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...

Bi-directional communication with the microenvironment is essential for homing and survival of cancer cells with implications for disease biology and behaviour. In chronic lymphocytic leukemia (CLL), the role of the microenvironment on malignant cell behaviour is well described. However, how CLL cells engage and recruit nurturing cells is poorly characterised. Here we demonstrate that CLL cells secrete exosomes that are nanovesicles originating from the fusion of multivesicular bodies with the plasma membrane, to shuttle proteins, lipids, microRNAs (miR) and mRNAs to recipient cells. We characterise and confirm the size (50–100 nm) and identity of the CLL-derived exosomes by Electron microscopy (EM), Atomic force microscopy (AFM), flow cytometry and western blotting using both exosome- and CLL-specific markers. Incubation of CLL-exosomes, derived either from cell culture supernatants or from patient plasma, with human stromal cells shows that they are readily taken up into endosomes, and induce expression of genes such as c-fos and ATM as well as enhance proliferation of recipient HS-5 cells. Furthermore, we show that CLL exosomes encapsulate abundant small RNAs and are enriched in certain miRs and specifically hsa-miR-202-3p. We suggest that such specific packaging of miR-202-3p into exosomes results in enhanced expression of ‘suppressor of fused’ (Sufu), a Hedgehog (Hh) signalling intermediate, in the parental CLL cells. Thus, our data show that CLL cells secrete exosomes that alter the transcriptome and behaviour of recipient cells. Such communication with microenvironment is likely to have an important role in CLL disease biology.

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