Characterization of the Expression of PKCalpha(Isoform) in DMH-induced Vascular Endothelial Proliferation

ABSTRACT

PURPOSE: DMH(1,2-dimethylhydrazine) has been known to induce vascular neoplasm such as malignant endothelioma in animal experiment, through induction of abnormal proliferation of HUVECs. In our previous studies, 11 types of PKC isoenzymes were determined by RT-PCR and the expression of PKCalpha, and mu was more prominent than other PKC isoenzymes in the DMH-treated group. However, this result was not based on objective assessment. In this study, we further evaluated the role of PKCalpha on the DMH-induced abnormal proliferation of HUVECs by two different methods to identify its presence with high relevance in objective view. PKCmu will be investigated in further study. METHODS: The study was conducted with the cultured HUVECs group(control) and the 0.75x10(-9)M DMH-treated group. After processing protein extraction in 0 and 24 hour, extracted protein was treated of quantitative test through BCA protein assay. In the western blot analysis, electrophoresis was performed in the order of gel preparation, sample preparation, and gel running. Electrotransfer to nitrocellulose membrane and reaction with antibody were done. Detection of PKCalpha was achieved through "Gel Image Analysis System". In the fluorescence immunocytochemical analysis, the grading of radiance of the intracellular PKCalpha particles was detected with confocal microscope after treating with primary and fluorescent secondary antibody in 0 and 24 hours. RESULTS: The Western blot analysis showed increased PKCalpha expression from the specimen obtained in 24 hour of the DMH treatment group when compared to those in control group. Under confocal fluorescence microscope, the emitting radiance in the DMH treated group was brighter at 24 hours as well. CONCLUSION: We believe that PKCalpha plays a role in DMH-induced abnormal proliferation of the vascular endothelium, which may provide insights in understanding the vascular neoplasm.