hTERT Splice Research Articles

Spotlight on hTERT Complex Regulation in Cutaneous T-Cell Lymphomas

As a major cancer hallmark, there is a sustained interest in understanding the telomerase contribution to carcinogenesis in order to therapeutically target this enzyme. This is particularly relevant in primary cutaneous T-cell lymphomas (CTCL), a malignancy showing telomerase dysregulation with few investigative data available. In CTCL, we examined the mechanisms involved in telomerase transcriptional activation and activity regulation. We analyzed 94 CTCL patients from a Franco-Portuguese cohort, as well as 8 cell lines, in comparison to 101 healthy controls. Our results showed that not only polymorphisms (SNPs) located at the promoter of human telomerase reverse transcriptase (hTERT) gene (rs2735940 and rs2853672) but also an SNP located within the coding region (rs2853676) could influence CTCL occurrence. Furthermore, our results sustained that the post-transcriptional regulation of hTERT contributes to CTCL lymphomagenesis. Indeed, CTCL cells present a different pattern of hTERT spliced transcripts distribution from the controls, mostly marked by an increase in the hTERT β+ variants proportion. This increase seems to be associated with CTCL development and progression. Through hTERT splicing transcriptome modulation with shRNAs, we observed that the decrease in the α-β+ transcript induced a decrease in the cell proliferation and tumorigenic capacities of T-MF cells in vitro. Taken together, our data highlight the major role of post-transcriptional mechanisms regulating telomerase non canonical functions in CTCL and suggest a new potential role for the α-β+ hTERT transcript variant.

Intronic Cis-Element DR8 in hTERT Is Bound by Splicing Factor SF3B4 and Regulates hTERT Splicing in Non-Small Cell Lung Cancer.

Manipulation of a core spliceosome protein reduces telomerase/hTERT splicing in lung cancer cells and results in slowed cancer cell growth and cell death, revealing a potential therapeutic strategy.

Acute Exercise Regulates hTERT Gene Expression and Alternative Splicing in the hTERT-BAC Transgenic Mouse Model.

Aerobic exercise maintains telomere length through increased human telomerase reverse transcriptase (hTERT) expression and telomerase enzyme activity. The impact of acute exercise on hTERT alternative splicing (AS) is unknown. This study aimed to examine hTERT AS in response to acute treadmill running. A bacterial artificial chromosome mouse model containing the 54-kilobase hTERT gene locus inserted into its genome (hTERT-BAC) was utilized. The gastrocnemius, left ventricle, and brain were excised before (Pre), upon cessation (Post), and during recovery (1, 24, 48, and 72 h; n = 5/time point) from treadmill running (30 min at 60% maximum speed). Full-length (FL) hTERT and the "minus beta" (-β) AS variant (skips exons 7 and 8 and does not code for active telomerase) were measured by gel-based and droplet digital reverse transcription-polymerase chain reaction methods. SF3B4 and SRSF2 protein expression were measured by Western blotting. Compared with Pre, FL hTERT increased at Post before decreasing during recovery in the gastrocnemius (48 and 72 h; P ≤ 0.001) and left ventricle (24 h; P = 0.004). The percentage of FL hTERT in the gastrocnemius also increased during recovery (1 and 72 h; P ≤ 0.017), whereas a decrease was observed in the left ventricle (1, 24, and 48 h; P ≤ 0.041). hTERT decreased in the brain (48 h), whereas FL hTERT percentage remained unaltered. SF3B4 protein expression decreased throughout recovery in the gastrocnemius and tended to be associated with FL hTERT (r = -0.348, P = 0.075) and -β in opposite directions (r = 0.345, P = 0.067). Endurance exercise increased hTERT gene expression, and altered FL hTERT splicing in contractile tissues and may maintain telomere length necessary to improve the function and health of the organism.

Acute Exercise Regulates Htert Alternative Splicing In Contractile Tissues Of Htert-bac Mice

Full length human telomerase reverse transcriptase (FL hTERT) is the rate-limiting, catalytic component necessary for telomerase enzyme activity and telomere length maintenance. Unlike rodents, humans evolved elongated introns with cis-elements that when bound by specific RNA binding proteins (RNAbp) splice hTERT to FL transcripts or produce inactive variants through alternative splicing (AS). Our working hypothesis is that hTERT AS regulates the amount of active telomerase. Evidence from human studies have shown that aerobic exercise maintains telomere length through increased hTERT expression and telomerase enzyme activity, however the impact of acute exercise on hTERT AS is unknown. PURPOSE: To examine hTERT AS regulation in response to acute treadmill running. METHODS: A bacterial artificial chromosome mouse model containing the full hTERT gene (5' and 3' regulatory elements; 16 exons and 15 introns) inserted into its genome (hTERT-BAC) was utilized. Gastrocnemius (Gas.) and heart (Hrt.) were excised from 10-week old hTERT-BAC mice prior to (PRE), upon cessation (POST), and during recovery (1, 24, 48, and 72H; n = 5/time point) from 30 min. of treadmill running (60% max. speed). Primers specific to hTERT were utilized to measure FL and the most common AS variant minus beta (-β, which skips exons 7 and 8) by two different RT-PCR methods - gel based and droplet digital PCR. Gene expression of the RNAbp PTBP1 was also measured. RESULTS: The expected human patterns of hTERT AS was recapitulated across all tissues examined. Compared to PRE, FL hTERT gene expression increased at POST before decreasing in the Gas. (48 and 72H; p ≤ 0.001) and Hrt. (24H; p = 0.004). Acute exercise also increased the percentage of FL hTERT in the Gas. at 1 and 72H (p ≤ 0.017), whereas a decrease was observed in the Hrt. at 1, 24, and 48H (p ≤ 0.041). Finally, Ptbp1 decreased at POST (Gas.) and at 1, 24, 48, and 72H (Gas. and Hrt.; p ≤ 0.05). CONCLUSIONS: Evolutionarily conserved mouse splicing machinery (RNAbp) recognize hTERT cis-elements to recapitulate human AS patterns and highlight the importance of the intronic elements in regulating hTERT splicing choice in humans. The hTERT-BAC mouse is a valuable resource to study the exercise-induced adaptive mechanisms that maintain telomere lengths in endurance trained individuals across the lifespan.

Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

The Role of Alternative RNA Splicing in the Regulation of hTERT, Telomerase, and Telomeres: Implications for Cancer Therapeutics.

Alternative RNA splicing impacts the majority (>90%) of eukaryotic multi-exon genes, expanding the coding capacity and regulating the abundance of gene isoforms. Telomerase (hTERT) is a key example of a gene that is alternatively spliced during human fetal development and becomes dysregulated in nearly all cancers. Approximately 90% of human tumors use telomerase to synthesize de novo telomere repeats and obtain telomere-dependent cellular immortality. Paradigm shifting data indicates that hTERT alternative splicing, in addition to transcription, plays an important role in the regulation of active telomerase in cells. Our group and others are pursuing the basic science studies to progress this emerging area of telomerase biology. Recent evidence demonstrates that switching splicing of hTERT from the telomerase activity producing full-length hTERT isoform to alternatively spliced, non-coding isoforms may be a novel telomerase inhibition strategy to prevent cancer growth and survival. Thus, the goals of this review are to detail the general roles of telomerase in cancer development, explore the emerging regulatory mechanisms of alternative RNA splicing of the hTERT gene in various somatic and cancer cell types, define the known and potential roles of hTERT splice isoforms in cancer cell biology, and provide insight into new treatment strategies targeting hTERT in telomerase-positive cancers.

Abstract 2724: A combination of short-read and long-read RNA sequencing reveals NOVA1’s role in telomere biology

Abstract Dysregulated alternative splicing underlies many aspects of cancer cell biology including tumor cell telomere maintenance. We recently described a role for the neuronal splicing factor, NOVA1, in non-small cell lung cancer in the regulation of full-length hTERT splicing, telomerase activity, maintenance of telomere length, and general tumor growth characteristics. We wanted to further define the role of NOVA1 in telomere biology. Cells that maintain telomeres with different mechanisms were studied to be able to decipher telomerase specific versus alternative lengthening of telomeres (ALT) specific perturbations by NOVA1. Both short-read (Illumina) and long-read (Pacific Biosciences) sequencing of control and NOVA1 siRNA knockdown cells in H920 non-small cell lung cancer cells (telomerase positive) and U2OS cells (ALT positive cells) were performed to detail the transcriptomes of these cells. A combination of rMATS analysis of the short-read sequencing, SQANTI analysis of the long-read sequencing, and pathway analysis was performed to identify genes related to telomere biology and alternative splicing related to telomerase. Skipped exon events were the primary focus of this study as this is NOVA1’s main role in alternative splicing. rMATS analysis identified 4,530 significant events mapping to 2,950 genes in H920 cells and 2,534 significant events in 1,863 genes in U2OS cells. SQANTI analyzed 15,443 full-length transcripts (7,530 genes) in control and 14,463 transcripts (7,240 genes) in NOVA1 siRNA treated samples of the H920 cells and 12,133 transcripts (6,618 genes) in control and 10,126 transcripts (5,959 genes) in NOVA1 siRNA treated U2OS cells. There were 2,762 and 2,470 novel transcripts in control and NOVA1 siRNA treated H920 cells, respectively. In U2OS cells there were 2,178 novel transcripts in control and 1,688 novel transcripts in NOVA1 siRNA treated cells. The pathway analysis of rMATS cross-referenced with SQANTI revealed five top candidates that were specifically altered in NOVA1 knockdown cells and related telomere length maintenance by telomerase (POLD1, SUN2, ERCC1, SRSF2, and PCBP2). Interestingly, SRSF2 and PCBP2 were identified in our recent hTERT minigene loss of function screen for splicing regulatory genes of hTERT and have binding sites in hTERT near NOVA1s motif in intron 8. Long-read sequencing determined the full-length nature of these splicing events pointed out by the rMATS analysis. These data indicate that telomerase positive cancer cells with dysregulated expression of NOVA1 utilize different isoforms compared to ALT cells with NOVA1 to maintain telomeres. These new observations reveal that alternative splicing regulated in part by NOVA1 plays a larger and unexpected role in telomere biology. Manipulation of alternative splicing to shorten/deprotect telomeres should be pursued as a means to inhibit survival and growth of cancer cells. Citation Format: Andrew T. Ludlow, Mohammed E. Sayed, Aaron L. Slusher, Mark Ribick, Anisha Pancholi, Brian Sereni, Yu Qui, Elizabeth Tseng, Meredith Ashby, David C. Corney. A combination of short-read and long-read RNA sequencing reveals NOVA1’s role in telomere biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2724.

Insights into Telomerase/hTERT Alternative Splicing Regulation Using Bioinformatics and Network Analysis in Cancer.

The reactivation of telomerase in cancer cells remains incompletely understood. The catalytic component of telomerase, hTERT, is thought to be the limiting component in cancer cells for the formation of active enzymes. hTERT gene expression is regulated at several levels including chromatin, DNA methylation, transcription factors, and RNA processing events. Of these regulatory events, RNA processing has received little attention until recently. RNA processing and alternative splicing regulation have been explored to understand how hTERT is regulated in cancer cells. The cis- and trans-acting factors that regulate the alternative splicing choice of hTERT in the reverse transcriptase domain have been investigated. Further, it was discovered that the splicing factors that promote the production of full-length hTERT were also involved in cancer cell growth and survival. The goals are to review telomerase regulation via alternative splicing and the function of hTERT splicing variants and to point out how bioinformatics approaches are leading the way in elucidating the networks that regulate hTERT splicing choice and ultimately cancer growth.

Correlations between the expression of hTERT and α and β splice variants in human brain tumors.

Astrocytomas are diffusible infiltrative and aggressive brain tumors that are extensive and heterogeneous clusters of neoplastic growths in the central nervous system (CNS). Meningioma tumors are commonly benign but may demonstrate an invasive pattern with frequent recurrences. Human telomerase reverse transcriptase (hTERT) is an unfavorable prognostic factor for several types of cancers, and there are controversies about its role. In the present study, we investigated the relative expression of hTERT splice variants in 2 groups of brain tumors compared to non-tumor samples. The mRNA of 40 brain tumor samples and 4 control samples was extracted; mRNA expression of hTERT α-deletion and β-deletion variants, as well as the wild type isoform, was quantified using quantitative reverse transcription polymerase chain reaction (RT-qPCR). The α-deletion variant was significantly expressed in primary benign meningeal tumors (p = 0.01). The results indicate a positive correlation between the relative expression of hTERT mRNA transcript and α-deletion and β-deletion variants in both groups of tumors (meningiomas and astrocytomas). A strong association between the expression of the full-length splice variant and the β-deletion variant was observed in astrocytoma tumors (p = 0.045). The most significant correlations were found between the hTERT full-length and β-deletion variants in high-grade meningiomas (p = 0.018, correlation coefficient (CC) = 0.964) and grade II astrocytomas (p = 0.015; CC = 0.580). In addition, in low grades of both types of tumors, the hTERT full-length variant and especially the α-deletion variant were the predominant isoforms. The overexpression of hTERT and β-deletion variants in high grades of these tumors was statistically significant. Our findings indicate that α-deletion and β-deletion isoforms are associated with high levels of full-length hTERT mRNA in both groups of brain tumor patients. Changes in the splicing pattern of hTERT splice variants in brain tumors and their correlation with pathological alterations in cells could be applied as diagnostic or prognostic biomarkers, or possibly as targets for cancer therapy. However, the function and biological role of hTERT splice variants remain to be clarified.

A novel antisense oligonucleotide anchored on the intronic splicing enhancer of hTERT pre-mRNA inhibits telomerase activity and induces apoptosis in glioma cells

Alternative splicing of hTERT pre-mRNA is an important step in the regulation of telomerase activity, but the regulation mechanisms and functions remain unclear. RT-PCR analysis was used to detect hTERT splicing in glioma cell lines and brain tissues. TRAP assay was used to detect the telomerase activity. Then, we designed and synthesized 2'-O-methyl-RNA phosphorothioate AONs and transfected them into glioma cells to detect the changes in telomerase activity. MTT assay, plate colony formation assay, western blotting and Annexin V/PI assay were used to detect cell proliferation and apoptosis. At last, bioinformatics analyses were used to predict the expression and function of splicing protein SRSF2 in gliomas. hTERT splicing occurs both in glioma cell lines and glioma patients' tissues. The telomerase activity was related to the expression level of the full-length hTERT, rather than the total hTERT transcript level. AON-Ex726 was complementary to the sequence of the intronic splicing enhancer (ISE) in intron six, and significantly altered the splicing pattern of hTERT pre-mRNA, reducing the expression level of the full-length hTERT mRNA and increasing the expression level of the -β hTERT mRNA. After transfection with AON-Ex726, the level of apoptosis was increased, while telomerase activity and cell proliferation were significantly decreased. By bioinformatic predictions, we found the AON-Ex726 anchoring sequence in ISE overlaps the binding site of SRSF2 protein, which is up-regulated during the development of gliomas. Our findings provided new targets and important clues for the gene therapy of gliomas by regulating the alternative splicing pattern of hTERT pre-mRNA.

NOVA1 directs PTBP1 to hTERT pre-mRNA and promotes telomerase activity in cancer cells

Alternative splicing is dysregulated in cancer cells, driving the production of isoforms that allow tumor cells to survive and continuously proliferate. Part of the reactivation of telomerase involves the splicing of hTERT transcripts to produce full-length (FL) TERT. Very few splicing factors to date have been described to interact with hTERT and promote the production of FL TERT. We recently described one such splicing factor, NOVA1, that acts as an enhancer of FL hTERT splicing, increases telomerase activity, and promotes telomere maintenance in cancer cells. NOVA1 is expressed primarily in neurons and is involved in neurogenesis. In the present studies, we describe that polypyrimidine-tract binding proteins (PTBPs), which are also typically involved in neurogenesis, are also participating in the splicing of hTERT to FL in cancer. Knockdown experiments of PTBP1 in cancer cells indicate that PTBP1 reduces hTERT FL splicing and telomerase activity. Stable knockdown of PTBP1 results in progressively shortened telomere length in H1299 and H920 lung cancer cells. RNA pulldown experiments reveal that PTBP1 interacts with hTERT pre-mRNA in a NOVA1 dependent fashion. Knockdown of PTBP1 increases the expression of PTBP2 which also interacts with NOVA1, potentially preventing the association of NOVA1 with hTERT pre-mRNA. These new data highlight that splicing in cancer cells is regulated by competition for splice sites and that combinations of splicing factors interact at cis regulatory sites on pre-mRNA transcripts. By employing hTERT as a model gene, we show the coordination of the splicing factors NOVA1 and PTBP1 in cancer by regulating telomerase that is expressed in the vast majority of cancer cell types.

NOVA1 regulates hTERT splicing and cell growth in non-small cell lung cancer

Alternative splicing is dysregulated in cancer and the reactivation of telomerase involves the splicing of TERT transcripts to produce full-length (FL) TERT. Knowledge about the splicing factors that enhance or silence FL hTERT is lacking. We identified splicing factors that reduced telomerase activity and shortened telomeres using a siRNA minigene reporter screen and a lung cancer cell bioinformatics approach. A lead candidate, NOVA1, when knocked down resulted in a shift in hTERT splicing to non-catalytic isoforms, reduced telomerase activity, and progressive telomere shortening. NOVA1 knockdown also significantly altered cancer cell growth in vitro and in xenografts. Genome engineering experiments reveal that NOVA1 promotes the inclusion of exons in the reverse transcriptase domain of hTERT resulting in the production of FL hTERT transcripts. Utilizing hTERT splicing as a model splicing event in cancer may provide new insights into potentially targetable dysregulated splicing factors in cancer.

Contact-independent suppressive activity of regulatory T cells is associated with telomerase inhibition, telomere shortening and target lymphocyte apoptosis

Regulatory T cells (Tregs) play a fundamental role in the maintenance of immunological tolerance by suppressing effector target T, B and NK lymphocytes. Contact-dependent suppression mechanisms have been well–studied, though contact-independent Treg activity is not fully understood. In the present study, we showed that human native Tregs, as well as induced ex vivo Tregs, can cause in vitro telomere-dependent senescence in target T, B and NK cells in a contact-independent manner. The co-cultivation of target cells with Tregs separated through porous membranes induced alternative splicing of the telomerase catalytic subunit hTERT (human Telomerase Reverse Transcriptase), which suppressed telomerase activity. Induction of the hTERT splicing variant was associated with increased expression of the apoptotic endonuclease EndoG, a splicing regulator. Inhibited telomerase in target cells co-cultivated with Tregs for a long period of time led to a decrease in their telomere lengths, cell cycle arrest, conversion of the target cells to replicative senescence and apoptotic death. Induced Tregs showed the ability to up-regulate EndoG expression, TERT alternative splicing and telomerase inhibition in mouse T, B and NK cells after in vivo administration. The results of the present study describe a novel mechanism of contact-independent Treg cell suppression that induces telomerase inhibition through the EndoG-provoked alternative splicing of hTERT and converts cells to senescence and apoptosis phenotypes.

Expression analysis of the MCPH1/BRIT1 and BRCA1 tumor suppressor genes and telomerase splice variants in epithelial ovarian cancer

AimsThe aim of this study was to explore the correlation of hTERT splice variant expression with MCPH1/BRIT1 and BRCA1 expression in epithelial ovarian cancer (EOC) samples. BackgroundTelomerase activation can contribute to the progression of tumors and the development of cancer. However, the regulation of telomerase activity remains unclear. MCPH1 (also known as BRIT1, BRCT-repeat inhibitor of hTERT expression) and BRCA1 are tumor suppressor genes that have been linked to telomerase expression. MethodsqPCR was used to investigate telomerase splice variants, MCPH1/BRIT1 and BRCA1 expression in EOC tissue and primary cultures. ResultsThe wild type α+/β+ hTERT variant was the most common splice variant in the EOC samples, followed by α+/β− hTERT, a dominant negative regulator of telomerase activity. EOC samples expressing high total hTERT demonstrated significantly lower MCPH1/BRIT1 expression in both tissue (p = 0.05) and primary cultures (p = 0.03). We identified a negative correlation between MCPH1/BRIT1 and α+/β+ hTERT (p = 0.04), and a strong positive association between MCPH1/BRIT1 and both α−/β+ hTERT and α−/β− hTERT (both p = 0.02). A positive association was observed between BRCA1 and α−/β+ hTERT and α−/β− hTERT expression (p = 0.003 and p = 0.04, respectively). ConclusionsThese findings support a regulatory effect of MCPH1/BRIT1 and BRCA1 on telomerase activity, particularly the negative association between MCPH1/BRIT1 and the functional form of hTERT (α+/β+).

Alternative splicing of human telomerase reverse transcriptase in gliomas and its modulation mediated by CX-5461

BackgroundGlioma is a heterogeneous, invasive primary brain tumor with a wide range of patient survival and a lack of reliable prognostic biomarkers. Human telomerase reverse transcriptase (hTERT) has been reported in the presence of multiple transcripts in various tumor systems. The biological function and precise regulatory mechanisms of hTERT transcripts remain uncertain.MethodsAlternative splicing of hTERT and telomerase activity were examined in 96 glioma specimens, including 38 glioblastomas (GBMs), 23 oligodendrogliomas (ODMs), and 35 oligoastrocytomas (OAMs). The correlation between telomerase activity or hTERT transcripts and patient clinical characteristics was investigated. We examined the regulation of alternative splicing of hTERT and telomerase activity by G-quadruplex stabilizer CX-5461 in GBM cells. The biological effects of CX-5461 on GBM cell lines, including inhibition of cell proliferation, effects on cell cycle/apoptosis, and telomere DNA damage were further explored.ResultsThe β splicing was verified in human gliomas and hTERT+β was significantly correlated with higher telomerase activity, higher KPS, larger tumor size, and higher tumor grades. Meanwhile, glioma patients lacking hTERT+β expression or telomerase activity showed a significant survival benefit. Notably, CX-5461 altered hTERT splicing patterns, leading to an increase of hTERT-β transcript and a decrease of hTERT+β transcript expression, which inhibits telomerase activity. In addition, CX-5461 had cytotoxic effects on GBM cells and caused telomere DNA damage response, induced G2/M arrest and apoptosis.ConclusionsThe hTERT+β is verified to be correlated with clinical parameters in gliomas, and could serve as a prognostic marker or possibly therapeutic target for gliomas. CX-5461 can regulate the splicing pattern of hTERT, inhibit telomerase activity, and kill GBM cells.

Induction of Apoptotic Endonuclease EndoG with DNA-Damaging Agents Initiates Alternative Splicing of Telomerase Catalytic Subunit hTERT and Inhibition of Telomerase Activity hTERT in Human CD4+ and CD8+ Т Lymphocytes

Induction of Apoptotic Endonuclease EndoG with DNA-Damaging Agents Initiates Alternative Splicing of Telomerase Catalytic Subunit hTERT and Inhibition of Telomerase Activity hTERT in Human CD4+ and CD8+ Т Lymphocytes

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.

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.

Cisplatin-induced apoptotic endonuclease EndoG inhibits telomerase activity and causes malignant transformation of human CD4+ T lymphocytes

Alternative splicing of telomerase catalytic subunit hTERT pre-mRNA (human Telomerase Reverse Transcriptase) regulates telomerase activity. Increased expression of non-active splice variant hTERT results in inhibition of telomerase. Apoptotic endonuclease EndoG is known to participate in hTERT alternative splicing. Expression of EndoG can be induced in response to DNA damages. The aim of this study was to determine the ability of a DNA-damaging compound, cisplatin, to induce EndoG and its influence on alternative splicing of hTERT and telomerase activity in human CD4+ Т lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of active full-length hTERT variant and upregulated its non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. Few cells survived and underwent malignant transformation. Transformed cells have increased telomerase activity and proliferative potential compare to initial CD4+ T cells. These cells have phenotype of T lymphoblastic leukemic cells and are able to form tumors and cause death in experimental mice.

Intracellular Localization of Apoptotic Endonuclease EndoG and Splice-Variants of Telomerase Catalytic Subunit hTERT.

The activity of telomerase catalytic subunit hTERT (human telomerase reverse transcriptase) can be regulated by alternative splicing of its mRNA. The mechanism of hTERT splicing is not understood in detail. Apoptotic endonuclease EndoG is known to participate in this process. In the present work, the intracellular colocalization and mRNA levels of EndoG and hTERT splice-variants in normal and apoptotic cancer cells were studied. We found that the development of apoptosis increased the expression of EndoG and changed the ratio of hTERT splice-variants, which decreased the telomerase activity in the cells. The development of apoptosis was accompanied by changes in the amount of mRNA and in the localization of EndoG and hTERT splice-variants in the cytoplasm, nuclei, and mitochondria of the cells. The suppression of EndoG expression using RNA interference prevented induction of the α+β- splice-variant of hTERT and inhibition of the telomerase activity. A high degree of the intracellular colocalization of EndoG and hTERT was shown. The changes in the expression and localization of EndoG corresponded with changes in the amount and localization of hTERT splice-variants. These data confirm the participation of EndoG in the alternative splicing of mRNA of the telomerase catalytic subunit and in regulation of the telomerase activity.