The Changes Of Resistant Gene Expression Profile In Homoharringtonine-Induced Multi-Drug Resistant Leukemia Cells K562/HHT

Leukemia is one of the most common clinical malignant tumors in the department of hematology,and chemotherapy is still the primary treatment now.Homoharringtonine (HHT) is a Cephalotoxin alkaloid with highly anti-leukemia activity.It had been mainly used for the treatment of acute myeloid leukemia,chronic myeloid leukemia and myelodysplastic syndrome.Recent research shows that,HHT can be used for the chronic myeloid leukemia patients after the failure of interferon therapy or imatinib-resistant.However,prolonged application of HHT will result in multi-drug resistance(MDR) phenomenon,with cross-resistance of anthracyclines,vincristine and podophyllotoxin and so on.MDR of leukemia cells is the main reason of the failure of chemotherapy for leukemia.The current primary study have confirmed that the multi-drug resistance's mechanisms involved in transmembrane glycoprotein,such as P-gp,GSH/GST system,DNA repair,and a variety of apoptosis-related gene,but still can not explain the MDR mechanisms to leukemia cells satisfactorily.Therefore,looking for a new resistance-associated genes revealed to the mechanism of resistance is necessary.SectionⅠScreening multi-drug resistance genes by Human whole-genome micro array in K562/HHT cell linesGene chip technology is a fast and high-throughput research method which can analyze the information of nucleic acid sequence in cells or organisms.The technology applying to tumor drug resistance contributes to the selection of new resistance associated genes and has an important role in promoting the diagnosis of drug resistance individuals and the mechanism of tumor drug resistance.To this end,we use gene chip technology to study the changes of gene expression profiles in HHT-induced multi-drug resistance leukemia cell lines K562/HHT and screen the multi-drug resistance genes associated K562/HHT cell lines.Objective Using gene chip technology to screen multi-drug resistance genes associated with K562/HHT cells lines induced by homoharringtonine(HHT).Methods On the basis of pre-established multi-drug resistant cell line K562/HHT,we detect the gene expression profile changes between K562/HHT,its parent cell line K562 and K562/HHT/RU486 cells which were reversed by RU486 on K562/HHT by using DNA micro array.Results Compared with K562,117 significantly differently expressed genes were screened out in the K562/HHT cell line,of which up- and down-regulated genes were 57 and 60 respectively,in which multi-drug resistant gene mdr-1 was significantly up-regulated(Ratio=4.2899).While compared with K562/HHT,50 significantly differently expressed genes were screened out in the K562/HHT/RU486 cells,of which up- and down-regulated genes were 13 and 37 respectively.These genes involved in resistance,cell signaling,cell differentiation,cell proliferation,transcription regulator, and so on.4 genes changed in the two group cells consistently,namely:NM-001721 (BMX),NM-031459(SESN2),NM-033642(FGF13) and AL-049309(SFRS12). Compared with K562,BMX gene expression increased in K562/HHT(Ratio=2.6036), while its expression reduced in K562/HHT/RU486 compared with K562/HHT (Ratio=0.4603).Conclusion We obtained the information of some genes associated with multi-drug resistance of leukemia cell line K562/HHT induced by homoharringtonine.SectionⅡPreliminary study on multi-drug resistance and its mechanisms of BMX involving in human leukemia cell lines K562/HHT induced by HHTBMX(bone marrow tyrosine kinase gene in chromosome X) is a gene cloned and isolated from human bone marrow cells by Tamagnone in 1994,located in chromosome Xp22.2,its mRNA length 2.7kb,encoding an 80kD protein,a member of non-receptor type tyrosine kinase Tec family.BMX expresses in arterial endothelial,extensive fetal endocardial,granulocyte,bone marrow cell lines and prostate cell lines and so on.It participates in a variety of cellular signal transductions both inside and outside,and its function is relevant to cell adhesion,migration,proliferation and survival and so on. However,there is no research on BMX and resistant leukemia cell.Part of the former study,we used gene chip technology to screen a lot of multi-drug resistance genes associated with K562/HHT cells and found that BMX may be associated with the formation of multi-drug resistance of leukemia cells K562/HHT HHT-induced,this part we will further study the role of BMX in formation of drug-resistant leukemia cell lines K562/HHT and explore its mechanism.Objective To further study the role of BMX gene in the formation of multi-drug resistance in human leukemia cell lines K562/HHT HHT-induced and its mechanism.Methods After the results about BMX in DNA micro array were confirmed by RT-PCR and Western blot,we transfected BMX plasmid into K562 and K562/HHT cells at the same time.When RT-PCR and Western blot confirmed BMX over expressed after transfection,we observe the drug resistance in the two cells to several commonly used chemotherapy and the changes of cell daunorubicin(DNR) content respectively by MTT(Tetrazolium blue assay) and FCM(Flow Cytometry).Results We found the expression of BMX increased in K562/HHT cells than K562 cells, reduced in K562/HHT/RU486 cells than K562/HHT cells by RT-PCR and Western blot. After BMX plasmid successful transfection,we found K562 cells thansfected with BMX plasmids to obtain varying degrees of resistances and the resistances of K562/HHT cells transfected with BMX plasmids had increased by varying degrees by MTT method.DNR contents were significantly lower(79.28±4.04,29.84±2.67) than before transfection (158.52±8.08,58.58±6.53) in both cell lines detected by FCM respectively.Conclusion BMX played a role in drug-resistant formation at K562/HHT cells by reducing intracellular drug accumulation,but how BMX reduced intracellular drug accumulation and whether there are other ways mediating resistance are worth further studying.