Figure 5.
Enhancement of PARP-1 activation by combination treatment of ionizing radiation with phytosphingosine. (A) Clone no. 6 was treated with 10 Gy of γ-radiation alone, 5 μg/mL of phytosphingosine alone, or combination of γ-radiation (10 Gy) and phytosphingosine (5 μg/mL) in the presence or absence of 30 μM DPQ or PARP-1 siRNA. After 3 hours, PARP enzyme activity was measured using a commercial kit under guidance of manufacturer (see “Materials and methods”). (B) Radiation-resistant cells were treated with 10 Gy of γ-radiation and/or 5 μg/mL of phytosphingosine in the presence or absence of 30 μM DPQ or PARP-1 siRNA. After 3 hours, cell lysates were subjected to Western blot analysis with anti-PARP-1 and β-actin antibodies. The data represent a typical experiment conducted at least 3 times with similar results. (C) Radiation-resistant cells were treated with 10 Gy of γ-radiation and 5 μg/mL of phytosphingosine in the presence or absence of 30 μM DPQ. After 3 hours, mitochondrial transmembrane potential of these cells was determined by retention of DioC3(6) added during the last 30 minutes of treatment. After removal of the medium, the amounts of retained DioC3(6) were measured by flow cytometry. (D) Radiation-resistant cells were treated with 10 Gy of γ-radiation and/or 5 μg/mL of phytosphingosine in the presence or absence of 30 μM DPQ. After 3 hours, cells were stained with Hoechst 33258, and apoptotic cells were quantitated by fluorescence microscopy. Results from 3 independent experiments are shown as means ± SEM. (E) Clone no. 6 was treated with 10 Gy of γ-radiation and/or 5 μg/mL of phytosphingosine in the presence or absence of 30 μM DPQ and 10 mM NAC. After 3 hours, mitochondrial or nuclear fractions were prepared. Mitochondrial protein fraction was subjected to Western blot analysis with anti-Bax and -HSP 60 antibodies, and nuclear protein fraction was subjected to Western blot analysis with anti-AIF and -Ref-1 antibodies. HSP 60 and Ref-1 were used as mitochondria and nuclear marker proteins, respectively.