Constitutively activated phosphatidylinositol-3 kinase (PI-3K) is involved in the defect of apoptosis in B-CLL: association with protein kinase Cδ by Ingo Ringshausen, Folker Schneller, Christian Bogner, Susanne Hipp, Justus Duyster, Christian Peschel, and Thomas Decker Blood Volume 100(10):3741-3748 November 15, 2002 ©2002 by American Society of Hematology

Effect of PI-3K inhibition on apoptosis in B-CLL cells Effect of PI-3K inhibition on apoptosis in B-CLL cells.B-CLL cells (1 × 106 cells/mL) were incubated in the absence or presence of LY294002 (10 μM) for 24 hours before the indicated tests were performed. Effect of PI-3K inhibition on apoptosis in B-CLL cells.B-CLL cells (1 × 106 cells/mL) were incubated in the absence or presence of LY294002 (10 μM) for 24 hours before the indicated tests were performed. (A) Annexin/PI staining revealed an increase in apoptotic cells (right panel) compared with medium control (left panel); 1 representative experiment of 24 is shown. (B) The proapoptotic effect was demonstrated in 24 of 24 different CLL samples. (C) A representative example of dose-response data as measured by annexin/PI staining is shown. As demonstrated in panel D, apoptosis was confirmed by measuring the mitochondrial membrane potential (Δψ); PI-3K inhibition resulted in an increase of Δψ-negative, apoptotic cells. The same experiment was repeated 9 times with similar results. Peripheral B cells of healthy donors (n = 5) were resistant to the proapoptotic effect of the PI-3K inhibitor (E). Ingo Ringshausen et al. Blood 2002;100:3741-3748 ©2002 by American Society of Hematology

PI-3K is constitutively activated in B-CLL cells PI-3K is constitutively activated in B-CLL cells.PI-3K activity was measured by incubation of anti-p85 antisera with PI and P32-γg-ATP. PI-3K is constitutively activated in B-CLL cells.PI-3K activity was measured by incubation of anti-p85 antisera with PI and P32-γg-ATP. P32-labeled enzymatic products of PI-3K were resolved by TLC using a silica gel, as described in “Materials and methods,” and visualized by autoradiography. Total cell lysate (200 μg) of freshly isolated B-CLL cells was used for immunoprecipitation. Lanes 1 to 7 show constitutive PI-3K activity of 7 different CLL samples. IL-3–stimulated (2 ng/mL) Ba/F3 cells were used for positive control (pc). Kinase reaction could be inhibited sufficiently by adding 300 nM wortmannin (Wn) to the reaction (lanes 8 and 10). Ingo Ringshausen et al. Blood 2002;100:3741-3748 ©2002 by American Society of Hematology

P38-and Erk-MAPK as well as p70S6 kinase are not involved in spontaneous apoptosis of B-CLL cells.Cells (1 × 106 cells/mL) were incubated with increasing amounts of SB203580, a specific inhibitor of p38-MAPK (A), PD98059, which inhibits Erk-MAPK pathway (B)... P38-and Erk-MAPK as well as p70S6 kinase are not involved in spontaneous apoptosis of B-CLL cells.Cells (1 × 106 cells/mL) were incubated with increasing amounts of SB203580, a specific inhibitor of p38-MAPK (A), PD98059, which inhibits Erk-MAPK pathway (B), or rapamycin, which blocks p70S6 kinase (C). Apoptosis was measured by annexin/PI staining after incubation for 24 hours. One representative result of 10 individual experiments is shown. Ingo Ringshausen et al. Blood 2002;100:3741-3748 ©2002 by American Society of Hematology

Antagonism of the proapoptotic effect of LY294002 by the pan-caspase inhibitor zvad.fmk and the caspase-3 inhibitor z-devd-fmk.B-CLL cells were incubated in medium alone or with 10 μM LY294002. Antagonism of the proapoptotic effect of LY294002 by the pan-caspase inhibitor zvad.fmk and the caspase-3 inhibitor z-devd-fmk.B-CLL cells were incubated in medium alone or with 10 μM LY294002. Cells were preincubated either with the broad-spectrum caspase inhibitor zvad.fmk (100 μM) or the caspase-3 inhibitor z-devd-fmk (200 μM) 30 minutes prior to adding LY294002 to the culture medium. Apoptosis was detected by annexin/PI staining after 24 hours. One representative experiment of 3 is shown. Ingo Ringshausen et al. Blood 2002;100:3741-3748 ©2002 by American Society of Hematology

Modulation of proapoptotic and antiapoptotic proteins in response to PI-3K inhibition.Representative immunoblot data show regulation of proapoptotic and antiapoptotic proteins. Modulation of proapoptotic and antiapoptotic proteins in response to PI-3K inhibition.Representative immunoblot data show regulation of proapoptotic and antiapoptotic proteins. B-CLL cells were cultured with or without 10 μM LY294002. Cells were harvested after 12 and 24 hours and whole cell lysates were prepared. A protein content of 40 μg was subjected to SDS-PAGE/immunoblot analysis by the use of specific antibodies for Bax, Bcl-2, XIAP, Mcl-1, and caspase-3. Ingo Ringshausen et al. Blood 2002;100:3741-3748 ©2002 by American Society of Hematology

PKB/Akt is expressed but not constitutively phosphorylated in B-CLL PKB/Akt is expressed but not constitutively phosphorylated in B-CLL.B-CLL cells were lysed immediately after separation from peripheral blood. PKB/Akt is expressed but not constitutively phosphorylated in B-CLL.B-CLL cells were lysed immediately after separation from peripheral blood. (A) Whole protein lysates (100 μg) from 7 different patients were analyzed by immunoblotting with the antibodies as indicated. NIH-3T3 cells treated with platelet-derived growth factor (PDGF) served as a positive control (pc); without PDGF as a negative control (nc). (B) Akt in vitro kinase assay was performed after immunoprecipitation of Akt from 7 different patients; GSK-3 fusion protein served as exogenous substrate for Akt. Kinase reaction was analyzed by immunoblotting with mAb specific for phospho-GSK-3 (Ser21/9). (C) To rule out in vitro phosphorylation of Akt, B-CLL cells were incubated with or without 10 μM LY294002 and lysed after 12 and 24 hours. Ingo Ringshausen et al. Blood 2002;100:3741-3748 ©2002 by American Society of Hematology

PKCδ is constitutively activated in B-CLL cells PKCδ is constitutively activated in B-CLL cells.(A) A sample of 300 μg total protein content of 8 different freshly isolated B-CLL cells was immunoprecipitated with an antibody against PKCδ and in vitro kinase assay was performed in the absence (lanes 1-8) ... PKCδ is constitutively activated in B-CLL cells.(A) A sample of 300 μg total protein content of 8 different freshly isolated B-CLL cells was immunoprecipitated with an antibody against PKCδ and in vitro kinase assay was performed in the absence (lanes 1-8) or presence (lane 9) of Rottlerin using histone H1 as an exogenous substrate. Proteins were analyzed by SDS-PAGE and a phosphorylated form of histone H1 was detected by autoradiography. (B) A sample of 100 μg total protein content of 7 different B-CLL cells was analyzed by immunoblot with an antiphosphothreonine 505 antibody specific for PKCδ. Samples from panel A were different from those shown in panel B. To investigate the influence of PI-3K activity on PKCδ, cells were incubated with 10 μM LY294002 and lysed after the indicated time period. Then PKCδ was immunoprecipitated with a specific antibody. (C) Tyrosine phosphorylation was detected by using an antiphosphotyrosine 4G10 antibody. (D) PKCδ kinase activity was measured by a in vitro kinase assay as described in (A). Ingo Ringshausen et al. Blood 2002;100:3741-3748 ©2002 by American Society of Hematology

Influence of PKCδ inhibition on viability of B-CLL cells Influence of PKCδ inhibition on viability of B-CLL cells.Cells (1 × 106 cells/mL) were incubated with or without Rottlerin (5 μM), which specifically inhibits PKCδ. Influence of PKCδ inhibition on viability of B-CLL cells.Cells (1 × 106 cells/mL) were incubated with or without Rottlerin (5 μM), which specifically inhibits PKCδ. After 24 hours apoptosis was measured by annexin/PI staining. Panel A shows the results of 12 different experiments. The proapototic effect was compared with peripheral B cells of 5 healthy donors (B). Ingo Ringshausen et al. Blood 2002;100:3741-3748 ©2002 by American Society of Hematology