Reversal Of Multidrug Resistance By Antisense Nucleotide Shandong University, School Of Medicine, Department Of Pathology

Objective Multidrug resistance(MDR) is a major obstacle in cancer treatment and has been closely associated with treatment failure. The overexpression of p-gp is the main mechanisms of multidrug resistance(MDR).P-glycoprotein (p-gp) is encoded by multidrug resistance genel(mdrl) and acts as an energy-dependent drug excretion from cell and reduces the intracellular drug contents. At present, it is thought to be a good strategy to reverse MDR of tumor cells by inhibiting the transcription of mdrl specially. In this study, two antisense techniques including antisense oligonucleotide (ASODN) and antisense ribozyme were investigated to reverse MDR in vitro.ASODN is short sequences of DNA that could form a specific hydrogen bond with complementary DNA or mRNA sequences and regulate the expression of gene. In this study, several transcription initiation sites and mian function zones of mdrl were blocked to select the ASODN with the best effort of reversal MDR. The effective dozen and dynamic observation were also investigated in the first 7 days after tranfecting ASODN.Ribozyme is a pair of RNA which has the nucleolytic activity and is able to cleave specific RNA sequences. Hammerhead ribozymes have been proven to be useful tools for inhibition expression of target gene. It could be engineered to cleave any triplet of GUC by changing the flanking sequence. In this study, the triplet of GUC was selected by RNA-folding program (m-fold version 3.0) and the ribozyme gene was constructed to a plasmid to reverase MDR of tumor cells stably by specific cleaving of mdr 1 mRNA.Methods 1. The phosphrothioate oligonucleotides complementary to four transcription initiation sites-major initiation start tone (MA zone), minor initiationzone (MI zone). CAAT box (C zone),GC box (G zone)and two main functional zone-mdr 1 mRNA start site code (S zone),ATP banding site code (A zone) were synthesized and transfectined into drug-resistant cells of breast carcinoma respectively. 48hours later, mdr 1 mRNA was detected by RT-PCR, the expression of p-gp was detected by immunohistochemistry. the function of p-gp was determined by intracellular Rhl23 efflux assay. T-test and the analysis of variance was used to evaluate the differences between two groups.2. RNA-folding program was used to choose the triplet of GUC in the surface of ribozyme. Two anti-mdrl ribozyme were synthesized with RNA polymerase trans cription termination signal at 3'end and the sticky end of EcoR I and Sal I at two ends. The two ribozymes were constructed into the multiclonal sites of pEGFP-cl and transducted into bacillus coli. After identification by restrictive enzymatic catalysis, the positive clone was selected and cultured. The two ribozyme plasmids were extracted and named pEGFP-RZ179, pEGFP-RZ196 respectively. Three days after transfecting the ribozyme plasmids into drug resistance cells, the expression of GFP was observed by fluorescence microscope and the transfection rate was detected by flow cytometry. After selected by G418 for 3 weeks, the survival cells were cultured for the detection of reversal effort. The changement of mdr 1 mRNA and p-gp was detected by RT-PCR, flow cytometry and Rhl23 efflus assay. T-test and analysis of variance was used to evaluate the differences between two groups. Results 1. 48hours after transfectined the ASODN complementary to MA zone, MI zone, C zone and G zone, the mdr 1 index of the treated cells was 1.4, 1.9, 1.6, 2.1 respectively, the expression rate of p-gp was 14%, 43%, 26%, 39%, the fluorescence intensity of Rhl23 was 75%, 31%, 63%, 27%. There was signicant difference between the cells treated by ASODN complementary to MA zone, C zone with the drug-resistant cells (p<0.05). 48 hours after transfectined the ASODN complementary to S zone and A zone, the content of mdrl mRNA and p-gp reduced significantly (p<0.05). The ASODN complementary to S zone had the best reversal effort. ASODN could reverse MDR of cells at the first day of tranfection and reach its best effort at the second day before the effort reduced in the following days. There was no...