Carcinogenic benzo(a)pyrene (BP), after activation to BP-diolepoxide, forms DNA adducts. This is associated with a p53 response in human cells in vitro and mouse tissues in vivo. Activation of p53 through phosphorylation has a pivotal role for its cellular functions. BP has been shown to induce ser15 phosphorylation and induction of apoptosis in murine cells. BP-induced p53 phosphorylation and p53 dependent cell death have been studied largely in animal cell lines (murine, rat). MCF-7 and A549 cells of human origin harbour wild type p53 protein enabling studies on the role of wt p53 in BP-exposed human cells. We studied whether and in what positions p53 in these cells is phosphorylated by BP-induced DNA-damage. A549 lung carcinoma and MCF-7 breast adenocarcinoma cells were treated with different concentrations of BP (1, 5, 10, 20 and 40 μM) for 24, 48 and 72 hours. BP was activated to BP-diolepoxide DNA-adducts in both cell lines, as studied by synchronous fluorescence spectrophotometry. In accordance with our earlier studies, the number of adducts was about ten times higher in MCF-7 cells. Cell death was clearly induced in MCF-7 cells while A549 cells were very resistant to the effects of BP. In MCF-7 viability decreased the most with 5 µM BP and seemingly increased towards higher concentrations of BP (10, 20 and 40 µM) at 48 and 72 hours. However, the relative cell number also decreased with higher BP concentrations already at 24 h. In A549 cells BP did not affect cell viability or relative cell number. Anti-phospho-p53 antibodies against p53 protein phosphorylated at serines 6, 9, 15, 20, 37, 46 and 392, and immunoblotting were used to analyze p53 phosphorylation. While no phosphorylation of p53 in A549 cells could be detected, in MCF-7 cells several sites of p53 protein were phosphorylated after BP induced DNA-damage. Ser392 phosphorylation was clear already at 24 hours in association with an increase in p53 protein, while ser15 and ser20 phosphorylations appeared later. In conclusion, in human cells p53 phosphorylation at ser392, rather than at ser15, seems to be related to p53 response after BP diolepoxide-DNA-damage.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA