Catalytic Component Of Telomerase Research Articles

Human artificial chromosome carrying 3p21.3-p22.2 region suppresses hTERT transcription in oral cancer cells

Telomerase is a ribonucleoprotein ribonucleic enzyme that elongates telomere repeat sequences at the ends of chromosomes and contributes to cellular immortalization. The catalytic component of telomerase, human telomerase reverse transcriptase (hTERT), has been observed to be reactivated in immortalized cells. Notably, most cancer cells have been found to have active hTERT mRNA transcription, resulting in continuous cell division, which is crucial for malignant transformation. Therefore, discovering mechanisms underlying the regulation of hTERT transcription is an attractive target for cancer-specific treatments.Loss of heterozygosity (LOH) of chromosome 3p21.3 has been frequently observed in human oral squamous cell carcinoma (OSCC). Moreover, we previously reported that HSC3 OSCC microcell hybrid clones with an introduced human chromosome 3 (HSC3#3) showed inhibition of hTERT transcription compared with the parental HSC3 cells. This study examined whether hTERT transcription regulators are present in the 3p21.3 region. We constructed a human artificial chromosome (HAC) vector (3p21.3-HAC) with only the 3p21.3-p22.2 region and performed functional analysis using the 3p21.3-HAC. HSC3 microcell hybrid clones with an introduced 3p21.3-HAC exhibited significant suppression of hTERT transcription, similar to the microcell hybrid clones with an intact chromosome 3. In contrast, HSC3 clones with truncated chromosome 3 with deletion of the 3p21.3 region (3delp21.3) showed no effect on hTERT expression levels. These results provide direct evidence that hTERT suppressor gene(s) were retained in the 3p21.3 region, suggesting that the presence of regulatory factors that control telomerase enzyme activity may be involved in the development of OSCC.

Short-Term TERT Inhibition Impairs Cellular Proliferation via a Telomere Length-Independent Mechanism and Can Be Exploited as a Potential Anticancer Approach.

Telomerase reverse transcriptase (TERT), the catalytic component of telomerase, may also contribute to carcinogenesis via telomere-length independent mechanisms. Our previous in vitro and in vivo studies demonstrated that short-term telomerase inhibition by BIBR1532 impairs cell proliferation without affecting telomere length. Here, we show that the impaired cell cycle progression following short-term TERT inhibition by BIBR1532 in in vitro models of B-cell lymphoproliferative disorders, i.e., Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines (LCLs), and B-cell malignancies, i.e., Burkitt's lymphoma (BL) cell lines, is characterized by a significant reduction in NF-κB p65 nuclear levels leading to the downregulation of its target gene MYC. MYC downregulation was associated with increased expression and nuclear localization of P21, thus promoting its cell cycle inhibitory function. Consistently, treatment with BIBR1532 in wild-type zebrafish embryos significantly decreased Myc and increased p21 expression. The combination of BIBR1532 with antineoplastic drugs (cyclophosphamide or fludarabine) significantly reduced xenografted cells' proliferation rate compared to monotherapy in the zebrafish xenograft model. Overall, these findings indicate that short-term inhibition of TERT impairs cell growth through the downregulation of MYC via NF-κB signalling and supports the use of TERT inhibitors in combination with antineoplastic drugs as an efficient anticancer strategy.

TERT promoter mutations and methylation for telomerase activation in urothelial carcinomas: New mechanistic insights and clinical significance.

Telomerase, an RNA-dependent DNA polymerase synthesizing telomeric TTAGGG sequences, is primarily silent in normal human urothelial cells (NHUCs), but widely activated in urothelial cell-derived carcinomas or urothelial carcinomas (UCs) including UC of the bladder (UCB) and upper track UC (UTUC). Telomerase activation for telomere maintenance is required for the UC development and progression, and the key underlying mechanism is the transcriptional de-repression of the telomerase reverse transcriptase (TERT), a gene encoding the rate-limiting, telomerase catalytic component. Recent mechanistic explorations have revealed important roles for TERT promoter mutations and aberrant methylation in activation of TERT transcription and telomerase in UCs. Moreover, these TERT-featured genomic and epigenetic alterations have been evaluated for their usefulness in non-invasive UC diagnostics, recurrence monitoring, outcome prediction and response to treatments such as immunotherapy. Importantly, the detection of the mutated TERT promoter and TERT mRNA as urinary biomarkers holds great promise for urine-based UC liquid biopsy. In the present article, we review recent mechanistic insights into altered TERT promoter-mediated telomerase activation in UCs and discuss potential clinical implications. Specifically, we compare differences in senescence and transformation between NHUCs and other types of epithelial cells, address the interaction between TERT promoter mutations and other factors to affect UC progression and outcomes, evaluate the impact of TERT promoter mutations and TERT-mediated activation of human endogenous retrovirus genes on UC immunotherapy including Bacillus Calmette-Guérin therapy and immune checkpoint inhibitors. Finally, we suggest the standardization of a TERT assay and evaluation system for UC clinical practice.

Abstract 5562: Development of a TERT-specific peptide/adjuvant vaccine in C57BL/6 mice

Abstract Telomerase plays a vital role in cell senescence and cellular replication and has been described as a leading regulator in several human cancers, including prostate, thyroid, breast, colon, and bladder cancer. Telomerase is responsible for elongating telomere length and prolonging cellular lifespan as well as acting as a transcriptional modulator in cancer signaling pathways. Because of its enhanced expression in primary tumor cells as well as incipient tumor-initiating stem cells and with limited distribution in normal somatic cells, the catalytic component of telomerase, TERT (telomerase reverse transcriptase), is regarded as a high-impact target for immunotherapeutic agents and vaccines. In order to derive a mouse TERT (mTERT)-specific vaccine which could be useful in a preventive approach, we implemented an epitope-mapping stratagem by which pools of 30-mer peptides overlapping by 5 spanning the entire mTERT sequence were used to immunize C57BL/6 mice. Several immunogenic peptides were identified by matrixed peptide pool IFN-ɣ ELISPOT screening in a 2-part process that first identified positive pools, then identified individual peptides. 15-mer and 31-mer versions of the top 7 immunogenic mTERT peptides were synthesized and used as a peptide pool combined separately with several adjuvants to optimize immunogenicity. Results suggested a peptide sequence-specific preference for TLR3-mediated Hiltonol versus TLR9-mediated CpG-based adjuvants. In addition, the majority of T cell responses specific to the TERT peptides was identified through flow cytometry as belonging to the CD4 compartment, although one class I-restricted epitope was also identified. Multifunctional CD4+ T cells expressing IFN-ɣ, TNF-α, IL-2, and CD107a were identified specific to several mTERT peptides. In vivo cytotoxic effects exerted on TERT peptide pool-loaded target cells were also uncovered that were specific to a CpG-adjuvanted version of the vaccine. Finally, multiple versions of the mTERT peptide/adjuvant vaccine with highest immunogenicity scores are being tested in a syngeneic graft mouse model using subcutaneous implantation of lung cancer-derived SPON10 cells expressing mTERT. If proven efficacious, these results will support the feasibility of a TERT peptide/adjuvant approach for prophylactically engendering broad antitumor immunity to TERT-expressing cancers. Funded by NCI Contract No. HHSN261200800001E Citation Format: Jason D. Marshall, Yurong Song, Hamid Zarkesh, Rebecca L. Matthews, Chelsea Sanders, Simone Difilippantonio, Ligia A. Pinto, Shizuko Sei, Robert H. Shoemaker. Development of a TERT-specific peptide/adjuvant vaccine in C57BL/6 mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5562.

Identification and characterization of novel ACD variants: modulation of TPP1 protein level offsets the impact of germline loss-of-function variants on telomere length.

Telomere biology disorders, largely characterized by telomere lengths below the first centile for age, are caused by variants in genes associated with telomere replication, structure, or function. One of these genes, ACD, which encodes the shelterin protein TPP1, is associated with both autosomal dominantly and autosomal recessively inherited telomere biology disorders. TPP1 recruits telomerase to telomeres and stimulates telomerase processivity. Several studies probing the effect of various synthetic or patient-derived variants have mapped specific residues and regions of TPP1 that are important for interaction with TERT, the catalytic component of telomerase. However, these studies have come to differing conclusions regarding ACD haploinsufficiency. Here, we report a proband with compound heterozygous novel variants in ACD (NM_001082486.1)—c.505_507delGAG, p.(Glu169del); and c.619delG, p.(Asp207Thrfs*22)—and a second proband with a heterozygous chromosomal deletion encompassing ACD: arr[hg19] 16q22.1(67,628,846-67,813,408)x1. Clinical data, including symptoms and telomere length within the pedigrees, suggested that loss of one ACD allele was insufficient to induce telomere shortening or confer clinical features. Further analyses of lymphoblastoid cell lines showed decreased nascent ACD RNA and steady-state mRNA, but normal TPP1 protein levels, in cells containing heterozygous ACD c.619delG, p.(Asp207Thrfs*22), or the ACD-encompassing chromosomal deletion compared to controls. Based on our results, we conclude that cells are able to compensate for loss of one ACD allele by activating a mechanism to maintain TPP1 protein levels, thus maintaining normal telomere length.

The Link between Chronic Stress and Accelerated Aging.

People exposed to chronic stress age rapidly. The telomeres in their cells of all types shorten faster. Inflammation is another important feature of stress that, along with aging, accounts for the phenomenon of inflammaging. In addition to aging itself, inflammaging can contribute to the development of several pathologies, including atherosclerosis, diabetes, hypertension, and others. Oxidative stress is one of the main mechanisms related to stress. Oxidative stress is caused by the over-production of reactive oxygen species (ROS) that can damage various tissues. The main source of ROS is mitochondria. Being suppressed by mitochondrial mutations, mitophagy can aggravate the situation. In this case, the aging-specific pro-inflammatory changes are amplified. It happens because of the inability of cells to maintain the normal state of mitochondria. Macrophages are the crucial element of the innate immunity associated with the chronic inflammation and, subsequently, with the inflammaging. In this review, we focus on the therapy approaches potentially reducing the deleterious effects of oxidative stress. These include stimulation of mitophagy, activation of mitochondrial uncoupling, induction of the expression of the telomerase catalytic component gene, and use of antioxidants. Any method reducing oxidative stress should improve post-traumatic stress disorder.

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.

ETS1, nucleolar and non-nucleolar TERT expression in nevus to melanoma progression.

TERT (telomerase reverse transcriptase) is the catalytic component of telomerase. TERT shows little expression in normal somatic cells but is commonly re-expressed in cancers, facilitating immortalization. Recently-discovered TERT promoter mutations create binding sites for ETS-family transcription factors to upregulate TERT. ETS1 is reported to be important for TERT upregulation in melanoma. However it is unclear when in melanoma progression TERT and ETS1 proteins are expressed. To elucidate this question, ETS1 and TERT immunohistochemistry were performed on a panel of benign (n=27) and dysplastic nevi (n=34), radial growth phase (n=29), vertical growth phase (n=25) and metastatic melanomas (n=27). Lesions were scored by percentage of positive cells. ETS1 was readily detectable in all lesions, but not in normal melanocytes. TERT was located in either the nucleolus, the nucleoplasm (non-nucleolar) or both. Non-nucleolar TERT increased in prevalence with progression, from 19% of benign nevi to 78% of metastases. It did not however correlate with cell proliferation (Ki-67 immunostaining), nor differ significantly in prevalence between primary melanomas with or without a TERT promoter mutation. These results demonstrate that ETS1 is expressed very early in melanoma progression, and interestingly only non-nucleolar TERT correlates clearly in prevalence with melanoma progression. It can be acquired at various stages and by mechanisms other than promoter mutations.

Transcriptional Regulation of Telomerase Reverse Transcriptase (TERT) by MYC.

Telomerase elongates telomeres and is crucial for maintaining genomic stability. While stem cells and cancer cells display high telomerase activity, normal somatic cells lack telomerase activity primarily due to transcriptional repression of telomerase reverse transcriptase (TERT), the catalytic component of telomerase. Transcription factor binding, chromatin status as well as epigenetic modifications at the TERT promoter regulates TERT transcription. Myc is an important transcriptional regulator of TERT that directly controls its expression by promoter binding and associating with other transcription factors. In this review, we discuss the current understanding of the molecular mechanisms behind regulation of TERT transcription by Myc. We also discuss future perspectives in investigating the regulation of Myc at TERT promoter during cancer development.

Analysis of Yeast Telomerase by Primer Extension Assays.

Telomeres are specialized nucleoprotein structures located at eukaryotic chromosomal termini, which are required for chromosome stability and are maintained by a reverse transcriptase named telomerase. Budding yeast has served as an extremely useful model system for analyzing telomere maintenance because the organism offers a wide range of genetic and biochemical tools. Several milestones in telomerase research have been reached through investigation of the yeast system. For example, the consequence of telomerase loss was first characterized in the budding yeast Saccharomyces cerevisiae. The catalytic component of telomerase (telomerase reverse transcriptase; TERT) was likewise initially cloned from this organism. Moreover, much of the current understanding of the structure and function of the telomerase complex was derived from yeast studies. In this chapter, we discuss one of the most useful tools for investigating yeast telomerase mechanisms and regulation: the primer extension assay. This assay can be used to examine the overall activity as well as the processivity of telomerase, which represents a unique aspect of telomerase enzymology. It can also be employed to analyze the mechanisms of telomerase regulatory proteins.

AZT exerts its antitumoral effect by telomeric and non-telomeric effects in a mammary adenocarcinoma model.

Limitless replicative potential is one of the hallmarks of cancer that is mainly due to the activity of telomerase. This holoenzyme maintains telomere length, adding TTAGGG repetitions at the end of chromosomes in each cell division. In addition to this function, there are extratelomeric roles of telomerase that are involved in cancer promoting events. It has been demonstrated that TERT, the catalytic component of telomerase, acts as a transcriptional modulator in many signaling pathways. Taking into account this evidence and our experience on the study of azidothymidine (AZT) as an inhibitor of telomerase activity, the present study analyzes the effect of AZT on some telomeric and extratelomeric activities. To carry out the present study, we evaluated the transcription of genes that are modulated by the Wnt/β-catenin pathway, such as c-Myc and cyclin-D1 (Cyc-D1) and cell processes related with their expression, such as, proliferation, modifications of the actin cytoskeleton, cell migration and cell cycle in a mammary carcinoma cell line (F3II). Results obtained after treatment with AZT (600µM) for 15passages confirmed the inhibitory effect on telomerase. Regarding extratelomeric activities, our results showed a decrease of 64, 38 and 25% in the transcription of c-Myc, Cyc-D1 and TERT, respectively (p<0.05) after AZT treatment. Furthermore, we found an effect on cell migration, reaching an inhibition of 48% (p<0.05) and a significant passage-dependent increase on cell doubling time during treatment. Finally, we evaluated the effect on cell cycle, obtaining a decline in G0/G1 in AZT-treated cells. These results allow us to postulate that AZT is not only an inhibitor of telomerase activity, but also a potential modulator of extratelomeric processes involved in cancer promotion.

Increased Stability of Nucleolar PinX1 in the Presence of TERT.

PinX1, a nucleolar protein of 328 amino acids, inhibits telomerase activity, which leads to the shortening of telomeres. The C-terminal region of PinX1 is responsible for its nucleolar localization and binding with TERT, a catalytic component of telomerase. A fraction of TERT localizes to the nucleolus, but the role of TERT in the nucleolus is largely unknown. Here, we report a functional connection between PinX1 and TERT regarding PinX1 stability. The C-terminal of PinX1205–328, a nucleolar fragment, was much more stable than the N-terminal of PinX11–204, a nuclear fragment. Interestingly, PinX1 was less stable in TERT-depleted cells and more stable in TERT-myc expressing cells. Stability assays for PinX1 truncation forms showed that both PinX11–328 and PinX1205–328, nucleolar forms, were more rapidly degraded in TERT-depleted cells, while they were more stably maintained in TERT-overexpressing cells, compared to each of the controls. However, PinX11–204 was degraded regardless of the TERT status. These results reveal that the stability of PinX1 is maintained in nucleolus in the presence of TERT and suggest a role of TERT in the regulation of PinX1 steady-state levels.

A telomerase dependent conditionally replicating adenovirus for treatment of hepatocellular carcinoma

Background: The catalytic component of telomerase (hTERT) is not expressed in most primary somatic human cells whereas a large variety of cancer cells and especially hepatoma cell lines reactivate telomerase by transcriptional upregulation of hTERT.

Cross talk between EBV and telomerase: the role of TERT and NOTCH2 in the switch of latent/lytic cycle of the virus.

Epstein–Barr virus (EBV)-associated malignancies, as well as lymphoblastoid cell lines (LCLs), obtained in vitro by EBV infection of B cells, express latent viral proteins and maintain their ability to grow indefinitely through inappropriate activation of telomere-specific reverse transcriptase (TERT), the catalytic component of telomerase. Our previous studies demonstrated that high levels of TERT expression in LCLs prevent the activation of EBV lytic cycle, which is instead triggered by TERT silencing. As lytic infection promotes the death of EBV-positive tumor cells, understanding the mechanism(s) by which TERT affects the latent/lytic status of EBV may be important for setting new therapeutic strategies. BATF, a transcription factor activated by NOTCH2, the major NOTCH family member in B cells, negatively affects the expression of BZLF1, the master regulator of viral lytic cycle. We therefore analyzed the interplay between TERT, NOTCH and BATF in LCLs and found that high levels of endogenous TERT are associated with high NOTCH2 and BATF expression levels. In addition, ectopic expression of TERT in LCLs with low levels of endogenous telomerase was associated with upregulation of NOTCH2 and BATF at both mRNA and protein levels. By contrast, infection of LCLs with retroviral vectors expressing functional NOTCH2 did not alter TERT transcript levels. Luciferase reporter assays, demonstrated that TERT significantly activated NOTCH2 promoter in a dose-dependent manner. We also found that NF-κB pathway is involved in TERT-induced NOTCH2 activation. Lastly, pharmacologic inhibition of NOTCH signaling triggers the EBV lytic cycle, leading to the death of EBV-infected cells. Overall, these results indicate that TERT contributes to preserve EBV latency in B cells mainly through the NOTCH2/BAFT pathway, and suggest that NOTCH2 inhibition may represent an appealing therapeutic strategy against EBV-associated malignancies.

Downregulation of Dyskerin Impairs Expansion of Normal Hematopoietic Progenitors and Erythroid Colony Formation in the Absence of Apparent Telomere Shortening

Dyskerin is an RNA binding protein that functions as a core component of the telomerase holoenzyme as well as in ribonuclear protein complexes involved in ribosome biogenesis and RNA processing. Within the telomerase holoenzyme, dyskerin stabilizes the telomerase RNA component (hTR) that functions as a template for synthesis of telomeric DNA at chromosome ends. Telomerase is active in malignant cells as well as normal hematopoietic stem and progenitor cells where it enables long-term cell replication.Mutation of the gene encoding dyskerin (DKC1) is the underlying cause of the inherited disease dyskeratosis congenita (DC), which is characterized by low levels of telomerase activity and shortened telomeres. Bone marrow failure is the most common cause of mortality in DC, however since dyskerin has telomerase-independent roles in fundamental cellular processes, it is not clear whether telomerase insufficiency is the sole cause of hematopoietic dysfunction in this disorder.To gain further insight to the consequences of deregulated expression of dyskerin in haematopoietic cells, we transduced normal human cord blood CD34+ progenitor cells with retroviral vectors encoding two different shRNAs targeting dyskerin. Transduced cells from seven independent experiments were FACS sorted then assayed for expansion and differentiation in vitro, clonogenic potential, telomerase activity and telomere length maintenance. Results from the functional assays showed that depletion of dyskerin impaired cell expansion and compromised the formation of BFU-E colonies. In contrast to BFU-E, CFU-GM colonies did not appear to be altered by dyskerin depletion. Analysis of non-transuduced cells subject to ex vivo expansion along erythroid and myelomonocytic lineages revealed that erythroid precursor cells expressed disproportionately high levels of dyskerin, and correspondingly high telomerase enzyme activity. shRNA-mediated downregulation of dyskerin lowered hTR levels and quenched telomerase activity in the erythroid precursors without altering expression of the telomerase catalytic component, hTERT. These results suggest that upregulation of dyskerin bolsters telomerase activity and renders erythroid precursors vulnerable to dyskerin depletion. However in spite of the reduction in telomerase activity induced by dyskerin shRNA, Southern blot analysis showed that the rate of telomere length shortening was not significantly altered by dyskerin depletion over the time course of these experiments. Together the data show that dyskerin is upregulated during erythroid differentiation, and has an essential function in the erythroid lineage that appears to be independent of its role in telomere length maintenance. DisclosuresNo relevant conflicts of interest to declare.

Knockdown of hTERT by siRNA inhibits cervical cancer cell growth in vitro and in vivo.

Human telomerase reverse transcriptase (hTERT) is the catalytic component of telomerase that facilitates tumor cell invasion and proliferation. It has been reported that telomerase and hTERT are significantly upregulated in majority of cancers including cervical cancer, thus, downregulation of hTERT is a promising target in malignant tumor treatment. We established a short interfering RNA (siRNA) targeting hTERT, and transfected it into HeLa cells (a cervical cancer cell line) to investi-gate the effect of cell proliferation, apoptosis, migration and invasion in cervical cancer cells. The results showed that siRNA targeting hTERT could effectively knock down hTERT expression, remarkably suppress telomerase activity, cell proliferation, migration and invasion, and induced cell apoptosis of cervical cancers cells in vitro. In addition, we evaluated whether siRNA targeting hTERT affects tumor growth in nude mice, and found that it dramatically inhibited tumorigenesis and growth of mice injected with siRNA targeting hTERT. Furthermore, we also found that knockdown of hTERT was able to significantly suppress constitutive phosphorylation of Akt, PI3K, which might imply that reduction of hTERT inhibited tumor growth via the PI3K/Akt signaling pathway to some extent. These results suggest that the suppression of hTERT expression by siRNA inhibits cervical cancer cell growth in vitro and in vivo, and may provide a novel target for anticancer gene therapy.

PinX1, a Telomere Repeat-binding Factor 1 (TRF1)-interacting Protein, Maintains Telomere Integrity by Modulating TRF1 Homeostasis, the Process in Which Human Telomerase Reverse Transcriptase (hTERT) Plays Dual Roles

TRF1, a telomere-binding protein, is important for telomere protection and homeostasis. PinX1 interacts with TRF1, but the physiological consequences of their interaction in telomere protection are not yet understood. Here we investigated PinX1 function on TRF1 stability in HeLa cells. PinX1 overexpression stabilized TRF1, but PinX1 depletion by siRNA led to TRF1 degradation, TRF1 ubiquitination, and less TRF1 telomere association. The depletion also induced DNA damage responses at telomeres and chromosome instability. These telomere dysfunctional phenotypes were in fact due to TRF1 deficiency. We also report that hTERT, a catalytic component of telomerase, plays dual roles in the TRF1 steady state pathway. PinX1-mediated TRF1 stability was not observed in hTERT-negative immortal cells, but was pronounced when hTERT was ectopically expressed in the cells, suggesting that hTERT may be needed in the PinX1-mediated TRF1 stability pathway. Interestingly, the knockdown of both PinX1 and hTERT in HeLa cells stabilized TRF1, suppressed DNA damage response activation, and restored chromosome stability. In summary, our findings suggested that PinX1 may maintain telomere integrity by regulating TRF1 stability and that hTERT may act as both a positive and a negative regulator of TRF1 homeostasis in a PinX1-dependent manner.

Prognostic significance of telomere maintenance mechanisms in pediatric high-grade gliomas

Children with high-grade glioma, including diffuse intrinsic pontine glioma (DIPG), have a poor prognosis despite multimodal therapy. Identifying novel therapeutic targets is critical to improve their outcome. We evaluated prognostic roles of telomere maintenance mechanisms in children with HGG, including DIPG. A multi-institutional retrospective study was conducted involving 50 flash-frozen HGG (35 non-brainstem; 15 DIPG) tumors from 45 children (30 non-brainstem; 15 DIPG). Telomerase activity, expression of hTERT mRNA (encoding telomerase catalytic component) and TERC (telomerase RNA template) and alternative lengthening of telomeres (ALT) mechanism were assayed. Cox Proportional Hazard regression analyses assessed association of clinical and pathological variables, TERC and hTERT levels, telomerase activity, and ALT use with progression-free or overall survival (OS). High TERC and hTERT expression was detected in 13/28 non-brainstem HGG samples as compared to non-neoplastic controls. High TERC and hTERT expression was identified in 13/15 and 11/15 DIPG samples, respectively, compared to controls. Evidence of ALT was noted in 3/11 DIPG and 10/19 non-brainstem HGG specimens. ALT and telomerase use were identified in 4/19 non-brainstem HGG and 2/11 DIPG specimens. In multivariable analyses, increased TERC and hTERT levels were associated with worse OS in patients with non-brainstem HGG, after controlling for tumor grade or resection extent. Children with HGG and DIPG, have increased hTERT and TERC expression. In children with non-brainstem HGG, increased TERC and hTERT expression levels are associated with a worse OS, making telomerase a promising potential therapeutic target in pediatric HGG.

TERT rs2736100T/G polymorphism upregulates interleukin 6 expression in non-small cell lung cancer especially in adenocarcinoma

Telomerase reverse transcriptase (TERT) is the catalytic component of telomerase, especially the rate-limiting determinant of telomerase activity. Accumulating evidence has suggested that TERT could modulate the expression of numerous genes including interleukin 6 (IL-6), an important cytokine for the development of lung cancer. It has been reported that TERT polymorphism rs2736100T/G is associated with increased susceptibility to non-small cell lung cancer (NSCLC). However, the mechanism remains unclear. In the current study, we investigated the association between rs2736100T/G and NSCLC in 1,552 NSCLC and 1,602 healthy controls. Data revealed that the prevalence of TG and GG genotypes were significantly elevated in patients than in controls (odds ratio (OR) = 1.18; 95% confidence interval (CI), 1.01-1.39; p = 0.040 and OR = 1.46; 95% CI, 1.19-1.78; p < 0.001, respectively). The association was more prominent in patients with lung adenocarcinoma than those with squamous cell carcinoma (p = 0.039). When analyzing the function of the polymorphism, we observed a significantly augmented level of IL-6 in subjects with GG genotype than those with GT and TT genotypes. Interestingly, the upregulation of IL-6 by GG genotype was 2.3-fold higher in lung adenocarcinoma compared to squamous cell carcinoma. These results suggest that the rs2736100T/G polymorphism modulates IL-6 expression and may play a unique role in lung adenocarcinoma.

Immortalized myogenic cells from congenital muscular dystrophy type1A patients recapitulate aberrant caspase activation in pathogenesis: a new tool for MDC1A research

BackgroundCongenital muscular dystrophy Type 1A (MDC1A) is a severe, recessive disease of childhood onset that is caused by mutations in the LAMA2 gene encoding laminin-α2. Studies with both mouse models and primary cultures of human MDC1A myogenic cells suggest that aberrant activation of cell death is a significant contributor to pathogenesis in laminin-α2-deficiency.MethodsTo overcome the limited population doublings of primary cultures, we generated immortalized, clonal lines of human MDC1A myogenic cells via overexpression of both CDK4 and the telomerase catalytic component (human telomerase reverse transcriptase (hTERT)). ResultsThe immortalized MDC1A myogenic cells proliferated indefinitely when cultured at low density in high serum growth medium, but retained the capacity to form multinucleate myotubes and express muscle-specific proteins when switched to low serum medium. When cultured in the absence of laminin, myotubes formed from immortalized MDC1A myoblasts, but not those formed from immortalized healthy or disease control human myoblasts, showed significantly increased activation of caspase-3. This pattern of aberrant caspase-3 activation in the immortalized cultures was similar to that found previously in primary MDC1A cultures and laminin-α2-deficient mice.ConclusionsImmortalized MDC1A myogenic cells provide a new resource for studies of pathogenetic mechanisms and for screening possible therapeutic approaches in laminin-α2-deficiency.