1 3040 116 GENOME HYPERMETHYLATION IN PINUS SILVESTRIS OF CHERNOBYL--A MECHANISM FOR RADIATION ADAPTATION? ADAPTATION IS A COMPLEX PROCESS BY WHICH POPULATIONS OF ORGANISMS RESPOND TO LONG-TERM ENVIRONMENTAL STRESSES BY PERMANENT GENETIC CHANGE. HERE WE PRESENT DATA FROM THE NATURAL "OPEN-FIELD" RADIATION ADAPTATION EXPERIMENT AFTER THE CHERNOBYL ACCIDENT AND PROVIDE THE FIRST EVIDENCE OF THE INVOLVEMENT OF EPIGENETIC CHANGES IN ADAPTATION OF A EUKARYOTE-SCOTS PINE (PINUS SILVESTRIS), TO CHRONIC RADIATION EXPOSURE. WE HAVE EVALUATED GLOBAL GENOME METHYLATION OF CONTROL AND RADIATION-EXPOSED PINE TREES USING A METHOD BASED ON CLEAVAGE BY A METHYLATION-SENSITIVE HPAII RESTRICTION ENDONUCLEASE THAT LEAVES A 5' GUANINE OVERHANG AND SUBSEQUENT SINGLE NUCLEOTIDE EXTENSION WITH LABELED [3H] DCTP. WE HAVE FOUND THAT GENOMIC DNA OF EXPOSED PINE TREES WAS CONSIDERABLY HYPERMETHYLATED. MOREOVER, HYPERMETHYLATION APPEARED TO BE DEPENDENT UPON THE RADIATION DOSE ABSORBED BY THE TREES. SUCH HYPERMETHYLATION MAY BE VIEWED AS A DEFENSE STRATEGY OF PLANTS THAT PREVENTS GENOME INSTABILITY AND RESHUFFLING OF THE HEREDITARY MATERIAL, ALLOWING SURVIVAL IN AN EXTREME ENVIRONMENT. FURTHER STUDIES ARE CLEARLY NEEDED TO ANALYZE IN DETAIL THE INVOLVEMENT OF DNA METHYLATION AND OTHER EPIGENETIC MECHANISMS IN THE COMPLEX PROCESS OF RADIATION STRESS AND ADAPTIVE RESPONSE. 2003 2 985 27 CHRONIC RADIATION EXPOSURE AS AN ECOLOGICAL FACTOR: HYPERMETHYLATION AND GENETIC DIFFERENTIATION IN IRRADIATED SCOTS PINE POPULATIONS. GENETIC AND EPIGENETIC CHANGES WERE INVESTIGATED IN CHRONICALLY IRRADIATED SCOTS PINE (PINUS SYLVESTRIS L.) POPULATIONS FROM TERRITORIES THAT WERE HEAVILY CONTAMINATED BY RADIONUCLIDES AS RESULT OF THE CHERNOBYL NUCLEAR POWER PLANT ACCIDENT. IN COMPARISON TO THE REFERENCE SITE, THE GENETIC DIVERSITY REVEALED BY ELECTROPHORETIC MOBILITY OF AFLPS WAS FOUND TO BE SIGNIFICANTLY HIGHER AT THE RADIOACTIVELY CONTAMINATED AREAS. IN ADDITION, THE GENOME OF PINE TREES WAS SIGNIFICANTLY HYPERMETHYLATED AT 4 OF THE 7 AFFECTED SITES. 2018 3 6777 20 [ASSOCIATION OF P-MOBILE ELEMENT ACTIVITY AND DNA METHYLATION PATTERN CHANGES IN THE CONDITIONS OF DROSOPHILA MELANOGASTER PROLONGED IRRADIATION]. ASSOCIATION OF THE RADIOSENSITIVITY AND EPIGENETIC PATTERN DNA CHANGES AT THE CONDITIONS OF PROLONGED IRRADIATION WAS INVESTIGATED. TWO LABORATORY DROSOPHILA MELANOGASTER STRAINS (CANTON-S AND RI) IRRADIATED FOR 20 GENERATIONS TO LOW DOSES RATE (1.2 X 10(-1), 0.8 X 10(-8) AND 0.12 X 10(-8) GY/S) WERE USED AS EXPERIMENTAL OBJECTS. DNA FOR THE ANALYSIS WAS EXTRACTED SEPARATELY FOR THE FLIES OF MALES AND FEMALES. RESTRICTION ENDONUCLEASES GLUL, GLAL WERE USED. RESTRICTION ANALYSIS HAS SHOWN THAT THERE ARE DIFFERENT DNA METHYLATED PATTERNS FOR MALES AND FEMALES AS FOR CONTROL AND EXPOSED VARIANTS. AT THE CHRONIC IRRADIATION THERE WAS THE DECLINE OF METHYLATION LEVEL AT THE ENZYMES GLUL, GLAL SITES RECOGNITION. 2010 4 6249 24 THE METHYLATION STATUS OF THE MAJOR BREAKPOINT CLUSTER REGION IN HUMAN LEUKEMIA CELLS, INCLUDING PHILADELPHIA CHROMOSOME-POSITIVE CELLS, IS LINKED TO THE LINEAGE OF HEMATOPOIETIC CELLS. THE PHILADELPHIA (PH) TRANSLOCATION [T(9;22)(Q34;Q11)] IS THE MOST COMMON GENETIC ABNORMALITY IN HUMAN LEUKEMIA; A TRANSPOSITION OF THE ABL GENE TO THE MAJOR-BREAKPOINT CLUSTER REGION (M-BCR) IS ASSOCIATED WITH THE PATHOGENESIS IN PH+ CHRONIC MYELOGENOUS LEUKEMIA (PH+ CML) AND IN SOME CASES OF PH+ ACUTE LEUKEMIA (PH+ AL). OUR CURRENT UNDERSTANDING OF THE METHYLATION OF HUMAN GENOMES ALLOWS US TO CONSIDER THE ASSOCIATION BETWEEN THE EPIGENETIC PHENOMENON AND THE CONTROL OF DIFFERENTIATION AND PROLIFERATION IN MAMMALIAN CELLS. IN ORDER TO DETERMINE WHETHER THE METHYLATION STATUS OF THE M-BCR IS ASSOCIATED WITH BREAKPOINT-LOCALIZATION IN THIS REGION AND WITH THE LINEAGE OF HEMATOPOIETIC CELLS, WE HAVE EXAMINED 28 PATIENTS WITH PH+ LEUKEMIAS, INCLUDING NINE WITH PH+ AL, SIX PATIENTS WITH ACUTE MYELOBLASTIC LEUKEMIA WITHOUT PH (PH- AML), AND FIVE PATIENTS WITH PH- ACUTE LYMPHOBLASTIC LEUKEMIA (PH- ALL); USING THE RESTRICTION ENDONUCLEASE ISOCHIZOMERS, MSPI AND HPAII. IN CML PATIENTS IN THE CHRONIC PHASE, THE HYPOMETHYLATED STATUS WITHIN THE NORMAL M-BCR ALLELE IS HETEROGENEOUS. IN CONTRAST, PATIENTS WITH PH+ CML IN THE LYMPHOID BLAST CRISIS PHASE EXHIBITED A 2.5/2.7 KB BAND WITH A COMPLETE DISAPPEARANCE OF THE GERMLINE M-BCR FRAGMENT (TYPE L). THIS PATTERN IS CONSISTENTLY NOTED IN PH- ALL CELLS, AND THE PATTERN IS QUITE DIFFERENT FROM THAT FOUND IN MYELOID BLAST CRISIS OR PH- AML (TYPE M). IN PATIENTS WITH M-BCR-NONREARRANGED PH+ ALL, IT IS SUGGESTED THAT THE M-BCR METHYLATION PATTERNS ARE CELL-LINEAGE SPECIFIC BUT SOME PH+ ALL CELLS HAD A HYPOMETHYLATION PATTERN THAT WAS IDENTICAL TO THAT OBSERVED IN PH- AML, SUGGESTING A DISTINCTION OF GENETIC DIVERSITY OF LEUKEMIA CELLS WITH THE PH CHROMOSOME, ESPECIALLY PH+ AL. 1993 5 5861 31 SUPER-ENHANCER LANDSCAPE REVEALS LEUKEMIA STEM CELL RELIANCE ON X-BOX BINDING PROTEIN 1 AS A THERAPEUTIC VULNERABILITY. RELAPSE OF PATIENTS WITH CHRONIC MYELOGENOUS LEUKEMIA (CML) MAY OCCUR AT LEAST PARTIALLY BECAUSE LEUKEMIA STEM CELLS (LSCS) LACK SENSITIVITY TO TYROSINE KINASE INHIBITORS (TKIS) SUCH AS IMATINIB. THE PRECISE REGULATION OF LSC STEMNESS IS INCOMPLETELY UNDERSTOOD. GIVEN THAT TRAITS OF LSCS ARE SUBJECT TO EPIGENETIC REGULATION, WE HYPOTHESIZED THAT LSCS MIGHT BE DEPENDENT ON CONTINUOUS ACTIVE TRANSCRIPTION OF GENES ASSOCIATED WITH SUPER-ENHANCERS (SES), WHICH MIGHT, IN TURN, SUGGEST AN OPPORTUNITY FOR INTERVENTION. IN THIS STUDY, WE TESTED THIS HYPOTHESIS AND DELINEATED THE SE LANDSCAPE IN LSCS FROM PATIENTS WITH CML. DISRUPTION OF THE SE-ASSOCIATED GENE TRANSCRIPTION BY THZ1, A COVALENT CYCLIN-DEPENDENT KINASE 7 (CDK7) INHIBITOR, EFFICIENTLY ERADICATED LSCS IN RETROVIRAL BCR-ABL-DRIVEN CML MICE WHILE SPARING NORMAL HEMATOPOIETIC STEM CELLS. FURTHERMORE, WE FOUND THAT X-BOX BINDING PROTEIN 1 (XBP1), A SUBSTRATE OF MRNA-SPLICING ENDONUCLEASE IRE1ALPHA IN THE UNFOLDED PROTEIN RESPONSE PATHWAY, WAS AN SE-ASSOCIATED ONCOGENE IN LSCS. KNOCKDOWN OF XBP1 REDUCED SURVIVAL AND SELF-RENEWAL CAPACITY IN PRIMARY CML CD34(+) CELLS AND ERADICATED LSCS IN CML MICE. SELECTIVELY BLOCKING GENERATION OF THE SPLICED FORM OF XBP1 BY HEMATOPOIETIC CELL-SPECIFIC IRE1 CONDITIONAL KNOCKOUT SUPPRESSED THE PROGRESSION OF CML AND IMPAIRED THE LEUKEMOGENESIS OF LSCS IN CML MICE. OVERALL, WE IDENTIFIED AN EPIGENETIC TRANSCRIPTIONAL PROGRAM IN LSCS, ADDING TO EVIDENCE FOR THE THEORY OF "ONCOGENE ADDICTION" AND SUGGESTING A POTENTIAL TARGETING STRATEGY FOR CML. 2021 6 1115 37 COMPARATIVE ANALYSIS OF EPIGENETIC VARIABILITY IN TWO PINE SPECIES EXPOSED TO CHRONIC RADIATION IN THE CHERNOBYL AND FUKUSHIMA AFFECTED ZONES. COMPARATIVE ANALYSIS OF EPIGENETIC VARIABILITY IN TWO PINE SPECIES AFFECTED AS A RESULT OF THE CHERNOBYL AND FUKUSHIMA ACCIDENTS IS PRESENTED. THE ABSORBED DOSE RATE WITHIN THE AFFECTED CHERNOBYL SITES VARIES OVER A WIDER RANGE (1.5-24.6 MUGY/H) THAN WITHIN THE FUKUSHIMA SITES (3.5-6.5 MUGY/H). IT WAS SHOWN THAT CHRONIC IRRADIATION CAN CHANGE THE LEVEL OF WHOLE GENOME METHYLATION IN PINE POPULATIONS, BUT IN DIFFERENT WAYS. THE GENOMES OF JAPANESE RED PINES ARE HYPOMETHYLATED, AND THE DEGREE OF METHYLATION AND HYDROXYMETHYLATION DECREASES WITH AN INCREASE IN THE LEVEL OF RADIATION EXPOSURE. IN CONTRAST, THE PERCENTAGES OF GENOME METHYLATION AND HYDROXYMETHYLATION IN SCOTS PINE POPULATIONS EXCEED THE REFERENCE LEVELS. THE OBSERVED DISCREPANCY IN THE PATTERNS OF GENOME-WIDE DNA METHYLATION CAN BE ATTRIBUTED PARTLY TO THE DESIGN OF THE STUDY (DIFFERENCES IN THE CLIMATE, RADIATION DOSE, AGE AND SPECIES OF THE PINES) WHICH COULD AFFECT THE RESULTS. IN THE FRAME OF IRAP ANALYSIS, A LARGER NUMBER OF DIFFERENT BANDS WAS OBSERVED IN THE CHERNOBYL POPULATIONS COMPARED TO THE JAPANESE POPULATIONS. BOTH THE JAPANESE AND CHERNOBYL POPULATIONS ARE CHARACTERIZED BY SIGNIFICANT GENETIC VARIABILITY. HOWEVER, THE MAIN PART OF THIS VARIABILITY IS OBSERVED WITHIN POPULATIONS. THE DENDROGRAMS, BASED ON PRESENCE/ABSENCE OF IRAP FRAGMENTS AND NEI'S GENETIC DISTANCES, REVEALED SUBDIVISIONS OF THE CHERNOBYL AND JAPANESE POPULATIONS ACCORDING TO THE LEVEL OF RADIOACTIVE CONTAMINATION. ANALYSIS OF THE RESULTS PRESENTED WILL IMPROVE OUR UNDERSTANDING OF THE MECHANISMS UNDERLYING THE RESPONSES OF PINE TREES TO CHRONIC RADIATION EXPOSURE. 2023 7 6248 22 THE METHYLATION STATUS OF THE DDX43 PROMOTER IN CHINESE PATIENTS WITH CHRONIC MYELOID LEUKEMIA. ABERRANT DNA METHYLATION IS A COMMON EPIGENETIC ALTERATION AND AN IMPORTANT FEATURE IN HUMAN CANCERS. THE DEAD BOX POLYPEPTIDE 43 (DDX43) HAS BEEN FOUND TO BE OVEREXPRESSED IN VARIOUS SOLID TUMORS AND SOME HEMATOLOGIC MALIGNANCIES. IN THE PRESENT STUDY, WE INVESTIGATED THE METHYLATION STATUS OF THE DDX43 PROMOTER IN 87 CHINESE PATIENTS WITH CHRONIC MYELOID LEUKEMIA (CML) USING REAL-TIME QUANTITATIVE METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION AND EXAMINED THE DDX43 TRANSCRIPT IN 35 PATIENTS USING REAL-TIME QUANTITATIVE POLYMERASE CHAIN REACTION. DDX43 PROMOTER HYPOMETHYLATION WAS OBSERVED IN 22 (25.3%) CML PATIENTS. NO SIGNIFICANT CORRELATION WAS FOUND BETWEEN THE HYPOMETHYLATION OF THE DDX43 PROMOTER WITH THE AGE, SEX, WHITE BLOOD CELL COUNTS, HEMOGLOBIN CONCENTRATION, PLATELET COUNTS, AND CHROMOSOMAL ABNORMALITIES OF CML PATIENTS (P>0.05). THE FREQUENCY OF DDX43 HYPOMETHYLATION IN PATIENTS IN THE CHRONIC PHASE, IN THE ACCELERATED PHASE, AND IN BLAST CRISIS WAS 23.4% (15/64), 25.0% (2/8), AND 33.3% (5/15), RESPECTIVELY (P>0.05). THERE WAS A SIGNIFICANT CORRELATION BETWEEN DDX43 HYPOMETHYLATION AND DDX43 TRANSCRIPT (R=0.469, P=0.004). OUR DATA SUGGEST THAT HYPOMETHYLATION OF THE DDX43 PROMOTER MAY BE AN EARLY AND FREQUENT MOLECULAR EVENT IN THE DEVELOPMENT OF CML IN CHINESE PATIENTS. 2013 8 156 24 ABERRANT METHYLATION OF THE DEATH-ASSOCIATED PROTEIN KINASE 1 (DAPK1) CPG ISLAND IN CHRONIC MYELOID LEUKEMIA. THE DEATH-ASSOCIATED PROTEIN KINASE 1 (DAPK1) GENE IS A CANDIDATE TUMOR SUPPRESSOR (TSG) AND THE ABNORMAL METHYLATION OF DAPK1 GENE HAS BEEN FOUND IN MANY CARCINOMAS. THE EPIGENETIC CHANGES OF TSGS ARE NOW RECOGNIZED AS A MECHANISM CONTRIBUTING TO THE DEVELOPMENT OF CHRONIC MYELOID LEUKEMIA (CML). TO CLARIFY THE ROLE OF DAPK1 IN CML, WE EXAMINED THE METHYLATION STATUS OF DAPK1 IN 49 PATIENTS WITH CML USING METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION. THE ABERRANT METHYLATION OF THE DAPK1 GENE WAS FOUND IN 25 OF 49 (51.0%) CML CASES, NOT IN ALL CONTROLS. NO CORRELATION WAS FOUND BETWEEN DAPK1 GENE METHYLATION AND THE AGE, HEMATOLOGIC PARAMETERS, CHROMOSOMAL ABNORMALITIES, THE TYPES AND LEVELS OF BCR/ABL TRANSCRIPTS OF CML PATIENTS. HOWEVER, CORRELATION COULD BE OBSERVED BETWEEN THE SEX AND THE STATUS OF DAPK1 METHYLATION IN CML PATIENTS (R = 0.374, P = 0.008). FURTHERMORE, THERE WAS A SIGNIFICANT CORRELATION BETWEEN DAPK1 METHYLATION AND THE STAGES OF CML (R = 0.354, P = 0.013). THE CML PATIENTS IN ACCELERATED PHASE (AP) AND BLAST CRISIS (BC) HAD HIGHER FREQUENCY OF DAPK1 METHYLATION THAN THOSE IN CHRONIC PHASE (CP) (75.0% VS. 34.5%) (CHI(2) = 7.776, P = 0.005). IN ONE PATIENT, THE STATUS OF DAPK1 METHYLATION BECAME POSITIVE ON THE TRANSITION FROM CP TO AP AND BC. THESE RESULTS SUGGESTED THAT DAPK1 PROMOTER METHYLATION MIGHT PLAY A SIGNIFICANT ROLE IN THE PROGRESSION OF CML. 2009 9 481 34 ARSENIC-INDUCED SUMOYLATION OF MUS81 IS INVOLVED IN REGULATING GENOMIC STABILITY. CHRONIC ENVIRONMENTAL EXPOSURE TO METAL TOXICANTS SUCH AS CHROMIUM AND ARSENIC IS CLOSELY RELATED TO THE DEVELOPMENT OF SEVERAL TYPES OF COMMON CANCERS. GENETIC AND EPIGENETIC STUDIES IN THE PAST DECADE REVEAL THAT POST-TRANSLATIONAL MODIFICATIONS OF HISTONES PLAY A ROLE IN METAL CARCINOGENESIS. HOWEVER, EXACT MOLECULAR MECHANISMS OF METAL CARCINOGENESIS REMAIN TO BE ELUCIDATED. IN THIS STUDY WE FOUND THAT AS(2)O(3), AN ENVIRONMENTAL METAL TOXICANT, UPREGULATED OVERALL MODIFICATIONS OF MANY CELLULAR PROTEINS BY SUMO2/3. SUMOYLATED PROTEINS FROM ARSENIC-TREATED CELLS CONSTITUTIVELY EXPRESSING HIS(6)-SUMO2 WERE PULLED DOWN BY NI-IDA RESIN UNDER DENATURING CONDITIONS. MASS SPECTROMETRIC ANALYSIS REVEALED OVER 100 PROTEINS THAT WERE POTENTIALLY MODIFIED BY SUMOYLATION. MUS81, A DNA ENDONUCLEASE INVOLVED IN HOMOLOGOUS RECOMBINATION REPAIR, WAS AMONG THE IDENTIFIED PROTEINS WHOSE SUMOYLATION WAS INCREASED AFTER TREATMENT WITH AS(2)O(3.) WE FURTHER SHOWED THAT K10 AND K524 WERE 2 LYSINE RESIDUES ESSENTIAL FOR MUS81 SUMOYLATION. MOREOVER, WE DEMONSTRATED THAT MUS81 SUMOYLATION IS IMPORTANT FOR NORMAL MITOTIC CHROMOSOME CONGRESSION AND THAT CELLS EXPRESSING SUMO-RESISTANT MUS81 MUTANTS DISPLAYED COMPROMISED DNA DAMAGE RESPONSES AFTER EXPOSURE TO METAL TOXINS SUCH AS CR(VI) AND ARSENIC. 2017 10 5940 31 TARGETING METHYLTRANSFERASE PRMT5 ELIMINATES LEUKEMIA STEM CELLS IN CHRONIC MYELOGENOUS LEUKEMIA. IMATINIB-INSENSITIVE LEUKEMIA STEM CELLS (LSCS) ARE BELIEVED TO BE RESPONSIBLE FOR RESISTANCE TO BCR-ABL TYROSINE KINASE INHIBITORS AND RELAPSE OF CHRONIC MYELOGENOUS LEUKEMIA (CML). IDENTIFYING THERAPEUTIC TARGETS TO ERADICATE CML LSCS MAY BE A STRATEGY TO CURE CML. IN THE PRESENT STUDY, WE DISCOVERED A POSITIVE FEEDBACK LOOP BETWEEN BCR-ABL AND PROTEIN ARGININE METHYLTRANSFERASE 5 (PRMT5) IN CML CELLS. OVEREXPRESSION OF PRMT5 WAS OBSERVED IN HUMAN CML LSCS. SILENCING PRMT5 WITH SHRNA OR BLOCKING PRMT5 METHYLTRANSFERASE ACTIVITY WITH THE SMALL-MOLECULE INHIBITOR PJ-68 REDUCED SURVIVAL, SERIAL REPLATING CAPACITY, AND LONG-TERM CULTURE-INITIATING CELLS (LTC-ICS) IN LSCS FROM CML PATIENTS. FURTHER, PRMT5 KNOCKDOWN OR PJ-68 TREATMENT DRAMATICALLY PROLONGED SURVIVAL IN A MURINE MODEL OF RETROVIRAL BCR-ABL-DRIVEN CML AND IMPAIRED THE IN VIVO SELF-RENEWAL CAPACITY OF TRANSPLANTED CML LSCS. PJ-68 ALSO INHIBITED LONG-TERM ENGRAFTMENT OF HUMAN CML CD34+ CELLS IN IMMUNODEFICIENT MICE. MOREOVER, INHIBITION OF PRMT5 ABROGATED THE WNT/BETA-CATENIN PATHWAY IN CML CD34+ CELLS BY DEPLETING DISHEVELLED HOMOLOG 3 (DVL3). THIS STUDY SUGGESTS THAT EPIGENETIC METHYLATION MODIFICATION ON HISTONE PROTEIN ARGININE RESIDUES IS A REGULATORY MECHANISM TO CONTROL SELF-RENEWAL OF LSCS AND INDICATES THAT PRMT5 MAY REPRESENT A POTENTIAL THERAPEUTIC TARGET AGAINST LSCS. 2016 11 3530 28 IMATINIB (ST1571) PROVIDES ONLY LIMITED SELECTIVITY FOR CML CELLS AND TREATMENT MIGHT BE COMPLICATED BY SILENT BCR-ABL GENES. VERY PROMISING RESULTS HAVE BEEN OBTAINED IN CLINICAL TRIALS ON CHRONIC-PHASE CHRONIC MYELOID LEUKEMIA (CP-CML) PATIENTS TREATED WITH IMATINIB MESYLATE (IM; GLEEVECR, STI571), A BCR-ABL TYROSINE KINASE INHIBITOR. HOWEVER, WE FOUND THAT IM CAUSED CONSIDERABLE INHIBITION OF NORMAL HEMATOPOIETIC PROGENITOR CELLS UPON TREATING CONTROL BONE MARROW (BM) CULTURES. IN VITRO IM TREATMENT GAVE A DECREASE IN THE YIELD AND SIZE OF COLONIES FROM BM OF UNTREATED CP-CML PATIENTS THAT WAS ONLY TWO TO THREE TIMES THAT FROM THE NORMAL SAMPLES. MOREOVER, ABOUT 30% OF MYELOID PROGENITORS (CFU-GM) FROM CML BM STILL FORMED COLONIES IN THE PRESENCE OF IM, MOST OF WHICH HAD BCR-ABL RNA. ABOUT HALF OF THESE TREATED COLONIES ALSO DISPLAYED METHYLATION OF THE INTERNAL ABL PA PROMOTER, A CML-SPECIFIC EPIGENETIC ALTERATION, WHICH WAS USED IN THIS STUDY AS A MARKER FOR BCR-ABL TRANSLOCATION-CONTAINING CELLS. HOWEVER, ~5-8% OF THE TREATED OR THE UNTREATED CML BM-DERIVED COLONIES HAD NO DETECTABLE BCR-ABL RNA BY TWO OR THREE ROUNDS OF RT-PCR DESPITE BEING POSITIVE FOR THE INTERNAL STANDARD RNA AND DISPLAYING HALLMARKS OF CML, EITHER T(9;22)(Q34;QL 1) OR ABL PA METHYLATION. OUR RESULTS INDICATE THAT IM IS ONLY PARTIALLY SPECIFIC FOR CML PROGENITOR CELLS COMPARED TO NORMAL HEMATOPOIETIC PROGENITOR CELLS AND SUGGEST THAT SOME CML CELLS MAY HAVE A SILENT BCR-ABL ONCOGENE THAT COULD INTERFERE WITH THERAPY. 2003 12 2327 27 EPIGENETIC REGULATION OF HUMAN CANCER/TESTIS ANTIGEN GENE, HAGE, IN CHRONIC MYELOID LEUKEMIA. BACKGROUND AND OBJECTIVES: CANCER TESTIS ANTIGENS (CTA) PROVIDE ATTRACTIVE TARGETS FOR CANCER-SPECIFIC IMMUNOTHERAPY. ALTHOUGH CTA GENES ARE EXPRESSED IN SOME NORMAL TISSUES, SUCH AS THE TESTIS, THIS IMMUNOLOGICALLY PROTECTED SITE LACKS MHC I EXPRESSION AND AS SUCH, DOES NOT PRESENT SELF ANTIGENS TO T CELLS. TO DATE, CTA GENES HAVE BEEN SHOWN TO BE EXPRESSED IN A RANGE OF SOLID TUMORS VIA DEMETHYLATION OF THEIR PROMOTER CPG ISLANDS, BUT RARELY IN CHRONIC MYELOID LEUKEMIA (CML) OR OTHER HEMATOLOGIC MALIGNANCIES. DESIGN AND METHODS: IN THIS STUDY, THE METHYLATION STATUS OF THE HAGE CTA GENE PROMOTER WAS ANALYZED BY QUANTITATIVE METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (MSP) AND SEQUENCING IN FOUR PHILADELPHIA-POSITIVE CELL LINES (TCC-S, K562, KU812 AND KYO-1) AND IN CML SAMPLES TAKEN FROM PATIENTS IN CHRONIC PHASE (CP N=215) OR BLAST CRISIS (BC N=47). HAGE EXPRESSION WAS ASSESSED BY QUANTITATIVE REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION. RESULTS: THE TCC-S CELL LINE SHOWED DEMETHYLATION OF HAGE THAT WAS ASSOCIATED WITH OVER-EXPRESSION OF THIS GENE. HAGE HYPOMETHYLATION WAS SIGNIFICANTLY MORE FREQUENT IN BC (46%) THAN IN CP (22%) (P=0.01) AND WAS CORRELATED WITH HIGH EXPRESSION LEVELS OF HAGE TRANSCRIPTS (P<0.0001). OF NOTE, IN CP-CML, EXTENSIVE HAGE HYPOMETHYLATION WAS ASSOCIATED WITH POORER PROGNOSIS IN TERMS OF CYTOGENETIC RESPONSE TO INTERFERON (P=0.01) OR IMATINIB (P=0.01), MOLECULAR RESPONSE TO IMATINIB (P=0.003) AND PROGRESSION-FREE SURVIVAL (P=0.05). INTERPRETATIONS AND CONCLUSION: THE METHYLATION STATUS OF THE HAGE PROMOTER DIRECTLY CORRELATES WITH ITS EXPRESSION IN BOTH CML CELL LINES AND PATIENTS AND IS ASSOCIATED WITH ADVANCED DISEASE AND POOR OUTCOME. 2007 13 3114 26 GERMLINE ALLELE-SPECIFIC EXPRESSION OF DAPK1 IN CHRONIC LYMPHOCYTIC LEUKEMIA. WE PREVIOUSLY REPORTED A RARE GERMLINE VARIANT (C.1-6531) THAT RESULTED IN ALLELE-SPECIFIC EXPRESSION (ASE) OF DEATH-ASSOCIATED PROTEIN KINASE 1 (DAPK1) AND PREDISPOSITION TO CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). WE INVESTIGATED A COHORT OF CLL PATIENTS LACKING THIS MUTATION FOR THE PRESENCE OF ASE OF DAPK1. WE DEVELOPED A NOVEL STRATEGY THAT COMBINES SINGLE-NUCLEOTIDE PRIMER EXTENSION (SNUPE) WITH MALDI-TOF MASS SPECTROMETRY, AND DETECTED GERMLINE DAPK1 ASE IN 17 OUT OF 120 (14.2%) CLL PATIENTS ASSOCIATED WITH A TREND TOWARDS YOUNGER AGE AT DIAGNOSIS. ASE WAS ABSENT IN 63 HEALTHY CONTROLS. GERMLINE CELLS OF CLL PATIENTS WITH ASE SHOWED INCREASED LEVELS OF DNA METHYLATION IN THE PROMOTER REGION, HOWEVER, NEITHER GENETIC NOR FURTHER EPIGENETIC ABERRATIONS COULD BE IDENTIFIED IN THE DAPK1 5' UPSTREAM REGULATORY REGION, WITHIN DISTINCT EXONS OR IN THE 3'-UTR. WE IDENTIFIED B-LYMPHOID MALIGNANCY RELATED CELL LINE MODELS HARBORING ALLELIC IMBALANCE AND FOUND THAT ALLELE-SPECIFIC METHYLATION IN DAPK1 IS ASSOCIATED WITH ASE. OUR DATA INDICATE THAT ASE AT THE DAPK1 GENE LOCUS IS A RECURRENT EVENT, MEDIATED BY EPIGENETIC MECHANISMS AND POTENTIALLY PREDISPOSING TO CLL. 2013 14 162 17 ABL1 METHYLATION IN PH-POSITIVE ALL IS EXCLUSIVELY ASSOCIATED WITH THE P210 FORM OF BCR-ABL. IN HUMAN PH-POSITIVE LEUKEMIA THERE IS A CLEAR ASSOCIATION OF DIFFERENT FORMS OF THE BCR-ABL ONCOGENE WITH DISTINCT TYPES OF LEUKEMIA. THE P190 FORM OF BCR-ABL IS RARELY OBSERVED IN CHRONIC MYELOID LEUKEMIA (CML) BUT IS PRESENT IN 50% OF PH-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA (ALL). IN CONTRAST, THE P210 FORM IS OBSERVED BOTH IN CML AND 50% OF PH-POSITIVE ALL. METHYLATION OF THE PROXIMAL PROMOTER OF THE ABL1 GENE HAS BEEN SHOWN TO BE A NEARLY UNIVERSAL EVENT ASSOCIATED WITH CLINICAL PROGRESSION OF CML. THIS RAISES THE QUESTION OF WHETHER METHYLATION OF THE ABL1 PROMOTER IS AN EPIGENETIC MODIFICATION ALSO ASSOCIATED WITH PH-POSITIVE ALL. TO STUDY THIS ISSUE, WE USED METHYLATION-SPECIFIC PCR AND BISULFITE SEQUENCING TO DETERMINE THE METHYLATION STATUS OF THE ABL1 PROMOTER IN 18 PH-POSITIVE ALL SAMPLES. WE REPORT HERE THAT GENE-SPECIFIC ABL1 PROMOTER METHYLATION IS ASSOCIATED MAINLY WITH THE P210 FORM OF BCR-ABL AND NOT THE P190 FORM. WHILE SIX OUT OF THE SEVEN P210-POSITIVE ALL SAMPLES HAD ABL1 PROMOTER METHYLATION, NONE OF THE 11 P190-POSITIVE ALL SAMPLES DEMONSTRATED ABL1 PROMOTER METHYLATION. IN ADDITION, WE ESTIMATED THE EXTENT AND RELATIVE ABUNDANCE OF ABL1 PROMOTER METHYLATION IN SEVERAL PH-POSITIVE ALL SAMPLES AND COMPARED IT TO THE METHYLATION PATTERN IN CHRONIC, ACCELERATED AND BLASTIC CRISIS PHASES OF CML. WE PUT FORTH A MODEL THAT CORRELATES THE DIFFERENT TYPES OF LEUKEMIAS WITH THE DIFFERENT LEVELS OF ABL1 PROMOTER METHYLATION. 2001 15 6366 26 THE ROLE OF METHYLATION IN CML. METHYLATION OF THE PROXIMAL PROMOTER OF THE ABL1 ONCOGENE IS COMMON EPIGENETIC ALTERATION ASSOCIATED WITH CLINICAL PROGRESSION OF CHRONIC MYELOID LEUKEMIA (CML). IN PRESENTED STUDY WE QUERIED WHETHER BOTH THE PH'-ASSOCIATED AND NORMAL ABL1 ALLELES UNDERGO METHYLATION; WHAT MAY BE THE PROPORTION OF HEMATOPOIETIC PROGENITORS BEARING METHYLATED ABL1 PROMOTERS IN CHRONIC VERSUS ACUTE PHASE DISEASE; WHETHER METHYLATION AFFECTS THE PROMOTER UNIFORMLY OR IN PATCHES WITH DISCRETE CLINICAL RELEVANCE; AND, FINALLY WHETHER METHYLATION OF ABL1 REFLECTS A GENERALIZED PROCESS OR IS GENE-SPECIFIC. TO ADDRESS THESE ISSUES, THE TECHNIQUE OF METHYLATION-SPECIFIC PCR AND BISULFITE-SEQUENCING WAS ADAPTED TO STUDY THE REGULATORY REGIONS OF ABL1 AND OTHER GENES. IN CELL LINES ESTABLISHED FROM CML BLAST CRISIS, WHICH ONLY CARRY A SINGLE ABL1 ALLELE NESTED WITHIN THE BCR-ABL FUSION GENE, ABL1 PROMOTERS WERE UNIVERSALLY METHYLATED. IN CLINICAL SAMPLES FROM PATIENTS AT ADVANCED STAGES OF THE DISEASE, BOTH METHYLATED AND UNMETHYLATED PROMOTER ALLELES WERE DETECTABLE. IN COLONIES DERIVED FROM SINGLE HEMATOPOIETIC PROGENITORS METHYLATED AND UNMETHYLATED PROMOTER ALLELES WERE REVEALED AS WELL. ABL1 METHYLATION WAS WAS NOTED IN THE VAST MAJORITY OF COLONIES FROM BLAST CRISIS, BUT NOT CHRONIC-PHASE CML. IT WAS SHOWN FINALLY THAT ABL1 METHYLATION DOES NOT REFLECT A GENERALIZED PROCESS AND MAY BE UNIQUE AMONG DNA REPAIR/GENOTOXIC STRESS RESPONSE GENES. THESE DATA SUGGEST THAT SPECIFIC METHYLATION OF THE PH'-ASSOCIATED ABL1 ALLELE ACCOMPANIES CLONAL EVOLUTION IN CML. 2000 16 2304 19 EPIGENETIC REGULATION OF CATHEPSIN L EXPRESSION IN CHRONIC MYELOID LEUKAEMIA. THE EXPRESSION AND SIGNIFICANCE OF CATHEPSIN L (CTSL) HAS BEEN EXTENSIVELY STUDIED IN SOLID TUMOURS. HOWEVER NO SUCH INFORMATION IN CHRONIC MYELOID LEUKAEMIA (CML) WAS AVAILABLE. WE INVESTIGATED THE ACTIVITY AND EXPRESSION OF THIS PROTEASE IN PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCS) OF 47 ADULT CML PATIENTS. THIRTY ADULTS SUFFERING FROM SYSTEMIC DISEASES AND 50 HEALTHY VOLUNTEERS SERVED AS CONTROLS. THE MRNA LEVELS OF CTSL, ITS SPECIFIC ENDOGENOUS INHIBITOR CYSTATIN C AND TRANSCRIPTIONAL UP-REGULATOR VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) WERE QUANTITATED BY REAL-TIME QPCR. CTSL PROTEASE ACTIVITY AND ITS MRNA EXPRESSION WERE SIGNIFICANTLY HIGHER IN CML CHRONIC PHASE (CP) PATIENTS COMPARED TO CML ACCELERATED PHASE/BLAST CRISIS (AP/BC) PATIENTS AND CONTROLS (P