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1.
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
2.
J Biomed Nanotechnol ; 17(7): 1284-1292, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34446132

RESUMO

This study aimed to introduce nano-gold PCR for detection of TERT methylation, and explore the correlation between TERT methylation and prognosis of hepatocellular carcinoma (HCC). From March 2016 to March 2018, 154 HBV carriers treated in our hospital were enrolled in the study and divided into HCC (68 cases), cirrhosis (45 cases) and chronic hepatitis (CH) groups (41 cases) based on clinical disease. HCC patients were further divided into methylation (30 cases) and non-methylation (38 cases) subgroup based on methylation status of the TERT. TERT methylation of HCC specimens were 44.12% and 35.24% by nano-PCR and conventional PCR, respectively. The TERT methylation and TERT expression in HCC specimens were higher than for cirrhosis and CH specimens. A significant positive correlation was observed between TERT methylation and TERT expression. AFP, Edmondson classification, tumor size, hilar lymph node and intrahepatic metastasis, and TNM staging in the methylation group were higher than in non-methylation group. Further, overall survival and progression-free survival were significantly shorter. Nano-gold PCR is more sensitive in detecting TERT methylation. As CHB progresses, TERT methylation increases. Greater methylation of the gene is associated with worse prognosis in HCC patients.


Assuntos
Carcinoma Hepatocelular , Hepatite B Crônica , Hepatite B , Neoplasias Hepáticas , Telomerase , Carcinoma Hepatocelular/genética , Metilação de DNA , Ouro , Vírus da Hepatite B/genética , Hepatite B Crônica/genética , Humanos , Neoplasias Hepáticas/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Telomerase/genética
3.
Zhonghua Bing Li Xue Za Zhi ; 50(7): 734-739, 2021 Jul 08.
Artigo em Chinês | MEDLINE | ID: mdl-34405606

RESUMO

Objective: To analyze the clinicopathological and molecular features and prognostic implications of adult isocitrate dehydrogenase wild type (IDH-wt) diffuse gliomas. Methods: A total of 87 cases of adult IDH-wt diffuse gliomas from 2016 to 2020 in Xuanwu Hospital of Capital Medical University were retrospectively collected. The clinicopathological characteristics and prognosis were analyzed. Molecular characteristics were also analyzed using Sanger sequencing and next generation sequencing. Results: There were 53 males and 34 females, aged from 19 to 78 years (mean 53 years). Histopathologically, there were 63 (72.4%) glioblastomas, 16 (18.4%) anaplastic astrocytomas, six (6.9%) diffuse astrocytomas, and one (1.1%) each of anaplastic oligodendrocytoma, and anaplastic oligodendroglioma. Common molecular genetic changes in IDH-wt gliomas included TERT promoter mutation which was found in 60 cases (69.0%); MGMT promoter methylation in 43 cases (49.4%); EGFR mutation in 38 cases (43.7%); PTEN mutation in 35 cases (40.2%) and TP53 mutation in 32 cases (36.8%). In addition, PDGFRA mutation was detected in 17 cases (19.5%), CDK4 amplification in 15 cases (17.2%) and MDM2 amplification in 11 cases (12.6%). In IDH-wt diffuse gliomas, there was no significant difference in the overall survival between TERT promoter, EGFR, PTEN, TP53, PDGFRA, CDK4, MDM2 mutations and the wild-type, since these gene mutations could co-occur in any case (P>0.05). Also there was no significant difference in the overall survival between the WHO grade Ⅱ/Ⅲ gliomas and glioblastoma patients with these gene mutations (P>0.05). Conclusions: TERT promoter, EGFR, PTEN, TP53, PDGFRA, CDK4 and MDM2 gene mutations are common molecular genetic changes in adult IDH-wt gliomas, and are associated with poor prognosis. It is suggested that these genes are potentially useful for predicting the prognosis and should be tested in adult IDH-wt gliomas.


Assuntos
Neoplasias Encefálicas , Glioma , Telomerase , Adulto , Neoplasias Encefálicas/genética , Feminino , Glioma/genética , Humanos , Isocitrato Desidrogenase/genética , Masculino , Biologia Molecular , Mutação , Prognóstico , Estudos Retrospectivos , Telomerase/genética
4.
Mol Biol (Mosk) ; 55(4): 606-616, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34432778

RESUMO

Tumor-specific promoters and cis-regulatory genetic elements are used for transcriptional control of therapeutic transgene expression in cancer gene therapy. HRE (hypoxia response element) and ARE (anti-oxidant response elements) cis-regulatory elements are targets for HIF1 and Nrf2 transcriptional factors, respectively, and mediate activation of gene transcription in a response to hypoxia and oxidative stress, characteristic of most solid tumors. Due to these features HREs and AREs are used in genetic constructs for cancer gene therapy to provide tumor-specific therapeutic transgene expression or replication of oncolytic adenovi-ruses. In this work on the basis of the tumor-specific promoter hTERT we have constructed hybrid promoters carrying combinations of HRE and ARE. We showed that upon imitation of hypoxia in human lung cancer cell lines the activity of the hybrid promoter HRE-ARE-hTERT is substantially higher compared to promoters carrying only ARE or HRE. Using in vitro suicide cancer gene therapy with the CD: UPRT/5-FC (cytosine deaminase; uracil phosphoribosyl transferase/5-fluorocytosine) enzyme-prodrug system as a model we showed an enhancement of the cytotoxic effect on human lung cancer cells upon imitation of hypoxia when cytosine deaminase: uracil phosphoribosyl transferase was expressed under the control of the HRE-ARE-hTERT promoter compared to HRE-hTERT and ARE-hTERT promoters. The novel hybrid promoter HRE-ARE-hTERT could be used for transcriptional targeting of therapeutic transgene expression or oncolytic adenovirus replication upon development of novel anti-cancer gene therapeutics.


Assuntos
Neoplasias Pulmonares , Telomerase , Adenoviridae , Carcinógenos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Telomerase/genética , Replicação Viral
5.
Ecotoxicol Environ Saf ; 223: 112558, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34333383

RESUMO

Telomeres are functional complexes at the ends of linear chromosomes, and telomerase aids in their maintenance and replication. Additionally, accumulating evidence suggests that telomerase-associated protein 1 (TEP1) is a component of the telomerase ribonucleoprotein complex and is responsible for catalyzing the addition of new synthetic telomere sequences to chromosome ends. In our previous study, we found that genetic variants of the TERT gene participated in the regulation of telomere length. Exposure to particulate matter, environmental pollutants, oxidative stress, and pesticides is associated with shortening of telomere length. However, it is unknown whether genetic variants in the TEP1 gene may affect telomere length (TL) in polycyclic aromatic hydrocarbon (PAH)-exposed workers. Therefore, we measured the peripheral leukocyte TL and genotyped the polymorphism loci in the TEP1 gene among 544 PAH-exposed workers and 238 healthy controls. Covariance analysis showed that the individuals carrying TEP1 rs1760903 CC and TEP1 rs1760904 TT had longer TL in the control group (P < 0.05). In the generalized linear model, we found that rs1760903 CC was a protective factor against TL shortening, and PAH exposure could promote telomere shortening (P < 0.05). Thus, this study reinforces the roles of environmental factors and genetic variations in telomere damage, and provides a theoretical foundation for the early detection of susceptible populations and the establishment of occupational standards.


Assuntos
Poluentes Ambientais , Hidrocarbonetos Policíclicos Aromáticos , Telomerase , Humanos , Hidrocarbonetos Policíclicos Aromáticos/análise , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Telomerase/genética , Telômero/genética , Encurtamento do Telômero
6.
Ageing Res Rev ; 70: 101411, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34284150

RESUMO

Telomeres protect genomic stability and shortening is one of the hallmarks of ageing. Telomerase reverse transcriptase (TERT) is the major protein component of telomerase, which elongates telomeres. Given that short telomeres are linked to a host of chronic diseases and the therapeutic potential of telomerase-based therapies as treatments and a strategy to extend lifespan, lifestyle factors that increase TERT gene expression and telomerase activity could attenuate telomere attrition and contribute to healthy biological ageing. Physical activity and maximal aerobic fitness are associated with telomere maintenance, yet the molecular mechanisms remain unclear. Therefore, the purpose of this systematic review and meta-analysis was to identify the influence of a single bout of exercise and long-term exercise training on TERT expression and telomerase activity. A search of human and rodent trials using the PubMed, Scopus, Science Direct and Embase databases was performed. Based on findings from the identified and eligible trials, both a single bout of exercise (n; standardised mean difference [95%CI]: 5; SMD: 1.19 [0.41-1.97], p = 0.003) and long-term exercise training (10; 0.31 [0.03-0.60], p = 0.03) up-regulates TERT and telomerase activity in non-cancerous somatic cells. As human and rodent studies were included in the meta-analyses both exhibited heterogeneity (I2 = 55-87%, p < 0.05). Endurance athletes also exhibited increased leukocyte TERT and telomerase activity compared to their inactive counterparts. These findings suggest exercise training as an inexpensive lifestyle factor that increases TERT expression and telomerase activity. Regular exercise training could attenuate telomere attrition through a telomerase-dependent mechanism and ultimately extend health-span and longevity.


Assuntos
Telomerase , Exercício Físico , Expressão Gênica , Telomerase/genética , Telomerase/metabolismo , Telômero/metabolismo , Encurtamento do Telômero
7.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209079

RESUMO

Although hepatitis B virus (HBV) integration into the cellular genome is well known in HCC (hepatocellular carcinoma) patients, its biological role still remains uncertain. This study investigated the patterns of HBV integration and correlated them with TERT (telomerase reverse transcriptase) alterations in paired tumor and non-tumor tissues. Compared to those in non-tumors, tumoral integrations occurred less frequently but with higher read counts and were more preferentially observed in genic regions with significant enrichment of integration into promoters. In HBV-related tumors, TERT promoter was identified as the most frequent site (38.5% (10/26)) of HBV integration. TERT promoter mutation was observed only in tumors (24.2% (8/33)), but not in non-tumors. Only 3.00% (34/1133) of HBV integration sites were shared between tumors and non-tumors. Within the HBV genome, HBV breakpoints were distributed preferentially in the 3' end of HBx, with more tumoral integrations detected in the preS/S region. The major genes that were recurrently affected by HBV integration included TERT and MLL4 for tumors and FN1 for non-tumors. Functional enrichment analysis of tumoral genes with integrations showed enrichment of cancer-associated genes. The patterns and functions of HBV integration are distinct between tumors and non-tumors. Tumoral integration is often enriched into both human-virus regions with oncogenic regulatory function. The characteristic genomic features of HBV integration together with TERT alteration may dysregulate the affected gene function, thereby contributing to hepatocarcinogenesis.


Assuntos
Carcinoma Hepatocelular/virologia , Vírus da Hepatite B/fisiologia , Hepatite B/genética , Neoplasias Hepáticas/virologia , Mutação , Telomerase/genética , Adulto , Idoso , Carcinoma Hepatocelular/genética , Estudos de Casos e Controles , DNA Viral/genética , Feminino , Fibronectinas/genética , Hepatite B/complicações , Histona-Lisina N-Metiltransferase/genética , Humanos , Neoplasias Hepáticas/genética , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Transativadores/genética , Proteínas Virais Reguladoras e Acessórias/genética , Integração Viral
8.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208629

RESUMO

The vast majority of the genetic variants (mainly SNPs) associated with various human traits and diseases map to a noncoding part of the genome and are enriched in its regulatory compartment, suggesting that many causal variants may affect gene expression. The leading mechanism of action of these SNPs consists in the alterations in the transcription factor binding via creation or disruption of transcription factor binding sites (TFBSs) or some change in the affinity of these regulatory proteins to their cognate sites. In this review, we first focus on the history of the discovery of regulatory SNPs (rSNPs) and systematized description of the existing methodical approaches to their study. Then, we brief the recent comprehensive examples of rSNPs studied from the discovery of the changes in the TFBS sequence as a result of a nucleotide substitution to identification of its effect on the target gene expression and, eventually, to phenotype. We also describe state-of-the-art genome-wide approaches to identification of regulatory variants, including both making molecular sense of genome-wide association studies (GWAS) and the alternative approaches the primary goal of which is to determine the functionality of genetic variants. Among these approaches, special attention is paid to expression quantitative trait loci (eQTLs) analysis and the search for allele-specific events in RNA-seq (ASE events) as well as in ChIP-seq, DNase-seq, and ATAC-seq (ASB events) data.


Assuntos
Sítios de Ligação , Suscetibilidade a Doenças , Regulação da Expressão Gênica , Herança Multifatorial , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/metabolismo , Alelos , Predisposição Genética para Doença , Genoma Humano , Estudo de Associação Genômica Ampla , Genômica/métodos , Humanos , Locos de Características Quantitativas , Telomerase/genética
9.
Aging (Albany NY) ; 13(13): 16957-16973, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253690

RESUMO

Many aging related diseases such as cancer implicate the myofibroblast in disease progression. Furthermore genesis of the myofibroblast is associated with manifestation of cellular senescence of unclear significance. In this study we investigated the role of a common regulator, namely telomerase reverse transcriptase (TERT), in order to evaluate the potential significance of this association between both processes. We analyzed the effects of TERT overexpression or deficiency on expression of CDKN2A and ACTA2 as indicators of senescence and differentiation, respectively. We assess binding of TERT or YB-1, a repressor of both genes, to their promoters. TERT repressed both CDKN2A and ACTA2 expression, and abolished stress-induced expression of both genes. Conversely, TERT deficiency enhanced their expression. Altering CDKN2A expression had no effect on ACTA2 expression. Both TERT and YB-1 were shown to bind the CDKN2A promoter but only YB-1 was shown to bind the ACTA2 promoter. TERT overexpression inhibited CDKN2A promoter activity while stimulating YB-1 expression and activation to repress ACTA2 gene. TERT repressed myofibroblast differentiation and senescence via distinct mechanisms. The latter was associated with TERT binding to the CDKN2A promoter, but not to the ACTA2 promoter, which may require interaction with co-factors such as YB-1.


Assuntos
Diferenciação Celular/fisiologia , Senescência Celular/fisiologia , Miofibroblastos/fisiologia , Telomerase/fisiologia , Actinas/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Masculino , Regiões Promotoras Genéticas , RNA Interferente Pequeno , Telomerase/biossíntese , Telomerase/genética
10.
Nucleic Acids Res ; 49(13): 7680-7694, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34181710

RESUMO

The enormous sequence heterogeneity of telomerase RNA (TR) subunits has thus far complicated their characterization in a wider phylogenetic range. Our recent finding that land plant TRs are, similarly to known ciliate TRs, transcribed by RNA polymerase III and under the control of the type-3 promoter, allowed us to design a novel strategy to characterize TRs in early diverging Viridiplantae taxa, as well as in ciliates and other Diaphoretickes lineages. Starting with the characterization of the upstream sequence element of the type 3 promoter that is conserved in a number of small nuclear RNAs, and the expected minimum TR template region as search features, we identified candidate TRs in selected Diaphoretickes genomes. Homologous TRs were then used to build covariance models to identify TRs in more distant species. Transcripts of the identified TRs were confirmed by transcriptomic data, RT-PCR and Northern hybridization. A templating role for one of our candidates was validated in Physcomitrium patens. Analysis of secondary structure demonstrated a deep conservation of motifs (pseudoknot and template boundary element) observed in all published TRs. These results elucidate the evolution of the earliest eukaryotic TRs, linking the common origin of TRs across Diaphoretickes, and underlying evolutionary transitions in telomere repeats.


Assuntos
Evolução Molecular , RNA de Plantas/química , RNA de Plantas/genética , RNA/química , RNA/genética , Telomerase/química , Telomerase/genética , Mutação , Conformação de Ácido Nucleico , RNA/biossíntese , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , RNA de Plantas/biossíntese , Alinhamento de Sequência , Telomerase/biossíntese , Telômero/química , Transcrição Genética , Transcriptoma , Viridiplantae/genética
11.
J Exp Biol ; 224(12)2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34142138

RESUMO

In wild animals, telomere attrition during early development has been linked with several fitness disadvantages throughout life. Telomerase enzyme can elongate telomeres, but it is generally assumed that its activity is suppressed in most somatic tissues upon birth. However, recent evidence suggests that this may not be the case for long-lived bird species. We have therefore investigated whether telomerase activity is maintained during the postnatal growth period in a wild yellow-legged gull (Larus michahellis) population. Our results indicate that telomerase activity is not negligible in the blood cells, but activity levels sharply decline from hatching to fledging following a similar pattern to the reduction observed in telomere length. Our results further suggest that the observed variation in telomere length may be the result of a negative effect of fast growth on telomerase activity, thus providing a new mechanism through which growth rates may affect telomere dynamics and potentially life-history trajectories.


Assuntos
Charadriiformes/crescimento & desenvolvimento , Telomerase , Telômero , Animais , Animais Selvagens , Telomerase/genética , Telômero/genética , Encurtamento do Telômero
12.
Nat Commun ; 12(1): 3308, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083519

RESUMO

The spatial partitioning of the transcriptome in the cell is an important form of gene-expression regulation. Here, we address how intron retention influences the spatio-temporal dynamics of transcripts from two clinically relevant genes: TERT (Telomerase Reverse Transcriptase) pre-mRNA and TUG1 (Taurine-Upregulated Gene 1) lncRNA. Single molecule RNA FISH reveals that nuclear TERT transcripts uniformly and robustly retain specific introns. Our data suggest that the splicing of TERT retained introns occurs during mitosis. In contrast, TUG1 has a bimodal distribution of fully spliced cytoplasmic and intron-retained nuclear transcripts. We further test the functionality of intron-retention events using RNA-targeting thiomorpholino antisense oligonucleotides to block intron excision. We show that intron retention is the driving force for the nuclear compartmentalization of these RNAs. For both RNAs, altering this splicing-driven subcellular distribution has significant effects on cell viability. Together, these findings show that stable retention of specific introns can orchestrate spatial compartmentalization of these RNAs within the cell. This process reveals that modulating RNA localization via targeted intron retention can be utilized for RNA-based therapies.


Assuntos
Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Telomerase/genética , Animais , Compartimento Celular , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Células HeLa , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Íntrons , Camundongos , Mitose , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Estabilidade de RNA , Especificidade da Espécie
13.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070219

RESUMO

Age-associated decline in oocyte quality is one of the dominant factors of low fertility. Aging alters several key processes, such as telomere lengthening, cell senescence, and cellular longevity of granulosa cells surrounding oocyte. To investigate the age-dependent molecular changes, we examined the expression, localization, and correlation of telomerase reverse transcriptase (TERT) and ß-Klotho (KLB) in bovine granulosa cells, oocytes, and early embryos during the aging process. Herein, cumulus-oocyte complexes (COCs) obtained from aged cows (>120 months) via ovum pick-up (OPU) showed reduced expression of ß-Klotho and its co-receptor fibroblast growth factor receptor 1 (FGFR1). TERT plasmid injection into pronuclear zygotes not only markedly enhanced day-8 blastocysts' development competence (39.1 ± 0.8%) compared to the control (31.1 ± 0.5%) and D-galactose (17.9 ± 1.0%) treatment groups but also enhanced KLB and FGFR1 expression. In addition, plasmid-injected zygotes displayed a considerable enhancement in blastocyst quality and implantation potential. Cycloastragenol (CAG), an extract of saponins, stimulates telomerase enzymes and enhances KLB expression and alleviates age-related deterioration in cultured primary bovine granulosa cells. In conclusion, telomerase activation or constitutive expression will increase KLB expression and activate the FGFR1/ß-Klotho pathway in bovine granulosa cells and early embryos, inhibiting age-related malfunctioning.


Assuntos
Blastocisto/metabolismo , Bovinos/embriologia , Bovinos/genética , Proteínas de Membrana/genética , Prenhez/genética , Telomerase/genética , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Bovinos/fisiologia , Células Cultivadas , Fase de Clivagem do Zigoto/metabolismo , Implantação do Embrião/genética , Implantação do Embrião/fisiologia , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Feminino , Expressão Gênica , Células da Granulosa/metabolismo , Proteínas de Membrana/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Gravidez , Prenhez/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética
14.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071371

RESUMO

The aim of this study was exploration of the genetic background of conjunctival melanoma (CM) and correlation with recurrent and metastatic disease. Twenty-eight CM from the Rotterdam Ocular Melanoma Study group were collected and DNA was isolated from the formalin-fixed paraffin embedded tissue. Targeted next-generation sequencing was performed using a panel covering GNAQ, GNA11, EIF1AX, BAP1, BRAF, NRAS, c-KIT, PTEN, SF3B1, and TERT genes. Recurrences and metastasis were present in eight (29%) and nine (32%) CM cases, respectively. TERT promoter mutations were most common (54%), but BRAF (46%), NRAS (21%), BAP1 (18%), PTEN (14%), c-KIT (7%), and SF3B1 (4%) mutations were also observed. No mutations in GNAQ, GNA11, and EIF1AX were found. None of the mutations was significantly associated with recurrent disease. Presence of a TERT promoter mutation was associated with metastatic disease (p-value = 0.008). Based on our molecular findings, CM comprises a separate entity within melanoma, although there are overlapping molecular features with uveal melanoma, such as the presence of BAP1 and SF3B1 mutations. This warrants careful interpretation of molecular data, in the light of clinical findings. About three quarter of CM contain drug-targetable mutations, and TERT promoter mutations are correlated to metastatic disease in CM.


Assuntos
Neoplasias da Túnica Conjuntiva/genética , Melanoma/genética , Mutação , Regiões Promotoras Genéticas/genética , Telomerase/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Túnica Conjuntiva/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Estimativa de Kaplan-Meier , Masculino , Melanoma/patologia , Pessoa de Meia-Idade , Biologia Molecular , Recidiva Local de Neoplasia , Prognóstico , Adulto Jovem
16.
Cancer Sci ; 112(8): 3293-3301, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34036669

RESUMO

Oncolytic virus therapy has emerged as a promising treatment option against cancer. To date, oncolytic viruses have been developed for malignant tumors, but the need for this new therapeutic modality also exists for benign and slow-growing tumors. G47∆ is an oncolytic herpes simplex virus type 1 (HSV-1) with an enhanced replication capability highly selective to tumor cells due to genetically engineered, triple mutations in the γ34.5, ICP6 and α47 genes. To create a powerful, but safe oncolytic HSV-1 that replicates efficiently in tumors regardless of growth speed, we used a bacterial artificial chromosome system that allows a desired promoter to regulate the expression of the ICP6 gene in the G47∆ backbone. Restoration of the ICP6 function in a tumor-specific manner using the hTERT promoter led to a highly capable oncolytic HSV-1. T-hTERT was more efficacious in the slow-growing OS-RC-2 and DU145 tumors than the control viruses, while retaining a high efficacy in the fast-growing U87MG tumors. The safety features are also retained, as T-hTERT proved safe when inoculated into the brain of HSV-1 sensitive A/J mice. This new technology should facilitate the use of oncolytic HSV-1 for all tumors irrespective of growth speed.


Assuntos
Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/genética , Telomerase/genética , Proteínas Virais/genética , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Cromossomos Artificiais Bacterianos/genética , Feminino , Glioblastoma/genética , Humanos , Camundongos , Mutação , Terapia Viral Oncolítica , Vírus Oncolíticos/fisiologia , Regiões Promotoras Genéticas , Células Vero , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Sci Data ; 8(1): 126, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33963195

RESUMO

Cellular immortalization enables indefinite expansion of cultured cells. However, the process of cell immortalization sometimes changes the original nature of primary cells. In this study, we performed expression profiling of poly A-tailed RNA from primary and immortalized corneal epithelial cells expressing Simian virus 40 large T antigen (SV40) or the combination of mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomere reverse transcriptase (TERT). Furthermore, we studied the expression profile of SV40 cells cultured in medium with or without serum. The profiling of whole expression pattern revealed that immortalized corneal epithelial cells with SV40 showed a distinct expression pattern from wild-type cells regardless of the presence or absence of serum, while corneal epithelial cells with combinatorial expression showed an expression pattern relatively closer to that of wild-type cells.


Assuntos
Antígenos Transformantes de Poliomavirus/genética , Epitélio Corneano/citologia , Epitélio Corneano/metabolismo , Transcriptoma , Ciclina D1/genética , Quinase 4 Dependente de Ciclina/genética , Células Epiteliais/metabolismo , Humanos , Cultura Primária de Células , Proteólise , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Telomerase/genética , Ubiquitina
19.
Nat Commun ; 12(1): 2584, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972520

RESUMO

Alternative Lengthening of Telomeres (ALT) is a telomere maintenance pathway utilised in 15% of cancers. ALT cancers are strongly associated with inactivating mutations in ATRX; yet loss of ATRX alone is insufficient to trigger ALT, suggesting that additional cooperating factors are involved. We identify H3.3G34R and IDH1/2 mutations as two such factors in ATRX-mutated glioblastomas. Both mutations are capable of inactivating histone demethylases, and we identify KDM4B as the key demethylase inactivated in ALT. Mouse embryonic stem cells inactivated for ATRX, TP53, TERT and KDM4B (KDM4B knockout or H3.3G34R) show characteristic features of ALT. Conversely, KDM4B over-expression in ALT cancer cells abrogates ALT-associated features. In this work, we demonstrate that inactivation of KDM4B, through H3.3G34R or IDH1/2 mutations, acts in tandem with ATRX mutations to promote ALT in glioblastomas.


Assuntos
Células-Tronco Embrionárias/metabolismo , Glioblastoma/genética , Histonas/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Homeostase do Telômero/genética , Proteína Nuclear Ligada ao X/genética , Adulto , Animais , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Replicação do DNA/genética , Técnicas de Inativação de Genes , Glioblastoma/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histonas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Isocitrato Desidrogenase/genética , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Mutação , Transdução de Sinais/genética , Telomerase/genética , Telomerase/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima
20.
Life Sci ; 277: 119621, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34004255

RESUMO

Effective telomerase-molecular targeted cancer therapy might be a promising approach for the efficient treatment of ovarian cancer. Therefore, folate-functionalized PLGA nanoparticles (NPs) were co-loaded with hTERT siRNA, Wortmannin (Wtmn), as a potent PI3K inhibitor, and magnetic nanoparticle (MNPs) as a theranostic agent to gain a multifunctional NPs for targeted drug delivery as well as molecular targeted therapy. 1HNMR, FTIR, DLS, FE-SEM and TEM were applied to characterize the synthesized NPs. In vitro discharge pattern for siRNA and Wtmn from the dual drug-loaded NPs showed an early fast release followed by a constant release up to 200 h. According to the MRI analysis, by increasing the concentration of Fe3O4 in NPs, the weaker T2 signal intensity was enhanced, and a considerable contrast was detected in the MRI images. MTT assay and median-effect analysis showed that the Wtmn/siRNA-loaded MNPs-PLGA-F2 NPs display the most synergistic cytotoxicity on the SKOV-3 ovarian cancer cells. Moreover, the Wtmn/siRNA-loaded MNPs-PLGA-FA NPs could significantly reduce the expression of hTERT, AKT, and p-AKT than the single drug-encapsulated NPs (P < 0.05). Taken together, the findings showed that the multifunctional NPs relying on combinatorial therapy might have considerable potential for effective telomerase-molecular targeted therapy of ovarian cancer.


Assuntos
Sistemas de Liberação de Medicamentos , Nanopartículas Magnéticas de Óxido de Ferro/química , Terapia de Alvo Molecular , Neoplasias Ovarianas/tratamento farmacológico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , RNA Interferente Pequeno/genética , Telomerase/antagonistas & inibidores , Wortmanina/farmacologia , Feminino , Ácido Fólico/química , Humanos , Imunossupressores/farmacologia , Nanopartículas/química , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Telomerase/genética , Células Tumorais Cultivadas
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