Human Telomerase Catalytic Subunit Research Articles

Potential of Telomerase in Age-Related Macular Degeneration-Involvement of Senescence, DNA Damage Response and Autophagy and a Key Role of PGC-1α.

Age-related macular degeneration (AMD), the main cause of vision loss in the elderly, is associated with oxidation in the retina cells promoting telomere attrition. Activation of telomerase was reported to improve macular functions in AMD patients. The catalytic subunit of human telomerase (hTERT) may directly interact with proteins important for senescence, DNA damage response, and autophagy, which are impaired in AMD. hTERT interaction with mTORC1 (mTOR (mechanistic target of rapamycin) complex 1) and PINK1 (PTEN-induced kinase 1) activates macroautophagy and mitophagy, respectively, and removes cellular debris accumulated over AMD progression. Ectopic expression of telomerase in retinal pigment epithelium (RPE) cells lengthened telomeres, reduced senescence, and extended their lifespan. These effects provide evidence for the potential of telomerase in AMD therapy. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) may be involved in AMD pathogenesis through decreasing oxidative stress and senescence, regulation of vascular endothelial growth factor (VEGF), and improving autophagy. PGC-1α and TERT form an inhibitory positive feedback loop. In conclusion, telomerase activation and its ectopic expression in RPE cells, as well as controlled clinical trials on the effects of telomerase activation in AMD patients, are justified and should be assisted by PGC-1α modulators to increase the therapeutic potential of telomerase in AMD.

The regulatory function of tandem repeat VNTR2-1 in hTERT gene involves basic Helix-loop-helix family transcription factors.

Telomerase is a ribonucleoprotein enzyme that synthesizes telomere end sequence. The expression of hTERT gene, encoding the catalytic subunit of human telomerase, is restricted to highly proliferative tissues and is undetectable in most somatic cells. Abnormal activation of hTERT gene is found in 90% of human tumors. Previously, we identified tandem repeat of 42-bp/unit, VNTR2-1, in intron 2 of the hTERT gene, as a novel regulatory element important for hTERT transcription in cancer cells. In the current study, we found that multiple 42-bp repeats of VNTR2-1 activated luciferase gene in reporter plasmids. Mutation of the predicted cis-regulatory elements within the 42-bp repeats, including a E-box motif, resulted in a partial or complete loss of its enhancer activity. Moreover, MYC family proteins, c-MYC, MAX, and MNT, regulated hTERT gene transcription through both VNTR2-1 and E-boxes at the proximal hTERT promoter. Chromatin segmentation analysis of published ChIP-sequencing data from K562 cells indicated that VNTR2-1 was a bivalent enhancer. In telomerase-expressing human melanoma cell line MelJuSo, deletion of VNTR2-1 caused the hTERT promoter chromatin status to change from an active state to a repressed state, accompanied by increases of H3K27me3 and H3K9me3 marks. Therefore, we provided additional evidence for VNTR2-1 as a functional regulatory element that regulated hTERT expression by MYC family transcription factors. These results have improved our knowledge on the functions of repetitive genomic DNAs and the regulatory mechanisms of human telomerase gene.

Alternative Splicing of Human Telomerase Reverse Transcriptase (hTERT) and Its Implications in Physiological and Pathological Processes.

Alternative splicing (AS) of human telomerase catalytic subunit (hTERT, human telomerase reverse transcriptase) pre-mRNA strongly regulates telomerase activity. Several proteins can regulate AS in a cell type-specific manner and determine the functions of cells. In addition to being involved in telomerase activity regulation, AS provides cells with different splice variants that may have alternative biological activities. The modulation of telomerase activity through the induction of hTERT AS is involved in the development of different cancer types and embryos, and the differentiation of stem cells. Regulatory T cells may suppress the proliferation of target human and murine T and B lymphocytes and NK cells in a contact-independent manner involving activation of TERT AS. This review focuses on the mechanism of regulation of hTERT pre-mRNA AS and the involvement of splice variants in physiological and pathological processes.

The Regulation of ROS- and BECN1-Mediated Autophagy by Human Telomerase Reverse Transcriptase in Glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant brain tumor with high morbidity and mortality. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, is overexpressed in most cancers including GBM. It is well known that hTERT can compensate telomere shortening to immortalize cells. However, in addition to the canonical function, hTERT has the roles beyond canonical telomere maintenance. To further understand the effects of hTERT on glioblastoma progression, we investigated the role of hTERT in regulating autophagy—a conserved pathway, by which cells deliver cellular organic material and impaired organelles to the lysosomes for degradation and recycle these cargos to produce energy under a stressful condition. Our results showed that downregulation of hTERT impaired autophagy levels by suppressing BECN1/beclin-1 and induced an increase of reactive oxygen species (ROS), which resulted in cell death ultimately. On the contrary, overexpression of BECN1 or treating cells with the antioxidant N-acetylcysteine (NAC) could restore the survival of hTERT knockdown cells. Our study will provide an additional basis of telomerase-targeting therapy for future clinical anticancer treatment.

Melatonin and doxorubicin co-delivered via a functionalized graphene-dendrimeric system enhances apoptosis of osteosarcoma cells

A functionalized graphene-dendrimeric system was designed via Fe3O4 nanoparticle (NP) as a magnetic nanocarrier for co-delivery of doxorubicin (DOX) and melatonin (MLT). Accordingly, β-Cyclodextrin (β-CD) was modified by creating amine functional groups. The modified β-CD was grafted with Graphene oxide (GO), and the resulting platform gain many functional groups, including the hydroxyl (-OH), carboxylic acid (-COOH), and amine functional groups (−NH2). Finally, magnetic NPs were synthesized on the prepared platform to efficiently controlling and targeting drugs to tumor sites. The human osteosarcoma cell lines including Saos-2 and MG-63, as well as Human Bone Marrow Mesenchymal Stem Cells (hBM-MSC) line, were used to determine the in vitro biological effects of the functionalized graphene-dendrimeric system. The magnetic nanocarrier has encapsulation efficiency (EE) values of 99.92% for DOX and 21.5% for MLT. The biocompatibility tests of the nanocarrier revealed that the magnetic nanocarrier was appropriate as a drug carrier. Co-delivery of DOX and MLT with an efficiently anticancer performance was also was confirmed by cellular uptake, 4′,6-diamidino-2-phenylindole (DAPI) staining, and apoptosis analysis in comparison with free DOX and MLT. Moreover, there was a synergy in the antitumor effect when MLT was combined with DOX, especially in the nano-formulation form, which may be due to the down-regulation of X-linked Inhibitor of Apoptosis (XIAP), survivin, and human telomerase catalytic subunit (hTERT) (p < 0.0001). Overall, the result of our study suggests that the designed carrier is a promising nanocarrier for targeted co-delivery of DOX and MLT with improved anticancer efficacy in cancer cells and thus reduced toxicity in normal cells.

Combining conservation and species-specific differences to determine how human telomerase binds telomeres

Telomerase catalyzes telomeric DNA synthesis at chromosome ends to allow for continued cell division. The telomeric protein TPP1 is essential for enhancing the processivity of telomerase and recruiting the enzyme to telomeres. The telomerase interaction surface on human TPP1 has been mapped to 2 regions of the N-terminal oligosaccharide/oligonucleotide-binding (OB) domain, namely the TPP1 glutamate (E) and leucine (L)-rich (TEL) patch and the N terminus of TPP1-oligosaccharide/oligonucleotide-binding (NOB) region. To map the telomerase side of the interface, we exploited the predicted structural similarities for human and Tetrahymena thermophila telomerase as well as the species specificity of human and mouse telomerase for their cognate TPP1 partners. We show that swapping in the telomerase essential N-terminal (TEN) and insertions in fingers domain (IFD)-TRAP regions of the human telomerase catalytic protein subunit TERT into the mouse TERT backbone is sufficient to bias the species specificity toward human TPP1. Employing a structural homology-based mutagenesis screen focused on surface residues of the TEN and IFD regions, we identified TERT residues that are critical for contacting TPP1 but dispensable for other aspects of telomerase structure or function. We present a functionally validated structural model for how human telomerase engages TPP1 at telomeres, setting the stage for a high-resolution structure of this interface.

Inactivating Mutations in Exonuclease and Polymerase Domains in DNA Polymerase Delta Alter Sensitivities to Inhibitors of dNTP Synthesis.

POLD1 encodes the catalytic subunit of DNA polymerase delta (Polδ), the major lagging strand polymerase, which also participates in DNA repair. Mutations affecting the exonuclease domain increase the risk of various cancers, while mutations that change the polymerase active site cause a progeroid syndrome called mandibular hypoplasia, deafness, progeroid features, and lipodystrophy (MDPL) syndrome. We generated a set of catalytic subunit of human telomerase (hTERT)-immortalized human fibroblasts expressing wild-type or mutant POLD1 using the retroviral LXSN vector system. In the resulting cell lines, expression of endogenous POLD1 was suppressed in favor of the recombinant POLD1. The siRNA screening of DNA damage-related genes revealed that fibroblasts expressing D316H and S605del POLD1 were more sensitive to knockdowns of ribonuclease reductase (RNR) components, RRM1 and RRM2 in the presence of hydroxyurea (HU), an RNR inhibitor. On the contrary, SAMHD1 siRNA, which increases the concentration of dNTPs, increased growth of wild type, D316H, and S605del POLD1 fibroblasts. Hypersensitivity to dNTP synthesis inhibition in POLD1 mutant lines was confirmed using gemcitabine. Our finding is consistent with the notion that reduced dNTP concentration negatively affects the cell growth of hTERT fibroblasts expressing exonuclease and polymerase mutant POLD1.

The inhibitory effects of ginsenoside Rd on the human glioma U251 cells and its underlying mechanisms.

The current study was designed to investigate the inhibitory effects of ginsenoside Rd (Gs-Rd) on human glioma U251 cells in vitro and its possible underlying mechanisms. The groups included blank control group, low concentration Gs-Rd treatment group (20 μM), mid concentration Gs-Rd treatment group (40 μM), and high concentration Gs-Rd treatment group (80 μM). The proliferative activity of human glioma U251 cells was detected by the MTT assay. Flow cytometry was performed to measure cell apoptosis of human glioma U251 cells. In addition, the ELISA assay was used to measure the telomerase activities in different groups on 24 hours, 48 hours, and 72 hours. Furthermore, real-time quantitative polymerase chain reaction (RT-PCR) and Western blot analysis were performed to measure the expression of Bcl-2, human telomerase catalytic subunit (hTERT), and caspase-3 in different groups on 48 hours at both messenger RNA (mRNA) and protein levels. The proliferation of U251 cells was inhibited by Gs-Rd with different concentrations in the dose- and time-dependent manners. In addition, Gs-Rd promoted U251 cell apoptosis rate in a dose-dependent manner. Gs-Rd with different concentrations (20 μM, 40 μM, and 80 μM) significantly enhanced the expression of teleomerase on 24 hours and 48 hours. In addition, Gs-Rd with different concentrations significantly increased caspase-3 and decreased Bcl-2 and hTERT expressions at both mRNA and protein levels. The Gs-Rd can remarkably inhibit the proliferation and promote cell apoptosis of human glioma U251 cells. The possible underlying mechanisms could be related to inhibiting telomerase activity, downregulating expression of Bcl-2 and hTERT, and upregulating expression of caspase-3 of human glioma U251 cells.

Induction of Alternative Splicing and Inhibition of Activity of Telomerase Catalytic Subunit by Apoptotic Endonuclease EndoG in Human T, B, and NK Cells.

The effect of apoptotic endonuclease EndoG on alternative splicing of mRNA of human telomerase catalytic subunit hTERT (human telomerase reverse transcriptase) and telomerase activity in normal human lymphocytes were studied. Human CD4+, CD8+, B, and NK cells were transfected with a plasmid pEndoG-GFP containing EndoG gene or control plasmid pGFP. The levels of mRNA of EndoG or hTERT splicing variants were analyzed by real-time PCR. Protein content was assessed by Western blotting. Telomerase activity was measured by the telomere repeats amplification protocol. EndoG overexpression reduced the expression of active full-length hTERT and increased the expression of inactive splice variant. Shifted balance of hTERT splice variants in cells led to a significant decrease in telomerase activity within 72 h after transfection.

Modulation of Telomerase Activity in Cancer Cells by Dietary Compounds: A Review.

Telomerase is expressed in ~90% of human cancer cell lines and tumor specimens, whereas its enzymatic activity is not detectable in most human somatic cells, suggesting that telomerase represents a highly attractive target for selective cancer treatment. Accordingly, various classes of telomerase inhibitors have been screened and developed in recent years. We and other researchers have successfully found that some dietary compounds can modulate telomerase activity in cancer cells. Telomerase inhibitors derived from food are subdivided into two groups: one group directly blocks the enzymatic activity of telomerase (e.g., catechin and sulfoquinovosyldiacylglycerol), and the other downregulates the expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, via signal transduction pathways (e.g., retinoic acid and tocotrienol). In contrast, a few dietary components, including genistein and glycated lipid, induce cellular telomerase activity in several types of cancer cells, suggesting that they may be involved in tumor progression. This review summarizes the current knowledge about the effects of dietary factors on telomerase regulation in cancer cells and discusses their molecular mechanisms of action.

Apoptotic Endonuclease EndoG Induces Alternative Splicing of Telomerase TERT Catalytic Subunit, Caspase-2, DNase I, and BCL-x in Human, Murine, and Rat CD4+ T Lymphocytes

Apoptotic endonuclease EndoG plays a key role in the alternative splicing of mRNA of human TERT telomerase catalytic subunit. The aim of this work was to test the ability of EndoG to induce alternative splicing of mRNA of other genes and in other organisms. To determine new mRNA splice-variants, EndoG overexpression was induced in human, mouse and rat CD4+-T-lymphocytes followed by sequencing of total RNA of these cells. Sequencing results showed that besides TERT, EndoG induced alternative splicing of deoxyribonuclease I (DNase I), caspase-2 (Casp-2) and BCL-x. The expression level of EndoG strongly correlated with mRNA splicing-variants of TERT, DNase I, Casp-2, and BCL-x in intact CD4+-T cells of healthy donors as well as different lines of mice and rats. EndoG overexpression induced down-regulation of fulllength mRNAs of TERT, DNase I, Casp-2, and BCL-x and up-regulation of their short-length mRNAs. Alternative splicing of studied mRNAs resulted in down-regulation of enzymatic activity of proteins in vitro and in vivo. The results of this work confirm the ability of endonuclease EndoG to induce alternative splicing of several mRNAs in human, mice and rats.

Alternative splicing of telomerase catalytic subunit hTERT generated by apoptotic endonuclease EndoG induces human CD4+ T cell death

Telomerase activity is regulated by alternative splicing of its catalytic subunit human Telomerase Reverse Transcriptase (hTERT) mRNA. Induction of a 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 the apoptotic endonuclease EndoG in alternative splicing of hTERT and telomerase activity. A strong correlation was identified between EndoG expression levels and hTERT splice variants in human CD4+ and CD8+ T lymphocytes. Overexpression of EndoG in CD4+ T cells down-regulated the expression of the active full-length hTERT variant and up-regulated expression of the non-active spliced variant. A reduction in full-length hTERT transcripts down-regulated telomerase activity. Long-term in vitro cultivation of EndoG-overexpressing CD4+ T cells led to dramatically shortened telomeres, conversion of cells into a replicative senescence state, and activation of the BCL2/BAX-associated apoptotic pathway finally leading to cell death. These data indicated the participation of EndoG in alternative mRNA splicing of the telomerase catalytic subunit hTERT, regulation of telomerase activity and determination of cell fate.

Establishment of a protocol to extend the lifespan of human hormone-secreting pituitary adenoma cells.

The aim of this study was to generate immortalized human anterior pituitary adenoma cells. Reliable cell models for the study of human pituitary adenomas are as yet lacking and studies performed so far used repeated passaging of freshly excised adenomas, with the attendant limitations due to limited survival in culture, early senescence, and poor reproducibility. We devised a technique based upon repeated co-transfections of two retroviral vectors, one carrying the catalytic subunit of human telomerase, hTERT, the other SV40 large T antigen. This approach extended the lifespan of cells derived from a human growth hormone-secreting adenoma up to 18 months while retaining morphology of primary cells, growth hormone synthesis and growth hormone secretion. Our attempt represents the first demonstration of successful lifespan extension of human growth hormone-secreting pituitary adenoma cells via co-transfection of hTERT and SV40T and paves the way to future attempts to obtain stable cell lines.

Injection of hTERT-Transduced Endothelial Progenitor Cells Promotes Beneficial Aortic Changes in a High-Fat Dietary Model of Early Atherosclerosis

Objectives: Cultured endothelial progenitor cells (EPCs) display troubling issues that adversely affect their applicability to endothelial regeneration. We hypothesized that transduction of the human telomerase catalytic subunit (hTERT) gene would enhance EPC function in treating dietary-induced early atherosclerosis (AS). Methods: A dietary-induced early AS model was successfully constructed in 90 healthy male rats, while 30 healthy control (HC) rats were normally fed. Four experimental groups were constructed: an untreated HC group; an untreated AS group injected with PBS; a null EPC AS group injected with null vector-transduced EPCs, and an hTERT EPC AS group injected with hTERT-transduced EPCs. Two months postinjection, abdominal aortas were extracted to validate EPC integration and comparatively assess mRNA and protein expression of the early atherosclerotic markers VCAM-1, ICAM-1, LFA-1, Mac-1, CD44, MCP-1, endothelial nitric oxide synthase (eNOS), and apolipoprotein E. Results: In vitro, hTERT transduction of EPCs resulted in a significantly superior proliferative capacity as well as significantly higher NO, iNOS, and LDH secretory capacity. In vivo injection of hTERT-transduced EPCs produced significant reductions in CD44 and MCP-1 expression as well as a significant increase in eNOS expression relative to injection with null vector-transduced EPCs (all p < 0.05). Conclusion: hTERT-transduced human EPCs may be useful in treating dietary-induced early AS.

Abstract 2731: Impact of hypomethylating agents on hTERT expression and synergistic effect in combination with imetelstat, a telomerase inhibitor, in AML cell lines

Abstract The catalytic subunit of human telomerase (hTERT) is highly expressed in acute myeloid leukemia (AML). Transcriptional factors and epigenetic modifications regulate this expression. Studies suggest hypermethylation promotes hTERT overexpression. Imetelstat is a competitive telomerase inhibitor. Decitabine (DAC) and azacitidine (AZA) are DNA methyltransferase inhibitors (DNMTIs) used in the treatment of AML. Combining imetelstat and DNMTI may be an improved treatment option for AML. We investigated effects of DAC and AZA on hTERT expression and cell viability in AML cell lines OCI-AML3 and OCI-AML5, and evaluated growth inhibition when combined with imetelstat. OCI-AML3 had higher baseline expression of hTERT compared with OCI-AML5. Dose-dependent downregulation of hTERT expression was observed with DAC and AZA treatments. OCI-AML5 was more sensitive to DAC (1 μM, 46% viability) than OCI-AML3 (1 μM, 95% viability) whereas sensitivity to AZA was similar in both cell lines (1 μM, 29% vs 33% viability). After removal of the drugs, growth inhibition was not sustained and cell proliferation rebounded. Single-agent imetelstat reduced both hTERT expression and telomerase activity but resulted in only limited growth inhibition in these cell lines. It was hypothesized that combining DNMTI with imetelstat may exert more potent growth inhibition. To test this, both cell lines were treated with DAC or AZA for 72 hours followed by different concentrations of imetelstat for 2 to 4 weeks. Dose-dependent synergistic activity was demonstrated for DAC followed by imetelstat: OCI-AML3 cell viability changed from 95% with DAC to 61% and 10% after 2 and 4 weeks of imetelstat, respectively; OCI-AML5 cell viability changed from 46% with DAC to 6% and 1% after 2 and 4 weeks of imetelstat, respectively. In contrast, no enhanced activity was observed in either cell line with AZA followed by imetelstat, but imetelstat was able to slow the growth rebound after AZA removal. Additional studies are ongoing to further understand the mechanism of action of this drug combination in AML and to evaluate different combination sequences of DNMTI with imetelstat for optimal anticancer activity. In summary, DAC and AZA reduced hTERT expression and differentially inhibited cell viability in AML cell lines. Although at the same concentration AZA was more active than DAC in OCI-AML3 and OCI-AML5 lines, the sustainability of growth inhibition after washout differed. Growth rebounded after removal of AZA or DAC treatment. Dose-dependent synergistic activity demonstrated by DAC followed by imetelstat was not seen with AZA followed by imetelstat. Distinctly different mechanisms of action of DAC and AZA may underlie their differential activity when combined with imetelstat, warranting further investigation. Citation Format: Joshua Rusbuldt, Jacqueline Bussolari, Aleksandra Rizo, Fei Huang. Impact of hypomethylating agents on hTERT expression and synergistic effect in combination with imetelstat, a telomerase inhibitor, in AML cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2731.

Apoptotic endonuclease EndoG regulates alternative splicing of human telomerase catalytic subunit hTERT

Human telomerase catalytic subunit hTERT is subjected to alternative splicing results in loss of its function and leads to decrease of telomerase activity. However, very little is known about the mechanism of hTERT pre-mRNA alternative splicing. Apoptotic endonuclease EndoG is known to participate this process. The aim of this study was to determine the role of EndoG in regulation of hTERT alternative splicing. Increased expression of b-deletion splice variant was determined during EndoG over-expression in CaCo-2 cell line, after EndoG treatment of cell cytoplasm and nuclei and after nuclei incubation with EndoG digested cell RNA. hTERT alternative splicing was induced by 47-mer RNA oligonucleotide in naked nuclei and in cells after transfection. Identified long non-coding RNA, that is the precursor of 47-mer RNA oligonucleotide. Its size is 1754 nucleotides. Based on the results the following mechanism was proposed. hTERT pre-mRNA is transcribed from coding DNA strand while long non-coding RNA is transcribed from template strand of hTERT gene. EndoG digests long non-coding RNA and produces 47-mer RNA oligonucleotide complementary to hTERT pre-mRNA exon 8 and intron 8 junction place. Interaction of 47-mer RNA oligonucleotide and hTERT pre-mRNA causes alternative splicing.

Aβ 1–40 enhances the proliferation of human diploid fibroblasts

There is a vast literature on the role of beta amyloid (Aβ) peptides in the pathogenesis of Alzheimer's disease. However, there is a paucity of research on the potential physiological functions of these evolutionarily conserved products of the Aβ precursor protein. Based on previous studies in neuroblastoma cells, we hypothesized that Aβ may contribute to the proliferation of somatic cells. We present evidence supporting this hypothesis for the case of cultured human skin fibroblasts immortalized with the catalytic subunit of human telomerase (hTERT). Optimal concentrations ranged from 100 pM–10 nM, depending on the nature of the assay.

Polo-like Kinase 1 (Plk1) Up-regulates Telomerase Activity by Affecting Human Telomerase Reverse Transcriptase (hTERT) Stability

Maintenance of telomere is regulated by active telomerase complex, including telomerase holoenzyme and its associated proteins. The activity of telomerase is precisely controlled in cells, and its dysregulation is one of the hallmarks of cancer. The telomerase catalytic subunit human telomerase reverse transcriptase (hTERT) plays a central role for telomerase activity. In this study, we indentified that Polo-like kinase 1 (Plk1) is a novel telomerase-associated protein. Plk1 can interact with hTERT independently of its kinase activity. More importantly, we found that Plk1 is associated with active telomerase complex. In addition, we demonstrated that knockdown of Plk1 caused the reduction of telomerase activity, whereas overexpression of Plk1 increased telomerase activity. Further analysis showed that overexpression of Plk1 led to a significant increase of hTERT protein by prolonging its half-life but did not affect the level of hTERT mRNA. Furthermore, we found that Plk1 enhanced the chromatin loading of hTERT and inhibited its ubiquitination. This implied that Plk1 affected hTERT stability by inhibiting its ubiquitin-mediated degradation. Collectively, these observations suggested that Plk1 is a positive modulator of telomerase by enhancing the stability of hTERT.

New NCI-N87-derived human gastric epithelial line after human telomerase catalytic subunit over-expression.

To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori (H. pylori) infection. Aiming to overcome this limitation, clones of the heterogenic cancer-derived NCI-N87 cell line were isolated, by stably-transducing it with the human telomerase reverse-transcriptase (hTERT) catalytic subunit gene. The clones were first characterized regarding their cell growth pattern and phenotype. For that we measured the clones' adherence properties, expression of cell-cell junctions' markers (ZO-1 and E-cadherin) and ability to generate a sustained transepithelial electrical resistance. The gastric properties of the clones, concerning expression of mucins, zymogens and glycan contents, were then evaluated by haematoxylin and eosin staining, Periodic acid Schiff (PAS) and PAS/Alcian Blue-staining, immunocytochemistry and Western blot. In addition, we assessed the usefulness of the hTERT-expressing gastric cell line for H. pylori research, by performing co-culture assays and measuring the IL-8 secretion, by ELISA, upon infection with two H. pylori strains differing in virulence. Compared with the parental cell line, the most promising NCI-hTERT-derived clones (CL5 and CL6) were composed of cells with homogenous phenotype, presented higher relative telomerase activities, better adhesion properties, ability to be maintained in culture for longer periods after confluency, and were more efficient in PAS-reactive mucins secretion. Both clones were shown to produce high amounts of MUC1, MUC2 and MUC13. NCI-hTERT-CL5 mucins were shown to be decorated with blood group H type 2 (BG-H), Lewis-x (Le(x)), Le(y) and Le(a) and, in a less extent, with BG-A antigens, but the former two antigens were not detected in the NCI-hTERT-CL6. None of the clones exhibited detectable levels of MUC6 nor sialylated Le(x) and Le(a) glycans. Entailing good gastric properties, both NCI-hTERT-clones were found to produce pepsinogen-5 and human gastric lipase. The progenitor-like phenotype of NCI-hTERT-CL6 cells was highlighted by large nuclei and by the apical vesicular-like distribution of mucin 5AC and Pg5, supporting the accumulation of mucus-secreting and zymogens-chief mature cells functions. These traits, in addition to resistance to microaerobic conditions and good responsiveness to H. pylori co-culture, in a strain virulence-dependent manner, make the NCI-hTERT-CL6 a promising model for future in vitro studies.

Subtelomeric demethylation deregulated hTERT expression, telomerase activity, and telomere length in four nasopharyngeal carcinoma cell lines.

Global DNA hypomethylation, in particular that of the gene promoter sequence in gene hypermethylation, is a well-known characteristic of human cancer. Subtelomeres are enriched CpG islands; methylation is believed to be a potential epigenetic regulator. However, regulation on the telomere length remains largely unknown. To demonstrate this correlation, four nasopharyngeal carcinoma cell lines (CNE, CNE1, CNE2, 5-8F) were treated for 72 hours with 0, 1, or 2.5 μM of the demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC). Subtelomeric (D4Z4) level methylation was evaluated with a bisulfite assay, the human telomerase catalytic subunit (hTERT) expression was assayed by reverse transcription-polymerase chain reaction, the telomerase activity was detected using a telomeric repeat amplification protocol assay, and the telomere length was measured by Southern blot terminal restriction fragment analysis. There was significant demethylation following 5-aza-dC treatment, and a strongly repressed hTERT expression decreased the telomerase activity and remarkably shortened telomeres. Thus, partial subtelomeric methylation does not repress hTERT expression; conversely, demethylation may downregulate hTERT expression and shorten telomeres.