scholarly journals Targeting Epigenetic Modifiers Can Reduce the Clonogenic Capacities of Sézary Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Alain Chebly ◽  
Martina Prochazkova-Carlotti ◽  
Yamina Idrissi ◽  
Laurence Bresson-Bepoldin ◽  
Sandrine Poglio ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Histone Deacetylase Inhibitors ◽  
Soft Agar ◽  
Tumor Formation ◽  
Human Telomerase Reverse Transcriptase ◽  
Hematological Disorders ◽  
Soft Agar Assay ◽  
Human Telomerase ◽  
Sézary Cells

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphomas (CTCL) in which the human Telomerase Reverse Transcriptase (hTERT) gene is re-expressed. Current available treatments do not provide long-term response. We previously reported that Histone deacetylase inhibitors (HDACi, romidespin and vorinostat) and a DNA methyltransferase inhibitor (DNMTi, 5-azacytidine) can reduce hTERT expression without altering the methylation level of hTERT promoter. Romidepsin and vorinostat are approved for CTCL treatment, while 5-azacytidine is approved for the treatment of several hematological disorders, but not for CTCL. Here, using the soft agar assay, we analyzed the functional effect of the aforementioned epidrugs on the clonogenic capacities of Sézary cells. Our data revealed that, besides hTERT downregulation, epidrugs’ pressure reduced the proliferative and the tumor formation capacities in Sézary cells in vitro.

scholarly journals Establishment and Characterization of a Telomerase-Immortalized Sheep Trophoblast Cell Line

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Yufei Zhang ◽  
Jing Shi ◽  
Shuying Liu
Keyword(s):  
Cell Line ◽  
Soft Agar ◽  
Trophoblast Cell ◽  
Placental Lactogen ◽  
Trophoblast Cells ◽  
Human Telomerase Reverse Transcriptase ◽  
Htert Gene ◽  
Human Telomerase ◽  
Trophoblast Cell Line

The primary sheep trophoblast cells (STCs) have a finite lifespan in culture. This feature limits the scope for long-termin vitrostudies with STCs. This study was an attempt to establish and characterize a telomerase-immortalized sheep trophoblast cell line. STCs were isolated and purified by using Percoll and specific immunoaffinity purification, respectively. The purified STCs were transfected with a plasmid carrying sequences of human telomerase reverse transcriptase (hTERT) to create immortalized sheep trophoblast cell line (hTERT-STCs). hTERT-STCs showed a stable expression of hTERT gene, serially passaged for a year, and showed active proliferation without signs of senescence. Cytokeratin 7 (CK-7), secreted human chorionic gonadotrophin subunitβ(CG-β), placental lactogen (PL), and endogenous jaagsiekte sheep retrovirus (enJSRV) envelope genes were expressed in hTERT-STCs. Transwell cell invasion assay indicated that hTERT-STCs still possessed the same invasive characteristics as normal primary sheep trophoblast cells. hTERT-STCs could not grow in soft agar and did not develop into tumors in nude mice. In this study, we established a strain of immortalized sheep trophoblast cell line which could be gainfully employed in the future as an experimental model to study trophoblast cells with secretory function, invasive features, and probable biological function of enJSRV envelope genes.

scholarly journals Mechanisms for the Inhibition of Colon Cancer Cells by Sulforaphane through Epigenetic Modulation of MicroRNA-21 and Human Telomerase Reverse Transcriptase (hTERT) Down-regulation

2017 ◽  
Vol 18 (1) ◽  
pp. 97-106 ◽  
Author(s):  
Samantha L. Martin ◽  
Rishabh Kala ◽  
Trygve O. Tollefsbol
Keyword(s):  
Reverse Transcriptase ◽  
Histone Deacetylase Inhibitors ◽  
Vital Role ◽  
Cruciferous Vegetables ◽  
Telomerase Reverse Transcriptase ◽  
Human Telomerase Reverse Transcriptase ◽  
Western Blot Assay ◽  
Cellular Density ◽  
Human Telomerase

Background: Epigenetic modulations such as histone modifications are becoming increasingly valued for their ability to modify genes without altering the DNA sequence. Many bioactive compounds have been shown to alter genetic and epigenetic profiles in various cancers. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables such as kale, cabbage and broccoli sprouts, is one of the most potent histone deacetylase inhibitors (HDACis) to date. Recently, it has been identified that HDACis may play a vital role in regulating microRNAs (miRs) and human telomerase reverse transcriptase (hTERT). Objective: The aim of our study was to identify if aberrant HDAC, hTERT and miR levels could be regulated through novel dietary-based approaches in colorectal cancer (CRC) cells. Methods: We evaluated the in vitro epigenetic effects of SFN on CRC cells by MTT assay, cellular density assay, real-time reverse transcriptase-polymerase chain reaction (RT-PCR), cell cycle analysis, western-blot assay, HDAC activity assay and teloTAGGG telomerase PCR Elisa assay. Results: We demonstrated the inhibitory effects of physiologically relevant concentrations of SFN in both HCT116 and RKO CRC cells, and showed for the first time that SFN treatment decreased cell density, significantly inhibited cell viability and induced apoptosis in CRC cells. We also found that practical doses of SFN significantly down-regulated oncogenic miR-21, HDAC and hTERT mRNA, protein and enzymatic levels in CRC cells. Conclusion: Our studies suggest that the regulation of HDAC, hTERT and miR-21 is a promising approach for delaying and/or preventing CRC and may be accomplished via the consumption of SFN in cruciferous vegetables.

scholarly journals MGMT-activated DUB3 stabilizes MCL1 and drives chemoresistance in ovarian cancer

2019 ◽  
Vol 116 (8) ◽  
pp. 2961-2966 ◽  
Author(s):  
Xiaowei Wu ◽  
Qingyu Luo ◽  
Pengfei Zhao ◽  
Wan Chang ◽  
Yating Wang ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Dna Methyltransferase ◽  
Essential Role ◽  
Histone Deacetylase Inhibitors ◽  
Combined Treatment ◽  
Ovarian Cancer Cells ◽  
Methylguanine Dna Methyltransferase

Chemoresistance is a severe outcome among patients with ovarian cancer that leads to a poor prognosis. MCL1 is an antiapoptotic member of the BCL-2 family that has been found to play an essential role in advancing chemoresistance and could be a promising target for the treatment of ovarian cancer. Here, we found that deubiquitinating enzyme 3 (DUB3) interacts with and deubiquitinates MCL1 in the cytoplasm of ovarian cancer cells, which protects MCL1 from degradation. Furthermore, we identified that O6-methylguanine-DNA methyltransferase (MGMT) is a key activator of DUB3 transcription, and that the MGMT inhibitor PaTrin-2 effectively suppresses ovarian cancer cells with elevated MGMT-DUB3-MCL1 expression both in vitro and in vivo. Most interestingly, we found that histone deacetylase inhibitors (HDACis) could significantly activate MGMT/DUB3 expression; the combined administration of HDACis and PaTrin-2 led to the ideal therapeutic effect. Altogether, our results revealed the essential role of the MGMT-DUB3-MCL1 axis in the chemoresistance of ovarian cancer and identified that a combined treatment with HDACis and PaTrin-2 is an effective method for overcoming chemoresistance in ovarian cancer.

scholarly journals Loss of Stag2 cooperates with EWS-FLI1 to transform murine Mesenchymal stem cells

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Marc El Beaino ◽  
Jiayong Liu ◽  
Amanda R. Wasylishen ◽  
Rasoul Pourebrahim ◽  
Agata Migut ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Soft Agar ◽  
Tumor Formation ◽  
Cellular Migration ◽  
Migration And Invasion ◽  
Sequencing Studies ◽  
Genetic Perturbations ◽  
Lentiviral Infection

Abstract Background Ewing sarcoma is a malignancy of primitive cells, possibly of mesenchymal origin. It is probable that genetic perturbations other than EWS-FLI1 cooperate with it to produce the tumor. Sequencing studies identified STAG2 mutations in approximately 15% of cases in humans. In the present study, we hypothesize that loss of Stag2 cooperates with EWS-FLI1 in generating sarcomas derived from murine mesenchymal stem cells (MSCs). Methods Mice bearing an inducible EWS-FLI1 transgene were crossed to p53−/− mice in pure C57/Bl6 background. MSCs were derived from the bone marrow of the mice. EWS-FLI1 induction and Stag2 knockdown were achieved in vitro by adenovirus-Cre and shRNA-bearing pGIPZ lentiviral infection, respectively. The cells were then treated with ionizing radiation to 10 Gy. Anchorage independent growth in vitro was assessed by soft agar assays. Cellular migration and invasion were evaluated by transwell assays. Cells were injected with Matrigel intramuscularly into C57/Bl6 mice to test for tumor formation. Results Primary murine MSCs with the genotype EWS-FLI1 p53−/− were resistant to transformation and did not form tumors in syngeneic mice without irradiation. Stag2 inhibition increased the efficiency and speed of sarcoma formation significantly in irradiated EWS-FLI1 p53−/− MSCs. The efficiency of tumor formation was 91% for cells in mice injected with Stag2-repressed cells and 22% for mice receiving cells without Stag2 inhibition (p < .001). Stag2 knockdown reduced survival of mice in Kaplan-Meier analysis (p < .001). It also increased MSC migration and invasion in vitro but did not affect proliferation rate or aneuploidy. Conclusion Loss of Stag2 has a synergistic effect with EWS-FLI1 in the production of sarcomas from murine MSCs, but the mechanism may not relate to increased proliferation or chromosomal instability. Primary murine MSCs are resistant to transformation, and the combination of p53 null mutation, EWS-FLI1, and Stag2 inhibition does not confer immediate conversion of MSCs to sarcomas. Irradiation is necessary in this model, suggesting that perturbations of other genes beside Stag2 and p53 are likely to be essential in the development of EWS-FLI1-driven sarcomas from MSCs.

scholarly journals Chronic Microcystin-LR Exposure Induces Hepatocarcinogenesis via Increased Gankyrin in Vitro and in Vivo

2018 ◽  
Vol 49 (4) ◽  
pp. 1420-1430 ◽  
Author(s):  
Lixiong He ◽  
Yujing Huang ◽  
Qiaonan Guo ◽  
Hui Zeng ◽  
Chuanfen Zheng ◽  
...  
Keyword(s):  
Low Dose ◽  
Soft Agar ◽  
Tumor Formation ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
L02 Cells ◽  
Insight Into

Background/Aims: Our recent study indicated that the serum microcystin-LR (MC-LR) level is positively linked to the risk of human hepatocellular carcinoma (HCC). Gankyrin is over-expressed in cancers and mediates oncogenesis; however, whether MC-LR induces tumor formation and the role of gankyrin in this process is unclear. Methods: We induced malignant transformation of L02 liver cells via 35 passages with exposure to 1, 10, or 100 nM MC-LR. Wound healing, plate and soft agar colony counts, and nude mice tumor formation were used to evaluate the tumorigenic phenotype of MC-LR-treated cells. Silencing gankyrin was used to confirm its function. We established a 35-week MC-LR exposure rat model by twice weekly intraperitoneal injection with 10 μg/kg body weight. In addition, 96 HCC patients were tested for tumor tissue gankyrin expression and serum MC-LR levels. Results: Chronic low-dose MC-LR exposure increased proliferation, mobility, clone and tumor formation abilities of L02 cells as a result of gankyrin activation, while silencing gankyrin inhibited the carcinogenic phenotype of MC-LR-treated cells. MC-LR also induced neoplastic liver lesions in Sprague-Dawley rats due to up-regulated gankyrin. Furthermore, a trend of increased gankyrin was observed in humans exposed to MC-LR. Conclusion: These results suggest that MC-LR induces hepatocarcinogenesis in vitro and in vivo by increasing gankyrin levels, providing new insight into MC-LR carcinogenicity studies.

Bioflavonoids protect cells against halogenated boroxine-induced genotoxic damage by upregulation of hTERT expression

2019 ◽  
Vol 74 (5-6) ◽  
pp. 125-129 ◽  
Author(s):  
Maida Hadzic ◽  
Sanin Haveric ◽  
Anja Haveric ◽  
Naida Lojo-Kadric ◽  
Borivoj Galic ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Housekeeping Genes ◽  
Inhibitory Effects ◽  
Human Telomerase Reverse Transcriptase ◽  
Cycle Progression ◽  
Genotoxic Damage ◽  
Malignant Skin ◽  
Human Telomerase

Abstract Plant bioflavonoids are widely present in the human diet and have various protective properties. In this study, we have demonstrated the capacity of delphinidin and luteolin to increase human telomerase reverse transcriptase (hTERT) expression level and act as protective agents against halogenated boroxine-induced genotoxic damage. Halogenated boroxine K2(B3O3F4OH) (HB), is a novel compound with potential for the treatment of both benign and malignant skin changes. In vivo and in vitro studies have confirmed the inhibitory effects of HB on carcinoma cell proliferation and cell cycle progression as well as enzyme inhibition. However, minor genotoxic effects of HB are registered in higher applied concentrations, but those can be suppressed by in vitro addition of delphinidin and luteolin in appropriate concentrations. Fresh peripheral blood samples were cultivated for 72 h followed by independent and concomitant treatments of HB with luteolin or delphinidin. We analyzed the differences in relative hTERT expression between series of treatments compared with controls, which were based on normalized ratios with housekeeping genes. The obtained results have shown that selected bioflavonoids induce upregulation of hTERT that may contribute to the repair of genotoxic damage in vitro.

scholarly journals Identification of Angiogenesis Inhibitors Using a Co-culture Cell Model in a High-Content and High-Throughput Screening Platform

2017 ◽  
Vol 23 (3) ◽  
pp. 217-225 ◽  
Author(s):  
Shuaizhang Li ◽  
Chia-Wen Hsu ◽  
Srilatha Sakamuru ◽  
Chaozhong Zou ◽  
Ruili Huang ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Cell Model ◽  
Hypoxia Inducible Factor ◽  
Angiogenesis Inhibitors ◽  
Culture Cell ◽  
Tube Formation ◽  
Tumor Formation ◽  
Human Telomerase Reverse Transcriptase

Angiogenesis is an important hallmark of cancer, contributing to tumor formation and metastasis. In vitro angiogenesis models for analyzing tube formation serve as useful tools to study these processes. However, current in vitro co-culture models using primary cells have limitations in usefulness and consistency. Therefore, in the present study, an in vitro co-culture assay system was optimized in a 1536-well format for high-throughput screening using human telomerase reverse transcriptase (hTERT)–immortalized mesenchymal stem cells and aortic endothelial cells. The National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (NPC) library containing 2816 drugs was evaluated using the in vitro co-culture assay. From the screen, 35 potent inhibitors (IC50 ≤1 µM) were identified, followed by 15 weaker inhibitors (IC50 1–50 µM). Moreover, many known angiogenesis inhibitors were identified, such as topotecan, docetaxel, and bortezomib. Several potential novel angiogenesis inhibitors were also identified from this study, including thimerosal and podofilox. Among the inhibitors, some compounds were proved to be involved in the hypoxia-inducible factor-1α (HIF-1α) and the nuclear factor-kappa B (NF-κB) pathways. The co-culture model developed by using hTERT-immortalized cell lines described in this report provides a consistent and robust in vitro system for antiangiogenic drug screening.

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