B-cell receptor signaling in chronic lymphocytic leukemia cells is regulated by overexpressed active protein kinase CβII

Abstract

One approach to understanding CLL is to investigate the nature of intracellular signals responsible for the development and prolonged survival of the malignant cells. 2 In this regard, signals generated by B-cell receptor (BCR) engagement are known to play an important role. 1 A key mediator of BCR-induced signaling is protein kinase C (PKC). [3][4][5][6][7][8][9] In CLL cells, this class of enzymes has been identified as a possible target of therapeutic intervention based on in vitro studies demonstrating that inhibition of these enzymes induces apoptosis. [10][11][12][13][14][15] Given the role of BCR signals in the survival and clonal expansion of CLL cells and the role of PKC(s) in the signaling pathways induced by BCR engagement, it follows that PKC(s) may play an important role in the BCR-induced survival of CLL cells.The PKC family is divided into 3 subgroups: the classical, which includes PKC␣, ␤I, ␤II, and ␥; the novel, which includes PKC␦, ⑀, , and ; and the atypical, which includes PKC and . These enzymes are activated by the presence of Ca 2ϩ , diacylglycerol, or other activating factors, 16 and they function in an array of cellular processes that can be specific for a particular cell type. In B cells, PKC, 5 PKC␤, 3,4,7,8 PKC␦,6 and PKC⑀ 9 play important roles in regulating signals generated by the BCR. With respect to CLL, active PKC␦ is thought to maintain cell survival downstream of phosphoinositol 3Ј-kinase. 15 Despite the potential of PKCs as therapeutic targets in CLL, [10][11][12][13][14][15] little is known about the relative levels and activities of the different isoforms known to be expressed within the malignant cells of this disease.In the present study, we show that PKC␤II is overexpressed in CLL cells and that the activity of this enzyme inversely correlates with CLL cell response to BCR engagement. Therefore, by regulating BCR signals important for malignant cell survival, PKC␤II may be a key factor in CLL progression. Materials and methods MaterialsMouse monoclonal and rabbit polyclonal anti-PKC␤II, monoclonal anti-PKC␤I, -PLC␥2, and -CD40 antibodies, rabbit anti-PKC␣, -PKC␦, -PKC, Mcl-1 and procaspase-8, and horseradish peroxidase-conjugated antimouse and anti-rabbit immunoglobulin antibodies were purchased from Santa Cruz Biotechnology (Insight Biotechnology, Middlesex, United Kingdom). Monoclonal anti-PKC␦ and anti-PKC⑀ antibodies were purchased from BD Biosciences (Oxford, United Kingdom). F(ab 2 )Ј fragments of goat anti-human IgM were purchased from Jackson ImmunoResearch Laboratories (Stratech, Soham, United Kingdom). Mouse anti-pS 180 -Bruton tyrosine kinase (Btk) and rabbit anti-Btk and anti-pY 759 -PLC␥2 antibodies were purchased from Cell Signaling Technology (New England Biolabs, Hitchin, Herts, United Kingdom). Purified recombinant PKC␣, PKC␤I, PKC␤II, PKC␦, PKC⑀, and PKC proteins and Ro32-0432 were purchased from Merck Biosciences (Nottingham, United Kingdom). Purified recombinant PKC and PKC proteins and mouse anti-ZAP-70 antibody were purchased from Upstate (Milton Ke...

LCK Is an Important Mediator of B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia Cells

“…Cell viability was assessed by flow cytometry using DiOC6 to measure mitochondrial integrity and PI incorporation (as a measure of dead cells) according to established protocol (29). Viable cell count was determined by taking an aliquot of cell suspension and measuring the number of DiOC6 bright /PI dim events (live cells) counted during a fixed time setting of 30 seconds where the flow cytometer (FACSCalibur) was set to medium flow.…”

“…Proteins within whole-cell lysates or within immunoprecipitates were separated by SDS-PAGE and then electroblotted on to Immobilon membranes. These membranes were then incubated with primary and secondary antibodies followed by visualization using enhanced chemiluminescence as we have previously described (29). Lck expression in CLL cells was quantitated using a modified procedure described by Abrams and colleagues (29).…”

“…The procedures used here are described in Abrams and colleagues (29). Briefly, whole-cell lysates were prepared by lysing CLL cell pellets with SDS-PAGE sample buffer, sonicating the lysate to shear DNA followed by incubation at 95 C for 5 minutes.…”

“…These membranes were then incubated with primary and secondary antibodies followed by visualization using enhanced chemiluminescence as we have previously described (29). Lck expression in CLL cells was quantitated using a modified procedure described by Abrams and colleagues (29). Briefly, purified CLL cells were lysed with clear SDS lysis buffer (50 mmol/L Tris, pH 6.8, 5 mmol/L EDTA, 1% SDS, and 10% glycerol), sonicated to disrupt released DNA, and incubated for 10 minutes at 95 C. Protein determination was carried out on the lysates, and 10 mg total protein from each lysate was subjected to SDS-PAGE and Western blotting with anti-LCK antibodies.…”

Protein kinase C (PKC) as a drug target in chronic lymphocytic leukemia

“…Recent studies have established that several PKC isoforms are overexpressed in CLL and are essential for CLL cell survival (21,52,54,55,77). Clinical trials using the PKC inhibitors PKC412 and enzastaurin have provided promising results with low side effects in CLL treatment (78)(79)(80).…”