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1.
Int J Mol Sci ; 23(11)2022 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-35682861

RESUMO

Non-coding RNAs are transcribed from telomeres and the telomeric repeat-containing RNAs (TERRA) are implicated in telomere homeostasis and in cancer. In this study, we aimed to assess in hepatocellular carcinoma (HCC) the cellular and extracellular expression of TERRA, the telomerase RNA subunit (TERC) and the telomerase catalytic subunit (TERT). We determined by qPCR the expression level of TERRA 1_2_10_13q, TERRA 15q, TERRA XpYp, TERC and of TERT mRNA in HCC tissues and in the plasma of HCC patients. Further, we profiled the same transcripts in the HCC cell lines, HA22T/VGH and SKHep1C3, and in the extracellular vesicles (EVs) derived from their secretomes. We found that the expression of TERRA and TERT mRNA was significantly deregulated in HCC, being TERRA downregulated and TERT mRNA upregulated in HCC tissues vs. the peritumoral (PT) ones, and the receiver operating characteristic (ROC) curve analyses revealed a significant ability in discriminating HCC from PT tissue. Further, the determinations of circulating TERRA and TERC showed higher amounts of these transcripts in the plasma of HCC patients vs. controls and ROC analyses gave significant results. The expression characterization of the cultured HCC cells showed their ability to produce and secrete TERRA and TERC into the EVs; the ability to produce TERT mRNA that was not detectable in the EVs; and the ability to respond to sorafenib treatment increasing TERRA expression. Our results highlight that: (i) both cellular and extracellular expressions of TERRA and TERC are dysregulated in HCC as well as the cellular expression of TERT mRNA and (ii) the combined detection of TERRA and TERC in plasma may represent a promising approach for non-invasive diagnostic molecular indicators of HCC.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , RNA Longo não Codificante , Telomerase , Carcinoma Hepatocelular/genética , Humanos , Neoplasias Hepáticas/genética , RNA , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/metabolismo
2.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 53(3): 457-465, 2022 May.
Artigo em Chinês | MEDLINE | ID: mdl-35642155

RESUMO

Objective: To investigate the association between WD40-encoding RNA antisense to p53 ( WRAP53 ß), a telomerase new core subunit, and the clinical, genomic and immune infiltration characteristics of squamous cell carcinoma of the head and neck (HNSC), and to explore for potential joint targeted therapy of HNSC. Methods: Tumor IMmune Estimation Resource (TIMER) online modules were adopted to predict the association between WRAP53 ß expression and the clinical features, oncogene, and immune infiltration of HNSC in the Cancer Genome Atlas (TCGA) cohort. Tumor Immune Single-cell Hub (TISCH) was used to analyze WRAP53 ß expression at the single cell level. Analysis of the small molecule inhibitors potentially targeting WRAP53 ß was carried out by Computational Analysis of REsistance (CARE). In the in vitro verification experiment, recombinant lentiviral particles with the sh WRAP53 ß sequence were synthesized. Then, the oral squamous cell carcinoma cell line Cal27 (the sh WRAP53 ßgroup) stably expressing sh WRAP53 ß were constructed, and two control groups were set up (the shNC group consisting of Cal27 cells added with lentiviral particles containing non-specific control sequences and the Con group consisting of untreated Cal27 cells). MTT assay was done to examine the proliferation of cells in the three groups. Cellular immunofluorescence assay was done for further qualitative examination of the expression of P53 protein in the cells of the sh WRAP53 ß group and the shNC group. Western blot was done to measure the expression of WRAP53ß and γ-H2AX, a DNA damage protein, in the 18 th, 23 rd and 28 th passages of the sh WRAP53 ß group and the shNC group. Finally, specimens of 13 cases of oral squamous cell carcinoma and 7 cases of oral mucosal inflammation were collected, and the expression of WRAP53ß and γ-H2AX in the clinical specimens of oral squamous cell carcinoma was verified with immunohistochemistry. Resluts: TIMER analysis revealed that the expression level of WRAP53 ß in HNSC tissues was significantly higher than that in normal tissues. There was a significant positive correlation between WRAP53 ß expression and multiple genes in the p53 pathway, including CCNB1, CCNB2 and CDK1. Although no significant correlation between WRAP53 ß expression and infiltrating immune cells was found, WRAP53 ß was significantly positively correlated with the inflammatory factors IFN-γ and IL23A, and negatively correlated with IL-1A and IL-6 in HPV-positive carcinoma of the head and neck. TISCH single cell sequencing datasets also showed higher expression of WRAP53 ß in malignant cells, and very low or zero expression in immune cells. According to the CARE scores, the most potent WRAP53 ß co-inhibitory drugs were ATM, CDK1 and MDM4 targeted inhibitors. In vitro cell experiments showed that the proliferation ability of Cal27 cells decreased significantly in the sh WRAP53 ß group as compared with that of the control group between Day 5 and Day 7 ( P<0.05). Furthermore, the expression of P53 decreased significantly in the sh WRAP53 ß group. As compared with the control group, the expression of WRAP53ß in sh WRAP53 ß group significantly decreased in the 18 th, 23 rd and 28 th passages ( P<0.05), while γ-H2AX expression only decreased in the 18 th and 28 th passages ( P<0.05) according to the results of Western blot. Clinical specimens showed rather high positive expression rate of γ-H2AX in oral squamous cell carcinoma tissues (12/13), while the expression of WRAP53ß was not detected in oral mucositis samples (0/7). Conclusions: WRAP53 ß showed significantly higher expression level in HSNC, and was significantly associated with p53 pathway genes. ATM, CDK1 and MDM4 inhibitors may be potential WRAP53 ß co-inhibitory agents. RNA interference of WRAP53 ß expression may cause inhibition of DNA damage, thereby indicating therapeutic potential for HNSC.


Assuntos
Chaperonas Moleculares , Neoplasias Bucais , Carcinoma de Células Escamosas de Cabeça e Pescoço , Telomerase , Linhagem Celular Tumoral , Biologia Computacional , Humanos , Chaperonas Moleculares/genética , Neoplasias Bucais/genética , Neoplasias Bucais/terapia , RNA , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Telomerase/genética
4.
Biomed Pharmacother ; 152: 113233, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35689861

RESUMO

Vascular remodeling is a significant feature of pulmonary artery hypertension (PAH), and is characterized by abnormal proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). Telomerase reverse transcriptase (TERT), as a determining factor for controlling telomerase activity, has been proven to be associated with cell proliferation. This study aims to explore whether TERT mediates the proliferation and migration of PASMCs and the underlying molecular mechanism. Primary PASMCs from Sprague-Dawley (SD) rats were used in this experiment. Cell proliferation and migration were evaluated by Cell Counting Kit-8, EdU incorporation assay and transwell assay, respectively. Telomerase activity was assessed with a rat TE ELISA kit. Small interfering RNA (siRNA) transfection was conducted to silence c-MYC expression. The protein levels of p-Akt, c-MYC, PPARγ and TERT were determined through western blotting. Our work demonstrates that PDGF upregulated TERT expression and telomerase activation by activating Akt and upregulating of c-MYC in PASMCs. Inhibition of Akt with LY294002, knockdown of c-MYC by siRNA or suppression of telomerase activity with BIBR1532 repressed PDGF-induced PASMC proliferation and migration. Furthermore, activation of peroxisome proliferator-activated receptor γ (PPARγ) with pioglitazone suppressed PDGF-induced TERT expression and telomerase activation, leading to inhibition of PASMC proliferation and migration.


Assuntos
Artéria Pulmonar , Telomerase , Animais , Proliferação de Células , Células Cultivadas , Miócitos de Músculo Liso/metabolismo , PPAR gama/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Artéria Pulmonar/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Telomerase/genética , Telomerase/metabolismo
5.
Biochem Biophys Res Commun ; 617(Pt 1): 22-29, 2022 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-35667242

RESUMO

As the core component of telomeres, the Shelterin complex interacts with telomerase and the CST complex and plays a crucial role in maintaining telomere structure. Perturbation of Shelterin subunits results in telomere damage and subsequent genomic instability, which leads to aging as well as multiple human diseases. Recently, zebrafish have been widely utilized to model human diseases. To establish appropriate zebrafish models of Shelterin-related human disorders, we generated knockout zebrafish of the Shelterin subunit genes acd, pot1, tinf2, terf1 and pinx1 using the CRISPR/Cas9 technology and analyzed the effects of gene deficiency on zebrafish development in detail. We discovered that tinf2, terf1 and pinx1 homozygous mutants could grow to adulthood normally, whereas acd and pot1 homozygous mutant larvae died between 12 and 15 dpf without obvious abnormalities. A few acd-/- mutants survived to adulthood and displayed several premature aging-like phenotypes, including male sterility, cachectic dwarfism and reduced lifespan. Overall, our study established a variety of telomere-deficient zebrafish mutant strains and provided novel animal models for further exploring the relationship between telomeres and aging as well as the pathogenesis of human diseases associated with telomere deficiency.


Assuntos
Telomerase , Peixe-Zebra , Animais , Masculino , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
6.
Oxid Med Cell Longev ; 2022: 2905663, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35707279

RESUMO

The upregulation telomerase activity is observed in over 85-90% of human cancers and provides an attractive target for cancer therapies. The high guanine content in the telomere DNA sequences and the hTERT promoter can form G-quadruplexes (G4s). Small molecules targeting G4s in telomeres and hTERT promoter could stabilize the G4s and inhibit hTERT expression and telomere extension. Several G4 ligands have shown inhibitory effects in cancer cells and xenograft mouse models, indicating these ligands have a potential for cancer therapies. The current review article describes the concept of the telomere, telomerase, and G4s. Moreover, the regulation of telomerase and G4s in telomeres and hTERT promoter is discussed as well. The summary of the small molecules targeting G4s in telomeric DNA sequences and the hTERT promoter will also be shown.


Assuntos
Quadruplex G , Telomerase , Animais , Humanos , Ligantes , Camundongos , Regiões Promotoras Genéticas/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo
7.
Artigo em Inglês | MEDLINE | ID: mdl-35661999

RESUMO

Evidence suggests antioxidant and anti-inflammatory properties of omega-3 polyunsaturated fatty acids (n-3 PUFA). However, the effect of supplementation of this fatty acid profile on the telomere length and the telomerase enzyme activity was not revised yet. The PubMed and Embase® databases were used to search for clinical trials. A total of six clinical trials were revised. Omega-3 PUFA supplementation did not statistically affect telomere length in three out of three studies but affected telomerase activity in two out of four studies. The supplementation increased telomerase enzyme activity in subjects with first-episode schizophrenia. Besides, it decreased telomerase enzyme activity without modulating the effects of Pro12Ala polymorphism on the PPARγ gene in type 2 diabetes subjects. The methodological differences between the studies and the limited number of studies on the theme suggest that further studies are needed to elucidate the effects of n-3 PUFA supplementation on telomere length and telomerase enzyme activity in humans.


Assuntos
Diabetes Mellitus Tipo 2 , Ácidos Graxos Ômega-3 , Telomerase , Ensaios Clínicos como Assunto , Diabetes Mellitus Tipo 2/tratamento farmacológico , Suplementos Nutricionais , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-3/uso terapêutico , Humanos , Telomerase/genética , Telomerase/metabolismo , Telômero
8.
DNA Repair (Amst) ; 115: 103342, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35588569

RESUMO

Activation of a telomere maintenance mechanism is key to achieving replicative immortality. Alternative Lengthening of Telomeres (ALT) is a telomerase-independent pathway that hijacks the homologous recombination pathways to elongate telomeres. Commitment to ALT is often associated with several hallmarks including long telomeres of heterogenous lengths, mutations in histone H3.3 or the ATRX/DAXX histone chaperone complex, and incorporation of non-canonical telomere sequences. The consequences of these genetic and epigenetic changes include enhanced replication stress and the presence of transcriptionally permissive chromatin, which can result in replication-associated DNA damage. Here, we detail the molecular mechanisms that are critical to repairing DNA damage at ALT telomeres, including the BLM Helicase, which acts at several steps in the ALT process. Furthermore, we discuss the emerging findings related to the telomere-associated RNA, TERRA, and its roles in maintaining telomeric integrity. Finally, we review new evidence for therapeutic interventions for ALT-positive cancers which are rooted in understanding the molecular underpinnings of this process.


Assuntos
Telomerase , Homeostase do Telômero , Cromatina , Histonas/genética , Telomerase/metabolismo , Telômero/metabolismo
9.
Placenta ; 123: 41-45, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35544964

RESUMO

INTRODUCTION: Considering that preeclampsia is characterized by oxidative stress, inflammation, and endothelial dysfunction, we hypothesized that preeclampsia and preeclampsia severity may affect the telomerase levels of the mother. METHODS: This cross-sectional case control study comprised 86 participants who were separated into three groups as severe preeclampsia, non-severe preeclampsia, and healthy control group. Venous blood samples were obtained from pregnant women with severe preeclampsia just before delivery for biochemical analysis and to evaluate maternal serum telomerase levels. Since gestational age, maternal age and BMI would have an effect on maternal serum telomerase levels, serum samples were taken in the control group and non-severe preeclampsia group at similar gestational age during clinical visits in order to homogenize these parameters, and these patients were followed up. Telomerase levels in maternal serum were evaluated using the enzyme-linked immune-sorbent assay. RESULTS: Maternal age, nulliparity, body mass index (kg/m2) at blood sampling day, ethnicity, smoking and history of caesarian section were statistically similar among the groups. The mean birth weight percentiles were the lowest in the severe preeclampsia group. Fetal growth restriction rates were significantly higher in the severe preeclampsia group than in the non-severe preeclampsia group. Gestational age at blood drawn was similar among groups. Neutrophil lymphocyte ratio, platelet lymphocyte ratio, mean platelet volume, red cell distribution width and white blood cell were statistically different among groups. The serum telomerase level was 1.137 ± 0.390 ng/mL in the severe preeclampsia group, 0.763 ± 0.390 ng/mL in the non-severe preeclampsia group, and 0.425 ± 0.160 ng/mL in the control group (p < .001). DISCUSSION: This study indicated that maternal serum telomerase levels were significantly increased in both preeclampsia groups.


Assuntos
Pré-Eclâmpsia , Telomerase , Estudos de Casos e Controles , Estudos Transversais , Feminino , Humanos , Paridade , Gravidez
10.
J Exp Clin Cancer Res ; 41(1): 173, 2022 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-35549739

RESUMO

BACKGROUND: The ETS transcription factor GABPA has long been thought of as an oncogenic factor and recently suggested as a target for cancer therapy due to its critical effect on telomerase activation, but the role of GABPA in clear cell renal cell carcinoma (ccRCC) is unclear. In addition, ccRCC is characterized by metabolic reprograming with aberrant accumulation of L-2-hydroxyglurate (L-2HG), an oncometabolite that has been shown to promote ccRCC development and progression by inducing DNA methylation, however, its downstream effectors remain poorly defined. METHODS: siRNAs and expression vectors were used to manipulate the expression of GABPA and other factors and to determine cellular/molecular and phenotypic alterations. RNA sequencing and ChIP assays were performed to identify GABPA target genes. A human ccRCC xenograft model in mice was used to evaluate the effect of GABPA overexpression on in vivo tumorigenesis and metastasis. ccRCC cells were incubated with L-2-HG to analyze GABPA expression and methylation. We carried out immunohistochemistry on patient specimens and TCGA dataset analyses to assess the effect of GABPA on ccRCC survival. RESULTS: GABPA depletion, although inhibiting telomerase expression, robustly enhanced proliferation, invasion and stemness of ccRCC cells, whereas GABPA overexpression exhibited opposite effects, strongly inhibiting in vivo metastasis and carcinogenesis. TGFBR2 was identified as the GABPA target gene through which GABPA governed the TGFß signaling to dictate ccRCC phenotypes. GABPA and TGFBR2 phenocopies each other in ccRCC cells. Higher GABPA or TGFBR2 expression predicted longer survival in patients with ccRCC. Incubation of ccRCC cells with L-2-HG mimics GABPA-knockdown-mediated phenotypic alterations. L-2-HG silenced the expression of GABPA in ccRCC cells by increasing its methylation. CONCLUSIONS: GABPA acts as a tumor suppressor by stimulating TGFBR2 expression and TGFß signaling, while L-2-HG epigenetically inhibits GABPA expression, disrupting the GABPA-TGFß loop to drive ccRCC aggressiveness. These results exemplify how oncometabolites erase tumor suppressive function for cancer development/progression. Restoring GABPA expression using DNA methylation inhibitors or other approaches, rather than targeting it, may be a novel strategy for ccRCC therapy.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Telomerase , Animais , Carcinogênese/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Epigênese Genética , Fator de Transcrição de Proteínas de Ligação GA/genética , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/patologia , Camundongos , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Telomerase/genética , Telomerase/metabolismo , Fator de Crescimento Transformador beta/metabolismo
11.
Proc Natl Acad Sci U S A ; 119(20): e2121499119, 2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35537048

RESUMO

As the global elderly population grows, it is socioeconomically and medically critical to provide diverse and effective means of mitigating the impact of aging on human health. Previous studies showed that the adeno-associated virus (AAV) vector induced overexpression of certain proteins, which can suppress or reverse the effects of aging in animal models. In our study, we sought to determine whether the high-capacity cytomegalovirus vector (CMV) can be an effective and safe gene delivery method for two such protective factors: telomerase reverse transcriptase (TERT) and follistatin (FST). We found that the mouse cytomegalovirus (MCMV) carrying exogenous TERT or FST (MCMVTERT or MCMVFST) extended median lifespan by 41.4% and 32.5%, respectively. We report CMV being used successfully as both an intranasal and injectable gene therapy system to extend longevity. Specifically, this treatment significantly improved glucose tolerance, physical performance, as well as preventing body mass loss and alopecia. Further, telomere shortening associated with aging was ameliorated by TERT and mitochondrial structure deterioration was halted in both treatments. Intranasal and injectable preparations performed equally well in safely and efficiently delivering gene therapy to multiple organs, with long-lasting benefits and without carcinogenicity or unwanted side effects. Translating this research to humans could have significant benefits associated with quality of life and an increased health span.


Assuntos
Infecções por Citomegalovirus , Terapia Genética , Expectativa de Vida , Telomerase , Administração por Inalação , Animais , Folistatina/genética , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos/genética , Injeções Intraperitoneais , Camundongos , Modelos Animais , Neoplasias , Telomerase/genética , Telomerase/metabolismo
12.
J Immunother Cancer ; 10(5)2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35613827

RESUMO

BACKGROUND: Therapeutic cancer vaccines represent a promising approach to improve clinical outcomes with immune checkpoint inhibition. UV1 is a second generation telomerase-targeting therapeutic cancer vaccine being investigated across multiple indications. Although telomerase is a near-universal tumor target, different treatment combinations applied across indications may affect the induced immune response. Three phase I/IIa clinical trials covering malignant melanoma, non-small cell lung cancer, and prostate cancer have been completed, with patients in follow-up for up to 8 years. METHODS: 52 patients were enrolled across the three trials. UV1 was given as monotherapy in the lung cancer trial and concurrent with combined androgen blockade in the prostate cancer trial. In the melanoma study, patients initiated ipilimumab treatment 1 week after the first vaccine dose. Patients were followed for UV1-specific immune responses at frequent intervals during vaccination, and every 6 months for up to 8 years in a follow-up period. Phenotypic and functional characterizations were performed on patient-derived vaccine-specific T cell responses. RESULTS: In total, 78.4% of treated patients mounted a measurable vaccine-induced T cell response in blood. The immune responses in the malignant melanoma trial, where UV1 was combined with ipilimumab, occurred more rapidly and frequently than in the lung and prostate cancer trials. In several patients, immune responses peaked years after their last vaccination. An in-depth characterization of the immune responses revealed polyfunctional CD4+ T cells producing interferon-γ and tumor necrosis factor-α on interaction with their antigen. CONCLUSION: Long-term immunomonitoring of patients showed highly dynamic and persistent telomerase peptide-specific immune responses lasting up to 7.5 years after the initial vaccination, suggesting a plausible functional role of these T cells in long-term survivors. The superior immune response kinetics observed in the melanoma study substantiate the rationale for future combinatorial treatment strategies with UV1 vaccination and checkpoint inhibition for rapid and frequent induction of anti-telomerase immune responses in patients with cancer.


Assuntos
Vacinas Anticâncer , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Melanoma , Neoplasias da Próstata , Telomerase , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Ensaios Clínicos Fase I como Assunto , Seguimentos , Humanos , Imunidade , Ipilimumab/uso terapêutico , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Melanoma/tratamento farmacológico , Peptídeos , Neoplasias da Próstata/patologia , Neoplasias Cutâneas , Vacinação , Vacinas de Subunidades
13.
Elife ; 112022 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-35559731

RESUMO

Dyskeratosis congenita (DC) is a rare genetic disorder characterized by deficiencies in telomere maintenance leading to very short telomeres and the premature onset of certain age-related diseases, including pulmonary fibrosis (PF). PF is thought to derive from epithelial failure, particularly that of type II alveolar epithelial (AT2) cells, which are highly dependent on Wnt signaling during development and adult regeneration. We use human induced pluripotent stem cell-derived AT2 (iAT2) cells to model how short telomeres affect AT2 cells. Cultured DC mutant iAT2 cells accumulate shortened, uncapped telomeres and manifest defects in the growth of alveolospheres, hallmarks of senescence, and apparent defects in Wnt signaling. The GSK3 inhibitor, CHIR99021, which mimics the output of canonical Wnt signaling, enhances telomerase activity and rescues the defects. These findings support further investigation of Wnt agonists as potential therapies for DC-related pathologies.


Assuntos
Disceratose Congênita , Células-Tronco Pluripotentes Induzidas , Telomerase , Células Epiteliais Alveolares/metabolismo , Disceratose Congênita/genética , Disceratose Congênita/patologia , Quinase 3 da Glicogênio Sintase , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Telomerase/genética , Telomerase/metabolismo , Telômero/metabolismo
14.
Cells ; 11(9)2022 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-35563795

RESUMO

Reverse transcriptase hTERT is essential to telomerase function in stem cells, as well as in 85-90% of human cancers. Its high expression in stem cells or cancer cells has made telomerase/hTERT an attractive therapeutic target for anti-aging and anti-tumor applications. In this study, we screened a natural product library containing 800 compounds using an endogenous hTERT reporter. Eight candidates have been identified, in which sanguinarine chloride (SC) and brazilin (Braz) were selected due to their leading inhibition. SC could induce an acute and strong suppressive effect on the expression of hTERT and telomerase activity in multiple cancer cells, whereas Braz selectively inhibited telomerase in certain types of cancer cells. Remarkably, SC long-term treatment could cause telomere attrition and cell growth retardation, which lead to senescence features in cancer cells, such as the accumulation of senescence-associated ß-galactosidase (SA-ß-gal)-positive cells, the upregulation of p16/p21/p53 pathways and telomere dysfunction-induced foci (TIFs). Additionally, SC exhibited excellent capabilities of anti-tumorigenesis, both in vitro and in vivo. In the mechanism, the compound down-regulated several active transcription factors including p65, a subunit of NF-κB complex, and reintroducing p65 could alleviate its suppression of the hTERT/telomerase. Moreover, SC could directly bind hTERT and inhibit telomerase activity in vitro. In conclusion, we identified that SC not only down-regulates the hTERT gene's expression, but also directly affects telomerase/hTERT. The dual function makes this compound an attractive drug candidate for anti-tumor therapy.


Assuntos
Produtos Biológicos , Telomerase , Benzofenantridinas/farmacologia , Produtos Biológicos/farmacologia , Humanos , Isoquinolinas , Telomerase/metabolismo , Telômero/metabolismo
15.
Analyst ; 147(11): 2405-2411, 2022 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-35579289

RESUMO

As a specific biological marker for the occurrence and progression of tumor cells, detection of telomerase activity is of great importance for the physiological research of tumors. However, in situ measurement of telomerase activity in living cells still remains a challenge. Herein, we report a precisely designed oligonucleotide-functionalized gold nanoparticle probe that has realized high-efficiency detection of telomerase activity for cellular imaging toward the identification of tumors. Our method has achieved intracellular imaging of telomerase activity and shows good performance towards the distinction of tumor cells from normal ones. Moreover, the method reported here for tracking tumor cells in blood has wide applications in cancer diagnosis. This strategy offers an opportunity for cancer diagnosis, guiding therapy and evaluating prognosis.


Assuntos
Nanopartículas Metálicas , Neoplasias , Telomerase , Ouro , Células HeLa , Humanos , Neoplasias/diagnóstico por imagem , Oligonucleotídeos , Imagem Óptica , Telomerase/metabolismo
16.
Nucleic Acids Res ; 50(10): 5652-5671, 2022 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-35639927

RESUMO

Homologous recombination is the predominant DNA repair pathway used in the gonad. Of the excess DNA double-strand breaks formed in meiosis, only a subset matures into crossovers, with the remainder repaired as non-crossovers. The conserved BTR complex (comprising Bloom helicase, topoisomerase 3 and RMI1/2 scaffold proteins) acts at multiple steps during recombination to dismantle joint DNA molecules, thereby mediating the non-crossover outcome and chromosome integrity. Furthermore, the complex displays a role at the crossover site that is less well understood. Besides catalytic and TOPRIM domains, topoisomerase 3 enzymes contain a variable number of carboxy terminal zinc finger (ZnF) domains. Here, we studied the Caenorhabditis elegans mutant, in which the single ZnF domain is deleted. In contrast to the gene disruption allele, the top-3-ZnF mutant is viable, with no replication defects; the allele appears to be a hypomorph. The TOP-3-ZnF protein is recruited into foci but the mutant has increased numbers of crossovers along its chromosomes, with minor defects in repressing heterologous recombination, and a marked delay in the maturation/processing of recombination intermediates after loading of the RAD-51 recombinase. The ZnF domain cooperates with the RMI1 homolog RMH-2 to stabilize association of the BTR complex with recombination intermediates and to prevent recombination between heterologous DNA sequences.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans , Proteínas Cromossômicas não Histona/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Reparo do DNA , Células Germinativas/metabolismo , Meiose/genética , RNA , Telomerase , Dedos de Zinco/genética
17.
Comput Math Methods Med ; 2022: 1721526, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35535227

RESUMO

Whether TERT promoter mutation is related to more aggressive clinicopathologic features and worse outcomes in papillary thyroid carcinoma patients (PTCs) is still variable and controversial. Our intention was to investigate the risk or prognostic factors that may additionally predict the TERT promoter mutation doable of these lesions and new prevention techniques in PTCs. A total of 2,539 PTC patients with 11.50% TERT mutation have been analyzed using Revman 5.3 software in this study. The PubMed and Embase databases were systematically searched for works published until November 9, 2021. The following variables had been associated with an extended chance of TERT promoter mutation in PTC patients: age < 45 years (MD = 10.93, 95%CI = 7.25-14.61); gender = male (pooled OR = 1.63, 95%CI = 1.17-2.28); tumor size > 1 cm (MD = 0.56, 95%CI = 0.34-0.77); lymph node metastasis (pooled OR = 1.29, 95%CI = 0.93-1.79); vascular invasion (pooled OR = 1.78, 95%CI = 0.83-3.84); extrathyroidal extension (pooled OR = 2.00, 95%CI = 1.32-3.02); distant metastasis (pooled OR = 1.46, 95%CI = 1.04-2.04); advanced TNM stage (pooled OR = 3.19, 95%CI = 2.28-4.45). In addition, multifocality (pooled OR = 0.67, 95%CI = 0.14-3.24) had no affiliation with TERT promoter mutation in PTC patients. Our finding showed that age < 45 years, male, tumor size > 1 cm, lymph node metastasis, vascular invasion, and superior/advanced TNM stage were dangerous elements for TERT promoter mutation of worse effect in PTCs while that multifocality was once negatively correlated. TERT promoter mutation is drastically associated with recurrence and PTC-related mortality.


Assuntos
Telomerase , Neoplasias da Glândula Tireoide , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Mutação , Prognóstico , Fatores de Risco , Telomerase/genética , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia
18.
Anal Chim Acta ; 1208: 339835, 2022 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-35525591

RESUMO

The development of methods to realize the on-site analysis of antibiotic pollutants is of great importance for food quality control and environmental monitoring. Herein, we designed a magnetic bead (MB)-based DNA walker and utilized its target-triggered and endonuclease-driven walking reaction to develop a novel colorimetric and electrochemical dual-mode biosensing method for the convenient detection of kanamycin (Kana) antibiotic. The colorimetric signal transduction strategy of the method was constructed on the telomerase extension of the DNA walking-released telomeric primer into G-quadruplex/hemin DNAzymes. Due to the DNA walking and telomerase dual signal amplification, a good linear relationship from 0.1 pg mL-1 to 1 ng mL-1 was obtained for this strategy with a detection limit of 22 fg mL-1. Meanwhile, the MB complex produced through the above DNA walking reaction was also used as a multipedal DNA walker to develop an electrochemical signal transduction strategy. By utilizing it to trigger another endonuclease-driven DNA walking at a DNA hairpin-modified electrode, ferrocene labels were quantitatively released from this electrode to cause the electrochemical signal decrease. Because of the dual endonuclease-driven DNA walking for signal amplification, a five-order of magnitude wide linear relationship from 0.01 pg mL-1 to 1 ng mL-1 was obtained with an ultralow detection limit of 8.4 fg mL-1. As the two strategies did not involve complicated manipulations and the requirement of expensive instruments, this biosensing method exhibits a high application value for the on-site semiquantitative screening and accurate analysis of antibiotic residues.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Telomerase , Antibacterianos/análise , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Colorimetria , DNA/química , Técnicas Eletroquímicas , Endonucleases , Limite de Detecção
19.
Cell Death Dis ; 13(5): 435, 2022 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-35508454

RESUMO

Idiopathic pulmonary fibrosis (IPF) was considered as a telomere-mediated disease. TERT and TERC correlated with telomere length. Although telomerase gene mutations were associated with IPF, majority patients did not carry mutations. The mechanism by which telomerase expression was regulated in IPF are still unclear. In this study, we aimed to delineate the mechanisms that how TERT protein expression were regulated in alveolar epithelial cells (AECs) in pulmonary fibrosis. Here, we found that P16, P21 and fibrosis markers (αSMA and Collagen-I) were prominently increased in lung tissues of IPF patients and bleomycin-induced mouse models, while the expression of KLF4 and TERT were decreased in AECs. In vivo experiments, AAV-6 vectors mediated KLF4 over-expression with specific SP-C promoter was constructed. Over-expression of KLF4 in AECs could protect TERT expression and suppress the development of pulmonary fibrosis in bleomycin-induced mouse models. In the mechanism exploration of TERT regulation, KLF4 and TERT were both down-regulated in bleomycin-induced senescent MLE-12 and BEAS-2B cells. Compared with control group, small-interfering RNA targeting KLF4 significantly reduced the TERT expression and telomerase activity, while overexpression of KLF4 can increased the expression of TERT and telomerase activity in senescent AECs. Furthermore, ChIP showed that KLF4 protein could bind to the TERT promoter region in MLE-12 cells, suggesting that KLF4 could implicate in pathogenesis of lung fibrosis through regulating TERT transcription in AECs. Taken together, this study identified that KLF4 might be a promising potential target for further understanding the mechanism and developing novel strategy for the treatment of lung fibrosis in IPF.


Assuntos
Fibrose Pulmonar Idiopática , Telomerase , Células Epiteliais Alveolares/metabolismo , Animais , Bleomicina/farmacologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Camundongos , Telomerase/metabolismo
20.
Stem Cells ; 40(1): 102-111, 2022 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-35511869

RESUMO

In adult tissues such as adipose tissue, post-mitotic cells like adipocytes can be replaced by differentiation of a population of tissue-resident stem cells. Expression of mouse telomerase reverse transcriptase (mTert) is a hallmark of stem cell populations, and previous efforts to identify tissue-resident adult stem cells by measuring mTert expression have increased our understanding of stem cell biology significantly. Here, we used a doxycycline-inducible mouse model to perform longitudinal, live-animal lineage-tracing of mTert-expressing cells for more than 1 year. We identified a rare (<2%) population of stem cells in different fat depots that express putative preadipocyte markers. The adipose-derived mTert-positive cells are capable of self-renewal and possess adipogenic potential. Finally, we demonstrate that high-fat diet (HFD) can initiate differentiation of these cells in vivo. These data identify a population of adipose stem cells that contribute to the depot-specific response to HFD.


Assuntos
Telomerase , Adipogenia/genética , Tecido Adiposo/metabolismo , Animais , Diferenciação Celular , Camundongos , Células-Tronco/metabolismo , Telomerase/genética , Telomerase/metabolismo
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