Hypomethylation and apoptosis in 5-azacytidine-treated myeloid cells.
OBJECTIVE
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.


METHODS
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).


RESULTS
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.


CONCLUSIONS
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.
Khan
Rasheed
R
Department of Medicine, Division of Hematology, Karolinska University Hospital Huddinge, Karolinska Institutet, Stockholm, Sweden. Rasheed.Zaki.Khan@ki.se
Schmidt-Mende
Jan
J
Karimi
Mohsen
M
Gogvadze
Vladimir
V
Hassan
Moustapha
M
Ekström
Tomas J
TJ
Zhivotovsky
Boris
B
Hellström-Lindberg
Eva
E
D017209:Apoptosis / Q000187:drug effects*; D001374:Azacitidine / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism / Q000473:pathology; D019175:DNA Methylation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007476:Ionophores / Q000494:pharmacology; D019169:Jurkat Cells; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology; D008985:Monensin / Q000494:pharmacology; D009190:Myelodysplastic Syndromes / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D022423:Myeloid Cells / Q000378:metabolism* / Q000473:pathology; D009942:Organometallic Compounds / Q000494:pharmacology; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013997:Time Factors
D004791:Enzyme Inhibitors; D007476:Ionophores; D009942:Organometallic Compounds; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; C110932:tetramethyl rhodamine ethyl ester; D008985:Monensin; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases; D001374:Azacitidine