| Background: Proto oncogene Bmi-1(B-cell specific muriney murine virus insertion site 1)is one of the key members of the Polycomb Group genes family.It is highly expressed in many malignant tumors,such as Chrorlic myeloid leukemia(CML).It is reported that Bmi-1 is closely related to the proliferation,differentiation,multidrug resistance and the sensitivity of radiotherapy and chemotherapy,which is one of the important factors leading to the failure of chemotherapy and tumor recurence.Recent studies have demonstrated that Bmi-1 plays an important role in the DNA damage response(DDR),which can enhance the repair ability of DNA damage and inhibit the DNA damage checkpoint.In solid tumors,Bmi-1 may be involved in chemotherapy induced DDR.However,in the leukemia,whether Bmi-1 can regulate the DDR induced by chemotherapeutic agents and how to regulate the biological mechanism of DNA damage checkpoint remains to be determined.In our study,chronic myeloid leukemia multidrug resistance cell line(K562/ADR)as the research object,using small interfering RNA(siRNA)technology,silencing Bmi-1 gene in K562/ADR cells,to observe whether enhancing the response of cells to the antitumor traditional Chinese medicine monomer-camptothecin(CPT,DSBs topoisomerase I inhibitor)induced DNA damage response,participating the regulation of cell DNA damage checkpoint,inhibiting the proliferation of leukemia cells,increasing the sensitivity of leukemic cells to chemotherapeutic agents.Our study further explore the possiblebiological mechanism.Methods: 1.The expression of Bmi-1 in K562/W and K562/ADR cells was compared by RT-PCR and Western blot.2.PGenesil-2-HK-pGenesil-2-Bmi-1 1 and pGenesil-2-Bmi-1 3 were transiently transfected into K562/ADR cell lines.After 48 hours we examined the expression of Bmi-1 gene and were named them K562/ADRCtr,K562/ADR-s1 and K562/ADR-s3,respectively At the same time,we transfected the plasmid pGenesil-1(supplied by Wuhan cell marker Biotechnology Co.,Ltd.)containing the EGFP gene into the cells and observed the transfection efficiency by fluorescence microscopy.3.Using 0,0.005,0.01,0.02,0.04,and 0.16?mol/L different concentration of CPT to treat K562/ADR cells for 1-4 days,using MTT method to detect the optimal drug concentration and dosing time of K562/ADR cells.4.With the optimal concentration and dosing time selection of(72h IC50: 0.016 ?mol/L)CPT in K562/ADR and K562/ADR-s1 cells,the proliferation of K562/ ADR-s1,K562/ADR +CPT and K562/ADR,K562/ADR-s1+CPT cells was detected by MTT during 1-5 days.5.Treating CPT(0.016?mol/L)in K562/ADR and K562/ADR-s1 cells 2 hours,harvesting the four groups in order to observe focusing point of the ?-H2AX(marker of cellular DSBs)by the cell immunofluorescence experiments.6.Western blot method was used to detect the expression of P-ATM,P-CHK2 and ?-H2 AX protein in K562/ADR,K562/ADR-s1,K562/ADR+CPT and K562/ ADR-s1 +CPT cells.7.Western blot method was used to detect the expression of P-ATM,P-CHK2 and ?-H2 AX protein in K562/ADR cells treated with Chloroquinediphosphate(72h,0.014 ?mol/L).8.Western blot method was used to detect the expression of P-ATM,PCHK2 and ?-H2 AX protein in K562/ADR-s1 cells treated with ATM Inhibitor(KU55933)(72h).Results:1.The expression of Bmi-1 in K562/ADR cells was higher than that in K562/W cells,and the K562/ADR cell line with high expression of Bmi-1 was selected for subsequent experiments.2.the transfection efficiency of pGenesil-1 was about 70%,and the two siRNA fragment was successfully inhibited the expression of Bmi-1,selected K562/ADR-s1 with better interference effect for subsequent experiments.3.Treating different concentrations 0,0.005,0.01,0.02,0.04,0.08 and 0.16 ?mol /LCPT in K562/ADR cells after 1-4 days showed a dose and time dependent,and effectively suppress the cell proliferation,the calculated 72 h IC50 value as a follow up experiment.4.the proliferation rate of K562/ADR-s1,K562/ADR+CPT and K562/ADR-s1+CPT cells were significantly lower than the K562/ADR,the cell proliferation rate decreased more significantly in the K562/ADR-s1+CPT group,the inhibit proliferation of CPT induced K562 cells was obviously enhanced by suppress expression of Bmi-1,and increase the sensitivity of K562 cells to chemotherapeutic agents CPT.5.K562/ADR-s1,K562/ADR+CPT and K562/ADR-s1+CPT groups have nuclei with different degrees of ?-H2 AX focusing points,the K562/ADR-s1+ CPT group of ?-H2 AX nuclear focus number increased significantly,indicating Bmi-1 knockdown enhanced the chemotherapy induced cell DSBs and the synergistic effect.6.Observed increased expression of Bmi-1,P-ATM,P-CHK2 and ?-H2 AX in K562/ADR-s1 and K562/ADR+CPT,and expression of Bmi-1,P-ATM,P-CHK2 and ?-H2 AX increased significantly in K562/ADR-s1+CPT compared with K562/ADRs1 and K562/ADR+CPT.7.The ATM activator cells for the detection of P-ATM,P-CHK2 and ?-H2 AX protein expression,that the expression of P-CHK2 and ?-H2 AX in ATM activator group cells were significantly increased.8.The ATM Inhibitor group cells was significantly lower in group K562/ADR-s1 cells after Bmi-1 silencing suppression.Conclusion:silencing Bmi-1 gene can enhance the sensitivity of K562/ADR cells to chemotherapeutic drug CPT,which may be related to the enhancement of the DNA damage response protein P-ATM activity induced by CPT,thus activating the DNA damage checkpoint CHK2. |
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