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Hypomethylation and apoptosis in 5-azacytidine-treated myeloid cells

Khan, Rasheed LU ; Schmidt-Mende, M; Karimi, M; Gogvadze, V; Hassan, M; Ekstrom, T J; Zhivotovsky, T and Hellstrom-Lindberg, E (2008) In Experimental Hematology 36(2). p.149-157
Abstract
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. MATERIALS AND 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 >/=0.5 muM. Hypomethylation... (More)
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. MATERIALS AND 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 >/=0.5 muM. Hypomethylation was observed in the sorted apoptotic fraction (41% decrease with 1 muM 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. (Less)
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author
publishing date
type
Contribution to journal
publication status
published
subject
in
Experimental Hematology
volume
36
issue
2
pages
149 - 157
publisher
Elsevier
external identifiers
  • scopus:38149088287
ISSN
1873-2399
DOI
10.1016/j.exphem.2007.10.002
language
English
LU publication?
no
id
58e56b2c-d000-4c68-9732-ee93940cba7a (old id 1299082)
date added to LUP
2009-07-09 13:57:56
date last changed
2017-08-06 03:39:21
@article{58e56b2c-d000-4c68-9732-ee93940cba7a,
  abstract     = {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. MATERIALS AND 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 >/=0.5 muM. Hypomethylation was observed in the sorted apoptotic fraction (41% decrease with 1 muM 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.},
  author       = {Khan, Rasheed and Schmidt-Mende, M and Karimi, M and Gogvadze, V and Hassan, M and Ekstrom, T J and Zhivotovsky, T and Hellstrom-Lindberg, E},
  issn         = {1873-2399},
  language     = {eng},
  number       = {2},
  pages        = {149--157},
  publisher    = {Elsevier},
  series       = {Experimental Hematology},
  title        = {Hypomethylation and apoptosis in 5-azacytidine-treated myeloid cells},
  url          = {http://dx.doi.org/10.1016/j.exphem.2007.10.002},
  volume       = {36},
  year         = {2008},
}