Effects of the protein kinase inhibitors wortmannin and KN62 on cellular radiosensitivity and radiation-activated S phase and G1/S checkpoints in normal human fibroblasts

Wortmannin is a potent inhibitor of phosphatidylinositol (PI) 3-kinase and PI 3-kinase-related proteins (e.g. ATM), but it does not inhibit the activi ty of purified calmodulin-dependent protein kinase II (CaMKII). In the pres ent study, we compared the effects of wortmannin and the CaMKII inhibitor K N62 on the response of normal human dermal fibroblast cultures to gamma rad iation, We demonstrate that wortmannin confers a phenotype on normal fibrob lasts remarkably similar to that characteristic of cells homozygous for the ATM mutation. Thus wortmannin-treated normal fibroblasts exhibit increased sensitivity to radiation-induced cell killing, lack of temporary block in transition from G1 to S phase following irradiation (i.e. impaired G1/S che ckpoint), and radioresistant DNA synthesis (i.e, impaired S phase checkpoin t). Wortmannin-treated cultures display a diminished capacity for radiation -induced up-regulation of p53 protein and expression of p21(WAF1), a p53-re gulated gene involved in cell cycle arrest at the G1/S border; the treated cultures also exhibit decreased capacity for enhancement of CaMKII activity post-irradiation, known to be necessary for triggering the S phase checkpo int. We further demonstrate that KN62 confers a radioresistant DNA synthesi s phenotype on normal fibroblasts and moderately potentiates their sensitiv ity to killing by gamma rays, without modulating G1/S checkpoint, p53 up-re gulation and p21(WAF1) expression following radiation exposure. We conclude that CaMKII is involved in the radiation responsive signalling pathway med iating S phase checkpoint but not in the p53-dependent pathway controlling G1/S checkpoint, and that a wortmannin-sensitive kinase functions upstream in both pathways. (C) 1999 Cancer Research Campaign.