1 2247 112 EPIGENETIC MODULATION OF MACROPHAGE POLARIZATION PREVENTS LUMBAR DISC DEGENERATION. INFLAMMATION PLAYS AN ESSENTIAL ROLE IN THE DEVELOPMENT OF LUMBAR DISC DEGENERATION (LDD), ALTHOUGH THE EXACT EFFECTS OF MACROPHAGE SUBTYPES ON LDD REMAIN UNCLEAR. BASED ON PREVIOUS STUDIES, WE HYPOTHESIZED THAT M2-POLARIZATION OF LOCAL MACROPHAGES AND SIMULTANEOUS SUPPRESSION OF THEIR PRODUCTION OF FIBROTIC TRANSFORMING GROWTH FACTOR BETA 1 (TGFBETA1) COULD INHIBIT PROGRESSION OF LDD. THUS, WE APPLIED AN ORTHOTOPIC INJECTION OF ADENO-ASSOCIATED VIRUS (AAV) CARRYING SHRNA FOR DNA METHYLTRANSFERASE 1 (DNMT1) AND/OR SHRNA FOR TGFBETA1 UNDER A MACROPHAGE-SPECIFIC CD68 PROMOTER TO SPECIFICALLY TARGET LOCAL MACROPHAGES IN A MOUSE MODEL FOR LDD. WE FOUND THAT SHDNMT1 SIGNIFICANTLY REDUCED LEVELS OF THE PRO-INFLAMMATORY CYTOKINES TNFALPHA, IL-1BETA AND IL-6, SIGNIFICANTLY INCREASED LEVELS OF THE ANTI-INFLAMMATORY CYTOKINES IL-4 AND IL-10, SIGNIFICANTLY INCREASED M2 MACROPHAGE POLARIZATION, SIGNIFICANTLY REDUCED CELL APOPTOSIS IN THE DISC DEGENERATION ZONE AND SIGNIFICANTLY REDUCED LDD-ASSOCIATED PAIN. THE ANTI-APOPTOTIC AND ANTI-PAIN EFFECTS WERE FURTHER STRENGTHENED BY CO-APPLICATION OF SHTGFBETA1. TOGETHER, THESE DATA SUGGEST THAT M2 POLARIZATION OF MACROPHAGES INDUCED BY BOTH EPIGENETIC MODULATION AND SUPPRESSED PRODUCTION AND RELEASE OF TGFBETA1 FROM POLARIZED M2 MACROPHAGES, MAY HAVE A DEMONSTRABLE THERAPEUTIC EFFECT ON LDD. 2020 2 5088 34 PIPERLONGUMINE REGULATES EPIGENETIC MODULATION AND ALLEVIATES PSORIASIS-LIKE SKIN INFLAMMATION VIA INHIBITION OF HYPERPROLIFERATION AND INFLAMMATION. PSORIASIS IS AN AUTOIMMUNE SKIN DISEASE, WHERE CHRONIC IMMUNE RESPONSES DUE TO EXAGGERATED CYTOKINE SIGNALING, ABNORMAL DIFFERENTIATION, AND EVASION OF KERATINOCYTES APOPTOSIS PLAYS A CRUCIAL ROLE IN MEDIATING ABNORMAL KERATINOCYTES HYPERPROLIFERATION. FROM THE THERAPEUTIC PERSPECTIVE, THE MOLECULES WITH STRONG ANTI-PROLIFERATIVE AND ANTI-INFLAMMATORY PROPERTIES COULD HAVE TREMENDOUS RELEVANCE. IN THIS STUDY, WE DEMONSTRATED THAT PIPERLONGUMINE (PPL) TREATMENT EFFECTIVELY ABROGATED THE HYPERPROLIFERATION AND DIFFERENTIATION OF KERATINOCYTES BY INDUCING ROS-MEDIATED LATE APOPTOSIS WITH LOSS OF MITOCHONDRIAL MEMBRANE POTENTIAL. BESIDES, THE ARREST OF CELL CYCLE WAS FOUND AT SUB-G1 PHASE AS A RESULT OF DNA FRAGMENTATION. MOLECULARLY, INHIBITION OF STAT3 AND AKT SIGNALING WAS OBSERVED WITH A DECREASE IN PROLIFERATIVE MARKERS SUCH AS PCNA, KI67, AND CYCLIN D1 ALONG WITH ANTI-APOPTOTIC BCL-2 PROTEIN EXPRESSION. KERATIN 17 IS A CRITICAL REGULATOR OF KERATINOCYTE DIFFERENTIATION, AND IT WAS FOUND TO BE DOWNREGULATED WITH PPL SIGNIFICANTLY. FURTHERMORE, PROMINENT ANTI-INFLAMMATORY EFFECTS WERE OBSERVED BY INHIBITION OF LIPOPOLYSACCHARIDE (LPS)/IMIQUIMOD (IMQ)-INDUCED P65 NF-KAPPAB SIGNALING CASCADE AND STRONGLY INHIBITED THE PRODUCTION OF CYTOKINE STORM INVOLVED IN PSORIASIS-LIKE SKIN INFLAMMATION, THUS LED TO THE RESTORATION OF NORMAL EPIDERMAL ARCHITECTURE WITH REDUCTION OF EPIDERMAL HYPERPLASIA AND SPLENOMEGALY. IN ADDITION, PPL EPIGENETICALLY INHIBITED HISTONE-MODIFYING ENZYMES, WHICH INCLUDE HISTONE DEACETYLASES (HDACS) OF CLASS I (HDAC1-4) AND CLASS II (HDAC6) EVALUATED BY IMMUNOBLOTTING AND HDAC ENZYME ASSAY KIT. IN ADDITION, OUR RESULTS SHOW THAT PPL EFFECTIVELY INHIBITS THE NUCLEAR TRANSLOCATION OF P65 AND A HISTONE MODULATOR HDAC3, THUS SEQUESTERED IN THE CYTOPLASM OF MACROPHAGES. FURTHERMORE, PPL EFFECTIVELY ENHANCED THE PROTEIN-PROTEIN INTERACTIONS OF HDAC3 AND P65 WITH IKAPPABALPHA, WHICH WAS DISRUPTED BY LPS STIMULATION AND WERE EVALUATED BY CO-IP AND MOLECULAR MODELING. COLLECTIVELY, OUR FINDINGS INDICATE THAT PIPERLONGUMINE MAY SERVE AS AN ANTI-PROLIFERATIVE AND ANTI-INFLAMMATORY AGENT AND COULD SERVE AS A POTENTIAL THERAPEUTIC OPTION IN TREATING PSORIASIS. 2020 3 3527 27 IL-6 ENHANCES THE NUCLEAR TRANSLOCATION OF DNA CYTOSINE-5-METHYLTRANSFERASE 1 (DNMT1) VIA PHOSPHORYLATION OF THE NUCLEAR LOCALIZATION SEQUENCE BY THE AKT KINASE. THE EPIGENETIC PROGRAMMING OF GENOMIC DNA IS ACCOMPLISHED, IN PART, BY SEVERAL DNA CYTOSINE-5-METHYLTRANSFERASES THAT ACT BY COVALENTLY MODIFYING CYTOSINES WITH THE ADDITION OF A METHYL GROUP. THIS COVALENT MODIFICATION IS MAINTAINED BY THE DNA CYTOSINE-5-METHYLTRANSFERASE-1 ENZYME (DNMT1), WHICH IS CAPABLE OF ACTING IN CONCERT WITH OTHER SIMILAR ENZYMES TO SILENCE IMPORTANT TUMOR SUPPRESSOR GENES. IL-6 IS A MULTIFUNCTIONAL MEDIATOR OF INFLAMMATION, ACTING THROUGH SEVERAL MAJOR SIGNALING CASCADES, INCLUDING THE PHOSPHATIDYLINOSITOL-3-KINASE PATHWAY (PI-3-K), WHICH ACTIVATES PROTEIN KINASE B (AKT/PKB) DOWNSTREAM. HERE, WE SHOW THAT THE SUBCELLULAR LOCALIZATION OF DNMT1 CAN BE ALTERED BY THE ADDITION OF IL-6, INCREASING THE RATE OF NUCLEAR TRANSLOCATION OF THE ENZYME FROM THE CYTOSOLIC COMPARTMENT. THE MECHANISM OF NUCLEAR TRANSLOCATION OF DNMT1 IS GREATLY ENHANCED BY PHOSPHORYLATION OF THE DNMT1 NUCLEAR LOCALIZATION SIGNAL (NLS) BY PKB/AKT KINASE. MUTAGENIC ALTERATION OF THE TWO AKT TARGET AMINO ACIDS WITHIN THE NLS RESULTS IN A MAJOR LOSS OF DNMT1 NUCLEAR TRANSLOCATION, WHILE THE CREATION OF A "PHOSPHO-MIMIC" AMINO ACID (MUTATION TO ACIDIC RESIDUES) RESTORES THIS COMPARTMENTATION ABILITY. THESE OBSERVATIONS SUGGEST AN INTERESTING HYPOTHESIS REGARDING HOW MEDIATORS OF CHRONIC INFLAMMATION MAY DISTURB THE DELICATE BALANCE OF CELLULAR COMPARTMENTALIZATION OF IMPORTANT PROTEINS, AND REVEALS A POTENTIAL MECHANISM FOR THE INDUCTION OR ENHANCEMENT OF TUMOR GROWTH VIA ALTERATION OF THE COMPONENTS INVOLVED IN THE EPIGENETIC PROGRAMMING OF A CELL. 2007 4 5972 21 TET REPRESSION AND INCREASED DNMT ACTIVITY SYNERGISTICALLY INDUCE ABERRANT DNA METHYLATION. CHRONIC INFLAMMATION IS DEEPLY INVOLVED IN VARIOUS HUMAN DISORDERS, SUCH AS CANCER, NEURODEGENERATIVE DISORDERS, AND METABOLIC DISORDERS. INDUCTION OF EPIGENETIC ALTERATIONS, ESPECIALLY ABERRANT DNA METHYLATION, IS ONE OF THE MAJOR MECHANISMS, BUT HOW IT IS INDUCED IS STILL UNCLEAR. HERE, WE FOUND THAT EXPRESSION OF TET GENES, METHYLATION ERASERS, WAS DOWNREGULATED IN INFLAMED MOUSE AND HUMAN TISSUES, AND THAT THIS WAS CAUSED BY UPREGULATION OF TET-TARGETING MIRNAS SUCH AS MIR20A, MIR26B, AND MIR29C, LIKELY DUE TO ACTIVATION OF NF-KAPPAB SIGNALING DOWNSTREAM OF IL-1BETA AND TNF-ALPHA. HOWEVER, TET KNOCKDOWN INDUCED ONLY MILD ABERRANT METHYLATION. NITRIC OXIDE (NO), PRODUCED BY NOS2, ENHANCED ENZYMATIC ACTIVITY OF DNA METHYLTRANSFERASES (DNMTS), METHYLATION WRITERS, AND NO EXPOSURE INDUCED MINIMAL ABERRANT METHYLATION. IN CONTRAST, A COMBINATION OF TET KNOCKDOWN AND NO EXPOSURE SYNERGISTICALLY INDUCED ABERRANT METHYLATION, INVOLVING GENOMIC REGIONS NOT METHYLATED BY EITHER ALONE. THE RESULTS SHOWED THAT A VICIOUS COMBINATION OF TET REPRESSION, DUE TO NF-KAPPAB ACTIVATION, AND DNMT ACTIVATION, DUE TO NO PRODUCTION, IS RESPONSIBLE FOR ABERRANT METHYLATION INDUCTION IN HUMAN TISSUES. 2020 5 5114 25 PORPHYROMONAS GINGIVALIS LIPOPOLYSACCHARIDE STIMULATION IN HUMAN PERIODONTAL LIGAMENT STEM CELLS: ROLE OF EPIGENETIC MODIFICATIONS TO THE INFLAMMATION. PERIODONTITIS IS A CHRONIC ORAL INFLAMMATORY DISEASE PRODUCED BY BACTERIA. GINGIVAL RETRACTION AND BONE AND CONNECTIVE TISSUES RESORPTION ARE THE HALLMARKS OF THIS DISEASE. CHRONIC PERIODONTITIS MAY CONTRIBUTE TO THE RISK OF ONSET OR PROGRESSION OF NEUROINFLAMMATORY PATHOLOGICAL CONDITIONS, SUCH AS ALZHEIMER'S DISEASE. THE MAIN GOAL OF THE PRESENT STUDY WAS TO INVESTIGATE IF THE ROLE OF EPIGENETIC MODULATIONS IS INVOLVED IN PERIODONTITIS USING HUMAN PERIODONTAL LIGAMENT STEM CELLS (HPDLSCS) AS AN IN VITRO MODEL SYSTEM. HPDLSCS WERE TREATED WITH LIPOPOLYSACCHARIDE OF PORPHYROMONAS GINGIVALIS AND THE EXPRESSION OF PROTEINS ASSOCIATED WITH DNA METHYLATION AND HISTONE ACETYLATION, SUCH AS DNMT1 AND P300, RESPECTIVELY, AND INFLAMMATORY TRANSCRIPTION FACTOR NF-KB, WERE EXAMINED. IMMUNOFLUORESCENCE, WESTERN BLOT AND NEXT GENERATION SEQUENCING RESULTS DEMONSTRATED THAT P. GINGIVALIS LIPOPOLYSACCHARIDE SIGNIFICANTLY REDUCED DNA METHYLASE DNMT1, WHILE IT MARKEDLY UPREGULATED THE LEVEL OF HISTONE ACETYLTRANSFERASE P300 AND NF-KB IN HPDLSCS. OUR RESULTS SHOWED THAT P. GINGIVALIS LIPOPOLYSACCHARIDE MARKEDLY REGULATE THE GENES INVOLVED IN EPIGENETIC MECHANISM, WHICH MAY RESULT IN INFLAMMATION INDUCTION. WE PROPOSE THAT P. GINGIVALIS LIPOPOLYSACCHARIDE-TREATED HPDLSCS COULD BE A POTENTIAL IN VITRO MODEL SYSTEM TO STUDY EPIGENETICS MODULATIONS ASSOCIATED WITH PERIODONTITIS, WHICH MIGHT BE HELPFUL TO IDENTIFY NOVEL BIOMARKERS LINKED TO THIS ORAL INFLAMMATORY DISEASE. 2017 6 1618 21 DNA METHYLTRANSFERASE INHIBITORS INCREASE NOD-LIKE RECEPTOR ACTIVITY AND EXPRESSION IN A MONOCYTIC CELL LINE. BACKGROUND: THE INTRACELLULAR NOD-LIKE RECEPTOR (NLR) FAMILY OF PATHOGEN RECOGNITION RECEPTORS (PRRA) IS INVOLVED IN INITIATING THE INNATE IMMUNE RESPONSE OF WHICH NOD1 AND NOD2 ARE THE BEST-CHARACTERIZED MEMBERS. ABERRANT EXPRESSION OF NOD1 AND NOD2 HAS BEEN UNCOVERED IN A NUMBER OF CHRONIC INFLAMMATORY DISEASES, SUCH AS INFLAMMATORY BOWEL DISEASE AND RHEUMATOID ARTHRITIS. HOWEVER, THE MECHANISM UNDERLYING NOD1/NOD2 GENE EXPRESSION REGULATION IS STILL IN ITS INFANCY. EPIGENETIC MODIFICATIONS SUCH AS DNA METHYLATION AND HISTONE ACETYLATION REGULATE THE EXPRESSION OF GENES AND ALTERATIONS IN THEIR PATTERNS HAVE BEEN LINKED TO MANY INFLAMMATORY DISEASES. THIS STUDY INVESTIGATED WHETHER EPIGENETIC MODIFYING DRUGS AFFECT THE REGULATION OF NOD1/NOD2 ACTIVITY AND EXPRESSION. DNA METHYLTRANSFERASE INHIBITORS HAVE RECENTLY BEEN USED IN THE TREATMENT OF MYELODYSPLASTIC SYNDROME AND AS COMBINATION THERAPY IN CANCER BUT THE FULL EXTENT OF THEIR EFFECTS HAS NOT BEEN QUANTIFIED.METHODS: PHARMACOLOGICAL INHIBITION OF EPIGENETIC ENZYMES IN A HUMAN MONOCYTIC THP-1 CELL LINE WAS CARRIED OUT AND NOD1/NOD2 EXPRESSION AND PRO-INFLAMMATORY RESPONSES WERE QUANTIFIED.RESULTS: CELLS PRIMED WITH A DNA METHYLTRANSFERASE INHIBITOR (BUT NOT A HISTONE DEACETYLASE [HDAC] INHIBITOR) WERE FOUND TO BE CONSISTENTLY MORE RESPONSIVE TO NOD1/NOD2 STIMULATION AND HAD INCREASED BASAL EXPRESSION.CONCLUSION: THE NOVEL EXPERIMENTATION CARRIED OUT HERE SUGGESTS FOR THE FIRST TIME THAT NOD1/NOD2 RECEPTOR ACTIVITY AND EXPRESSION IN MONOCYTES ARE POSSIBLY REGULATED DIRECTLY BY DNA METHYLATION. 2022 7 4210 25 METFORMIN AND VITAMIN D MODULATE INFLAMMATION AND AUTOPHAGY DURING ADIPOSE-DERIVED STEM CELL DIFFERENTIATION. ADIPOSE-DERIVED STEM CELLS (ADSCS) CAME OUT FROM THE REGENERATIVE MEDICINE LANDSCAPE FOR THEIR ABILITY TO DIFFERENTIATE INTO SEVERAL PHENOTYPES, CONTRIBUTING TO TISSUE REGENERATION BOTH IN VITRO AND IN VIVO. DYSREGULATION IN STEM CELL RECRUITMENT AND DIFFERENTIATION DURING ADIPOGENESIS IS LINKED TO A CHRONIC LOW-GRADE INFLAMMATION AND MACROPHAGE INFILTRATION INSIDE THE ADIPOSE TISSUE, INSULIN RESISTANCE, CARDIOVASCULAR DISEASE AND OBESITY. IN THE PRESENT PAPER WE AIMED TO EVALUATE THE ROLE OF METFORMIN AND VITAMIN D, ALONE OR IN COMBINATION, IN MODULATING INFLAMMATION AND AUTOPHAGY IN ADSCS DURING ADIPOGENIC COMMITMENT. ADSCS WERE CULTURED FOR 21 DAYS IN THE PRESENCE OF A SPECIFIC ADIPOGENIC DIFFERENTIATION MEDIUM, TOGETHER WITH METFORMIN, OR VITAMIN D, OR BOTH. WE THEN ANALYZED THE EXPRESSION OF FOXO1 AND HEAT SHOCK PROTEINS (HSP) AND THE SECRETION OF PROINFLAMMATORY CYTOKINES IL-6 AND TNF-ALPHA BY ELISA. AUTOPHAGY WAS ALSO ASSESSED BY SPECIFIC WESTERN BLOT ANALYSIS OF ATG12, LC3B I, AND LC3B II EXPRESSION. OUR RESULTS SHOWED THE ABILITY OF THE CONDITIONED MEDIA TO MODULATE ADIPOGENIC DIFFERENTIATION, FINELY TUNING THE INFLAMMATORY RESPONSE AND AUTOPHAGY. WE OBSERVED A MODULATION IN HSP MRNA LEVELS, AND A SIGNIFICANT DOWNREGULATION IN CYTOKINE SECRETION. TAKEN TOGETHER, OUR FINDINGS SUGGEST THE POSSIBLE APPLICATION OF THESE MOLECULES IN CLINICAL PRACTICE TO COUNTERACT UNCONTROLLED LIPOGENESIS AND PREVENT OBESITY AND OBESITY-RELATED METABOLIC DISORDERS. 2021 8 194 30 ACETYLSHIKONIN SUPPRESSES INVASION OF PORPHYROMONAS GINGIVALIS?INFECTED YD10B ORAL CANCER CELLS BY MODULATING THE INTERLEUKIN-8/MATRIX METALLOPROTEINASE AXIS. THE DEVELOPMENT OF PHARMACEUTICAL AGENTS POSSESSING ANTI?INVASIVE AND ANTI?METASTATIC ABILITIES, AS WELL AS APOPTOTIC ACTIVITY, IS IMPORTANT IN DECREASING THE INCIDENCE AND RECURRENCE OF ORAL CANCER. CANCER CELLS ARE KNOWN TO ACQUIRE INVASIVENESS NOT ONLY THROUGH EPIGENETIC CHANGES, BUT ALSO FROM INFLAMMATORY STIMULI WITHIN THE TUMOR MICROENVIRONMENT. ACCORDINGLY, THE IDENTIFICATION OF AGENTS THAT CAN SUPPRESS THE INFLAMMATION?PROMOTED INVASIVENESS OF CANCER CELLS MAY BE IMPORTANT IN TREATING CANCER AND IMPROVING THE PROGNOSIS OF PATIENTS WITH CANCER. ACETYLSHIKONIN, A FLAVONOID WITH ANTI?INFLAMMATORY ACTIVITY, INHIBITS PROLIFERATION AND INDUCES APOPTOSIS OF ORAL CANCER CELLS. IN THE PRESENT STUDY, THE ANTI?INVASIVE EFFECT OF ACETYLSHIKONIN ON YD10B ORAL CANCER CELLS INFECTED WITH PORPHYROMONAS GINGIVALIS, A MAJOR PATHOGEN OF CHRONIC PERIODONTITIS, AND THE MECHANISMS INVOLVED WERE INVESTIGATED. FIRSTLY, WE EXAMINED WHETHER P. GINGIVALIS INFECTION INCREASED THE INVASIVENESS OF YD10B CELLS. RESULTS SUGGESTED THAT YD10B ORAL CANCER CELLS BECOME MORE AGGRESSIVE WHEN THEY ARE INFECTED WITH P. GINGIVALIS. SECONDLY, ACETYLSHIKONIN SIGNIFICANTLY INHIBITED THE INVASION OF P. GINGIVALIS?INFECTED YD10B CELLS BY SUPPRESSING IL?8 RELEASE AND IL?8?DEPENDENT MMP RELEASE. THESE DATA SUGGEST THAT ACETYLSHIKONIN MAY BE A USEFUL PREVENTIVE AND THERAPEUTIC CANDIDATE FOR ORAL CANCER THAT IS CHRONICALLY INFECTED WITH PERIODONTAL PATHOGENS. 2018 9 164 26 ABNORMAL HISTONE METHYLATION IS RESPONSIBLE FOR INCREASED VASCULAR ENDOTHELIAL GROWTH FACTOR 165A SECRETION FROM AIRWAY SMOOTH MUSCLE CELLS IN ASTHMA. VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF), A KEY ANGIOGENIC MOLECULE, IS ABERRANTLY EXPRESSED IN SEVERAL DISEASES INCLUDING ASTHMA WHERE IT CONTRIBUTES TO BRONCHIAL VASCULAR REMODELING AND CHRONIC INFLAMMATION. ASTHMATIC HUMAN AIRWAY SMOOTH MUSCLE CELLS HYPERSECRETE VEGF, BUT THE MECHANISM IS UNCLEAR. IN THIS STUDY, WE DEFINED THE MECHANISM IN HUMAN AIRWAY SMOOTH MUSCLE CELLS FROM NONASTHMATIC AND ASTHMATIC PATIENTS. WE FOUND THAT ASTHMATIC CELLS LACKED A REPRESSION COMPLEX AT THE VEGF PROMOTER, WHICH WAS PRESENT IN NONASTHMATIC CELLS. RECRUITMENT OF G9A, TRIMETHYLATION OF HISTONE H3 AT LYSINE 9 (H3K9ME3), AND A RESULTANT DECREASE IN RNA POLYMERASE II AT THE VEGF PROMOTER WAS CRITICAL TO REPRESSION OF VEGF SECRETION IN NONASTHMATIC CELLS. AT THE ASTHMATIC PROMOTER, H3K9ME3 WAS ABSENT BECAUSE OF FAILED RECRUITMENT OF G9A; RNA POLYMERASE II BINDING, IN ASSOCIATION WITH TATA-BINDING PROTEIN-ASSOCIATED FACTOR 1, WAS INCREASED; H3K4ME3 WAS PRESENT; AND SP1 BINDING WAS EXAGGERATED AND SUSTAINED. IN CONTRAST, DNA METHYLATION AND HISTONE ACETYLATION WERE SIMILAR IN ASTHMATIC AND NONASTHMATIC CELLS. THIS IS THE FIRST STUDY, TO OUR KNOWLEDGE, TO SHOW THAT AIRWAY CELLS IN ASTHMA HAVE ALTERED EPIGENETIC REGULATION OF REMODELING GENE(S). HISTONE METHYLATION AT GENES SUCH AS VEGF MAY BE AN IMPORTANT NEW THERAPEUTIC TARGET. 2012 10 2389 28 EPIGENETIC REPOLARIZATION OF T LYMPHOCYTES FROM CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS USING 5-AZA-2'-DEOXYCYTIDINE. T CELL IMMUNE DYSFUNCTION HAS AN IMPORTANT ROLE IN THE PROFOUND IMMUNE SUPPRESSION THAT CHARACTERIZES CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). IMPROPER POLARIZATION OF T CELLS HAS BEEN PROPOSED AS ONE OF THE MECHANISM INVOLVED. MOUNTING DATA IMPLICATES CHROMATIN REGULATION, NAMELY PROMOTER METHYLATION, IN THE PLASTICITY OF NAIVE HUMAN T CELLS. RECENT IN VITRO EVIDENCE INDICATES THAT THIS PLASTICITY MAY BE PHENOTYPICALLY ALTERED BY USING METHYLATION INHIBITORS WHICH ARE APPROVED FOR CLINICAL USE IN CERTAIN TYPES OF CANCER. THESE RESULTS BEG THE QUESTION: CAN THE INEFFECTIVE POLARIZATION OF T LYMPHOCYTES IN THE CONTEXT OF CLL BE EFFECTIVELY MODULATED USING METHYLATION INHIBITORS IN A SUSTAINABLE THERAPEUTIC FASHION? TO ANSWER THIS QUESTION OUR LABORATORY HAS STUDIED THE EFFECTS OF 5-AZA-2'-DEOXYCYTIDINE (5A2) IN HELPER AND CYTOTOXIC T LYMPHOCYTES FROM HEALTHY DONORS AND CLL PATIENTS IN WELL CHARACTERIZED MOLECULAR AND EPIGENETIC SIGNALING PATHWAYS INVOLVED IN EFFECTIVE POLARIZATION. MOREOVER, WE SOUGHT TO INVESTIGATE THE CONSEQUENCES OF METHYLATION INHIBITOR TREATMENT ON LYMPHOCYTE SURVIVAL, ACTIVATION INTENSITY, AND NAIVE CELL POLARIZATION. OUR DATA INDICATES THAT 5A2 TREATMENT CAN DEPOLARIZE TH2 CELLS TO EFFECTIVELY SECRETE INTERFERON GAMMA, SIGNAL VIA T-BET, AND ACHIEVE DEMETHYLATION OF CRITICAL TH1 SPECIFIC PROMOTERS. MOREOVER, WE DEMONSTRATE THAT 5A2 CAN FORCE TH1 POLARIZATION OF NAIVE T CELLS DESPITE A STRONG IL-4 STIMULI AND A LACK OF IL-12. IN CONCLUSION OUR DATA SEEKS TO DEFINE A MODALITY IN WHICH IMPROPER OR INEFFECTIVE T CELL POLARIZATION CAN BE ALTERED BY 5AZA AND COULD BE INCORPORATED IN FUTURE THERAPEUTIC INTERVENTIONS. 2011 11 5975 25 TET1 IS AN IMPORTANT TRANSCRIPTIONAL ACTIVATOR OF TNFALPHA EXPRESSION IN MACROPHAGES. ACTIVATION OF MACROPHAGES AND OVEREXPRESSION OF TNFALPHA IS ASSOCIATED WITH THE PATHOGENESIS OF CHRONIC INFLAMMATORY DISEASES. HOWEVER, THE MECHANISMS LEADING TO TNFALPHA OVEREXPRESSION ARE STILL UNKNOWN. 5-METHYLOCYTOSINE (5-MC) IS AN EPIGENETIC MODIFICATION THAT IS ASSOCIATED WITH SILENCED GENES. RECENT STUDIES SHOWED THAT IT IS CONVERTED TO 5-HYDROXYLMETHYLOCYTOSINE (5-HMC) AND REACTIVATES GENE EXPRESSION THROUGH THE ACTION OF THE FAMILY OF TEN-ELEVEN-TRANSLOCATION (TET1-3) ENZYMES. IN THIS STUDY, WE SHOW THAT 5-HMC LEVELS ARE INCREASED GLOBALLY AND SPECIFICALLY IN THE TNFALPHA PROMOTER DURING THE DIFFERENTIATION OF MONOCYTES TO MACROPHAGES. IN ADDITION, THE LEVELS OF 5-HMC ARE INCREASED UPON LPS STIMULATION OF MACROPHAGES. FURTHERMORE, CRIPSR STABLE KNOCKOUT OF TET1 DECREASES THE EXPRESSION OF TNFALPHA AND OTHER PRO-INFLAMMATORY CYTOKINES. IN CONCLUSION, WE SHOWED THAT TET1 CONTRIBUTES TO THE ACTIVATION OF MACROPHAGES POSSIBLY THROUGH REGULATION OF 5-HYDROXYMETHYLATION IN THE PROMOTER OF PRO-INFLAMMATORY CYTOKINE GENES. THE TET1 ENZYME COULD BE A PROMISING THERAPEUTIC TARGET TO INHIBIT THE PERSISTENT INFLAMMATION CAUSED BY MACROPHAGES IN CHRONIC INFLAMMATORY DISEASES. 2019 12 4742 25 NOVEL HISTONE MODIFICATIONS IN MICROGLIA DERIVED FROM A MOUSE MODEL OF CHRONIC PAIN. AS THE RESIDENT IMMUNE CELLS IN THE CENTRAL NERVOUS SYSTEM, MICROGLIA PLAY AN IMPORTANT ROLE IN THE MAINTENANCE OF ITS HOMEOSTASIS. DYSREGULATION OF MICROGLIA HAS BEEN ASSOCIATED WITH THE DEVELOPMENT AND MAINTENANCE OF CHRONIC PAIN. HOWEVER, THE RELEVANT MOLECULAR PATHWAYS REMAIN POORLY DEFINED. IN THIS STUDY, WE USED A MASS SPECTROMETRY-BASED PROTEOMIC APPROACH TO SCREEN POTENTIAL CHANGES OF HISTONE PROTEIN MODIFICATIONS IN MICROGLIA ISOLATED FROM THE BRAIN OF CONTROL AND CISPLATIN-INDUCED NEUROPATHIC PAIN ADULT C57BL/6J MALE MICE. WE IDENTIFIED SEVERAL NOVEL MICROGLIAL HISTONE MODIFICATIONS ASSOCIATED WITH PAIN, INCLUDING STATISTICALLY SIGNIFICANTLY DECREASED HISTONE H3.1 LYSINE 27 MONO-METHYLATION (H3.1K27ME1, 54.8% OF CONTROL) AND H3 LYSINE 56 TRI-METHYLATION (7.5% OF CONTROL), AS WELL AS A TREND SUGGESTING INCREASED H3 TYROSINE 41 NITRATION. WE FURTHER INVESTIGATED THE FUNCTIONAL ROLE OF H3.1K27ME1 AND FOUND THAT TREATMENT OF CULTURED MICROGLIAL CELLS FOR 4 CONSECUTIVE DAYS WITH 1-10 MUM OF NCDM-64, A POTENT AND SELECTIVE INHIBITOR OF LYSINE DEMETHYLASE 7A, AN ENZYME RESPONSIBLE FOR THE DEMETHYLATION OF H3K27ME1, DOSE-DEPENDENTLY ELEVATED ITS LEVELS WITH A GREATER THAN A TWO-FOLD INCREASE OBSERVED AT 10 MUM COMPARED TO VEHICLE-TREATED CONTROL CELLS. MOREOVER, PRETREATMENT OF MICE WITH NCDM-64 (10 OR 25 MG/KG/DAY, I.P.) PRIOR TO CISPLATIN TREATMENT PREVENTED THE DEVELOPMENT OF NEUROPATHIC PAIN IN MICE. THE IDENTIFICATION OF SPECIFIC CHROMATIN MARKS IN MICROGLIA ASSOCIATED WITH CHRONIC PAIN MAY YIELD CRITICAL INSIGHT INTO THE CONTRIBUTION OF MICROGLIA TO THE DEVELOPMENT AND MAINTENANCE OF PAIN, AND OPENS NEW AVENUES FOR THE DEVELOPMENT OF NOVEL NONOPIOID THERAPEUTICS FOR THE EFFECTIVE MANAGEMENT OF CHRONIC PAIN. 2022 13 6765 29 ZINC DEFICIENCY LEADS TO REDUCED INTERLEUKIN-2 PRODUCTION BY ACTIVE GENE SILENCING DUE TO ENHANCED CREMALPHA EXPRESSION IN T CELLS. BACKGROUND & AIMS: THE MICRONUTRIENT ZINC IS ESSENTIAL FOR PROPER IMMUNE FUNCTION. CONSEQUENTLY, ZINC DEFICIENCY LEADS TO IMPAIRED IMMUNE FUNCTION, AS SEEN IN DECREASED SECRETION OF INTERLEUKIN (IL)-2 BY T CELLS. ALTHOUGH THIS ASSOCIATION HAS BEEN KNOWN SINCE THE LATE 1980S, THE UNDERLYING MOLECULAR MECHANISMS ARE STILL UNKNOWN. ZINC DEFICIENCY AND REDUCED IL-2 LEVELS ARE ESPECIALLY FOUND IN THE ELDERLY, WHICH IN TURN ARE PRONE TO CHRONIC DISEASES. HERE, WE DESCRIBE A NEW MOLECULAR LINK BETWEEN ZINC DEFICIENCY AND REDUCED IL-2 EXPRESSION IN T CELLS. METHODS: THE EFFECTS OF ZINC DEFICIENCY WERE FIRST INVESTIGATED IN VITRO IN THE HUMAN T CELL LINES JURKAT AND HUT-78 AND COMPLEMENTED BY IN VIVO DATA FROM ZINC-SUPPLEMENTED PIGS. A SHORT- AND LONG-TERM MODEL FOR ZINC DEFICIENCY WAS ESTABLISHED. ZINC LEVELS WERE DETECTED BY FLOW CYTOMETRY AND EXPRESSION PROFILES WERE INVESTIGATED ON THE MRNA AND PROTEIN LEVEL. RESULTS: THE EXPRESSION OF THE TRANSCRIPTION FACTOR CAMP-RESPONSIVE-ELEMENT MODULATOR ALPHA (CREMALPHA) IS INCREASED DURING ZINC DEFICIENCY IN VITRO, DUE TO INCREASED PROTEIN PHOSPHATASE 2A (PP2A) ACTIVITY, RESULTING IN DECREASED IL-2 PRODUCTION. ADDITIONALLY, ZINC SUPPLEMENTATION IN VIVO REDUCED CREMALPHA LEVELS CAUSING INCREASED IL-2 EXPRESSION. ON EPIGENETIC LEVELS INCREASED CREMALPHA BINDING TO THE IL-2 PROMOTER IS MEDIATED BY HISTONE DEACETYLASE 1 (HDAC1). THE HDAC1 ACTIVITY IS INHIBITED BY ZINC. MOREOVER, DEACETYLATION OF THE ACTIVATING HISTONE MARK H3K9 WAS INCREASED UNDER ZINC DEFICIENCY, RESULTING IN REDUCED IL-2 EXPRESSION. CONCLUSIONS: WITH THE TRANSCRIPTION FACTOR CREMALPHA A MOLECULAR LINK WAS UNCOVERED, CONNECTING ZINC DEFICIENCY WITH REDUCED IL-2 PRODUCTION DUE TO ENHANCED PP2A AND HDAC1 ACTIVITY. 2021 14 3660 30 INDUCTION OF HEPATIC DIFFERENTIATION OF MOUSE BONE MARROW STROMAL STEM CELLS BY THE HISTONE DEACETYLASE INHIBITOR VPA. BONE MARROW STROMAL STEM CELLS (BMSSCS) MAY HAVE POTENTIAL TO DIFFERENTIATE IN VITRO AND IN VIVO INTO HEPATOCYTES. HERE, WE INVESTIGATED THE EFFECTS OF VALPROIC ACID (VPA) INVOLVED IN EPIGENETIC MODIFICATION, A DIRECT INHIBITOR OF HISTONE DEACETYLASE, ON HEPATIC DIFFERENTIATION OF MOUSE BMSSCS. FOLLOWING THE TREATMENT OF 2.5 MM VPA FOR 72 HRS, THE IN VITRO EXPANDED, HIGHLY PURIFIED AND FUNCTIONALLY ACTIVE MOUSE BMSSCS FROM BONE MARROW WERE EITHER EXPOSED TO SOME WELL-DEFINED CYTOKINES AND GROWTH FACTORS IN A SEQUENTIAL WAY (FIBROBLAST GROWTH FACTOR-4 [FGF-4], FOLLOWED BY HGF, AND HGF + OSM + ITS + DEXAMETHASONE, RESEMBLING THE ORDER OF SECRETION DURING LIVER EMBRYOGENESIS) OR TRANSPLANTED (CAUDAL VEIN) IN MICE SUBMITTED TO A PROTOCOL OF CHRONIC INJURY (CHRONIC I.P. INJECTION OF CCL4). ADDITIONAL EXPOSURE OF THE CELLS TO VPA CONSIDERABLY IMPROVED THE IN VITRO DIFFERENTIATION, AS DEMONSTRATED BY A MORE HOMOGENEOUS CELL POPULATION EXHIBITED EPITHELIAL MORPHOLOGY, INCREASING EXPRESSION OF HEPATIC SPECIAL GENES AND ENHANCED HEPATIC FUNCTIONS. FURTHER MORE, IN VIVO RESULTS INDICATE THAT THE PRE-TREATMENT OF VPA SIGNIFICANTLY INCREASED THE HOMING EFFICIENCY OF BMSSCS TO THE SITE OF LIVER INJURY AND, ADDITIONALLY, FOR SUPPORTING HEPATIC DIFFERENTIATION AS WELL AS IN VITRO. WE HAVE DEMONSTRATED THE USEFULNESS OF VPA IN THE TRANSDIFFERENTIATION OF BMSSCS INTO HEPATOCYTES BOTH IN VITRO AND IN VIVO, AND REGULATION OF FIBROBLAST GROWTH FACTOR RECEPTORS (FGFRS) AND C-MET GENE EXPRESSION THROUGH POST-TRANSLATIONAL MODIFICATION OF CORE HISTONES MIGHT BE THE PRIMARY INITIATING EVENT FOR THESE EFFECTS. THIS MODE COULD BE HELPFUL FOR LIVER ENGINEERING AND CLINICAL THERAPY. 2009 15 3893 25 LACTATE INDUCES METABOLIC AND EPIGENETIC REPROGRAMMING OF PRO-INFLAMMATORY TH17 CELLS. INCREASED LACTATE LEVELS IN THE TISSUE MICROENVIRONMENT ARE A WELL-KNOWN FEATURE OF CHRONIC INFLAMMATION. HOWEVER, THE ROLE OF LACTATE IN REGULATING T CELL FUNCTION REMAINS CONTROVERSIAL. HERE, WE DEMONSTRATE THAT EXTRACELLULAR LACTATE PREDOMINANTLY INDUCES DEREGULATION OF THE TH17-SPECIFIC GENE EXPRESSION PROGRAM BY MODULATING THE METABOLIC AND EPIGENETIC STATUS OF TH17 CELLS. FOLLOWING LACTATE TREATMENT, TH17 CELLS SIGNIFICANTLY REDUCED THEIR IL-17A PRODUCTION AND UPREGULATED FOXP3 EXPRESSION THROUGH ROS-DRIVEN IL-2 SECRETION. MOREOVER, WE OBSERVED INCREASED LEVELS OF GENOME-WIDE HISTONE H3K18 LACTYLATION, A RECENTLY DESCRIBED MARKER FOR ACTIVE CHROMATIN IN MACROPHAGES, IN LACTATE-TREATED TH17 CELLS. IN ADDITION, WE SHOW THAT HIGH LACTATE CONCENTRATIONS SUPPRESS TH17 PATHOGENICITY DURING INTESTINAL INFLAMMATION IN MICE. THESE RESULTS INDICATE THAT LACTATE IS CAPABLE OF REPROGRAMMING PRO-INFLAMMATORY T CELL PHENOTYPES INTO REGULATORY T CELLS. 2022 16 5872 17 SUSTAINED TNF-ALPHA STIMULATION LEADS TO TRANSCRIPTIONAL MEMORY THAT GREATLY ENHANCES SIGNAL SENSITIVITY AND ROBUSTNESS. TRANSCRIPTIONAL MEMORY ALLOWS CERTAIN GENES TO RESPOND TO PREVIOUSLY EXPERIENCED SIGNALS MORE ROBUSTLY. HOWEVER, WHETHER AND HOW THE KEY PROINFLAMMATORY CYTOKINE TNF-ALPHA MEDIATES TRANSCRIPTIONAL MEMORY ARE POORLY UNDERSTOOD. USING HEK293F CELLS AS A MODEL SYSTEM, WE REPORT THAT SUSTAINED TNF-ALPHA STIMULATION INDUCES TRANSCRIPTIONAL MEMORY DEPENDENT ON TET ENZYMES. THE HYPOMETHYLATED STATUS OF TRANSCRIPTIONAL REGULATORY REGIONS CAN BE INHERITED, FACILITATING NF-KAPPAB BINDING AND MORE ROBUST SUBSEQUENT ACTIVATION. A HIGH INITIAL METHYLATION LEVEL AND CPG DENSITY AROUND KAPPAB SITES ARE CORRELATED WITH THE FUNCTIONAL POTENTIAL OF TRANSCRIPTIONAL MEMORY MODULES. INTERESTINGLY, THE CALCB GENE, ENCODING THE PROVEN MIGRAINE THERAPEUTIC TARGET CGRP, EXHIBITS THE BEST TRANSCRIPTIONAL MEMORY. A NEIGHBORING PRIMATE-SPECIFIC ENDOGENOUS RETROVIRUS STIMULATES MORE RAPID, MORE STRONG, AND AT LEAST 100-FOLD MORE SENSITIVE CALCB INDUCTION IN SUBSEQUENT TNF-ALPHA STIMULATION. OUR STUDY REVEALS THAT TNF-ALPHA-MEDIATED TRANSCRIPTIONAL MEMORY IS GOVERNED BY ACTIVE DNA DEMETHYLATION AND GREATLY SENSITIZES MEMORY GENES TO MUCH LOWER DOSES OF INFLAMMATORY CUES. 2020 17 2926 27 GENERATION OF AN EPIGENETIC SIGNATURE BY CHRONIC HYPOXIA IN PROSTATE CELLS. INCREASING LEVELS OF TISSUE HYPOXIA HAVE BEEN REPORTED AS A NATURAL FEATURE OF THE AGING PROSTATE GLAND AND MAY BE A RISK FACTOR FOR THE DEVELOPMENT OF PROSTATE CANCER. IN THIS STUDY, WE HAVE USED PWR-1E BENIGN PROSTATE EPITHELIAL CELLS AND AN EQUIVALENTLY AGED HYPOXIA-ADAPTED PWR-1E SUB-LINE TO IDENTIFY PHENOTYPIC AND EPIGENETIC CONSEQUENCES OF CHRONIC HYPOXIA IN PROSTATE CELLS. WE HAVE IDENTIFIED A SIGNIFICANTLY ALTERED CELLULAR PHENOTYPE IN RESPONSE TO CHRONIC HYPOXIA AS CHARACTERIZED BY INCREASED RECEPTOR-MEDIATED APOPTOTIC RESISTANCE, THE INDUCTION OF CELLULAR SENESCENCE, INCREASED INVASION AND THE INCREASED SECRETION OF IL-1 BETA, IL6, IL8 AND TNFALPHA CYTOKINES. IN ASSOCIATION WITH THESE PHENOTYPIC CHANGES AND THE ABSENCE OF HIF-1 ALPHA PROTEIN EXPRESSION, WE HAVE DEMONSTRATED SIGNIFICANT INCREASES IN GLOBAL LEVELS OF DNA METHYLATION AND H3K9 HISTONE ACETYLATION IN THESE CELLS, CONCOMITANT WITH THE INCREASED EXPRESSION OF DNA METHYLTRANSFERASE DMNT3B AND GENE-SPECIFIC CHANGES IN DNA METHYLATION AT KEY IMPRINTING LOCI. IN CONCLUSION, WE HAVE DEMONSTRATED A GENOME-WIDE ADJUSTMENT OF DNA METHYLATION AND HISTONE ACETYLATION UNDER CHRONIC HYPOXIC CONDITIONS IN THE PROSTATE. THESE EPIGENETIC SIGNATURES MAY REPRESENT AN ADDITIONAL MECHANISM TO PROMOTE AND MAINTAIN A HYPOXIC-ADAPTED CELLULAR PHENOTYPE WITH A POTENTIAL ROLE IN TUMOUR DEVELOPMENT. 2009 18 1584 24 DNA METHYLATION PROFILES OF SELECTED PRO-INFLAMMATORY CYTOKINES IN ALZHEIMER DISEASE. BY MEANS OF FUNCTIONAL GENOMICS ANALYSIS, WE RECENTLY DESCRIBED THE MRNA EXPRESSION PROFILES OF VARIOUS GENES INVOLVED IN THE NEUROINFLAMMATORY RESPONSE IN THE BRAINS OF SUBJECTS WITH LATE-ONSET ALZHEIMER DISEASE (LOAD). SOME OF THESE GENES, NAMELY INTERLEUKIN (IL)-1BETA AND IL-6, SHOWED DISTINCT EXPRESSION PROFILES WITH PEAK EXPRESSION DURING THE FIRST STAGES OF THE DISEASE AND CONTROL-LIKE LEVELS AT LATER STAGES. IL-1BETA AND IL-6 GENES ARE MODULATED BY DNA METHYLATION IN DIFFERENT CHRONIC AND DEGENERATIVE DISEASES; IT IS ALSO WELL KNOWN THAT LOAD MAY HAVE AN EPIGENETIC BASIS. INDEED, WE AND OTHERS HAVE PREVIOUSLY REPORTED GENE-SPECIFIC DNA METHYLATION ALTERATIONS IN LOAD AND IN RELATED ANIMAL MODELS. BASED ON THESE DATA, WE STUDIED THE DNA METHYLATION PROFILES, AT SINGLE CYTOSINE RESOLUTION, OF IL-1BETA AND IL-6 5'-FLANKING REGION BY BISULPHITE MODIFICATION IN THE CORTEX OF HEALTHY CONTROLS AND LOAD PATIENTS AT 2 DIFFERENT DISEASE STAGES: BRAAK I-II/A AND BRAAK V-VI/C. OUR ANALYSIS PROVIDES EVIDENCE THAT NEUROINFLAMMATION IN LOAD IS ASSOCIATED WITH (AND POSSIBLY MEDIATED BY) EPIGENETIC MODIFICATIONS. 2017 19 1117 29 COMPARATIVE AND EXPERIMENTAL STUDIES ON THE GENES ALTERED BY CHRONIC HYPOXIA IN HUMAN BRAIN MICROENDOTHELIAL CELLS. BACKGROUND : HYPOXIA INDUCIBLE FACTOR 1 ALPHA (HIF1A) IS A MASTER REGULATOR OF ACUTE HYPOXIA; HOWEVER, WITH CHRONIC HYPOXIA, HIF1A LEVELS RETURN TO THE NORMOXIC LEVELS. IMPORTANTLY, THE GENES THAT ARE INVOLVED IN THE CELL SURVIVAL AND VIABILITY UNDER CHRONIC HYPOXIA ARE NOT KNOWN. THEREFORE, WE TESTED THE HYPOTHESIS THAT CHRONIC HYPOXIA LEADS TO THE UPREGULATION OF A CORE GROUP OF GENES WITH ASSOCIATED CHANGES IN THE PROMOTER DNA METHYLATION THAT MEDIATES THE CELL SURVIVAL UNDER HYPOXIA. RESULTS : WE EXAMINED THE EFFECT OF CHRONIC HYPOXIA (3 DAYS; 0.5% OXYGEN) ON HUMAN BRAIN MICRO ENDOTHELIAL CELLS (HBMEC) VIABILITY AND APOPTOSIS. HYPOXIA CAUSED A SIGNIFICANT REDUCTION IN CELL VIABILITY AND AN INCREASE IN APOPTOSIS. NEXT, WE EXAMINED CHRONIC HYPOXIA ASSOCIATED CHANGES IN TRANSCRIPTOME AND GENOME-WIDE PROMOTER METHYLATION. THE DATA OBTAINED WAS COMPARED WITH 16 OTHER MICROARRAY STUDIES ON CHRONIC HYPOXIA. NINE GENES WERE ALTERED IN RESPONSE TO CHRONIC HYPOXIA IN ALL 17 STUDIES. INTERESTINGLY, HIF1A WAS NOT ALTERED WITH CHRONIC HYPOXIA IN ANY OF THE STUDIES. FURTHERMORE, WE COMPARED OUR DATA TO THREE OTHER STUDIES THAT IDENTIFIED HIF-RESPONSIVE GENES BY VARIOUS APPROACHES. ONLY TWO GENES WERE FOUND TO BE HIF DEPENDENT. WE SILENCED EACH OF THESE 9 GENES USING CRISPR/CAS9 SYSTEM. DOWNREGULATION OF EGLN3 SIGNIFICANTLY INCREASED THE CELL DEATH UNDER CHRONIC HYPOXIA, WHEREAS DOWNREGULATION OF ERO1L, ENO2, ADRENOMEDULLIN, AND SPAG4 REDUCED THE CELL DEATH UNDER HYPOXIA. CONCLUSIONS : WE PROVIDE A CORE GROUP OF GENES THAT REGULATES CELLULAR ACCLIMATIZATION UNDER CHRONIC HYPOXIC STRESS, AND MOST OF THEM ARE HIF INDEPENDENT. 2017 20 3525 28 IL-1BETA, IL-8, AND MATRIX METALLOPROTEINASES-1, -2, AND -10 ARE ENRICHED UPON MONOCYTE-BREAST CANCER CELL COCULTIVATION IN A MATRIGEL-BASED THREE-DIMENSIONAL SYSTEM. BREAST CANCER REMAINS THE FIRST CANCER-RELATED CAUSE OF DEATH IN WOMEN WORLDWIDE, PARTICULARLY IN DEVELOPING COUNTRIES IN WHICH MOST CASES ARE DIAGNOSED IN LATE STAGES. ALTHOUGH MOST CANCER STUDIES ARE BASED IN THE GENETIC OR EPIGENETIC CHANGES OF THE TUMOR CELLS, IMMUNE CELLS WITHIN THE TUMOR STROMA OFTEN COOPERATE WITH CANCER PROGRESSION. PARTICULARLY, MONOCYTES ARE ATTRACTED TO THE TUMOR PRIMARY SITE IN WHICH THEY ARE DIFFERENTIATED INTO TUMOR-ASSOCIATED MACROPHAGES THAT FACILITATE TUMOR CELL INVASION AND METASTASIS. IN THIS STUDY, WE USED THREE-DIMENSIONAL CULTURES TO FORM ACINI-LIKE STRUCTURES TO ANALYZE THE INFLAMMATORY SECRETION PROFILE OF TUMOR CELLS INDIVIDUALLY OR IN CO-CULTURE WITH MONOCYTES. BREAST CANCER CELL LINES AND PRIMARY ISOLATES FROM EIGHT MEXICAN PATIENTS WITH BREAST CANCER WERE USED. WE FOUND HIGH LEVELS OF RANTES/CCL5, MCP-1/CCL2, AND G-CSF IN THE BREAST CANCER INDIVIDUAL CULTURES, SUPPORTING AN IMPORTANT RECRUITMENT CAPACITY OF MONOCYTES, BUT ALSO OF NEUTROPHILS. THE CO-CULTURES OF THE TUMOR CELLS AND MONOCYTES WERE SIGNIFICANTLY ENRICHED WITH THE POTENT PRO-INFLAMMATORY CYTOKINES INTERLEUKIN (IL)-1BETA AND IL-8, KNOWN TO SUPPORT MALIGNANT PROGRESSION. WE ALSO FOUND THAT THE INTERACTION OF TUMOR CELLS WITH MONOCYTES PROMOTED HIGH LEVELS OF MATRIX METALLOPROTEINASES (MMP)-1, MMP-2, AND MMP-10. OUR STUDY SUPPORTS THAT A KEY EVENT FOR MALIGNANT PROGRESSION IS THE RECRUITMENT OF DIFFERENT IMMUNE CELL POPULATIONS, WHICH HELP TO SUSTAIN AND ENHANCE A CHRONIC INFLAMMATORY MICROENVIRONMENT THAT HIGHLY FAVORS TUMOR MALIGNANCY. 2017