5-Aza-2'-deoxycytidine sensitizes busulfan-resistant myeloid leukemia cells by regulating expression of genes involved in cell cycle checkpoint and apoptosis.
Busulfan (Bu) is a DNA-alkylating drug used in myeloablative pretransplant conditioning therapy for patients with myeloid leukemia (ML). A major obstacle to successful treatment is cellular Bu-resistance. To investigate the possible contribution of DNA hypermethylation to Bu-resistance, we examined the cytotoxic activity of combined 5-aza-2'-deoxycytidine (DAC) and Bu. Exposure of Bu-resistant B5/Bu250(6) ML cells to 0.5 microM DAC resulted in G2-arrest and apoptosis. The observed G2-arrest was associated with hypomethylation and subsequent expression of epigenetically controlled genes including p16(INK4A), activation of the p53 pathway, and phosphorylation of CDC2. The DAC-mediated apoptosis was partly due to hypomethylation and up-regulation of XAF1, which resulted in down-regulation of the anti-apoptotic proteins XIAP, cIAP1 and cIAP2. The pro-apoptotic PUMA and BNIP3 proteins were up-regulated while pro-survival STAT3 and c-MYC were suppressed. Combination of 0.05 microM DAC and 5 microg/ml Bu resulted in synergistic cytotoxicity, which was associated with PARP1 cleavage and activation of caspases 3 and 8, suggesting induction of an apoptotic response. P53 inhibition in B5/Bu250(6) cells using pifithrin-alpha alleviated these effects, suggesting a role for p53 therein; this observation was supported by the relative resistance of p53-null K562 cells to [DAC+Bu] combinations and by the effects of an anti-p53 shRNA on the OCI-AML3 cell line. We conclude that the synergistic effects of [DAC+Bu] are p53-dependent and involve cell cycle arrest, apoptosis induction and down-regulation of pro-survival genes. Our results suggest that, depending on tumor p53 status, incorporation of DAC might synergistically improve the cytoreductive efficacy of Bu-based pretransplant regimen in patients with ML.
Valdez
Benigno C
BC
Department of Stem Cell Transplantation and Cellular Therapy, University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA. bvaldez@mdanderson.org
Li
Yang
Y
Murray
David
D
Corn
Paul
P
Champlin
Richard E
RE
Andersson
Borje S
BS
D000964:Antimetabolites, Antineoplastic / Q000008:administration & dosage; D018906:Antineoplastic Agents, Alkylating / Q000008:administration & dosage; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001374:Azacitidine / Q000008:administration & dosage / Q000031:analogs & derivatives; D015153:Blotting, Western; D002066:Busulfan / Q000008:administration & dosage; D045744:Cell Line, Tumor; D002469:Cell Separation; D000077209:Decitabine; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects* / Q000235:genetics; D018816:Genes, cdc / Q000187:drug effects*; D006801:Humans; D020287:In Situ Nick-End Labeling; D007951:Leukemia, Myeloid / Q000188:drug therapy*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects
D000964:Antimetabolites, Antineoplastic; D018906:Antineoplastic Agents, Alkylating; D000077209:Decitabine; D002066:Busulfan; D001374:Azacitidine