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1.
In Vivo ; 36(1): 94-102, 2022.
Article in English | MEDLINE | ID: mdl-34972704

ABSTRACT

BACKGROUND/AIM: The promoter region of the telomerase reverse transcriptase (TERT) gene is a regulatory element capable of affecting TERT expression, telomerase activity, and telomerase length. Mutations within the TERT promoter region are the most common mutations in many cancers. In this study, we characterized the TERT promoter mutation status in hepatobiliary, pancreatic, and gastrointestinal cancer cell lines. MATERIALS AND METHODS: TERT promoter mutation status was assessed by digital PCR in 12 liver cancer, 5 cholangiocarcinoma (CCA), 12 pancreatic cancer, 17 gastrointestinal cancer, and 3 healthy control cell lines. RESULTS: The C228T promoter mutation was detected in 9 liver cancer lines, and the C250T TERT mutation was detected in 1 oesophageal squamous cell carcinoma line. CONCLUSION: The C228T promoter mutation is specific to liver cancer cell lines among various gastrointestinal cancer cell lines. These data will contribute to future research on the tumorigenic mechanisms and clinical use of digital PCR to detect mutations.


Subject(s)
Gastrointestinal Neoplasms , Liver Neoplasms , Telomerase , Cell Line , Gastrointestinal Neoplasms/genetics , Humans , Liver Neoplasms/genetics , Mutation , Promoter Regions, Genetic , Telomerase/genetics , Telomerase/metabolism
2.
In Vivo ; 36(1): 111-120, 2022.
Article in English | MEDLINE | ID: mdl-34972706

ABSTRACT

BACKGROUND/AIM: Poorly differentiated thyroid carcinoma (PDTC), anaplastic thyroid carcinoma (ATC), and advanced DTC have poor outcomes. MATERIALS AND METHODS: We performed next-generation sequencing in nine selected aggressive thyroid cancers. RESULTS: Among the nine patients, the driver gene mutations BRAF V600E (3/9) and NRAS Q61K (1/9) were detected. Other oncogenic mutations included ERBB2 (1/9) and CDK4 (1/9). Telomerase reverse transcriptase (TERT) promoter mutation was found in five cases. Among tumor suppressor genes, mutations in TP53 (3/9), ARID1A (1/9), APC (1/9), MEN1 (1/9), DICER1 (1/9), and MED12 (1/9) were identified. RET fusions were found in two cases, one with PTDC and the other with ATC. The ATC with RET fusion also harbored TP53 and TERT promoter mutations. None of the PDTC cases had BRAF or RAS gene alterations. CONCLUSION: Since genetic alterations with therapeutic and prognostic implications were detected using next-generation sequencing, this technique is recommended to be performed for patients with aggressive thyroid cancer.


Subject(s)
Telomerase , Thyroid Carcinoma, Anaplastic , Thyroid Neoplasms , DEAD-box RNA Helicases , Genomics , Humans , Mutation , Proto-Oncogene Proteins B-raf/genetics , Ribonuclease III , Telomerase/genetics , Thyroid Carcinoma, Anaplastic/genetics , Thyroid Neoplasms/genetics
3.
In Vivo ; 36(1): 140-152, 2022.
Article in English | MEDLINE | ID: mdl-34972709

ABSTRACT

BACKGROUND/AIM: Human dermal fibroblasts (HDFs) are widely used as a skin model in cosmetic and pharmaceutical industry due their advantages for the cosmetic industry and medical aspects. Telomeres are key players in controlling cellular aging, in which telomeres and the telomerase enzyme (hTERT) can maintain proliferative capacity and prolong cellular senescence. The primary aim of the study was to elucidate the underlying mechanisms of hTERT/SV40 immortalization of human dermal fibroblasts. MATERIALS AND METHODS: Transgenic expression of hTERT and SV40 large antigen, as well as co-transfection of both factors was performed and their significance evaluated in terms of HDF immortalization efficiency. RESULTS: The results showed that the immortalized fibroblasts of all conditions can be cultured in over 60 passages and maintain their telomere length. Further, key markers of skin cells, such as COL1A1, KRT18 and ELASTIN, were up-regulated in immortalized cells. In addition, p53 expression was enhanced in all immortalized cells, in accordance with activation of the SIRT1 gene upon transgenic immortalization. The significant role of SIRT1 in fibroblast proliferation was assessed by shRNA-knockdown, and it was found that SIRT1 silencing led to loss of Ki67, a proliferation marker. Moreover, miR-93, a SIRT1-targeted miRNA, also had a significantly reduced expression in the co-transfected immortalized cells, highlighting the linkage of the miRNA and SIRT1 pathway in the immortalization of human dermal fibroblasts. CONCLUSION: This evidence from this study could benefit the efficient development of human skin cell lines for use in the cosmetic industry in the future.


Subject(s)
MicroRNAs , Telomerase , Cells, Cultured , Cellular Senescence/genetics , Fibroblasts/metabolism , Humans , MicroRNAs/genetics , Sirtuin 1/genetics , Telomerase/genetics , Telomerase/metabolism
4.
Eur J Cancer ; 161: 99-107, 2022 01.
Article in English | MEDLINE | ID: mdl-34936949

ABSTRACT

BACKGROUND: Around 50% of cutaneous melanomas harbour therapeutically targetable BRAF V600 mutations. Reliable clinical biomarkers predicting duration of response to BRAF-targeted therapies are still lacking. Recent in vitro studies demonstrated that BRAF-MEK inhibitor therapy response is associated with tumour TERT promoter mutation status. We assessed this potential association in a clinical setting. METHODS: The study cohort comprised 232 patients with metastatic or unresectable BRAF V600-mutated melanoma receiving combined BRAF/MEK inhibitor treatment, including a single-centre retrospective discovery cohort (N = 120) and a prospectively collected multicenter validation cohort (N = 112). Patients were excluded if they received BRAF or MEK inhibitors in an adjuvant setting, as monotherapy, or in combination with immunotherapy. Kaplan-Meier and univariate/multivariate Cox regression analyses were performed as appropriate. RESULTS: median age at first diagnosis was 54 years (range 16-84 years). The majority of patients were men 147/232 (63.4%). Most tumours harboured TERT promoter mutations (72%, N = 167). A survival advantage was observed in both progression-free survival (PFS) and overall survival (OS) for patients with TERT promoter-mutant versus wild-type tumours in both the discovery cohort (mPFS of 9.6 months [N = 87] vs 5.0 months [N = 33]; hazard ratio [HR] = 0.56 [95% confidence interval {CI} 0.33-0.96] and mOS of 33.6 months vs 15.0 months; HR = 0.47 [95%CI 0.32-0.70]) as well as the validation cohort (mPFS of 7.3 months [N = 80] vs 5.8 months [N = 32]; HR = 0.67 [95%CI 0.41-1.10] and mOS of 51.1 months vs 15.0 months; HR = 0.33 [95%CI 0.18-0.63]). In the pooled cohort of TERT promoter-mutant (N = 167) versus wild-type (N = 65) tumours, respectively, PFS was 8.9 versus 5.5 months, (HR = 0.62; 95%CI 0.45-0.87; P = 0.004), and OS was 33.6 versus 17.0 months, (HR = 0.51; 95%CI 0.35-0.75, P = 0.0001). CONCLUSIONS: In patients with melanoma receiving BRAF/MEK-targeted therapies, TERT promoter mutations are associated with longer survival. If validated in larger studies, TERT promoter mutation status should be included as a predictive biomarker in treatment algorithms for advanced melanoma.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Melanoma/drug therapy , Melanoma/genetics , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mutation/genetics , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Telomerase/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/genetics , Female , Humans , Male , Middle Aged , Progression-Free Survival , Retrospective Studies , Young Adult
5.
Tumour Biol ; 43(1): 327-340, 2021.
Article in English | MEDLINE | ID: mdl-34957975

ABSTRACT

BACKGROUND: The inhibition of the enzyme telomerase (TERT) has been widely investigated as a new pharmacological approach for cancer treatment, but its real potential and the biochemical consequences are not totally understood. OBJECTIVE: Here, we investigated the effects of the telomerase inhibitor MST-312 on a human glioma cell line after both short- and long-term (290 days) treatments. METHODS: Effects on cell growth, viability, cell cycle, morphology, cell death and genes expression were assessed. RESULTS: We found that short-term treatment promoted cell cycle arrest followed by apoptosis. Importantly, cells with telomerase knock-down revealed that the toxic effects of MST-312 are partially TERT dependent. In contrast, although the long-term treatment decreased cell proliferation at first, it also caused adaptations potentially related to treatment resistance and tumor aggressiveness after long time of exposition. CONCLUSIONS: Despite the short-term effects of telomerase inhibition not being due to telomere erosion, they are at least partially related to the enzyme inhibition, which may represent an important strategy to pave the way for tumor growth control, especially through modulation of the non-canonical functions of telomerase. On the other hand, long-term exposure to the inhibitor had the potential to induce cell adaptations with possible negative clinical implications.


Subject(s)
Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Brain Neoplasms/drug therapy , Glioma/drug therapy , Telomerase/antagonists & inhibitors , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans
6.
BMC Med ; 19(1): 277, 2021 11 12.
Article in English | MEDLINE | ID: mdl-34763698

ABSTRACT

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.


Subject(s)
Glioma , Neoplastic Cells, Circulating , Telomerase , Biomarkers, Tumor , Glioma/diagnosis , Humans , Neutrophils/metabolism , Prognosis , Telomerase/genetics , Telomerase/metabolism , Tumor Microenvironment
7.
BMC Neurol ; 21(1): 460, 2021 Nov 24.
Article in English | MEDLINE | ID: mdl-34814870

ABSTRACT

BACKGROUND: The aim of this study was to investigate the relationship between tumor biology and values of cerebral blood volume (CBV), cerebral blood flow (CBF), mean transit time (MTT), time to peak (TTP), permeability surface (PS) of tumor in patients with glioma. METHODS: Forty-six patients with glioma were involved in the study. Histopathologic and molecular pathology diagnoses were obtained by tumor resection, and all patients accepted perfusion computed tomography (PCT) before operation. Regions of interests were placed manually at tumor and contralateral normal-appearing thalamus. The parameters of tumor were divided by those of contralateral normal-appearing thalamus to normalize at tumor (relative [r] CBV, rCBF, rMTT, rTTP, rPS). The relationships of the parameters, world health organization (WHO) grade, molecular pathological findings were analysed. RESULTS: The rCBV, rMTT and rPS of patients are positively related to the pathological classification (P < 0.05). The values of rCBV and rPS in IDH mutated patients were lower than those IDH wild-type. The values of rCBF in patients with MGMT methylation were lower than those MGMT unmethylation (P < 0.05). The MVD of TERT wild-type group was lower than TERT mutated group (P < 0.05). The values of rCBV were significant difference in the four molecular groups divided by the combined IDH/TERT classification (P < 0.05). The progression free survival (PFS) and overall survival (OS) were significant difference in the four molecular groups divided by the combined IDH/TERT classification (P < 0.05). CONCLUSIONS: Our study introduces and supports the changes of glioma flow perfusion may be closely related to its biological characteristics.


Subject(s)
Brain Neoplasms , Glioma , Telomerase , Brain Neoplasms/diagnostic imaging , Brain Neoplasms/genetics , Cerebrovascular Circulation , DNA Modification Methylases/genetics , DNA Repair Enzymes/genetics , Glioma/diagnostic imaging , Glioma/genetics , Humans , Perfusion , Perfusion Imaging , Tomography, X-Ray Computed , Tumor Suppressor Proteins/genetics
8.
Anal Chim Acta ; 1184: 339036, 2021 Nov 01.
Article in English | MEDLINE | ID: mdl-34625244

ABSTRACT

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.


Subject(s)
Metal Nanoparticles , Metal-Organic Frameworks , Telomerase , Allosteric Regulation , Electrochemical Techniques , Electrochemistry , Silver , Telomerase/metabolism
9.
Int J Mol Sci ; 22(19)2021 Oct 01.
Article in English | MEDLINE | ID: mdl-34639008

ABSTRACT

Background: Mesenchymal stromal cells (MSCs) have the capacity for self-renewal and multi-differentiation, and for this reason they are considered a potential cellular source in regenerative medicine of cartilage and bone. However, research on this field is impaired by the predisposition of primary MSCs to senescence during culture expansion. Therefore, the aim of this study was to generate and characterize immortalized MSC (iMSC) lines from aged donors. Methods: Primary MSCs were immortalized by transduction of simian virus 40 large T antigen (SV40LT) and human telomerase reverse transcriptase (hTERT). Proliferation, senescence, phenotype and multi-differentiation potential of the resulting iMSC lines were analyzed. Results: MSCs proliferate faster than primary MSCs, overcome senescence and are phenotypically similar to primary MSCs. Nevertheless, their multi-differentiation potential is unbalanced towards the osteogenic lineage. There are no clear differences between osteoarthritis (OA) and non-OA iMSCs in terms of proliferation, senescence, phenotype or differentiation potential. Conclusions: Primary MSCs obtained from elderly patients can be immortalized by transduction of SV40LT and hTERT. The high osteogenic potential of iMSCs converts them into an excellent cellular source to take part in in vitro models to study bone tissue engineering.


Subject(s)
Mesenchymal Stem Cells/cytology , Tissue Donors , Aged , Cell Culture Techniques , Cell Differentiation , Cell Line , Cell Proliferation , Cells, Cultured , Gene Expression , Humans , Immunohistochemistry , Mesenchymal Stem Cells/metabolism , Osteogenesis , Telomerase , Transduction, Genetic
10.
Bull Cancer ; 108(11): 1044-1056, 2021 Nov.
Article in French | MEDLINE | ID: mdl-34593218

ABSTRACT

Refractory thyroid cancers include radio-iodine-refractory cancers, metastatic or locally advanced unresectable medullary and anaplastic thyroid cancers. Their management has been based for several years on the use of multi-target kinase inhibitors, with anti-angiogenic action, with the exception of anaplastic cancers usually treated with chemo- and radiotherapy. The situation has recently evolved due to the availability of molecular genotyping techniques allowing the discovery of rare but targetable molecular abnormalities. New treatment options have become available, more effective and less toxic than the previously available multi-target kinase inhibitors. The management of refractory thyroid cancers is therefore becoming more complex both at a diagnosis level with the need to know when, how and why to look for these molecular abnormalities but also at a therapeutic level, innovative treatments being hardly accessible. The cost of molecular analyzes and the access to treatments need also to be homogenized because disparities could lead to inequality of care at a national or international level. Finally, the strategy of identifying molecular alterations and treating these rare tumors reinforces the importance of a discussion in a multidisciplinary consultation meeting.


Subject(s)
Carcinoma, Medullary/genetics , Genotype , Mutation , Thyroid Carcinoma, Anaplastic/genetics , Thyroid Neoplasms/genetics , Anaplastic Lymphoma Kinase/genetics , Carcinoma, Medullary/pathology , Carcinoma, Medullary/therapy , Genes, ras , Humans , Immunotherapy , Molecular Targeted Therapy , Protein Kinase Inhibitors/adverse effects , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins c-ret/genetics , Receptor, trkA/genetics , Telomerase/genetics , Thyroid Carcinoma, Anaplastic/pathology , Thyroid Carcinoma, Anaplastic/therapy , Thyroid Neoplasms/pathology , Thyroid Neoplasms/therapy
11.
Braz J Med Biol Res ; 54(11): e11352, 2021.
Article in English | MEDLINE | ID: mdl-34495249

ABSTRACT

Diabetes mellitus is associated with neural and micro- and macrovascular complications. Therapeutic options for these complications are limited and the delivery of mesenchymal stem cells into lesions have been reported to improve the healing process. In this work, the effects of the administration of a lineage of human bone marrow mesenchymal stem cells immortalized by the expression of telomerase (hBMSC-TERT) as a potential therapeutic tool for wound healing in diabetic rats were investigated. This is the first description of the use of these cells in diabetic wounds. Dorsal cutaneous lesions were made in streptozotocin-induced diabetic rats and hBMSC-TERT were subcutaneously administered around the lesions. The healing process was evaluated macroscopically, histologically, and by birefringence analysis. Diabetic wounded rats infused with hBMSC-TERT (DM-TERT group) and the non-diabetic wounded rats not infused with hBMSC-TERT (CW group) had very similar patterns of fibroblastic response and collagen proliferation indicating improvement of wound healing. The result obtained by birefringence analysis was in accordance with that obtained by the histological analysis. The results indicated that local administration of hBMSC-TERT in diabetic wounds improved the wound healing process and may become a therapeutic option for wounds in individuals with diabetes.


Subject(s)
Diabetes Mellitus, Experimental , Mesenchymal Stem Cells , Telomerase , Animals , Humans , Rats , Streptozocin , Wound Healing
12.
Am J Physiol Heart Circ Physiol ; 321(5): H985-H1003, 2021 11 01.
Article in English | MEDLINE | ID: mdl-34559580

ABSTRACT

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.


Subject(s)
Endothelial Cells/enzymology , Hyperoxia/complications , Lung Injury/enzymology , Lung/blood supply , Microvessels/enzymology , Mitochondria/enzymology , Mitophagy , Telomerase/metabolism , Animals , Autophagy-Related Proteins/metabolism , Capillary Permeability , Cells, Cultured , Disease Models, Animal , Endothelial Cells/pathology , Female , Gene Knockout Techniques , Inflammation Mediators/metabolism , Lung Injury/etiology , Lung Injury/genetics , Lung Injury/pathology , Male , Membrane Transport Proteins/metabolism , Microvessels/pathology , Mitochondria/genetics , Mitochondria/pathology , Rats, Sprague-Dawley , Rats, Transgenic , Receptors, Cell Surface/metabolism , Telomerase/deficiency , Telomerase/genetics , Toll-Like Receptor 4/metabolism , Toll-Like Receptor 9/metabolism
13.
Anal Methods ; 13(36): 4063-4068, 2021 09 23.
Article in English | MEDLINE | ID: mdl-34555130

ABSTRACT

Osteosarcoma is the most frequent primary malignant bone tumor, composed of mesenchymal cells producing osteoid and immature bone. The sensitive detection of telomerase plays a pivotal role in the early diagnosis and therapeutic treatment of osteosarcoma. We report here an in vitro strategy for sensitive telomerase activity detection through the integration of rolling circle amplification (RCA) and a clustered regularly spaced short palindrome repeats (CRISPR)-Cas12a system. In the proposed strategy, telomerase substrate (TS) primers are easily controlled to extend five bases (GGGTT) to give short telomerase extension products (TEP) with definite lengths without adding dATP. The resulting short TEPs can then cyclize the padlock through hybridizing with its two terminals and thus initiate the following RCA. To obtain an improved sensitivity, the CRISPR-Cas12a system is attached to collaterally cut surrounding DNA reporter probes after recognizing the target single strand DNA sequence in the RCA products. The highlights of this strategy are as follows: (i) the short TEP triggered strategy is excellent at detecting low telomerase activity and thus contributes to the early diagnosis of malignant tumors; (ii) highly sensitive telomerase activity detection which is easy to operate from RCA initiated CRISPR-Cas12a; (iii) opening up of a new avenue for telomerase activity detection with a CRISPR-Cas12a system. Finally, the proposed strategy exhibited sensitive telomerase activity detection under optimized experimental parameters and has great application potential for the clinical diagnosis of malignant tumors and the development of anti-cancer drugs.


Subject(s)
Osteosarcoma , Telomerase , CRISPR-Cas Systems/genetics , Humans , Osteosarcoma/diagnosis , Telomerase/genetics
14.
BMC Cancer ; 21(1): 1025, 2021 Sep 15.
Article in English | MEDLINE | ID: mdl-34525976

ABSTRACT

BACKGROUND: Mutations in driver genes such as IDH and BRAF have been identified in gliomas. Meanwhile, dysregulations in the p53, RB1, and MAPK and/or PI3K pathways are involved in the molecular pathogenesis of glioblastoma. RAS family genes activate MAPK through activation of RAF and PI3K to promote cell proliferation. RAS mutations are a well-known driver of mutation in many types of cancers, but knowledge of their significance for glioma is insufficient. The purpose of this study was to reveal the frequency and the clinical phenotype of RAS mutant in gliomas. METHODS: This study analysed RAS mutations and their clinical significance in 242 gliomas that were stored as unfixed or cryopreserved specimens removed at Kyoto University and Osaka National Hospital between May 2006 and October 2017. The hot spots mutation of IDH1/2, H3F3A, HIST1H3B, and TERT promoter and exon 2 and exon 3 of KRAS, HRAS, and NRAS were analysed with Sanger sequencing method, and 1p/19q codeletion was analysed with multiplex ligation-dependent probe amplification. DNA methylation array was performed in some RAS mutant tumours to improve accuracy of diagnosis. RESULTS: RAS mutations were identified in four gliomas with three KRAS mutations and one NRAS mutation in one anaplastic oligodendroglioma, two anaplastic astrocytomas (IDH wild-type in each), and one ganglioglioma. RAS-mutant gliomas were identified with various types of glioma histology. CONCLUSION: RAS mutation appears infrequent, and it is not associated with any specific histological phenotype of glioma.


Subject(s)
Brain Neoplasms/genetics , Genes, ras/genetics , Glioma/genetics , Mutation , Adolescent , Adult , Aged , Aged, 80 and over , Astrocytoma/genetics , Brain Neoplasms/pathology , Child , Child, Preschool , DNA Methylation , DNA Modification Methylases/metabolism , DNA Mutational Analysis/methods , DNA Repair Enzymes/metabolism , Exons/genetics , Female , Glioblastoma/genetics , Glioma/pathology , Histones/genetics , Humans , Isocitrate Dehydrogenase/genetics , Male , Middle Aged , Oligodendroglioma/genetics , Phenotype , Promoter Regions, Genetic , Telomerase/genetics , Tumor Suppressor Proteins/metabolism , Young Adult
15.
Nat Commun ; 12(1): 5423, 2021 09 20.
Article in English | MEDLINE | ID: mdl-34538872

ABSTRACT

Hepatoblastoma (HB) is the most common pediatric liver malignancy; however, hereditary predisposition and acquired molecular aberrations related to HB clinicopathological diversity are not well understood. Here, we perform an integrative genomic profiling of 163 pediatric liver tumors (154 HBs and nine hepatocellular carcinomas) based on the data acquired from a cohort study (JPLT-2). The total number of somatic mutations is precious low (0.52/Mb on exonic regions) but correlated with age at diagnosis. Telomerase reverse transcriptase (TERT) promoter mutations are prevalent in the tween HBs, selective in the transitional liver cell tumor (TLCT, > 8 years old). DNA methylation profiling reveals that classical HBs are characterized by the specific hypomethylated enhancers, which are enriched with binding sites for ASCL2, a regulatory transcription factor for definitive endoderm in Wnt-pathway. Prolonged upregulation of ASCL2, as well as fetal-liver-like methylation patterns of IGF2 promoters, suggests their "cell of origin" derived from the premature hepatoblast, similar to intestinal epithelial cells, which are highly proliferative. Systematic molecular profiling of HB is a promising approach for understanding the epigenetic drivers of hepatoblast carcinogenesis and deriving clues for risk stratification.


Subject(s)
DNA Methylation , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Hepatoblastoma/genetics , Liver Neoplasms/genetics , Child, Preschool , Chromatin Immunoprecipitation Sequencing/methods , Cohort Studies , DNA Copy Number Variations , Female , Humans , Infant , Kaplan-Meier Estimate , Male , Mutation , Promoter Regions, Genetic/genetics , Telomerase/genetics , Whole Exome Sequencing/methods , beta Catenin/genetics
16.
Appl Microbiol Biotechnol ; 105(19): 7353-7365, 2021 Oct.
Article in English | MEDLINE | ID: mdl-34515845

ABSTRACT

The telomerase reverse transcriptase (TERT) is the core catalytic subunit of telomerase. Its canonical function is synthesizing telomeric repeats to maintain telomere length and chromosomal stability. Accumulating evidence suggests that TERT has other important fundamental functions in addition to its catalytic telomere repeat synthesis activity. However, the non-canonical roles of TERT independent of its enzymatic activity are not clear in filamentous fungi. In the present study, we characterized the GlTert gene in Ganoderma lucidum. The non-canonical roles of GlTert were explored using GlTert-silenced strains (Terti8 and Terti25) obtained by RNA interference. Silencing GlTert delayed the fungal growth, decreased the length between hyphal branches, and induced fungal resistance to oxidative stress in G. ludicum. Further examination revealed that the intracellular ROS (reactive oxygen species) levels were increased while the enzyme activities of the antioxidant systems (superoxide dismutase, catalase, glutathione peroxidase, and ascorbate peroxidase) were decreased in GlTert-silenced strains. In addition, silencing GlTert decreased the ganoderic acid (GA) biosynthesis of G. lucidum. Taken together, our results indicate that GlTert plays a fundamental function on fungal growth, oxidative stress, and GA biosynthesis in G. lucidum, providing new insights for the canonical functions of TERT in filamentous fungi. KEY POINTS: • GlTert affected fungal growth and hyphal branching of G. lucidum. • Silencing GlTert increased the intracellular ROS levels of G. lucidum. • GlTert regulated GA biosynthesis of G. lucidum.


Subject(s)
Reishi , Telomerase , Oxidative Stress , Telomerase/genetics , Triterpenes
17.
Talanta ; 235: 122814, 2021 Dec 01.
Article in English | MEDLINE | ID: mdl-34517670

ABSTRACT

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.


Subject(s)
Biosensing Techniques , Telomerase , Copper , DNA , HeLa Cells , Humans , Nucleic Acid Amplification Techniques , Telomerase/genetics , Telomerase/metabolism
18.
Life Sci ; 285: 119947, 2021 Nov 15.
Article in English | MEDLINE | ID: mdl-34530016

ABSTRACT

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.


Subject(s)
Adipose Tissue/cytology , Cell- and Tissue-Based Therapy/methods , Cellular Senescence , Curcumin/administration & dosage , Drug Liberation , Mesenchymal Stem Cells/drug effects , Nanofibers/chemistry , Nanoparticles/chemistry , Animals , Cell Adhesion/drug effects , Cell Proliferation , Cells, Cultured , Curcumin/pharmacology , Fibroblasts/drug effects , Fibroblasts/metabolism , Gelatin/chemistry , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/physiology , Mice , Polyesters/chemistry , RNA, Messenger/biosynthesis , Silicon Dioxide/chemistry , Telomerase/biosynthesis
19.
Drug Alcohol Depend ; 227: 108982, 2021 10 01.
Article in English | MEDLINE | ID: mdl-34482039

ABSTRACT

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.


Subject(s)
Telomerase , Telomere , Down-Regulation , Humans , Morphine/pharmacology , RNA, Messenger/genetics , Telomerase/genetics , Telomerase/metabolism , Telomere/genetics , Telomere/metabolism , Telomeric Repeat Binding Protein 2
20.
Elife ; 102021 09 06.
Article in English | MEDLINE | ID: mdl-34486523

ABSTRACT

In cancer, telomere maintenance is critical for the development of replicative immortality. Using genome sequences from the Cancer Cell Line Encyclopedia and Genomics of Drug Sensitivity in Cancer Project, we calculated telomere content across 1299 cancer cell lines. We find that telomerase reverse transcriptase (TERT) expression correlates with telomere content in lung, central nervous system, and leukemia cell lines. Using CRISPR/Cas9 screening data, we show that lower telomeric content is associated with dependency of CST telomere maintenance genes. Increased dependencies of shelterin members are associated with wild-type TP53 status. Investigating the epigenetic regulation of TERT, we find widespread allele-specific expression in promoter-wildtype contexts. TERT promoter-mutant cell lines exhibit hypomethylation at PRC2-repressed regions, suggesting a cooperative global epigenetic state in the reactivation of telomerase. By incorporating telomere content with genomic features across comprehensively characterized cell lines, we provide further insights into the role of telomere regulation in cancer immortality.


Subject(s)
Telomerase/metabolism , Telomere Homeostasis , Telomere/metabolism , Cell Line, Tumor , Epigenesis, Genetic , Humans , Neoplasms/genetics , Telomerase/genetics
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