scholarly journals Manipulating Histone Acetylation Leads to Adverse Effects in Hemangiosarcoma Cells

2021 ◽  
Author(s):  
Tamami Suzuki ◽  
Keisuke Aoshima ◽  
Jumpei Yamazaki ◽  
Atsushi Kobayashi ◽  
Takashi Kimura
Keyword(s):  
Histone Acetylation ◽  
Cell Lines ◽  
Histone Deacetylase Inhibitors ◽  
Epigenetic Modification ◽  
Malignant Tumour ◽  
Tumour Immunity ◽  
Cancer Pathogenesis ◽  
Induced Apoptosis

AbstractCanine hemangiosarcoma (HSA) is a malignant tumour derived from endothelial cells. No effective treatment has yet been developed because of the lack of understanding of its pathogenesis. Histone acetylation, an epigenetic modification, is highly associated with cancer pathogenesis. Manipulating histone acetylation by histone deacetylase inhibitors (HDACi) or bromodomain and extraterminal domain inhibitors (BETi) is one approach to treat various cancers. However, the role of histone acetylation in HSA remains unknown. This study aimed to investigate how histone acetylation functions in HSA pathogenesis using two HDACi, suberanilohydroxamic acid (SAHA) and valproic acid (VPA), and one BETi, JQ1, in vitro and in vivo. Histone acetylation levels were high in cell lines and heterogeneous in clinical cases. SAHA and JQ1 induced apoptosis in HSA cell lines. SAHA and VPA treatment in HSA cell lines upregulated inflammatory-related genes, thereby attracting macrophages. This implies that SAHA and VPA can induce anti-tumour immunity. JQ1 stimulated autophagy and inhibited the cell cycle. Finally, JQ1 suppressed HSA tumour cell proliferation in vivo. These results suggest that HDACi and BETi can be alternative drugs for HSA treatment. Although further research is required, this study provides useful insights for developing new treatments for HSA.

NPI-2358, a Novel Vascular Disrupting Agent Blocks Growth and Angiogenesis in Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3842-3842
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Madhavi Bandi ◽  
Noopur Raje ◽  
Robert L Schlossman ◽  
...  
Keyword(s):  
Cell Lines ◽  
Annexin V ◽  
Vascular Endothelial ◽  
Caspase 9 ◽  
Migration Assays ◽  
Induced Apoptosis ◽  
Rpmi 8226 ◽  
Anti Tumor Activity

Abstract Abstract 3842 Poster Board III-778 Background and Rationale Vascular disrupting agents (VDAs) act via selectively disrupting established tumor vasculature and have shown remarkable clinical success as anti-cancer therapies. NPI-2358 is a novel VDA with a distinct structure and mechanism of action from other available VDAs. NPI-2358 binds to the colchicine-binding site of beta-tubulin preventing polymerization and disrupting the cytoplasmic microtubule network, thereby causing loss of vascular endothelial cytoskeletal function, and inducing cytotoxicity in cancer cells. Here, we examined the anti-angiogenic and anti-tumor activity of NPI-2358 in multiple myeloma (MM) cells using both in vitro and in vivo model systems. Material and Methods We utilized MM.1S, MM.1R, RPMI-8226, U266, and INA-6 human MM cell lines, as well as purified tumor cells from MM patients relapsing after prior anti-MM therapies. Cell viability/apoptosis assays were performed using MTT, trypan blue exclusion, and Annexin V/PI staining. Angiogenesis was measured in vitro using Matrigel capillary-like tube structure formation assays: Since human vascular endothelial cells (HUVECs) plated onto Matrigel differentiate and form capillary-like tube structures similar to in vivo neovascularization, this assay measures anti-angiogenic effects of drugs/agents. Migration assays were performed using transwell insert assays. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, Bax, pJNK and GAPDH. Statistical significance was determined using a Student t test. Results Treatment of MM.1S, RPMI-8226, MM.1R, INA-6, and KMS-12BM with NPI-2358 for 24h induces a dose-dependent significant (P < 0.005) decrease in viability of all cell lines (IC50 range: 5-8 nM; n=3). To determine whether NPI-2358-induced decrease in viability is due to apoptosis, MM cell lines were treated with NPI-2358 for 24h; harvested, and analyzed for apoptosis using Annexin V/PI staining. A significant increase in NPI-2358-induced apoptosis was observed in all MM cell lines (% Annexin V+/PI- apoptotic cells: MM.1S, 48 ± 2.3%; MM.1R, 46.6 ± 3.1%; RPMI-8226, 61.7 ± 4.5%; and INA-6, 59.9 ± 3.2%; P < 0.05; n=3). Importantly, NPI-2358 decreased viability of freshly isolated MM cells from patients (IC50 range: 3-7 nM; P < 0.005), without affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for NPI-2358. Examination of in vitro angiogenesis using capillary-like tube structure formation assay showed that even low doses of NPI-2358 (7 nM treatment for 12h; IC50: 20 nM at 24h) significantly decreased tubule formation in HUVECs (70-80% decrease; P < 0.05). Transwell insert assays showed a marked reduction in serum-dependent migration of NPI-2358-treated MM cells (42 ± 2.1% inhibition in NPI-2358-treated vs. control; P < 0.05). NPI-2358 at the concentrations tested (5 nM for 12h) in the migration assays did not affect survival of MM cells (> 95% viable cells). A similar anti-migration activity of NPI-2358 was noted against HUVEC cells (48 ± 1.7% decrease in migration; P < 0.05). Mechanistic studies showed that NPI-2358-induced apoptosis was associated with activation of caspase-8, caspase-9, caspase-3 and PARP. Importantly, treatment of MM.1S cells with NPI-2358 (5 nM) triggered phosphorylation of c-Jun amino-terminal kinase (JNK), a classical stress response protein, without affecting Bcl-2 family members Bax and Bcl-2. Blockade of JNK using dominant negative strategy markedly abrogated NPI-2358-induced apoptosis. Conclusion Our preclinical data provide evidence for remarkable anti-angiogenic and anti-tumor activity of NPI-2358 against MM cells, without significant toxicity in normal cells. Ongoing studies are examining in vivo anti-MM activity of NPI-2358 in animal models. Importantly, a Phase-1 study of NPI-2358 as a single agent in patients with advanced malignancies (lung, prostrate and colon cancer) has already established a favorable pharmacokinetic, pharmacodynamic and safety profile; and, a Phase-2 study of the combination of NPI-2358 and docetaxel in non-small cell lung cancer showed encouraging safety, pharmacokinetic and activity data. These findings, coupled with our preclinical studies, provide the framework for the development of NPI-2358-based novel therapies to improve patient outcome in MM. Disclosures: Chauhan: Nereus Pharmaceuticals, Inc: Consultancy. Lloyd:Nereus Pharmaceuticals, In: Employment. Palladino:Nereus Pharmaceuticals, Inc: Employment. Anderson:Nereus Pharmaceuticals, Inc: Consultancy.

The Combination of Histone Deacetylase Inhibitors and Hypomethylating Agents Exhibits Marked Synergy In Preclinical Models of T-Cell Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3937-3937 ◽  
Author(s):  
Enrica Marchi ◽  
Danielle C Bongero ◽  
Matko Kalac ◽  
Luigi Scotto ◽  
Owen A. O'Connor
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Histone Deacetylase Inhibitors ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Hypomethylating Agents ◽  
Cytotoxicity Assays ◽  
Nanomolar Range

Abstract Abstract 3937 CHOP and CHOP-like chemotherapy programs remain the most commonly used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite often sub-optimal results. Histone deacetylase inhibitors (HDACIs) are epigenetic agents known to be active in T-cell lymphoma. Recently romidepsin (R) was approved for patients with relapsed or refractory CTCL. Both R and belinostat (B) are being investigated in patients with relapsed or refractory PTCL. We have previously shown that hypomethylating agents as decitabine (D) produce synergistic interactions with HDACIs in B-cell lymphomas. We investigated the in vitro and in vivo activity of D, R and B alone or in combination in different T-cell lymphoma and leukemia cell lines including CTCL (H9, HH), and T- acute lymphoblastic leukemia (T-ALL) lines resistant to gamma-secretase inhibitors (GSI) (P12, PF-382). For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-Glo™ followed by acquisition on a Biotek Synergy HT. The IC50s for D, B and R were calculated using the Calcusyn software (Biosoft). Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR) based on the GraphPad software (RRR<1 are defining synergism). Apoptosis was assessed by staining with Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition. Whole cell lysate proteins were extracted and quantified according to Bradford assay. After electrophoresis on a gradient 4–20% SDS-PAGE gels the proteins were transferred to nitrocellulose membrane. After blocking and incubation with the primary and the secondary antibodies, the chemiluminescent agent was added and the x-ray films were exposed to the membranes. The IC50s for belinostat alone at 24, 48 and 72 hours were generally in the nanomolar range: H9: 108.1nM – 35.7nM – 29.1nM; HH: 240.1nM - 67.6nM – 39.01nM; P12: 386.9nM – 99.9nM – 99.8nM; PF 382: 267.1nM – 135nM – 118.3nM. The IC50s for romidepsin alone at 24, 48 and 72 hours were generally in the low nanomolar range: H9: 5nM – 2.1nM – 2.2nM; HH: 14nM – 2.6nM - 2.5nM; P12: 6.2nM – 2.4nM – 2.1nM; PF382: 6.1nM – 1.7nM – 1.5nM. The IC50s for D alone at 72 and 96 hours were in the micromolar range: H9: 7.4uM – 3.7uM; HH: > 20 uM. In the cytotoxicity assays, the combination of D and B or R at 72 hours showed synergism in all the cell lines studied. The most representative RRRs are showed in table 1. Table 1 D 0.5 uM 1uM B (nM) RRR H9 50 0.7 0.7 70 0.6 0.6 100 0.4 0.5 PF 382 150 0.8 0.7 0.5 uM 1 uM R (nM) RRR H9 0.5 0.9 0.9 1 0.8 0.8 2 0.3 0.3 PF 382 1 0.8 0.7 1.5 0.4 0.4 2 0.1 0.1 When H9, HH, P12 and PF382 cell lines were treated with D and B or R for 72 hours, all the combination groups showed significantly more apoptosis than the single drug exposures and controls. Table 2 displays the range of apoptosis induction for B, R and D or for them used in combination and the RRR value after the analysis for the most significant data. Table 2 B D B + D RRR (% Apoptotic + Dead Cells) H9 100nM (22.9%) 500nM (17.9%) 51.5% 0.7 HH 100nM (42.9%) 1uM (46.9%) 61.3% 0.8 P 12 150nM (16%) 1uM (42.7%) 80.1% 0.4 PF 382 100nM (8.3%) 1uM (27.9%) 40.1% 0.8 R D R + D H9 2nM (22.2%) 500nM (17.9%) 63.6% 0.5 HH 2nM (80%) 1uM (46.9%) 89.7% 0.6 P 12 2nM (9.9%) 10uM (58.7%) 98% 0.03 PF 382 2nM (54.5%) 500nM (17.9%) 88.7% 0.2 Increased acetylation of H3 was observed when H9 cells were treated with R alone and synergistically increased after exposing cells to the combination of D + B and D + R. The expression of phosphorylated Stat3 was decreased after exposure of H9 cells to the combination of D and R. Additional interrogation of the effects of this epigenetic therapy on the JAK-STAT signaling pathway are now underway. An in vivo xenograft study in six to eight weeks old female SCID beige mice injected subcutaneously with 2 × 107 HH cells has also begun and will be reported. Mice were separated into different cohorts and treated with intraperitoneal injections of D or B or their combination according to the following schedules: D alone at 1.5 mg/kg on days 1, 5; B alone at 35 mg/Kg/day for 7 days. Collectively, the data suggest that the combination of a hypomethylating agent like D and a HDACI (B and R) are synergistic in in vitro models of human T-cell lymphoma, and may lead to a new platform for the treatment of these diseases. Disclosures: O'Connor: Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Pralatrexate Has Potent Activity Against Multiple Myeloma In Vitro and In Vivo, and Activity Correlates with Tumor RFC-1 and DHFR Expression

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1831-1831 ◽  
Author(s):  
Michael Mangone ◽  
Luigi Scotto ◽  
Enrica Marchi ◽  
Owen A. O'Connor ◽  
Hearn J. Cho
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Research Funding ◽  
Folate Metabolism ◽  
Nog Mice ◽  
Induced Apoptosis ◽  
Dose Dependent ◽  
Functional Biomarkers

Abstract Abstract 1831 Multiple myeloma (MM) is the second most common hematologic malignancy. Although there are effective new agents that can induce remission, relapse is inevitable and the disease is currently incurable. Progress in the treatment of this disease demands development of novel therapeutics and identification of functional biomarkers that may be used to distinguish tumors that are susceptible to specific targeted agents, creating a “personalized” therapeutic strategy for individual patients. We investigated these principles with anti-folates, which are not commonly used in MM but have demonstrated activity in this disease. Pralatrexate (PDX, 10-propargyl 10-deazaaminopterin) is a folate analogue that was rationally designed to have high affinity for Reduced Folate Carrier (RFC)-1, an oncofetal protein expressed in many cancers that actively transports folates into cells. PDX induced dose-dependent apoptotic cell death in a subset of human myeloma cell lines (HMCL) and CD138+ MM cells isolated from a clinical specimen. In sensitive cell lines, PDX exhibited 10-fold greater potency compared to the structurally related drug methotrexate (MTX). PDX induced dose-dependent, intrinsic apoptosis in sensitive HMCLs, characterized by cleavage of caspase-3 and -9 and accompanied by the loss of full-length Mcl-1, a Bcl-2 family protein that plays a critical role in drug-induced apoptosis in MM. Furthermore, the activity of PDX is not abrogated by the presence of exogenous interleukin-6 or by co-culture with HS-5 bone marrow stromal cells, both of which exert powerful survival effects on MM cells and can antagonize apoptosis in response to some cytotoxic chemotherapy drugs. Sensitivity to PDX-induced apoptosis correlated with higher relative levels of RFC-1 mRNA in sensitive compared to resistant HMCL. Resistant HMCL also exhibited a dose-dependent up-regulation of dihydrofolate reductase (DHFR) protein, a primary molecular target for anti-folates, in response to PDX exposure, whereas sensitive HMCL did not. These changes in functional folate metabolism biomarkers, high baseline RFC-1 expression and upregulation of DHFR in response to PDX, appeared to be mutually exclusive to sensitive or resistant HMCL, respectively. Importantly, PDX was also effective against sensitive HMCL in vivo in a novel mouse xenograft model. NOD/Shi-scid/IL-2Rγnull (NOG) mice were inoculated with MM.1s HMCL stably transduced to express both GFP and luciferase (GFP-luc). GFP-luc MM.1s cells engrafted into the long bones, pelvis, and vertebral column of NOG mice within 4–7 days after injection of cells, as assessed by in vivo bioluminescent imaging. Treatment with PDX resulted in a significant reduction in tumor burden after two doses. These results demonstrate that PDX has potent anti-myeloma activity in vitro and in vivo, and that RFC-1 expression and DHFR upregulation are robust functional biomarkers that may identify patients who are likely to benefit from PDX therapy. These data support further exploration of PDX therapy in clinical trials for MM and investigation of folate metabolism biomarkers as indices for treatment with this class of drugs. Improved anti-folates such as PDX are a promising class of agents that may be a valuable addition to the arsenal against MM. Disclosures: O'Connor: Celgene: Consultancy, Research Funding; Merck: Research Funding; Novartis: Research Funding; Spectrum: Research Funding.

scholarly journals Dual MAPK and HDAC inhibition rewires the apoptotic rheostat to trigger colorectal cancer cell death

2021 ◽  
Author(s):  
Laura J. Jenkins ◽  
Ian Y. Luk ◽  
Michelle Palmieri ◽  
W. Douglas Fairlie ◽  
Erinna F. Lee ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Histone Deacetylase Inhibitors ◽  
Hdac Inhibition ◽  
Erk Mapk ◽  
Cancer Cell Death ◽  
Treatment Paradigm ◽  
Apoptotic Proteins

AbstractThe EGFR/RAS/MEK/ERK signalling pathway (ERK/MAPK) is hyper-activated in most colorectal cancers (CRCs). A current limitation of inhibitors of this pathway is that they primarily induce cytostatic effects in CRC cells. Nevertheless, these drugs do induce expression of pro-apoptotic factors, suggesting they may prime CRC cells to undergo apoptosis. As histone deacetylase inhibitors (HDACi) induce expression of multiple pro-apoptotic proteins, we examined whether they could synergize with ERK/MAPK inhibitors to trigger CRC cell apoptosis. Combined MEK/ERK and HDAC inhibition synergistically induced apoptosis in CRC cell lines and patient-derived tumour organoids in vitro, and attenuated Apc-initiated adenoma formation in vivo. Mechanistically this effect was mediated through induction of the BH3-only pro-apoptotic proteins BIM and BMF. Importantly, we demonstrate that this treatment paradigm can be tailored to specific MAPK genotypes in CRCs, by combining HDACi with EGFR, KRASG12C or BRAFV600 inhibitors in KRAS/BRAFWT; KRASG12C, BRAFV600E CRC cell lines respectively. These findings identify a novel ERK/MAPK genotype-targeted treatment paradigm for colorectal cancer.

scholarly journals CBMT-31. HDAC INHIBITION DIMINISHES THE GROWTH OF ENDOGENOUS IDH MUTANT GLIOMAS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi39-vi40
Author(s):  
Lubayna Elahi ◽  
Matthew Garrett ◽  
Lea Guo ◽  
Michael Condro ◽  
Riki Kawaguchi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Transcriptional Repression ◽  
Glioma Cell ◽  
Histone Deacetylase Inhibitors ◽  
Sequencing Analysis ◽  
Hdac Inhibition ◽  
In Vivo Experiments ◽  
Glioma Cell Lines

Abstract Histone deacetylase inhibitors (HDACi’s) have emerged as a promising class of drugs for treatment of malignancies such as glioblastoma (GBM). Several studies have demonstrated the anti-tumor property of HDACi’s against GBM in both in vitro and in vivo experiments. Nonetheless, in clinical trials, HDACi only marginally increased overall survival of patients with GBM. The mixed results of trials with HDACi’s in glioma have prompted us to hypothesize that improved selection of patients by tumor characteristics could enhance the efficacy of therapy. We specifically tested the effects of valproic acid (VPA), a HDACi and an antiepileptic drug against IDH mutant gliomas. We have previously demonstrated that our IDH mutant glioma cell lines have gene expression and methylation patterns highly similar to IDH mutant tumors in situ. Mutant IDH1 alters the epigenetic landscape of gliomas leading to the hypermethylation phenotype and transcriptional repression of genes. This repression of genes may contribute to tumorigenesis and progression of IDH mutant gliomas. We found that VPA inhibits the growth of patient-derived IDH1 mutant glioma lines. In addition, RNA sequencing analysis of vehicle and VPA-treated IDH1 mutant glioma cells showed de-repression of several genes previously shown to be downregulated in IDH1 mutant glioma cell lines. We also treated cells with another HDACi LBH589 and found that both VPA and LBH589 upregulates similar gene sets suggesting that HDAC inhibition promotes de-repression of previously repressed genes. Ongoing studies are aimed at determining the molecular mechanism by which VPA regulates the growth of IDH1 mutant tumors.

scholarly journals Inhibition of Cyclin D1 Expression in Human Glioblastoma Cells is Associated with Increased Temozolomide Chemosensitivity

2018 ◽  
Vol 51 (6) ◽  
pp. 2496-2508 ◽  
Author(s):  
Danfeng Zhang ◽  
Dawei Dai ◽  
Mengxia Zhou ◽  
Zhenxing Li ◽  
Chunhui Wang ◽  
...  
Keyword(s):  
Malignant Glioma ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Effective Means ◽  
Malignant Gliomas ◽  
Glioma Cells ◽  
Normal Brain ◽  
Induced Apoptosis

Background/Aims: Cyclin D1 (CCND1) is frequently overexpressed in malignant gliomas. We have previously shown ectopic overexpression of CCND1 in human malignant gliomas cell lines. Methods: Quantitative reverse transcriptase PCR (qRT-PCR) and Western Blot (WB) was performed to investigate the expression of CCND1 in glioma tissues and cell lines. The biological function of CCND1 was also investigated through knockdown and overexpression of BCYRN1 in vitro. Results: Here we reported that CCND1 expression was positively associated with the pathological grade and proliferative activity of astrocytomas, as the lowest expression was found in normal brain tissue (N = 3) whereas the highest expression was in high-grade glioma tissue (N = 25). Additionally, we found that the expression level of CCND1 was associated with IC50 values in malignant glioma cell lines. Forced inhibition of CCND1 increased temozolomide efficacy in U251 and SHG-44 cells. After CCND1 overexpression, the temozolomide efficacy decreased in U251 and SHG-44 cells. Colony survival assay and apoptosis analysis confirmed that CCND1 inhibition renders cells more sensitive to temozolomide treatment and temozolomide-induced apoptosis in U251 and SHG-44 cells. Inhibition of P-gp (MDR1) by Tariquidar overcomes the effects of CCND1 overexpression on inhibiting temozolomide-induced apoptosis. Inhibition of CCND1 inhibited cell growth in vitro and in vivo significantly more effectively after temozolomide treatments than single temozolomide treatments. Finally, inhibition of CCND1 in glioma cells reduced tumor volume in a murine model. Conclusion: Taken together, these data indicate that CCND1 overexpression upregulate P-gp and induces chemoresistance in human malignant gliomas cells and that inhibition of CCND1 may be an effective means of overcoming CCND1 associated chemoresistance in human malignant glioma cells.

scholarly journals In Vivo and In Vitro Studies of Cytosolic Phospholipase A2 Expression in Helicobacter pyloriInfection

2001 ◽  
Vol 69 (9) ◽  
pp. 5857-5863 ◽  
Author(s):  
Gerardo Nardone ◽  
Eileen L. Holicky ◽  
James R. Uhl ◽  
Lina Sabatino ◽  
Stefania Staibano ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Protein Expression ◽  
Cell Lines ◽  
Cellular Adhesion ◽  
Cancer Pathogenesis ◽  
Cellular Expression ◽  
Epithelial Cell Lines ◽  
H Pylori

ABSTRACT Modifications of mucosal phospholipids have been detected in samples from patients with Helicobacter pylori-positive gastritis. These alterations appear secondary to increased phospholipase A2 activity (PLA2). The cytosolic form of this enzyme (cPLA2), normally involved in cellular signaling and growth, has been implicated in cancer pathogenesis. The aim of this study was to investigate cPLA2 expression and PLA2 activity in the gastric mucosae of patients with and without H. pylori infection. In gastric biopsies from 10H. pylori-positive patients, cPLA2 levels, levels of mRNA as determined by reverse transcriptase PCR, levels of protein as determined by immunohistochemistry, and total PLA2 activity were higher than in 10 H. pylori-negative gastritis patients. To clarify whether H. pylori had a direct effect on the cellular expression of cPLA2, we studied cPLA2 expression in vitro with different human epithelial cell lines, one from a patient with larynx carcinoma (i.e., HEp-2 cells) and two from patients with gastric adenocarcinoma (i.e., AGS and MKN 28 cells), incubated with differentH. pylori strains. The levels of cPLA2, mRNA, and protein expression were unchanged in Hep-2 cells independently of cellular adhesion or invasion of the bacteria. Moreover, no change in cPLA2 protein expression was observed in AGS or MKN 28 cells treated with wild-type H. pylori. In conclusion, our study shows increased cPLA2 expression and PLA2activity in the gastric mucosae of patients with H. pyloriinfection and no change in epithelial cell lines exposed to H. pylori.

scholarly journals Cped1 promotes chicken SSCs formation with the aid of histone acetylation and transcription factor Sox2

2018 ◽  
Vol 38 (5) ◽  
Author(s):  
Chen Zhang ◽  
Fei Wang ◽  
Qisheng Zuo ◽  
Changhua Sun ◽  
Jing Jin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Histone Acetylation ◽  
Histone Deacetylase Inhibitors ◽  
Embryonic Stem ◽  
Control Area ◽  
Luciferase Reporter ◽  
Marker Genes

Spermatogonial stem cells (SSCs) may apply to gene therapy, regenerative medicine in place of embryonic stem cells (ESCs). However, the application of SSCs was severely limited by the low induction efficiency and the lack of thorough analysis of the regulatory mechanisms of SSCs formation. Current evidences have demonstrated multiple marker genes of germ cells, while genes that specifically regulate the formation of SSCs have not been explored. In our study, cadherin-like and PC-esterase domain containing 1 (Cped1) expressed specifically in SSCs based on RNA-seq data analysis. To study the function of Cped1 in the formation of SSCs, we successfully established a CRISPR/Cas9 knockout system. The gene disruption frequency is 37% in DF1 and 25% in ESCs without off-target effects. Knockout of Cped1 could significantly inhibit the formation of SSCs in vivo and in vitro. The fragment of −1050 to −1 bp had the activity as Cped1 gene promoter. Histone acetylation could regulate the expression of Cped1. We added 5-azaeytidi (DNA methylation inhibitors) and TSA (histone deacetylase inhibitors) respectively during the cultivation of SSCs. TSA was validated to promote the transcription of Cped1. Dual-luciferase reporter assay revealed that active control area of the chicken Cped1 gene is −296 to −1 bp. There are Cebpb, Sp1, and Sox2 transcription factor binding sites in this region. Point-mutation experiment results showed that Sox2 negatively regulates the transcription of Cped1. Above results demonstrated that Cped1 is a key gene that regulates the formation of SSCs. Histone acetylation and transcription factor Sox2 participate in the regulation of Cped1.

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