Telomerase Reverse Transcriptase Expression Research Articles

EXTH-68. EPIGENETIC EDITING BY MRNA-BASED CRISPROFF ENABLES FLEXIBLE AND DURABLE TARGETING OF GLIOBLASTOMA DRIVERS MGMT AND TERT

Abstract Alterations in cis-regulatory elements, such as hypomethylation of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter and activating mutations within the telomerase reverse transcriptase (TERT) promoter, are pervasive drivers of glioblastoma (GBM) tumorigenesis that are challenging to target. CRISPRoff is an engineered CRISPR/Cas9 system that can potently and heritably silence cis-regulatory elements through DNA methylation. Here, we develop an mRNA-based CRISPRoff platform to target the MGMT and TERT promoters in GBM, and we establish proof-of-principal in vivo targeting through lipid nanoparticles (LNP). Epigenetic editing in GBM cells and primary GBM organoids was performed using either electroporation or LNP encapsulation of CRISPRoff mRNA and sgRNAs. Target gene silencing and functional phenotypes were assessed by bisulfite sequencing, RT-qPCR, RNA-seq, western blot, cell viability/apoptosis, and telomere restriction fragment assays. In vivo efficacy was demonstrated using intracranial GBM xenografts, first through transplantation of cells with CRISPRoff delivered ex vivo, then with direct delivery of LNPs using convection enhanced delivery (CED) coupled with immunofluorescence/ immunohistochemistry. CRISPRoff targeting of MGMT promoter resulted in >99% reduction in MGMT expression in MGMT unmethylated primary GBM organoids, inducing up to 150-fold sensitization to temozolomide. Temozolomide sensitization was retained after intracrania transplantation, and silencing was durable in clonally isolated GBM cells continuously passaged for over 8 months after transient delivery of CRISPRoff. CRISPRoff targeting of the TERT promoter in primary GBM cultures harboring the G228A TERT promoter mutation silenced TERT expression by up to 99%, overcoming constitutive activation of TERT, and induced telomere shortening sufficient for complete replicative senescence. CED of mRNAs encapsulated by LNPs based on cKK-E12 or Lipid A9 resulted in tumor-selective uptake of mRNA transcripts, establishing a foundation for direct delivery of CRISPRoff in vivo bypassing the blood-brain-barrier. In summary, we establish a flexible and durable epigenetic editing system against multiple regulatory elements driving GBM, leveraging LNPs with potential for in vivo application.

Aged Gut Microbiome Induces Metabolic Impairment and Hallmarks of Vascular and Intestinal Aging in Young Mice.

Aging, an independent risk factor for cardiometabolic diseases, refers to a progressive deterioration in physiological function, characterized by 12 established hallmarks. Vascular aging is driven by endothelial dysfunction, telomere dysfunction, oxidative stress, and vascular inflammation. This study investigated whether aged gut microbiome promotes vascular aging and metabolic impairment. Fecal microbiome transfer (FMT) was conducted from aged (>75 weeks old) to young C57BL/6 mice (8 weeks old) for 6 weeks. Wire myography was used to evaluate endothelial function in aortas and mesenteric arteries. ROS levels were measured by dihydroethidium (DHE) staining and lucigenin-enhanced chemiluminescence. Vascular and intestinal telomere function, in terms of relative telomere length, telomerase reverse transcriptase expression and telomerase activity, were measured. Systemic inflammation, endotoxemia and intestinal integrity of mice were assessed. Gut microbiome profiles were studied by 16S rRNA sequencing. Some middle-aged mice (40-42 weeks old) were subjected to chronic metformin treatment and exercise training for 4 weeks to evaluate their anti-aging benefits. Six-week FMT impaired glucose homeostasis and caused vascular dysfunction in aortas and mesenteric arteries in young mice. FMT triggered vascular inflammation and oxidative stress, along with declined telomerase activity and shorter telomere length in aortas. Additionally, FMT impaired intestinal integrity, and triggered AMPK inactivation and telomere dysfunction in intestines, potentially attributed to the altered gut microbial profiles. Metformin treatment and moderate exercise improved integrity, AMPK activation and telomere function in mouse intestines. Our data highlight aged microbiome as a mechanism that accelerates intestinal and vascular aging, suggesting the gut-vascular connection as a potential intervention target against cardiovascular aging and complications.

TERT Expression and Clinical Outcome in Pulmonary Carcinoids.

The clinical course of pulmonary carcinoids ranges from indolent to fatal disease, suggesting that specific molecular alterations drive progression toward the fully malignant state. A similar spectrum of clinical phenotypes occurs in pediatric neuroblastoma, in which activation of telomerase reverse transcriptase (TERT) is decisive in determining the course of disease. We therefore investigated whether TERT expression defines the clinical fate of patients with pulmonary carcinoid. TERT expression was examined by RNA sequencing in a test cohort and a validation cohort of pulmonary carcinoids (n = 88 and n = 105, respectively). A natural TERT expression cutoff was determined in the test cohort on the basis of the distribution of TERT expression, and its prognostic value was assessed by Kaplan-Meier survival estimates and multivariable analyses. Telomerase activity was validated by telomere repeat amplification protocol assay. Similar to neuroblastoma, TERT expression exhibited a bimodal distribution in pulmonary carcinoids, separating tumors into TERT-high and TERT-low subgroups. A natural TERT cutoff discriminated unfavorable from favorable clinical courses with high accuracy both in the test cohort (5-year overall survival [OS], 0.547 ± 0.132 v 1.0; P < .001) and the validation cohort (5-year OS, 0.788 ± 0.063 v 0.913 ± 0.048; P < .001). In line with these findings, telomerase activity was largely absent in TERT-low tumors, whereas it was readily detectable in TERT-high carcinoids. In multivariable analysis considering TERT expression, histology (typical v atypical carcinoid), and stage (≤IIA v ≥IIB), high TERT expression was an independent prognostic marker for poor survival, with a hazard ratio of 5.243 (95% CI, 1.943 to 14.148; P = .001). Our data demonstrate that high TERT expression defines clinically aggressive pulmonary carcinoids with fatal outcome, similar to neuroblastoma, indicating that activation of TERT may be a defining feature of lethal cancers.

Astragalus polysaccharide ameliorates bone marrow haematopoietic failure in mice with aplastic anaemia via the Hippo/TERT signalling pathway

ObjectiveTo study the effects of astragalus polysaccharide (APS) on aplastic anaemia (AA) in mice through modulation of the upstream and downstream effectors of the Hippo pathway. Further, we investigated the underlying mechanisms of APS in the treatment of AA to provide possible therapeutic strategies and drugs for clinical use. MethodsBALB/c mice were subjected to 4 Gy X-ray irradiation and subsequently injected with lymphocytes from DBA/2 donor mice by using the tail vein to establish an AA mouse model. Next BALB/c mice were randomly divided into five groups: control (untreated), AA (model), and three APS remedy groups - LA (low-dose APS, 200 mg/kg), MA (medium-dose APS, 400 mg/kg), and HA (high-dose APS, 800 mg/kg). The mice were sacrificed after 14 d of continuous gavage with different doses of APS or saline, and the bone marrow, spleen and liver were obtained. The haematopoietic condition, apoptosis levels, and the expression of Large Tumour Suppressor 1/2 (LATS 1/2), transcriptional co-activator of activation (TCA) Yes-associated protein (YesAP), transcriptional enhancer of adherence domain (TEAD) protein, and Telomerase Reverse Transcriptase (TERT), were analysed. ResultsCompared with control mice, mice in the AA group exhibited a reduction in peripheral blood cell counts, a significant decrease in the haematopoietic area, increased adipocyte infiltration, and overall haematopoietic failure, mirroring the clinical presentation of AA; After 14 days of treatment with different doses of APS, the APS-treated group, in comparison to the untreated AA group, showed an increase in peripheral blood cell counts (P < 0.05), an expansionof the haematopoietic area in the bone marrow, restoration of haematopoietic function (P < 0.05), and a marked reduction in adipocyte infiltration within the bone marrow(P < 0.05). Cell infiltration into the bone marrow was also reduced. Further experiments revealed that the expression levels of TERT, LATS 1/2, YAP, and TEAD were elevated in the treated mice (P < 0.05). ConclusionX-ray irradiation combined with T-lymphocyte infusion successfully established an AA mouse model, and APS administration modulated the expression of TERT and the Hippo pathway effectors LATS 1/2, YAP, and TEAD, which are implicated in the pathogenesis of AA. This modulation resulted in the restoration of bone marrow haematopoietic function in AA mice, suggesting APS as a promising therapeutic agent for the treatment of AA.

TERT upregulation promotes cell proliferation via degradation of p21 and increases carcinogenic potential.

Telomerase reverse transcriptase (TERT) gene aberration is detectable in >80% of cases with hepatocellular carcinoma (HCC). TERT reactivation is essential for cellular immortalization because it stabilizes telomere length, although the role of TERT in hepatocarcinogenesis remains unelucidated. To elucidate the significance of aberrant TERT expression in hepatocytes in inflammation-associated hepatocarcinogenesis, we generated Alb-Cre;TertTg mice, which overexpress TERT in the liver and examined their phenotype during chronic inflammation. Based on transcriptome data from the liver tissue of Alb-Cre;TertTg mice, we examined the role of TERT in hepatocarcinogenesis in vitro. We also evaluated the relationship between TERT and cell-cycle-related molecules, including p21, in HCC samples. The liver tumor development rate was increased by TERT overexpression during chronic inflammation, especially in the absence of p53 function. Gene set enrichment analysis of liver tissues revealed that gene sets related to TNF-NFκB signaling, cell cycle, and apoptosis were upregulated in Alb-Cre;TertTg liver. A luciferase reporter assay and immunoprecipitation revealed that TERT interacted with NFκB p65 and enhanced NFκB promoter activity. On the other hand, TERT formed protein complexes with p21, cyclin A2, and cyclin E and promoted ubiquitin-mediated degradation of p21, specifically in the G1 phase. In the clinical HCC samples, TERT was highly expressed but p21 was conversely downregulated, and TERT expression was associated with the upregulation of molecules related to the cell cycle. Taken together, the aberrant upregulation of TERT increased NFκB promoter activity and promoted cell cycle progression via p21 ubiquitination, leading to hepatocarcinogenesis. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Annexin A1 protects periodontal ligament cells against lipopolysaccharide-induced inflammatory response and cellular senescence: An implication in periodontitis.

Periodontitis is an inflammatory condition that affects the tooth-supporting structures, triggered by the host's immune response toward the bacterial deposits around the teeth. Annexin A1 (AnxA1), a vital member of the annexin superfamily, is known for its diverse physiological functions, particularly its anti-inflammatory and anti-senescence properties. We hypothesized that AnxA1 has a protective effect against lipopolysaccharide (LPS)-induced inflammatory responses and cellular damage in periodontal ligament cells (PDLCs). In this study, we demonstrate that LPS stimulation significantly reduced telomerase activity in PDLCs, a decline that was dose-dependently reversed by AnxA1. Importantly, AnxA1 protected the cells from LPS-induced cellular senescence and the downregulation of human telomerase reverse transcriptase (hTERT) expression. In line with this, AnxA1 suppressed the LPS-induced expression of p21 and p16 at both the mRNA and protein levels. Furthermore, AnxA1 demonstrated potent anti-inflammatory effects by inhibiting the secretion of interleukin 6 (IL-6), interleukin 8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1). It also mitigated LPS-induced oxidative stress by reducing the levels of phosphorylated Foxo3a (Ser253) and restored sirtuin 1 (SIRT1) expression. Notably, SIRT1 silencing abolished AnxA1's protective effects on Foxo3a phosphorylation and cellular senescence, suggesting that SIRT1 mediates AnxA1's actions. In conclusion, AnxA1 protected PDLCs against LPS-triggered inflammation and cell senescence by activating SIRT1 signal pathway. These findings indicate that AnxA1 could serve as a promising therapeutic strategy for the treatment of periodontitis.

Telomere biology and its maintenance in schizophrenia spectrum disorders: Exploring links to cognition

ObjectiveContemporary research suggests reduced telomere length in schizophrenia spectrum disorders (SZ) compared to age-adjusted non-affected individuals. However, the role of telomere maintenance and telomere repair in SZ is poorly understood as well as the involvement of telomere biology in cognitive abnormalities in SZ. MethodsThe study consisted of 758 participants (SZ [n = 357] and healthy controls, HC [n = 401]) collected as part of the Norwegian TOP study. Participants were assessed with standardized neuropsychological tests measuring five cognitive domains. Leucocyte telomere length (TL) was measured via blood and determined by quantitative real-time Polymerase Chain Reaction (qPCR) providing a telomere to single copy ratio (T/S ratio), used to estimate the mean telomere length. Telomerase activity was assessed by the expression levels of the Telomerase Reverse Transcriptase (TERT) and Telomerase RNA Component (TERC) genes. To assess telomere maintenance and telomere repair we calculated the telomerase expression to TL ratio (TERT/TL and TERC/TL respectively). ResultsPatients had reduced TERT (F = 5.03, p = 0.03), but not TERC expression (F = 1.04, p = 0.31), and higher TERT/TL (F = 6.68, p = 0.01) and TERC/TL (F = 6.71, p = 0.01), adjusted for age, sex, and ethnicity. No statistically significant association was observed between any of the telomere biology markers and the cognitive domains (p > 0.05). ConclusionOur study shows changes in TERT expression and telomere maintenance and telomere repair in SZ compared HC. However, the role of telomere biology in the mechanism underlying cognitive impairment in psychosis seems limited.

Exploring the impact of diabetes on aging: insights from TERT and COL1A1 methylation.

Aging, a multifaceted biological process, leads to diminished physical performance, especially in older adults with diabetes, where a mismatch between biological and chronological age is noticeable. Numerous studies have demonstrated that diabetes accelerates aging at the cellular and organ levels. Notable aging markers are telomerase reverse transcriptase (TERT), related to telomere length, and type 1 chain collagen (COL1A1), a key component of skin collagen. Additionally, age-related methylation increases, as revealed through methylation analysis, augmenting aspects of aging. However, the detailed interplay between aging and diabetes, particularly regarding methylation, remains underexplored and warrants further study to elucidate the biological links between the two. In this study, we elucidate the modulatory influence of diabetes on the aging process, focusing specifically on the modifications in TERT in the kidney and COL1A1 in the skin using mice of Swiss Webster strain as the diabetes model. Specimens were categorized into three distinct chronological cohorts: chronologically young (16 weeks; n = 5), chronologically old (40 weeks; n = 5), and a periodically assessed group (16 weeks; n = 30), from which five mice were systematically sacrificed on a weekly basis. Our findings reveal a marked impact of diabetes on the methylation statuses of TERT and COL1A1, characterized by an elevation in methylation levels within the periodic group (1st-6th week) and a simultaneous, progressive attenuation in the expression of TERT and COL1A1 genes. The observed alterations in the methylation levels of TERT and COL1A1 propound the hypothesis that diabetes potentially expedites the aging process, concomitantly impinging on the production of TERT and COL1A, ostensibly through the mechanism of promoter gene hypermethylation.

Promoter mutation-independent TERT expression is related to the immune-enriched milieu in papillary thyroid cancer.

Telomerase reverse transcriptase promoter mutation (pTERT MT) promotes human carcinogenesis via aberrant expression of telomerase reverse transcriptase (TERT). However, the tumorigenic impact of TERT expression independent of pTERT MT remains unclear despite numerous mechanisms of TERT being suggested. To tackle this issue, we employed comprehensive bioinformatics to assess biological variations noticed among different TERT expression mechanisms. Papillary thyroid cancer (PTC) with pTERT MT (pTERT MT PTC) presented aggressive clinical behavior and exhibited biological profiles associated with cellular immortality and genomic instability. PTC with TERT expression but without pTERT MT (TERT (+) PTC), also exhibited poor clinicopathological characteristics and was enriched with immune responses. In accordance, c-MYC/E2F and nuclear factor kappa B (NFκB) were dominant transcription factors in pTERT MT PTC and TERT (+) PTC, respectively. Notably, we revealed TERT hypermethylated oncological region (THOR) as a potential TERT expressing mechanism in TERT (+) PTC patients. Furthermore, three unique subtypes of papillary thyroid cancer were deciphered using a combination of machine learning-based scoring systems. Our proposed scoring system was clinically significant, especially in microcarcinoma, predicting survival outcomes and inferring therapeutic responses to radioactive iodine therapy. Finally, our analysis was expanded to endocrine-related cancers, unveiling various regulatory mechanisms of TERT with poor clinical outcomes and biological behaviors.

TERT upstream promoter methylation regulates TERT expression and acts as a therapeutic target in TERT promoter mutation-negative thyroid cancer

BackgroundDNA hypermethylation and hotspot mutations were frequently observed in the upstream and core promoter of telomerase reverse transcriptase (TERT), respectively, and they were associated with increased TERT expression and adverse clinical outcomes in thyroid cancer. In TERT promoter mutant cancer cells, the hypomethylated TERT mutant allele was active and the hypermethylated TERT wild-type allele was silenced. However, whether and how the upstream promoter methylation regulates TERT expression in TERT mutation-negative cells were largely unknown.MethodsDNA demethylating agents 5-azacytidine and decitabine and a genomic locus-specific demethylation system based on dCas9-TET1 were used to assess the effects of TERT upstream promoter methylation on TERT expression, cell growth and apoptosis of thyroid cancer cells. Regulatory proteins binding to TERT promoter were identified by CRISPR affinity purification in situ of regulatory elements (CAPTURE) combined with mass spectrometry. The enrichments of selected regulatory proteins and histone modifications were evaluated by chromatin immunoprecipitation.ResultsThe level of DNA methylation at TERT upstream promoter and expression of TERT were significantly decreased after treatment with 5-azacytidine or decitabine in TERT promoter wild-type thyroid cancer cells. Genomic locus-specific demethylation of TERT upstream promoter induced TERT downregulation, along with cell apoptosis and growth inhibition. Consistently, demethylating agents sharply inhibited the growth of thyroid cancer cells harboring hypermethylated TERT but had little effect on cells with TERT hypomethylation. Moreover, we identified that the chromatin remodeling protein CHD4 binds to methylated TERT upstream promoter and promotes its transcription by suppressing the enrichment of H3K9me3 and H3K27me3 at TERT promoter.ConclusionsThis study uncovered the mechanism of promoter methylation mediated TERT activation in TERT promoter mutation-negative thyroid cancer cells and indicated TERT upstream promoter methylation as a therapeutic target for thyroid cancer.

Circ_0000263 improves radiosensitivity of Hela cells by inhibiting the activity of telomerase protein through miR-338-3p/TERT

Objective: To explore the effect and molecular mechanism of circ_0000263 on HeLa cell activity, apoptosis, telomerase activity, and radiosensitivity. Methods: The Hela cells were divided into si-NC, si-circ, vector, circ_0000263, anti-NC, anti-miR-338-3p, miR-NC, miR-338-3p, si-circ+anti-NC, si-circ+ anti-miR-338-3p, si-circ+vector, si-circ+TERT, sh-NC, sh-circ groups. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expressions of circ_0000263 and miR-338-3p. Cell clone formation array was used to detect cell survival; cell counting kit-8 (CCK-8) to detect cell proliferation; flow cytometry to detect apoptosis; western blot method to detect the expressions of proliferating cell nuclear antigen (PCNA), Cleaved-casp3, telomerase reverse transcriptase (TERT) proteins; double luciferase assay to detect the targeting relationships of circ_0000263 and miR-338-3p, miR-338-3p and TERT; telomere repeat amplification enzyme linked immunosorbent assay (TRAR-ELISA) to detect telomerase activity. Results: Circ_0000263 was highly expressed in Hela cells, miR-338-3p was low expressed, and TERT was highly expressed; circ_0000263 was also highly expressed in Hela cells treated with radiation (P＜0.05). Knockdown of circ_0000263 inhibited the clone formation and cell proliferation ability of HeLa cells, and enhanced the radiosensitivity and apoptosis of HeLa cells. In contrast, knockdown of circ_0000263 decreased PCNA protein expression level and enhanced Cleaved-casp3 protein expression level in HeLa cells (P＜0.05). The apoptosis rate in the si-circ group was (13.19±1.12)%, which was higher than (6.80±0.62)% of si-NC group (P＜0.05). The apoptosis rate in the si-circ+4 Gy group was (24.82±1.57)%, which was higher than (17.00±0.96)% of si-NC+4 Gy group (P＜0.05). Circ_0000263 targeted regulated miR-338-3p, and miR-338-3p targeted regulated TERT. MiR-338-3p was lowly expressed in HeLa cells, and knockdown of circ_0000263 elevated miR-338-3p expression level in HeLa cells. Circ_0000263 regulated TERT expression and inhibited telomerase activity through miR-338-3p. MiR-338-3p/TERT can restore the effect of circ_0000263 on the radiosensitivity of Hela cells. The apoptosis rate in the si-circ+anti-NC group was (27.37±0.89)%, which was higher than (18.22±1.18)% of the si-circ+anti-miR-338-3p group (P＜0.05). The apoptosis rate in the si-circ+vector group was (27.55±0.48)%, which was higher than (20.10±0.68)% of si-circ+TERT group (P＜0.05). After 72 hours of radiation by 4 Gy, the cell survival fraction of si-circ+anti-NC group was 0.41±0.02, which was lower than 0.66±0.03 of the si-circ+anti-miR-338-3p group (P＜0.05); the cell survival fraction of si-circ+vector group was 0.42±0.05, which was lower than 0.70±0.03 of si-circ+TERT group (P＜0.05). Conclusion: Inhibiting the expression of circ_0000263 supresses the proliferation of Hela cells by regulating miR-338-3p/TERT, promotes apoptosis, inhibits telomerase activity, increases the radiosensitivity of cancer cells, and provides a theoretical basis for improving the radiosensitivity of Hela cells.

Effect and mechanism of curcumin on colon cancer cell senescence through early growth response 1 (EGR1).

The expression level of early growth response 1 (EGR1) is elevated in colon cancer (CC) tissues and is closely associated with poor prognosis in colorectal cancer. However, the role of EGR1 as a transcription factor (TF) influencing cell senescence in the progression of CC remains largely unexplored. This study aims to investigate the impact of curcumin on colorectal cancer cell senescence by modulating EGR1. Genes associated with cell senescence were obtained from a public database, and ChIP-X predicted TFs were utilized. The R2 database was employed to examine the relationship between gene expression and survival. CC cell lines were transfected with plasmids to achieve stable expression. Stable transfected cell lines were screened, and changes in RNA and protein expression were assessed using real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blot (WB) analysis. Senescence levels were measured by SA-β-Gal staining. Cell proliferation and invasion capabilities were evaluated through soft agar and Matrigel invasion assays. Molecular docking was used to predict the interaction between curcumin and EGR1. Gene activity changes were detected using a dual luciferase reporter gene assay. The results indicated that EGR1 was overexpressed in CC tissues and correlated with poor prognosis. As a TF, EGR1 negatively regulated the expression of telomerase reverse transcriptase (TERT) and sirtuin 6 (SIRT6) genes associated with cell senescence. Knocking down EGR1 increased the rate of cell senescence and inhibited cell proliferation and invasion. Curcumin inhibited the transcriptional activity of EGR1, thereby promoting cell senescence and inhibiting tumor progression. In conclusion, curcumin hampers the activity of TF EGR1, affecting the transcription and translation of target genes TERT and SIRT6, thus promoting cell senescence and inhibiting CC cell proliferation. These findings provide potential insights for targeted therapy of CC.

Transcript level of telomerase reverse-transcriptase (TERT) gene in the rainbow trout (Oncorhynchus mykiss) eggs with different developmental competence for gynogenesis.

Expression of the telomerase reverse-transcriptase (TERT) gene and activity of telomerase have been reported in the somatic tissues and gonads in fish irrespective of their age and size. Nevertheless, little is known about TERT expression in the fish eggs. In the current study, the presence of the TERT transcripts was confirmed in the rainbow trout ovulated eggs before and after activation with nonirradiated and UV-irradiated (gynogenesis) sperm. Eggs originating from eight females had high and comparable quality expressed by similar hatching rates. However, survival of the gynogenetic larvae that hatched from eggs activated with UV-irradiated sperm and further exposed to the high hydrostatic pressure (HHP) shock for duplication of the maternal chromosomes varied between females from 2.1 ± 0.4 to 40.5 ± 2.2%. Increased level of TERT transcripts was observed in eggs originating from two females, and gametes from only one of them showed improved competence for gynogenesis (27.3 ± 1.9%). In turn, eggs from the female that exhibited the highest survival after gynogenetic activation were characterized by the lowest expression of the TERT gene. Telomerase in rainbow trout eggs may compensate erosion of the telomeres during early embryonic development; however, its upregulation does not assure better development after gynogenetic activation.

Nesfatin-1 attenuated lipopolysaccharide-induced inflammatory response and senescence in human dental pulp cells

Lipopolysaccharide (LPS)-triggered damage in human dental pulp cells (hDPCs) is associated with the progression of gingivitis, which is inflammation of the gingival tissue. Nesfatin-1 is a peptide secreted by neurons and peripheral tissues. Here, we report a novel property of Nesfatin-1 in ameliorating LPS-induced inflammatory response and senescence in hDPCs. First, we demonstrate that Nesfatin-1 repressed LPS-triggered expression of inflammatory factors. Secondly, Nesfatin-1 restored telomerase activity and the expression of human telomerase reverse transcriptase (hTERT) and telomeric repeat binding factor 2 (TERF2) against LPS. Senescence-associated β-galactosidase (SA-β-gal) staining assay revealed that Nesfatin-1 attenuated LPS-induced cellular senescence in hDPCs. We also found that Nesfatin-1 increased telomerase activity in LPS-challenged hDPCs. It is also shown that Nesfatin-1 reduced the expression of plasminogen activator inhibitor-1 (PAI-1) and p16. Additionally, LPS stimulation reduced the expression of SIRT1, which was rescued by Nesfatin-1. However, the silencing of sirtuin1 (SIRT1) abrogated the protective property of Nesfatin-1 in preventing cellular senescence, implying that the function of Nesfatin-1 is regulated by SIRT1. Taken together, our findings suggest that Nesfatin-1 might possess a protective effect against gingivitis.

IMPDH Inhibition Decreases TERT Expression and Synergizes the Cytotoxic Effect of Chemotherapeutic Agents in Glioblastoma Cells.

IMP dehydrogenase (IMPDH) inhibition has emerged as a new target therapy for glioblastoma multiforme (GBM), which remains one of the most refractory tumors to date. TCGA analyses revealed distinct expression profiles of IMPDH isoenzymes in various subtypes of GBM and low-grade glioma (LGG). To dissect the mechanism(s) underlying the anti-tumor effect of IMPDH inhibition in adult GBM, we investigated how mycophenolic acid (MPA, an IMPDH inhibitor) treatment affected key oncogenic drivers in glioblastoma cells. Our results showed that MPA decreased the expression of telomerase reverse transcriptase (TERT) in both U87 and U251 cells, and the expression of O6-methylguanine-DNA methyltransferase (MGMT) in U251 cells. In support, MPA treatment reduced the amount of telomere repeats in U87 and U251 cells. TERT downregulation by MPA was associated with a significant decrease in c-Myc (a TERT transcription activator) in U87 but not U251 cells, and a dose-dependent increase in p53 and CCCTC-binding factor (CTCF) (TERT repressors) in both U87 and U251 cells. In U251 cells, MPA displayed strong cytotoxic synergy with BCNU and moderate synergy with irinotecan, oxaliplatin, paclitaxel, or temozolomide (TMZ). In U87 cells, MPA displayed strong cytotoxic synergy with all except TMZ, acting primarily through the apoptotic pathway. Our work expands the mechanistic potential of IMPDH inhibition to TERT/telomere regulation and reveals a synthetic lethality between MPA and anti-GBM drugs.

Controlled cell proliferation and immortalization of human dental pulp stem cells with a doxycycline-inducible expression system.

Human dental pulp stem cells are a potentially useful resource for cell-based therapies and tissue repair in dental and medical applications. However, the primary culture of isolated dental pulp stem cells has notably been limited. A major requirement of an ideal human dental pulp stem cell culture system is the preservation of efficient proliferation and innate stemness over prolonged passaging, while also ensuring ease of handling through standard, user-friendly culture methods. In this study, we have engineered a novel human dental pulp stem cell line, distinguished by the constitutive expression of telomerase reverse transcriptase (TERT), and the conditional expression of the R24C mutant cyclin-dependent kinase 4 (CDK4R24C) and Cyclin D1. We have named this cell line Tet-off K4DT hDPSCs. Furthermore, we have conducted a comprehensive comparative analysis of their biological attributes in relation to a previously immortalized human dental pulp stem cells, hDPSC-K4DT, which were immortalized by the constitutive expression of CDK4R24C, Cyclin D1 and TERT. In Tet-off K4DT cells, the expression of the K4D genes can be precisely suppressed by the inclusion of doxycycline. Remarkably, Tet-off K4DT cells demonstrated an extended cellular lifespan, increased proliferative capacity, and enhanced osteogenic differentiation potential when compared to K4DT cells. Moreover, Tet-off K4DT cells had no observable genomic aberrations and also displayed a sustained expression of stem cell markers even at relatively advanced passages. Taken together, the establishment of this new cell line holds immense promise as powerful experimental tool for both fundamental and applied research involving dental pulp stem cells.

Pan-cancer experimental characteristic of human transcriptional patterns connected with telomerase reverse transcriptase (TERT) gene expression status.

The TERT gene encodes the reverse transcriptase subunit of telomerase and is normally transcriptionally suppressed in differentiated human cells but reactivated in cancers where its expression is frequently associated with poor survival prognosis. Here we experimentally assessed the RNA sequencing expression patterns associated with TERT transcription in 1039 human cancer samples of 27 tumor types. We observed a bimodal distribution of TERT expression where ∼27% of cancer samples did not express TERT and the rest showed a bell-shaped distribution. Expression of TERT strongly correlated with 1443 human genes including 103 encoding transcriptional factor proteins. Comparison of TERT- positive and negative cancers showed the differential activation of 496 genes and 1975 molecular pathways. Therein, 32/38 (84%) of DNA repair pathways were hyperactivated in TERT+ cancers which was also connected with accelerated replication, transcription, translation, and cell cycle progression. In contrast, the level of 40 positive cell cycle regulator proteins and a set of epithelial-to-mesenchymal transition pathways was specific for the TERT- group suggesting different proliferation strategies for both groups of cancer. Our pilot study showed that the TERT+ group had ∼13% of cancers with C228T or C250T mutated TERT promoter. However, the presence of promoter mutations was not associated with greater TERT expression compared with other TERT+ cancers, suggesting parallel mechanisms of its transcriptional activation in cancers. In addition, we detected a decreased expression of L1 retrotransposons in the TERT+ group, and further decreased L1 expression in promoter mutated TERT+ cancers. TERT expression was correlated with 17 genes encoding molecular targets of cancer therapeutics and may relate to differential survival patterns of TERT- positive and negative cancers.

The protective effects of Chromofungin in oligomeric amyloid β42 (Aβ42)-induced toxicity in neurons in Alzheimer's disease.

Oligomeric Aβ42 is considered to play a harmful role in the pathophysiology of Alzheimer's disease (AD). Prolonged exposure to oligomeric Aβ42 could induce neuronal damage including cellular senescence. Amelioration of Aβ42-induced cellular senescence has been considered as a promising strategy for the treatment of AD. Chromofungin, a chromogranin A-derived peptide, has displayed various biological functions in different types of cells and tissues. However, the effects of Chromofungin on oligomeric Aβ42-induced cellular senescence have not been previously reported. In the current study, we report a novel function of Chromofungin by showing that treatment with Chromofungin could ameliorate Aβ42-induced neurotoxicity in M17 neuronal cells. The Cell Counting Kit-8 (CCK-8) assay and the lactate dehydrogenase (LDH) release experiments revealed that 0.5 and 1 mM are the optimal concentrations of Chromofungin for cell culture in M17 cells. Challenging with oligomeric Aβ42 (5 μM) for 7 and 14 days led to a significant decrease in telomerase activity, which was rescued by Chromofungin dose-dependently. Additionally, the senescence-associated β-galactosidase (SA-β-gal) staining assay demonstrated that Chromofungin mitigated oligomeric Aβ42-induced cellular senescence. Correspondingly, treatment with Chromofungin reversed the gene expression of human telomerase reverse transcriptase (hTERT), telomeric repeat-binding factor 2 (TERF2), and p21 against oligomeric Aβ42 in M17 neurons. Interestingly, Chromofungin attenuated oligomeric Aβ42-induced oxidative stress (OS) in M17 cells by reducing the production of intracellular reactive oxygen species (ROS) but increasing the levels of intracellular superoxide dismutase (SOD). Importantly, the presence of Chromofungin reduced the expression of cyclooxygenase2 (COX-2) as well as the generation of prostaglandin E2 (PGE2). Transduction with Ad-COX-2 impaired the effects of Chromofungin on telomerase activity and the profile of cellular senescence. Our findings suggest that Chromofungin might act as a potential agent for the treatment of AD.

An association between elevated telomerase reverse transcriptase expression and the immune tolerance disruption of dendritic cells

BackgroundTo elucidate the mechanism of dysfunction of tolerogenic dendritic cells (DCs) is of significance. Telomerase involves the regulation of the cell fate and activities. The objective of this study is to investigate the role of telomerase reverse transcriptase (TERT) in regulating the tolerogenic feature of DCs.MethodsThe telomerase was assessed in DCs, which were collected from patients with allergic rhinitis (AR), healthy control (HC) subjects, and mice. RNAs were extracted from DCs, and analyzed by RNA sequencing (RNAseq), real-time quantitative RT-PCR, and Western blotting.ResultsThe results showed that expression of TERT was higher in peripheral DCs of AR patients. The expression of IL10 in DCs was negatively correlated with the levels of TERT expression. Importantly, the levels of TERT mRNA in DCs were associated with the AR response in patients with AR. Endoplasmic reticulum (ER) stress promoted the expression of Tert in DCs. Sensitization with the ovalbumin-aluminum hydroxide protocol increased the expression of Tert in DCs by exacerbating ER stress. TERT interacting with c-Maf (the transcription factor of IL-10) inducing protein (CMIP) in DCs resulted in CMIP ubiquitination and degradation, and thus, suppressed the production of IL-10. Inhibition of Tert in DCs mitigated experimental AR.ConclusionsElevated amounts of TERT were detected in DCs of patients with AR. The tolerogenic feature of DCs was impacted by TERT. Inhibited TERT attenuated experimental AR.

Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine.

Stem cells are undifferentiated cells that possess the potential for self-renewal with the capacity to differentiate into multiple lineages. In humans, their limited numbers pose a challenge in fulfilling the necessary demands for the regeneration and repair of damaged tissues or organs. Studies suggested that mesenchymal stem cells (MSCs), necessary for repair and regeneration via transplantation, require doses ranging from 10 to 400 million cells. Furthermore, the limited expansion of MSCs restricts their therapeutic application. To optimize a novel protocol to achieve qualitative and quantitative expansion of MSCs to reach the targeted number of cells for cellular transplantation and minimize the limitations in stem cell therapy protocols. Human umbilical cord (hUC) tissue derived MSCs were obtained and re-cultured. These cultured cells were subjected to the following evaluation procedures: Immunophenotyping, immunocytochemical staining, trilineage differentiation, population doubling time and number, gene expression markers for proliferation, cell cycle progression, senescence-associated β-galactosidase assay, human telomerase reverse transcriptase (hTERT) expression, mycoplasma, cytomegalovirus and endotoxin detection. Analysis of pluripotent gene markers Oct4, Sox2, and Nanog in recultured hUC-MSC revealed no significant differences. The immunophenotypic markers CD90, CD73, CD105, CD44, vimentin, CD29, Stro-1, and Lin28 were positively expressed by these recultured expanded MSCs, and were found negative for CD34, CD11b, CD19, CD45, and HLA-DR. The recultured hUC-MSC population continued to expand through passage 15. Proliferative gene expression of Pax6, BMP2, and TGFb1 showed no significant variation between recultured hUC-MSC groups. Nevertheless, a significant increase (P < 0.001) in the mitotic phase of the cell cycle was observed in recultured hUC-MSCs. Cellular senescence markers (hTERT expression and β-galactosidase activity) did not show any negative effect on recultured hUC-MSCs. Additionally, quality control assessments consistently confirmed the absence of mycoplasma, cytomegalovirus, and endotoxin contamination. This study proposes the development of a novel protocol for efficiently expanding stem cell population. This would address the growing demand for larger stem cell doses needed for cellular transplantation and will significantly improve the feasibility of stem cell based therapies.