C-ABL, FREE RADICALS, AND OSTEOPOROSIS

RESULTS. The c-Abl kinase is activated by treatment with oxidants in addition to genotoxic stress, and is likely to participate in the response to oxidative stress. We tested whether induction of Prx I was affected by the absence of c-Abl by using primary calvarial osteoblasts isolated from day 20 c-abl-/- and control embryos. The osteoblasts were treated with different concentrations of sodium arsenate for 12 hours and expression of Prx I mRNA was assessed by Northern blot. We observed Prx I mRNA up-regulation at lower concentration of arsenate, and to higher maximal levels, in the c-abl-/- cells. Western blot analysis showed similar increases in Prx I protein in the mutant lines. Thus, the c-abl-/- cells are hypersensitive to arsenate, suggesting that c-Abl normally limits or negatively regulates Prx I induction. To test whether the absence of c-Abl affects the rate of induction of cell death caused by oxidative stress, we treated c-abl-/- and control osteoblasts for 24 hours with different concentrations of arsenate and the percentage of dead cells was scored by trypan blue exclusion. Untreated cell populations, as well as cells treated with only 0.05 mM arsenate, contained less than 10% dead cells. Mutant cells treated with 0.1 mM arsenate were hypersensitive: whereas wild-type cultures showed less than 30% dead cells, c-abl-/- cultures showed more than 60% dead cells. At all concentrations, c-abl-/- osteoblasts consistently showed more cell death than control cells. Treatment with H2O2, and CDDP, a DNA damage drug that also generates free radicals, produced similar results (data not shown). These results suggest that c-abl-/- osteoblasts are more sensitive than the wild-type cells to several oxidative stresses.