Quantification of change in phosphorylation of BCR-ABL kinase and its substrates in response to Imatinib treatment in human chronic myelogenous leukemia cells

Phosphorylation by the constitutively activated BCR-ABL tyrosine kinase is associated with the pathogenesis of the human chronic myelogenous leukemia (CML). It is difficult to characterize kinase response to stimuli or drug treatment because regulatory phosphorylation events are largely transient changes affecting low abundance proteins. Stable isotope labeling with amino acids in cell culture (SILAC) has emerged as a pivotal technology for quantitative proteomics. By metabolically labeling proteins with light or heavy tyrosine, we are able to quantify the change in phosphorylation of BCR-ABL kinase and its substrates in response to drug treatment in human CML cells. In this study, we observed that BCR-ABL kinase is phosphorylated at tyrosines 393 and 644, and that SH2-domain containing inositol phosphatase (SHIP)-2 and downstream of kinase (Dok)-2 are phosphorylated at tyrosine 1135 and 299, respectively. Based on the relative intensity of isotopic peptide pairs, we demonstrate that the level of phosphorylation of BCR-ABL kinase as well as SHIP-2 and Dok-2 is reduced approximately 90% upon treatment with Imatinib, a specific inhibitor of BCR-ABL kinase. Furthermore, proteins, such as SHIP-1, SH2-containing protein (SHC) and Casitas B-lineage lymphoma proto-oncogene (CBL), are also regulated by Imatinib. These results demonstrate the simplicity and utility of SILAC as a method to quantify dynamic changes in phosphorylation at specific sites in response to stimuli or drug treatment in cell culture.     

Receptors and signaling mechanisms for B-lymphocyte activation, proliferation and differentiation - Insights from both in vivo and in vitro approaches

During the last three decades, a number of B-lymphocyte specific surface antigens have been defined some of which may also show activation/differentiation specific expression. Here, we review the various signaling events and the receptor-ligand interactions for B-cell development, activation and differentiation. Our discussion and presentation include reviewing the in vivo and in vitro mechanisms. Focus is on the experiments that give us valuable insights into the B cell signaling mechanisms in vitro. Three significant pathways in B-cell development - c-Kit, FLT-3 and IL-7 signaling pathways are elucidated upon. Both antigen dependent and antigen independent mechanisms of B cell stimulation are also reviewed.     

Deletion of ABL/BCR on der(9) associated with severe basophilia

Chronic basophilic leukemia is a rare form in chronic myeloid leukemia patients. Only limited number of reports are available. Herein, we describe a patient who presented with fatigue, weight loss, leucocytosis, prominent basophilia, and mild eosinophilia. On biopsy, bone marrow was hypercellular with marked basophils. The immunophenotype showed abnormal expression of CD7, which is suggestive of basophilic maturation. Chromosomal analysis from GTG-banded metaphases revealed Ph positivity, and fluorescence in situ hybridization (FISH) with BCR/ABL dual color, dual fusion probe showed single fusion on the der(22) chromosome and ABL/BCR fusion was deleted on the der(9) chromosome. The deletion (ABL/BCR) on der(9) may be associated with basophilia which may be also indicative of the transformation of CML to acute myeloid leukemia.     

Phosphoinositide 3-kinase delta (PI3Kδ) in leukocyte signaling and function

PI3Kδ is a lipid kinase of the PI3K class IA family involved in early signaling events of leukocytes responding to a wide variety of stimuli. The leukocyte specificity of PI3Kδ is defined by its expression, whereas its signaling function is via the production of phosphoinositide 3,4,5-triphosphates at the proximity of activated receptors for recruiting other signaling molecules. The importance of PI3Kδ in B cell development and function is most apparent, and its role in other leukocyte cell types can be easily demonstrated as well. PI3Kδ participates in the development, activation and migration of T cells and NK cells. The role of PI3Kδ in myeloid cell activities, such as inflammation driven cell infiltration, neutrophil oxidative burst, immune complex mediated macrophage activation, as well as mast cell maturation and degranulation, has been well illustrated in various studies. As a result of the broad effects of PI3Kδ in leukocyte functions, the disruption of PI3Kδ expression or activity leads to decreased inflammatory and immune responses in vivo. The protective role of PI3Kδ inactivation in animal models of arthritis, asthma or obstructive respiratory diseases has been demonstrated. These findings suggest the potential efficacy achievable with PI3Kδ inhibitors in the treatment of autoimmune and respiratory diseases.     

The growth of B cell receptor microcluster is a universal response of B cells encountering antigens with different motion features

B lymphocyte cell senses and acquires foreign antigens through clonal distributed B cell receptors (BCRs) expressed on the surface of plasma membrane. The presentation formats of antigens are quite diverse. Based on their Brownian diffusion mobility, there are three forms: free mobile soluble antigens, lateral mobile membrane bound antigens, and fixed immobile antigens. Here, using high resolution high speed live cell imaging approaches, we provide evidence that BCR microclusters are formed on the surface of B cells shortly after B cell’s encountering of antigens with each format of motion features. Through high speed live cell imaging, we determine that these BCR microclusters show dynamic growth feature and by doing so function as the basic platforms for B cells to acquire the antigens. We propose that the formation and dynamic growth of BCR microcluster is a universal mechanism for B cell to response to antigens with diverse motion features.     

Solution structure of the complex formed between human complement C3d and full-length complement receptor type 2

Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response during the activation of B-cells through its binding to C3d, a cleavage fragment of the major complement component C3. The extracellular portion of CR2 comprises 15 or 16 short complement regulator (SCR) domains in a partially folded-back but flexible structure. Here, the effect of C3d binding to CR2 was determined by analytical ultracentrifugation and X-ray scattering. The sedimentation coefficient of unbound CR2 is 4.03 S in 50 mM NaCl. Because this agrees well with a value of 3.93 S in 137 mM NaCl, the overall CR2 structure is unaffected by change in ionic strength. Unbound C3d exists in monomer-dimer and monomer-trimer equilibria in 50 mM NaCl, but as a monomer only in 137 mM NaCl. In c(s) size-distribution analyses, an equimolar mixture of the CR2-C3d complex in 50 mM NaCl revealed a single peak shifted to 4.52 S when compared to unbound CR2 at 4.03 S to show that the complex had formed. The CR2-C3d complex in 137 mM NaCl showed two peaks at 2.52 S and 4.07 S to show that this had dissociated. Solution structural models for the CR2 SCR-1/2 complex with C3d and CR2 SCR-1/15 were superimposed. These gave an average sedimentation coefficient of 4.57 S for the complex, in good agreement with the observed value of 4.52 S. It is concluded that CR2 does not detectably change conformation when C3d is bound to it. Consistent with previous analyses, its C3d complex is not formed in physiological salt conditions. The implications of these solution results for its immune role are discussed. To our knowledge, this is the first solution structural study of a large multidomain SCR protein CR2 bound to its physiological ligand C3d.     

Antigen-B Cell Receptor Complexes Associate with Intracellular major histocompatibility complex (MHC) Class II Molecules

Antigen processing and MHC class II-restricted antigen presentation by antigen-presenting cells such as dendritic cells and B cells allows the activation of naïve CD4+ T cells and cognate interactions between B cells and effector CD4+ T cells, respectively. B cells are unique among class II-restricted antigen-presenting cells in that they have a clonally restricted antigen-specific receptor, the B cell receptor (BCR), which allows the cell to recognize and respond to trace amounts of foreign antigen present in a sea of self-antigens. Moreover, engagement of peptide-class II complexes formed via BCR-mediated processing of cognate antigen has been shown to result in a unique pattern of B cell activation. Using a combined biochemical and imaging/FRET approach, we establish that internalized antigen-BCR complexes associate with intracellular class II molecules. We demonstrate that the M1-paired MHC class II conformer, shown previously to be critical for CD4 T cell activation, is incorporated selectively into these complexes and loaded selectively with peptide derived from BCR-internalized cognate antigen. These results demonstrate that, in B cells, internalized antigen-BCR complexes associate with intracellular MHC class II molecules, potentially defining a site of class II peptide acquisition, and reveal a selective role for the M1-paired class II conformer in the presentation of cognate antigen. These findings provide key insights into the molecular mechanisms used by B cells to control the source of peptides charged onto class II molecules, allowing the immune system to mount an antibody response focused on BCR-reactive cognate antigen.     

Cisplatin-evoked DNA fragmentation in normal and cancer cells and its modulation by free radical scavengers and the tyrosine kinase inhibitor STI571

Cis-diamminedichloroplatinum(II) (cisplatin, cis-DDP) is well studied anticancer drug, whose activity can be attributed to its ability to form adducts with DNA, but this drug can also form DNA-damaging free radicals, however this mechanism of cisplatin action is far less explored. Using the comet assay we studied cisplatin-induced DNA damage in the presence of spin traps: DMPO and PBN, Vitamins A, C and E as well as the tyrosine kinases inhibitor STI571 in normal human lymphocytes and leukemic K562 cells. The latter cells express the BCR/ABL fusion protein, which can be a target of the tyrosine kinase inhibitor STI571. A 20 h incubation with cisplatin at 1-10 microM induced DNA cross-links and DNA fragmentation in normal and cancer cells. Cisplatin could induce intra- and interstrand DNA-DNA cross-links as well as DNA-protein cross-links. DNA damage in K562 cells was more pronounced than in normal lymphocytes. In the presence of spin traps and vitamins we noticed a decrease in the DNA fragmentation in both cell types. Co-treatment of the lymphocytes with cisplatin at 10 microM and STI571 at 0.25 microg/ml caused an increase of DNA fragmentation in comparison with DNA fragmentation induced by cisplatin alone. In the case of K562 cells, an increase of DNA fragmentation was observed after treatment with cisplatin at 1 microM. Our results indicate that the free radicals scavengers could decrease DNA fragmentation induced by cisplatin in the normal and cancer cells, but probably they have no effect on DNA cross-linking induced by the drug. The results obtained with the BCR/ABL inhibitor suggest that K562 cells could be more sensitive towards co-treatment of cisplatin and STI571. Our results suggest also that aside from the BCR/ABL other factors such as p53 level, signal transduction pathways and DNA repair processes can be responsible for the increased sensitivity of K562 cells to cisplatin compared with normal lymphocytes.