title: 5-Aza-2'-deoxycytidine induces p21WAF expression by demethylation of p73 leading to p53-independent apoptosis in myeloid leukemia.
abstract: The DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) has significant therapeutic value for the treatment of patients with myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). The demethylating effect of 5-Aza-CdR has been well characterized. In contrast, less is known about the molecular events downstream of the methylation inhibition. Here, 5-Aza-CdR induced apoptosis in AML cells (both p53 mutant and wild-type) but not in epithelial or normal PBMCs. Cell death was accompanied by activation of the mitochondrial apoptosis pathway, as shown by release of cytochrome c and AIF and loss of mitochondrial membrane potential (DeltaPsim). Activation of caspase-3 (but not -6 and -8) was detectable using Western blot analysis and measurement of caspase enzymatic activity. 5-Aza-CdR treatment resulted in the induction of p21, which correlated with the arrest of AML cells in the G1 cell cycle phase. Induction of p21 expression was independent of its promoter methylation status but mediated by 5-Aza-CdR-induced reexpression of the tumor-suppressor p73, a known upstream regulator of p21. The p73 promoter was hypermethylated in AML cell lines and in primary AML cells but not in epithelial cells, which were resistant toward 5-Aza-CdR. Therefore, 5-Aza-CdR-mediated specific killing of myeloid cells might be dependent on its ability to revert p73 promoter methylation and to reexpress p73 mRNA. In addition, exogenous expression of p73 rendered epithelial cells sensitive to apoptosis induced by 5-Aza-CdR or other cytostatic drugs. We therefore conclude that p73 is a relevant target for methylation-dependent efficacy of 5-Aza-CdR in AML cells.
authors.affiliation: Department of Hematology and Oncology, Universitätsmedizin Berlin, Charité, Campus Virchow, Berlin, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Schmelz
authors.forename: Mandy
authors.initials: M
authors.lastname: Wagner
authors.forename: Bernd
authors.initials: B
authors.lastname: Dörken
authors.forename: Ingo
authors.initials: I
authors.lastname: Tamm
mesh_terms: D017209:Apoptosis; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000096:biosynthesis*; D045744:Cell Line, Tumor; D018899:CpG Islands; D050759:Cyclin-Dependent Kinase Inhibitor p21; D045304:Cytochromes c / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000077209:Decitabine; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D016193:G1 Phase; D016147:Genes, Tumor Suppressor; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy*; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D009190:Myelodysplastic Syndromes / Q000188:drug therapy*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013447:Sulfites / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins; D020298:U937 Cells; D015854:Up-Regulation
chemical_list: C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D013447:Sulfites; C000606460:TP73 protein, human; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D012313:RNA; D000077209:Decitabine; D045304:Cytochromes c; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D001374:Azacitidine