Role of reactive oxygen species in the radiation response of human hematopoietic stem/progenitor cells

Abstract

Hematopoietic stem/progenitor cells (HSPCs), which are present in small numbers in hematopoietic tissues, can differentiate into all hematopoietic lineages and self-renew to maintain their undifferentiated phenotype. HSPCs are extremely sensitive to oxidative stressors such as anti-cancer agents, radiation, and the extensive accumulation of reactive oxygen species (ROS). The quiescence and stemness of HSPCs are maintained by the regulation of mitochondrial biogenesis, ROS, and energy homeostasis in a special microenvironment called the stem cell niche. The present study evaluated the relationship between the production of intracellular ROS and mitochondrial function during the proliferation and differentiation of X-irradiated CD34(+) cells prepared from human placental/umbilical cord blood HSPCs. Highly purified CD34(+) HSPCs exposed to X-rays were cultured in liquid and semi-solid medium supplemented with hematopoietic cytokines. X-irradiated CD34(+) HSPCs treated with hematopoietic cytokines, which promote their proliferation and differentiation, exhibited dramatically suppressed cell growth and clonogenic potential. The amount of intracellular ROS in X-irradiated CD34(+) HSPCs was significantly higher than that in non-irradiated cells during the culture period. However, neither the intracellular mitochondrial content nor the mitochondrial superoxide production was elevated in X-irradiated CD34(+) HSPCs compared with non-irradiated cells. Radiation-induced gamma-H2AX expression was observed immediately following exposure to 4 Gy of X-rays and gradually decreased during the culture period. This study reveals that X-irradiation can increase persistent intracellular ROS in human CD34(+) HSPCs, which may not result from mitochondrial ROS due to mitochondrial dysfunction, and indicates that substantial DNA double-strand breakage can critically reduce the stem cell function.

Figure 1. The radiation dose–response curves of human umbilical myeloid hematopoietic progenitors.

CD34⁺ HSPCs were X-irradiated and assayed in methylcellulose cultures for 14 days. The values represent the means ± SD of three separate experiments performed in three wells. Curves were fitted as described in the Materials and Methods. Panel A and B: Radiation dose-response curves for each of the cell types and for the total hematopoietic myeloid progenitor cells were shown, respectively.

Figure 2. The relationship between the incubation period and ratio of viable cells.

CD34⁺ HSPCs exposed to 0, 2 or 4 Gy of X-rays were cultured in liquid medium supplemented with a combination of G-CSF, GM-CSF, IL3, SCF and EPO. Cells were harvested from the culture on days 1, 3 and 7. The viable cells were counted using trypan blue dye exclusion and the results are relative to the initial value. The values represent the means ± SD of three separate experiments. * P<0.05 vs. non-irradiated controls.

Figure 3. The relationships among the incubation periods, intracellular ROS and mitochondrial superoxide detected in CD34⁺ HSPCs.

Cells were cultured for 3 h, 6 h, 12 h, 1 day, 3 days or 7 days. The intracellular ROS (panel A) and mitochondrial superoxide levels (panel B) were analyzed by flow cytometry. The values represent the means ± SD of three separate experiments. * P<0.05 or ** P<0.01 vs. non-irradiated controls.

Figure 4. The relationship between the incubation period and intracellular mitochondrial contents detected in CD34⁺ HSPCs.

Cells were cultured for 3 h, 6 h, 12 h, 1 day, 3 days or 7 days. After cultivation, the intracellular mitochondrial contents were analyzed by flow cytometry. The values represent the means ± SD of three separate experiments.

Figure 5. The effects of X-irradiation on DNA double-strand breaks in CD34⁺ HSPCs.

CD34⁺ HSPCs exposed to 0 or 4 Gy of X-rays were cultured in liquid medium supplemented with a combination of G-CSF, GM-CSF, IL3, SCF and EPO. Cells were harvested at 24, 78 and 168 hours, and the expression of gamma-H2AX was evaluated as described in the Materials and Methods. * P<0.05 or ** P<0.01 vs. non-irradiated controls and vs. X-ray irradiated cells.