Ultrastructural study of protein kinase C-betaII localization during the cell cycle of human glioma cells.
Transmission electron microscopy and immunogold labeling were used to determine how PKC-betaII is localized at stages in the cell cycle of the human glioma cell line U-373MG. Results show that immunogold particles in both dimethylsulfoxide (DMSO) and calphostin C (0.5 microM)-treated cells were mainly located in the cytoplasm. The concentration of gold particles in the nucleus was relatively small and constant throughout the cell cycle of both DMSO and calphostin C treated cells. Micrographs revealed changes in PKC-betaII during the cell cycle. The concentration of gold particles in the DMSO-treated cells was constant until 8 h. Subsequently, cytoplasmic PKC-betaII oscillated with an increased at 10 h, a rapid decrease at 12 h, and a rise at 14 h. The concentration of the gold particles then gradually decreased. In contrast, immunogold labeling in calphostin C-treated cells increased gradually up to 10 h. Subsequently, the pattern of PKC-betaII labeling in calphostin C-treated cells recapitulated those of control cells as seen by the rapid decline of PKC-betaII labeling at 12 h and its re-accumulation at 14 h. Additionally, there was a rapid increase at 20 h. Western blots of PKC-betaII showed constant PKC-betaII immunoreactivity throughout the cell cycle. In comparison to Western blots, in-situ immunogold labeling revealed changes in PKC-II immunoreactivity at 10 h and 14 h. This technique may represent intracellular immunoreactivity of PKC-betaII. The results from the immunogold labeling technique suggest that binding of calphostin C to the regulatory domain of PKC-betaII provokes a conformation change in PKC-betaII, preventing its activation and degradation.
Biçaku
E
E
James A. Haley Veterans Hospital, Tampa, Florida 33612, USA.
Acevedo-Duncan
M
M
D015153:Blotting, Western; D001932:Brain Neoplasms / Q000201:enzymology*; D002453:Cell Cycle; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005910:Glioma / Q000201:enzymology*; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000032:analysis*; D008854:Microscopy, Electron; D009281:Naphthalenes / Q000494:pharmacology*; D011493:Protein Kinase C / Q000032:analysis*; D064546:Protein Kinase C beta; D013194:Staining and Labeling; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D009281:Naphthalenes; D011493:Protein Kinase C; D064546:Protein Kinase C beta; C058819:calphostin C; D004121:Dimethyl Sulfoxide