various clinicopathological parameters in breast tumorigenesis. We used in situ hybridization (ISH) to examine hTERT gene expression, and immunohistochemistry (IHC) to examine expression of hTERT protein, ER and PR, in breast tissues including 64 adenocarcinomas, 2 phyllode tumors and their adjacent normal breast tissues. hTERT gene expression was detected by ISH in 56 (88%) carcinomas, but in neither of the 2 phyllode tumors. hTERT protein expression was detected by IHC in 52 (81%) carcinomas, but in neither of the 2 phyllode tumors. Moreover, ER and PR were expressed in 42 (66%) and 42 (66%) carcinomas, respectively, and in neither of the 2 phyllode tumors. In 4 cases of breast carcinoma that strongly expressed hTERT gene and protein before treatment, neo-adjuvant chemotherapy led to disappearance of gene and protein expression in all cases. There was a strong correlation between detection of hTERT gene expression by ISH and of hTERT protein by ICH in tissue specimens from breast tumors. These results suggest that detection of hTERT protein by ICH can be used to distinguish breast cancers as a potential diagnostic and therapeutic marker.

Tumor necrosis factor-α inhibits hTERT gene expression in human myeloid normal and leukemic cells

AbstractTelomerase catalytic subunit (hTERT) has been shown to play a critical role not only in telomere homeostasis but also in cellular survival, DNA repair, and genetic stability. In a previous study, we described that tumor necrosis factor-×α (TNF×α) induced in the leukemic KG1 cells a senescence state characterized by decreased hTERT activity followed by prolonged growth arrest, increased× β-galactosidase activity, telomere shortening, and major chromosomal instability. Interestingly, granulocyte-macrophage colony-stimulating factor (GM-CSF) abrogated all these events. In the present study, we show for the first time that TNF×α acts by inhibiting the hTERT gene in both normal CD34×+ cells and fresh leukemic cells. Using KG1 cells as a representative cellular model, we show that TNF×α induced sphingomyelin hydrolysis, ceramide production, and c-Jun N-terminal kinase (JNK) activation, all of which are critical components of TNF×α signaling, resulting in hTERT gene inhibition. Moreover, we provide evidence that the protective effect of GM-CSF is related to its capacity to interfere with both ceramide generation and ceramide signaling. Negative regulation of the hTERT gene may represent one mechanism by which TNF×α interferes with normal hemopoiesis.

Expression of a recombinant vector of a mutant human telomerase reverse transcriptase gene in human bladder cancer cell line T24, and its clinical significance

To construct a mutant enhanced green fluorescence protein (pEGFP) human telomerase reverse transcriptase (hTERT) gene expression vector (pEGFP-hTERT), to observe its expression in transfected human bladder cancer cell line T24 and its role in the molecular regulatory mechanisms of telomerase, and to provide a new target gene for bladder cancer therapy. Polymerase chain reaction (PCR) amplification was performed using primers based on the gene sequence of hTERT. The PCR product was cloned into plasmid pGEMT-T Easy and the sequence of mutant hTERT gene analysed. A recombinant mutant hTERT vector (pEGFP-hTERT) was constructed at the EcoR I and Sal I sites of the pEGFP-C1 vector. After transfecting the fusion gene into T24 cells by the method of calcium phosphate-DNA co-precipitation, we detected steady expression of the GFP-hTERT fusion protein by fluorescent-light microscopy. Changes in the proliferation of T24 cells were detected by light microscopy, and beta-galactosidase staining correlated with senescence. Identification of pEGFP-hTERT by enzyme digestion showed that the mutant hTERT fragment had been cloned into EcoR I and Sal I sites of the pEGFP-C1 vector. Steady expression of GFP-hTERT fusion protein was located in the nucleus of transfected cells. Positive expression senescence-associated beta-galactosidase staining in transfected cells increased gradually with extended cultured time, and their growth was suppressed. The recombinant mutant vector (pEGFP-hTERT) was successfully constructed and expressed steadily in T24 cells. The mutant-type hTERT gene suppresses the proliferation of T24 cells by a competitive effect on telomerase activity. This suggests that the hTERT gene might be a suitable gene target for bladder cancer therapy.

Doxorubicin modulates telomerase activity in Ewing's sarcoma in vitro and in vivo

Since most tumours escape replicative senescence by re-activation of the enzyme telomerase, telomerase is a promising target in the treatment of cancer and a promising marker for diagnosis and therapeutic response. We evaluated the effects of doxorubicin, one of the most active drugs in the treatment of Ewing's sarcoma, on telomerase in the human Ewing's sarcoma cell line STA-ET-1 in vitro and in STA-ET-1 xenografts in vivo. Telomerase activity (TA) was examined by TRAP-assay and real-time PCR. Real-time PCR was also used to quantify the mRNA expression of the catalytic subunit of telomerase (hTERT). In vitro growth inhibition was determined by the MTT-assay. Tumour xenografts were analyzed for tumour volume, apoptosis, necrosis, and proliferation. Doxorubicin concentrations that inhibited in vitro growth of STA-ET-1 by 50% compared to untreated controls ranged between 0.14 microM after 24 h and 0.01 microM after 72 h. Compared to untreated controls doxorubicin reduced TA in STA-ET-1 at toxic concentrations, but increased TA at non-toxic concentrations. In comparison with untreated xenografts, TA was reduced to 65% and hTERT expression dropped to 25% within 72 h in xenografts treated with 17.5 mg/kg doxorubicin i.p.; both recovered to initial values after 264 h. The rate of proliferating cells dropped to 70% within 96 h and increased thereafter. The highest rates of necrosis and apoptosis were seen after 96 h. hTERT expression co-varied significantly with proliferation but not with TA, apoptosis, and necrosis. No correlation was observed between TA, proliferation, apoptosis and necrosis. The results suggest doxorubicin induces down-regulation of hTERT gene expression that at least in part modulates TA in these tumours.

Study on Expression of hTERT Gene in Human Lung Cancers

目的 探讨端粒酶逆转录酶(hTERT)在肺癌临床诊断中的价值.方法41例支气管黏膜上皮不典型增生患者经纤维支气管镜取气管上皮组织、150例肺癌患者手术标本及40例正常肺组织,用免疫组化的方法检测端粒酶逆转录酶(hTERT)基因在上述肺组织中的表达.结果5%(2/40)的正常肺组织中可检测到端粒酶逆转录酶(hTERT)基因的蛋白表达;61%(25/41)的支气管黏膜上皮不典型增生组织和90%(135/150)的肺癌组织中可检出端粒酶逆转录酶(hTERT)基因表达,组间差异有高度显著性,x2=111.9,P＜0.01;且随着肺组织癌变过程的变化,端粒酶逆转录酶(hTERT)基因的表达呈逐渐增高的趋势,x2trend=107.7,P＜0.01.结论端粒酶逆转录酶(hTERT)基因的表达与肺癌变的发生、发展有关;hTERT基因检测有望成为诊断肺癌变的早期生物标志物。

Difference in hTERT gene expressions between HbsAg-positive and HbsAg-negative hepatocellular carcinoma.

To investigate the difference in expression of hTERT gene between HbsAg-positive human hepatocellular carcinoma (HCC) and HbsAg-negative HCC and to explore the relationship between HBV infection and hTERT gene expression in HCC. The expression of hTERT protein in 30 cases of HbsAg positive HCC and 17 cases of HbsAg negative HCC was detected by immunohistochemistry (SP method), and the expression of hTERT mRNA was analyzed by reverse transcription polymerase chain reaction (RT-PCR). t-test, Chi-squared test and cochran- armitage trend test were used to see whether there was an interrelation between HBsAg and hTERT gene in HCC. The expression of hTERT protein was mostly located in plasm and occasionally in the nucleus of liver cancer cells. The positive rate of hTERT protein and hTERT mRNA in HbsAg positive HCC- 93.33% (28/30) and 83.33% (25/30) respectively which were much higher than those in HbsAg negative HCC- 52.94% (9/17), 47.06% (8/17) (P<0.01) respectively. HbsAg is related to hTERT gene expression in human hepatocellular carcinoma. The hTERT gene activated by the efficacious ingredient of HBV may play an important role in hepatocellular transformation and carcinogenesis.

Tectorigenin and other phytochemicals extracted from leopard lily Belamcanda chinensis affect new and established targets for therapies in prostate cancer

Isoflavones have been shown to exert antiproliferative effects on cancer cells by steroid receptor signaling. In this study, we demonstrate the potential of plant constituents extracted from Belamcanda chinensis as anticancer drugs, which regulate the aberrant expression of genes relevant in proliferation, invasion, immortalization and apoptosis. LNCaP cells were treated with B.chinensis extract, tectorigenin or other isoflavones and mRNA expression was quantified by using real time RT-PCR. In addition, ELISA, TRAP assays and western blots were used to measure protein expression or activity. Male nude mice (n=18) were injected subcutaneously with LNCaP cells and were fed with extracts from B.chinensis, and tumor development was monitored versus a control animal group (n=18). Tectorigenin and several other phytochemicals downregulated PDEF, PSA and IGF-1 receptor mRNA expression in vitro. Furthermore, PSA secretion and IGF-1 receptor protein expression were diminished, and hTERT mRNA expression and telomerase activity decreased after tectorigenin treatments. However, TIMP-3 mRNA was upregulated on tectorigenin treatment. Growth of subcutaneous tumors in nude mice was delayed and diminished in animals fed with extracts from B.chinensis. The downregulation of PDEF, PSA, hTERT and IGF-1 receptor gene expression by tectorigenin demonstrates the antiproliferative potential of these agents. The upregulation of TIMP-3 gene expression indicates a pro-apoptotic function of the drug and a reduction of the invasiveness of tumors. The animal experiments demonstrate that B.chinensis markedly inhibited the development of tumors in vivo. Thus, these compounds may be useful for the prevention or treatment of human prostate cancer.

Effective siRNA targets screening for human telomerase reverse transcriptase

To study the inhibitory effects of siRNAs targeting different hTERT sequences and to screen the effective siRNA sequence. Five double-stranded siRNAs targeting coding and non-coding regions of hTERT gene were designed and synthesized by T7 transcription system in vitro. siRNA4 sequence was screened by full length gene targeting technique and the rest of the siRNA sequences were selected randomly. After being purified by ethanol precipitation, the siRNAs were transfected to the human hepatocellular carcinoma cell (HepG2) by Lipofectamine 2000. At 48-72 h after siRNAs transfection, MTT assay, RT-PCR and Western-blot were applied to evaluate the effects of siRNAs on cell growth, mRNA and protein expression level of hTERT gene, respectively. Compared to the control cells, the cells treated with the five double-stranded siRNAs exhibited different degrees of inhibition of cell proliferation in a dose-dependent manner. siRNA2 and siRNA4, exhibited obvious effects of inhibiting hTERT mRNA and protein expression in HepG2 cells. siRNAs targeting different hTERT sequences have significantly various inhibitory effects on hTERT gene expression. The siRNA sequence screened by full length gene targeting technique has comparable inhibitory effect with the rest siRNA sequences screened by random selection, suggesting that siRNAs and antisense oligonucleic acids may have the same effective target sites. Compared with chemical synthesis method, synthesizing double-stranded siRNA by T7 transcription system in vitro is a rapid, simple, and inexpensive method suitable for screening high-effect siRNA targeting site for specific gene.

Identification of two distinct elements mediating activation of telomerase (hTERT) gene expression in association with cell growth in human T cells

Lymphocytes are exceptional among normal somatic cells in that they express high telomerase activity like germline and malignant cells. We investigated the induction of telomerase in human T cells in association with cell growth. IL-2 significantly augmented the expression of mRNA for human telomerase reverse transcriptase (hTERT), a rate-limiting component of telomerase, in PHA-activated human peripheral blood leukocytes. An isolated 5'-flanking sequence (-3927-+51) of the hTERT gene was examined for its promoter activity in an IL-2-dependent human T cell line Kit 225. Addition of IL-2 into quiescent Kit 225 cells induced activation of the hTERT promoter. Reporter assays with mutant fragments of the hTERT promoter further revealed that IL-2-dependent activation was independently mediated by two elements within the +9-+51 regions. The two elements showed similar kinetics of activation in response to IL-2, which coincided with the G1 to S phase transition of the cell cycle. Interestingly, introduction of mutation in the elements increased background promoter activities in resting T cells in the absence of IL-2. Our results demonstrate that the hTERT promoter may be suppressed by the elements and IL-2 may signal for de-suppression in association with promotion of cell growth. IL-2-dependent activation of the hTERT promoter may be necessary for prevention from senescence induced by extraordinary cell division during immune reactions.

Demonstration of human telomerase reverse transcriptase by in situ hybridization in lung carcinoma

Telomerase activity is present in most malignant tumors and provides a mechanism for unlimited replication of neoplastic cells. This study was undertaken to investigate the expression of the gene encoding human telomerase reverse transcriptase (hTERT), the telomerase catalytic subunit gene in lung carcinoma, by in situ hybridization (ISH). hTERT is associated with telomerase activity, and overexpressed in most lung carcinomas. We assayed hTERT gene expression by ISH to study telomerase activity in lung carcinomas of 27 patients, and compared these results with those of telomerase activity by telomeric repeat amplification protocol (TRAP) assay. TERT gene expression by ISH was detected mainly in the nuclei of lung carcinoma cells. Some specimens showed a significant expression in only infiltrating lymphocytes. hTERT gene expression by ISH was found in 16 of 27 (59%) cases. On the other hand, telomerase activity by TRAP assay was detected in 21 of 27 (78%) cases. In 7 cases, TRAP assay detected telomerase activity, but ISH did not detect hTERT expression. TRAP assay might detect telomerase activity in not only carcinoma cells, but also in infiltrating lymphocytes. Our findings therefore suggest that ISH-based analysis of hTERT gene expression is superior to TRAP assay as a means of determining telomerase status during carcinogenesis.

Telomerase activity and hTERT mRNA expression in acute leukemia

Objective: To investigate the clinical implications of telomerase activity and human telomerase reverse transcriptase (hTERT) expression as useful diagnostic marker in acute leukemia. Methods: Expression of hTERT was detected by reverse transcription-polymerase chain reaction (RT-PCR) in 24 cases with acute leukemia and in 12 normal persons. Quantitative levels of telomerase activity were examined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). Results: In the bone marrow and peripheral blood of 24 acute leukemia, telomerase activity was detected in 75% of the samples, with absorbances (A) of 0.538±0.062 and 0.463±0.054, respectively. Whereas in 12 normal peripheral blood, telomerase activity had only a positive rate of 8.3%, with A value of 0.16±0.012. telomerase activities in the bone marrow and peripheral blood of acute leukemia were significantly higher than in normal control (P<0.05). RT-PCR analysis revealed that hTERT mRNA was expressed in 79.17%(19/24) of acute leukemia, but in only 1 of 12 normal peripheral blood. In 24 acute leukemias, 17 cases had both positive telomerse activity and hTERT mRNA expression. The expression of hTERT mRNA is correlated with telomerase activity (P<0.01). Conclusion: Telomerase and hTERT mRNA could be useful in diagnosis of acute leukemia. hTERT gene expression was strongly associated with telomerase activity in acute leukemia.

Study on Mechanisms of Telomerase Regulation in Arsenic Trioxide Inducing Apoptosis in Myelodysplasia Cell Line.

The telomerase activity can be down regulated by arsenic trioxide (As2O3), which is regarded as an apoptotic induction agent, is confirmed in many kinds of tumor cells. To investigate the mechanisms of telomerase regulation and to explore the correlation of As2O3 inducing apoptosis and telomerase regulation in MUTZ-1 cells, which are established as a high-risk myelodysplasia Cell line that derived from a MDS patient (FAB subtype refractory anemia with excess of blasts), a quantitative assessment of the telomerase activity by TRAP-ELISA and detection of the expression levels of hTERT, TRF1 (TTAGGG repeat binding factor 1), TRF2 (TTAGGG repeat binding factor 2), bcl-2, bax mRNA were performed, together with the assessment of the apoptosis by means of translocation of phosphatidylserine (PS) through flow cytometry assay. The results indicated that a typical apoptotic cell group distribution of DNA content was represented in the MUTZ-1 cells after being exposed to As2O3 at the range of concentration from 1μmol/L to 8μmol/L in a dose-dependent manner (r=0.736, P<0.001) and time-dependent manner (r=0.674, p<0.05), and the telomerase activity was down-regulated in a time-dependent manner (r=−0.976,P=0.024), and the expression level of hTERT mRNA in MUTZ-1 cells was represented in a dose-dependent manner (r=−0.892,P=0.042) and time-dependent manner (r=−1.000,P=0.04), after the cells were treated by As2O3 at the dosage as above. It was showed that a significant correlation between the decreased telomerase activity and the increased percentage of apoptotic cells in the treated cells (r=0.938,P=0.018), and there was a strong relationship between the telomerase activity and the mRNA expression of hTERT gene (r=0.783,P=0.022). However, As2O3 has no obvious effect on the expression level of TRF1 mRNA and TRF2 mRNA, which were regarded as two telomere-binding proteins. Further findings indicated that the inhibition of telomerase activity in MUTZ-1 cells was accompanied with down-regulated mRNA expression of bcl-2 gene (densitometry readings: 0.255±0.017 vs 0.466±0.069, P<0.05) and decreased ration of bcl-2/bax (densitometry reading ratios: 0.890±0.083 vs 0.546±0.014, P<0.05) at the dosage of 4μmol/L for 24 hours. These observations suggest that the apoptosis induced by As2O3 on MUTZ- 1 cells might be mediated through the inhibition of telomerase activity regulated by expression of hTERT gene, which implies that may be one of the mechanisms of As2O3 inducing apoptosis in MUTZ-1 cells.

Unusual distribution of DNA methylation within the hTERT CpG island in tissues and cell lines

The promoter region of the gene encoding the human telomerase reverse transcriptase ( hTERT) is located in a CpG island and was shown to be regulated, at least in part, by DNA methylation. However, the observed correlation between hTERT methylation and gene expression was opposite to the general model of regulation by DNA methylation. We established a detailed mapping of methylcytosines at the CpG island (−1539 to +1732) surrounding the hTERT promoter in tissues and cell lines. In telomerase-positive samples, a methylation of all the CpG sites was observed for the hTERT promoter region (−500 to +1), whereas the exonic part (+1 to +450) revealed an unstable methylation pattern. Incomplete methylation of the proximal exon region could be necessary for, at least, a low level of hTERT transcription. In conclusion, hypermethylation of the CpG island plays a complex but essential role in the expression of hTERT in telomerase-positive cells.

HTERT expression and prognosis in B-chronic lymphocytic leukemia

In B-chronic lymphocytic leukemia (B-CLL), there is a need for molecular markers to predict the evolution of this heterogeneous disease in individual patients. The level of expression of the human telomerase reverse transcriptase (hTERT) gene has been associated with disease aggressiveness in human cancers. The purpose of the present study was to examine the prognostic significance of hTERT expression in B-CLL. We used real-time reverse transcription-PCR to quantitate the amount of hTERT transcripts in mononuclear blood cells from 90 B-CLL patients. In addition, samples were analyzed for somatic mutations in the immunoglobulin V (IgV) genes. The expression of hTERT gene was detected in 59% of patients. The level of expression increased with advancing B-CLL stage (P=0.0064). Patients expressing hTERT showed significantly shorter survival than hTERT-negative patients (P=0.000034), irrespective of the disease stage. On average, the level hTERT mRNA expression was seven-fold higher in the poor-prognosis B-CLL group with unmutated IgV than in the Ig-mutated group (P<10(-7)). The level of hTERT expression discriminated the Ig-unmutated from Ig-mutated B-CLL in 89% of cases. Our data indicate that hTERT expression in B-CLL may serve as a molecular prognostic marker.

Telomerase inhibition by retinoids precedes cytodifferentiation of leukemia cells and may contribute to terminal differentiation

Human promyelocytic leukemia HL60 cells display high telomerase activity, a phenotype related to their immortal status. All-trans retinoic acid (ATRA) is a clinically effective cytodifferentiating agent. To understand the mechanism underlying ATRA-induced cytodifferentiation, we did a kinetic analysis of the role of ATRA in inhibiting telomerase in HL60 cells. Our studies indicate that telomerase inhibition by ATRA occurred relatively early after treatment of HL60 cells due to a rapid decrease in hTERT gene expression. More importantly, however, we found through monitoring the expression of CD11b, a marker for granulocytic differentiation of HL60 cells, that down-regulation of telomerase preceded the differentiation of HL60 cells. These observations suggest that the hTERT gene may be a primary target of ATRA regulation of cellular differentiation and the antileukemia activity of ATRA may be mediated by its ability to induce the differentiation of the promyelocytic leukemia cells through down-regulation of the hTERT gene.

Methylation status and expression of human telomerase reverse transcriptase in ovarian and cervical cancer

Objectives. Activation of telomerase, the enzyme that synthesizes the telomere ends of linear chromosomes, has been implicated in human cell immortalization and cancer cell pathogenesis. The expression pattern of human telomerase reverse transcriptase ( hTERT), the telomerase catalytic subunit gene, is correlated with telomerase activity. The promotor region of the hTERT gene has been located in a CpG island and may therefore be regulated, at least in part, by DNA methylation. The potential for methylation-mediated regulation of hTERT gene expression in ovarian and cervical cancer tissue has not been investigated up to now. The aim of this study was to investigate the expression and methylation pattern of hTERT in ovarian and cervical cancer tissue and their correlation with clinicopathological features and outcome of the disease. Methods. A total of 223 tissues were analyzed for hTERT methylation using MethyLight: 65 patients with cervical cancer and 124 with ovarian cancer were studied. The control group consisted of 20 normal ovarian tissues and 14 normal cervical tissues. Quantitative hTERT expression analysis was carried out in a subgroup of patients using real time PCR. Results. hTERT expression was statistically significantly higher in ovarian and cervical cancer tissue in comparison to normal tissue. While methylation of hTERT in cervical cancer was significantly more frequent in comparison to normal cervical tissue, the difference between ovarian cancer and normal ovarian tissue was not significant. No correlation was detected between hypermethylation of hTERT and hTERT mRNA expression. Both ovarian cancer and normal ovary showed an increase in hTERT methylation with increasing age. hTERT expression was not correlated with prognosis, whereas cervical and ovarian cancer patients with unmethylated hTERT had significantly better overall survival. Conclusion. At least in some tumor entities, hTERT methylation is a function of age and is associated with a poorer outcome, irrespective of hTERT expression.

Detection of telomerase hTERT gene expression and its splice variants by RT-PCR.

Detection of telomerase hTERT gene expression and its splice variants by RT-PCR.