| OBJECTIVE: Acquisition of a multidrug resistance (MDR) phenotype in tumor cells is a major obstacle to successful therapy. The tumor often becomes resistant not only to the drug used in the therapy, but also to a broad spectrum of structurally and functionally unrelated cytotoxic drugs as well. The mechanisms of MDR are complicated in tumors. One of the mechanisms involved in the development of multidrug resistance in human cancer cells is the overexpression of P-glycoprotein(P-gp), a product of the mdrl gene. P-gp is an ATP-dependent transmembrane efflux pump. The expression of P-gp has been demonstrated in many types of solid tumors, such as KBV200 cells, MCF-7/adr cells etc. There is sufficient evidence that P-gp overpression is mainly regulated at the transcriptional level. Recently, the role of PKC in the transcriptional regulation of mdrl gene arised attention. PKC is an important element of cellular signal transduction pathways that regulate cell growth, survival, differentiation and gene expression. PKC can influence the MDR phenotype by two mechanisms, firstly by direct P-gp phosphorylation and, secondly, through modulation of mdrl gene transcription via transcription factor phosphorylation. Currently many studys have been demonstrated that PKC plays an important role in MDR, but the mechanisms of PKC regulating expression of mdrl generemains unclear. In this study, we investigated PKC activity, expression of P-gp and regulation of mdrl promoter activity. The aim of the study was to elucidate the importance and mechanism of PKC in MDR, and to construct the expression vector for mdrl promoter-GFP fusion protein, which might be instrumental in the study of transcription regulation of mdrl gene.METHODS: The cell inhibitory rate was evaluated by MTT. PKC activity was assayed by the measurement of the incorporation of 32P from [ Y -32P] ATP into peptide substrates. Western blot was used for the assessment of P-gp expression. Primers were devised according to published mdrl gene sequence. PCR was used for the amplification of mdrl gene promoter, and amplified DNA was purified using 3S DNA Gel Purification Kit V 3. 1. Then subcloned into the report gene vector pEGFP-N2- The recombinant plasmid was verified by enzyme digestion, PCR and sequence analysis. Plasmid was transiently transfected to cells by Lipofectamine 2000. The activity of mdrl gene promoter was determined by flow cytometry(FCM). And SPSS 8. 0 was used to analyze data and make charts.RESULTS: The values of IC5o of vincristine(VCR) and adriamycin(ADR) in KB cells were 21.84 nmol/L and 5.13 nmol/L, and those in KBV200 cells were 1458.60 nmol/L and 110.37 nmol/L respectively^ < 0. 01, vs KB cells). The resistance indexes of VCR and ADR were 66.79 and 21.51 respectively. P-glycoprotein (p-gp) was overexpressed in KBV200 cells. There was no P-glycoprotein (p-gp) expression in KB cells. PKC activity was higher in KBV200 cells than in KB cells. Total PKC activity in KBV200 cells was 2.16-fold higher than that in KB cells.The anticipated DNA fragment band of mdrl gene promoter which is about 440 base pairs was showed clearly by agarose gel electrophoresis. There was no non-specific amplification. Enzyme digestion and PCR were used to verify the recombinant plasmid , and the electrophoresis bands were right. The result of sequence analysis was the same to the published results.In order to elucidate the role of PKC in mdrl gene promotor, theKBV200 cells were divided into three groups:Control group (no activitor or inhibitor). PMA group (200 nmol/L PMA preincubation) and SP group (100 nmol/L SP preincubation). The total PKC activity were higher in PMA group than that in control group (p=0.001), The total PKC activity was lower in SP group than that in control group(P= 0.001). The results of Western blot showed that PMA could imcrease the expression of p-gp and SP could decrease it. The percentage of GFP positive cells was increased after PMA preincubation and it is decreased by SP preincubation.CONCLUSION: 1. The multidrug resis... |
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