The Expression Of Multi-drug Resistance Gene And Proportion Of Leukemia Stem Cells In Heavy Ion Beam-and X-ray-irradiated Leukemic Cells

OBJECTIVE:To investigate the effects of heavy ion and X-ray irradiation on expression of multidrug resistance gene(mdr1) and P-glycoprotein(P-gp),the proportion of leukemia stem cells (LSC) in multi-drug resistant leukemia K562/ADM cells and the drug-sensitive parent K562 cells,and their sensitivity to adriamycin;To explore the correlation between radioresistance and chemoresistance of leukemia and LSC.METHODS:The multidrug resistant K562/ADM cells of drug-sensitive K562 cells were used as target cells.The target cells were irradiated with of X-ray from the linear accelerator and heavy ion (carbon ion,¹²C⁶⁺) beam from the heavy ion accelerator,the morphological changes of apoptosis were observed under inverted phase-contrast microscope;The mdr1 gene mRNA was measured by quantitative real-time reverse transiption-polymerase chain reaction (real-time RT-PCR);Flow cytometry(FCM) techenique was employed to detect the LSC surface markers(CD34⁺CD38^-CD123⁺) and P-gp expression;MTT colormetric assay was used to detect the sensitivity to the chemotherapeutic agent adriamycin.RESULTS:1.K562 cells only weakly expressed mdr1 and P-gp,and the proportion of LSC in K562 cell population was extremely low,about 1.1%-1.4%.After 1 to 8 Gy of X-ray radiation exposure,the expression of mdr1 gene mRNA of K562 cells increased 2.7 to 2.8 folds,and for P-gp expression 2.6 to 3.8 times,the proportion of LSC in K562 cell population increased 6.3 to 22.5 folds,and then the mdr1/P-gp expression and LSC proportion gradually reduced with the prolong of the culturing period to the level of the un-irradiated cells.The sensitivity of X-ray-irradiated K562 cells to adriamycin was obviously decreased.K562/ADM cells over-expressed mdr1 and P-gp,and LSC in K562/ADM cell population was 6-time higher than that in K562 cells.The X-ray-irradiated K562/ADM cells expressed 2.5 to 2.9-fold mdr1 mRNA and 4.0 to 6.5-fold P-gp higher over the un-irradiated cells;The proportion of LSC in X-ray-irradiated K562/ADM cell population increased 5.4 to 11.9 folds.The mdr1/P-gp expression and LSC proportion gradually reduced with the prolong of the culturing period but the high level remained long-drawn.The resistance of K562/ADM cells to adriamycin was significantly enhanced after X-ray irradiation.2.After 0.5 to 6 Gy of heavy ion beam irradiation,the positive rate of P-gp expression in K562 cells increased 6.0 to 10.0 folds,and the mean fluorescence intensity(MFI) of P-gp expression in K562/ADM cells increased 6.0 to 10.0 times,and then the expressing level gradually reduced to that of the un-irradiated cells.The proportion of LSC in heavy ion-irradiated K562 cells increased nearly 21 folds at peak-peak,and gradually decreased to the level of the control cells with the prolong of the period.After heavy ion beam irradiation, The proportion of LSC in K562 cell population also increased 5.5 times,which was lower than that in K562 cells,and the higher level last for a long time.The sensivity of both K562 cells and K562/ADM cells to adriamycin were reduced after heavy ion beam irradiation. Compared with X-ray radiation,the heavy ion beam irradiation had obvious hysteresis effect on the increase of mdr1/P-gp expression and LSC content in the cells.These results suggested that X-ray mainly injured the more differentiated leukemia cells and have a weak killing effect on leukemia stem cells,and the heavy ion beam irradiation could damage both differentiated cells and LSCs.The leuukemia cell killing effect of the heavy ion beam was higher than that of X-ray.The irradiation may mediate the secondary drug-resistance via increasing the proportion of LSC and stimulating mdr1/P-gp expression in leukemia cells.CONCULSlON:Heave ion beam and X-ray irradiation are capable of enhancing mdr1/P-gp expression and increasing the proportion of LSC in drug-sensitive and -resistant leukemia cells,and then secondly derease the sensitivity of the cells to the chemotherapeutic agent adriamycin.Heave ion beam irradiation hold much higher efficacy of killing leukemia over X-ray,and potential to destroy the drug-resitant leukemia cells and leukemia stem cells.