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

Abstract

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 A-difluoromethylornithine, a specific inhibitor of ODC activity, blocks its phosphorylation. Ker/ ODC also displays increased generation of H 2 O 2 , acroleinlysine 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 H 2 O 2 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 H 2 O 2 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]

Elevated ornithine decarboxylase activity promotes skin tumorigenesis by stimulating the recruitment of bulge stem cells but not via toxic polyamine catabolic metabolites

“…Previously we reported that elevated ODC activity in keratinocytes increases levels of the catabolic enzyme spermine oxidase with no detectable increase in SSAT, APAO, or AdoMetDC (Wei et al 2008). This is consistent with our finding that cutaneous spermine levels are often slightly decreased and putrescine and spermidine levels increased following ODC induction in ODC-ER transgenic mice compared to polyamine levels in normal littermate skin (Lan et al 2005).…”

“…Previous studies have shown that ODC activity and cutaneous putrescine levels rapidly increase and are significantly elevated within 2 days of 4OHT treatment, continuing to rise with daily 4OHT treatments (Lan et al 2005). Studies with ODC-overexpressing primary keratinocytes isolated from ODC transgenic mice have confirmed high levels of putrescine with small increases in spermidine levels and decreased levels of spermine compared to keratinocytes from normal littermate mice (Wei et al 2008). …”

“…Whereas elevated ODC activity provides a strong proliferative stimulus, it can also paradoxically induce the expression of p53 (Gilmour et al 1999; Wei et al 2008), stimulate ROS generation via induction of polyamine catabolic oxidases, and activate the ATM-DNA damage response pathway, resulting in apoptotic cell death in keratinocytes (Wei et al 2008). In contrast to DFMO-inhibition of ODC activity that completely blocks skin tumor formation in DMBA-initiated K6/ODC (Peralta Soler et al 1998) and ODC-ER transgenic mice (Lan et al 2005), we have shown that inhibition of polyamine cata-bolic oxidases with MDL72527 in DMBA-initiated ODC-ER mice leads to increased skin tumor growth and conversion to carcinomas.…”

“…Elevated ODC activity not only stimulates proliferation of normal keratinocytes but also induces apoptotic cell death via generation of reactive aldehydes and H 2 O 2 , due at least in part to the induction of SMO, followed by the subsequent activation of the ATM-DNA damage response pathway (Wei et al 2008). These findings suggest that cells with high ODC activity will be selectively sensitized to die when exposed to further oxidative or metabolic stress.…”

Elevated Epidermal Ornithine Decarboxylase Activity Suppresses Contact Hypersensitivity

“…The supernatants were cleared of debris by centrifugation and cytokine analysis was performed using flow cytometry as above. Keratinocyte cultures were prepared as described (Wei et al , 2008). …”