Effect of serum starvation on expression and phosphorylation of PKC‐α and p53 in V79 cells: Implications for cell death

• Published in: International journal of cancer 1999-01, Vol.80 (3), p.400-405
• Main Authors: Hasan, Nael M., Adams, Gerald E., Joiner, Michael C.
• Format: Article
• Language: English
• Subjects: Ageing, cell death; Animals; Apoptosis; Biological and medical sciences; Cell physiology; Cells, Cultured; Cricetinae; Cricetulus; Culture Media, Serum-Free; Cycloheximide - pharmacology; Dactinomycin - pharmacology; Enzyme Inhibitors - pharmacology; Fibroblasts - drug effects; Fibroblasts - metabolism; Fundamental and applied biological sciences. Psychology; Isoenzymes - antagonists & inhibitors; Isoenzymes - metabolism; Molecular and cellular biology; Phosphorylation; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - metabolism; Protein Kinase C-alpha; Staurosporine - pharmacology; Tetradecanoylphorbol Acetate - pharmacology; Tumor Suppressor Protein p53 - metabolism
• ISSN: 0020-7136; 1097-0215
• DOI: 10.1002/(SICI)1097-0215(19990129)80:3<400::AID-IJC11>3.0.CO;2-U

The effect of serum starvation on the expression and phosphorylation of PKC‐α and p53 in Chinese hamster V79 cells was investigated. Serum starvation led to growth arrest, rounding up of cells and the appearance of new PKC‐α and p53 bands on Western blots. Prolonged incubation (≥48 hr) in serum‐deprived medium led to cell detachment and death. Moving cells to fresh medium containing 10% serum before, but not after, cell detachment reversed the changes observed in PKC‐α and p53, and also prevented later cell detachment. Radiolabelling studies showed that the higher‐molecular‐weight PKC‐α and p53 bands result from increased phosphorylation, while a lower‐molecular‐weight PKC‐α band reflects newly synthesized protein. Immunocomplex kinase assays have shown that the increased phosphorylation of PKC‐α is associated with its increased activity. To study the relationship between PKC‐α, p53 and cell death, cells were treated either with TPA, to down‐regulate PKC or with staurosporine, to inhibit PKC activity. Staurosporine, a potent PKC inhibitor and inducer of programmed cell death, caused the appearance of new PKC‐α and p53 bands similar to those induced by serum starvation. If serum starvation was preceded by prolonged (48 hr) TPA treatment to down‐regulate PKC‐α, cell detachment and death did not take place within the same time frame. Intracellular fractionation of cells demonstrated that increased expression of PKC‐α and the appearance of the associated higher and lower molecular‐weight bands occurred in the nucleus. These data highlight the association of PKC‐α and p53 with cellular events leading to cell death. Int. J. Cancer 80:400–405, 1999. © 1999 Wiley‐Liss, Inc.