Ultrastructural study of protein kinase C-beta II localization during the cell cycle of human glioma cells

Transmission electron microscopy and immunogold labeling were used to deter mine how PKC-beta II is localized at stages in the cell cycle of the human glioma cell line U-373MG, Results show that immunogold particles in both di methylsulfoxide (DMSO) and calphostin C (0.5 muM)-treated cells were mainly located in the cytoplasm, The concentration of gold particles in the nucle us was relatively small and constant throughout the cell cycle of both DMSO and calphostin C treated cells. Micrographs revealed changes in PKC-beta I I during the cell cycle. The concentration of gold particles in the DMSO-tr eated cells was constant until 8 h, Subsequently, cytoplasmic PKC-beta II o scillated with an increased at 10 h, a rapid decrease at 12 h, and a rise a t 14 h, The concentration of the gold particles then gradually decreased. I n contrast, immunogold labeling in calphostin C-treated cells increased gra dually up to 10 h, Subsequently, the pattern of PKC-beta II labeling in cal phostin C -treated cells recapitulated those of control cells as seen by th e rapid decline of PKC-beta II labeling at 12 h and its re-accumulation at 14 h, Additionally, there was a rapid increase at 20 h. Western blots of PK C-beta II showed constant PKC-beta II immunoreactivity throughout the cell cycle. In comparison to Western blots, in-situ immunogold labeling revealed changes in PKC-beta II immunoreactivity at 10 h and 14 h. This technique m ay represent intracellular immunoreactivity of PKC-beta II, The results fro m the immunogold labeling technique suggest that binding of calphostin C to the regulatory domain of PKC-beta II provokes a conformation change in PKC -beta II, preventing its activation and degradation. (C) 2001 Harcourt Publ ishers Ltd.