Lymphocytes from chronic lymphocytic leukaemia undergo ABL1-linked amoeboid motility and homotypic interaction as an early adaptive change to ex vivo culture
© Hutchinson et al.; licensee BioMed Central Ltd. 2014
Received: 19 February 2014
Accepted: 19 February 2014
Published: 11 March 2014
Those stimuli that together promote the survival, differentiation and proliferation of the abnormal B-lymphocytes of chronic lymphocytic leukaemia (CLL) are encountered within tissues, where together they form the growth-supporting microenvironment. Different tissue-culture systems promote the survival of the neoplastic lymphocytes from CLL, partly replicating the in vivo tissue environment of the disorder. In the present study, we focussed on the initial adaptive changes to the tissue culture environment focussing particularly on migratory behaviour and cellular interactions.
A high-density CLL culture system was employed to test CLL cell-responses using a range of microscopic techniques and flow cytometric analyses, supported by mathematical measures of cell shape-change and by biochemical techniques. The study focussed on the evaluation of changes to the F-actin cytoskeleton and cell behaviour and on ABL1 signalling processes.
We showed that the earliest functional response by the neoplastic lymphocytes was a rapid shape-change caused through rearrangement of the F-actin cytoskeleton that resulted in amoeboid motility and promoted frequent homotypic interaction between cells. This initial response was functionally distinct from the elongated motility that was induced by chemokine stimulation, and which also characterised heterotypic interactions between CLL lymphocytes and accessory cells at later culture periods. ABL1 is highly expressed in CLL lymphocytes and supports their survival, it is also recognised however to have a major role in the control of the F-actin cytoskeleton. We found that the cytoplasmic fraction of ABL1 became co-localised with F-actin structures of the CLL lymphocytes and that the ABL1 substrate CRKL became phosphorylated during initial shape-change. The ABL-inhibitor imatinib mesylate prevented amoeboid movement and markedly reduced homotypic interactions, causing cells to acquire a globular shape to rearrange F-actin to a microvillus form that closely resembled that of CLL cells isolated directly from circulation.
We suggest that ABL1-induced amoeboid motility and homotypic interaction represent a distinctive early response to the tissue environment by CLL lymphocytes. This response is separate from that induced by chemokine or during heterotypic cell-contact, and may play a role in the initial entry and interactions of CLL lymphocytes in tissues.
KeywordsCLL ABL1 Actin Cytoskeleton Motility Homotypic
Those stimuli that promote the survival, differentiation and proliferation of the abnormal B-lymphocytes of chronic lymphocytic leukaemia (CLL) are encountered within tissues, where together they form the growth-supporting microenvironment (reviewed in ). The soluble and structural elements of the CLL microenvironment include accessory cells , chemokines  and perhaps antigenic stimuli [4, 5], as well as contact with other CLL cells , and cases with significant infiltration of tissues have adverse clinical outcome . The effective interaction between CLL lymphocytes and their microenvironment therefore depends on directed migration and appropriate cellular contact [3, 8, 9].
When cultured ex vivo at standard cell density, pure preparations of neoplastic CLL-lymphocytes from CLL patients enter apoptotic cell death within 24 hours . Apoptosis is however prevented by the additional presence of relevant accessory cells [3, 11], or by culture of the CLL lymphocytes together at high cell density with blood mononuclear cells . The enhanced cell survival within these cultures is believed to derive, at least in part, from direct interaction between cells . Such interactions resemble the behaviour of normal B-lymphocytes at particular developmental stages in vivo, in which migration through tissues involves repeated brief homotypic and heterotypic cell contacts, as well as more sustained intercellular interactions . The processes that control movement, interaction and adhesion for cells of the immune system require the dynamic and coordinated rearrangements of the cell cytoskeleton mediated through specific filamentous actin (F-actin) structures. In this regard, there is an emerging recognition that the Abelson kinase (ABL1) has a central role in the organisation of F-actin structures that promote particular aspects of cell motility and interaction in cells of the immune system . ABL1 is highly expressed in the lymphocytes of CLL, and the inhibitor of ABL-family protein tyrosine kinases imatinib mesylate (imatinib) has been shown to induce the apoptosis of neoplastic CLL B-lymphocytes in vitro . In CLL, imatinib targets are largely confined to the ABL kinases . We postulated therefore that imatinib might alter F-actin mediated motility or adhesion of CLL lymphocytes.
Recent reports that a redistribution of CLL lymphocytes between blood and tissues accompanies the therapeutic effect of signal-inhibitor agents as part of their clinical effect , have further highlighted the importance of understanding how tissue migration and adhesion in CLL is controlled. In the present paper we describe very specific rearrangements of F-actin that are induced in ex vivo when CLL lymphocytes are maintained in high-density culture. We show that these rearrangements mediate frequent, but transient, homotypic interactions between the neoplastic CLL B-lymphocytes, and that the morphological appearances of the CLL lymphocytes in high-density culture resemble those of CLL lymphocytes within tissues, and are consistent with the interactions between cells seen in tissues . The F-actin rearrangements we describe are shown to be functionally distinct from those induced by chemokine, and from those that drive heterotypic interactions between CLL cells and accessory cells. We suggest that ABL-dependent adhesion and migration represents a separate pathway of tissue interaction for CLL lymphocytes that may be important in the initial adaptation to the tissue environment.
High cell-density culture of CLL B-lymphocytes promotes early shape-change and the formation of transient homotypic interactions
The shape-change of cultured CLL lymphocytes reflects dynamic F-actin rearrangement supporting amoeboid motility and interactive filopodia formation
ABL1 supports the amoeboid motility and homotypic interactions of CLL lymphocytes, but is not required for the separate elongated motility directed by chemokines
In vivo, CLL lymphocytes adapt to the tissue environment, undergoing functional and phenotypic changes that support their migratory and interactive behaviour [8, 11, 24]. It is probable that similar principles underlie the response of CLL lymphocytes to the ex vivo tissue culture environment, and culture systems have successfully been used to model those responses . We were interested particularly in the cytoskeletal changes of the cells within tissues. In the present study we have shown that when fixed directly in whole blood the neoplastic B-lymphocytes of CLL have a uniform globular cell-shape with numerous cell-surface microvilli resembling the features of resting lymphocytes . However, soon after entering high-density culture the cells undergo a rearrangement of their F-actin cytoskeleton that promotes their dynamic motility and homotypic interactions. Using sections of CLL-involved tissues we observed that similar morphological appearances typify CLL lymphocytes within tissues in vivo. While, shape change occurring within the tissue environment in vivo is likely also to reflect the effects of heterotypic adhesion and chemokine stimulus, the presence of cellular projections extended between CLL cells in tissues was demonstrated. We postulated therefore that the behaviour we observed in vitro may in part reflect similar adaptive functional responses occurring in vivo.
The mononuclear cell high-density preparations of CLL lymphocytes employed in this study have previously been reported to replicate features of the in vivo tissue-environment, and share characteristics with other culture systems used in CLL . In the present study, we observed that a series of dynamic changes affecting cytoskeleton and cell behaviour developed as cells became established in culture. Detailed analysis showed that these morphological forms in fact represented a process of dynamic amoeboid shape-change and “random walk” motility, during which individual cells underwent periodic transition between two morphological forms, a globular cell population and an irregular polarised form in which F-actin polymerisation was increased: the two of apparently different forms in fact representing a single population in dynamic equilibrium. Additionally, during this behaviour the cells extended “exploratory” filopodial processes that mediated specific homotypic contact and which supported the formation of cell-groups. These homotypic cell groups were transient, but comprised cells that had extensive filopodial interactions accompanied by distinctive accumulation of F-actin and integrin receptors at points of contact. When Rho kinase was inhibited to prevent cytoplasmic process retraction , extensive interacting networks of extended filopodia were seen to connect the cells, consistent with the involvement of Rho kinase in maintaining the observed structures.
Chemokine induced motility by CLL lymphocytes has been reported and characterised in detail by others, and has been shown to be important during the formation of heterotypic cell interactions, particularly those between CLL-lymphocytes and NLCs . The changes that we observed however were induced at early culture points prior to NLC formation or demonstrable chemokine effect. Furthermore, the morphological and functional characteristics of the culture-associated movement differed from those induced by cytokine. The different cell forms and behavioural responses we have reported are recognised in other lymphocyte types, where they are reported to have a different signal basis and specific functional significance. Elongated-type motility is a directed response that is characteristically induced by chemokine and integrin adhesion has been linked to Rac1-mediated signalling processes associated with directed movement in two dimensions. By contrast, amoeboid motility is a Rho/CDC42 controlled form of cell movement characteristically occurring within three-dimension lattices (reviewed in ), and frequently underpins an “exploratory phase” of transient homotypic interactions that accompany early cell activation in tissues. This pattern of movement has been described for normal T lymphocytes and B lymphocytes both in vivo and in vitro [12, 28], and in T lymphocytes the brief homotypic contacts are recognised to mediate cell activation as a prelude to antigen-mediated cell events .
Modifying the motility and interactions between CLL lymphocytes and the supportive microenvironment is an attractive therapeutic option in CLL. We therefore explored the signalling basis of the behaviour we observed. In this regard, ABL1 is highly expressed and active in CLL lymphocytes. ABL family kinases bind to F-actin, and are increasingly recognised to play a central role in the control of the F-actin cytoskeletal structures [13, 30]. ABL-mediated intracellular signalling affects cell migration in many cell types , and specifically mediates interactions of T cells within the immune environment . In the present study the neoplastic cells of CLL were confirmed to express ABL1 as described , and the cytoplasmic fraction of ABL1 protein was shown to become co-localised with F-actin within the large projections of motile cells. Inhibition of ABL1 using imatinib caused a rapid dose-dependent inhibition of CRKL-phosphorylation. In parallel with the inhibition of CRKL-phosphorylation the neoplastic cells became globular, with retraction of their extended cytoplasmic projections and a reappearance of their surface microvilli, inducing appearances similar to those of cells fixed in whole blood. Imatinib treated CLL lymphocytes were prevented from undergoing spontaneous motility and filopodial contact between the CLL cells was greatly reduced. Consistent with this a separate signal basis for elongated-type motility, imatinib had significantly less effect on chemokine-induced behaviour, such that cell polarisation and elongation continued to occur even in the presence of the drug and significant chemotaxis was observed at imatinib concentrations that significantly inhibited CRKL phosphorylation.
Several threads link our own findings to pathways and behaviours that have recognised importance to CLL biology and to CLL cytoskeletal-function. ABL-family kinases interact directly with the F-actin control complex that includes WASP/WAVE2 proteins , and through these proteins to activation of actin-nucleation proteins (ARP1 and ARP2) that mediate cytoskeletal reorganisation. The microvillus F-actin structure of lymphocytes in circulation is independent of WASP/WAVE2 and therefore would not be affected by ABL1 inhibition, and may be regarded as a “default” organisation of lymphocyte cytoskeleton . However, ABL1 activation is central to the formation of structures that include lamellipodia and filopodia [32, 33]. The reversion of CLL cytoskeleton to microvillus structure following imatinib-treatment of CLL lymphocytes therefore, is entirely consistent with an importance for ABL1-dependent signalling in the cells. In this regard, ABL-family kinases also interact with several other pathways that have emerging importance in CLL biology. The LYN kinase substrate protein HS1 that has a major role in cytoskeletal activation in CLL is recognised to act together with ABL1/CRKL and ZAP70 to induce cytoskeletal activation in lymphoid cells . This study reinforces the complexity of the motile behaviour and cellular interactions made by CLL lymphocytes identifying separate motile and interactive behaviours linked either to spontaneous movement or to chemokine. ABL1 signals may therefore be considered as one part of a system of signal processes and cellular interactions that act together to drive the pathophysiology of the disorder –.
Interest in CLL motility and migration has recently increased following the recognition that the therapeutic effect of signal inhibitors including BTK inhibitors is accompanied by a compartment shift of cells between tissue and blood, suggesting that effects on cell interactions or migration may contribute to the therapeutic benefit of these agents . However, despite ABL1 inhibition having significant activity against CLL in vitro affecting a range of cell behaviours [14, 37, 38], single agent inhibition of ABL1 by imatinib or dasatinib has not emerged as having major clinical activity in vivo  The present study however emphasises however, that the interaction between CLL cells and their tissue microenvironment is complex, and that ABL1 may form part of a coordinated behaviour pattern that together with other pathways promotes the migration and tissue interactions of the neoplastic cells. The findings of in vitro studies may not be directly applicable the more complex microenvironment of the cells in vivo, and the functional significance of our findings to the pathobiology of CLL remains to be established in patients. However, awareness of how these different pathways contribute to the microenvironment interactions in CLL is therefore important, and should be considered as coordinated strategies designed to disrupt environment interactions are developed.
The present study demonstrates for the first time that activation of ABL1-linked signal pathways is an early response by CLL lymphocytes within the cell culture environment, that is associated with amoeboid-pattern migration and with frequent brief homotypic interactions between the neoplastic cells. This behaviour is distinct from the responses of CLL lymphocytes to chemokine, and from those which occur during heterotypic interactions. We suggest that ABL1 mediated migration and interactions may be an early adaptive response by CLL cells leaving the circulation, and may resemble similar behaviour by T-lymphocytes where such interactions promotes their activation, and enhance their subsequent response to antigen or other stimuli. Awareness of the different pathways that together coordinate the motility and interaction in CLL within the tissue microenvironment is highly relevant to the development of new treatment strategies, and targeting ABL1 may have a role as part of such approaches.
Cells and preparation
Detailed evaluation employed samples from 8 patients, confirmatory study of morphology and of further biochemical aspects used samples from 10 additional patients. Patient samples were collected with informed consent with approval of the local Research Ethics Committee (reference 10/H1017/73). All samples had typical clinical presentation and immunophenotype with no adverse cytogenetic features, and comprised >95% CLL B-lymphocytes as determined by flow cytometry. Specific samples differing in the presence or absence of somatic mutation of IgVH genes were kindly provided from the Leukaemia Lymphoma Research -Cell Bank Facility at the Royal Liverpool University Hospital. Tissues from diagnostic use were employed to examine cell interactions within tissue sections. Mononuclear cells were isolated by Ficoll-Paque gradient centrifugation (GE Healthcare, Buckinghamshire, UK) and were used freshly or were cryopreserved until use in 90% fetal bovine serum, 10% DMSO cells.
Cells were maintained in culture at 1 × 107/ml in 24 well plates unless otherwise indicated using RPMI medium with L-glutamine (Invitrogen, Paisley, UK) and 10% FBS supplemented with penicillin and streptomycin. Where adherent cells were required, the cells were washed and cells were induced to adhere directly to fibronectin (FN)-coated glass coverslips (40 μg/ml overnight) within the 24 well plates in serum-free conditions for the duration of the experiment. Chemotaxis experiments employed filter migration plates with pore size 5 μm (Transwell Plates, Corning Life Sciences, Amsterdam, The Netherlands) initial cell density in the upper chamber was 5 × 106/ml. Cells migrating into the bottom plate were counted after the indicated time. For all culture assays, reagents and drugs were added directly to culture medium at the indicated final concentrations.
Antibodies and biochemicals
General biochemicals and reagents were obtained from Sigma Aldridge (Sigma Aldridge, Poole, UK) unless otherwise specified. Fluorochrome-conjugated antibodies recognising the following antigens were employed: CD19, CD5, CD23, CD14, CD3, CD4 all from (BD Pharmingen, Oxford, UK). Unconjugated antibodies recognised: CD29 (clone P5D2 Abcam, Cambridge, UK), CRKL (ab32018 Abcam), P-CRKL (Y207) ( #3181 Cell Signaling Technology, New England Biolabs, Hitchin, UK), ABL (ABL clone 8E9 Abcam), ARG (181B11 Abcam). Secondary antibodies used for immunofluorescence were (Goat anti-mouse FITC Ab5999 and goat anti-mouse tr ab6003 Abcam). Phalloidin probes for F-actin were nitrobenzoxadiazole (NBD)-pallacidin (Invitrogen) and Texas Red phalloidin (Invitrogen). Other agents were CXCL12 (Peprotech EC Ltd, London, UK); Y27632 (Tocris Bioscience, Bristol, UK); and imatinib (LKT Laboratories, Alexis Corporation, Lausen, Switzerland). Annexin V FITC Staining kit (BD Pharmingen) was used with manufacturers protocols to determine apoptotic cell death. Immunoperoxidase staining used standard reagents and protocols (DAKO UK Ltd, Ely, UK).
Slide imaging and slide immunofluorescence
Live cell observation employed enclosed, temperature and CO2 controlled environment. Cells were taken from high density culture and added to glass-bottomed 24 well culture plates for the period of the experiment only. Cell density was reduced to allow optimal viewing within optimal areas. Established shape transition was not reversed during the culture period allowing cells to be observed in detail. Image capture as single image or time-lapse series employed a dLeica DMIRE2 using × 63 oil lens (Leica Microsystems, Milton Keynes, UK) image capture employed Image-Proplus (Media Cybernetics Inc., Bethesda, MD) with bright field or interference settings, image series were analysed using ImageJ (public domain open source software). General viewing of fixed-stained cells used bright field or immunofluorescence using an Nikon Eclipse 80i microscope with camera Hamamatsu C4742-95 camera or Nikon DSFi1 equipped with NIS-Elements BR 2.30 software/image analysis modules (supplied by Nikon, Nikon Instruments Europe, Amstelveen, The Netherlands). Plane images of brightfield sections used a Zeiss Axio Imager M.1 with AxioCam and Plan-apochromatic 63× 1.4 oil lens and AxioVision software 4.8.1 (Karl Zeiss Ltd, Herts, UK). Confocal microscopy employed a Zeiss LSM510META Confocal microscope with × 100 (1.4NA0) lens (Zeiss LSM 501META/LSM software, Karl Zeiss Ltd). Cells from adherence culture were fixed for 10 min in 4% w/v paraformaldehyde then washed in PBS, coverslips were then extracted for individual staining. Image analysis samples were stained using Rose Bengal 1% in PBS. Cell shape measures used approaches previously described , and were made using NIS-Elements BR 2.30 (Nikon). Optimal shape measures to discriminate shape of adherent cells were tested and cell area and cell elongation provided best discrimination (and was comparable to aspect ratio intensity measured by flow cytometry described below). Measures were exported for further analysis (SPSS/Microsoft Excel). Samples for immunostaining or F-actin probes were permeablised in 0.2% v/v Triton-X for 5 min before blocking in PBS/1%BSA (w/v). Primary antibodies were diluted in buffer and titrated to optimal concentration and used alone or in combination as indicated followed by appropriate secondary antibodies. All slides were mounted in Prolong Gold Antifade reagent with DAPI (4′,6-diamidino-2-phenylindole)(Invitrogen) for examination. Additional image processing employed ImageJ/JAVA software (ImageJ FIJI v1.48 k NIH, US) using Colocalization Plugin for pixel co-localisation analysis.
Standard flow cytometery used a FACSCanto II with BD FACSDiva Software using standard protocols and reagents (BD Biosciences). Morphological analysis by flow cytometry employed the ImageStream Imaging Flow Cytometer (Amnis, Seattle, WA) using simultaneous imaging facility and analysis by IDEAS v4 software (Amnis). To allow determination of shape change live cells were simultaneously fixed and stained using NBD phallacidin. Different shape parameters were tested for their capability to discriminate cell populations. Aspect ratio intensity (a measure that combines weighted actin intensity distribution and elongated cell shape) provided the most effective discrimination, between cell populations. Parameters were exported for detailed analysis using spreadsheet software (Microsoft Excel).
Cell lysates were prepared from washed cultured cells using RIPA buffer and inhibitors (Halt protease and phosphatase Thermo scientific/Pierce, Cramlington, UK). Samples (5 μg) were separated using SDS-PAGE and 10% gels. The separated proteins were transferred onto polyvinlidene fluoride (PDVF) membrane (GE Healthcare) then washed with PBS-Tween (1xPBS with 1% Tween 20) and blocked (5% w/v non-fat dry milk). Washed membranes were incubated with primary antibody as indicated. HRP-conjugated secondary antibody (GE Healthcare) was detected using Enhanced Chemiluminescence (GE Healthcare).
Statistical testing was performed as indicated in individual experiments using SPSS software (IBM-SPSS, Portsmouth, UK).
Chronic lymphocytic leukaemia
The authors would like to thank Leukaemia & Lymphoma Research UK who funded the research described in this study. We also would like to thank the Robert Whiteson Memorial Trust for the support of JAA and the Gene Machine Charity who supported SN.
- Bertilaccio MT, Scielzo C, Muzio M, Caligaris-Cappio F: An overview of chronic lymphocytic leukaemia biology. Best Pract Res Clin Haematol 2010,23(1):21–32. 10.1016/j.beha.2009.12.005PubMedView ArticleGoogle Scholar
- Ghia P, Circosta P, Scielzo C, Vallario A, Camporeale A, Granziero L, Caligaris-Cappio F: Differential effects on CLL cell survival exerted by different microenvironmental elements. Current topics in microbiology and immunology 2005, 294: 135–145.PubMedGoogle Scholar
- Burger JA, Tsukada N, Burger M, Zvaifler NJ, Dell’Aquila M, Kipps TJ: Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1. Blood 2000,96(8):2655–2663.PubMedGoogle Scholar
- Catera R, Silverman GJ, Hatzi K, Seiler T, Didier S, Zhang L, Hervé M, Meffre E, Oscier DG, Vlassara H, Scofield RH, Chen Y, Allen SL, Kolitz J, Rai KR, Chu CC, Chiorazzi N: Chronic lymphocytic leukemia cells recognize conserved epitopes associated with apoptosis and oxidation. Molecular Medicine 2008,14(11–12):665–674.PubMedPubMed CentralGoogle Scholar
- Ghiotto F, Fais F, Valetto A, Albesiano E, Hashimoto S, Dono M, Ikematsu H, Allen SL, Kolitz J, Rai KR, Nardini M, Tramontano A, Ferrarini M, Chiorazzi N: Remarkably similar antigen receptors among a subset of patients with chronic lymphocytic leukemia. The Journal of clinical investigation 2004,113(7):1008–1016. 10.1172/JCI19399PubMedView ArticlePubMed CentralGoogle Scholar
- Pettitt AR, Moran EC, Cawley JC: Homotypic interactions protect chronic lymphocytic leukaemia cells from spontaneous death in vitro. Leuk Res 2001,25(11):1003–1012. 10.1016/S0145-2126(01)00067-4PubMedView ArticleGoogle Scholar
- Norin S, Kimby E, Lundin J: Tumor burden status evaluated by computed tomography scan is of prognostic importance in patients with chronic lymphocytic leukemia. Med Oncol 2010,27(3):820–825. 10.1007/s12032-009-9292-yPubMedView ArticleGoogle Scholar
- Soma LA, Craig FE, Swerdlow SH: The proliferation center microenvironment and prognostic markers in chronic lymphocytic leukemia/small lymphocytic lymphoma. Hum Pathol 2006,37(2):152–159. 10.1016/j.humpath.2005.09.029PubMedView ArticleGoogle Scholar
- Till KJ, Spiller DG, Harris RJ, Chen H, Zuzel M, Cawley JC: CLL, but not normal, B cells are dependent on autocrine VEGF and alpha4beta1 integrin for chemokine-induced motility on and through endothelium. Blood 2005,105(12):4813–4819. 10.1182/blood-2004-10-4054PubMedView ArticleGoogle Scholar
- Schulz A, Toedt G, Zenz T, Stilgenbauer S, Lichter P, Seiffert M: Inflammatory cytokines and signaling pathways are associated with survival of primary chronic lymphocytic leukemia cells in vitro: a dominant role of CCL2. Haematologica 2011,96(3):408–416. 10.3324/haematol.2010.031377PubMedView ArticlePubMed CentralGoogle Scholar
- Herishanu Y, Pérez-Galán P, Liu D, Biancotto A, Pittaluga S, Vire B, Gibellini F, Njuguna N, Lee E, Stennett L, Raghavachari N, Liu P, McCoy JP, Raffeld M, Stetler-Stevenson M, Yuan C, Sherry R, Arthur DC, Maric I, White T, Marti GE, Munson P, Wilson WH, Wiestner A: The lymph node microenvironment promotes B-cell receptor signaling, NF-kappaB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood 2011,117(2):563–574. 10.1182/blood-2010-05-284984PubMedView ArticlePubMed CentralGoogle Scholar
- Carrasco YR: Molecular and cellular dynamics at the early stages of antigen encounter: the B-cell immunological synapse. Curr Top Microbiol Immunol 2010, 340: 51–62.PubMedGoogle Scholar
- Bradley WD, Koleske AJ: Regulation of cell migration and morphogenesis by Abl-family kinases: emerging mechanisms and physiological contexts. J Cell Sci 2009,122(Pt 19):3441–3454.PubMedView ArticlePubMed CentralGoogle Scholar
- Lin K, Glenn MA, Harris RJ, Duckworth AD, Dennett S, Cawley JC, Zuzel M, Slupsky JR: c-Abl expression in chronic lymphocytic leukemia cells: clinical and therapeutic implications. Cancer research 2006,66(15):7801–7809. 10.1158/0008-5472.CAN-05-3901PubMedView ArticleGoogle Scholar
- Okuda K, Weisberg E, Gilliland DG, Griffin JD: ARG tyrosine kinase activity is inhibited by STI571. Blood 2001,97(8):2440–2448. 10.1182/blood.V97.8.2440PubMedView ArticleGoogle Scholar
- Ponader S, Chen SS, Buggy JJ, Balakrishnan K, Gandhi V, Wierda WG, Keating MJ, O'Brien S, Chiorazzi N, Burger JA: The Bruton tyrosine kinase inhibitor PCI-32765 thwarts chronic lymphocytic leukemia cell survival and tissue homing in vitro and in vivo. Blood 2012,119(5):1182–1189. 10.1182/blood-2011-10-386417PubMedView ArticleGoogle Scholar
- Terzakis JA: Distinguishing B and T lymphocytes by scanning electron microscopy. Ultrastruct Pathol 2000,24(4):205–209. 10.1080/01913120050176653PubMedView ArticleGoogle Scholar
- Vincent AM, Cawley JC, Burthem J: Integrin function in chronic lymphocytic leukaemia. Blood 1995,86(10):3366.Google Scholar
- Redondo-Munoz J, Escobar-Diaz E, Samaniego R, Terol MJ, Garcia-Marco JA, Garcia-Pardo A: MMP-9 in B-cell chronic lymphocytic leukemia is up-regulated by alpha4beta1 integrin or CXCR4 engagement via distinct signaling pathways, localizes to podosomes, and is involved in cell invasion and migration. Blood 2006,108(9):3143–3151. 10.1182/blood-2006-03-007294PubMedView ArticleGoogle Scholar
- Gorgun G, Holderried TA, Zahrieh D, Neuberg D, Gribben JG: Chronic lymphocytic leukemia cells induce changes in gene expression of CD4 and CD8 T cells. J Clin Invest 2005,115(7):1797–1805. 10.1172/JCI24176PubMedView ArticlePubMed CentralGoogle Scholar
- Worthylake RA, Lemoine S, Watson JM, Burridge K: RhoA is required for monocyte tail retraction during transendothelial migration. J Cell Biol 2001,154(1):147–160. 10.1083/jcb.200103048PubMedView ArticlePubMed CentralGoogle Scholar
- Schrottner P, Leick M, Burger M: The role of chemokines in B cell chronic lymphocytic leukaemia: pathophysiological aspects and clinical impact. Ann Hematol 2010,89(5):437–446. 10.1007/s00277-009-0876-6PubMedView ArticleGoogle Scholar
- Huang Y, Comiskey EO, Dupree RS, Li S, Koleske AJ, Burkhardt JK: The c-Abl tyrosine kinase regulates actin remodeling at the immune synapse. Blood 2008,112(1):111–119. 10.1182/blood-2007-10-118232PubMedView ArticlePubMed CentralGoogle Scholar
- Brown MJ, Nijhara R, Hallam JA, Gignac M, Yamada KM, Erlandsen SL, Delon J, Kruhlak M, Shaw S: Chemokine stimulation of human peripheral blood T lymphocytes induces rapid dephosphorylation of ERM proteins, which facilitates loss of microvilli and polarization. Blood 2003,102(12):3890–3899. 10.1182/blood-2002-12-3807PubMedView ArticleGoogle Scholar
- Majstoravich S, Zhang J, Nicholson-Dykstra S, Linder S, Friedrich W, Siminovitch KA, Higgs HN: Lymphocyte microvilli are dynamic, actin-dependent structures that do not require Wiskott-Aldrich syndrome protein (WASp) for their morphology. Blood 2004,104(5):1396–1403. 10.1182/blood-2004-02-0437PubMedView ArticleGoogle Scholar
- Tsukada N, Burger JA, Zvaifler NJ, Kipps TJ: Distinctive features of “nurselike” cells that differentiate in the context of chronic lymphocytic leukemia. Blood 2002,99(3):1030–1037. 10.1182/blood.V99.3.1030PubMedView ArticleGoogle Scholar
- Parri M, Chiarugi P: Rac and Rho GTPases in cancer cell motility control. Cell Commun Signal 2010, 8: 23. 10.1186/1478-811X-8-23PubMedView ArticlePubMed CentralGoogle Scholar
- Doh J, Krummel MF: Immunological synapses within context: patterns of cell-cell communication and their application in T-T interactions. Curr Top Microbiol Immunol 2010, 340: 25–50.PubMedGoogle Scholar
- Mempel TR, Henrickson SE, von Andrian UH: T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature 2004,427(6970):154–159. 10.1038/nature02238PubMedView ArticleGoogle Scholar
- Miller AL, Wang Y, Mooseker MS, Koleske AJ: The Abl-related gene (Arg) requires its F-actin-microtubule cross-linking activity to regulate lamellipodial dynamics during fibroblast adhesion. J Cell Biol 2004,165(3):407–419. 10.1083/jcb.200308055PubMedView ArticlePubMed CentralGoogle Scholar
- Echarri A, Lai MJ, Robinson MR, Pendergast AM: Abl interactor 1 (Abi-1) wave-binding and SNARE domains regulate its nucleocytoplasmic shuttling, lamellipodium localization, and wave-1 levels. Mol Cell Biol 2004,24(11):4979–4993. 10.1128/MCB.24.11.4979-4993.2004PubMedView ArticlePubMed CentralGoogle Scholar
- Ideses Y, Brill-Karniely Y, Haviv L, Ben-Shaul A, Bernheim-Groswasser A: Arp2/3 branched actin network mediates filopodia-like bundles formation in vitro. PLoS One 2008,3(9):e3297. 10.1371/journal.pone.0003297PubMedView ArticlePubMed CentralGoogle Scholar
- Nolz JC, Nacusi LP, Segovis CM, Medeiros RB, Mitchell JS, Shimizu Y, Billadeau DD: The WAVE2 complex regulates T cell receptor signaling to integrins via Abl- and CrkL-C3G-mediated activation of Rap1. J Cell Biol 2008,182(6):1231–1244. 10.1083/jcb.200801121PubMedView ArticlePubMed CentralGoogle Scholar
- McCaig AM, Cosimo E, Leach MT, Michie AM: Dasatinib inhibits B cell receptor signalling in chronic lymphocytic leukaemia but novel combination approaches are required to overcome additional pro-survival microenvironmental signals. British Journal of Haematology 2011,153(2):199–211. 10.1111/j.1365-2141.2010.08507.xPubMedView ArticleGoogle Scholar
- Song Z, Lu P, Furman RR, Leonard JP, Martin P, Tyrell L, Lee FY, Knowles DM, Coleman M, Wang YL: Activities of SYK and PLCgamma2 predict apoptotic response of CLL cells to SRC tyrosine kinase inhibitor dasatinib. Clin Cancer Res 2010,16(2):587–599. 10.1158/1078-0432.CCR-09-1519PubMedView ArticleGoogle Scholar
- Veldurthy A, Patz M, Hagist S, Pallasch CP, Wendtner CM, Hallek M, Krause G: The kinase inhibitor dasatinib induces apoptosis in chronic lymphocytic leukemia cells in vitro with preference for a subgroup of patients with unmutated IgVH genes. Blood 2008,112(4):1443–1452. 10.1182/blood-2007-11-123984PubMedView ArticleGoogle Scholar
- Chow KU, Nowak D, Hofmann W, Schneider B, Hofmann WK: Imatinib induces apoptosis in CLL lymphocytes with high expression of Par-4. Leukemia 2005,19(6):1103–1105. author reply 5–6; discussion 6–7 10.1038/sj.leu.2403739PubMedView ArticleGoogle Scholar
- Allen JC, Talab F, Zuzel M, Lin K, Slupsky JR: c-Abl regulates Mcl-1 gene expression in chronic lymphocytic leukemia cells. Blood 2010,117(8):2414–2422.View ArticleGoogle Scholar
- Fisher DC, Lacasce AS, Jacobsen ED, Armand P, Hasserjian RP, Werner L, Neuberg D, Brown JR: Phase II study of dasatinib in relapsed or refractory chronic lymphocytic leukemia. Clin Cancer Res 2011,17(9):2977–2986. 10.1158/1078-0432.CCR-10-2879PubMedView ArticlePubMed CentralGoogle Scholar
- Burthem J, Baker PK, Hunt JA, Cawley JC: Hairy cell interactions with extracellular matrix: expression of specific integrin receptors and their role in the cell’s response to specific adhesive proteins. Blood 1994,84(3):873–882.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.