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 (MP) respond to temozolomide (TMZ)-induced DNA damage.
Experimental Design
We developed an ex vivo primary human MP 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 caspase-3 activities following 6BG/TMZ treatment. These events correlated with decreases in activated AKT, downregulation of the DNA repair protein O6methylguanine-DNA methyltransferase (MGMT), and increased cell death. During MP cell expansion, FAS/CD95/APO1(FAS) expression increased over time and was present on ~100% of the cells following exposure to 6BG/TMZ. While 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. Additionally, there were no changes in the extent of cell death in MP 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 MP cells, 6BG/TMZ-initiated apoptosis occurred by intrinsic, mitochondrial-mediated and not extrinsic, FAS-mediated apoptosis. Human MP 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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