title: Temozolomide-mediated DNA methylation in human myeloid precursor cells: differential involvement of intrinsic and extrinsic apoptotic pathways.
abstract: OBJECTIVE
  An understanding of how hematopoietic cells respond to therapy that causes myelosuppression will help develop approaches to prevent this potentially life-threatening toxicity. The goal of this study was to determine how human myeloid precursor cells respond to temozolomide (TMZ)-induced DNA damage.
  
  
  METHODS
  We developed an ex vivo primary human myeloid precursor cells model system to investigate the involvement of cell-death pathways using a known myelosuppressive regimen of O(6)-benzylguanine (6BG) and TMZ.
  
  
  RESULTS
  Exposure to 6BG/TMZ led to increases in p53, p21, γ-H2AX, and mitochondrial DNA damage. Increases in mitochondrial membrane depolarization correlated with increased caspase-9 and -3 activities following 6BG/TMZ treatment. These events correlated with decreases in activated AKT, downregulation of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), and increased cell death. During myeloid precursor cell expansion, FAS/CD95/APO1(FAS) expression increased over time and was present on approximately 100% of the cells following exposure to 6BG/TMZ. Although c-flipshort, an endogenous inhibitor of FAS-mediated signaling, was decreased in 6BG/TMZ-treated versus control, 6BG-, or TMZ alone-treated cells, there were no changes in caspase-8 activity. In addition, there were no changes in the extent of cell death in myeloid precursor cells exposed to 6BG/TMZ in the presence of neutralizing or agonistic anti-FAS antibodies, indicating that FAS-mediated signaling was not operative.
  
  
  CONCLUSIONS
  In human myeloid precursor cells, 6BG/TMZ-initiated apoptosis occurred by intrinsic, mitochondrial-mediated and not extrinsic, FAS-mediated apoptosis. Human myeloid precursor cells represent a clinically relevant model system for gaining insight into how hematopoietic cells respond to chemotherapeutics and offer an approach for selecting effective chemotherapeutic regimens with limited hematopoietic toxicity.
authors.affiliation: Department of Pediatrics, Section of Pediatric Hematology/Oncology, Herman B Wells Center for Pediatric Research, Riley Hospital for Children at Indiana University Health, Indianapolis, Indiana 46202, USA.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Wang
authors.forename: Shanbao
authors.initials: S
authors.lastname: Cai
authors.forename: Aaron
authors.initials: A
authors.lastname: Ernstberger
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Bailey
authors.forename: Michael Z
authors.initials: MZ
authors.lastname: Wang
authors.forename: Wenjing
authors.initials: W
authors.lastname: Cai
authors.forename: W Scott
authors.initials: WS
authors.lastname: Goebel
authors.forename: Magdalena B
authors.initials: MB
authors.lastname: Czader
authors.forename: Colin
authors.initials: C
authors.lastname: Crean
authors.forename: Attaya
authors.initials: A
authors.lastname: Suvannasankha
authors.forename: Inna
authors.initials: I
authors.lastname: Shokolenkoc
authors.forename: Glenn L
authors.initials: GL
authors.lastname: Wilson
authors.forename: Arthur R
authors.initials: AR
authors.lastname: Baluyut
authors.forename: Lindsey D
authors.initials: LD
authors.lastname: Mayo
authors.forename: Karen E
authors.initials: KE
authors.lastname: Pollok
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D015153:Blotting, Western; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D019175:DNA Methylation / Q000187:drug effects*; D004272:DNA, Mitochondrial / Q000235:genetics; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D020869:Gene Expression Profiling; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D023461:Myeloid Progenitor Cells / Q000187:drug effects* / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D000077204:Temozolomide; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism
chemical_list: D018906:Antineoplastic Agents, Alkylating; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004272:DNA, Mitochondrial; C468783:H2AX protein, human; D006657:Histones; D016159:Tumor Suppressor Protein p53; D019014:fas Receptor; C064976:O(6)-benzylguanine; D006147:Guanine; D003606:Dacarbazine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D000077204:Temozolomide
references.citation: J Immunol. 2004 Sep 1;173(5):2976-84
references.pmid: 15322156
references.citation: Clin Cancer Res. 2011 Apr 15;17(8):2195-206
references.pmid: 21487065
references.citation: Cancers (Basel). 2011 Jun;3(2):1639-71
references.pmid: 22348197
references.citation: Carcinogenesis. 1992 Nov;13(11):1967-73
references.pmid: 1423864
references.citation: DNA Repair (Amst). 2009 Jun 4;8(6):704-19
references.pmid: 19272840
references.citation: Cancer Res. 2005 Apr 15;65(8):3319-27
references.pmid: 15833865
references.citation: Curr Neurol Neurosci Rep. 2012 Jun;12(3):286-93
references.pmid: 22437507
references.citation: Cell Stem Cell. 2010 Aug 6;7(2):186-97
references.pmid: 20619763
references.citation: Nucleic Acids Res. 1997 Nov 1;25(21):4362-9
references.pmid: 9336469
references.citation: Cancer Res. 2007 Jan 1;67(1):26-31
references.pmid: 17210680
references.citation: Prog Nucleic Acid Res Mol Biol. 2001;68:255-6
references.pmid: 11554301
references.citation: Int Rev Neurobiol. 2002;53:519-34
references.pmid: 12512351
references.citation: Nucleic Acids Res. 2002 Jan 1;30(1):207-10
references.pmid: 11752295
references.citation: Mutat Res. 1999 Jul 30;434(3):149-59
references.pmid: 10486589
references.citation: Oncogene. 2007 Jan 11;26(2):186-97
references.pmid: 16819506
references.citation: J Biol Chem. 2001 Jun 8;276(23):20633-40
references.pmid: 11279218
references.citation: Mutat Res. 1990 Sep-Nov;236(2-3):269-75
references.pmid: 2398914
references.citation: Toxicology. 2010 Jan 12;267(1-3):112-7
references.pmid: 19883721
references.citation: Toxicol Sci. 2003 Oct;75(2):355-67
references.pmid: 12883091
references.citation: Cell. 1997 Oct 17;91(2):231-41
references.pmid: 9346240
references.citation: J Clin Oncol. 2005 Oct 1;23(28):7178-87
references.pmid: 16192602
references.citation: J Cell Sci. 2007 Aug 1;120(Pt 15):2479-87
references.pmid: 17646672
references.citation: Nucleic Acids Res. 2003 Feb 1;31(3):1052-8
references.pmid: 12560503
references.citation: Science. 1998 Nov 13;282(5392):1318-21
references.pmid: 9812896
references.citation: Biochemistry. 1994 Aug 9;33(31):9045-51
references.pmid: 8049205
references.citation: J Clin Oncol. 1999 Aug;17(8):2604-13
references.pmid: 10561328
references.citation: Blood. 2005 Sep 1;106(5):1581-9
references.pmid: 15905188
references.citation: Cell Death Differ. 2003 Mar;10(3):335-44
references.pmid: 12700633
references.citation: Free Radic Biol Med. 2005 Jan 1;38(1):2-11
references.pmid: 15589366
references.citation: Mol Cell. 2004 Apr 9;14(1):105-16
references.pmid: 15068807
references.citation: Cell. 1982 Apr;28(4):855-63
references.pmid: 6807553
references.citation: J Biol Chem. 1988 May 15;263(14):6854-6
references.pmid: 3360810
references.citation: Annu Rev Biochem. 1997;66:863-917
references.pmid: 9242927
references.citation: PLoS One. 2012;7(6):e39956
references.pmid: 22768182
references.citation: Crit Rev Oncog. 2012;17(1):69-95
references.pmid: 22471665
references.citation: Oncogene. 2004 Jan 15;23(2):359-67
references.pmid: 14724564
references.citation: DNA Repair (Amst). 2007 Aug 1;6(8):1170-8
references.pmid: 17507295
references.citation: DNA Repair (Amst). 2007 Aug 1;6(8):1079-99
references.pmid: 17485253
references.citation: J Biol Chem. 2005 Jul 22;280(29):27345-55
references.pmid: 15886205
references.citation: J Cell Biol. 2010 Aug 9;190(3):377-89
references.pmid: 20696707
references.citation: Cancer Res. 2000 Oct 15;60(20):5815-24
references.pmid: 11059778
references.citation: Blood. 1996 Sep 1;88(5):1649-55
references.pmid: 8781420
references.citation: J Biol Chem. 2000 Dec 1;275(48):37518-23
references.pmid: 10982789
references.citation: Carcinogenesis. 1988 Feb;9(2):285-92
references.pmid: 3338112
references.citation: Cell Stem Cell. 2012 Feb 3;10(2):120-36
references.pmid: 22305562
references.citation: EMBO J. 1996 Dec 2;15(23):6541-51
references.pmid: 8978681
references.citation: Clin Cancer Res. 2007 Nov 15;13(22 Pt 1):6712-8
references.pmid: 18006772
references.citation: Cancer Res. 2011 Mar 1;71(5):1836-48
references.pmid: 21242281
references.citation: N Engl J Med. 2005 Mar 10;352(10):987-96
references.pmid: 15758009
references.citation: Nucleic Acids Res. 1999 Nov 15;27(22):4510-6
references.pmid: 10536162