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Elevated Ornithine Decarboxylase Levels Activate Ataxia Telangiectasia Mutated–DNA Damage Signaling in Normal Keratinocytes

¹ Lankenau Institute for Medical Research, Wynnewood, Pennsylvania and ² Department of Pharmaceutical Sciences, Wayne State University, Detroit, Michigan

Requests for reprints: Susan K. Gilmour, Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096. Phone: 610-645-8429; Fax: 610-645-2205; E-mail: gilmours{at}mlhs.org.

Key Words: p53 • polyamines • ornithine decarboxylase • reactive oxygen species • ATM activation • spermine oxidase

We examined the effect of increased expression of ornithine decarboxylase (ODC), a key rate-limiting enzyme in polyamine biosynthesis, on cell survival in primary cultures of keratinocytes isolated from the skin of K6/ODC transgenic mice (Ker/ODC) and their normal littermates (Ker/Norm). Although elevated levels of ODC and polyamines stimulate proliferation of keratinocytes, Ker/ODC undergo apoptotic cell death within days of primary culture unlike Ker/Norm that continue to proliferate. Phosphorylation of ataxia telangiectasia mutated (ATM) and its substrate p53 are significantly induced both in Ker/ODC and in K6/ODC transgenic skin. Chromatin immunoprecipitation analyses show that the increased level of p53 in Ker/ODC is accompanied by increased recruitment of p53 to the Bax proximal promoter. ATM activation is polyamine dependent because -difluoromethylornithine, a specific inhibitor of ODC activity, blocks its phosphorylation. Ker/ODC also displays increased generation of H₂O₂, acrolein-lysine conjugates, and protein oxidation products as well as polyamine-dependent DNA damage, as measured by the comet assay and the expression of the phosphorylated form of the histone variant H2AX. Both reactive oxygen species generation and apoptotic cell death of Ker/ODC may, at least in part, be due to induction of a polyamine catabolic pathway that generates both H₂O₂ and cytotoxic aldehydes, because spermine oxidase (SMO) levels are induced in Ker/ODC. In addition, treatment with MDL 72,527, an inhibitor of SMO, blocks the production of H₂O₂ and increases the survival of Ker/ODC. These results show a novel activation of the ATM-DNA damage signaling pathway in response to increased ODC activity in nontumorigenic keratinocytes. [Cancer Res 2008;68(7):2214–22]