1 2844 133 FREQUENT EPIGENETIC INACTIVATION OF SFRP GENES IN HEPATOCELLULAR CARCINOMA. BACKGROUND: ACTIVATION OF THE WNT SIGNALING PATHWAY IS FREQUENTLY OBSERVED IN HEPATOCELLULAR CARCINOMA (HCC), THOUGH MUTATION OF THREE OF ITS COMPONENTS, CTNNB1, AXIN1, AND AXIN2, IS OBSERVED SUBSTANTIALLY LESS OFTEN. METHODS: WE EXAMINED THE RELATIONSHIP BETWEEN WNT SIGNALING AND EPIGENETIC ALTERATION OF SECRETED FRIZZLED-RELATED PROTEIN (SFRP) GENES IN HCC. RESULTS: WE FREQUENTLY DETECTED THE ACTIVE FORM OF BETA-CATENIN AND ACCUMULATION OF NUCLEAR BETA-CATENIN IN LIVER CANCER CELL LINES. WE DETECTED METHYLATION OF SFRP FAMILY GENES IN LIVER CANCER CELL LINES (SFRP1, 9/12, 75%; SFRP2, 7/12, 58%; SFRP4, 3/12, 25%; SFRP5, 7/12, 58%) AND PRIMARY HCCS (SFRP1, 9/19, 47%; SFRP2, 12/19, 63%; SFRP5, 8/19, 42%), THOUGH METHYLATION OF SFRP4 WAS NOT FOUND IN PRIMARY HCCS. SFRP METHYLATION ALSO WAS DETECTED IN HEPATITIS B OR C VIRUS-ASSOCIATED CHRONIC HEPATITIS (SFRP1, 6/37, 16%; SFRP2, 14/37, 38%; SFRP5, 5/37, 14%) AND LIVER CIRRHOSIS (SFRP1, 10/28, 36%; SFRP2, 9/28, 32%; SFRP5, 3/28, 11%), SUGGESTING THAT METHYLATION OF THESE GENES IS AN EARLY EVENT IN LIVER CARCINOGENESIS. ECTOPIC EXPRESSION OF SFRPS DOWNREGULATED T-CELL FACTOR/LYMPHOCYTE ENHANCER FACTOR (TCF/LEF) TRANSCRIPTIONAL ACTIVITY IN LIVER CANCER CELLS, WHILE OVEREXPRESSION OF A BETA-CATENIN MUTANT AND DEPLETION OF SFRP1 USING SIRNA SYNERGISTICALLY UPREGULATED TCF/LEF TRANSCRIPTIONAL ACTIVITY. CONCLUSIONS: OUR RESULTS CONFIRM THE FREQUENT METHYLATION AND SILENCING OF WNT ANTAGONIST GENES IN HCC, AND SUGGEST THAT THEIR LOSS OF FUNCTION CONTRIBUTES TO ACTIVATION OF WNT SIGNALING DURING HEPATOCARCINOGENESIS. 2008 2 388 40 AN INTEGRATED ANALYSIS OF SOCS1 DOWN-REGULATION IN HBV INFECTION-RELATED HEPATOCELLULAR CARCINOMA. PERSISTENT INFLAMMATION TOGETHER WITH GENETIC/EPIGENETIC ABERRATIONS IS STRONGLY ASSOCIATED WITH CHRONIC HEPATITIS B VIRUS (HBV) INFECTION-RELATED HEPATOCARCINOGENESIS. HERE, WE INVESTIGATED THE ALTERATIONS OF THE SUPPRESSOR OF CYTOKINE SIGNALLING (SOCS) FAMILY GENES IN HBV-RELATED HEPATOCELLULAR CARCINOMA (HCC). A TOTAL OF 116 PATIENTS WITH HCC WERE ENROLLED IN THIS STUDY. THE METHYLATION STATUSES OF SOCS1-7 AND CISH GENES WERE QUANTITATIVELY MEASURED AND CLINICOPATHOLOGICAL SIGNIFICANCE OF SOCS1 METHYLATION WAS STATISTICALLY ANALYSED. THE GENE COPY NUMBER VARIATION WAS ASSAYED BY ACGH. LUCIFERASE REPORTER ASSAY AND WESTERN BLOT WERE USED TO DETECT THE INVOLVEMENT OF SOCS1 IN P53 SIGNALLING. WE FOUND HIGH FREQUENCIES OF SOCS1 GENE HYPERMETHYLATION IN BOTH TUMOUR (56.03%) AND ADJACENT NONTUMOUR TISSUES (54.31%), BUT TUMOUR TISSUES EXHIBITED INCREASED METHYLATION INTENSITY (24.01% VS 13.11%, P < 0.0001), PARTICULARLY IN PATIENTS WITH LARGER TUMOUR SIZE OR CIRRHOSIS BACKGROUND (P < 0.0001). IN ADDITION, THE FREQUENCY AND INTENSITY OF SOCS1 HYPERMETHYLATION IN TUMOUR TISSUES WERE BOTH SIGNIFICANTLY HIGHER THAN THOSE IN NONTUMOUR TISSUES IN MALE GENDER PATIENTS AND IN PATIENTS >/=45 YEARS OLD (P = 0.0214 AND P < 0.0001, P = 0.0232 AND P < 0.0001, RESPECTIVELY). SOCS1 GENE DELETION WAS FOUND IN 8 OF 25 ACGH ASSAYED TUMOUR SPECIMENS, WHICH WAS ASSOCIATED WITH LOWER SOCS1 MRNA EXPRESSION (P = 0.0448). FURTHERMORE, ECTOPIC SOCS1 OVEREXPRESSION COULD ACTIVATE THE P53 SIGNALLING PATHWAY IN HCC CELL LINES. HYPERMETHYLATION OF SOCS2-7 AND CISH GENES WAS SELDOM FOUND IN HCC. OUR RESULTS SUGGESTED THAT THE GENE LOSS AND EPIGENETIC SILENCING OF SOCS1 WERE STRONGLY ASSOCIATED WITH HBV-RELATED HCC. 2014 3 2088 42 EPIGENETIC DYSREGULATION OF SECRETED FRIZZLED-RELATED PROTEINS IN MYELOPROLIFERATIVE NEOPLASMS COMPLEMENTS THE JAK2V617F-MUTATION. BACKGROUND: SECRETED FRIZZLED-RELATED PROTEINS (SFRPS) ARE ANTAGONISTS OF THE WNT SIGNALING PATHWAY, WHICH PLAYS A CENTRAL ROLE IN STEM CELL MAINTENANCE AND DIFFERENTIATION OF STEM CELLS AND HEMATOPOIETIC PROGENITORS. EPIGENETIC DOWNREGULATION OF SFRPS BY PROMOTER HYPERMETHYLATION HAS BEEN DESCRIBED TO BE INVOLVED IN THE PATHOGENESIS OF HEMATOPOIETIC MALIGNANCIES. THERE IS AN ASSOCIATION BETWEEN ABERRANT WNT SIGNALING AND THE ESTABLISHED CANCER STEM CELL CONCEPT. IN CONTRAST TO BCR-ABL1-POSITIVE CHRONIC MYELOID LEUKEMIA CML, BCR-ABL1-NEGATIVE MYELOPROLIFERATIVE NEOPLASMS (PH-MPN) ARE CHARACTERIZED BY THE FREQUENT OCCURRENCE OF AN AUTOACTIVATING MUTATION IN THE JAK2 TYROSINE KINASE (JAK2V617F) OR OTHER MUTATIONS IN THE JAK-STAT PATHWAY. HOWEVER, PATHOGENETIC MECHANISMS OF JAK2 MUTATED OR UNMUTATED PH-MPN REMAIN NOT COMPLETELY UNDERSTOOD. WE DETERMINED THE PROMOTER METHYLATION STATUS OF SFRP-1, -2, -4, AND -5 IN 57 MPN PATIENT SAMPLES BY METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (PCR) (MSP). JAK2V617F WAS ASSESSED BY ALLELE-SPECIFIC PCR. RESULTS: ABERRANT METHYLATION AMONG PRIMARY MPN SAMPLES WAS 4% FOR SFRP-1, 25% FOR SFRP-2, 2% FOR SFRP-4, AND 0% FOR SFRP-5. HYPERMETHYLATION OF SFRP-2, WHICH WAS THE MOST FREQUENTLY HYPERMETHYLATED GENE IN OUR STUDY, COULD NOT BE CORRELATED TO ANY SPECIFIC MPN SUBTYPE. HOWEVER, WE DETECTED A SIGNIFICANT CORRELATION BETWEEN SFRP-2 METHYLATION AND PRESENCE OF A JAK2V617F MUTATION (P = 0.008). NONE OF THE 10 CML SAMPLES SHOWED ANY SFRP-METHYLATION. CONCLUSIONS: OUR DATA INDICATE THAT EPIGENETIC DYSREGULATION OF THE WNT SIGNALING PATHWAY IS A COMMON EVENT IN MPN WITH ABERRANT METHYLATION OF AT LEAST ONE SFRP BEING DETECTED IN 25% OF THE PRIMARY PATIENT SAMPLES AND IN 30% IF ONLY ACCOUNTING FOR PH-MPN. A SIGNIFICANT CORRELATION BETWEEN SFRP-2 METHYLATION AND PRESENCE OF JAK2V617F IN OUR DATA SUPPORTS THE HYPOTHESIS THAT EPIGENETIC DYSREGULATION MAY BE A COMPLEMENTARY MECHANISM TO GENETIC ABERRATIONS. ABERRANT METHYLATION OF CRUCIAL STEM CELL MAINTENANCE GENES SEEMS TO CONTRIBUTE TO DISEASE PATHOGENESIS IN PH-MPN. 2012 4 5668 39 SFRP1 EXPRESSION REGULATES WNT SIGNALING IN CHRONIC MYELOID LEUKEMIA K562 CELLS. BACKGROUND: WNT SIGNALING CASCADES PLAY IMPORTANT ROLES IN CELL FATE DECISIONS AND THEIR DEREGULATION HAS BEEN DOCUMENTED IN MANY DISEASES, INCLUDING MALIGNANT TUMORS AND LEUKEMIA. ONE MECHANISM OF ABERRANT WNT SIGNALING IS THE SILENCING OF WNT INHIBITORS THROUGH EPIGENETIC MECHANISMS. THE SFRPS ARE ONE OF THE MOST STUDIED WNT INHIBITORS; AND THE SFRP1 LOSS IS KNOWN IN MANY HEMATOLOGICAL MALIGNANCIES. THEREFORE, WE AIMED TO COMPARE THE EXPRESSION OF WNT RELATED GENES IN THE PRESENCE AND ABSENCE OF SFRP1 IN A CHRONIC MYELOID LEUKEMIA (CML) CELL LINE. OBJECTIVE: IT IS IMPORTANT TO UNDERSTAND HOW SFRP1 AND SFRP1 PERFORM THEIR EFFECTS ON CML TO DESIGN NEW AGENTS AND STRATEGIES FOR RESISTANT AND ADVANCED FORMS OF CML. MATERIALS AND METHODS: WE USED K562 CELLS, WHICH NORMALLY DO NOT EXPRESS SFRP1 AND ITS SFRP1 EXPRESSING SUBCLONE K562S. TOTAL RNA WAS ISOLATED FROM K562 AND K562S CELL LINES AND CONVERTED TO CDNA. PCR ARRAY EXPERIMENTS WERE PERFORMED USING HUMAN WNT SIGNALING PATHWAY PLUS RT2 PROFILER KIT. WNT SIGNALING PATHWAY ACTIVATION WAS STUDIED BY WESTERN BLOT FOR DOWNSTREAM SIGNALING TARGETS. RESULTS: THE WNT3, LRP6, PRICKLE1 AND BTRC EXPRESSIONS WERE SIGNIFICANTLY DECREASED IN THE PRESENCE OF SFRP1; WHILE WNT5B INCREASED. THE SFRP1 EXPRESSION INHIBITED STABILIZATION OF TOTAL BETA-CATENIN PROTEIN AND DOWNSTREAM EFFECTOR PHOSPHORYLATION OF NONCANONICAL WNT/PCP SIGNALING; WHEREAS CA2+/PKC SIGNALING REMAINED ACTIVE. CONCLUSION: THE RESULTS SUGGEST THAT SFRP1 COULD BE A PROMISING THERAPEUTIC ANTICANCER AGENT. DEFINING THESE PATHWAY INTERACTIONS IS CRUCIAL FOR DESIGNING NEW AGENTS RESISTANT AND ADVANCED FORMS OF CML. 2022 5 494 25 ASSESSMENT OF PROMOTER METHYLATION IDENTIFIES PTCH AS A PUTATIVE TUMOR-SUPPRESSOR GENE IN HUMAN CLL. BACKGROUND: CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS CHARACTERIZED BY A CLONAL ACCUMULATION OF NEOPLASTIC LYMPHOCYTES, INDICATING DISRUPTION OF APOPTOSIS. PATIENTS AND METHODS: DIFFERENTIAL METHYLATION HYBRIDIZATION ANALYSIS WAS PERFORMED TO IDENTIFY NOVEL TARGET GENES SILENCED BY CPG ISLAND METHYLATION IN PATIENTS WITH CLL. RESULTS: PATCHED (PTCH), A TUMOR-SUPPRESSOR GENE, WAS FOUND TO BE FREQUENTLY METHYLATED IN CLL SAMPLES COMPARED TO SAMPLES DERIVED FROM HEALTHY INDIVIDUALS. DE NOVO METHYLATION OF A CPG ISLAND REGION LOCATED UPSTREAM OF PTCH EXON 1 WAS CONFIRMED BY PYROSEQUENCING IN 17/37 (46%) OF PERIPHERAL BLOOD MONONUCLEAR CELLS OF PATIENTS WITH CLL, BUT IN NONE ISOLATED FROM SEVEN HEALTHY INDIVIDUALS. NO ASSOCIATION WAS FOUND BETWEEN PTCH HYPERMETHYLATION AND CURRENTLY USED PROGNOSTIC CLL FACTORS. CONCLUSION: OUR INVESTIGATION SUGGESTS THAT EPIGENETIC SILENCING OF PTCH IS A MECHANISM CONTRIBUTING TO CLL TUMORIGENESIS. 2016 6 5669 24 SFRP1 PROMOTER METHYLATION IS ASSOCIATED WITH PERSISTENT PHILADELPHIA CHROMOSOME IN CHRONIC MYELOID LEUKEMIA. EPIGENETIC SILENCING OF SFRP GENES HAS BEEN SHOWN TO LEAD TO CONSTITUTIVE ACTIVATION OF THE CANONICAL WNT-SIGNALING PATHWAY. THE FIRST DESCRIPTION OF DEREGULATED WNT-SIGNALING ACTIVATION IN A HEMATOLOGICAL MALIGNANCY WAS REPORTED IN CHRONIC MYELOID LEUKEMIA (CML). TO INVESTIGATE WHETHER EPIGENETIC SILENCING OF SFRP IS RESPONSIBLE FOR THE OBSERVED WNT ACTIVATION IN CML, WE STUDIED THE METHYLATION AND MUTATIONAL STATUS OF THE SFRP1 PROMOTER IN 48 CHRONIC PHASE CML PATIENTS. OF THE 48 CML PATIENTS 41 WERE SHOWN TO BE UNMETHYLATED, 6 PATIENTS HEMI-METHYLATED AND 1 PATIENT FULLY METHYLATED AT THE SFRP1 PROMOTER. ALBEIT OBSERVED INFREQUENTLY IN CHRONIC PHASE CML, SFRP1 PROMOTER METHYLATION CORRELATED WITH PRIMARY CYTOGENETIC RESISTANCE TO IMATINIB MESYLATE. SFRP1 PROMOTER METHYLATION MAY INDICATE A GENETICALLY MORE UNSTABLE FORM OF DISEASE RESISTANT TO THERAPY AND PROVIDE A KEY BIOLOGICAL DIFFERENCE IN THERAPY RESISTANT PATIENTS, IN ADDITION TO A POSSIBLE MECHANISM FOR THE OBSERVED ACTIVATION OF CANONICAL WNT SIGNALING IN CML. 2009 7 4220 27 METHYLATED CYSTEINE DIOXYGENASE-1 GENE PROMOTER IN THE SERUM IS A POTENTIAL BIOMARKER FOR HEPATITIS B VIRUS-RELATED HEPATOCELLULAR CARCINOMA. HEPATOCELLULAR CARCINOMA (HCC) IS THE THIRD LEADING CAUSE OF CANCER-RELATED MORTALITY WORLDWIDE. EPIGENETIC ANALYSIS HAS ATTRACTED INCREASING ATTENTION IN THE MOLECULAR DIAGNOSIS OF HCC. CYSTEINE DIOXYGENASE 1 (CDO1) IS A KEY ENZYME IN THE TAURINE BIOSYNTHETIC PATHWAY AND CONVERTS CYSTEINE TO CYSTEINE SULFINATE. THE CDO1 GENE IS A TUMOR SUPPRESSOR GENE AND IS USUALLY SILENCED BY THE METHYLATION OF ITS PROMOTER IN CARCINOGENESIS. IN THIS STUDY, WE EVALUATED WHETHER THE METHYLATION STATUS OF CDO1 GENE PROMOTER IS OF DIAGNOSTIC VALUE FOR HEPATITIS B VIRUS (HBV)-RELATED HCC. THE CDO1 PROMOTER METHYLATION STATUS WAS DETERMINED IN SERUM SAMPLES USING METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (MSP) IN A COHORT OF 123 PATIENTS WITH HBV-RELATED HCC, 28 WITH LIVER CIRRHOSIS (LC), 29 WITH CHRONIC HEPATITIS B (CHB) AND 20 HEALTHY CONTROLS. THE FREQUENCY OF THE CDO1 PROMOTER METHYLATION IN HBV-RELATED HCC (42.3%) WAS SIGNIFICANTLY HIGHER THAN THAT IN LC (14.3%), CHB (6.9%) AND HEALTHY CONTROLS (0%) (P = 0.006; P < 0.0001; P < 0.0001; RESPECTIVELY). FURTHERMORE, IN HCC PATIENTS, THE FREQUENCY OF CDO1 PROMOTER METHYLATION WAS HIGHER IN ADVANCED STAGES (III-IV) (53%) THAN THE EARLY STAGES (I-II) (20%) (P = 0.001). EVALUATION OF THE CDO1 PROMOTER METHYLATION STATUS IN SERUM, IN COMBINATION WITH AFP (> 20 NG/ML), SIGNIFICANTLY IMPROVED THE DIAGNOSTIC VALUE, WITH SENSITIVITY AND SPECIFICITY OF 82.9% AND 75.4%, RESPECTIVELY IN DISTINGUISHING HCC FROM LC AND CHB. IN CONCLUSION, METHYLATION STATUS OF SERUM CDO1 GENE PROMOTER MAY BE HELPFUL IN THE DIAGNOSIS OF HCC AND THE ESTIMATION OF THE HCC STAGES. 2014 8 155 33 ABERRANT METHYLATION OF POLO-LIKE KINASE CPG ISLANDS IN PLK4 HETEROZYGOUS MICE. BACKGROUND: HEPATOCELLULAR CARCINOMA (HCC), ONE OF THE MOST COMMON CANCERS WORLD-WIDE OCCURS TWICE AS OFTEN IN MEN COMPARED TO WOMEN. PREDISPOSING CONDITIONS SUCH AS ALCOHOLISM, CHRONIC VIRAL HEPATITIS, AFLATOXIN B1 INGESTION, AND CIRRHOSIS ALL CONTRIBUTE TO THE DEVELOPMENT OF HCC. METHODS: WE USED A COMBINATION OF METHYLATION SPECIFIC PCR AND BISULFITE SEQUENCING, QREAL-TIME PCR (QPCR), AND WESTERN BLOT ANALYSIS TO EXAMINE EPIGENETIC CHANGES FOR THE POLO-LIKE KINASES (PLKS) DURING THE DEVELOPMENT OF HEPATOCELLULAR CARCINOMA (HCC) IN PLK4 HETEROZYGOUS MICE AND MURINE EMBRYONIC FIBROBLASTS (MEFS). RESULTS: HERE WE REPORT THAT THE PROMOTER METHYLATION OF PLK4 CPG ISLANDS INCREASES WITH AGE, WAS MORE PREVALENT IN MALES AND THAT PLK4 EPIGENETIC MODIFICATION AND SUBSEQUENT DOWNREGULATION OF EXPRESSION WAS ASSOCIATED WITH THE DEVELOPMENT OF HCC IN PLK4 MUTANT MICE. INTERESTINGLY, THE OPPOSITE OCCURS WITH ANOTHER PLK FAMILY MEMBER, PLK1 WHICH WAS TYPICALLY HYPERMETHYLATED IN NORMAL LIVER TISSUE BUT BECAME HYPOMETHYLATED AND UPREGULATED IN LIVER TUMOURS. FURTHERMORE, UPON ALCOHOL EXPOSURE MURINE EMBRYONIC FIBROBLASTS EXHIBITED INCREASED PLK4 HYPERMETHYLATION AND DOWNREGULATION ALONG WITH INCREASED CENTROSOME NUMBERS AND MULTINUCLEATION. CONCLUSIONS: THESE RESULTS SUGGEST THAT ABERRANT PLK METHYLATION IS CORRELATED WITH THE DEVELOPMENT OF HCC IN MICE. 2011 9 4221 30 METHYLATION AND SILENCING OF PROTEIN TYROSINE PHOSPHATASE RECEPTOR TYPE O IN CHRONIC LYMPHOCYTIC LEUKEMIA. PURPOSE: PREVIOUS STUDIES IN OUR LABORATORY HAVE SHOWN THE PROGRESSIVE METHYLATION AND SUPPRESSION OF THE GENE ENCODING PROTEIN TYROSINE PHOSPHATASE, PTPRO, IN THE LIVERS OF RATS FED A METHYL-DEFICIENT DIET THAT INDUCES HEPATOCARCINOGENESIS. SUBSEQUENTLY, WE OBSERVED THE METHYLATION OF PTPRO IN PRIMARY HUMAN LUNG TUMORS AND ALSO SHOWED ITS POTENTIAL TUMOR SUPPRESSOR CHARACTERISTICS. THE PRESENT STUDY WAS UNDERTAKEN TO INVESTIGATE WHETHER THE TRUNCATED FORM OF PTPRO (PTPROT), SPECIFICALLY EXPRESSED IN NAIVE B LYMPHOCYTES, WAS ALSO METHYLATED AND SUPPRESSED IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), A DISEASE GENERALLY AFFECTING B LYMPHOCYTES. EXPERIMENTAL DESIGN AND RESULTS: INITIAL SCREENING SHOWED THAT 60% OF THE 52 CLL SAMPLES ANALYZED USING METHYLATION-SPECIFIC PCR ASSAY WERE METHYLATED COMPARED WITH B LYMPHOCYTES FROM NORMAL INDIVIDUALS, WHICH WERE NOT METHYLATED. THE EXPRESSION OF PTPROT, AS MEASURED BY SEMIQUANTITATIVE REVERSE TRANSCRIPTION-PCR, INVERSELY CORRELATED WITH METHYLATION IN THE FEW SAMPLES TESTED. ANALYSIS OF ADDITIONAL SAMPLES (N = 50) BY COMBINED BISULFITE RESTRICTION ANALYSIS SHOWED THAT THE PTPRO CPG ISLAND WAS METHYLATED IN 82% OF PATIENTS WITH CLL COMPARED WITH B LYMPHOCYTES FROM NORMAL INDIVIDUALS. FURTHERMORE, OVERALL EXPRESSION OF PTPRO WAS REDUCED IN CLL RELATIVE TO NORMAL LYMPHOCYTES. THE PTPRO GENE WAS ALSO SUPPRESSED BY METHYLATION IN THE CLL CELL LINE WAC3CD5, WHERE IT COULD BE REACTIVATED UPON TREATMENT WITH THE DNA HYPOMETHYLATING AGENT 5-AZAC. ECTOPIC EXPRESSION OF PTPROT IN A NONEXPRESSING CELL LINE INCREASED GROWTH INHIBITION WITH FLUDARABINE TREATMENT, A THERAPY COMMONLY USED FOR CLL. CONCLUSION: THIS STUDY REVEALS THE POTENTIAL ROLE OF PTPRO METHYLATION AND SILENCING IN CLL TUMORIGENESIS AND ALSO PROVIDES A NOVEL MOLECULAR TARGET IN THE EPIGENETIC THERAPY. 2007 10 5349 28 RASSF1A AND DOK1 PROMOTER METHYLATION LEVELS IN HEPATOCELLULAR CARCINOMA, CIRRHOTIC AND NON-CIRRHOTIC LIVER, AND CORRELATION WITH LIVER CANCER IN BRAZILIAN PATIENTS. HEPATOCELLULAR CARCINOMA (HCC) IS THE SECOND MOST COMMON CAUSE OF CANCER MORTALITY WORLDWIDE. MOST CASES OF HCC ARE ASSOCIATED WITH CIRRHOSIS RELATED TO CHRONIC HEPATITIS B VIRUS OR HEPATITIS C VIRUS INFECTIONS. HYPERMETHYLATION OF PROMOTER REGIONS IS THE MAIN EPIGENETIC MECHANISM OF GENE SILENCING AND HAS BEEN INVOLVED IN HCC DEVELOPMENT. THE AIM OF THIS STUDY WAS TO DETERMINE WHETHER ABERRANT METHYLATION OF RASSF1A AND DOK1 GENE PROMOTERS IS ASSOCIATED WITH THE PROGRESSION OF LIVER DISEASE IN BRAZILIAN PATIENTS. METHYLATION LEVELS WERE MEASURED BY PYROSEQUENCING IN 41 (20 HCC, 9 CIRRHOTIC, AND 12 NON-CIRRHOTIC) LIVER TISSUE SAMPLES. MEAN RATES OF METHYLATION IN RASSF1A AND DOK1 WERE 16.2% AND 12.0% IN NON-CIRRHOTIC, 26.1% AND 19.6% IN CIRRHOTIC, AND 59.1% AND 56.0% IN HCC TISSUES, RESPECTIVELY, SHOWING A GRADUAL INCREASE ACCORDING TO THE PROGRESSION OF THE DISEASE, WITH SIGNIFICANTLY HIGHER LEVELS IN TUMOR TISSUES. IN ADDITION, HYPERMETHYLATION OF RASSF1A AND DOK1 WAS FOUND IN THE VAST MAJORITY (88%) OF THE HCC CASES. INTERESTINGLY, DOK1 METHYLATION LEVELS IN HCC SAMPLES WERE SIGNIFICANTLY HIGHER IN THE GROUP OF YOUNGER (<40 YEARS) PATIENTS, AND HIGHER IN MODERATELY DIFFERENTIATED THAN IN POORLY DIFFERENTIATED TUMORS (P < 0.05). OUR RESULTS REINFORCE THE HYPOTHESIS THAT HYPERMETHYLATION OF RASSF1A AND DOK1 CONTRIBUTES TO HEPATOCARCINOGENESIS AND IS ASSOCIATED TO CLINICOPATHOLOGICAL CHARACTERISTICS. RASSF1A AND DOK1 PROMOTER HYPERMETHYLATION MAY BE A VALUABLE BIOMARKER FOR EARLY DIAGNOSIS OF HCC AND A POTENTIAL MOLECULAR TARGET FOR EPIGENETIC-BASED THERAPY. 2016 11 5458 26 RESEARCH ON EPIGENETIC MECHANISM OF SFRP2 IN ADVANCED CHRONIC MYELOID LEUKEMIA. SECRETED FRIZZLED-RELATED PROTEIN 2 (SFRP2) HAS BEEN REPORTED TO ACT AS A TUMOR SUPPRESSORS. THIS STUDY AIMS TO DETECT THE BIOLOGICAL ROLE OF SFRP2 IN ADVANCED CHRONIC MYELOID LEUKEMIA (CML). IN THIS STUDY WE EXAMINED BONE MARROW SAMPLES FROM 45 CML PATIENTS AND 10 HEALTHY DONORS. K562 AND KCL22 CELLS WERE CULTURED AND TREATED WITH DEMETHYLATION DRUG AND HISTONE DEACETYLASE INHIBITOR (HDACI). KCL22 AND K562 CELLS WERE TRANSFECTED WITH LENTIVIRAL VECTOR (LV)-SFRP2, LV-CONTROL. THE CELLS WERE THEN SUBJECTED TO PROLIFERATION AND APOPTOSIS ASSAYS, REAL TIME POLYMERASE CHAIN REACTION (PCR), METHYLATION-SPECIFIC PCR (MSP), WESTERN BLOTTING, CO-IMMUNOPRECIPITATION (COIP) AND CHROMATIN IMMUNOPRECIPITATION (CHIP), WE FOUND THAT SFRP2 WAS DOWN-REGULATED IN THE ACCELERATED AND BLAST PHASE OF CML, WHEREAS, THE LEVELS OF WNT1, WNT3 AND WNT5A WERE UP-REGULATED IN THE ACCELERATED AND BLAST PHASE OF CML. OVEREXPRESSION SFRP2 INHIBITED PROLIFERATION, PROMOTED APOPTOSIS AND ACTIVATED THE WNT PATHWAY. COIP-MS RESULTS SHOWED THAT SFRP2 INTERACTED WITH WNT1 AND WNT5A. CHIP-SEQ RESULT INDICATED THAT THE PROMOTER OF H3K4ME3 AND H3K27ME3 WERE ABLE TO INTERACT WITH SFRP2. IN CONCLUSION, OUR FINDINGS DEMONSTRATED THE SFRP2 ACT AS A POTENTIAL THERAPEUTIC TARGET FOR ADVANCED CML. FURTHERMORE, OUR RESULTS SUPPORT THE USE OF DEMETHYLATION DRUGS AND HDACI AS A POTENTIAL CML TREATMENT STRATEGY. 2018 12 2453 25 EPIGENETIC SUPPRESSION OF THE IMMUNOREGULATOR MZB1 IS ASSOCIATED WITH THE MALIGNANT PHENOTYPE OF GASTRIC CANCER. PREDICTION OF TUMOR RECURRENCE AFTER CURATIVE RESECTION IS CRITICAL FOR DETERMINING THE PROGNOSIS OF PATIENTS WITH GASTRIC CANCER (GC). THE INITIATION AND PROGRESSION OF GC ARE ASSOCIATED WITH INAPPROPRIATE IMMUNE RESPONSES CAUSED BY CHRONIC INFLAMMATION OF THE GASTRIC MUCOSA. TO IDENTIFY IMMUNOREGULATORY MOLECULES INVOLVED IN GC PROGRESSION, GC CELL LINES AND 200 PAIRS OF TUMOR AND NORMAL TISSUES FROM PATIENTS WITH GC WERE ANALYZED FOR GENE EXPRESSION, AMPLIFICATION AND METHYLATION AS WELL AS FUNCTION OF A DIFFERENTIALLY EXPRESSED GENE. THE TRANSCRIPTOME ANALYSIS REVEALED THAT MARGINAL ZONE B AND B1 CELL SPECIFIC PROTEIN (MZB1) WAS EXPRESSED AT SIGNIFICANTLY DECREASED LEVELS IN PRIMARY GC TISSUES WHEN COMPARED WITH THE CORRESPONDING NORMAL GASTRIC MUCOSA. PCR ARRAY ANALYSIS EXPLORING GENES EXPRESSED COOPERATIVELY WITH MZB1 REVEALED THAT DIFFERENTIAL EXPRESSION OF MZB1 MRNA IN GC CELL LINES CORRELATED POSITIVELY WITH THE LEVELS OF THE MRNAS ENCODING ESTROGEN RECEPTOR 1 AND DESUMOYLATING ISOPEPTIDASE 1. HYPERMETHYLATION OF THE MZB1 PROMOTER WAS FREQUENT IN CELL LINES WITH DECREASED LEVELS OF MZB1 MRNA. SIRNA-MEDIATED KNOCKDOWN OF MZB1 SIGNIFICANTLY INCREASED PROLIFERATION, INVASION AND MIGRATION OF GC CELL LINES. LOW MZB1 EXPRESSION WAS AN INDEPENDENT PROGNOSTIC FACTOR FOR RECURRENCE AFTER CURATIVE GASTRECTOMY AND WAS ASSOCIATED SIGNIFICANTLY WITH INCREASED HEMATOGENOUS RECURRENCE. MZB1 ACTS AS A SUPPRESSOR OF GC. LOW MZB1 EXPRESSION IN THE PRIMARY GC TISSUE IS PREDICTIVE OF RECURRENCE AFTER CURATIVE RESECTION. 2016 13 2090 36 EPIGENETIC DYSREGULATION OF THE WNT SIGNALLING PATHWAY IN CHRONIC LYMPHOCYTIC LEUKAEMIA. BACKGROUND: WNT SIGNALLING HAS RECENTLY BEEN IMPLICATED IN THE PATHOGENESIS OF CANCER. METHODS: THIS STUDY INVESTIGATED THE ACTIVITY OF WNT SIGNALLING IN PERIPHERAL BLOOD CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL) LYMPHOCYTES, AND THE METHYLATION STATUS OF SEVEN SOLUBLE WNT ANTAGONIST GENES, INCLUDING WIF1, DKK3, APC, SFRP1, SFRP2, SFRP4 AND SFRP5, BY USING METHYLATION-SPECIFIC PCR IN THE PERIPHERAL BLOOD CLL LYMPHOCYTES AND BONE MARROW SAMPLES OF PATIENTS WITH CLL AT DIAGNOSIS. RESULTS: IN THE PERIPHERAL BLOOD CLL LYMPHOCYTES, CONSTITUTIVE ACTIVATION OF WNT SIGNALLING WAS DETECTED, ASSOCIATED WITH HYPERMETHYLATION OF THE SOLUBLE WNT INHIBITOR GENES. IN THE DIAGNOSTIC CLL MARROW SAMPLES, METHYLATION OF THE SEVEN GENES WAS DETECTED IN UP TO 36.4% OF SAMPLES. MOREOVER, 23 (52.3%) PATIENTS HAD METHYLATION OF AT LEAST ONE OF THE SEVEN GENES, OF WHOM 14 (60.8%) HAD METHYLATION OF TWO OR MORE WNT INHIBITOR GENES. APART FROM AN ASSOCIATION OF ADVANCED AGE WITH DKK3 METHYLATION, THERE WAS NO ASSOCIATION OF GENE HYPERMETHYLATION WITH EITHER CLINICAL CHARACTERISTICS (INCLUDING AGE, GENDER, LYMPHOCYTE COUNT AT DIAGNOSIS, RAI STAGE AND POOR-RISK KARYOTYPE) OR SURVIVAL. CONCLUSION: WNT SIGNALLING IS CONSTITUTIVELY ACTIVATED IN CLL B LYMPHOCYTES IN ASSOCIATION WITH METHYLATION OF MULTIPLE SOLUBLE WNT ANTAGONIST GENES. METHYLATION OF THESE SOLUBLE WNT ANTAGONIST GENES, OCCASIONALLY MULTIPLE GENES, IN PRIMARY CLL MARROW SAMPLES SUGGESTS AN IMPORTANT ROLE IN CLL PATHOGENESIS. MOREOVER, THIS STUDY UNDERSCORED THE IMPORTANCE OF STUDYING METHYLATION OF A PANEL OF, BUT NOT INDIVIDUAL, GENES REGULATING A CELLULAR PATHWAY. 2008 14 5844 32 STUDY OF PROMOTER HYPOMETHYLATION PROFILES OF RAS ONCOGENES IN HEPATOCELLULAR CARCINOMA DERIVED FROM HEPATITIS C VIRUS GENOTYPE 3A IN PAKISTANI POPULATION. EPIGENETIC MODIFICATIONS SUCH AS DNA METHYLATION CONTRIBUTE TO PROGRESSION OF HEPATITIS C VIRUS (HCV) INFECTION TO LIFE-THREATENING HEPATOCELLULAR CARCINOMA (HCC) BY PROMOTING THE SILENCING OF TUMOR SUPPRESSOR GENES THROUGH DNA HYPERMETHYLATION AND BY CAUSING GENOMIC INSTABILITY THROUGH GLOBAL HYPOMETHYLATION. HOWEVER FEW STUDIES HAVE ADDRESSED THE PROMOTER REGION HYPOMETHYLATION STATUS OF THE ONCOGENES INVOLVED IN HCV DERIVED HCC. IN THIS STUDY, WE ANALYZED THE PROMOTER REGION METHYLATION PATTERN OF RAS ONCOGENES (HRAS, KRAS, AND NRAS) USING METHYLATION-SPECIFIC PCR FOR 50 CHRONIC HCV PATIENTS INFECTED WITH GENOTYPE 3A (27 HCC PATIENTS AND 23 CONTROL NON-HCC PATIENTS). METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION ANALYSIS REVEALED THAT THE NRAS ONCOGENE PROMOTER (P = .0025) WAS SIGNIFICANTLY HYPOMETHYLATED IN HCC PATIENTS COMPARED TO THE NON-HCC PATIENTS SUGGESTING ITS CONTRIBUTION TO THE PROGRESSION OF HCV TOWARDS HCC. TO IDENTIFY THE AGENT FOR ALTERATION IN THE RAS ONCOGENE EXPRESSION, 7 HCV GENES WERE EXPRESSED IN THE HUH-7 CELL LINE FOLLOWED BY MEASUREMENT OF THE NRAS EXPRESSION LEVEL IN HUH-7 BY A QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION. AN INCREASE IN THE MESSENGER RNA LEVEL OF THE NRAS GENE WAS DETECTED WHEN HUH-7 WERE TRANSFECTED WITH CORE, NS5A, AND NS2 GENES. OUR FINDINGS SUGGEST THE INVOLVEMENT OF NRAS ONCOGENE IN THE PATHOGENESIS OF HCV3A DERIVED HCC IN PAKISTANI POPULATION AND ALSO IDENTIFIES THE HCV GENES RESPONSIBLE FOR ITS ENHANCED EXPRESSION. OUR STUDY RAISES THE HYPOTHESIS THAT A SINGLE HCV GENE MAY INCREASE THE CHANCES OF MALIGNANCY. THEREFORE, OUR STUDY MAY HAVE IDENTIFIED A USEFUL EPIGENETIC BIOMARKER OF HCC PROGRESSION IN HCV PATIENTS AND MAY HELP TO DEVELOP NOVEL DIAGNOSTIC TOOLS. 2018 15 4232 41 METHYLATION OF RUNX3 IN VARIOUS TYPES OF HUMAN CANCERS AND PREMALIGNANT STAGES OF GASTRIC CARCINOMA. ACCUMULATING EVIDENCE HAS IDENTIFIED A MECHANISM POTENTIALLY RESPONSIBLE FOR THE INACTIVATION OF TUMOR SUPPRESSOR GENES, NAMELY TRANSCRIPTIONAL SILENCING BY ABERRANT METHYLATION OF CPG ISLANDS. A PREVIOUS STUDY HAS SHOWN THE LOSS OF RUNX3 EXPRESSION, DUE TO ABERRANT METHYLATION OF ITS CPG ISLAND, IN GASTRIC CANCER CELL LINES, SUGGESTING THAT RUNX3 IS A TARGET FOR EPIGENETIC GENE SILENCING IN GASTRIC CARCINOGENESIS. HOWEVER, THERE ARE LIMITED DATA ON THE METHYLATION STATUS OF RUNX3 IN THE NEOPLASTIC AND NON-NEOPLASTIC TISSUES IN VARIOUS TYPES OF HUMAN CANCERS, INCLUDING GASTRIC CANCER. HERE, WE REPORT THAT 60% OF GASTRIC CANCER CELL LINES AND 64% OF PRIMARY GASTRIC CARCINOMAS (N=75) WERE METHYLATED AT THE RUNX3 CPG ISLAND. RUNX3 METHYLATION WAS ALSO DETECTED IN HEPATOCELLULAR CARCINOMAS (73%, N=48), LARYNX CANCERS (62%, N=37), LUNG CANCERS (46%, N=24), BREAST CANCERS (25%, N=25), PROSTATE CANCERS (23%, N=44), ENDOMETRIAL CANCERS (12.5%, N=24), COLON CANCERS (4.9%, N=61) AND UTERINE CERVICAL CANCERS (2.5%, N=40), SHOWING THAT RUNX3 METHYLATION IS NOT RESTRICTED TO GASTRIC CANCER. INTERESTINGLY, THE RUNX3 METHYLATION WAS ESPECIALLY FREQUENT IN TUMORS FROM TISSUES OF A FOREGUT DERIVATIVE, THAT IS, THE STOMACH, LIVER, LARYNX AND LUNG. NEXT, THE METHYLATION STATUS OF RUNX3 IN VARIOUS NON-NEOPLASTIC TISSUES WAS EXAMINED, INCLUDING THE PREMALIGNANT LESIONS OF GASTRIC CARCINOMAS. THE RUNX3 METHYLATION WAS FOUND IN 8.1% OF CHRONIC GASTRITIS (N=99), 28.1% OF INTESTINAL METAPLASIA (N=32), 27.3% OF GASTRIC ADENOMAS (N=77) AND 64% OF GASTRIC CARCINOMAS (N=75), BUT NOT IN CHRONIC HEPATITIS B, NORMAL PROSTATE AND COLON MUCOSA, EVEN THOUGH IN CASES OF CHRONIC HEPATITIS, THE METHYLATION FREQUENCY OF ITS NEOPLASTIC TISSUES WAS VERY HIGH. IN CONCLUSION, RUNX3 METHYLATION IS FREQUENTLY FOUND IN HUMAN CANCERS, INCLUDING GASTRIC CANCER, AND IS MOSTLY CANCER SPECIFIC, WITH THE EXCEPTION OF THE STOMACH, AND THUS, MIGHT BE USEFUL AS A POTENTIAL DIAGNOSTIC BIOMARKER OF CANCER. 2004 16 4225 21 METHYLATION DEGREE OF METALLOPROTEINASE INHIBITOR RECK GENE: LINKS TO RECK PROTEIN LEVEL AND HEPATOCELLULAR CARCINOMA IN CHRONIC HCV INFECTION PATIENTS. THE RECK GENE, A TUMOR SUPPRESSOR GENE, INHIBITS ANGIOGENESIS, INVASION, AND TUMOR METASTASIS. EPIGENETIC REGULATION OF THE RECK GENE CONSTITUTES A POTENT APPROACH TO THE MOLECULAR BASIS OF LIVER MALIGNANCY. THIS STUDY AIMS TO EVALUATE THE PROMOTER METHYLATION STATUS OF THE RECK GENE AND ITS SERUM LEVEL IN PATIENTS WITH HEPATITIS C VIRUS (HCV)-RELATED HEPATOCELLULAR CARCINOMA (HCC) AND THE POTENTIAL ASSOCIATION OF RECK GENE METHYLATION WITH CLINICAL CRITERIA OF HCC. ONE HUNDRED AND FIFTY-FIVE SUBJECTS WERE INCLUDED (HEALTHY CONTROL [55], CHRONIC HCV PATIENTS [55], HCV-RELATED HCC PATIENTS [45]). THE METHYLATION STATUS OF THE RECK GENE PROMOTER AND SERUM RECK LEVEL WERE INVESTIGATED BY METHYLATION-SPECIFIC PCR AND ENZYME-LINKED IMMUNOSORBENT ASSAY TECHNIQUES, RESPECTIVELY. RECK GENE PROMOTER HYPERMETHYLATION WAS RECORDED IN 46.7% OF HCC PATIENTS, AND 10.9% OF HCV PATIENTS, BUT NOT IN CONTROL SUBJECTS (0%). IT WAS RELATED TO RECK PROTEIN LEVEL, VARICES, EDEMA, ASCITES, LYMPH NODE METASTASIS, VASCULAR INVASION, AND THE LARGEST DIAMETER OF FOCAL LESIONS. MEANWHILE, IT WAS NOT ASSOCIATED WITH FOCAL LESION NUMBER NOR DISTANT METASTASIS OF HCC. IN CONCLUSION, RECK GENE PROMOTER HYPERMETHYLATION IS LINKED TO HCV GENOTYPE-4-RELATED HCC. MOREOVER, DIFFERENT DEGREES OF RECK GENE PROMOTER METHYLATION ARE ASSOCIATED WITH SERUM RECK LEVEL, LYMPH NODE METASTASIS, AND VASCULAR INVASION, WHICH COULD PROVE ITS PATHOGENIC ROLE IN HEPATOCARCINOGENESIS IN CHRONIC HCV-INFECTED PATIENTS. 2021 17 2128 28 EPIGENETIC INACTIVATION OF PLCD1 IN CHRONIC MYELOID LEUKEMIA. PHOSPHOLIPASE C DELTA1 (PLCD1), IS LOCATED AT THE IMPORTANT TUMOR SUPPRESSOR LOCUS 3P22. IT ENCODES AN ENZYME THAT MEDIATES REGULATORY SIGNALING OF ENERGY METABOLISM, CALCIUM HOMEOSTASIS AND INTRACELLULAR MOVEMENTS. PLCD1 HAS BEEN STUDIED IN SOME HUMAN SOLID TUMORS RELATING TO THE CPG ISLAND METHYLATION OF THE GENE PROMOTER AS A FUNCTIONAL TUMOR SUPPRESSOR. HOWEVER, NO SUCH INFORMATION IS AVAILABLE IN CHRONIC MYELOID LEUKEMIA (CML). IN THIS STUDY, WE INVESTIGATED PLCD1 EXPRESSION IN THE CML K562 CELL LINE (0/1) AND 15% (2/13) OF BONE MARROW MONONUCLEAR CELLS WITH CML BY USING SEMI-QUANTITATIVE PCR. THE CPG ISLAND (CGI) METHYLATION STATUS OF THE PLCD1 PROMOTER WAS DETECTED IN K562 (0/1) AND 56% (23/41) OF CML PATIENTS BY METHYLATION-SPECIFIC PCR (MSP), BUT NOT IN THE NORMAL ADULT BONE MARROW MONONUCLEAR CELLS. FURTHERMORE, THE DNA DEMETHYLATION AGENT 5'-AZA-2'DEOXYCYTIDINE RESTORED THE EXPRESSION OF PLCD1 IN K562 CELLS. FUNCTIONAL STUDIES SHOWED THAT ECTOPIC EXPRESSION OF PLCD1 IN K562 CELLS WAS ABLE TO DRAMATICALLY INHIBIT THEIR COLONY FORMATION AND INDUCE CELL CYCLE G1 ARREST, SUGGESTING THAT PLCD1 ACTS AS A FUNCTIONAL TUMOR SUPPRESSOR AND MAY SERVE AS A BIOMARKER FOR POSSIBLE EARLY DETECTION AND PROGNOSIS OF CML. 2012 18 6645 31 UP-REGULATION OF DBPA MRNA IN HEPATOCELLULAR CARCINOMA ASSOCIATED WITH METABOLIC SYNDROME. PURPOSE: METABOLIC SYNDROME (MS) IS A GROUP OF RECOGNIZED RISK FACTORS FOR THE DEVELOPMENT OF HEPATOCELLULAR CARCINOMA (HCC) IN PATIENTS WITH CHRONIC LIVER DISEASE. THE AIM OF THIS STUDY WAS TO ANALYZE THE CLINICOPATHOLOGICAL CHARACTERISTICS OF HCC PATIENTS WITH MS AND THE RISK FACTORS FOR RECURRENCE. ALSO, THE AIM WAS TO INVESTIGATE THE COLD SHOCK PROTEIN: DNA-BINDING PROTEIN A (DBPA) EXPRESSION IN HCC PATIENTS WITH MS. METHODS: A TOTAL OF 243 PATIENTS WHO UNDERWENT CURATIVE RESECTIONS FOR HCC WERE CLASSIFIED INTO TWO GROUPS. DBPA EXPRESSION WAS INVESTIGATED IN 66 HCC PATIENTS WITH MS AND IN 30 PATIENTS WITHOUT MS BY USING REAL-TIME RT-PCR. PROMOTER METHYLATION STATUS WAS EXAMINED BY USING MS-PCR. RESULTS: THE INCIDENCE OF METABOLIC FACTORS AFFECT THE HCC SIGNIFICANTLY HIGHER IN NON-B NON-C PATIENTS THAN IN HEPATITIS B VIRUS (HBV) OR HEPATITIS C VIRUS (HCV) PATIENTS (P < 0.001). UNIVARIATE ANALYSIS OF HCC PATIENTS WITH MS RECURRENCE REVEALED ASPARTATE AMINO TRANSFERASE (AST), MULTIPLE TUMORS, LIVER DAMAGE, HEPATIC VEIN INVASION, ADVANCED CANCER STAGES (P < 0.01), ALPHA-FETOPROTEIN (AFP) AND DIABETES MELLITUS TYPE II (P < 0.05) AS RISK FACTORS. MULTIVARIATE ANALYSIS, AST, MULTIPLE TUMORS, AND HEPATIC VEIN INVASION (P < 0.01) WERE IDENTIFIED AS INDEPENDENT FACTORS FOR THE RECURRENCE. DBPA MRNA WAS HIGHER IN PATIENTS WITH MS THAN IN THOSE WITHOUT MS (P = 0.016), AND IT WAS MOSTLY UPREGULATED IN NON-B NON-C HCC PATIENTS WITH MS THAN IN NON-B NON-C HCC PATIENTS WITHOUT HBV OR HCV. ESPECIALLY, IN HCC PATIENTS WITH DIABETES MELLITUS TYPE II, THE MRNA AND PROTEIN LEVELS WERE HIGHLY UPREGULATED. THE DBPA EXPRESSION WAS REGULATED BY PROMOTER METHYLATION STATUS (P < 0.05). CONCLUSIONS: THIS STUDY IDENTIFIES THAT DBPA MAY ACCELERATE THE HEPATOCARCINOGENESIS IN HCC PATIENTS WITH MS VIA INFLAMMATION-INDUCED AND OXIDATIVE STRESS PATHWAYS. THE DEMETHYLATION-RELATED EPIGENETIC ACTIVATION MAY BE ONE OF THE REGULATING FACTORS FOR HCC PATIENTS WITH MS. 2013 19 5275 31 PROMOTER METHYLATION OF THE BONE MORPHOGENETIC PROTEIN-6 GENE IN ASSOCIATION WITH ADULT T-CELL LEUKEMIA. BONE MORPHOGENETIC PROTEINS (BMP), BELONGING TO THE TRANSFORMING GROWTH FACTOR-BETA SUPERFAMILY, ARE MULTIFUNCTIONAL REGULATORS OF CELL PROLIFERATION, DIFFERENTIATION AND APOPTOSIS IN VARIOUS TYPES OF MALIGNANT CELLS. IN THIS STUDY, WE INVESTIGATED BMP-6 PROMOTER METHYLATION IN PATIENTS WITH VARIOUS TYPES OF LEUKEMIAS. THE BMP-6 METHYLATION WAS FOUND PREFERENTIALLY IN ADULT T-CELL LEUKEMIA (ATL) (49 OF 60, 82%) COMPARED WITH OTHER TYPES OF LEUKEMIAS STUDIED INCLUDING ACUTE MYELOID LEUKEMIA (3 OF 67, 5%), ACUTE LYMPHOBLASTIC LEUKEMIA (6 OF 38, 16%) AND CHRONIC LYMPHOCYTIC LEUKEMIA (1 OF 21, 5%). AMONG SUBTYPES OF ATL, THE BMP-6 GENE WAS MORE FREQUENTLY METHYLATED IN AGGRESSIVE ATL FORMS OF ACUTE (96%) AND LYMPHOMA (94%) TYPES THAN LESS MALIGNANT CHRONIC ATL (44%) AND SMOLDERING ATL (20%). WE ALSO ANALYZED THE METHYLATION STATUS OF PERIPHERAL BLOOD MONONUCLEAR CELLS FROM HEALTHY DONORS AND NONMALIGNANT LYMPH NODES WITH REACTIVE LYMPHADENOPATHY, NONE OF WHICH SHOWED DETECTABLE BMP-6 METHYLATION IN THIS STUDY. THE BMP-6 METHYALTION WAS CORRELATED WITH DECREASED MRNA TRANSCRIPT AND PROTEIN EXPRESSION. EXPRESSION OF BMP-6 WAS RESTORED BY THE DEMETHYLATING AGENT 5-AZA-2'-DEOXYCYTIDINE, SUGGESTING THAT METHYLATION WAS ASSOCIATED WITH THE TRANSCRIPTIONAL SILENCING. SERIAL ANALYSIS DEMONSTRATED AN INCREASING METHYLATION OF CPG SITES IN THE BMP-6 PROMOTER AND THE RESULTANT SUPPRESSION OF BMP-6 EXPRESSION AS ATL PROGRESSED. THESE FINDINGS SUGGESTED THAT BMP-6 PROMOTER METHYLATION IS LIKELY TO BE A COMMON EPIGENETIC EVENT AT LATER STAGES OF ATL AND THAT THE METHYLATION PROFILES MAY BE USEFUL FOR THE STAGING OF ATL AS WELL AS FOR EVALUATION OF THE INDIVIDUAL RISK OF DEVELOPING THE DISEASE. 2008 20 2262 27 EPIGENETIC PROFILING IN CHRONIC LYMPHOCYTIC LEUKEMIA REVEALS NOVEL METHYLATION TARGETS. CPG ISLAND METHYLATION IS AN EPIGENETIC ALTERATION THAT CONTRIBUTES TO TUMORIGENESIS BY TRANSCRIPTIONAL INACTIVATION OF GENES. LITTLE IS KNOWN ABOUT THE OVERALL LEVELS OF CPG ISLAND METHYLATION IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). TO PROVIDE A BASELINE ESTIMATE OF GLOBAL ABERRANT METHYLATION AND IDENTIFY TARGET SEQUENCES FOR ADDITIONAL INVESTIGATION, WE PERFORMED RESTRICTION LANDMARK GENOMIC SCANNING ON 10 CLL SAMPLES. TWO METHYLATION-SENSITIVE LANDMARK ENZYMES WERE USED (NOTI AND ASCI), ALLOWING ASSESSMENT OF OVER 3000 CPG ISLANDS IN EACH SAMPLE. TUMOR-DERIVED RESTRICTION LANDMARK GENOMIC SCANNING PROFILES WERE COMPARED WITH PROFILES FROM CD19-SELECTED B CELLS FROM NORMAL VOLUNTEERS AND MATCHED NORMAL NEUTROPHILS FROM 4 CLL PATIENTS. WE FOUND 2.5-8.1% (MEAN 4.8%) OF THE CPG ISLANDS IN CLL SAMPLES WERE ABERRANTLY METHYLATED COMPARED WITH CONTROLS, AND THE METHYLATION EVENTS HAD A NONRANDOM DISTRIBUTION (P < 0.0001). FURTHERMORE, WE IDENTIFIED 193 ABERRANTLY METHYLATED SEQUENCES, OF WHICH 93% HAVE CPG ISLAND CHARACTERISTICS AND 90% HAVE HOMOLOGY TO GENES OR EXPRESSED SEQUENCES. ONE SUCH GENE, THE G PROTEIN-COUPLED METABOTROPIC GLUTAMATE RECEPTOR 7 (GRM7), POSSIBLY INHIBITS CYCLIC AMP SIGNALING IN THE INDUCTION OF APOPTOSIS. BISULFITE SEQUENCING OF GRM7 CONFIRMED EXTENSIVE CPG ISLAND METHYLATION, AND TREATMENT WITH 5-AZA-2'-DEOXYCYTIDINE (DECITABINE) RESULTED IN UP-REGULATED EXPRESSION OF SEVERAL GENES IN VITRO WITH CONCURRENT CELLULAR DEPLETION OF DNMT1 PROTEIN. OUR DUAL-ENZYME GLOBAL METHYLATION STUDY SHOWS THAT CLL IS CHARACTERIZED BY WIDESPREAD NONRANDOM CPG ISLAND METHYLATION SIMILAR TO OTHER TUMORS AND PROVIDES A PANEL OF NOVEL METHYLATION TARGETS THAT CAN BE USED IN LARGER STUDIES DESIGNED TO ASSESS IMPACT ON DISEASE PROGRESSION AND SURVIVAL. 2004