R.L.Warters1, M.H.Barcellos-Hoff2 and A.E.Cress3
1University of Utah Health Sciences Center, Salt Lake City,
UT 84132, 2Lawrence Berkeley National Laboratory, Berkeley,
CA 94720 and 3Arizona Cancer Center, Tucson, AZ 85724
A major stress response in cells exposed to ionizing radiation (IR) is the p53-directed, DNA damage response pathway. The p53 protein, activated indirectly by the ATM protein kinase, transactivates target genes and thus induces cell cycle progression blocks or apoptosis. We examined elements of the p53 stress response pathway in two human cell lines; the SK-N-SH neuroblastoma and the HCA-2 fibroblasts. Cells were exposed to a range of gamma ray doses from 0.5-500 cGy, and examined either by western analysis of cell lysates, or by immunofluorescence of fixed cells. Progressive increases were observed in cellular levels of the p53 and p21/WAF1 proteins over the first 4-6 hours after irradiation. These protein increases were induced in a dose dependent manner at all IR doses in excess of 5-10 cGy. Since this is the dose range within which we would expect a yield of about 1-2 dsb/cell, our results are consistent with the production of DNA dsb activating the p53-directed, DNA damage stress response. Increased levels of p53 and p21/WAF1 decayed within 24 hours after cell irradiation.
In contrast, we observe a post-translational modification of the p53 protein at much lower IR doses. Indirect immunofluorescence microscopy detects a dose dependent increase in the nuclear level of the PAb421 antibody epitope, an anti-p53 antibody that recognizes amino acids 370-380. Increases in nuclear PAb421 epitope were detected at all IR doses > 0.5 cGy. IR-induced increases in nuclear PAb421 epitope result from an ATM dependent dephosphorylation of p53 serine 376 by a PP1 or PP2A type phosphatase. IR-induced dephosphorylation of p53 serine 376 is induced in cells exposed to H2O2, and is inhibited in the presence of an aminothiol radical scavenger. Our results are consistent with there being ATM directed responses to IR that occur at doses well below doses that induce conventional p53 DNA damage responses. These responses to low IR doses appear to be a response to the production of intracellular reactive oxygen species.