Gossypol-mediated CLL lymphocyte cell death and effect of caspase inhibitor or an antioxidant. (A) Gossypol induces cell death in chronic lymphocytic leukemia (CLL) lymphocytes. CLL primary cells were cultured for 24 hours either (i) alone (control) or (ii) with 30 μM gossypol, and the cell viability was determined by flow cytometry after double staining with FITC-conjugated annexin V and propidium iodide. (iii) Percentage of cell death in CLL primary cells from 9 individual patients treated with gossypol for the indicated times. (iv) CLL primary cells were incubated with 10 and 30 μM gossypol for the indicated times. Cells were lysed and cleavage of PARP was measured by immunoblotting. Actin was used as a loading control. Similar experiments were done in 8 patients, and data from 2 representative patients are shown. (B) Effect of caspase inhibitor on gossypol-induced cell death. CLL primary cells were incubated (i) for 24 hours alone (control), (ii) with Z-VAD.fmk, (iii) with 30 μM gossypol plus Z-VAD.fmk, or (iv) with 30 μM gossypol for 24 hours, and then the cell viability was determined as described in panel A. (v) Percentage of cell death in CLL primary cells from 9 individual patients treated with Z-VAD.fmk and gossypol (C, untreated control; Z, Z-VAD.fmk; Z + G, Z-VAD.fmk plus gossypol; G, gossypol). (C) Effect of antioxidant (NAC) on gossypol (G)–induced cell death. CLL primary cells were incubated (i) for 24 hours alone (control), (ii) with NAC, (iii) with 30 μM gossypol plus NAC, or (iv) with 30 μM gossypol, and then the cell viability was determined as described in panel A. (v) Percentage of cell death in CLL primary cells from 6 individual patients treated with NAC plus gossypol (Con, untreated control).