Calcium Ionophore And PMA Induce K562 Cells Eliciting Anti-leukemia Immunity

Objective: To investigate(1) the effect of calcium ionophore(CI) and phorbol myristate acetate(PMA) inducing the leukemia cell line K562 differentiation into activated dendritic cells(DC),(2) the effect of these K562-DCs to activate bone marrow cells' cytotoxicity against K562 cells.Methods: K562 cells in logarithmic phase were cultured in common medium, containing CI(375 ng/ml) and PMA(10 ng/ml)(group 1), CI(375 ng/ml) alone(group 2), PMA(10 ng/ml) alone(group 3), and neither CI nor PMA(control group). After 96 h treatment, the cells morphology was observed under light phase contrast microscope and the ultrastructure under electron microscope. Cell surface markers were analyzed with flow cytometry and the proliferation reaction of allogeneic T cells was detected by MTT colorimetry. In cytotocixity assay, normal bone marrow cells serve as effector cells, which were stimulated with those K562 cells after PMA+CI induction. The bone marrow cells without stimulation serve as control effector cells. K562 cells serve as target cells. Effector and target cells were mixed in the ratio of 1:1, 2:1, 10:1. After 96 h of culture, RT-PCR was used to detect the expression of BCR/ABL of effector-target mixture cells. Besides, we used PKH67 green fluorescent to mark target cells before they were mixed with effector cells. After 96 h of culture, the percentage of the target cells in effector-target mixture cells was measured by the flow cytometry and kill rate was calculated.Results: After 96 h treatment of CI and PMA, the K562 cells exhibited characteristic DCs morphology, flow cytometry showed that the expression of CD83, CD86, CD80, CD40,HLA-DR and CD1a was significantly upregulated, and the cells were found to strongly stimulate proliferation of allogeneic T cells when cultured with them. After the target cells were mixed and cultured with those effector cells with K562 cells stimulation, the expression of BCR/ABL in the effector-target mixture cells became lower. The flow cytometry showed that the percent of the target cells was reduce obviously. In the effector:target ratio of 1:1, 2:1 and 10:1, the kill rate were 29.6%, 55.2% and 65.8%, respectively.Conclusion: CI and PMA appear to quickly induce K562 cells differentiation into DC. The harvested cells showed similar morphology with DC, expressed the molecules necessary for T cell activation and stimulated proliferation of allogeneic T cells. When co-cultured with bone marrow cells, these K562-DC cells might stimulate T lymphocytes developing specific cytotoxicity against leukemia.