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1.
BMC Med ; 19(1): 277, 2021 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-34763698

RESUMO

BACKGROUND: Gliomas are the most common aggressive cancer in the central nervous system. Considering the difficulty in monitoring glioma response and progression, an approach is needed to evaluate the progression or survival of patients with glioma. We propose an application to facilitate clinical detection and treatment monitoring in glioma patients by using telomerase-positive circulating tumor cells (CTCs) and to further evaluate the relationship between the immune microenvironment and CTCs in glioma patients. METHODS: From October 2014 to June 2017, 106 patients newly diagnosed with glioma were enrolled. We used the telomerase reverse transcriptase CTC detection method to detect and analyze the CTC statuses of glioma patients before and after surgery. FlowSight and FISH confirmed the CTCs detected by the telomerase-based method. To verify the correlation between CTCs and the immune response, peripheral white blood cell RNA sequencing was performed. RESULTS: CTCs were common in the peripheral blood of glioma patients and were not correlated with the pathological classification or grade of patients. The results showed that the presence of postoperative CTCs but not preoperative CTCs in glioma patients was a poor prognostic factor. The level of postoperative CTCs, which predicts a poor prognosis after surgery, may be associated with neutrophils. RNA sequencing suggested that postoperative CTCs were positively correlated with innate immune responses, especially the activation of neutrophils and the generation of neutrophil extracellular traps, but negatively correlated with the cytotoxic response. CONCLUSIONS: Our results showed that telomerase-positive CTCs can predict a poor prognosis of patients with glioma. Our results also showed a correlation between CTCs and the immune macroenvironment, which provides a new perspective for the treatment of glioma.


Assuntos
Glioma , Células Neoplásicas Circulantes , Telomerase , Biomarcadores Tumorais , Glioma/diagnóstico , Humanos , Neutrófilos/metabolismo , Prognóstico , Telomerase/genética , Telomerase/metabolismo , Microambiente Tumoral
2.
Anal Chim Acta ; 1184: 339036, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34625244

RESUMO

A direct electrochemistry of silver nanoparticles (AgNPs)-anchored metal-organic frameworks (MOFs) is developed for detection of telomerase activity based on allosteric activation of an aptamer hairpin. AgNPs in situ decorated on PCN-224 (AgNPs/PCN-224) constituted the direct electrochemical labels that were further biofunctionalized by recognition moiety of streptavidin (SA). To achieve the target biosensing, an allosteric hairpin-structured DNA was elaborately designed for signal transduction. The presence of telomerase elongated its primer in the hairpin to displace partial stem strand, thus resulted in the formation of SA aptamer-open structure. Through the specific interaction with aptamer, SA-biofunctionalized AgNPs/PCN-224 probe was attached onto the electrode surface, generating electrochemical signal at + 0.072 V of AgNPs centralized by MOF structure. The direct electrochemical biosensor showed target activity-dependent response from 1.0 × 10-7 to 1.0 × 10-1 IU L-1 with a detection limit of 5.4 × 10-8 IU L-1. Moreover, the sensor was applied in evaluation of telomerase activity in living cancer cells. The established electrochemical detection approach in this work avoids the critical deoxygenation conditions and additional electrocatalytic reagents, which opens a novel biosensing perspective for direct electrochemistry of MOF-based nanocomposites.


Assuntos
Nanopartículas Metálicas , Estruturas Metalorgânicas , Telomerase , Regulação Alostérica , Técnicas Eletroquímicas , Eletroquímica , Prata , Telomerase/metabolismo
3.
Nat Commun ; 12(1): 5514, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535663

RESUMO

Human shelterin components POT1 and TPP1 form a stable heterodimer that protects telomere ends from ATR-dependent DNA damage responses and regulates telomerase-dependent telomere extension. Mice possess two functionally distinct POT1 proteins. POT1a represses ATR/CHK1 DNA damage responses and the alternative non-homologous end-joining DNA repair pathway while POT1b regulates C-strand resection and recruits the CTC1-STN1-TEN1 (CST) complex to telomeres to mediate C-strand fill-in synthesis. Whether POT1a and POT1b are involved in regulating the length of the telomeric G-strand is unclear. Here we demonstrate that POT1b, independent of its CST function, enhances recruitment of telomerase to telomeres through three amino acids in its TPP1 interacting C-terminus. POT1b thus coordinates the synthesis of both telomeric G- and C-strands. In contrast, POT1a negatively regulates telomere length by inhibiting telomerase recruitment to telomeres. The identification of unique amino acids between POT1a and POT1b helps us understand mechanistically how human POT1 switches between end protective functions and promoting telomerase recruitment.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Análise Mutacional de DNA , Camundongos , Ligação Proteica , Rad51 Recombinase/metabolismo , Sarcoma/patologia
4.
Talanta ; 235: 122814, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517670

RESUMO

Simple and sensitive detection of telomerase activity is of vital importance for both early diagnosis and therapy of malignant tumors. Inspired by DNA-biobarcode amplification reported by Chad A. Mirkin, we developed a facile DNA-biobarcode-like SERS-based copper-mediated signal amplification strategy for sensitive detection of telomerase activity. In this strategy, a duplex DNA constructed by hybridization of a copper oxide nanoparticle (CuO NP)-labeled reporting sequence (RS) with the telomerase primer sequence (TS) is ingeniously designed, and anchored on the magnetic bead (MB) to build the CuO NPs-encoded magnetic bead (MB-CuO NPs) detection probe. Upon selective sensing of telomerase, telomerase elongation reaction and structure change of TS products make the CuO NP-RS displace and separate from MB. The separated CuO NPs are dissolved into a mass of Cu2+, which prompt monodisperse dopamine-functionalized AgNPs (D-AgNPs) signal probe into aggregation, resulting in color changes and significantly enhancing of SERS signal. The SERS signal increases with the increase of Cu2+, which is directly proportional to the telomerase. Benefiting from the transformation of CuO NP to Cu2+ with a high amplification effect, this strategy could realize the telomerase activity measurement down to 3 HeLa cells and a dynamic range of 10-10000 cells. It shows a significant improvement of sensitivity without need for other enzymes and elaborate design, which escapes from the complicated manipulations and design in polymerase chain reaction (PCR) and DNA amplification techniques. Moreover, with this strategy, telomerase activities of different cell lines and telomerase inhibitors screening were successfully performed. Significantly, it can also be utilized for visual detection of telomerase, which validates the potential on-site application and its application as point-of-care testing (POCT) for efficient monitoring. Given the high-performance for telomerase analysis, the strategy has a promising application in biological detection and clinical diagnosis, as well as point-of-care tests.


Assuntos
Técnicas Biossensoriais , Telomerase , Cobre , DNA , Células HeLa , Humanos , Técnicas de Amplificação de Ácido Nucleico , Telomerase/genética , Telomerase/metabolismo
5.
Am J Physiol Heart Circ Physiol ; 321(5): H985-H1003, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34559580

RESUMO

Ventilation with gases containing enhanced fractions of oxygen is the cornerstone of therapy for patients with hypoxia and acute respiratory distress syndrome. Yet, hyperoxia treatment increases free reactive oxygen species (ROS)-induced lung injury, which is reported to disrupt autophagy/mitophagy. Altered extranuclear activity of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), plays a protective role in ROS injury and autophagy in the systemic and coronary endothelium. We investigated interactions between autophagy/mitophagy and TERT that contribute to mitochondrial dysfunction and pulmonary injury in cultured rat lung microvascular endothelial cells (RLMVECs) exposed in vitro, and rat lungs exposed in vivo to hyperoxia for 48 h. Hyperoxia-induced mitochondrial damage in rat lungs [TOMM20, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], which was paralleled by increased markers of inflammation [myeloperoxidase (MPO), IL-1ß, TLR9], impaired autophagy signaling (Beclin-1, LC3B-II/1, and p62), and decreased the expression of TERT. Mitochondrial-specific autophagy (mitophagy) was not altered, as hyperoxia increased expression of Pink1 but not Parkin. Hyperoxia-induced mitochondrial damage (TOMM20) was more pronounced in rats that lack the catalytic subunit of TERT and resulted in a reduction in cellular proliferation rather than cell death in RLMVECs. Activation of TERT or autophagy individually offset mitochondrial damage (MTT). Combined activation/inhibition failed to alleviate hyperoxic-induced mitochondrial damage in vitro, whereas activation of autophagy in vivo decreased mitochondrial damage (MTT) in both wild type (WT) and rats lacking TERT. Functionally, activation of either TERT or autophagy preserved transendothelial membrane resistance. Altogether, these observations show that activation of autophagy/mitophagy and/or TERT mitigate loss of mitochondrial function and barrier integrity in hyperoxia.NEW & NOTEWORTHY In cultured pulmonary artery endothelial cells and in lungs exposed in vivo to hyperoxia, autophagy is activated, but clearance of autophagosomes is impaired in a manner that suggests cross talk between TERT and autophagy. Stimulation of autophagy prevents hyperoxia-induced decreases in mitochondrial metabolism and sustains monolayer resistance. Hyperoxia increases mitochondrial outer membrane (TOMM20) protein, decreases mitochondrial function, and reduces cellular proliferation without increasing cell death.


Assuntos
Células Endoteliais/enzimologia , Hiperóxia/complicações , Lesão Pulmonar/enzimologia , Pulmão/irrigação sanguínea , Microvasos/enzimologia , Mitocôndrias/enzimologia , Mitofagia , Telomerase/metabolismo , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Permeabilidade Capilar , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/patologia , Feminino , Técnicas de Inativação de Genes , Mediadores da Inflamação/metabolismo , Lesão Pulmonar/etiologia , Lesão Pulmonar/genética , Lesão Pulmonar/patologia , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Microvasos/patologia , Mitocôndrias/genética , Mitocôndrias/patologia , Ratos Sprague-Dawley , Ratos Transgênicos , Receptores de Superfície Celular/metabolismo , Telomerase/deficiência , Telomerase/genética , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo
6.
Drug Alcohol Depend ; 227: 108982, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34482039

RESUMO

BACKGROUND: Drug dependence promotes accelerated aging and higher mortality compare with the general population. Telomere length is a biomarker of determination of cellular aging. Telomere attrition has been reported in heroin dependent patients. To investigate whether telomere length is affected by morphine or not, the expressions of hTERT and TERF2 in morphine treated human SH-SY5Y cells were determined and compared with untreated cells. METHODS: The SH-SY5Y cells were treated with 1 and 5 µM concentrations of morphine for different exposure times (1d, 2d, 3d, 7d and 60 days). The mRNA levels of hTERT and TERF2 were determined using quantitative real-time RCR. The relative telomere length was measured as the ratio of telomere/36B4. RESULTS: The hTERT and TERF2 mRNA levels were down regulated in morphine treated cells as a function of exposure duration. These alterations were reversible if morphine was removed from the culture medium. No reduction in the relative expression of hTERT and TERF2 in the cells exposed to N-acetyl cysteine (NAC) plus morphine was observed. In the SH-SY5Y cells treated by 5 µM morphine for 60 consecutive days, the hTERT and TERF2 mRNA levels and relative telomere lengths remarkably decreased. CONCLUSIONS: Reversible alteration of mRNA levels by removing morphine from culture medium, and effect of NAC in co-treatment of morphine plus NAC, emphasize the role of reactive oxygen species in down-regulation of the expression of hTERT and TERF2 by morphine. Telomere attrition in morphine treated cells is a consequence of down-regulation of the expression of hTERT and TERF2.


Assuntos
Telomerase , Telômero , Regulação para Baixo , Humanos , Morfina/farmacologia , RNA Mensageiro/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas
7.
Sci Rep ; 11(1): 17726, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489496

RESUMO

Telomere, the terminus of linear chromosome in eukaryotes, is composed of specific repeat DNA which is mainly synthesized by a protein complex called telomerase. The maintenance of telomere DNA is important for unlimited proliferative capacity of cancer cells. The telomerase activity is controlled by the expression level of telomerase reverse transcriptase (TERT), a catalytic unit of telomerase, in some species including human. Therefore, to reveal the regulatory mechanisms of the transcription of TERT gene is important for understanding the tumor development. We found that template activating factor-I (TAF-I), a multifunctional nuclear protein, is involved in the transcriptional activation of TERT for the maintenance of telomere DNA in HeLa cells. TAF-I maintains the histone H3 modifications involved in transcriptional activation and hypomethylated cytosines in CpG dinucleotides around the transcription start site (TSS) in the TERT gene locus. Collectively, TAF-I is involved in the maintenance of telomere DNA through the regulation of TERT transcription, then consequently the occurrence and/or recurrence of cancer cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Chaperonas de Histonas/metabolismo , Telomerase/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Células HeLa , Chaperonas de Histonas/genética , Histonas/metabolismo , Humanos , Telomerase/genética
8.
Sci Rep ; 11(1): 17100, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429452

RESUMO

To better understand the pathogenesis of nasal polyps (NPs) and sinonasal inverted papillomas (SIPs), we aimed to establish cell lines from fresh tissues of NPs and SIPs and characterize them. Primary cell cultures were obtained from two NP tissues (NP2 and NP3) and one SIP tissue (IP4). All the cells were polygonal in shape, expressed cytokeratin 14, and had normal diploid chromosome status. HPV58 DNA was detected in NP3. To obtain immortal primary cells, NP2 and IP4 cells were transduced with a combination of mutant CDK4, cyclinD1 and TERT. These cells were thereafter named NP2/K4DT and IP4/K4DT, respectively. HPV58-positive NP3 cells were transduced with TERT alone, the resulting cells named NP3/T. Phenotypic and genotypic identity of original tissues and derived cells was investigated. All the cell cultures with transgenes were confirmed to be derived from their parental cells and primary tumor tissues by analysis of short tandem repeats (STR) and maintained in vitro growth, genetic profiles and gene expression characteristics of the primary cells. These virtually immortalized cells, as well as the primary cells, have potential as in vitro models for studying the pathogenesis of NPs and SIPs and for preclinical study to develop new therapeutic agents.


Assuntos
Técnicas de Cultura de Células/métodos , Pólipos Nasais/genética , Neoplasias Nasais/genética , Papiloma Invertido/genética , Adolescente , Idoso , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Criança , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Humanos , Masculino , Repetições de Microssatélites , Pólipos Nasais/patologia , Neoplasias Nasais/patologia , Papiloma Invertido/patologia , Telomerase/genética , Telomerase/metabolismo , Transdução Genética/métodos
9.
Biomolecules ; 11(8)2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34439779

RESUMO

Telomere maintenance plays important roles in genome stability and cell proliferation. Tumor cells acquire replicative immortality by activating a telomere-maintenance mechanism (TMM), either telomerase, a reverse transcriptase, or the alternative lengthening of telomeres (ALT) mechanism. Recent advances in the genetic and molecular characterization of TMM revealed that telomerase activation and ALT define distinct neuroblastoma (NB) subgroups with adverse outcomes, and represent promising therapeutic targets in high-risk neuroblastoma (HRNB), an aggressive childhood solid tumor that accounts for 15% of all pediatric-cancer deaths. Patients with HRNB frequently present with widely metastatic disease, with tumors harboring recurrent genetic aberrations (MYCN amplification, TERT rearrangements, and ATRX mutations), which are mutually exclusive and capable of promoting TMM. This review provides recent insights into our understanding of TMM in NB tumors, and highlights emerging therapeutic strategies as potential treatments for telomerase- and ALT-positive tumors.


Assuntos
Genoma Humano , Proteína Proto-Oncogênica N-Myc/genética , Neoplasias do Sistema Nervoso/genética , Neuroblastoma/genética , Telomerase/genética , Telômero/química , Proteína Nuclear Ligada ao X/genética , Antineoplásicos/uso terapêutico , Criança , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Humanos , Mutação , Proteína Proto-Oncogênica N-Myc/metabolismo , Metástase Neoplásica , Neoplasias do Sistema Nervoso/tratamento farmacológico , Neoplasias do Sistema Nervoso/mortalidade , Neoplasias do Sistema Nervoso/patologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Fatores de Risco , Transdução de Sinais , Análise de Sobrevida , Telomerase/metabolismo , Telômero/efeitos dos fármacos , Telômero/patologia , Homeostase do Telômero , Proteína Nuclear Ligada ao X/metabolismo
10.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(4): 1056-1064, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34362482

RESUMO

OBJECTIVE: To investigate the cytotoxic effect and its mechanism of the micromolecule compound on the leukemia cells. METHODS: The cytotoxic effects of 28 Nilotinib derivatives on K562, KA, KG, HA and 32D cell lines were detected by MTT assays, and the compound Nilo 22 was screen out. Cell apoptosis and cell cycle on leukemia cells were detected by flow cytometry. The effect of compound screened out on leukemogenesis potential of MLL-AF9 leukemia mice GFP+ cells was tested by colony-forming units assays (CFU). The cytotoxic effect was further detected by transplant assays ex vivo. Telomerase activity assay, C-circle assay were used to measure the effects of compound on the length mechanism of telomere, RT-PCR was used to detected the changes of telomere. RESULTS: Nilo 22 serves as the most outstanding candidate out of 28 Nilotinib derivatives, which impairs leukemia cell lines, but spares normal hematopoietic cell line. Comparing with Nilotinib, Nilo 22 could induce the apoptosis of GFP+ cells significantly, slightly arrests the cell cycle at G0/G1 phase, and significantly inhibits colony formation and prolong the progression in MLL-AF9 leukemia mice model. The expression showed that the compound could slow the disease progression in MLL-AF9 leukemia mice significantly. Mechanistically, Nilo 22 could reduce the length of telomere by inhibiting telomerase activity and alternative lengthening of telomere (ALT). CONCLUSION: Nilo 22 shows a significant cytotoxic effect on mice and human leukemia cells, especially for drug resistance cells. Nilo 22 is a promising anti-leukemia agent to solve the common clinical problems of drug resistance and relapse of leukemia.


Assuntos
Leucemia , Telomerase/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Camundongos , Proteína de Leucina Linfoide-Mieloide/genética , Telomerase/metabolismo , Telômero/metabolismo
11.
Molecules ; 26(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34443381

RESUMO

The nutrients and their potential benefits are a new field of study in modern medicine for their positive impact on health. Curcumin, the yellow polyphenolic compound extracted from Curcuma longa species, is widely used in traditional Ayurvedic medicine to prevent and contrast many diseases, considering its antioxidant, immunomodulatory, anti-inflammatory, anti-microbial, cardio-protective, nephron-protective, hepato-protective, anti-neoplastic, and anti-rheumatic proprieties. In recent years, the investigations of curcumin have been focused on its application to aging and age-associated diseases. Aging is a physiological process in which there is a decreasing of cellular function due to internal or external stimuli. Oxidative stress is one of the most important causes of aging and age-related diseases. Moreover, many age-related disorders such as cancer, neuroinflammation, and infections are due to a low-grade chronic systemic inflammation. Curcumin acting on different proteins is able to contrast both oxidative stress than inflammation. In the brain, curcumin is able to modulate inflammation induced by microglia. Finally in brain tumors curcumin is able to reduce tumor growth by inhibition of telomerase activity. This review emphasizes the anti-aging role of curcumin focusing on its mechanism to counteract aging in the brain. Moreover, new formulations to increase the bioavailability of curcumin are discussed.


Assuntos
Envelhecimento/fisiologia , Produtos Biológicos/farmacologia , Encéfalo/fisiologia , Curcumina/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Humanos , Fármacos Neuroprotetores/farmacologia , Telomerase/metabolismo
12.
Biomed Res Int ; 2021: 3456321, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34458366

RESUMO

Inflammation response is an important reason for disc cell senescence during disc degeneration. Recently, melatonin is suggested to protect against disc degeneration. However, the effects of melatonin on annulus fibrosus (AF) cell senescence are not fully studied. The main purpose of this study was to investigate the effects of melatonin on AF cell senescence in an inflammatory environment and the underlying mechanism. Rat disc AF cells were cultured in a medium with tumor necrosis factor-α (TNF-α). Melatonin was added along with the medium to observe its protective effects. Compared with the control AF cells, TNF-α significantly declined cell proliferation potency and telomerase activity, elevated senescence-associated ß-galactosidase (SA-ß-Gal) activity, upregulated protein expression of senescence markers (p16 and p53), and increased reactive oxygen species (ROS) content and activity of the NF-κB pathway. However, when the TNF-α-treated AF cells were incubated with melatonin, ROS content and activity of the NF-κB pathway were decreased, and those parameters reflecting cell senescence indicated that AF cell senescence was also partly alleviated. Together, melatonin suppresses AF cell senescence through regulating the ROS/NF-κB pathway in an inflammatory environment. This study sheds a new light that melatonin may be promising to retard inflammation-caused disc degeneration.


Assuntos
Anel Fibroso/efeitos dos fármacos , Inflamação/tratamento farmacológico , Degeneração do Disco Intervertebral/tratamento farmacológico , Melatonina/farmacologia , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Anel Fibroso/metabolismo , Anel Fibroso/patologia , Antioxidantes/farmacologia , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Inflamação/metabolismo , Inflamação/patologia , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Masculino , Modelos Animais , NF-kappa B/genética , Ratos , Ratos Sprague-Dawley , Telomerase/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
13.
Vet Res ; 52(1): 110, 2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34412690

RESUMO

This study aimed to explore the mutual regulation between chicken telomerase reverse transcriptase (chTERT) and the Wnt/ß-catenin signalling pathway and its effects on cell growth and avian leukosis virus subgroup J (ALV-J) replication in LMH cells. First, LMH cells stably overexpressing the chTERT gene (LMH-chTERT cells) and corresponding control cells (LMH-NC cells) were successfully constructed with a lentiviral vector expression system. The results showed that chTERT upregulated the expression of ß-catenin, Cyclin D1, TCF4 and c-Myc. chTERT expression level and telomerase activity were increased when cells were treated with LiCl. When the cells were treated with ICG001 or IWP-2, the activity of the Wnt/ß-catenin signalling pathway was significantly inhibited, and chTERT expression and telomerase activity were also inhibited. However, when the ß-catenin gene was knocked down by small interfering RNA (siRNA), the changes in chTERT expression and telomerase activity were consistent with those in cells treated with ICG001 or IWP-2. These results indicated that chTERT and the Wnt/ß-catenin signalling pathway can be mutually regulated. Subsequently, we found that chTERT not only shortened the cell cycle to promote proliferation but also inhibited apoptosis by downregulating the expression of Caspase 3, Caspase 9 and BAX; upregulating BCL-2 and BCL-X expression; and promoting autophagy. Moreover, chTERT significantly enhanced the migration ability of LMH cells, upregulated the protein and mRNA expression of ALV-J and increased the virus titre. ALV-J replication promoted chTERT expression and telomerase activity.


Assuntos
Apoptose/genética , Vírus da Leucose Aviária/fisiologia , Proteínas Aviárias/genética , Movimento Celular , Galinhas/fisiologia , Telomerase/genética , Replicação Viral , Via de Sinalização Wnt , Animais , Leucose Aviária/patologia , Proteínas Aviárias/metabolismo , Carcinogênese , Linhagem Celular , Galinhas/genética , Doenças das Aves Domésticas/patologia , Telomerase/metabolismo
14.
Sci Rep ; 11(1): 14175, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34239032

RESUMO

Retinal pigment epithelial (RPE) cells that underlie the neurosensory retina are essential for the maintenance of photoreceptor cells and hence vision. Interactions between the RPE and their basement membrane, i.e. the inner layer of Bruch's membrane, are essential for RPE cell health and function, but the signals induced by Bruch's membrane engagement, and their contributions to RPE cell fate determination remain poorly defined. Here, we studied the functional role of the soluble complement regulator and component of Bruch's membrane, Factor H-like protein 1 (FHL-1). Human primary RPE cells adhered to FHL-1 in a manner that was eliminated by either mutagenesis of the integrin-binding RGD motif in FHL-1 or by using competing antibodies directed against the α5 and ß1 integrin subunits. These short-term experiments reveal an immediate protein-integrin interaction that were obtained from primary RPE cells and replicated using the hTERT-RPE1 cell line. Separate, longer term experiments utilising RNAseq analysis of hTERT-RPE1 cells bound to FHL-1, showed an increased expression of the heat-shock protein genes HSPA6, CRYAB, HSPA1A and HSPA1B when compared to cells bound to fibronectin (FN) or laminin (LA). Pathway analysis implicated changes in EIF2 signalling, the unfolded protein response, and mineralocorticoid receptor signalling as putative pathways. Subsequent cell survival assays using H2O2 to induce oxidative stress-induced cell death suggest hTERT-RPE1 cells had significantly greater protection when bound to FHL-1 or LA compared to plastic or FN. These data show a non-canonical role of FHL-1 in protecting RPE cells against oxidative stress and identifies a novel interaction that has implications for ocular diseases such as age-related macular degeneration.


Assuntos
Integrina alfa5beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Musculares/metabolismo , Estresse Oxidativo , Epitélio Pigmentado da Retina/metabolismo , Comunicação Celular , Morte Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas Imobilizadas/metabolismo , Ligação Proteica , Telomerase/metabolismo
15.
J Med Chem ; 64(15): 11432-11444, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34283610

RESUMO

Human (h) telomerase (TL; EC 2.7.7.49) plays a key role in sustaining cancer cells by means of elongating telomeric repeats at the 3' ends of chromosomes. Since TL-inhibitor (TI) stand-alone cancer therapy has been proven to be remarkably challenging, a polypharmacological approach represents a valid alternative. Here we consider a series of compounds able to inhibit both hTL and the tumor-associated carbonic anhydrases (CAs; EC 4.2.1.1) IX and XII. Compounds 7 and 9 suppressed hTL activity in both cell lysates and human colon cancer cell lines, and prolonged incubation with either 7 or 9 resulted in telomere shortening, cell cycle arrest, replicative senescence, and apoptosis. Enzyme kinetics showed that 7 and 9 are mixed-type inhibitors of the binding of DNA primers and deoxynucleoside triphosphate (dNTP) to the TL catalytic subunit hTERT, which is in agreement with docking experiments. Compound 9 showed antitumor activity in Colo-205 mouse xenografts and suppressed telomerase activity by telomere reduction.


Assuntos
Antineoplásicos/farmacologia , Anidrases Carbônicas/metabolismo , Inibidores Enzimáticos/farmacologia , Sulfonamidas/farmacologia , Telomerase/antagonistas & inibidores , Zidovudina/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Relação Estrutura-Atividade , Sulfonamidas/química , Telomerase/metabolismo , Células Tumorais Cultivadas , Zidovudina/química
16.
Cell Prolif ; 54(9): e13101, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34296479

RESUMO

OBJECTIVE: Osteosarcoma (OS) is characterized by high levels of the tumour-associated inflammatory microenvironment. Moreover, in approximately 60% of OS, telomere length is maintained by alternative lengthening of telomeres (ALT) pathway. Whether the ALT pathway can be exploited for OS therapeutic treatment and how the OS inflammatory microenvironment influences the anti-cancer drug effect remains unknown. Here, we examined the biological effects of TMPyP4 and cisplatin in the inflammatory microenvironment of OS cells. MATERIALS AND METHODS: Immunofluorescence in situ hybridization (IF-FISH) and C-circle experiments were used to detect the G-quadruplex and ALT activity. The redox potential of single guanine, G-quadruplex and G-quadruplex/TMPyP4 was evaluated by the lowest unoccupied molecular orbital energy (LUMO), zeta potential and cyclic voltammetry. Cell viability, flow cytometry and apoptosis, Western blot, comet assay, adhesion, transwell and scratch experiments were performed to compare the anti-tumour proliferation and migration effects of TMPyP4 and cisplatin in the inflammatory microenvironment. RESULTS: This study indicated that compared with cisplatin, TMPyP4 could induce the formation of human telomeres and FAK G-quadruplex in vitro and in vivo, and TMPyP4-treated OS cells showed fewer extrachromosomal C-circles and fewer ALT-associated promyelocytic leukaemia bodies. Consequently, the ALT activity and FAK-related cell migration were suppressed by TMPyP4. Mechanistically, the formation of G-quadruplex resulted in both lower redox potential than G within the genome and FAK transcription inhibition, and TMPyP4 could enhance this phenomenon, especially in the inflammatory microenvironment. CONCLUSIONS: Our results reveal that TMPyP4 is more suitable for OS treatment than cisplatin.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Quadruplex G/efeitos dos fármacos , Osteossarcoma/tratamento farmacológico , Porfirinas/farmacologia , Microambiente Tumoral/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Osteossarcoma/metabolismo , Telomerase/metabolismo , Telômero/efeitos dos fármacos
17.
Cell Death Dis ; 12(7): 665, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215721

RESUMO

It has been reported that growth differentiation factor 11 (GDF11) protects against myocardial ischemia/reperfusion (IR) injury, but the underlying mechanisms have not been fully clarified. Considering that GDF11 plays a role in the aging/rejuvenation process and that aging is associated with telomere shortening and cardiac dysfunction, we hypothesized that GDF11 might protect against IR injury by activating telomerase. Human plasma GDF11 levels were significantly lower in acute coronary syndrome patients than in chronic coronary syndrome patients. IR mice with myocardial overexpression GDF11 (oe-GDF11) exhibited a significantly smaller myocardial infarct size, less cardiac remodeling and dysfunction, fewer apoptotic cardiomyocytes, higher telomerase activity, longer telomeres, and higher ATP generation than IR mice treated with an adenovirus carrying a negative control plasmid. Furthermore, mitochondrial biogenesis-related proteins and some antiapoptotic proteins were significantly upregulated by oe-GDF11. These cardioprotective effects of oe-GDF11 were significantly antagonized by BIBR1532, a specific telomerase inhibitor. Similar effects of oe-GDF11 on apoptosis and mitochondrial energy biogenesis were observed in cultured neonatal rat cardiomyocytes, whereas GDF11 silencing elicited the opposite effects to oe-GDF11 in mice. We concluded that telomerase activation by GDF11 contributes to the alleviation of myocardial IR injury through enhancing mitochondrial biogenesis and suppressing cardiomyocyte apoptosis.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Mitocôndrias Cardíacas/enzimologia , Infarto do Miocárdio/enzimologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Miócitos Cardíacos/enzimologia , Biogênese de Organelas , Telomerase/metabolismo , Aminobenzoatos/farmacologia , Animais , Apoptose , Proteínas Morfogenéticas Ósseas/genética , Estudos de Casos e Controles , Células Cultivadas , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Fatores de Diferenciação de Crescimento/genética , Humanos , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/patologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Naftalenos/farmacologia , Ratos , Transdução de Sinais , Telomerase/antagonistas & inibidores
18.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206297

RESUMO

Increased cell proliferation is a hallmark of acute lymphoblastic leukemia (ALL), and genetic alterations driving clonal proliferation have been identified as prognostic factors. To evaluate replicative history and its potential prognostic value, we determined telomere length (TL) in lymphoblasts, B-, and T-lymphocytes, and measured telomerase activity (TA) in leukocytes of patients with ALL. In addition, we evaluated the potential to suppress the in vitro growth of B-ALL cells by the telomerase inhibitor imetelstat. We found a significantly lower TL in lymphoblasts (4.3 kb in pediatric and 2.3 kb in adult patients with ALL) compared to B- and T-lymphocytes (8.0 kb and 8.2 kb in pediatric, and 6.4 kb and 5.5 kb in adult patients with ALL). TA in leukocytes was 3.2 TA/C for pediatric and 0.7 TA/C for adult patients. Notably, patients with high-risk pediatric ALL had a significantly higher TA of 6.6 TA/C compared to non-high-risk patients with 2.2 TA/C. The inhibition of telomerase with imetelstat ex vivo led to significant dose-dependent apoptosis of B-ALL cells. These results suggest that TL reflects clonal expansion and indicate that elevated TA correlates with high-risk pediatric ALL. In addition, telomerase inhibition induces apoptosis of B-ALL cells cultured in vitro. TL and TA might complement established markers for the identification of patients with high-risk ALL. Moreover, TA seems to be an effective therapeutic target; hence, telomerase inhibitors, such as imetelstat, may augment standard ALL treatment.


Assuntos
Oligonucleotídeos/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Telomerase/antagonistas & inibidores , Telômero/metabolismo , Adolescente , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Biomarcadores Tumorais/análise , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Oligonucleotídeos/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatologia , Prognóstico , Telomerase/metabolismo , Homeostase do Telômero
19.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203694

RESUMO

Proper functioning of cells-their ability to divide, differentiate, and regenerate-is dictated by genomic stability. The main factors contributing to this stability are the telomeric ends that cap chromosomes. Telomere biology and telomerase activity have been of interest to scientists in various medical science fields for years, including the study of both cancer and of senescence and aging. All these processes are accompanied by telomere-length modulation. Maintaining the key levels of telomerase component (hTERT) expression and telomerase activity that provide optimal telomere length as well as some nontelomeric functions represents a promising step in advanced anti-aging strategies, especially in dermocosmetics. Some known naturally derived compounds contribute significantly to telomere and telomerase metabolism. However, before they can be safely used, it is necessary to assess their mechanisms of action and potential side effects. This paper focuses on the metabolic potential of natural compounds to modulate telomerase and telomere biology and thus prevent senescence and skin aging.


Assuntos
Envelhecimento/metabolismo , Produtos Biológicos/farmacologia , Pele/patologia , Telomerase/metabolismo , Telômero/metabolismo , Animais , Humanos , Pele/efeitos dos fármacos
20.
Int J Mol Sci ; 22(13)2021 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-34281248

RESUMO

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


Assuntos
Degeneração Macular/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Telomerase/metabolismo , Envelhecimento/metabolismo , Autofagia/fisiologia , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Humanos , Degeneração Macular/fisiopatologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/fisiologia , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais , Telomerase/fisiologia , Telômero/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
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