Ischemic preconditioning activates MAPKAPK2 in the isolated rabbit ...
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2000 Feb 4;86(2):144-51.Ischemic preconditioning activates MAPKAPK2 in the isolated rabbit heart: evidence for involvement of p38 MAPK.1, , , , , , .1Department of Physiology, University of South Alabama, Mobile, AL , USA.AbstractRecent studies suggest that p38 mitogen-activated protein kinase (MAPK) may be involved in ischemic preconditioning (PC). To further test this possibility, the regulation of MAPK-activated protein kinase 2 (MAPKAPK2), a kinase immediately downstream from p38 MAPK, and the activity of c-Jun NH(2)-terminal kinase (JNK), a second MAPK, were examined in preconditioned hearts. Isolated, perfused rabbit hearts were subjected to 20 to 30 minutes of global ischemia. Ventricular biopsies before treatment and after 20 minutes of ischemia were homogenized, and the activities of MAPKAPK2 and JNK were evaluated. For the MAPKAPK2 experiments, 7 groups were studied, as follows: p hearts treated with 500 nmol/L R(-) N(6)-(2-phenylisopropyl) adenosine (PIA), an A(1)-adenos preconditioned hearts pretreated with 100 micromol/L 8-(p-sulfophenyl) theophylline (SPT), an adenosine preconditioned hearts also treated with SB 203580, a potent inhibitor of p38 MAPK hearts treated with 50 ng/mL anisomycin (a p38 MAPK/JNK activator); and hearts treated with both anisomycin (50 ng/mL) and the tyrosine kinase inhibitor genistein (50 micromol/L). MAPKAPK2 activity was not altered in control hearts after 20 minutes of global ischemia. By contrast, there was a 3.8-fold increase in activity during ischemia in preconditioned hearts. Activation of MAPKAPK2 in preconditioned hearts was blocked by both SPT and SB 203580. MAPKAPK2 activity during ischemia increased 3.5-fold and 3.3-fold in hearts pretreated with PIA or anisomycin, respectively. MAPKAPK2 activation during ischemia in hearts pretreated with anisomycin was blocked by genistein. In separate hearts, anisomycin mimicked the anti-infarct effect of PC, and that protection was abolished by genistein. JNK activity was measured in control and preconditioned hearts. There was a comparable, modest decline in activity during 30 minutes of global ischemia in both groups. As a positive control, a third group of hearts was treated with anisomycin before global ischemia, and in these, JNK activity increased by 290% above baseline. These results confirm that the p38 MAPK/MAPKAPK2 pathway is activated during ischemia only if the heart is in a preconditioned state. These data further support p38 MAPK as an important signaling component in ischemic PC.PMID:
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External link. Please review our .The MDS1-EVI1 gene complex as a retrovirus integration site: impact...
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):439-49. doi: 10.1038/sj.mt.6300372. Epub
2008 Jan 29.The MDS1-EVI1 gene complex as a retrovirus integration site: impact on behavior of hematopoietic cells and implications for gene therapy.1, .1Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.AbstractGene therapy trials have been performed with virus-based vectors that have the ability to integrate permanently into genomic DNA and thus allow prolonged expression of corrective genes after transduction of hematopoietic stem and progenitor cells. Adverse events observed during the X-linked severe combined immunodeficiency gene therapy trial revealed a significant risk of genotoxicity related to retrovirus vector integration and activation of adjacent proto-oncogenes, with several cases of T-cell leukemia linked to vector activation of the LMO2 gene. In patients with chronic granulomatous disease (CGD), rhesus macaques, and mice receiving hematopoietic stem and progenitor cells transduced with retrovirus vectors, a highly non-random pattern of vector integration has been reported. The most striking finding has been overrepresentation of integrations in one specific genomic locus, a complex containing the MDS1 and the EVI1 genes. Most evidence suggests that this overrepresentation is primarily due to a modification of primitive myeloid cell behavior by overexpression of EVI1 or MDS1-EVI1, as opposed to a specific predilection for integration at this site. Three different proteins can be produced from this complex locus: MDS1, MDS1-EVI1, and EVI1. This review will summarize current knowledge regarding this locus and its gene products, with specific focus on issues with relevance to gene therapy, leukemogenesis, and hematopoiesis. Insights into the mechanisms that result in altered hematopoiesis and leukemogenesis when this locus is dysregulated could improve the safety of gene therapy in the future.PMID:
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External link. Please review our .EVI1 splice variants modulate functional responses in ovarian cance...
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):647-68. doi: 10.1016/j.molonc.. Epub
2013 Mar 5.EVI1 splice variants modulate functional responses in ovarian cancer cells.1, , , , , .1Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL 33620, USA.AbstractAmplification of 3q26.2, found in many cancer lineages, is a frequent and early event in ovarian cancer. We previously defined the most frequent region of copy number increase at 3q26.2 to EVI1 (ecotropic viral integration site-1) and MDS1 (myelodysplastic syndrome 1) (aka MECOM), an observation recently confirmed by the cancer genome atlas (TCGA). MECOM is increased at the DNA, RNA, and protein level and likely contributes to patient outcome. Herein, we report that EVI1 is aberrantly spliced, generating multiple variants including a Del(190-515) variant (equivalent to previously reported) expressed in &90% of advanced stage serous epithelial ovarian cancers. Although EVI1(Del190-515) lacks ~70% of exon 7, it binds CtBP1 as well as SMAD3, important mediators of TGFβ signaling, similar to wild type EVI1. This contrasts with EVI1 1-268 which failed to interact with CtBP1. Interestingly, the EVI1(Del190-515) splice variant preferentially localizes to PML nuclear bodies compared to wild type and EVI1(Del427-515). While wild type EVI1 efficiently repressed TGFβ-mediated AP-1 (activator protein-1) and plasminogen activator inhibitor-1 (PAI-1) promoters, EVI1(Del190-515) elicited a slight increase in both promoter activities. Expression of EVI1 and EVI1(Del427-515) (but not EVI1(Del190-515)) in OVCAR8 ovarian cancer cells increased cyclin E1 LMW expression and cell cycle progression. Furthermore, knockdown of specific EVI1 splice variants (both MDS1/EVI1 and EVI1(Del190-515)) markedly increased claudin-1 mRNA and protein expression in HEY ovarian and MDA-MB-231 breast cancer cells. Changes in claudin-1 were associated with alterations in specific epithelial-mesenchymal transition markers concurrent with reduced migratory potential. Collectively, EVI1 is frequently aberrantly spliced in ovarian cancer with specific forms eliciting altered functions which could potentially contribute to ovarian cancer pathophysiology.Copyright (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.PMID:
[PubMed - indexed for MEDLINE] PMCID: PMC3805042 Identification of aberrant EVI1 splice forms in ovarian cancers. (A) Schematic representation of the MECOM locus displaying generation of the MDS1/EVI1 and EVI1 splice forms. MDS1 is composed of 4 exons whereas EVI1 is composed of 16 exons. MDS1/EVI1 is comprised of Exon I and II from MDS1 as well as Exon II – XVI from EVI1. EVI1 is comprised of Exon III to Exon XVI. (B) Various aberrant splice forms identified in ovarian cancer patients are shown. Exon arrangement of EVI1 (16 exons) with the various zinc finger regions, CtBP1 binding motif, repressor domain, potential nuclear localization signals (NLS), as well as start and stop codon are indicated. Regions that were found to be deleted in the splice variants identified are indicated by hatched boxes in Exon VII. (C) A sequence chromatogram for EVI1Del190–515 is shown on the left panel and the vertical line denotes the Exon VI to the cryptic internal region of Exon VII splice junction site compared to the wild type EVI1 sequence chromatograms where the vertical line denotes the Exon VI to Exon VII splice junction. The right panel depicts the exon arrangement of EVI1Del190–515 showing the donor splice site and the cryptic acceptor splice site resulting in loss of a region containing a portion of the repressor domain and the SMAD2/3 binding site. (D) A sequence chromatogram for EVI1Del427–515 is shown on the left panel and the vertical line denotes the internal cryptic donor site in internal region of Exon VII to cryptic acceptor site in internal region of Exon VII compared to the wild type EVI1 sequence chromatograms where the vertical line shows the wild type Exon VII read-through sequence. The right panel depicts the exon arrangement of EVI1Del427–515 showing the cryptic donor splice site and cryptic acceptor splice site resulting in loss of a region containing partial of the repressor domain and the SMAD2/3 binding site.Mol Oncol. ):647-668.Increased EVI1Del190–515 splice variant in advanced stage serous epithelial ovarian cancers. (A) The location of the qPCR probes specific for wild type EVI1, EVI1Del427–515, and EVI1Del190–515 are shown (top panel). Validation of the custom-made qPCR probes was performed using RNA isolated from stable retrovirally infected T29 cells with control pLEGFP-C1, wild type EVI1 EGFP, EVI1Del190–515 EGFP, or EVI1Del427–515 EGFP (bottom panels). (B) Measurement of wild type EVI1, EVI1Del190–515, and EVI1Del427–515 in serous epithelial ovarian cancers was performed by qPCR with the specific qPCR probes/primers. Results are expressed as RNA fold increases relative to normal ovarian surface epithelium (OSE) (left panel). In the top right panel, the same data is presented but ungrouped to indicate stage I, stage II, stage III, and stage IV serous epithelial ovarian cancers as well as relative expression changes in individual cancers. The bottom right panel represents the absolute RNA quantitation of wild type EVI1 (using Exon VII probe) and Del190–515 for OSE, stage I, stage II, stage III, and stage IV ovarian serous epithelial carcinomas. (C) Western analysis of ovarian cell lines (T80, T29, OVCAR8, OVCA429, HEY, and SKOV3 cells) using a rabbit polyclonal antibody against EVI1 detecting multiple forms of EVI1 including Del190–515 (upper panel). Real-time PCR analyses of EVI1 splice forms in the ovarian cell lines (middle panel). Western analysis of various stages of ovarian cancer (stage II, stage III, and stage IV serous epithelial ovarian cancers) using a rabbit polyclonal EVI1 antibody (generated by Dr. James Ihle). Irrelevant lanes were deletes as marked in gray dotted vertical lines (bottom panel).Mol Oncol. ):647-668.EVI1Del190–515 binds CtBP1 and SMAD3 with increased localization to PML nuclear subdomains in ovarian cells. (A) Western analysis of transient overexpression of EGFP fusion proteins of wild type EVI1, EVI1Del427–515, EVI1Del190–515, EVI1 Zn Del 1–7, EVI1 Zn Del 8–10, and EVI1 1–268 (expressing amino acids 268) mutants in T29 cells was performed using GFP mouse monoclonal antibody (top panel) and an N-terminal EVI1 antibody (bottom panel). (B) Localization of EVI1 and splice/deletion forms in T29 cells by immunofluorescence staining for CtBP1. (C) Co-immunoprecipitation of CtBP1 with EGFP-tagged EVI1 was performed using an anti-GFP polyclonal antibody with lysates from cells expressing EVI1 and deletion/mutant EVI1 forms (as EGFP fusions) followed by western blotting analyses using monoclonal antibody against GFP and CtBP1 monoclonal antibodies. (D) Overexpression of PML4 and EVI1 splice variants in T29 cells and immunofluorescence staining for PML4.Mol Oncol. ):647-668.EVI1Del190–515 alters TGFβ-mediated transcriptional regulation of the PAI-1 promoter in ovarian cells. (A) Co-immunoprecipitation of SMAD3 with EVI1 was performed using an anti-FLAG antibody with lysates from cells expressing EVI1 and EVI1Del190–515 (as EGFP fusions) and FLAG-SMAD3 followed by western analysis using EVI1 and SMAD3 polyclonal antibodies. The inputs represent 10% of the total. Based on densitometric analyses, between 1 and 5% of SMAD2/3 immunoprecipitated with EVI1. (B–C) Nucleofector transfection was performed on T29 cells with EVI1, EVI1Del427–515, or EVI1Del190–515 (5 μg) in combination with CAGA (1 μg) (B) or AP-1 (1 μg) (C) using Renilla luciferase to normalize (0.05 μg). Cells were re-seeded 6 h post-transfection, allowed to adhere for 6 h, and serum starved/treated with 50 pM TGFβ. 50 pM TGFβ was chosen as being on the linear portion of the dose curve. The following day (24 h post-transfection), cells were harvested in passive lysis buffer and assessed for luciferase activity using Dual Luciferase Assay kit.Mol Oncol. ):647-668.High expressing EVI1 and EVI1Del427–515 modulate cyclin E1 expression. (A) Western analyses is shown from lysates isolated from overexpressed EVI1 splice variants in T29 and OVCAR8 cells (retrovirally infected to generate two independent parental cell populations). (B) Reverse phase protein array results displayed as heatmaps with T29 (left panel) and OVCAR8 (right panel). (C) Western analysis of cyclin E1 levels in OVCAR8 retroviral cell lines together with positive controls in MCF7 cells overexpressing full length cyclin E1, cyclin E1 LMW T1, and cyclin E1 LMW T2. (D) Cell cycle analyses results are shown graphically (left panel) and in tabular format (right panel) as a combination of two independent experiments.Mol Oncol. ):647-668.Knockdown of specific splice variants of EVI1 via siRNAs. (A) HEY cells were seeded at 325,000 cells/well and transfected with (1) non-targeting (control) siRNA (Ctsi), (2) siB, (3) si2kb, (4) si04, or (5) siME. Following 72 h post-transfection, cell lysates were collected and western analyses performed using the indicated antibodies. The data shown are representative of three independent experiments. (B) Densitometric analyses of N-cadherin and claudin-1 westerns presented in (A). (C) MDA-MB-231 cells were seeded at 1 million cells/well and transfected with (1) non-targeting (control) siRNA (Ctsi), (2) siB, (3) si2kb, (4) si04, or (5) siME. Following 72 h post-transfection, cell lysates were collected and western analyses performed using the indicated antibodies. The data shown are representative of three independent experiments. (D) Densitometric analyses of claudin-1 westerns presented in (C). (E) RNA was isolated from HEY cells transfected with control siRNA, siB, or siME for real-time PCR analyses for claudin-1, Twist, Snail, Slug, ZEB-1, ZEB-2, and N-cadherin. The data shown are representative of three independent experiments. (F) RNA was isolated from MDA-MB-231 cells transfected with control siRNA, siB, or siME for real-time PCR analyses for claudin-1, Snail, Slug, ZEB-1, ZEB-2, N-cadherin, and E-cadherin. The data shown are representative of three independent experiments.Mol Oncol. ):647-668.Reduced expression of MDS1/EVI1 and EVI1Del190–515 modulates cellular motility in HEY and MDA-MB-231 cells. (A) HEY cells were transfected with control siRNA or siME. Seventy-two hours post-transfection, Triton X-100 soluble and insoluble fractions were extracted and western analyses performed using indicated antibodies. The data shown are representative of two independent experiments. (B) MDA-MB-231 cells were transfected with control siRNA or siME. Seventy-two hours post-transfection, Triton X-100 soluble and insoluble fractions were extracted and western analyses performed using indicated antibodies. The data shown are representative of two independent experiments. (C) HEY and MDA-MB-231 cells were treated with control siRNA or siME followed by seeding into Boyden chambers to assess migratory potential. Following 18 h, migrated cells were stained with crystal violet and counted.Mol Oncol. ):647-668.Publication TypesMeSH TermsSubstancesSupplementary ConceptsGrant SupportFull Text SourcesOther Literature SourcesMedicalMiscellaneous
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