Temozolomide-mediated DNA methylation in human myeloid precursor cells: Differential involvement of intrinsic and extrinsic apoptotic pathways

Haiyan Wang, Shanbao Cai, Aaron Ernstberger, Barbara J. Bailey, Michael Z. Wang, Wenjing Cai, W. Goebel, Magdalena Czader, Colin Crean, Attaya Suvannasankha, Inna Shokolenkoc, Glenn L. Wilson, Arthur R. Baluyut, Lindsey Mayo, Karen Pollok

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Purpose: 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. Experimental Design: 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 O6-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 O6-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.

Original languageEnglish
Pages (from-to)2699-2709
Number of pages11
JournalClinical Cancer Research
Volume19
Issue number10
DOIs
StatePublished - May 15 2013

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temozolomide
Myeloid Cells
DNA Methylation
Cell Death
DNA Damage
Apoptosis
Caspase 9
Caspase 8
Methyltransferases
Mitochondrial Membranes

ASJC Scopus subject areas

  • Cancer Research
  • Oncology
  • Medicine(all)

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Temozolomide-mediated DNA methylation in human myeloid precursor cells : Differential involvement of intrinsic and extrinsic apoptotic pathways. / Wang, Haiyan; Cai, Shanbao; Ernstberger, Aaron; Bailey, Barbara J.; Wang, Michael Z.; Cai, Wenjing; Goebel, W.; Czader, Magdalena; Crean, Colin; Suvannasankha, Attaya; Shokolenkoc, Inna; Wilson, Glenn L.; Baluyut, Arthur R.; Mayo, Lindsey; Pollok, Karen.

In: Clinical Cancer Research, Vol. 19, No. 10, 15.05.2013, p. 2699-2709.

Research output: Contribution to journalArticle

Wang, Haiyan ; Cai, Shanbao ; Ernstberger, Aaron ; Bailey, Barbara J. ; Wang, Michael Z. ; Cai, Wenjing ; Goebel, W. ; Czader, Magdalena ; Crean, Colin ; Suvannasankha, Attaya ; Shokolenkoc, Inna ; Wilson, Glenn L. ; Baluyut, Arthur R. ; Mayo, Lindsey ; Pollok, Karen. / Temozolomide-mediated DNA methylation in human myeloid precursor cells : Differential involvement of intrinsic and extrinsic apoptotic pathways. In: Clinical Cancer Research. 2013 ; Vol. 19, No. 10. pp. 2699-2709.
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abstract = "Purpose: 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. Experimental Design: 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 O6-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 O6-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.",
author = "Haiyan Wang and Shanbao Cai and Aaron Ernstberger and Bailey, {Barbara J.} and Wang, {Michael Z.} and Wenjing Cai and W. Goebel and Magdalena Czader and Colin Crean and Attaya Suvannasankha and Inna Shokolenkoc and Wilson, {Glenn L.} and Baluyut, {Arthur R.} and Lindsey Mayo and Karen Pollok",
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T1 - Temozolomide-mediated DNA methylation in human myeloid precursor cells

T2 - Differential involvement of intrinsic and extrinsic apoptotic pathways

AU - Wang, Haiyan

AU - Cai, Shanbao

AU - Ernstberger, Aaron

AU - Bailey, Barbara J.

AU - Wang, Michael Z.

AU - Cai, Wenjing

AU - Goebel, W.

AU - Czader, Magdalena

AU - Crean, Colin

AU - Suvannasankha, Attaya

AU - Shokolenkoc, Inna

AU - Wilson, Glenn L.

AU - Baluyut, Arthur R.

AU - Mayo, Lindsey

AU - Pollok, Karen

PY - 2013/5/15

Y1 - 2013/5/15

N2 - Purpose: 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. Experimental Design: 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 O6-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 O6-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.

AB - Purpose: 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. Experimental Design: 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 O6-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 O6-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.

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