by Beatriz Bellosillo, Mireia Dalmau, Dolors Colomer, and Joan Gil Involvement of CED-3/ICE Proteases in the Apoptosis of B-Chronic Lymphocytic Leukemia Cells by Beatriz Bellosillo, Mireia Dalmau, Dolors Colomer, and Joan Gil Blood Volume 89(9):3378-3384 May 1, 1997 ©1997 by American Society of Hematology

PARP cleavage in B-CLL cells. PARP cleavage in B-CLL cells. (A) Time course of dexamethasone-induced PARP cleavage. B-CLL lymphocytes were incubated with or without 10 μmol/L dexamethasone for the indicated times. (B) Dose response of PARP cleavage. Cells were incubated for 48 hours with various concentrations of dexamethasone as indicated. PARP cleavage was analyzed by Western blot as described in Materials and Methods. The position of the native PARP (116 kD) and the proteolytic fragment (85 kD) is indicated. Beatriz Bellosillo et al. Blood 1997;89:3378-3384 ©1997 by American Society of Hematology

Correlation between cell viability and PARP degradation. Correlation between cell viability and PARP degradation. The values of cell viability for cells incubated without (○) or with 10 μmol/L dexamethasone (•) are shown in Table 1. PARP degradation was quantified by densitometric scanning of the immunoblots. Numbers beside the symbols indicate patient numbers. Beatriz Bellosillo et al. Blood 1997;89:3378-3384 ©1997 by American Society of Hematology

Inhibition of PARP proteolysis by TPA and IL-4. Inhibition of PARP proteolysis by TPA and IL-4. (A) Inhibition of dexamethasone-induced PARP proteolysis by TPA. B-CLL lymphocytes from three patients were incubated with 100 nmol/L TPA in the presence or absence of 10 μmol/L dexamethasone for 48 hours. (B) Inhibition of spontaneous PARP proteolysis. Cells from patient 6 were incubated for 48 hours either in the absence of any factor (C) or with 100 nmol/L TPA, or IL-4 (10 ng/mL). (C) Inhibition of fludarabine-induced PARP cleavage. Cells from patient 5 were incubated with the indicated factors for 24 hours. The concentrations used were 100 nmol/L TPA and 5 μg/mL fludarabine. Western blot of PARP was performed with protein extracts from these cells as previously described. Beatriz Bellosillo et al. Blood 1997;89:3378-3384 ©1997 by American Society of Hematology

Effect of the CED-3/ICE–like protease inhibitor Z-VAD Effect of the CED-3/ICE–like protease inhibitor Z-VAD.fmk on PARP proteolysis in B-CLL cells. Effect of the CED-3/ICE–like protease inhibitor Z-VAD.fmk on PARP proteolysis in B-CLL cells. (A) Dose response of the inhibitory effect of Z-VAD.fmk on PARP proteolysis. Cells from patient 6 were incubated without dexamethasone (○) and cells from patient 9 were incubated with 10 μmol/L dexamethasone (•), in the presence of various concentrations of Z-VAD.fmk for 48 hours. Western blots of PARP were performed as described in Materials and Methods and the inhibition of PARP cleavage was quantified by densitometric scanning of the immunoblots. (B) B lymphocytes from four B-CLL patients were incubated for 48 hours with 10 μmol/L dexamethasone in the presence or absence of 200 μmol/L Z-VAD.fmk. Z-VAD.fmk was added 1 hour before dexamethasone administration. Cells were lysed and analyzed by Western blot as described in Materials and Methods. Beatriz Bellosillo et al. Blood 1997;89:3378-3384 ©1997 by American Society of Hematology

Effect of the CED-3/ICE–like protease inhibitor Z-VAD Effect of the CED-3/ICE–like protease inhibitor Z-VAD.fmk on DNA fragmentation in B-CLL cells. Effect of the CED-3/ICE–like protease inhibitor Z-VAD.fmk on DNA fragmentation in B-CLL cells. Cells from patients 5 and 9 were incubated for 24 hours with 10 μmol/L dexamethasone in the presence or absence of 200 μmol/L Z-VAD.fmk. Z-VAD.fmk was added 1 hour before dexamethasone administration. DNA was extracted and subjected to agarose gel electrophoresis as described in Materials and Methods. Similar results were obtained with cells from patients 7 and 8. Beatriz Bellosillo et al. Blood 1997;89:3378-3384 ©1997 by American Society of Hematology

Effect of Z-VAD.fmk on the viability of B-CLL cells. Effect of Z-VAD.fmk on the viability of B-CLL cells. Cells from five patients were seeded in 96-microwell plates and incubated for 48 hours with the indicated factors. The concentrations used were 10 μmol/L dexamethasone and 200 μmol/L Z-VAD.fmk. Z-VAD.fmk was added 1 hour before dexamethasone administration. Cytotoxicity was analyzed by the MTT method as described in Materials and Methods. Data are shown as the mean value ± SD of two (patients 6, 8, and 9) or four (patients 5 and 7) independent experiments. Statistical significance of differences between treatments without or with Z-VAD.fmk was assayed by analysis of variance (ANOVA) (Fisher's Protected Least Significant Difference [PLSD]): * = P < .12, ** = P < .05, *** = P < .01. Beatriz Bellosillo et al. Blood 1997;89:3378-3384 ©1997 by American Society of Hematology