monensin and Myelodysplastic-Syndromes

Although clinically approved for myelodysplastic syndromes (MDS), the mode of action of 5-azacytidine has not been well understood at the cellular level. The present study aimed at characterizing the mechanisms for 5-azacytidine-induced apoptosis, as well as the presence of a possible link between apoptosis and DNA hypomethylation.. We investigated the effects of 5-azacytidine on a spectrum of specific apoptotic pathways, as well as on global DNA methylation, assessed by luminometric methylation assay, in myeloid (P39, HL60) and T cells (Jurkat).. 5-Azacytidine induced dose-dependent apoptosis as well as non-dose-dependent global DNA hypomethylation at concentrations >or=0.5 microM. Hypomethylation was observed in the sorted apoptotic fraction (41% decrease with 1 microM after 24 hours), while nonapoptotic cells retained a methylation pattern similar to untreated cells (+/-6%). The induced apoptotic pattern involved several pathways: cleavage of Bcl-2 family proteins, activation of caspase-2 and -3-like, mitochondrial involvement characterized by loss of transmembrane potential (tetramethylrhodamine ethyl ester [TMRE]) and cytochrome release, and acidification of cytosol. Selective inhibition of caspase-3-like, -2, -8, -9, and pan-caspase activity, as well as stabilization of cytosolic pH by monensin completely failed to block apoptosis. Poly(ADP-ribose) polymerase (PARP) inhibitors only partially inhibited loss of TMRE (32% reduction) and caspase-2 activity (38% reduction); indicative of PARP operation (or action) upstream of caspase-2. Moreover, cytosine arabinoside induced a similar degree of apoptosis, while leaving methylation status mainly unaffected.. 5-Azacytidine acts via multiple and separately regulated pathways, including parallel induction of hypomethylation. The broad action of 5-azacytidine may explain its therapeutic effects in poor-prognostic MDS.

Topics: Apoptosis; Azacitidine; Caspases; Cytosol; DNA Methylation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Hydrogen-Ion Concentration; Ionophores; Jurkat Cells; Membrane Potential, Mitochondrial; Mitochondria; Monensin; Myelodysplastic Syndromes; Myeloid Cells; Organometallic Compounds; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Time Factors