Abstract
The effect of ionising radiation on the regulation of gene and protein expression is complex. This study focuses on the translational regulational of the epsilon isoform of protein kinase C by ionising radiation. We found that protein kinase C epsilon is rapidly increased in the human lung adenocarcinoma cell line A549 following irradiation. Western blots showed increased accumulation of this protein at doses as low as 75 cGy after 15 min post irradiation. Maximal induction (11-fold over unirradiated cells) of PKC epsilon occurred at 150 cGy within 1 h after treatment by X-rays in A549 cells. The increased levels of PKC epsilon protein after X-rays does not require de novo protein or RNA synthesis, suggesting that this increase is post-translationally controlled. In contrast to A549 cells PKC epsilon levels in the large cell lung carcinoma cell line NCI H661 were not induced by radiation. In the small cell lung carcinoma cell line NCI N417, PKC epsilon was also not induced but a higher molecular weight PKC epsilon protein, suggestive of phosphorylation, appeared at 2 h after irradiation. The variation in induction or phosphorylation of PKC epsilon by ionising radiation in the cell lines tested in this study suggested that no clear correlation existed between intrinsic radiation sensitivity and PKC epsilon induction. To determine whether PKC epsilon does play a role in cell survival to irradiation, we used the protein kinase inhibitor staurosporin to decrease PKC activity and found that staurosporin sensitised cells to killing by ionising radiation. Pulsed field gel electrophoresis, however, indicated that DNA double-strand break repair was not decreased, suggesting that PKC epsilon is modifying the fidelity of rejoining and not the overall magnitude of repair. The regulation of PKC by ionising radiation will be discussed with respect to the biological consequences of gene induction by DNA damage agents.
Highlights
This study focuses on the epsilon (e) isoform of protein kinase C which is found in brain (Heidenreich et al, 1990), thymocytes (Strulovici et al, 1990), and small cell carcinomas of the lung (Baxter et al, 1992)
Human lung adenocarcinoma A549 cells were obtained from the American Type Culture Collection (ATCC) and routinely cultured in alpha MEM containing 10% foetal calf serum
We analysed the induction of PKC epsilon protein in lung carcinoma cell lines using a specific antibody against this isoform
Summary
Cell lines and culture conditionsHuman lung adenocarcinoma A549 cells were obtained from the American Type Culture Collection (ATCC) and routinely cultured in alpha MEM containing 10% foetal calf serum. The large cell line used was a NCI H66 1 These cell lines were established by Adi Gazdar and generously provided by Dr John Minna (NCI, Bethedsa, Md) and cultured in RPMI 1640 medium with 10% heat inactivated serum. These cell lines have been previously described by Carmichael et al with respect to their radiation sensitivity (Carmichael et al, 1989) and biochemical and morphological properties (Carney et al, 1985; Gazdar et al, 1985). To reduce the effects of serum on PKC induction, cells were refed 16 h before the experiment with alpha MEM or RPMI containing 0.1% BSA before induction experiments. To determine whether induction required de novo protein or RNA synthesis, cells were preincubated for 1 h with either 1 Ag ml-' or cyclohexamide or
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