Biological Characterization And Mechanism Of Resistant Cell Sublines Of Induced K562/ADM And Transfer K562/MDR

Objective: It is still unrevealed that why many tumors failed to respond to chemotherapy agents and then fall into a bad prognosis. Multidrug resistance(MDR)is cells selected for resistance to a single cytostatic drug may become cross-resistant to a wide spectrum of drugs that have different structures and functional mechanisms. Although there has been steady progress in the studies of MDR, it is still unclear that how to reverse such a process in clinic. MDR of tumors is multi-factor drug resistant phenotype. It may vary with different kinds of induced drugs, different families of cells, various cell differential phage and microenviroments. MDR is a result of expression of one drug resistant gene or the co-expression of the many drug resistant genes. Heretofore most reseaches on multidrug resistance use models established by drug inducement. Many studies have shown that the transfection of drug resistant gene will cause MDR in drug sensitive tumor cells. But the reseach concerning the comparison of biological characterization and drug resistant mechanism between induced drug resistant and gene transfected drug resistant leukaemia cell sublines have not been reported by now. We here take chronic myelocytic erythroblastoid crsis K562, K562 resistant to Adriamycin(K562/ADM) and mdrl gene transfected K562(K562/MDR) cells as the objectives for the study. Our goal is to compare the biological characterization of these three different cell lines and drug resistance mechanisms of later two cell sublines. We are also going to explore the feasibility of K562/MDR as a model possessing single drug resistant mechanism in order to make a foundation for the studies of tumor's MDR and its reverse.Method: Our work is composed of three parts: (1) Observing the biological characters of K562, K562/ADM and K562/MDR. It consists of observing the morphology of these three cell lines under light and electronic microscope; determining the multiplication time, Using flow cytometry to determine the cell cycle distributions,counting the chromosome. To study the effects of drug inducement and gene transferring on the biological characters of K562/ADM and K562/MDR cell sublines. (2) Using K562 as a control, comparing the grade of drug resistance beween K562/MDR and K562/ADM and trying reversing their resistance by Arsenic trioxide(As203). In order to compare the grade and spectrum of drug resistance and the effect of reversing between K562/ADM and K562/MDR, applying MTT method to examine the ICjo value of three cell lines to As2O3, Adriamycin, Daunorubicin and Vincristin and low cytotoxic dose to As2O3. Then determining the IC50 value of three cell lines to ADM, DNR and VCR under the present of low cytotoxic dose As2O3, respectively. (3) Detection of multi-drug resistance related genes and proteins. Immunocytochemistry is used to detect the change of expression of P-glycoprotein encodened by mdrl and GST-rc, and the results were by semi-qualification analyzed. Using flow cytometry to qualificationally determine the expression of P-gp and bcl-2. Biochemistry methods are used to detect the change of intracellular GSTs and Topo II activity. Using RT-PCR to determine the Topo II mRNA expression level.Results: (1) There is no obvious diference in morphology among these three cell lines. The multiplication time of K562/ADM is longer than that of K562 and K562/MDR. The cell cycle distributions of three cell lines are almost the same.There are much more chromosomes in K562/ADM and the number of K562/MDR is between the other two cells lines. (2) The ICso value and low cytotoxic dose of K562 and K562/MDR to As2O3 have no significant diference(P>0.05). The IC5o value and low cytotoxic dose of K562/ADM to As2O3 are significantly different from that of K562 and K562/MDR(P<0.05). The resisting folds of K562/ADM to ADM, DNR and VCR are higher than that of K562/MDR. As2O3 has effect on reversing the drug resistance of K562/ADM and K562/MDR. (3) The result of immunocytochemistry shows that K562/ADM and K562/MDR have overexpressed P-gp compared with K562.