In situ effects of interferon on human glioma protein kinase C-alpha and -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 showed that except for nuclear localization, there were no specific cytoplasmic organelles that PKC-alpha or -beta translocated to following HuIFN alpha-2b treatment. Electron micrographs of PKC-beta in proliferating cells depicted 1.34-fold more PKC-beta in the nucleus than in the cytoplasm and a 1-min HuIFN alpha-2b (500 units/ml) treatment transiently increased PKC-beta immunoreactivity in the cytoplasm (1.95-fold) and nucleus (1.97-fold). In confluent cells, incubation with HuIFN alpha-2b for 2 min significantly decreased cytoplasmic PKC-beta immunoreactivity by 37%, and no change was observed in nuclear PKC-beta labeling. PKC-alpha labeling in proliferating cells showed similar immunoreactivity in both control cytoplasm and nucleus. Treatment of proliferating cells with HuIFN alpha-2b for 2 min decreased PKC-alpha in the cytoplasm (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 HuIFN 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. Western blots of total PKC-alpha in proliferating and confluent cells and PKC-beta in confluent cells showed similar results. However, Western blots of total PKC-alpha and -beta in proliferating cells did not demonstrate any significant changes in either PKC-alpha or -beta immunoreactivity following 1-min HuIFN alpha-2b treatment. These results suggest that treatment of proliferating U-373 cells with HuIFN alpha-2b for 1 min unfolds and exposes PKC-beta antigenic sites (hinge region) and increases in situ PKC-beta immunogold labeling.
Acevedo-Duncan
M
M
Department of Radiology, Moffitt Cancer Center and Research Institute, James A. Haley Veterans Hospital, Tampa, Florida, USA.
Collins
J
J
Zhang
R
R
Haller
E
E
Chalfant
C E
CE
Cooper
D R
DR
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D015153:Blotting, Western; D005455:Fluorescent Antibody Technique; D005910:Glioma / Q000201:enzymology*; D006801:Humans; D007150:Immunohistochemistry; D016898:Interferon-alpha / Q000494:pharmacology*; D007527:Isoenzymes / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D012333:RNA, Messenger / Q000032:analysis; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000648:ultrastructure
D016898:Interferon-alpha; D007527:Isoenzymes; D012333:RNA, Messenger; C496628:PRKCA protein, human; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha