Synergistic effect of hydrogen peroxide on polyploidization during the megakaryocytic differentiation of K562 leukemia cells by PMA

Abstract

The human myelogenous cell line, K562 has been extensively used as a model for the study of megakaryocytic (MK) differentiation, which could be achieved by exposure to phorbol 12-myristate 13-acetate (PMA). In this study, real-time PCR analysis revealed that the expression of catalase (cat) was significantly repressed during MK differentiation of K562 cells induced by PMA. In addition, PMA increased the intracellular reactive oxygen species (ROS) concentration, suggesting that ROS was a key factor for PMA-induced differentiation. PMA-differentiated K562 cells were exposed to hydrogen peroxide (H2O2) to clarify the function of ROS during MK differentiation. Interestingly, the percentage of high-ploidy (DNA content >4N) cells with H2O2 was 34.8±2.3% at day 9, and was 70% larger than that without H2O2 (21.5±0.8%). Further, H2O2 addition during the first 3 days of PMA-induced MK differentiation had the greatest effect on polyploidization. In an effort to elucidate the mechanisms of enhanced polyploidization by H2O2, the BrdU assay clearly indicated that H2O2 suppressed the division of 4N cells into 2N cells, followed by the increased polyploidization of K562 cells. These findings suggest that the enhancement in polyploidization mediated by H2O2 is due to synergistic inhibition of cytokinesis with PMA. Although H2O2 did not increase ploidy during the MK differentiation of primary cells, we clearly observed that cat expression was repressed in both immature and mature primary MK cells, and that treatment with the antioxidant N-acetylcysteine effectively blocked and/or delayed the polyploidization of immature MK cells. Together, these findings suggest that MK cells are more sensitive to ROS levels during earlier stages of maturation.

Keywords: Catalase down-regulation; Hydrogen peroxide; K562 cell; Megakaryocytic differentiation; Polyploidization; Reactive oxygen species.

Fig. 1

Catalase expression is repressed by PMA. mRNA expression levels of selected genes at day 1 in the culture of PMA-induced or control K562 cells in the presence or absence of 60 μmol/l H₂O₂. The error bars represent standard deviation. Based on a paired t-test, values of p < 0.05 (*) are indicated. Abbreviations: cat catalase, sod1 superoxide dismutase 1, gpx1 glutathione peroxidase 1, gata1 globin transcription factor 1.

Fig. 2

Intracellular ROS content of K562 cells is increased by PMA and further increased by H₂O₂. ROS content was measured using oxidized DCF-DA at day 1 in the culture of PMA-induced or control K562 cells in the presence or absence of 60 μmol/l H₂O₂. The error bars represent standard deviation. Based on a paired t-test, values of p < 0.05 (*) are indicated.

Fig. 3

H₂O₂ greatly decreases expansion of PMA-treated K562 cells. Time courses of total cell concentration, percentage of viable cells, and percentage of apoptotic cells during the PMA-induced differentiation of K562 cells in the presence or absence of 60 μmol/l H₂O₂. The error bars represent standard deviation. Based on a paired t-test, values of p < 0.05 (*) are indicated for the various time points in comparison to the PMA-only culture.

Fig. 4

H₂O₂ increases ploidy of PMA-induced K562 cells. (A) DNA histograms of K562 cells in the culture with PMA at day 1 and day 9 with or without 60 μmol/l H₂O₂. The gate shows high-ploidy K562 cells with DNA content >4N. (B) Time courses of the percentage of high-ploidy K562 cells throughout the culture period with or without H₂O₂. (C) Time courses of the mean fluorescence intensity of CD41 of K562 cells in the absence or presence of H₂O_2. The error bars represent standard deviation. Based on a paired t-test, values of p < 0.05 (*) are indicated for the various time points in comparison to the PMA-only culture.

Fig. 5

H₂O₂ decreases the percentage of 2N cells and transiently increases the percentage of 4N cells. Time courses of the percentage of 2N and 4N cells throughout the culture period with or without 60 μmol/l H₂O₂. The error bars represent standard deviation. Based on a paired t-test, values of p < 0.05 (*) are indicated for the various time points in comparison to the PMA-only culture.

Fig. 6

The percentage of high-ploidy cells is inversely proportional to the total-cell concentration. Correlation between total-cell concentration and percentage of high-ploidy cells at day 9 for the experiments shown in Table 1, in which 60 μmol/l H₂O₂ was added in the culture at day 0 and/or day 3 and/or day 6. The solid line represents the linear regression curve. In this experiment, total-cell concentrations were determined using a hemocytometer.

Fig. 7

H₂O₂ blocks the division of 4N K562 cells after PMA addition. (A) Representative dot plot showing BrdU–FITC vs. 7–AAD fluorescence intensities at day 1 with or without 60 μmol/l H₂O₂ after incubation with BrdU for 12 h. The indicated regions were used to discriminate the BrdU–negative (BrdU⁻) 2N cells (R1), BrdU–positive (BrdU⁺) 2N cells (R2), BrdU⁻ ≥ 4N cells (R3), and BrdU⁺ ≥ 4N cells (R4). (B) Percentages of BrdU⁺ and BrdU⁻ cell populations in terms of ploidy. The data were from three independent cultures. The error bars represent standard deviation.

Fig. 8

Primary human MK cells derived from mobilized peripheral blood CD34⁺ cells in culture were sorted to isolate immature and mature populations. Immature CD41⁺CD42⁻, and mature CD41⁺CD42⁺ MK cells were selected by immuo-labeled magnetic beads. Purity of the selected populations was verified by flow cytometry. This analysis was performed for two independent donors (donor 1 selected at day 7, donor 2 selected at day 8).

Fig. 9

Catalase expression is repressed by MK maturation. A fraction of each selected population was used for RNA collection and subsequent analysis by real-time PCR. Expression of the antioxidant genes in the 3 selected fractions (CD41⁺CD42⁻, CD41⁺CD42⁺, CD41⁻CD42⁻) was compared to the expression in CD34⁺ progenitor cells after 3 days of culture. This analysis was performed for two independent donors (donor 1 selected at day 7, donor 2 selected at day 8).

Fig. 10

The antioxidant NAC effectively delayed the polyploidization of immature human MK cells. Culture-derived (A) CD41⁺CD42⁺ and (B) CD41⁺CD42⁻ MK cells were selected (see Figure 8) and returned to culture with various doses of NAC or H₂O₂. Subsequent polyploidization of (A) the mature and (B) immature fractions were monitored for 3 days. This analysis was performed for two independent donors (donor 1 selected at day 7, donor 2 selected at day 8).