IN-SITU EFFECTS OF INTERFERON ON HUMAN GLIOMA PROTEIN-KINASE-C-ALPHA AND PROTEIN-KINASE-C-BETA ULTRASTRUCTURAL-LOCALIZATION

Transmission electron microscopy was used to determine how immunogold labeling of PKC-alpha or -beta is modulated by the antitumor drug IFN (HuIFN alpha-2b) in the cytoplasm, membrane structures, and nucleus of rapidly dividing and confluent human glioma U-373 cells. Results show ed that except for nuclear localization, there were no specific cytopl asmic organelles that PKC-alpha or -beta translocated to following HuI FN alpha-2b treatment. Electron micrographs of PKC-beta in proliferati ng cells depicted 1.34-fold more PKC-beta in the nucleus than in the c ytoplasm and a 1-min HuIFN alpha-2b (500 units/ml) treatment transient ly increased PKC-beta immunoreactivity in the cytoplasm (1.95-fold) an d nucleus (1.97-fold). In confluent cells, incubation with HuIFN alpha -2b for 2 min significantly decreased cytoplasmic PKC-beta immunoreact ivity by 37%, and no change was observed in nuclear PKC-beta labeling. PKC-alpha labeling in proliferating cells showed similar immunoreacti vity in both control cytoplasm and nucleus. Treatment of proliferating cells with HuIFN alpha-2b for 2 min decreased PKC-alpha in the cytopl asm (59%) and nucleus (44%). In confluent cells, cytoplasmic PKC-alpha labeling decreased 59% at 1 min, 61% at 2 min, and 76% at 10 min of H uIFN alpha-2b treatment. Nuclear PKC-alpha decreased by 65% at 1 min, 80% at 2 min, and 62% at 10 min after HuIFN alpha-2b treatment. Wester n blots of total PKC-alpha in proliferating and confluent cells and PK C-beta in confluent cells showed similar results. However, Western blo ts of total PKC-alpha and -beta in proliferating cells did not demonst rate any significant changes in either PKC-alpha or -beta immunoreacti vity following 1-min HuIFN alpha-2b treatment. These results suggest t hat treatment of proliferating U-373 cells with HuIFN alpha-2b for 1 m in unfolds and exposes PKC-beta antigenic sites (hinge region) and inc reases in situ PKC-beta immunogold labeling.