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

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 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.

Figure 1. Correlation of MGMT status, cell viability, and DNA damage response in MP cells following exposure to 6BG/TMZ

(A) Human MP cells were exposed to 6BG in the absence or presence of TMZ (200 uM and 400 uM) and 6BG/TMZ for one day and probed for MGMT expression. Beta-actin served as loading (B) MP cells were treated in triplicate and viability of myeloid cultures determined over time. (C-E). Analysis of 6BG/TMZ-induced p53 stabilization, p21, AKT phosphorylation, and cell cycle arrest at Day 3 post-treatment. GAPDH served as a loading control. *p < 0.05, 6BG/TMZ vs. control, 6BG, or TMZ. Data are representative of 3 independent experiments.

Figure 2. Determination of mitochondrial-genome specific and global DNA damage exposure to 6BG/TMZ

(A) Human MP cells were exposed to increasing doses of TMZ (100-500 uM) in absence or presence of 6BG for 2 hours and the level of mitochondrial DNA strand breaks determined by alkaline Southern Blot. A mitochondrial-specific probe recognizing the CoII-ATPase 6 sequences was used to detect mitochondrial DNA damage (B) Human MP cells were exposed to 6BG in the absence or presence of 200 uM TMZ for 3 days and the levels of γ-H2AX determined by Western analysis. GAPDH served as a loading control. Data are representative of 2 independent experiments.

Figure 3. Analysis of mitochondrial membrane depolarization and apoptosis following exposure to myelosuppressive chemotherapy

Human MP cells were treated with 6BG +/- TMZ for 4 days. (A) Mitochondrial membrane depolarization was determined by DiIC₁ staining. Increased depolarization correlates with decreased DiC₁ stain. Insert on control histogram is DiIC₁ staining of myeloid cells treated with the membrane depolarization agent, CCCP. (B) Apoptotic cell death was determined by Annexin V and Sytox- green stain. (C) Caspase-9 activity was determined in the +/- of the caspase-9 specific inhibitor. (D) Caspase-3/7 activation was determined by flow cytometry. Data are representative of 3 independent experiments for A-C and 2 independent experiments for D.

Figure 4. Modulation of FAS and c-FLIP expression in MP cells exposed to 6BG/TMZ

(A) Time course of FAS expression was performed during the expansion period. FAS expression was analyzed in triplicate via flow cytometry on cells in the early expansion and differentiation phase - days 0-8 post-expansion. (B) At 10 days post-expansion, control MP cells (C) or MP cells were exposed to 6BG, TMZ, or combination of 6BG/TMZ for 3 days. The percent of FAS positive and the relative density of FAS expression was determined via flow cytometry. (C) MP cells were exposed to 6BG, TMZ, or 6BG/TMZ for 3 days and cellular lysates analyzed via Western blot for c-FLIP_short and c-FLIP _long. Flow cytometric data are expressed as the mean and standard deviation of triplicate data points for each group. Flow cyotmetric data and Western analysis were repeated twice with similar results.

Figure 5. Relevance of FAS-mediated signaling in MP cells exposed to 6BG/TMZ

(A) As a control for antibody-mediated signaling through FAS, Jurkat T cells were incubated in triplicate with agonistic (Ag), neutralizing (Neut), isotype (Iso) or combinations of antibodies for 24 hours and the percent of nonviable cells determined via Annexin V staining and flow cytometry. MP cells that had been expanded in culture for 10 days were treated with 6BG/TMZ for 3 days in the presence of combinations of agonistic (Ag), neutralizing (Neut), and isotype (Iso) antibodies. Antibody was added each day of the 3-day culture and nonviable cells determined via Annexin V staining and flow cytometry on day 4 post-treatment. (B) Caspase-8 activity was determined in lysates from control and 6BG/TMZ-treated cells in the +/- of a caspase-8 specific inhibitor. Data are representative of two independent experiments (A) or 3 independent experiments (B).