Vascular endothelial growth factor stimulates protein kinase CβII expression in chronic lymphocytic leukemia cells by Simon T. Abrams, Benjamin R. B. Brown,

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Vascular endothelial growth factor stimulates protein kinase CβII expression in chronic lymphocytic leukemia cells by Simon T. Abrams, Benjamin R. B. Brown, Mirko Zuzel, and Joseph R. Slupsky Blood Volume 115(22):4447-4454 June 3, 2010 ©2010 by American Society of Hematology

PKCβII gene transcription and protein kinase activity in CLL cells decrease after 24-hour culture. PKCβII gene transcription and protein kinase activity in CLL cells decrease after 24-hour culture. (A) Quantitative RT-PCR analysis of PKCβ mRNA levels in CLL cells showing the effect of in vitro culture in the presence and absence of 100nM LY379196 on PKCβ expression. A 24-hour incubation of CLL cells resulted in a significant drop in PKCβ mRNA levels (P = .002, n = 9). Results are reported as a percentage of PKCβ mRNA levels in CLL cells at T = 0. (B) Western blot analysis of Btk in anti–pS180-Btk immunoprecipitates from CLL cells (top panel), and in whole-cell lysates (bottom panel). Where CLL cells were incubated with LY379196, a concentration of 100nM was used. (C) Quantitative RT-PCR analysis of PKCβ mRNA levels in CLL cells exposed to 200nM mithramycin for 24 hours. Treatment of CLL cells with mithramycin resulted in a significant reduction in PKCβ mRNA levels compared with untreated (UT) cells (P = .013, n = 5). PKCβ mRNA amounts are reported in femtograms (fg) of PKCβ plasmid DNA equivalents. Simon T. Abrams et al. Blood 2010;115:4447-4454 ©2010 by American Society of Hematology

VEGF stimulates PKCβ mRNA expression in CLL cells. VEGF stimulates PKCβ mRNA expression in CLL cells. Quantitative RT-PCR analysis of PKCβ mRNA levels in CLL cells reported as a percentage of PKCβ mRNA levels in control (T = 0) cells. (A) The effect of 24-hour treatment of CLL cells with 100 ng/mL VEGF (n = 11) or with 100 ng/mL bFGF (n = 10) on PKCβ mRNA levels. (B) Effect of 100nM LY379196 on VEGF-induced PKCβ mRNA expression in CLL cells (n = 4). (C) Effect of 10μM SU5416 on VEGF-induced PKCβ mRNA expression in CLL cells (n = 4). (B-C) Error bars represent SD. Simon T. Abrams et al. Blood 2010;115:4447-4454 ©2010 by American Society of Hematology

VEGF stimulates PKCβII activity in CLL cells. VEGF stimulates PKCβII activity in CLL cells. (A) Western blot analysis of Btk in whole-cell lysates and in anti–pS180-Btk immunoprecipitates from CLL cells treated with 100 ng/mL VEGF in the presence and absence of 100nM LY379196. This experiment is representative of 3 using different CLL cases. (B) Effect of 100 ng/mL VEGF on BCR-induced intracellular Ca2+ release in CLL cells. Fura-2-loaded CLL cells were stimulated with 20 μg/mL anti-IgM antibody (BCR-XL) in the presence and absence of VEGF. LY379196 (100nM) was used to reverse the effects of VEGF. (C) Data presented in panel B, highlighting that the incubation of CLL cells with VEGF induced significant inhibition of intracellular Ca2+ release (P = .05, n = 6). Simon T. Abrams et al. Blood 2010;115:4447-4454 ©2010 by American Society of Hematology

VEGF does not stimulate PKCβ gene expression in normal B cells. VEGF does not stimulate PKCβ gene expression in normal B cells. Quantitative RT-PCR analysis of PKCβ mRNA levels in CLL (n = 6) and normal peripheral B cells (n = 3) incubated in the presence and absence of 100 ng/mL VEGF for 24 hours and compared with control uncultured cells. The PKCβ mRNA levels are reported relative to β-actin mRNA levels in the same cell in arbitrary units. (Inset) RT-PCR analysis of VEGF-R2 expression in normal B cells and in an endothelial cell line (EAhy926; top panel) and Western blot analysis of VEGF-R2 protein expression in CLL- and normal B-cell lysates (equal amounts of protein were loaded onto the gel). Simon T. Abrams et al. Blood 2010;115:4447-4454 ©2010 by American Society of Hematology

Role of overexpressed PKCβII in VEGF-stimulated induction of PKCβ mRNA expression. Role of overexpressed PKCβII in VEGF-stimulated induction of PKCβ mRNA expression. (A) Western blot analysis of PKCβII and GFP expression in an endothelial cell line (EAhy926) transfected to overexpress GFP-PKCβII or GFP. (B) Effect of 100 ng/mL VEGF on PKCβ promoter-driven luciferase activity in GFP-PKCβII- and GFP-transfected endothelial cells (n = 4). Simon T. Abrams et al. Blood 2010;115:4447-4454 ©2010 by American Society of Hematology