RESUMO
Aberrant activation of telomerase in human cancer is achieved by various alterations within the TERT promoter, including cancer-specific DNA hypermethylation of the TERT hypermethylated oncological region (THOR). However, the impact of allele-specific DNA methylation within the TERT promoter on gene transcription remains incompletely understood. Using allele-specific next-generation sequencing, we screened a large cohort of normal and tumor tissues (n = 652) from 10 cancer types and identified that differential allelic methylation (DAM) of THOR is restricted to cancerous tissue and commonly observed in major cancer types. THOR-DAM was more common in adult cancers, which develop through multiple stages over time, than in childhood brain tumors. Furthermore, THOR-DAM was especially enriched in tumors harboring the activating TERT promoter mutations (TPMs). Functional studies revealed that allele-specific gene expression of TERT requires hypomethylation of the core promoter, both in TPM and TERT WT cancers. However, the expressing allele with hypomethylated core TERT promoter universally exhibits hypermethylation of THOR, while the nonexpressing alleles are either hypermethylated or hypomethylated throughout the promoter. Together, our findings suggest a dual role for allele-specific DNA methylation within the TERT promoter in the regulation of TERT expression in cancer.
Assuntos
Metilação de DNA , DNA de Neoplasias/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Neoplasias/metabolismo , Regiões Promotoras Genéticas , Telomerase/biossíntese , DNA de Neoplasias/genética , Humanos , Proteínas de Neoplasias/genética , Neoplasias/genética , Telomerase/genéticaRESUMO
Electrospun nanofibers (NFs) were utilized to realize the dual-stage release of curcumin (Curc) to fully support the attachment, viability and proliferation of adipose-derived stem cells (hADSCs) with a delay in cellular senescence. For this purpose, both free Curc and Curc-loaded mesoporous silica nanoparticles (Curc@MSNs) were integrated into the electrospun polycaprolactone/gelatin (PCL/GEL) nanofibrous scaffolds and characterized via FTIR, BET, FE-SEM and TEM. In vitro drug release results demonstrated strong dual stage-discharge of Curc from the Curc/Curc@MSNs-NFs. Because of the combination of initial rapid release and late extended drug release, hADSCs cultured on the Curc/Curc@MSNs-NFs showed the greatest adhesion, metabolic activity and proliferation rate with a fibroblastic phenotype after 28 days of culture. Besides, a significant reduction in senescence-associated lysosomal α-L-fucosidase (SA-α-Fuc) expression and activity was also measured in hADSCs cultured on the Curc/Curc@MSNs-NFs. Moreover, not only the expression of hTERT in mRNA and protein levels was considerably increased in hADSCs seeded on the Curc/Curc@MSNs-NFs, but also the telomerase activity and telomere length were significantly enhanced in the scaffolds compared to the other types of scaffolds and control group. These results uncovered the potential of the two-stage discharge profile of Curc from Curc/Curc@MSNs-NFs to provide the biofunctionality of long-term cultured hADSCs for efficient stem cell-based regenerative therapies.
Assuntos
Tecido Adiposo/citologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Senescência Celular , Curcumina/administração & dosagem , Liberação Controlada de Fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Nanofibras/química , Nanopartículas/química , Animais , Adesão Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Curcumina/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Gelatina/química , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Poliésteres/química , RNA Mensageiro/biossíntese , Dióxido de Silício/química , Telomerase/biossínteseRESUMO
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/genéticaRESUMO
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 Gênica , Transcriptoma , Viridiplantae/genéticaRESUMO
BACKGROUND: Immortalization of primary prostate epithelial cells (PrEC) with just hTERT expression is particularly inefficient in the absence of DNA tumor viral proteins or p16INK4A knockdown. MATERIALS AND METHODS: Here, we describe the establishment of immortalized normal prostate epithelial cell line models using CRISPR technology to inactivate the CDKN2A locus concomitantly with ectopic expression of an hTERT transgene. RESULTS: Using this approach, we have obtained immortal cell clones that exhibit fundamental characteristics of normal cells, including diploid genomes, near normal karyotypes, normal p53 and pRB cell responses, the ability to form non-invasive spheroids, and a non-transformed phenotype. Based on marker expression, these clones are of basal cell origin. CONCLUSIONS: Use of this approach resulted in the immortalization of independent clones of PrEC that retained normal characteristics, were stable, and non-transformed. Thus, this approach could be used for the immortalization of normal primary prostate cells. This technique could also be useful for establishing cell lines from prostate tumor tissues of different tumor grades and/or from patients of diverse ethnicities to generate cell line models that facilitate the study of the molecular basis of disease disparity.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , RNA Neoplásico/genética , Telomerase/genética , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Telomerase/biossínteseRESUMO
Long non-coding RNAs can often fold into different conformations. Telomerase RNA, an essential component of the telomerase ribonucleoprotein (RNP) enzyme, must fold into a defined structure to fulfill its function with the protein catalytic subunit (TERT) and other accessory factors. However, the mechanism by which the correct folding of telomerase RNA is warranted in a cell is still unknown. Here we show that La-related protein Pof8 specifically recognizes the conserved pseudoknot region of telomerase RNA and instructs the binding of the Lsm2-8 complex to its mature 3' end, thus selectively protecting the correctly folded RNA from exonucleolytic degradation. In the absence of Pof8, TERT assembles with misfolded RNA and produces little telomerase activity. Therefore, Pof8 plays a key role in telomerase RNA folding quality control, ensuring that TERT only assembles with functional telomerase RNA to form active telomerase. Our finding reveals a mechanism for non-coding RNA folding quality control.
Assuntos
Dobramento de RNA , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA/genética , RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Telomerase/biossíntese , Domínio Catalítico , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Controle de Qualidade , RNA/química , Proteínas com Motivo de Reconhecimento de RNA/química , Proteínas com Motivo de Reconhecimento de RNA/genética , RNA Fúngico , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , Motivos de Ligação ao RNA , Ribonucleoproteínas Nucleares Pequenas/química , Schizosaccharomyces/química , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Telomerase/química , Telomerase/genética , Telomerase/metabolismoRESUMO
Telomerase is a cancer promoting ribonucleoprotein complex and is a potential therapeutic target for cancer. In this study, the effects of telomerase downregulation on the whole cell proteome were investigated. Understanding how the effect of downregulation on the whole proteome profile will generate a greater understanding of the possible roles played by telomerase in cancer. Downregulation was achieved by RNA interference (RNAi), targeting the telomerase reverse transcriptase (TERT) subunits of telomerase. Transfection of TERT siRNA downregulates TERT gene expression and induced downregulation of telomerase activity. Investigation of the effect of silencing TERT in telomerase was further validated through proteomic analysis by performing 2-dimension electrophoresis (2DE) coupled with MALDI-TOF/TOF. 12 protein spots in HeLa cells were reported to be significantly differentially expressed with 11 of them were upregulated and 1 downregulated. Through STRING analysis, differentially expressed proteins demonstrated strong associations with endoplasmic reticulum stress marker and mitochondrial energy production marker. In conclusions, the result exhibited novel integrated proteomic response involving endoplasmic reticulum stress and mitochondrial energy production in response to the TERT downregulation in cervical cancer cells.
Assuntos
Regulação para Baixo , Estresse do Retículo Endoplasmático , Metabolismo Energético , Mitocôndrias/metabolismo , Interferência de RNA , Telomerase/biossíntese , Células HeLa , Humanos , Telomerase/genéticaRESUMO
The human telomerase gene (hTERT) is repressed in most somatic cells. How transcription factors activate the hTERT promoter in its repressive chromatin environment is unknown. Here, we report that the ETS family protein ETS variant transcription factor 5 (ETV5) mediates epidermal growth factor (EGF)-induced hTERT expression in MCF10A cells. This activation required MYC proto-oncogene bHLH transcription factor (c-Myc) and depended on the chromatin state of the hTERT promoter. Using chromatinized bacterial artificial chromosome (BAC) reporters in human fibroblasts, we found that ETV5 and c-Myc/MYC-associated factor X (MAX) synergistically activate the hTERT promoter via two identical, but inverted, composite Ets/E-box motifs enclosing the core promoter. Mutations of Ets or E-box sites in either DNA motif abolished the activation and reduced or eliminated the synergism. ETV5 and c-Myc facilitated each other's binding to the hTERT promoter. ETV5 bound to the hTERT promoter in both telomerase-negative and -positive cells, but it activated the repressed hTERT promoter and altered histone modifications only in telomerase-negative cells. The synergistic ETV5/c-Myc activation disappeared when hTERT promoter repression became relieved because of the loss of distal regulatory elements in chimeric human/mouse BAC reporters. Our results suggest that the binding of c-Myc and ETS family proteins to the Ets/E-box motifs derepresses the hTERT promoter by inducing an active promoter configuration, providing a mechanistic insight into hTERT activation during tumorigenesis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Elementos de Resposta , Telomerase/biossíntese , Fatores de Transcrição/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Proteínas de Ligação a DNA/genética , Humanos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/genética , Telomerase/genética , Fatores de Transcrição/genéticaRESUMO
Transcriptional reactivation of telomerase catalytic subunit (TERT) is a frequent hallmark of cancer, occurring in 90% of human malignancies. However, specific mechanisms driving TERT reactivation remain obscure for many tumor types and in particular gastric cancer (GC), a leading cause of global cancer mortality. Here, through comprehensive genomic and epigenomic analysis of primary GCs and GC cell lines, we identified the transcription factor early B cell factor 1 (EBF1) as a TERT transcriptional repressor and inactivation of EBF1 function as a major cause of TERT upregulation. Abolishment of EBF1 function occurs through 3 distinct (epi)genomic mechanisms. First, EBF1 is epigenetically silenced via DNA methyltransferase, polycomb-repressive complex 2 (PRC2), and histone deacetylase activity in GCs. Second, recurrent, somatic, and heterozygous EBF1 DNA-binding domain mutations result in the production of dominant-negative EBF1 isoforms. Third, more rarely, genomic deletions and rearrangements proximal to the TERT promoter remobilize or abolish EBF1-binding sites, derepressing TERT and leading to high TERT expression. EBF1 is also functionally required for various malignant phenotypes in vitro and in vivo, highlighting its importance for GC development. These results indicate that multimodal genomic and epigenomic alterations underpin TERT reactivation in GC, converging on transcriptional repressors such as EBF1.
Assuntos
Epigenômica , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Neoplasias Gástricas/metabolismo , Telomerase/biossíntese , Transativadores/metabolismo , Linhagem Celular Tumoral , Humanos , Mutação , Proteínas de Neoplasias/genética , Elementos de Resposta , Neoplasias Gástricas/genética , Telomerase/genética , Transativadores/genéticaRESUMO
Background: Telomerase reverse transcriptase (TERT) promoter mutations play a role in carcinogenesis and are found in both tumors and cancer cell lines. TERT promoter methylation, transcription factor binding, chromatin remodeling, and alternative splicing are also known to play an integral role in TERT regulation. Methods: Using nanopore Cas9 targeted sequencing, we characterized allele-specific methylation in thyroid cancer cell lines heterozygous for the TERT promoter mutation. Furthermore, using chromatin immunoprecipitation followed by Sanger sequencing, we probed allele-specific binding of the transcription factors GABPA (GA binding protein transcription factor subunit alpha) and MYC, as well as the chromatin marks H3K4me3 and H3K27me3. Finally, using coding single nucleotide polymorphisms and the long-read sequencing, we examined complementary DNA for monoallelic expression (MAE). Results: We found the mutant TERT promoter allele to be significantly less methylated than wild type, while more methylated in the gene body in heterozygous TERT mutant cell lines. We demonstrated that the transcriptional activators GABPA and MYC bind only to the mutant TERT allele. In addition, the activating and repressive chromatin marks H3K4me3 and H3K27me3, respectively, bind mutant and wild-type alleles exclusively. Finally, in heterozygous mutant cell lines, TERT exhibits MAE from the mutant allele only. Conclusions: In summary, by employing new long-read sequencing methods, we were able to definitively demonstrate allele-specific DNA methylation, histone modifications, transcription factor binding, and the resulting monoallelic transcription in cell lines with heterozygous TERT mutations.
Assuntos
Alelos , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas , Telomerase/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Proteína 9 Associada à CRISPR , Linhagem Celular Tumoral , Cromatina/metabolismo , Ilhas de CpG , Metilação de DNA , DNA Complementar/metabolismo , Fator de Transcrição de Proteínas de Ligação GA/genética , Heterozigoto , Histonas/metabolismo , Humanos , Imunoprecipitação , Mutação , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Telomerase/biossíntese , Fatores de Transcrição/metabolismoRESUMO
Telomerase activity and (human) Telomerase Reverse Transcriptase (hTERT) expression are considered hallmarks in oncogenesis of neoplasms and are upregulated by alterations of the hTERT promoter. In meningiomas, numerous studies investigated hTERT expression, telomerase activity, promoter mutations, and methylations. Moreover, reports about hTERT-targeted chemotherapy in meningiomas have recently been published. We provide a systematic review of the literature about the role of hTERT in meningiomas. TERT expression and telomerase activity is found in benign and high-grade meningiomas and increase with WHO grade. Remarkably, rates of TERT expression/telomerase activity usually exceed mutation frequency and both telomerase activity and TERT expression have also been found in hTERT promoter wildtype meningiomas, indicating further mechanisms of TERT upregulation. Although hTERT promoter methylation has been reported in the vast majority of meningiomas, correlation with TERT expression remains controversial. Rates of promoter mutations, and methylation were shown to increase with rising WHO grade. Moreover, promoter methylation and mutations strongly correlate with prognosis. Although mutations predicted malignant progression, de novo mutations in high-grade recurrences of former benign lesions were also observed. Retroviral transduction of the TERT gene enabled immortalization in several grade I-III meningioma cell lines. In vitro analyses revealed significant effects on viability in hTERT-mutated meningioma cells after targeted treatment. Alternative mechanisms of telomere lengthening are usually absent in meningiomas. TERT and hTERT promoter alterations play a major role during oncogenesis of meningiomas with implications for prognosis and potentially treatment.
Assuntos
Neoplasias Encefálicas/genética , Meningioma/genética , Telomerase/genética , Telomerase/metabolismo , Homeostase do Telômero/genética , Telômero/genética , Neoplasias Encefálicas/ultraestrutura , Humanos , Meningioma/ultraestrutura , Telomerase/biossíntese , Telômero/ultraestruturaRESUMO
Human telomerase reverse transcriptase enzyme, the catalytic subunit of telomerase are seen to be frequently reactivated in cancers including Oral squamous cell carcinoma (OSCC). Increased hTERT expression have been seen in potentially malignant conditions including Oral submucous fibrosis (OSMF). The aim of the study was to evaluate the expression levels in OSMF, OSCC in the background of OSMF and OSCC using immunohistochemistry and also to correlate hTERT expression with clinicopathologic parameters. A total of 50 histopathologically diagnosed cases of 20 OSMF, 20 OSCC wherein 5 were OSCC in the background of OSMF and 10 Normal oral mucosae were retrieved from the departmental archives and subjected to immunohistochemical analysis of hTERT. The expression of hTERT increased from normal, OSMF, to OSCC with statistically significant differences in mean labelling score (LS). We also found a shift in cellular localization of stain where, normal mucosal tissues showed a nuclear stain unlike OSMF, where combined nuclear and cytoplasmic staining as noted. The tumor cells in OSCC showed predominant cytoplasmic staining. There was no correlation between hTERT expression and clinicopathological parameters of OSMF. However, a significant increase of hTERT expression was seen with increasing histological grading of OSCC. These results suggest the role of hTERT in the early event of malignant transformation of OSMF. Telomerase could be used as a potent diagnostic marker to identify high-risk group of OSMF.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Bucais/patologia , Fibrose Oral Submucosa/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Telomerase/biossíntese , Adulto , Biomarcadores Tumorais/análise , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/enzimologia , Fibrose Oral Submucosa/enzimologia , Lesões Pré-Cancerosas/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/enzimologiaRESUMO
The BRAF gene and the TERT promoter are among the most frequently altered genomic loci in low-grade (LGG) and high-grade-glioma (HGG), respectively. The coexistence of BRAF and TERT promoter aberrations characterizes a subset of aggressive glioma. Therefore, we investigated interactions between those alterations in malignant glioma. We analyzed co-occurrence of BRAFV600E and TERT promoter mutations in our clinical data (n = 8) in addition to published datasets (n = 103) and established a BRAFV600E-positive glioma cell panel (n = 9) for in vitro analyses. We investigated altered gene expression, signaling events and TERT promoter activity upon BRAF- and E-twenty-six (ETS)-factor inhibition by qRT-PCR, chromatin immunoprecipitation (ChIP), Western blots and luciferase reporter assays. TERT promoter mutations were significantly enriched in BRAFV600E-mutated HGG as compared to BRAFV600E-mutated LGG. In vitro, BRAFV600E/TERT promoter double-mutant glioma cells showed exceptional sensitivity towards BRAF-targeting agents. Remarkably, BRAF-inhibition attenuated TERT expression and TERT promoter activity exclusively in double-mutant models, while TERT expression was undetectable in BRAFV600E-only cells. Various ETS-factors were broadly expressed, however, only ETS1 expression and phosphorylation were consistently downregulated following BRAF-inhibition. Knock-down experiments and ChIP corroborated the notion of a functional role for ETS1 and, accordingly, all double-mutant tumor cells were highly sensitive towards the ETS-factor inhibitor YK-4-279. In conclusion, our data suggest that concomitant BRAFV600E and TERT promoter mutations synergistically support cancer cell proliferation and immortalization. ETS1 links these two driver alterations functionally and may represent a promising therapeutic target in this aggressive glioma subgroup.
Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Inibidores de Proteínas Quinases/farmacologia , Proteína Proto-Oncogênica c-ets-1/genética , Proteínas Proto-Oncogênicas B-raf/genética , Telomerase/genética , Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Células HEK293 , Humanos , Indóis/farmacologia , Mutação/genética , Regiões Promotoras Genéticas/genética , Proteína Proto-Oncogênica c-ets-1/antagonistas & inibidores , Proteína Proto-Oncogênica c-ets-1/biossíntese , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/biossíntese , Telomerase/biossínteseRESUMO
Phage N15 protelomerase (TelN) cleaves double-stranded circular DNA containing a telomerase-occupancy-site (tos) and rejoins the resulting linear-ends to form closed-hairpin-telomeres in Escherichia coli (E. coli). Continued TelN expression is essential to support resolution of the linear structure. In mammalian cells, no enzyme with TelN-like activities has been found. In this work, we show that phage TelN, expressed transiently and stably in human and mouse cells, recapitulates its native activities in these exogenous environments. We found TelN to accurately resolve tos-DNA in vitro and in vivo within human and mouse cells into linear DNA-containing terminal telomeres that are resistant to RecBCD degradation, a hallmark of protelomerase processing. In stable cells, TelN activity was detectable for at least 60 days, which suggests the possibility of limited silencing of its expression. Correspondingly, linear plasmid containing a 100â¯kb human ß-globin gene expressed for at least 120â¯h in non-ß-globin-expressing mouse cells with TelN presence. Our results demonstrate TelN is able to cut and heal DNA as hairpin-telomeres within mammalian cells, providing a tool for creating novel structures by DNA resolution in these hosts. The TelN protelomerase may be useful for exploring novel technologies for genome interrogation and chromosome engineering.
Assuntos
Replicação do DNA/fisiologia , DNA/metabolismo , Precursores Enzimáticos , Telomerase , Proteínas Virais , Globinas beta/genética , Animais , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/fisiologia , Escherichia coli , Engenharia Genética/métodos , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Telomerase/biossíntese , Telomerase/fisiologia , Proteínas Virais/biossíntese , Proteínas Virais/fisiologiaRESUMO
A hybrid 6XRE-hTERT promoter consisting of the hTERT tumor-specific promoter and six copies of the XRE element from the CYP1A1 human gene promoter was created. Using a human lung cancer cells as a model, we showed that XRE elements in the hybrid promoter greatly increase the activity of the hTERT promoter and ensure the reporter gene transcriptional activation in response to the treatment of the cells with the AhR ligand benzo(a)pyrene. However, similar effects were also observed in normal human bronchial epithelial cells HBEpC, which indicates the loss of the tumor-specific activity by the 6XRE-hTERT hybrid promoter. XRE elements can be used for nonspecific transcription enhancement but are unsuitable for the creation of tumor-specific promoters with enhanced activity.
Assuntos
Citocromo P-450 CYP1A1 , Elementos de Resposta , Telomerase , Ativação Transcricional/efeitos dos fármacos , Células A549 , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Benzo(a)pireno/toxicidade , Brônquios/metabolismo , Brônquios/patologia , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Telomerase/biossíntese , Telomerase/genéticaRESUMO
Alzheimer's disease (AD) represents the most common form of neurodegenerative disorders with only palliative treatments currently available. Amyloid plaque formation caused by amyloid-ß (Aß) aggregation and neurofibrillary tangle formation caused by hyperphosphorylated tau are hallmarks for the development of AD. The 37âkDa/67âkDa laminin receptor (LRP/LR) has been implicated in AD and tools blocking or downregulating LRP/LR impede amyloid plaque formation in vitro and in vivo. We have recently shown that LRP::FLAG enhances telomerase activity with a concomitant reduction of senescent markers. Here, we overexpressed LRP::FLAG in HEK293 and SH-SY5Y cells, which resulted in an increase in hTERT levels as well as increased telomerase activity and increased cell viability in the presence of cytotoxic levels of exogenous Aß. LRP::FLAG overexpression decreased Aß shedding and intracellular Aß levels in HEK293 cells. This suggests that LRP::FLAG rescues cells from Aß-induced cytotoxicity through increased telomerase activity. This study recommends LRP::FLAG as a novel alternative therapeutic for AD treatment through activation of telomerase activity.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Receptores de Laminina/metabolismo , Proteínas Ribossômicas/metabolismo , Telomerase/biossíntese , Doença de Alzheimer/induzido quimicamente , Peptídeos beta-Amiloides/biossíntese , Sobrevivência Celular , Células HEK293 , Humanos , Fragmentos de Peptídeos/toxicidadeRESUMO
INTRODUCTION: Elevated mRNA expression of human telomerase reverse transcriptase (hTERT mRNA) is common in many types of tumors, participating in tumor growth and progression. Such expression has not been sufficiently examined in renal cancer. The goal of the present study was to quantify it and analyze its possible clinical value in the management of this pathology. PATIENTS AND METHODS: The study included 111 patients who underwent surgery for renal cell carcinoma (RCC) between 2015 and 2017. Tumor samples were taken from all patients and, in 94 of them, healthy renal tissue adjacent to the tumor was also sampled. The 2 types of tissue were histologically confirmed, after which mRNA was extracted. Using real-time quantitative PCR, the expression of hTERT and glyceraldehyde-3-phosphate dehydrogenase (as endogenous control) were indirectly quantified using the crossing point (CP), which is inversely correlated with the number of sample replicates yielding positive results. These values were correlated with patient socio-demographic variables and clinical-pathological factors of the RCC. RESULTS: The majority of patients were males, with an average age of 60.5 years (SD: 14.02). Most tumors (69.4%) were clear cell carcinomas. The most frequent stages were pT2 or lower (73%), while 5% were pN1 and 12% pM1. The majority of tumors (58%) were Fuhrman grades 1 or 2 (low grade). All samples of tumor and nontumor tissue expressed glyceraldehyde-3-phosphate dehydrogenase mRNA, with the CP in the tumor sample significantly lower than in the nontumor tissue (P < 0.001). The expression of hTERT mRNA was detected in 68% of tumor tissues and significantly correlated with histopathology: 100% in sarcomatoid RCC and 77.9% in clear cell carcinomas (P < 0.0001). The CP was lower in pN1 (Pâ¯=â¯0.018), pM1 (Pâ¯=â¯0.046), and TNM IV stages (Pâ¯=â¯0,041). A greater number of hTERT mRNA replicas were detected in M1 patients (Pâ¯=â¯0.0005) and TNM IV stage (Pâ¯=â¯0.017). There was no correlation of hTERT mRNA expression with Fuhrman grade. CONCLUSIONS: The quantitation of hTERT mRNA expression in RCC might be useful as a complementary diagnostic tool as well as for assessing aggressiveness of the tumor.
Assuntos
Neoplasias Renais/enzimologia , Neoplasias Renais/genética , RNA Mensageiro/biossíntese , Telomerase/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Feminino , Humanos , Neoplasias Renais/diagnóstico , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Telomerase/biossíntese , Telomerase/metabolismoAssuntos
Antígenos CD34 , Regulação Enzimológica da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/enzimologia , Leucemia Mieloide Aguda/enzimologia , Síndromes Mielodisplásicas/enzimologia , Proteínas de Neoplasias/biossíntese , Telomerase/biossíntese , Feminino , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Proteínas de Neoplasias/genética , Telomerase/genéticaRESUMO
The maintenance of stem cells often requires the support of feeder cells. Primary mouse embryonic fibroblasts (MEFs) have traditionally been used as feeder cells, and although these MEF-derived feeder cells have exhibited a reasonable performance, they require repeated cell isolation, since MEFs cannot expand indefinitely. To overcome this limitation, immortalized cells, such as STO cells, have been used. However, one major disadvantage is that previously reported immortalized cells can only support stem cell cultures for a relatively short period, typically 4 to 7â¯days. In this study, we found that our newly established rat-derived fibroblasts immortalized by the expression of mutant cyclin-dependent kinase 4, cyclin D, and telomerase reverse transcriptase, can function as feeder cells for relatively long cell culture periods of approximately 14â¯days. The rat-derived immortalized cells developed in this study should be a useful source of feeder cells to support stem cell research.
Assuntos
Ciclina D/biossíntese , Quinase 4 Dependente de Ciclina/biossíntese , Células Alimentadoras/metabolismo , Fibroblastos/metabolismo , Mutação , Células-Tronco/metabolismo , Telomerase/biossíntese , Animais , Linhagem Celular Transformada , Técnicas de Cocultura , Ciclina D/genética , Quinase 4 Dependente de Ciclina/genética , Células Alimentadoras/citologia , Fibroblastos/citologia , Camundongos , Ratos , Células-Tronco/citologia , Telomerase/genéticaRESUMO
BACKGROUND AND OBJECTIVES: Telomerase is an enzyme involved in maintaining the length of telomeres and cell senescence. Numerous studies have shown that in more than 90% of malignant tumors telomerase activity is detected. MATERIAL AND METHODS: Retrospective observational study in a series of 85 cases of primary melanomas, 12 metastatic melanomas, and 22 melanocytic nevi. We used the monoclonal antibody hTERT (human telomerase reverse transcriptase, Rockland) to assess telomerase activity. The SPSS software package was used to analyze data. RESULTS: Telomerase expression was present in all the melanocytic neoplasms analyzed. Expression was heterogenous and moderate or high in the melanomas. In contrast, expression was homogeneous and lower in the nevi. Heterogeneous expression was associated with rapid melanoma growth (P=.028), a Breslow thickness of more than 4 mm (P=.004), mitosis (P=.032), and mutations in the TERT gene (P=.002). Activity was less intense in intradermal nevi, and more intense in compound and dysplastic nevi (P=.054). CONCLUSIONS: Telomerase expression is found in all melanocytic neoplasms but is higher in melanomas than in nevi. A heterogeneous pattern of expression in melanomas is associated with more aggressive tumors.