Abl In Cells Research Articles

Verbascoside potentiates the effect of tyrosine kinase inhibitors on the induction of apoptosis and oxidative stress via the Abl-mediated MAPK signalling pathway in chronic myeloid leukaemia.

Verbascoside (Verb) may exhibit potential antitumour activities in leukaemia. The present study investigated the effect of Verb, in combination with imatinib (IM), dasatinib (Das), lipopolysaccharide (LPS) and TNF, on cell survival, Abl expression, apoptosis, oxidative stress and the MAPK pathway in chronic myeloid leukaemia (CML) cells. Cell viability was determined using the WST-8 assay in K562 and R-K562 cells treated with Verb and/or IM, Das, LPS and TNF. Apoptosis and DNA damage in CML cells was detected by caspase-3 and comet analysis. The protein levels of Abl (Phospho-Tyr412), and total/phosphorylated p38, JNK and ERK in CML cells were analysed using a Colorimetric Cell-Based Assay. Oxidative stress was examined using total antioxidant and oxidant status assays. Treatment with Verb and/or tyrosine kinase inhibitors (TKIs), LPS and TNF resulted in a significant decrease in the Tyr-412 phosphorylation of Abl in K562 and R-K562 cells. In addition, cotreatment with Verb and IM or Das additively induced apoptosis by activating caspase-3 levels in both cell lines. Activation of p38 and JNK can result in growth arrest and cell death, whereas ERK stimulation results in cell division and differentiation. The present study demonstrated that cotreatment with Verb and TKIs suppressed phosphorylated-ERK1/2, whereas the levels of phosphorylated-p-38 and phosphorylated-JNK were significantly elevated by Verb and/or IM, Das, LPS and TNF, thus suggesting that Abl and Src inhibition could be involved in the effects of Verb on MAPK signalling in R-K562 cells. Furthermore, Verb elevated reactive oxygen species levels additively with TKIs in both cell lines by increasing the oxidant capacity and decreasing the antioxidant capacity. In conclusion, anti-leukemic mechanisms of Verb may be mediated by Abl protein and regulation of its downstream p38-MAPK/JNK pathway, caspase-3 and oxidative stress in CML cells.

Inhibition of Abl or Src tyrosine kinase decreased porcine circovirus type 2 production in PK15 cells

Porcine circovirus type 2 (PCV2) causes huge economic losses in the global swine industry and has a complex and poorly understood virus-host interaction mechanism. We reported that the C-terminal of the capsid protein of all PCV2 isolates shared a strictly conserved PXXP motif that may interact with SH3 domain-containing tyrosine kinases; however, its roles in PCV2 cell entry and replication remain unknown. In this study, we determined that mRNA levels of two SH3 domain-containing tyrosine kinases family (Abl and Src) had distinct profiles (wild-type and PXXP-mutated) during PCV2 infections of PK15 cells. Therefore, we hypothesized that activities of tyrosine kinases (Abl and Fyn) in PK15 cells may be hijacked by PCV2 via its PXXP motif of the Cap, to favor virus replication. Specific inhibitors PP2 of Lck/Fyn and STI-571 of Abl family kinases decreased viral production through suppression of DNA and Cap synthesis at the replication stage. However, based on indirect immunofluorescence assay (IFA), entry of PCV2 virus-like particles (VLPs) into PK15 cells was not altered. Elucidating mechanisms of PCV2-host interactions should provide new insights for development of new compounds to prevent or reduce PCV2 infections.

Coordination of signalling networks and tumorigenic properties by ABL in glioblastoma cells

The cytoplasmic tyrosine kinase ABL exerts positive or negative effects in solid tumours according to the cellular context, thus functioning as a “switch modulator”. The therapeutic effects of drugs targeting a set of signals encompassing ABL have been explored in several solid tumours. However, the net contribution of ABL inhibition by these agents remains elusive as these drugs also act on other signalling components. Here, using glioblastoma (GBM) as a cellular paradigm, we report that ABL inhibition exacerbates mesenchymal features as highlighted by down-regulation of epithelial markers and up-regulation of mesenchymal markers. Cells with permanent ABL inhibition exhibit enhanced motility and invasive capabilities, while proliferation and tumorigenic properties are reduced. Intriguingly, permanent ABL inhibition also interferes with GBM neurosphere formation and with expression of stemness markers in sphere-cultured GBM cells. Furthermore, we show that the molecular and biological characteristics of GBM cells with impaired ABL are reversible by restoring ABL levels, thus uncovering a remarkable plasticity of GBM cells to ABL threshold. A phospho-signalling screen revealed that loss of tumorigenic and self-renewal properties in GBM cells under permanent ABL inhibition coincide with drastic changes in the expression and/or phosphorylation levels of multiple signalling components. Our findings identify ABL as a crucial player for migration, invasion, proliferation, tumorigenic, and stem-cell like properties of GBM cells. Taken together, this work supports the notion that the oncogenic role of ABL in GBM cells is associated with its capability to coordinate a signalling setting that determines tumorigenic and stem-cell like properties.

Reproductive fitness advantage of BCR– ABL expressing leukemia cells

Mutations in oncogenes and tumor suppressor genes confer a fitness advantage to cells that can lead to cancer. The tumor phenotype normally results from the interaction of many mutant genes making it difficult to estimate the fitness advantage provided by any oncogene, except when tumors depend on one oncogene only. We utilize a model of chronic myeloid leukemia (CML), to quantitate the fitness advantage conferred by expression of BCR– ABL in hematopoietic cells from in vivo patient data. We show that BCR– ABL expression provides a high fitness advantage, which explains why this single mutation drives the chronic phase of CML.

C-Abl-mediated Phosphorylation of WAVE3 Is Required for Lamellipodia Formation and Cell Migration

The activity of the Wiskott-Aldrich syndrome-related WAVE3 protein is critical for the regulation of the Arp2/3-dependent cytoskeleton organization downstream of Rac-GTPase. The Ableson (Abl) non-receptor tyrosine kinase is also involved in the remolding of actin cytoskeleton in response to extracellular stimuli. Here we show that platelet-derived growth factor stimulation of cultured cells results in WAVE3-Abl interaction and localization to the cell periphery. WAVE3-Abl interaction promotes the tyrosine phosphorylation of WAVE3 by Abl, and STI-571, a specific inhibitor of Abl kinase activity, abrogates the Abl-mediated phosphorylation of WAVE3. We have also shown that Abl targets and phosphorylates four tyrosine residues in WAVE3 and that the Abl-dependent phosphorylation of WAVE3 is critical for the stimulation of lamellipodia formation and cell migration. Our results show that the activation of WAVE3 to promote actin remodeling is enhanced by the c-Abl-mediated tyrosine phosphorylation of WAVE3.

An enhanced association of RACK1 with Abl in cells transfected with oncogenic ras

The cellular RACK1 was shown in association with Abl in BALB/3T3 cells transfected with S- ras(Q 61K) by immunoprecipitation. An identical finding was demonstrated with cells transfected with the embryonic E- ras, but not in cells without transformation. The Abl-RACK1 of transformed cells as resolvable with Triton X-114 was found with little affinity for FAK, PY 397-FAK and integrin. Of interests, PY 397-FAK in the membrane skeleton of transformed cells was shown in significant quantities on the Western blot. However the PY 397-FAK of transformed cells was not functionally able to react with RACK1 and recruit cytokeratin-1, a substrate of Src, indicating that PY 397-FAK is not operative to transmit integrin signals. In other words, the Abl-RACK1 of transformed cells cannot replace the Src-RACK1 of cells without transformation to bridge PY 397-FAK and cytokeratin-1 for integrin signals, and the formation of Abl-RACK1 in transformed cells may block the association of PY 397-FAK-RACK1. We characterized Abl and RACK1 from transformed cells by chromatography on a HiTrap-PEP Taxol affinity column, constructed from a β-tubulin peptide specific for Taxol binding (PEP Taxol). However, the Triton X-100 cannot achieve the same resolution of Abl-RACK1 from plasma membrane as is shown with Triton X-114. A significant fraction of Abl was deposited at the membrane skeleton and was therefore not accessible with Triton X-100. Half of Abl resolved with Triton X-100 was demonstrated to have catalytic activity as shown with positive phosphotyrosine staining on the Western blot and competitive elution with a specific phosphate, such as sodium β-glycerophosphate, from HiTrap-PEP Taxol, but this was not associated with RACK1. No significant difference of RACK1 was found in Triton X-100 resolvable membrane preparations from cells with and without transformations. Future studies are planned to differentiate the mechanism operative for RACK1 associated and RACK1 freed Abl in cells transformed with oncogenic ras.

Conformational Variations of a Key Enzyme Offer Clues to Cancer-Drug Resistance

The smooth operation of a cell requires that critical changes in molecular activity take place when and where they're supposed to. One family of enzymes, called tyrosine kinases, plays an important role in effecting these changes by attaching phosphate groups to other proteins in a process called phosphorylation. This chemical reaction initiates a tag team of signals that lead from a stimulus, through a series of intervening molecules, to a protein controlling a specific cellular process. To phosphorylate the recipient protein, a tyrosine kinase must first grab onto it with a highly specific lock-and-key connection—each kinase tailored to its unique target. This happens only when the kinase is in an active conformation—when the key has been twisted or bent by a signal from the previous molecule in the tag team into a conformation that will fit the shape of its target protein. C-Abl is a tyrosine kinase that helps convey messages about cell growth and movement. Normally it does so only when it is activated by the preceding protein in the signaling pathway. But when cells possess a mutated form of c-Abl called BCR-Abl, the tyrosine kinase gets stuck in its active form, and sends the cell constant encouragement to grow. The miscommunication manifests itself as the blood cancer chronic myeloid leukemia, or CML. A common treatment for CML is a drug called imatinib (known commercially as Gleevec or Glivec). Imatinib binds to a conformation of Abl (either c- or BCR-) in which a section called DFG has been rotated about 180 degrees from the active form, “DFG-Asp In,” to the inactive “DFG-Asp Out” conformation, preventing the tyrosine kinase from doing its phosphate-transfer job. But in some CML cells, imatinib doesn't work. A better understanding of the various conformations of the kinase domain of Abl will shed light on not only the molecule's function, but also how imatinib does—and sometimes doesn't—inhibit it. Nicholas M. Levinson, Olga Kuchment, John Kuriyan, and colleagues explored the ins and outs of the kinase portion of normal and imatinib-resistant (mutant) Abl using crystallography, a process that makes it possible to take “snapshots” of proteins in various conformations. With the use of a novel synthetic bisubstrate analog inhibitor, the researchers found four previously undescribed forms of the kinase domain of Abl. They analyzed the position of various key amino acids and groups of amino acids within these conformations, as well as within a fifth, already-known conformation: that of Abl with imatinib attached. In doing so, the researchers uncovered a big surprise: an inactive Abl conformation that differs dramatically from the rest of the conformations studied—and from all previously known conformations of Abl. This unprecedented inactive conformation was very similar to the inactive form of another kinase called Src, in which DFG is not flipped but another part of the kinase, an alpha helix, is swung out from the active conformation, known as αC-Glu In, into the inactive form, αC-Glu Out. The researchers explored the functional significance of the odd inactive conformation using clues garnered from the other conformations they observed and from sophisticated computer simulations that allowed them to model changes from one configuration to another. The results of the simulations supported their speculation that the Src-like form might be an intermediate that facilitates the DFG flip. Could this be a clue to imatinib resistance? Some forms of imatinib resistance are known to result from mutations that block the binding of imatinib to Abl. More commonly, however, mutations prevent Abl from adopting the conformation that imatinib binds to. The researchers noted that such imatinib-resistant mutations tend to destabilize the Src-like conformation of Abl more than the active or imatinib-bound conformations of Abl, suggesting that this conformation does indeed play a role in whether imatinib is effective in blocking the activity of Abl in CML cells.

COMBO-FISH for focussed fluorescence labelling of gene domains: 3D-analysis of the genome architecture of abl and bcr in human blood cells

Structural analysis and nanosizing of gene domains requires not only high-resolution microscopy but also improved techniques of fluorescence labelling strongly focussed on the gene domains. To investigate the architecture of abl and bcr in blood cell nuclei forming the Philadelphia chromosome in CML, we applied COMBO-FISH using specifically colocalising combinations of triple strand forming oligonucleotide probes for abl on chromosome 9 and bcr on chromosome 22. Each probe set consisting of 31 homopyrimidine oligonucleotides was computer selected from the human genome database. Measurements by 3D microscopy were compared to results obtained after standard FISH using commercially available abl/bcr BAC probes. The relative radial fluorescence distributions in lymphocyte cell nuclei of healthy donors in comparison to cell nuclei of blood cells of CML patients showed a strong correlation in the location of abl and bcr for both labelling techniques. The absolute distances of the homologous bcr domains and the abl domain-nuclear center-abl domain angles in cell nuclei of CML donors differed significantly from those of healthy donors only when COMBO-FISH was applied. These results indicate that COMBO-FISH may be more sensitive than standard FISH in case of slight modifications in the genome architecture.

Regulation of Vasodilator-stimulated Phosphoprotein Phosphorylation and Interaction with Abl by Protein Kinase A and Cell Adhesion

Members of the vasodilator-stimulated phosphoprotein (VASP) family are important regulators of actin cytoskeletal dynamics whose functions and protein-protein interactions are regulated by phosphorylation by the cAMP-dependent protein kinase (PKA). Herein, we show that phosphorylation of VASP is dynamically regulated by cellular adhesion to extracellular matrix. Detachment of cells stimulated PKA activity and induced PKA-dependent phosphorylation of VASP and the related murine-Enabled (Mena) protein. VASP and Mena were rapidly dephosphorylated immediately following reattachment but showed an intermediate level of phosphorylation during active cell spreading. This pattern correlated closely with adhesion-dependent changes in PKA activity. The in vivo interaction of VASP with the Abl tyrosine kinase, shown here for the first time, was readily apparent in adherent cells, lost following cellular detachment, and induced upon reattachment to matrix. Importantly, inhibition of PKA activity prevented phosphorylation of VASP and dissociation of VASP-Abl complexes after cellular detachment, whereas activation of PKA completely eliminated the co-immunoprecipitation of Abl activity with VASP. These data establish a new biochemical link between cell adhesion and regulation of VASP proteins and provide the first demonstration of a regulated interaction between VASP and Abl in mammalian cells.

Abelson kinase regulates epithelial morphogenesis in Drosophila.

Activation of the nonreceptor tyrosine kinase Abelson (Abl) contributes to the development of leukemia, but the complex roles of Abl in normal development are not fully understood. Drosophila Abl links neural axon guidance receptors to the cytoskeleton. Here we report a novel role for Drosophila Abl in epithelial cells, where it is critical for morphogenesis. Embryos completely lacking both maternal and zygotic Abl die with defects in several morphogenetic processes requiring cell shape changes and cell migration. We describe the cellular defects that underlie these problems, focusing on dorsal closure as an example. Further, we show that the Abl target Enabled (Ena), a modulator of actin dynamics, is involved with Abl in morphogenesis. We find that Ena localizes to adherens junctions of most epithelial cells, and that it genetically interacts with the adherens junction protein Armadillo (Arm) during morphogenesis. The defects of abl mutants are strongly enhanced by heterozygosity for shotgun, which encodes DE-cadherin. Finally, loss of Abl reduces Arm and α-catenin accumulation in adherens junctions, while having little or no effect on other components of the cytoskeleton or cell polarity machinery. We discuss possible models for Abl function during epithelial morphogenesis in light of these data.

The substrate specificity of the catalytic domain of Abl plays an important role in directing phosphorylation of the adaptor protein Crk

c-Abl preferentially phosphorylates peptide substrates that contain proline at the P+3 site (relative to the phosphorylated tyrosine). We previously described a mutant form of the Abl catalytic domain (Y569W) with altered substrate specificity at the P+3 position, as measured using synthetic peptides. In this study, we examine the phosphorylation of Crk, a protein substrate of Abl that is phosphorylated in the sequence Tyr221-Ala-Gln-Pro. In vitro, phosphorylation of Crk by Y569W Abl is greatly reduced relative to wild-type Abl. Overexpression of Y569W mutant Abl in 293T kidney cells produces a similar overall pattern of tyrosine phosphorylation as wild-type Abl, indicating that not all cellular proteins depend on Pro at P+3 for Abl recognition. However, phosphorylation of Crk by Y569W Abl in these cells is markedly reduced relative to wild-type Abl. A truncated form of Abl lacking the C-terminal polyproline region is not able to phosphorylate Crk in these assay conditions. Thus, proper phosphorylation of Crk by Abl depends not only on the interaction of the Crk SH3 domain with the Abl polyproline region, but also on the recognition of amino acids surrounding tyrosine by the Abl catalytic domain.

Bcl-2-independent Bcr-Abl-mediated resistance to apoptosis: protection is correlated with up regulation of Bcl-xL.

Bcr - Abl is the molecule responsible for both the transformation phenotype and the resistance to chemotherapeutic drugs found in chronic myelogenous leukemia (CML) cells. Wild-type HL-60, a transformed pro-myelocytic cell line, is very susceptible to apoptosis-inducing agents. We show here that expression of Bcr - Abl in HL-60 cells rendered them extremely resistant to apoptosis induced by a wide variety of agents. The anti-apoptotic effect of Bcr - Abl was found to be independent of the phase of the cell cycle. Treatment with antisense oligonucleotides directed to bcr decreased the expression of the ectopic bcr - abl and restored susceptibility to apoptosis. Double mutations affecting the autophosphorylation site and the phosphotyrosine-binding motif (FLVRES) have been previously shown to impair the transforming activity of Bcr - Abl in fibroblasts and hematopoietic cells, however HL-60 cells expressing this double mutant molecule exhibited the same level of resistance to apoptosis as those expressing the wild-type Bcr - Abl. Interestingly, wild type and mutant Bcr - Abl induced in HL-60 cells a dramatic down regulation of Bcl-2 and increased the levels of Bcl-xL. The level of Bax did not change in response to the presence of Bcr - Abl. Antisense oligonucleotides targeted to bcl-x downregulated the expression of Bcl-x, and increased the susceptibility of HL-60. Bcr - Abl cells to staurosporine. Importantly, HL-60 cells overexpressing Bcl-xL showed higher expression of Bcl-xL but lower resistance to apoptosis when compared to HL-60. Bcr - Abl cells. The results described here show that Bcr - Abl is a powerful mammalian anti-apoptotic molecule and can act independently of Bcl-2. Bcl-xL, however, seems to participate in part in Bcr - Abl-mediated resistance to apoptosis in HL-60 cells.

Bcr - Abl-mediated resistance to apoptosis is independent of PI 3-kinase activity.

The Bcr - Abl tyrosine kinase is responsible for the oncogenic phenotype observed in Philadelphia chromosome-positive leukemia and induces resistance to apoptotic cell death in a variety of cell types. Recent evidence supports the hypothesis that these two properties of Bcr - Abl are derived from cooperative but distinct signaling pathways. Phosphatidylinositol 3-kinase (PI3K), which has been suggested to associate with and become activated by Bcr - Abl, has been shown to be required for Bcr - Abl-mediated cell growth. Also, PI3K has been implicated in resistance to apoptosis induced by some growth factors. We therefore examined the role of PI 3-kinase in the anti-apoptotic effect of Bcr - Abl. First, we confirmed that expression of p185(bcr - abl) in HL-60 cells, which renders these cells resistant to apoptosis, induces tyrosine phosphorylation of the p85 subunit of PI3K. Consistent with this result, we observed a 20-fold increase in PI3K activity upon immunoprecipitation of tyrosine-phosphorylated proteins from cells expressing Bcr - Abl versus control cells. Nevertheless, treatment of HL-60.p185(bcr - abl) cells with wortmannin, a potent inhibitor of PI3K, eliminated PI3K activity but did not interfere with the resistance of these cells to apoptosis. Similar results were obtained with the CML line K562 and with the BaF3.p185 (bcr - abl) line. We conclude that while PI3K participates in the anti-apoptotic response mediated by some growth factors and also seems to be important for the growth of Bcr-Abl-positive cells, it does not play any role in Bcr - Abl-mediated resistance to apoptosis.

An 8-kilobase abl RNA transcript in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a clonal hematologic malignancy characterized by a reciprocal translocation between chromosomes 9 and 22 [t(9;22)] in greater than 90% of cases. This translocation results in a short chromosome 22, termed the Philadelphia (Ph1 or 22q-) chromosome. Recently, the cellular oncogenes abl and sis were mapped to human chromosomes 9 and 22, respectively. Moreover, abl was shown to be translocated from chromosome 9 to 22 and sis from chromosome 22 to 9 in CML patients with t(9;22). These findings raised the possibility that one or both of these oncogenes is activated and directly involved in the development of the disease. We analyzed expression of the abl and sis oncogenes in leukemic cells from CML patients with t(9;22). We found that sis is not expressed but that abl is transcribed into an 8-kilobase RNA. This abl RNA is also present in two leukemic cell lines (EM2 and K562), which were derived from CML patients and contain the t(9;22). This 8-kilobase RNA is not detected in normal cells, in other human leukemias without t(9;22), or in human cell lines that lack t(9;22). The consistent presence of this abl RNA transcript in CML with t(9;22) suggests that it is a consequence of abl translocation and that it plays a role in the development of this leukemia.