Early apoptosis of HL-60 cells induced by anti-CD44 McAb A3D8 inhibiting ERK1/2-upregulated Bim expression / 中国实验血液学杂志

This study was aimed to investigate the effects of anti-CD44 mAb A3D8 on proliferation and apoptosis of AML cells, to explore the mechanism of ERK1/2 and Bim in this process. Effect of the anti-CD44 mAb A3D8 on the HL-60 cell proliferation was assayed with MTT method, the change of mitochondrial transmembrane potential of HL-60 cells was analyzed by flow cytometry. The mRNA expression of Bim was determined by real-time quantitative RT-PCR. Western blot was used to detect the protein expression of p-ERK1/2. The results showed that mAb A3D8 could remarkably inhibit the proliferation capacity of the HL-60 cells in a dosage- and time-dependent ways. The mitochondrial transmembrane potential in HL-60 cells treated with A3D8 (3.0 µg/ml) was significantly decreased as compared with the control cells. Furthermore, the mRNA expression of Bim was much higher than that in controls. Expression of the p-ERK was much lower than that of the controls. It is concluded that anti-CD44 mAb A3D8 can inhibit the proliferation and induce the apoptosis of HL-60 cells, mechanism of which is enhancing the expression of Bim via inhibiting p-ERK1/2.

Reversal of multidrug resistance in HL-60, MSC and CD34(+) cells from umbilical cord blood by sustained intracellular acidification / 中国实验血液学杂志

The aim of this study was to investigate the effect of intracellular acidification on accumulation of rhodamine 123 (rh123) in non-mature cells with none or low expression of multidrug resistance MDR1. The expression of MDR1 mRNA was detected by real-time quantitative RT-PCR. Confocal laser microscopy was used to determine the calibration curve of intracellular acidification (pHi). MTT assay was used to detect the cytotoxicity of intracellular acidification on HL-60, MSC and CD34(+) cells from umbilical cord blood. Flow cytometry was applied to measure the influence of intracellular acidification. The results indicated that the intracellular acidification had no obvious cytotoxicity on HL-60, MSC and CD34(+) cells. The acidification resulted in the increased rhodamine 123 accumulation in HL-60, MSC and CD34(+) cells without P-gp activity. Moreover, the more primitive cells, the less accumulation of intracellular Rh123 were observed. It is concluded that the intracellular acidification can reverse the MDR of HL-60, MSC and CD34(+) cells.

Na(+)/H(+) exchanger 1 expression and its effect on apoptosis in K562 and HL-60 cells with DNA damage / 中国实验血液学杂志

This study was aimed to investigate the expression of Na(+)/H(+) exchanger 1 (NHE1) in K562 and HL-60 cells undergoing DNA damage induced by etoposide and to elucidate the regulating mechanism. Real-time quantitative PCR (RQ-PCR) and Western blot methods were used to determine the expression of NHE1 in K562 cells after the treating with etoposide. Meanwhile, the flow cytometry was used to detect the apoptosis of leukemic cells. The luciferase reporter vector containing NHE1 promoter was constructed to measure relative luciferase activity after treating with different etoposide concentrations. The results showed that the mRNA and protein of NHE1 increased in accordance with apoptosis ratio in HL-60 cells after treated with etoposide (p < 0.05), but no such obvious increase in K562 cells. Treatment with NHE1 specific inhibitor could block etoposide induced alkalization and reduce the apoptosis ratio of HL-60 cells. The expression pattern and apoptosis alteration was not similar in K562 cells. Relative luciferase activity of reporter vector containing NHE1 promoter however increased in K562 cells after treated with etoposide. It is concluded that the expression of NHE1 is up-regulated in the process of apoptosis of HL-60 cells induced by etoposide and depends on the pHi increasing caused by NHE1 up-regulation which is not found in K562 cells although the transcriptional activity increased.