Experimental Studies On CDNA Of PKC-α From Multidrug Resistance KBV200 Human Carcinoma Cells

Background and Objective:Multidrug resistance (MDR) is characterized by a cross-resistance to numerous antineoplastic drugs derived from natural products. The MDR phenotype induces cross-resistance to many chemotherapeutic agents in cancer cells. The emergence of drug resistance remains a major obstacle to the success of chemotherapy of human cancers. Drug-resistant sublines of human cells developed in vitro have served as useful tools for the understanding of the mechanisms that control this phenomenon, and for the development of methods aimed at its circumvention.Here, we have known that the PKC a -isoform (PKC- a) is overexpressed in multidrug-resistant KB-V human carcinoma cell lines, and suggest that this isozyme may be expressed in a modified form or be subject to an altered regulation in MDR cells. It is directly activated by tumor-promoting phorbol esters and is a key enzyme thatmodulates growth, differentiation, and apoptosis in a cell-specific fashion.In order to determine the role of specific PKC isoenzymes in regulating the MDR phenotype, the sequence of PKC- a in the human cervical carcinoma cell line, KB cell, and an MDR subline, KBV cell, were examined. And comparison of the expression of the PKC- a mRNA after the exposure of each cell type to 100nM TPA for 2 hour was performed. Methods:(1) The cell inhibitory rate was evaluated by MTT. The resistance factor was estimated from the ratio of the IC50 of vinblastine in the resistant line to that in the sensitive one.(2) The RNA was extracted with TRIzol reagent.(3) RT - PCR. To perform sequence analysis of subcloned polymerase chain reaction (PCR)-amplified cDNA , first strand cDNA was produced in a reaction using the AMV reverse transcriptase kit . The total RNA sequence was amplified in two overlapping fragments of 1.3 kilobase.(4) Amplified DNA was purified on agarose gel . Then the two fragments were cloned into the cloning vector pMD 18-T. The recombinant vectors were transformed into DH-5 a Escherichia coli, and plasmid preparations were performed by restriction enzyme digestion.(5) DNA sequencing was performed in both fractions on four separate clones to obtain the complete sequence information. Thecomparison of cDNA sequence of KB cell and KBV cell was performed according to manufacturer s instructions.(6) To compare the expression of the PKC- a mRNA, the intracellular mRNA of PKC- a was detected by RT-PCR after the initiation of TPA treatment.Results:The values of IC50 of VCR in KB cells was 25.44 nmol/L and that in KBV cells was 1477.85 nmol/L .The resistance indexes of VCR in KBV and KB cells were 58.09.The products of RT-PCR was purified and analyzed by polyacrylamide gel electrophoresis. The gel-purified products were inserted into the pMD-18T vector and the plasmids were transfected into colon bacillus. DNA sequencing show that no point mutation or base deletion was detected in the cDNA from the MDR cells, KBV cell.The further studies indicated that TPA, the activator of protein kinase C, could increase the expression of PKC?a mRNA in KB and KBV cell. We could amplify the PKC- a cDNA by RT-PCR in TPA-treated KB and KBV cells, but in the cells that were not treated with TPA , the PKC?a cDNA could not be amplified by RT-PCR. Comparable TPA-induced alteration in mRNA levels were observed between KB cell and the MDR cell, KBV cell. The efficiency of activation of TPA was more obviously in KBV cell than in KB cell. Conclusions:That the multidrug resistance of KBV cells is stable was confirmed by MTT assay. The residual resistance factor in the presence of a modulator was determined as the ratio of the IC50 of a cytotoxic drug with the reverter in the resistant line and without reverter in the sensitive line.It is well known that PKC- a was modestly increased in the multidrug resistant cells, the KBV cells, and this finding highlights the importance of PKC- a in the MDR. But at the gene level, there is no differentiation between the cDNA of PKC- a from the KB cell and the MD...