Molecular profile reveals immune-associated markers of medulloblastoma for different subtypes.

Medulloblastoma, a common pediatric malignant tumor, has been recognized to have four molecular subgroups [wingless (WNT), sonic hedgehog (SHH), group 3, group 4], which are defined by the characteristic gene transcriptomic and DNA methylomic profiles, and has distinct clinical features within each subgroup. The tumor immune microenvironment is integral in tumor initiation and progression and might be associated with therapeutic responses. However, to date, the immune infiltrative landscape of medulloblastoma has not yet been elucidated. Thus, we proposed MethylCIBERSORT to estimate the degree of immune cell infiltration and weighted correlation network analysis (WGCNA) to find modules of highly correlated genes. Synthesizing the hub genes in the protein-protein interaction (PPI) network and modules of the co-expression network, we identify three candidate biomarkers [GRB2-associated-binding protein 1 (GAB1), Abelson 1 (ABL1), and CXC motif chemokine receptor type 4 (CXCR4)] via the molecular profiles of medulloblastoma. Given this, we investigated the correlation between these three immune hub genes and immune checkpoint blockade response and the potential of drug prediction further. In addition, this study demonstrated a higher presence of endothelial cells and infiltrating immune cells in Group 3 tumor bulk. The above results will be conducive to better comprehending the immune-related pathogenesis and treatment of medulloblastoma.

ABL1 and Cofilin1 promote T-cell acute lymphoblastic leukemia cell migration.

The fusion gene of ABL1 is closely related to tumor proliferation, invasion, and migration. It has been reported recently that ABL1 itself is required for T-cell acute lymphoblastic leukemia (T-ALL) cell migration induced by CXCL12. Further experiments revealed that ABL1 inhibitor Nilotinib inhibited leukemia cell migration induced by CXCL12, indicating the possible application of Nilotinib in T-ALL leukemia treatment. However, the interacting proteins of ABL1 and the specific mechanisms of their involvement in this process need further investigation. In the present study, ABL1 interacting proteins were characterized and their roles in the process of leukemia cell migration induced by CXCL12 were investigated. Co-immunoprecipitation in combination with mass spectrometry analysis identified 333 proteins that interact with ABL1, including Cofilin1. Gene ontology analysis revealed that many of them were enriched in the intracellular organelle or cytoplasm, including nucleic acid binding components, transfectors, or co-transfectors. Kyoto Encyclopedia of Genes and Genomes analysis showed that the top three enriched pathways were translation, glycan biosynthesis, and metabolism, together with human diseases. ABL1 and Cofilin1 were in the same complex. Cofilin1 binds the SH3 domain of ABL1 directly; however, ABL1 is not required for the phosphorylation of Cofilin1. Molecular docking analysis shows that ABL1 interacts with Cofilin1 mainly through hydrogen bonds and ionic interaction between amino acid residues. The mobility of leukemic cells was significantly decreased by Cofilin1 siRNA. These results demonstrate that Cofilin1 is a novel ABL1 binding partner. Furthermore, Cofilin1 participates in the migration of leukemia cells induced by CXCL12. These data indicate that ABL1 and Cofilin1 are possible targets for T-ALL treatment.

Mebendazole-induced M1 polarisation of THP-1 macrophages may involve DYRK1B inhibition.

OBJECTIVE: We recently showed that the anti-helminthic compound mebendazole (MBZ) has immunomodulating activity by inducing a M2 to M1 phenotype switch in monocyte/macrophage models. In the present study we investigated the potential role of protein kinases in mediating this effect. RESULTS: MBZ potently binds and inhibits Dual specificity tyrosine-phosphorylation-regulated kinase 1B (DYRK1B) with a Kd and an IC50 of 7 and 360 nM, respectively. The specific DYRK1B inhibitor AZ191 did not mimic the cytokine release profile of MBZ in untreated THP-1 monocytes. However, in THP-1 cells differentiated into macrophages, AZ191 strongly induced a pro-inflammatory cytokine release pattern similar to MBZ and LPS/IFNγ. Furthermore, like MBZ, AZ191 increased the expression of the M1 marker CD80 and decreased the M2 marker CD163 in THP-1 macrophages. In this model, AZ191 also increased phospho-ERK activity although to a lesser extent compared to MBZ. Taken together, the results demonstrate that DYRK1B inhibition could, at least partly, recapitulate immune responses induced by MBZ. Hence, DYRK1B inhibition induced by MBZ may be part of the mechanism of action to switch M2 to M1 macrophages.

Nonreceptor Tyrosine Kinase c-Abl- and Arg-Mediated IRF3 Phosphorylation Regulates Innate Immune Responses by Promoting Type I IFN Production.

The nonreceptor tyrosine kinase c-Abl plays important roles in T cell development and immune responses; however, the mechanism is poorly understood. IFN regulatory factor 3 (IRF3) is a key transcriptional regulator of type I IFN-dependent immune responses against DNA and RNA viruses. The data in this study show that IRF3 is physically associated with c-Abl in vivo and directly binds to c-Abl in vitro. IRF3 is phosphorylated by c-Abl and c-Abl-related kinase, Arg, mainly at Y292. The inhibitor AMN107 inhibits IFN-ß production induced by poly(dA:dT), poly(I:C), and Sendai virus in THP-1 and mouse bone marrow-derived macrophage cells. IRF3-induced transcription of IFN-ß is significantly reduced by the mutation of Y292 to F. Moreover, AMN107 suppresses gene expression of absent in melanoma 2 (AIM2) and subsequently reduces inflammasome activation induced by cytosolic bacteria, dsDNA, and DNA viruses. Consistent with this finding, Francisella tularensis subsp. holarctica live vaccine strain (Ft LVS), which is known as an activator of AIM2 inflammasome, induces death in significantly more C57BL/6 mice treated with the Abl inhibitor AMN107 or c-Abl/Arg small interfering RNA than in untreated mice. This study provides new insight into the function of c-Abl and Arg in regulating immune responses and AIM2 inflammasome activation, especially against Ft LVS infection.

The Lupus-Associated Fcγ Receptor IIb-I232T Polymorphism Results in Impairment in the Negative Selection of Low-Affinity Germinal Center B Cells Via c-Abl in Mice.

OBJECTIVE: Fcγ receptor IIb (FcγRIIb) is an essential negative regulator of B cells that blocks B cell receptor (BCR) signaling and triggers c-Abl-dependent apoptosis of B cells. FcγRIIb-deficient mice display splenomegaly with expansion of B cells, leading to lupus. FcγRIIb-I232T is a hypofunctional polymorphism associated with lupus susceptibility in humans, an autoimmune disease linked to diminished deletion of autoreactive B cells. In the context of the FcγRIIb-I232T polymorphism, we investigated the role of FcγRIIb in the deletion of low-affinity germinal center (GC) B cells, an important mechanism for preventing autoimmunity. METHODS: We generated FcγRIIb232T/T mice to mimic human FcγRIIb-I232T carriers and immunized mice with chicken gamma globulin (CGG)-conjugated NP, a T cell-dependent antigen, to examine the response of GC B cells. RESULTS: Compared to wild-type (WT) mice, FcγRIIb232T/T mice showed increased numbers of low-affinity NP-specific IgG and NP-specific B cells and plasma cells; additionally, the expression of a somatic mutation (W33L) in their VH 186.2 genes encoding high-affinity BCR was reduced. Notably, FcγRIIb232T/T mice had a higher number of GC light zone B cells and showed less apoptosis than WT mice, despite having equivalent follicular helper T cell numbers and function. Moreover, phosphorylation of c-Abl was reduced in FcγRIIb232T/T mice, and treatment of WT mice with the c-Abl inhibitor nilotinib during the peak of GC response resulted in reduced affinity maturation reminiscent of FcγRIIb232T/T mice. CONCLUSION: Our findings provide evidence of a critical role of FcγRIIb/c-Abl in the negative selection of GC B cells in FcγRIIb232T/T mice. Importantly, our findings indicate potential benefits of up-regulating FcγRIIb expression in B cells for treatment of systemic lupus erythematosus.

The vascular endothelial specific IL-4 receptor alpha-ABL1 kinase signaling axis regulates the severity of IgE-mediated anaphylactic reactions.

BACKGROUND: Severe IgE-mediated, food-induced anaphylactic reactions are characterized by pulmonary venous vasodilatation and fluid extravasation, which are thought to lead to the life-threatening anaphylactic phenotype. The underlying immunologic and cellular processes involved in driving fluid extravasation and the severe anaphylactic phenotype are not fully elucidated. OBJECTIVE: We sought to define the interaction and requirement of IL-4 and vascular endothelial (VE) IL-4 receptor α chain (IL-4Rα) signaling in histamine-abelson murine leukemia viral oncogene homology 1 (ABL1)-mediated VE dysfunction and fluid extravasation in the severity of IgE-mediated anaphylactic reactions in mice. METHODS: Mice deficient in VE IL-4Rα and models of passive and active oral antigen- and IgE-induced anaphylaxis were used to define the requirements of the VE IL-4Rα and ABL1 pathway in severe anaphylactic reactions. The human VE cell line (EA.hy926 cells) and pharmacologic (imatinib) and genetic (short hairpin RNA knockdown of IL4RA and ABL1) approaches were used to define the requirement of this pathway in VE barrier dysfunction. RESULTS: IL-4 exacerbation of histamine-induced hypovolemic shock in mice was dependent on VE expression of IL-4Rα. IL-4- and histamine-induced ABL1 activation in human VE cells and VE barrier dysfunction was ABL1-dependent. Development of severe IgE-mediated hypovolemia and shock required VE-restricted ABL1 expression. Treatment of mice with a history of food-induced anaphylaxis with the ABL kinase inhibitor imatinib protected the mice from severe IgE-mediated anaphylaxis. CONCLUSION: IL-4 amplifies IgE- and histamine-induced VE dysfunction, fluid extravasation, and the severity of anaphylaxis through a VE IL-4Rα/ABL1-dependent mechanism. These studies implicate an important contribution by the VE compartment in the severity of anaphylaxis and identify a new pathway for therapeutic intervention of IgE-mediated reactions.

Neutrophil FcγRIIA promotes IgG-mediated glomerular neutrophil capture via Abl/Src kinases.

The kidney glomerular capillaries are frequent sites of immune complex deposition and subsequent neutrophil accumulation in post-infectious and rapidly progressive glomerulonephritis. However, the mechanisms of neutrophil recruitment remain enigmatic, and there is no targeted therapeutic to avert this proximal event in glomerular inflammation. The uniquely human activating Fc receptor FcγRIIA promotes glomerular neutrophil accumulation and damage in anti-glomerular basement membrane-induced (anti-GBM-induced) glomerulonephritis when expressed on murine neutrophils. Here, we found that neutrophils are directly captured by immobilized IgG antibodies under physiological flow conditions in vitro through FcγRIIA-dependent, Abl/Src tyrosine kinase-mediated F-actin polymerization. Biophysical measurements showed that the lifetime of FcγRIIA-IgG bonds increased under mechanical force in an F-actin-dependent manner, which could enable the capture of neutrophils under physiological flow. Kidney intravital microscopy revealed that circulating neutrophils, which were similar in diameter to glomerular capillaries, abruptly arrested following anti-GBM antibody deposition via neutrophil FcγRIIA and Abl/Src kinases. Accordingly, inhibition of Abl/Src with bosutinib reduced FcγRIIA-mediated glomerular neutrophil accumulation and renal injury in experimental, crescentic anti-GBM nephritis. These data identify a pathway of neutrophil recruitment within glomerular capillaries following IgG deposition that may be targeted by bosutinib to avert glomerular injury.

Frontline Science: Tumor necrosis factor-α stimulation and priming of human neutrophil granule exocytosis.

Neutrophil granule exocytosis plays an important role in innate and adaptive immune responses. The present study examined TNF-α stimulation or priming of exocytosis of the 4 neutrophil granule subsets. TNF-α stimulated exocytosis of secretory vesicles and gelatinase granules and primed specific and azurophilic granule exocytosis to fMLF stimulation. Both stimulation and priming of exocytosis by TNF-α were dependent on p38 MAPK activity. Bioinformatic analysis of 1115 neutrophil proteins identified by mass spectrometry as being phosphorylated by TNF-α exposure found that actin cytoskeleton regulation was a major biologic function. A role for p38 MAPK regulation of the actin cytoskeleton was confirmed experimentally. Thirteen phosphoproteins regulated secretory vesicle quantity, formation, or release, 4 of which-Raf1, myristoylated alanine-rich protein kinase C (PKC) substrate (MARCKS), Abelson murine leukemia interactor 1 (ABI1), and myosin VI-were targets of the p38 MAPK pathway. Pharmacologic inhibition of Raf1 reduced stimulated exocytosis of gelatinase granules and priming of specific granule exocytosis. We conclude that differential regulation of exocytosis by TNF-α involves the actin cytoskeleton and is a necessary component for priming of the 2 major neutrophil antimicrobial defense mechanisms: oxygen radical generation and release of toxic granule contents.

Tyrosine kinase c-Abl regulates the survival of plasma cells.

Tyrosine kinase c-Abl plays an important role in early B cell development. Its deletion leads to reduced pro- and pre-B cell generation in mice. However, its function in B cell terminal differentiation remains unexplored. Here, we used c-Ablf/f Aicdacre/+ mice, in which c-Abl is ablated only in antigen-activated B cells, to study the role of c-Abl in germinal center (GC) B and antibody-secreting plasma cell formation. Upon challenge with a model antigen, we found normal GC and memory B but reduced plasma cells and antigen-specific antibody response in the mutant mice. In-vitro studies revealed that plasma cells lacking c-Abl could be generated but did not accumulate in culture, indicative of survival defect. They also exhibited impaired STAT3 phosphorylation. The plasma cell defects could be rectified by introduction of Bim-deficiency or delivery of colivelin, a STAT3 activator, into c-Ablf/f Aicdacre/+ mice. Hence, c-Abl signalling regulates the survival of plasma cells.

Sos1 Regulates Macrophage Podosome Assembly and Macrophage Invasive Capacity.

Podosomes are protrusive structures implicated in macrophage extracellular matrix degradation and three-dimensional migration through cell barriers and the interstitium. Podosome formation and assembly are regulated by cytoskeleton remodeling requiring cytoplasmic tyrosine kinases of the Src and the Abl families. Considering that Abl has been reported to phosphorylate the guanine nucleotide exchange factor Sos1, eliciting its Rac-guanine nucleotide exchange factor activity, and Rac regulates podosome formation in myeloid cells and invadopodia formation in cancer cells, we addressed whether Sos1 is implicated in podosome formation and function in macrophages. We found that ectopically expressed Abl or the Src kinase Fgr phosphorylate Sos1, and the Src kinases Hck and Fgr are required for Abl and Sos1 phosphorylation and Abl/Sos1 interaction in macrophages. Sos1 localizes to podosomes in both murine and human macrophages, and its silencing by small interfering RNA results in disassembly of murine macrophage podosomes and a marked reduction of GTP loading on Rac. Matrix degradative capacity, three-dimensional migration through Matrigel, and transmigration through an endothelial cell monolayer of Sos1-silenced macrophages were inhibited. In addition, Sos1- or Abl-silenced macrophages, or macrophages treated with the selective Abl inhibitor imatinib mesylate had a reduced capability to migrate into breast tumor spheroids, the majority of cells remaining at the margin and the outer layers of the spheroid itself. Because of the established role of Src and Abl kinases to regulate also invadopodia formation in cancer cells, our findings suggest that targeting the Src/Abl/Sos1/Rac pathway may represent a double-edged sword to control both cancer-invasive capacities and cancer-related inflammation.

c-Abl tyrosine kinase plays a critical role in ß2 integrin-dependent neutrophil migration by regulating Vav1 activity.

The recruitment and migration of neutrophils are critical for innate immunity and acute inflammatory responses. However, the mechanism that regulates the recruitment and migration of neutrophils has not been well characterized. We here reveal a novel function of c-Abl kinase in regulating neutrophil migration. Our results demonstrate that c-Abl kinase is required for neutrophil recruitment in vivo and migration in vitro, and the inhibition of c-Abl kinase activity has a significant impact on neutrophil migratory behavior. Moreover, c-Abl kinase activation depends on ß2 integrin engagement, and the activated c-Abl kinase further regulates actin polymerization and membrane protrusion dynamics at the extended leading edges during neutrophil migration. In addition, we identify the Rho GEF Vav1 as a major downstream effector of c-Abl kinase. The C-terminal SH3-SH2-SH3 domain and proline-rich region of Vav1 are required for its interaction with c-Abl kinase, and c-Abl kinase probably regulates the activity of Vav1 by direct phosphorylation at Tyr-267 in the DH domain. Together, these results indicate that c-Abl kinase plays a critical role in ß2 integrin-dependent neutrophil migration by regulating Vav1 activity.

Abl family kinases regulate FcγR-mediated phagocytosis in murine macrophages.

Phagocytosis of Ab-coated pathogens is mediated through FcγRs, which activate intracellular signaling pathways to drive actin cytoskeletal rearrangements. Abl and Arg define a family of nonreceptor tyrosine kinases that regulate actin-dependent processes in a variety of cell types, including those important in the adaptive immune response. Using pharmacological inhibition as well as dominant negative and knockout approaches, we demonstrate a role for the Abl family kinases in phagocytosis by macrophages and define a mechanism whereby Abl kinases regulate this process. Bone marrow-derived macrophages from mice lacking Abl and Arg kinases exhibit inefficient phagocytosis of sheep erythrocytes and zymosan particles. Treatment with the Abl kinase inhibitors imatinib and GNF-2 or overexpression of kinase-inactive forms of the Abl family kinases also impairs particle internalization in murine macrophages, indicating Abl kinase activity is required for efficient phagocytosis. Further, Arg kinase is present at the phagocytic cup, and Abl family kinases are activated by FcγR engagement. The regulation of phagocytosis by Abl family kinases is mediated in part by the spleen tyrosine kinase (Syk). Loss of Abl and Arg expression or treatment with Abl inhibitors reduced Syk phosphorylation in response to FcγR ligation. The link between Abl family kinases and Syk may be direct, as purified Arg kinase phosphorylates Syk in vitro. Further, overexpression of membrane-targeted Syk in cells treated with Abl kinase inhibitors partially rescues the impairment in phagocytosis. Together, these findings reveal that Abl family kinases control the efficiency of phagocytosis in part through the regulation of Syk function.

Endogenous cAbl regulates receptor endocytosis.

There are two key processes underlying ligand-induced receptor endocytosis: receptor ubiquitylation and remodeling of the actin cytoskeleton. Tyrosine kinases play critical roles in both receptor endocytosis and actin reorganization. Interestingly, members of the Abl family are the only known tyrosine kinases that possess an actin-binding domain and thus have the potential to directly regulate the actin cytoskeleton. However, the role of non-transforming cAbl in receptor endocytosis remains undefined. We report that cAbl promotes ligand-induced antigen receptor endocytosis in B lymphocytes. We show that pharmacologic inhibition or genetic deletion of cAbl causes a defect in tyrosine phosphorylation of the cytoskeletal adapter CrkII. cAbl inhibition or ablation also impairs Rac activation downstream of CrkII, as well as antigen receptor capping and endocytosis. Although phosphorylation of CrkII has been suggested to maintain it in a closed inactive conformation, we demonstrate that it is in fact essential for the activation of Rac. On the other hand, association of CrkII with cCbl, a key mediator of receptor ubiquitylation, does not require CrkII phosphorylation and is cAbl-independent. Phosphorylation of cCbl itself is also cAbl-independent. Our results thus indicate that CrkII links receptor engagement to cytoskeletal remodeling by coupling cCbl- and cAbl-mediated signaling pathways that cooperatively regulate ligand-induced receptor endocytosis.

Abl tyrosine kinases in T-cell signaling.

Stimulation of the T-cell antigen receptor (TCR) leads to the activation of signaling pathways that are essential for T-cell development and the response of mature T cells to antigens. The TCR has no intrinsic catalytic activity, but TCR engagement results in tyrosine phosphorylation of downstream targets by non-receptor tyrosine kinases. Three families of tyrosine kinases have long been recognized to play critical roles in TCR-dependent signaling. They are the Src, zeta-associated protein of 70 kDa, and Tec families of kinases. More recently, the Abelson (Abl) tyrosine kinases have been shown to be activated by TCR engagement and to be required for maximal TCR signaling. Using T-cell conditional knockout mice deficient for Abl family kinases, Abl (Abl1) and Abl-related gene (Arg) (Abl2), it was recently shown that loss of Abl kinases results in defective T-cell development and a partial block in the transition to the CD4(+)CD8(+) stage. Abl/Arg double null T cells exhibit impaired TCR-induced signaling, proliferation, and cytokine production. Moreover, conditional knockout mice lacking Abl and Arg in T cells exhibit impaired CD8(+) T-cell expansion in vivo upon Listeria monocytogenes infection. Thus, Abl kinase signaling is required for both T-cell development and mature T-cell function.

c-Abl-deficient mice exhibit reduced numbers of peritoneal B-1 cells and defects in BCR-induced B cell activation.

A role for c-Abl in B cell development and signaling has been suggested by previous work showing that c-Abl-deficient mice have defects in bone marrow B cell development and that c-Abl-deficient B cells are hypoproliferative in response to antigen receptor stimulation. Here we show that in addition to defects in early B cell development, c-Abl-deficient mice have defects in peripheral B cell development, including reduced percentages of peritoneal B-1 cells as well as transitional and marginal zone B cells in the spleen. It has been shown that c-Abl kinase activity increases upon B cell receptor (BCR) stimulation and that one of the targets of tyrosine phosphorylation by c-Abl is CD19. However, the consequences of c-Abl activity on B cell activation and CD19 signaling remain unknown. Here, we show that c-Abl-deficient splenic B cells exhibit reduced calcium flux in response to CD19 cross-linking, consistent with a role for c-Abl in CD19-dependent signaling. Additionally, we show that c-Abl-deficient B cells are defective in their ability to be activated in response to antigen receptor engagement, suggesting a functional role for c-Abl in BCR-dependent activation signaling pathways.

Inhibitory receptor signaling via tyrosine phosphorylation of the adaptor Crk.

Many cellular responses, such as autoimmunity and cytotoxicity, are controlled by receptors with cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). Here, we showed that binding of inhibitory natural killer (NK) cell receptors to human leukocyte antigen (HLA) class I on target cells induced tyrosine phosphorylation of the adaptor Crk, concomitant with dephosphorylation of the guanine exchange factor Vav1. Furthermore, Crk dissociated from the guanine exchange factor C3G and bound to the tyrosine kinase c-Abl during inhibition. Membrane targeting of a tyrosine-mutated form of Crk could overcome inhibition of NK cell cytotoxicity, providing functional evidence that Crk phosphorylation contributes to inhibition. The specific phosphorylation of Crk and its dissociation from a signaling complex, observed here with two types of inhibitory receptors, expands the signaling potential of the large ITIM-receptor family and reveals an unsuspected component of the inhibitory mechanism.

A single-chain antibody/epitope system for functional analysis of protein-protein interactions.

Protein-protein interactions play a critical role in cellular processes such as signal transduction. Although many methods for identifying the binding partners of a protein of interest are available, it is currently difficult or impossible to assess the functional consequences of a specific interaction in vivo. To address this issue, we propose to modify proteins by addition of an artificial protein binding interface, thereby forcing them to interact in the cell in a pairwise fashion and allowing the functional consequences to be determined. For this purpose, we have developed an artificial binding interface consisting of a anti-Myc single-chain antibody (ScFv) and its peptide epitope. We found that the binding of an ScFv derived from anti-Myc monoclonal antibody 9E10 was relatively weak in vivo, so we selected an improved clone, 3DX, by in vitro mutagenesis and phage display. 3DX bound well to its epitope in a yeast two-hybrid system, and GST-fused 3DX also bound to several Myc-tagged proteins in mammalian cells. In vivo binding was relatively insensitive to the position of the ScFv in a fusion protein, but was improved by including multiple tandem copies of the Myc epitope in the binding partner. To test the system, we successfully replaced the SH3 domain-mediated interaction between the Abl tyrosine kinase and adaptor proteins Crk and Nck with an engineered interaction between 3DX and multiple Myc tags. We expect that this approach, which we term a functional interaction trap, will be a powerful proteomic tool for investigating protein-protein interactions.

Intracellular antibody capture technology: application to selection of intracellular antibodies recognising the BCR-ABL oncogenic protein.

The expression of antibodies inside cells to ablate protein function has the potential for disease therapy and for target validation in functional genomics. However, due to inefficient expression or folding, only a few antibodies or antibody fragments, usually as single-chain Fv antibody fragments (scFv), bind their antigens in an intracellular environment. We have established a genetic-selection technology (intracellular antibody capture, IAC) to facilitate the isolation of functional intracellular scFv from a diverse repertoire. This approach comprises an in vitro library screen with scFv-expressing bacteriophage, employing bacterially expressed antigen, followed by a yeast in vivo antibody-antigen interaction screen of the sub-library of in vitro scFv antigen-binders. Accordingly, we have isolated panels of scFv that bind intracellularly to the BCR or the ABL parts of the BCR-ABL oncogenic protein. Sequence analysis of the intracellular antibody scFv panels revealed a sequence conservation indicating an intracellular antibody consensus for both VH and VL, which could form the basis for the de novo synthesis of intracellular antibody libraries to be used with intracellular antibody-capture technology.

[Effects of anti-ABL tyrosine kinase intrabody on the growth of K562 cells in nude mice].

OBJECTIVE: To study the effects of anti-ABL tyrosine kinase intrabody on the growth of human chronic myelogenous leukemia (CML) cells in nude mice. METHODS: A recombinant retroviral vector MSCV-ibE-IRES-eGFP was constructed to express intracellular single-chain antibody (intrabody) against ABL tyrosine kinase domain in CML cells. K562 cells were transduced with the retrovirus, eGFP+ cells were then selected by fluorescence-activated cell sorting (FACS). The intrabody mRNA expression was determined by reverse transcription (RT)-polymerase chain reaction (PCR). BCR/ABL and c-ABL protein tyrosine kinase (PTK) activity in the cells was examined. Transduced cells and control group K562 cells were transplanted into nude mice respectively and the tumor sizes were dynamically observed. RESULTS: K562-ibE cell was obtained. Expression of the BCR/ABL and c-ABL protein tyrosine kinase activity of harvested K562-ibE cells were markedly inhibited. At 14, 21 and 28 days after cell injection, the tumor volumes of experimental mice were obviously smaller than that of control mice, about one half of the control groups (P < 0.05). CONCLUSION: The growth of K562-ibE cells was significantly inhibited in vivo. It is possible that inhibition of the BCR/ABL protein tyrosine kinase activity by the intrabody blocked BCR/ABL signal transduction pathway, promoted apoptosis and reduced tumorigenicity of K562 cells in vivo.