Phytosphingosine in combination with ionizing radiation enhances apoptotic cell death in radiation-resistant cancer cells. (A) Selection of radiation-resistant Jurkat clones. Single cells in limiting dilution condition (0.2 cells/well) were incubated in 96-well plates for 2 months and then individual clones were irradiated with increasing doses of γ-radiation. Cells were allowed to grow on soft agar for 10 to 14 days and were stained with 0.5% crystal violet and scored for colony formation. Results are given as means ± SEM of 3 independent experiments. (B) Individual clones were treated with 10 Gy of γ-radiation and cultured for 24 or 48 hours. Cells were stained with Hoechst 33258, and apoptotic cells were analyzed by fluorescence microscopy. Apoptotic cells containing condensed chromatin fragments were scored and expressed as a percentage of the total cell numbers measured. Results from 3 independent experiments are shown as means ± SEM. IR indicates ionizing radiation. (C) Cross-resistance to the phytosphingosine in radiation-resistant clones. Radiation-resistant Jurkat clones were treated with 5 μg/mL of phytosphingosine. 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. ▪ indicates control; □, phytosphingosine. (D) Phytosphingosine sensitizes radiation-resistant Jurkat clones to radiation-induced apoptotic cell death. Radiation-resistant Jurkat clones were 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). 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) Mitochondrial transmembrane potential 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. (F) Cell lysates of clone no. 6 treated with 10 Gy of γ-radiation and/or 5 μg/mL of phytosphingosine were subjected to Western blot analysis with anti-caspase-8, -Bid, -caspase-9, -caspase-3, and -PARP antibodies. The data represent a typical experiment conducted at least 3 times with similar results. (G) Cells were treated with 10 Gy of γ-radiation and/or 5 μg/mL of phytosphingosine in the presence or absence of 30 μM z-VAD-fmk. 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.