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SUMMARY Targeting mitochondrial metabolism has emerged as a treatment option for cancer patients. The ABL tyrosine kinases promote metastasis, and enhanced ABL signaling is associated with a poor prognosis in lung adenocarcinoma patients. Here we show that ABL kinase allosteric inhibitors impair mitochondrial integrity and decrease oxidative phosphorylation. To identify metabolic vulnerabilities that enhance this phenotype, we utilized a CRISPR/Cas9 loss-of-function screen and identified HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway and target of statin therapies, as a top-scoring sensitizer to ABL inhibition. Combination treatment with ABL allosteric inhibitors and statins decreases metastatic lung cancer cell survival in vitro in a synergistic manner. Notably, combination therapy in mouse models of lung cancer brain metastasis and therapy resistance impairs metastatic colonization with a concomitant increase in animal survival. Thus, metabolic combination therapy might be effective to decrease metastatic outgrowth, leading to increased survival for lung cancer patients with advanced disease., Graphical Abstract, In brief Metabolic reprogramming in tumors is an adaptation that generates vulnerabilities that can be exploited for developing new therapies. Here Luttman et al. identify synergism between ABL allosteric inhibitors and lipophilic statins to impair metastatic lung cancer cell outgrowth and colonization, leading to increased survival in mouse models of advanced disease.

Background The mechanism underlying chromosome rearrangement in nasopharyngeal carcinoma (NPC) remains elusive. It is known that most of the aetiological factors of NPC trigger oxidative stress. Oxidative stress is a potent apoptotic inducer. During apoptosis, chromatin cleavage and DNA fragmentation occur. However, cells may undergo DNA repair and survive apoptosis. Non-homologous end joining (NHEJ) pathway has been known as the primary DNA repair system in human cells. The NHEJ process may repair DNA ends without any homology, although region of microhomology (a few nucleotides) is usually utilised by this DNA repair system. Cells that evade apoptosis via erroneous DNA repair may carry chromosomal aberration. Apoptotic nuclease was found to be associated with nuclear matrix during apoptosis. Matrix association region/scaffold attachment region (MAR/SAR) is the binding site of the chromosomal DNA loop structure to the nuclear matrix. When apoptotic nuclease is associated with nuclear matrix during apoptosis, it potentially cleaves at MAR/SAR. Cells that survive apoptosis via compromised DNA repair may carry chromosome rearrangement contributing to NPC tumourigenesis. The Abelson murine leukaemia (ABL) gene at 9q34 was targeted in this study as 9q34 is a common region of loss in NPC. This study aimed to identify the chromosome breakages and/or rearrangements in the ABL gene in cells undergoing oxidative stress-induced apoptosis. Results In the present study, in silico prediction of MAR/SAR was performed in the ABL gene. More than 80% of the predicted MAR/SAR sites are closely associated with previously reported patient breakpoint cluster regions (BCR). By using inverse polymerase chain reaction (IPCR), we demonstrated that hydrogen peroxide (H2O2)-induced apoptosis in normal nasopharyngeal epithelial and NPC cells led to chromosomal breakages within the ABL BCR that contains a MAR/SAR. Intriguingly, we detected two translocations in H2O2-treated cells. Region of microhomology was found at the translocation junctions. This observation is consistent with the operation of microhomology-mediated NHEJ. Conclusions Our findings suggested that oxidative stress-induced apoptosis may participate in chromosome rearrangements of NPC. A revised model for oxidative stress-induced apoptosis mediating chromosome rearrangement in NPC is proposed. Electronic supplementary material The online version of this article (10.1186/s40246-018-0160-8) contains supplementary material, which is available to authorized users.

Discoidin, CUB, and LCCL domain containing 2 (DCBLD2) is a neuropilin-like transmembrane scaffolding receptor with known and anticipated roles in vascular remodeling and neuronal positioning. DCBLD2 is also up-regulated in several cancers and can drive glioblastomas downstream of activated epidermal growth factor receptor. While a few studies have shown either a positive or negative role for DCBLD2 in regulating growth factor receptor signaling, little is known about the conserved signaling features of DCBLD family members that drive their molecular activities. We previously identified DCBLD2 tyrosine phosphorylation sites in intracellular YxxP motifs that are required for the phosphorylation-dependent binding of the signaling adaptors CRK and CRKL (CT10 regulator of kinase and CRK-like). These intracellular YxxP motifs are highly conserved across vertebrates and between DCBLD family members. Here, we demonstrate that, as for DCBLD2, DCBLD1 YxxP motifs are required for CRKL–SH2 (Src homology 2) binding. We report that Src family kinases (SFKs) and Abl differentially promote the interaction between the CRKL–SH2 domain and DCBLD1 and DCBLD2, and while SFKs and Abl each promote DCBLD1 and DCBLD2 binding to the CRKL–SH2 domain, the effect of Abl is more pronounced for DCBLD1. Using high-performance liquid chromatography coupled with tandem mass spectrometry, we quantified phosphorylation at several YxxP sites in DCBLD1 and DCBLD2, mapping site-specific preferences for SFKs and Abl. Together, these data provide a platform to decipher the signaling mechanisms by which these novel receptors drive their biological activities.

Heterotrimeric G-proteins are essential cellular signal transducers. One of the G-proteins, Gα13, is critical for actin cytoskeletal reorganization, cell migration, cell proliferation and apoptosis. Previously we have shown that Gα13 is essential for both G protein-coupled receptor (GPCR) and receptor tyrosine kinase (RTK)-induced actin cytoskeletal reorganization such as dynamic dorsal ruffle turnover and cell migration. However, the mechanism by which Gα13 signals to actin cytoskeletal reorganization is not completely understood. Here we show that Gα13 directly interacts with Abl tyrosine kinase which is a critical regulator of actin cytoskeleton. This interaction is critical for Gα13-induced dorsal ruffle turnover, endothelial cell remodeling, and cell migration. Our data uncover a new molecular signaling pathway by which Gα13 controls actin cytoskeletal reorganization.

Background MicroRNAs (miRNAs) have emerged as crucial factors that regulate proliferation and apoptosis of cardiac c-kit+ cells. Although much is known about their role in maintaining cardiac c-kit+ cell pluripotency, the mechanisms by which they affect cell fate decisions that are an essential part of the repair of heart failure remain poorly understood. Methods Cardiac c-kit+ cells were obtained from Balb/c mice and cultured in vitro. Lentiviral vectors of miR199a-3p, its corresponding anti-miRNA, or short hairpin RNA against Cables1 were transfected into cells. The proliferation of cardiac c-kit+ cells was evaluated using EdU and flow cytometry. Furthermore, we examined cell apoptosis by flow cytometry under treatment with 200nM angiotensin II for 48 h. The levels of miR199a-3p and Cables1 mRNA were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was performed to examine the expression of Cables1 and P53 proteins. Results We demonstrated a significantly decreased expression of miR199a-3p in heart failure samples compared with healthy donors. Meanwhile, we identified miR199a-3p as a proliferation- and apoptosis-associated regulator impacted through Cdk5 and Abl enzyme substrate 1 (CABLES1) targeting, and also attributed their repression to P53 protein expression. We further demonstrated that P53 induced miR199a-3p expression and, in turn, miR199-3p decreased P53 activity. Conclusion Collectively, our findings uncover one new mechanism by which P53 induced miR199a-3p expression and, in turn, miR199-3p decreased P53 activity. Therefore, miR199a-3p and P53 are coupled through CABLES1 and comprise a novel negative feedback loop that likely contributes to cardiac c-kit+ cell proliferation and apoptosis.

Background Dysregulation of the Src pathway has been shown to be important at various stages of cancer. Dasatinib is a potent Src/Abl inhibitor and has demonstrated to have anti-proliferative and anti-invasive activity in many preclinical models. The objective of this study was to determine the anti-tumor activity of dasatinib using in vitro and in vivo preclinical colorectal (CRC) models. Methods CRC cell lines and patient-derived tumor explant (PDX) models were used to investigate the efficacy of dasatinib. We treated 50 CRC cell lines with dasatinib for 72 hours and proliferation was assayed by a sulforhodamine B (SRB) assay; an IC50 ≤ 0.08 μmol/L was considered sensitive. We treated 17 patient-derived CRC explants with dasatinib (50 mg/kg/day, administered once-daily) for 28 days to determine in vivo efficacy. Tumor growth inhibition (TGI) ≥ 50% was considered sensitive. Results We found that 8 out of 50 CRC cell lines reached an IC50 ≤ 0.08 μmol/L with dasatinib treatment. In addition, of 17 CRC explants grown in the xenograft mouse model, 2 showed sensitivity to dasatinib. The anti-tumor effects observed in this study were a result of G1 cell cycle arrest as the dasatinib sensitive CRC cell lines exhibited G1 inhibition. Moreover, those CRC cell lines that were responsive (0.08 μmol/L) to treatment demonstrated a significant baseline increase in Src and FAK gene expression. Conclusion Dasatinib demonstrated significant anti-proliferative activity in a subset of CRC cell lines in vitro, especially in those with increased Src expression at baseline, but only showed modest efficacy in CRC explants. Dasatinib is currently being studied in combination with chemotherapy in patients with advanced CRC, as its use as a single agent appears limited.

Crk, the prototypical member of a class of Src homology-2 (SH2) and Src homology-3 (SH3) domain containing proteins that controls the coordinated assembly of signaling complexes, is regulated by phosphorylation of Y221 in the linker region, which forms an intramolecular SH2-pY221 auto-clamp to interrupt SH2-N-terminal SH3 domain (SH3N) signaling. Here, we show using LC-MS/MS and by generating phospho-specific antibodies that, iteratively with Y221, the Crk C-terminal SH3 domain (SH3C) is routinely phosphorylated on Y239 and/or Y251 by several extracellular stimuli known to engage Crk. Although phosphorylation at Y221 auto-inhibits the Crk SH2, phosphorylation of the SH3C generates an unconventional phosphoSH3C-SH3N unit in which the SH3N is fully functional to bind polyproline type II ligands and the phosphoSH3C binds de novo to other SH2 domains. Using high-throughput SH2 domain profiling, artificial neural network and position-specific scoring matrix-based bioinformatics approaches, and unbiased mass spectometry, we found that the phosphoSH3C binds several SH2 domain containing proteins, including specific non-receptor tyrosine kinases-Abl via pY251 and C-terminal Src kinase via pY239. Functionally, we show that the phosphoSH3C modulates the Abl-mediated phenotypes of cell spreading and motility. Together, these studies describe a versatile mechanism wherein phosphorylation of Crk at Y221 is not an off switch but redirects signaling from the SH2-SH3N axis to a phosphoSH3C-SH3N axis, with the SH3N as a common denominator.

The mammalian ABL1 gene encodes the ubiquitously expressed nonreceptor tyrosine kinase ABL. In response to growth factors, cytokines, cell adhesion, DNA damage, oxidative stress, and other signals, ABL is activated to stimulate cell proliferation or differentiation, survival or death, retraction, or migration. ABL also regulates specialized functions such as antigen receptor signaling in lymphocytes, synapse formation in neurons, and bacterial adhesion to intestinal epithelial cells. Although discovered as the proto-oncogene from which the Abelson leukemia virus derived its Gag-v-Abl oncogene, recent results have linked ABL kinase activation to neuronal degeneration. This body of knowledge on ABL seems confusing because it does not fit the one-gene-one-function paradigm. Without question, ABL capabilities are encoded by its gene sequence and that molecular blueprint designs this kinase to be regulated by subcellular location-dependent interactions with inhibitors and substrate activators. Furthermore, ABL shuttles between the nucleus and the cytoplasm where it binds DNA and actin--two biopolymers with fundamental roles in almost all biological processes. Taken together, the cumulated results from analyses of ABL structure-function, ABL mutant mouse phenotypes, and ABL substrates suggest that this tyrosine kinase does not have its own agenda but that, instead, it has evolved to serve a variety of tissue-specific and context-dependent biological functions.

Brain tumors frequently recur or progress as focal masses after treatment with ionizing radiation. However, the mechanisms underlying the repopulation of tumor cells after radiation have remained unclear. In this study, we show that cellular signaling from Abelson murine leukemia viral oncogene homolog (Abl) to protein kinase Cδ (PKCδ) is crucial for fractionated-radiation-induced expansion of glioma-initiating cell populations and acquisition of resistance to anticancer treatments. Treatment of human glioma cells with fractionated radiation increased Abl and PKCδ activity, expanded the CD133-positive (CD133+) cell population that possesses tumor-initiating potential and induced expression of glioma stem cell markers and self-renewal-related proteins. Moreover, cells treated with fractionated radiation were resistant to anticancer treatments. Small interfering RNA (siRNA)-mediated knockdown of PKCδ expression blocked fractionated-radiation-induced CD133+ cell expansion and suppressed expression of glioma stem cell markers and self-renewal-related proteins. It also suppressed resistance of glioma cells to anticancer treatments. Similarly, knockdown of Abl led to a decrease in CD133+ cell populations and restored chemotherapeutic sensitivity. It also attenuated fractionated-radiation-induced PKCδ activation, suggesting that Abl acts upstream of PKCδ. Collectively, these data indicate that fractionated radiation induces an increase in the glioma-initiating cell population, decreases cellular sensitivity to cancer treatment and implicates activation of Abl–PKCδ signaling in both events. These findings provide insights that might prove pivotal in the context of ionising-radiation-based therapeutic interventions for brain tumors.

Sialylated lipids serve as cellular receptors for polyomaviruses. Using pharmacological inhibitors and cell lines derived from knockout mice, we demonstrate that Abl family tyrosine kinases are required for replication of mouse polyomavirus and BK virus, a human polyomavirus associated with allograft failure following kidney transplantation. We show that decreasing Abl family kinase activity results in low levels of cell surface ganglioside receptors for mouse polyomavirus and that inhibition of sialidase activity promotes virion binding in the absence of Abl family kinase activity. These data provide evidence that Abl family kinases reduce ganglioside turnover in the plasma membrane by inhibiting host cell sialidase activity. Thus, Abl family kinases regulate the susceptibility of cells to polyomavirus infection by modulating gangliosides required for viral attachment.

In the present study, we have examined the effect of perforin (pfp) deficiency in 4 models of mouse B-cell lymphomagenesis. We have examined pfp loss on the background of either Mlh1 tumor suppressor allele loss or oncogene expression [Ig heavy chain (Eμ)-v-Abl, Eμ-myc, and vav-bcl2]. Pfp was shown to act as a suppressor of B-cell malignancies characteristically driven by v-Abl or bcl-2, whereas Mlh loss cooperated in accelerating spontaneous B-cell lymphomas characteristic of pfp loss. No protective role for pfp was observed in the more aggressive Eμ-myc model of B-cell lymphoma. These transgenic models have allowed us to distinguish the role of pfp in surveillance of B-cell lymphomagenesis, as opposed to its loss simply driving the onset of a spontaneous lymphoma characteristic of pfp deficiency.

The precise mechanisms by which Abl oncogenes transform hematopoietic cells are unknown. We have examined the role of Pim kinases in v-Abl–mediated transformation. In v-Abl transformants, expression of Pim-1 and Pim-2, but not Pim-3, is dependent on Abl kinase activity. Transformation assays demonstrate that v-Abl cannot efficiently transform bone marrow cells derived from Pim-1−/−/Pim-2−/− mice. Ectopic expression of either Pim-1 or Pim-2 in Pim-1−/−/Pim-2−/− cells restores transformation by v-Abl, strongly suggesting that either Pim-1 or Pim-2 is required for v-Abl–mediated tumorigenesis. Interestingly, the combined deficiency of Pim-1, Pim-2, and Suppressor of Cytokine Signalling (SOCS)-1 resulted in partial restoration of v-Abl transformation efficiency. In addition, Pim kinases are involved in modification of SOCS-1 and in regulating SOCS-1 protein levels in v-Abl–transformed cells. Furthermore, Pim kinases regulate the proapoptotic proteins Bcl-XS and BAD. Pim kinases inhibit the expression of Bcl-XS. Pim deficiency decreases the phosphorylation levels of BAD, whereas ectopic expression of Pim-1 increases the amount of phospho-BAD. This correlates with an increased protection from apoptosis in Abl transformants expressing Pim kinases. Together, these data suggest that Pim kinases play a key role in the v-Abl transformation, possibly via participating in modulation of SOCS-1 and via regulating the apoptotic signaling.

Like the v-Onc proteins encoded by many transforming retroviruses, the v-Abl protein is expressed as a Gag-Onc fusion. Although the Gag-derived myristoylation signal targets the v-Abl protein to the plasma membrane, the protein contains the entire MA and p12 sequences and a small number of CA-derived residues. To understand the role of Gag sequences in transformation, mutants lacking portions of these sequences were examined for the effects of these deletions on v-Abl function and localization. Deletion of the N-terminal third of p12 or all of p12 enhanced the transformation of both pre-B cells and NIH 3T3 cells. In contrast, deletions in MA or a deletion removing all of Gag except the first 34 amino acids important for myristoylation highly compromised the ability to transform either cell type. Although all of the mutant proteins retained kinase activity, those defective in transformation were reduced in their ability to activate Erk, suggesting a role for Gag sequences in v-Abl signaling. Immunofluorescence analysis revealed that a v-Abl protein retaining only the first 34 amino acids of Gag localized to the nucleus. These data indicate that Gag sequences are important for normal v-Abl signaling and that they suppress nuclear localization of the molecule.

The multi-functional protein c-Cbl is an important modulator of actin cytoskeletal dynamics in diverse biological systems. We had previously reported that c-Cbl facilitates cell spreading and adhesion and suppresses anchorage-independent growth of v-Abl-transformed fibroblasts. To assess the importance of membrane localization of c-Cbl for the observed effects of c-Cbl in v-Abl-3T3 cells, we first mapped the membrane interactive domain(s) of c-Cbl. Our studies indicate that localization of c-Cbl to the membrane is likely to be mediated by the tyrosine kinase binding (TKB) domain and the proline-rich region of c-Cbl, whereas C-terminal tyrosine phosphorylation does not play a role. The association of v-Cbl, which encompasses the TKB domain, with the membrane was unusual as it was not entirely dependent on SH2-phosphotyrosine interactions. Our studies further demonstrate that Src-like adaptor protein (SLAP), which binds to v-Cbl in a tyrosine phosphorylation-independent manner, facilitates membrane association of Cbl. The interaction between c-Cbl and SLAP in v-Abl-3T3 cells positively influenced c-Cbl-mediated spreading and adhesion of these cells. SLAP appears to exert its effects not simply by increasing the amount of c-Cbl in the membrane but by facilitating binding of p85-phosphatidylinositol-3-kinase (PI3K) with membrane-associated c-Cbl.

The DNA damage response network stimulates microRNA (miRNA) biogenesis to coordinate repair, cell cycle checkpoints, and apoptosis. The multistep process of miRNA biogenesis involves the cleavage of primary miRNAs by the microprocessor complex composed of the ribonuclease Drosha and the RNA binding protein DGCR8. We found that the tyrosine kinase ABL phosphorylated DGCR8, a modification that was required for the induction of a subset of miRNAs after DNA damage. Focusing on the miR-34 family, ABL stimulated the production of miR-34c, but not miR-34a, through Drosha/DGCR8-dependent processing of primary miR-34c (pri-miR-34c). This miRNA-selective effect of ABL required the sequences flanking the precursor miR-34c (pre-miR-34c) stem-loop. In pri-miRNA processing, DGCR8 binds the pre-miR stem-loop and recruits Drosha to the miRNA. RNA cross-linking assays showed that DGCR8 and Drosha interacted with pri-miR-34c, but we found an inverse correlation between ABL-stimulated processing and DGCR8 association with pri-miR-34c. When coexpressed in HEK293T cells, ABL phosphorylated DGCR8 at Tyr(267). Ectopic expression of a Y267F-DGCR8 mutant reduced the recruitment of Drosha to pri-miR-34c and prevented ABL or Drosha from stimulating the processing of pri-miR-34c. In mice engineered to express a nuclear import-defective mutant of ABL, miR-34c, but not miR-34a, expression was reduced in the kidney, and apoptosis of the renal epithelial cells was impaired in response to cisplatin. These results reveal a new pathway in the DNA damage response wherein ABL-dependent tyrosine phosphorylation of DGCR8 stimulates the processing of selective primary miRNAs.

The tumor suppressor STAT1 is considered a key regulator of the surveillance of developing tumors. Here, we describe an unexpected tumor-promoting role for STAT1 in leukemia. STAT1(-/-) mice are partially protected from leukemia development, and STAT1(-/-) tumor cells induce leukemia in RAG2(-/-) and immunocompetent mice with increased latency. The low MHC class I protein levels of STAT1(-/-) tumor cells enable efficient NK cell lysis and account for the enhanced tumor clearance. Strikingly, STAT1(-/-) tumor cells acquire increased MHC class I expression upon leukemia progression. These findings define STAT1 as a tumor promoter in leukemia development. Furthermore, we describe the upregulation of MHC class I expression as a general mechanism that allows for the escape of hematopoietic malignancies from immune surveillance.

N-acetyl-cysteine (NAC) has been reported to have anticancer properties such as counteractions against mutagens and prevention of tumor progression by scavenging reactive oxygen species (ROS). However, here we report that NAC can enhance the anchorage-independent growth of cells transformed by activated ABL tyrosine kinases or Ras. This effect was not dependent on loss of focal adhesion kinase activation. NAC rescued cell growth that was suppressed by heat shock protein (Hsp) 90 inhibitors possibly by chemical modification of their quinone moiety. NAC rendered Rat1/BCR-ABL cells resistance to a Ras inhibitor manumycin in soft agar colony formation. In the absence of Hsp90 inhibitors, NAC stimulated the activation of MAP kinase in BCR-ABL-transformed but not in the parental Rat1 cells. We propose that NAC should be used carefully in cancer treatment. (Cancer Sci 2005; 96: 240 –244)

The v-Abl tyrosine kinase activates several signaling pathways during transformation of bone marrow cells in mice. Because the SH2-containing inositol 5'-phosphatase (SHIP) and Downstream of tyrosine kinase 1 (Dok1) have been shown to interact with Abl, the effect of SHIP and Dok1 deficiency on v-Abl transformation was investigated. Bone marrow cells from either Dok1- or SHIP-deficient mice are more susceptible to transformation by v-Abl. v-Abl-transformed preB cells from these knockout mice show Abl kinase-dependent hyperproliferation and moderate resistance to apoptosis. Elevated activation of Ras, Raf-1, and Erk, but not of Akt, was observed in either SHIP(-/-) or Dok1(-/-) v-Abl-transformed cells. This activation is sensitive to treatment with STI571. Furthermore, treatment of these cells with either a farnesyltransferase inhibitor or a MEK1/2 inhibitor abrogates the increased proliferation of SHIP(-/-) or Dok1(-/-) cells in a dose-dependent manner. Complementation of SHIP(-/-) or Dok1(-/-) cells abrogates their hyperproliferation and intracellular Erk activation. These data indicate that both SHIP and Dok1 functionally regulate the activation of Ras-Erk pathway by v-Abl and affect the mitogenic activity of v-Abl transformed bone marrow cells.

The v-Abl oncogene activates Jak-Stat signaling during transformation of pre-B cells in mice. Disrupting Jak activation by deleting the Jak binding domain of v-Abl or by expressing a dominant-negative Jak1 decreases v-Abl transformation efficiency. As SOCS-1 is a known potent inhibitor of Jak kinases, the mechanism by which v-Abl bypasses SOCS-1 regulation to constitutively activate Jak kinases was investigated. SOCS-1 is expressed in v-Abl-transformed cells but is unable to inhibit v-Abl-mediated Jak-Stat signaling. In v-Abl transformants, SOCS-1 can inhibit cytokine signals, but it is more efficient at doing so when the cells are treated with STI571, an Abl kinase inhibitor. Downstream effects of v-Abl signaling include phosphorylation of SOCS-1 on nontyrosine residues, disruption of the interaction between SOCS-1 and the Elongin BC complex, and inhibition of SOCS-1-mediated proteasomal targeting of activated Jaks. These findings reveal a mechanism by which Jak-dependent oncogenes may bypass SOCS-1 inhibition.

The v-Abl protein encoded by Abelson murine leukemia virus (Ab-MLV) induces transformation of pre-B cells via a two-stage process. An initial proliferative phase during which cells with limited tumorigenic potential expand is followed by a crisis period marked by high levels of apoptosis and erratic growth. Transformants that survive this phase emerge as fully malignant cells and usually contain mutations that disable the p53 tumor suppressor pathway. Consistent with the importance of p53 in this process, pre-B cells from p53 null animals bypass crisis. Thus, the transformation process reflects a balance between signals from the v-Abl protein that drive transformation and those coming from the cellular response to inappropriate growth. One prediction of this hypothesis is that Ab-MLV mutants that are compromised in their ability to transform cells may be less equipped to overcome the effects of p53. To test this idea, we examined the ability of the P120/R273K mutant to transform pre-B cells from wild-type, p53 null, and Ink4a/Arf null mice. The SH2 domain of the v-Abl protein encoded by this mutant contains a substitution that affects the phosphotyrosine-binding pocket, and this mutant is compromised in its ability to transform NIH 3T3 and pre-B cells, especially at 39.5°C. Our data reveal that loss of p53 or Ink4a/Arf locus products complements the transforming defect of the P120/R273K mutant, but it does not completely restore wild-type function. These results indicate that one important transforming function of v-Abl proteins is overcoming the effects of a functional p53 pathway.

Bcr-Abl constitutes a deregulated tyrosine kinase involved in the pathogenesis of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL). Although activation of the transcription factor NF-kappaB/Rel has been demonstrated, mechanisms of NF-kappaB/Rel activation by Bcr-Abl remain obscure. In this paper we demonstrate activation of NF-kappaB/Rel by Bcr-Abl and for the first time by v-Abl. Furthermore, we investigated mechanisms of NF-kappaB/Rel induction by Bcr-Abl and v-Abl. Both Bcr-Abl and v-Abl induced NF-kappaB/Rel DNA binding in Ba/F3 cells. DNA binding was a result of nuclear translocation of p65/RelA, whereas p65/RelA expression was unaffected. Nuclear translocation of p65/RelA is at least partially due to increased IkappaBalpha degradation, which is independent of IkappaB kinase (IKK) activity. IKK activity is not deregulated by Bcr-Abl and v-Abl. NF-kappaB/Rel transactivation was dependent on abl kinase activity but independent of Grb2 and Grb10 binding tobcr sequences. In addition, NF-kappaB/Rel activation was dependent on Ras activity. Primary CML blasts showed constitutive p65/RelA NF-kappaB/Rel DNA binding activity. Thus NF-kappaB/Rel represents a potential target for molecular therapies in CML.

We have molecularly cloned TPP36, a novel 36 kDa protein with 281 amino acids that was identified as a protein phosphorylated in B progenitor cells following stimulation with pervanadate/H2O2. Analysis with anti-TPP36 antiserum revealed that TPP36 was expressed ubiquitously and had an isoform with 236 amino acids, designated TPP32. TPP36/32 were localized mainly in cytoplasm despite the presence of a typical nuclear localization signal sequence. These proteins were phosphorylated preferentially by Abl among a panel of tyrosine kinases examined. Phosphorylation of tyrosine 120 in TPP36/32 led to an apparent mobility shift in sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting conformational change in the phosphorylated protein. Thus, TPP36/32 appear to be novel substrates of Abl tyrosine kinase.

Abelson murine leukemia virus-transformed cell lines have provided a critical model system for studying the regulation of B cell development. However, transformation by v-Abl blocks B cell development, resulting in the arrest of these transformants in an early pre-B cell-like state. We report here that treatment of Abelson virus-transformed pre-B cell lines with the small molecule Abl kinase inhibitor (STI571) results in their differentiation to a late pre-B cell-like state characterized by induction of immunoglobulin (Ig) light chain gene rearrangement. DNA microarray analyses enabled us to identify two genes inhibited by v-Abl that encode the Igk 3' enhancer-binding transcription factors Spi-B and IRF-4. We show that enforced expression of these two factors is sufficient to induce germline Igk transcription in Abelson-transformed pro-B cell lines. This suggests a key role for these factors, and perhaps for c-Abl itself, in the regulated activation of Ig light chain gene rearrangement.

The α6β4 integrin is the receptor for various laminin isoforms and is a component of the hemidesmosome. Increased expression levels of this integrin correlate with the aggressive phenotype of many epithelial tumors compared with surrounding normal tissue. Furthermore, the long cytoplasmic tail of the β4 integrin subunit has been implicated in several signal transduction pathways that are involved not only in invasion, but also in proliferation and apoptosis. Here we report that the exogenous expression of β4 integrin in 32D/v-abl–transformed cells reduces tumor aggressiveness in vivo and strongly inhibits cell proliferation in vitro by inducing monocytic differentiation. These effects are accompanied by growth arrest and p73 protein accumulation. The hypothesis that the inhibition of v-Abl oncogenic capacity could allow the activation of the endogenous c-Abl was tested in RKO cells. The results clearly demonstrated a strong increase of c-Abl phosphorylation that is accompanied by its association with p73 protein. Overall, the reported findings indicate that α6β4 integrin promotes growth arrest and differentiation by modulating Abl kinases and p73 protein pathway(s).

SOCS-1 is an inducible SH2-containing inhibitor of Jak kinases and as such can potently suppress cytokine signaling. SOCS-1 deficient mice die within the first three weeks of life from a myeloproliferative disorder driven by excessive interferon signaling. We report here that SOCS-1 inhibits proliferation signals induced by a variety of oncogenes active within the hematopoietic system. Ectopic expression of SOCS-1 abolished proliferation mediated by a constitutively active form of the KIT receptor, TEL-JAK2, and v-ABL, and reduced metastasis from BCR-ABL transformed cells. SOCS-1, however, did not interfere with v-SRC or RASV12 mediated cellular transformation. A mutant form of SOCS-1 unable to bind through its SH2 domain to tyrosine phosphorylated proteins could still inhibit KIT, but not TEL-JAK2, indicating multiple mechanisms for SOCS-1-mediated tumor suppression. We show that the steady state levels of TEL-JAK2 and to a greater extent v-ABL are diminished in the presence of SOCS-1. Lastly, we show that SOCS-1 -/- fibroblasts are more sensitive than wild type fibroblasts to either spontaneous or oncogene-induced transformation. These data suggest that loss-of-function of SOCS-1 may collaborate with a variety of hematopoietic oncogenes to facilitate tumor progression.

Enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium are attaching-and-effacing (A/E) pathogens that cause intestinal inflammation and diarrhea. The bacteria adhere to the intestinal epithelium, destroy microvilli, and induce actin-filled membranous pedestals but do not invade the mucosa. Adherence leads to activation of several host cell kinases, including FYN, n-SRC, YES, ABL, and ARG, phosphorylation of the bacterial translocated intimin receptor, and actin polymerization and pedestal formation in cultured cells. However, marked functional redundancy appears to exist between kinases, and their physiological importance in A/E pathogen infections has remained unclear. To address this question, we employed a novel dynamic in vitro infection model that mimics transient and short-term interactions in the intestinal tract. Screening of a kinase inhibitor library and RNA interference experiments in vitro revealed that ABL and platelet-derived growth factor (PDGF) receptor (PDGFR) kinases, as well as p38 MAP kinase, have unique, indispensable roles in early attachment of EPEC to epithelial cells under dynamic infection conditions. Studies with mutant EPEC showed that the attachment functions of ABL and PDGFR were independent of the intimin receptor but required bacterial bundle-forming pili. Furthermore, inhibition of ABL and PDGFR with imatinib protected against infection of mice with modest loads of C. rodentium, whereas the kinases were dispensable for high inocula or late after infection. These results indicate that ABL and PDGFR have indispensable roles in early A/E pathogen attachment to intestinal epithelial cells and for in vivo infection with limiting inocula but are not required for late intimate bacterial attachment or high inoculum infections. © 2014 the American Physiological Society.

The abi-1 gene encodes a protein that binds and is phosphorylated by the Abelson protein tyrosine kinase. Constructs expressing a full-length abi-1 cDNA, and a smaller cDNA arising from an alternatively spliced form, were generated and tested for their effect on transformation of NIH3T3 cells by the Abelson murine leukemia virus. Overexpression of both forms of the protein strongly inhibited transformation by the wild-type P160 strain of the virus, but not by the non-interacting mutant P90A strain. The inhibition required the SH3 domain of Abi-1, suggesting that a direct interaction was required for the effect. Rare breakthrough P160 transformants of the Abi-1 overexpressing lines were found to have downregulated Abi-1 protein levels by a post-transcriptional mechanism.

WAVE proteins are members of the Wiskott-Aldrich syndrome protein (WASP) family of scaffolding proteins that coordinate actin reorganization by coupling Rho-related small molecular weight GTPases to the mobilization of the Arp2/3 complex. We identified WAVE-1 in a screen for rat brain A kinase-anchoring proteins (AKAPs), which bind to the SH3 domain of the Abelson tyrosine kinase (Abl). Recombinant WAVE-1 interacts with cAMP-dependent protein kinase (PKA) and Abl kinases when expressed in HEK-293 cells, and both enzymes co-purify with endogenous WAVE from brain extracts. Mapping studies have defined binding sites for each kinase. Competition experiments suggest that the PKA-WAVE-1 interaction may be regulated by actin as the kinase binds to a site overlapping a verprolin homology region, which has been shown to interact with actin. Immunocytochemical analyses in Swiss 3T3 fibroblasts suggest that the WAVE-1 kinase scaffold is assembled dynamically as WAVE, PKA and Abl translocate to sites of actin reorganization in response to platelet-derived growth factor treatment. Thus, we propose a previously unrecognized function for WAVE-1 as an actin-associated scaffolding protein that recruits PKA and Abl.

Transformation of 3T3 fibroblasts by the v-Abl tyrosine kinase replaces mitogenic and adhesion signals normally required for cell cycle progression. A 3T3 cell line conditionally transformed with v-Abl has been used to study v-Abl's effects on cell cycle in the context of either serum depletion or absence of adhesion signals. We show that E2F-dependent mRNAs, encoding proteins required for cell cycle progression, are induced by v-Abl. In addition, we identify two previously unknown targets of v-Abl signaling: (1) cyclin D1 and D2 mRNAs are induced upon v-Abl activation; and (2) the CDK inhibitor p27 is decreased upon v-Abl activation.

Abelson murine leukemia virus (A-MuLV) is an acute transforming retrovirus that preferentially transforms early B-lineage cells both in vivo and in vitro. Its transforming protein, v-Abl, is a tyrosine kinase related to v-Src but containing an extended C-terminal domain. Many mutations affecting the C-terminal portion of the molecule block the pre-B-transforming activity of v-Abl without affecting the fibroblast-transforming ability. In this study we have determined the abilities of both wild-type and C-terminally truncated (p90) forms of v-Abl to transform cells from p53(-/-) mice. Lack of p53 increases the susceptibility of bone marrow cells to transformation by v-Abl by a factor of more than 7 but does not alter v-Abl's preference for B220(+) IgM(-) pre-B cells. p53-deficient mice have earlier tumor onset, more rapid tumor progression, and decreased survival time following A-MuLV infection, but all of the tumors are pre-B lymphomas. Thus, p53-dependent pathways inhibit v-Abl transformation but play no role in conferring preferential transformation of pre-B cells. Surprisingly, the C-terminally truncated form of v-Abl (p90) transforms pre-B cells very efficiently in mice lacking p53, thus demonstrating that the C terminus of v-Abl does not determine preB tropism but is necessary to overcome p53-dependent inhibition of transformation.

Tumorigenesis is a multistep process that involves the activation of oncogenes and the inactivation of tumor suppressor genes. The transforming activity of the v-Abl oncogene of Abelson murine leukemia virus (A-MuLV) in immortal cell lines has been well studied, while the effects of v-Abl in primary fibroblasts are less clear. Here we show that v-Abl causes cell cycle arrest in primary mouse embryonic fibroblasts (MEFs) and elevated levels of both p53 and the cyclin-dependent kinase inhibitor p21Cip. p53-/- or p19ARF-/- MEFs were resistant to v-Abl-induced cell cycle arrest. Although wild-type MEFs were resistant to v-Abl transforming activity, p53-/- or p19ARF-/- MEFs were susceptible. The results indicate that loss of p19ARF and p53 function plays an important role during the transformation of primary cells by v-Abl. We suggest that although v-Abl is a potent oncogene, its full potential transforming activity cannot be realized until the ARF-, and p53-dependent growth inhibitory pathway is disabled. We also show that p53 is not the mediator of v-Abl toxicity in immortal fibroblasts and does not determine the susceptibility of immortal fibroblasts to v-Abl transformation.

Human and mouse Abelson interacting proteins (Abi) are SH3-domain containing proteins that bind to the proline-rich motifs of the Abelson protein tyrosine kinase. We report a new member of this gene family, a Drosophila Abi (dAbi) that is a substrate for Abl kinase and that co-immunoprecipitates with Abl if the Abi SH3 domain is intact. We have identified a new function for both dAbi and human Abi-2 (hAbi-2). Both proteins activate the kinase activity of Abl as assayed by phosphorylation of the Drosophila Enabled (Ena) protein. Removal of the dAbi SH3 domain eliminates dAbi's activation of Abl kinase activity. dAbi is an unstable protein in cells and is present at low steady state levels but its protein level is increased coincident with phosphorylation by Abl kinase. Expression of the antisense strand of dAbi reduces dAbi protein levels and abolishes activation of Abl kinase activity. Modulation of Abi protein levels may be an important mechanism for regulating the level of Abl kinase activity in the cell.

To study the effects of the murine scid mutation and DNA ploidy on the susceptibility of lymphoid cell lines to induction of apoptosis by ionizing radiation and thereby to determine whether DNA lesions are critical initiators of apoptosis.Sensitivity to killing and rapidity of induction of apoptosis following y-irradiation or DNA-associated 125I decays were compared in pre-B and pre-T cell lines derived from wild-type mice and from mice homozygous or heterozygous for the scid mutation. Effects of differences in DNA ploidy on the same endpoints were studied using pseudo-diploid and -tetraploid clones of a murine pre-T cell line.Pairs of pre-B- and pre-T cell-derived lines that expressed wild-type p53 and underwent rapid interphase apoptosis after irradiation were identified. In both cases, the scid homozygous cell lines were more sensitive to killing, suggesting that DNA repair capability influences susceptibility to induction of apoptosis. Increasing DNA ploidy in a cell line that undergoes rapid interphase apoptosis produced a corresponding increase in the number of DNA lesions required to produce a lethal event; again consistent with DNA being the target for radiation action.DNA damage is an important, if not the sole, initiator of external beam ionizing radiation-induced apoptosis.

The mechanism by which early lymphoid cells are selectively transformed by v-Abl is currently unknown. Previous studies have shown constitutive activation of IL-4 and IL-7 signaling pathways, as measured by activation of Janus protein kinase (JAK)1, JAK3, STAT5, and STAT6, in pre-B cells transformed by v-Abl. To determine whether activation of these cytokine signaling pathways by v-Abl is important in the cellular events induced by the Abelson murine leukemia virus, the effects of IL-4 and IL-7 on pre-B cells transformed with a temperature-sensitive v-Abl mutant were examined. Whereas IL-4 had little or no effect, IL-7 delayed both the apoptosis and cell cycle arrest that occur upon v-Abl kinase inactivation. IL-7 also delayed the decreases in the levels of c-Myc, Bcl-2, and Bcl-xL that occur upon loss of v-Abl kinase activity. IL-7 did not maintain v-Abl-mediated differentiation arrest of the pre-B cells, as activation of NF-κB and RAG gene transcription was unaffected by IL-7. These results identify a potential role for IL-7 signaling pathways in transformation by v-Abl while demonstrating that a combination of IL-4 and IL-7 signaling cannot substitute for an active v-Abl kinase in transformed pre-B cells.

Many of the key decisions in lymphocyte differentiation and activation are dependent on integration of antigen receptor and co-receptor signals. Although there is significant understanding of these receptors and their signaling pathways, little is known about the molecular requirements for signal integration at the level of activation of gene expression. Here we show that in primary B cells, expression of the B-cell specific transcription coactivator OCA-B (also known as OBF-1 or Bob-1) is regulated synergistically by the B-cell antigen receptor, CD40L and interleukin signaling pathways. Consistent with the requirement for multiple T cell-dependent signals to induce OCA-B, we find that OCA-B protein is highly expressed in germinal center B cells. Accordingly, germinal center formation is blocked completely in the absence of OCA-B expression in B cells, whereas the helper functions of OCA-B-deficient T cells are indistinguishable from controls. The requirement for OCA-B expression in B cells is germinal center specific since the development of primary B cell follicles, the marginal zone and plasma cells are all intact. Thus, OCA-B is the first example of a transcriptional coactivator that is both synergistically induced by and required for integration of signals that mediate cell fate decisions.

CGP 57148 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the ABL and the platelet-derived growth factor receptor (PDGFR) protein tyrosine kinases. We previously showed that CGP 57148 selectively kills p210BCR-ABL–expressing cells. To extend these observations, we evaluated the ability of CGP 57148 to inhibit other activated ABL tyrosine kinases, including p185BCR-ABL and TEL-ABL. In cell-based assays of ABL tyrosine phosphorylation, inhibition of ABL kinase activity was observed at concentrations similar to that reported for p210BCR-ABL. Consistent with the in vitro profile of this compound, the growth of cells expressing activated ABL protein tyrosine kinases was inhibited in the absence of exogenous growth factor. Growth inhibition was also observed with a p185BCR-ABL–positive acute lymphocytic leukemia (ALL) cell line generated from a Philadelphia chromosome–positive ALL patient. As CGP 57148 inhibits the PDGFR kinase, we also showed that cells expressing an activated PDGFR tyrosine kinase, TEL-PDGFR, are sensitive to this compound. Thus, this compound may be useful for the treatment of a variety of BCR-ABL–positive leukemias and for treatment of the subset of chronic myelomonocytic leukemia patients with a TEL-PDGFR fusion protein.

Biochemical and genetic evidence suggests that the tyrosine kinase activity of c-Abl is tightly regulated in vivo by a cellular factor binding to the Src homology 3 (SH3) domain of Abl. We used the yeast two-hybrid system to identify a gene, PAG, whose protein product (Pag) interacts specifically with the Abl SH3 domain. Pag, also known as macrophage 23-kD stress protein (MSP23), is a member of a novel family of proteins with antioxidant activity implicated in the cellular response to oxidative stress and in control of cell proliferation and differentiation. In a co-expression assay, Pag associates with c-Abl in vivo and inhibits tyrosine phosphorylation induced by overexpression of c-Abl. Inhibition requires the Abl SH3 and kinase domains and is not observed with other Abl SH3-binding proteins. Expression of Pag also inhibits the in vitro kinase activity of c-Abl, but not SH3-mutated Abl or v-Abl. When transfected in NIH-3T3 cells, Pag is localized to nucleus and cytoplasm and rescues the cytostatic effect induced by c-Abl. These observations suggest Pag is a physiological inhibitor of c-Abl in vivo.

Here we investigate the role of the Raf-1 kinase in transformation by the v-abl oncogene. Raf-1 can activate a transforming signalling cascade comprising the consecutive activation of Mek and extracellular-signal-regulated kinases (Erks). In v-abl-transformed cells the endogenous Raf-1 protein was phosphorylated on tyrosine and displayed high constitutive kinase activity. The activities of the Erks were constitutively elevated in both v-raf- and v-abl-transformed cells. In both cell types the activities of Raf-1 and v-raf were almost completely suppressed after activation of the cyclic AMP-dependent kinase (protein kinase A [PKA]), whereas the v-abl kinase was not affected. Raf inhibition substantially diminished the activities of Erks in v-raf-transformed cells but not in v-abl-transformed cells, indicating that v-abl can activate Erks by a Raf-1-independent pathway. PKA activation induced apoptosis in v-abl-transformed cells while reverting v-raf transformation without severe cytopathic effects. Overexpression of Raf-1 in v-abl-transformed cells partially protected the cells from apoptosis induced by PKA activation. In contrast to PKA activators, a Mek inhibitor did not induce apoptosis. The diverse biological responses correlated with the status of c-myc gene expression. v-abl-transformed cells featured high constitutive levels of expression of c-myc, which were not reduced following PKA activation. Myc activation has been previously shown to be essential for transformation by oncogenic Abl proteins. Using estrogen-regulated c-myc and temperature-sensitive Raf-1 mutants, we found that Raf-1 activation could protect cells from c-myc-induced apoptosis. In conclusion, these results suggest (i) that Raf-1 participates in v-abl transformation via an Erk-independent pathway by providing a survival signal which complements c-myc in transformation, and (ii) that cAMP agonists might become useful for the treatment of malignancies where abl oncogenes are involved, such as chronic myeloid leukemias.

The E2F family of transcription factors regulates cell cycle progression, and deregulated expression of E2F-1 can lead to neoplastic transformation. In myeloid cells, introduction and expression of the Abelson leukemia virus causes growth factor independence. Here, the p120 v-Abl protein activates E2F-1-mediated transcription through a physical interaction with the E2F-1 transcription factor. BCR-Abl and c-Abl also stimulate E2F-1-mediated transcription. Our results suggest a new mechanism by which v-Abl leads to factor-independent myeloid cell proliferation: the activation of E2F-1-mediated transcription.

v-Abl is an oncogenic form of the c-Abl nonreceptor tyrosine kinase. v-Abl induces transcription of c-myc, and c-Myc function is a necessary but not sufficient component of the v-Abl transformation program. Previously we showed that the E2F site in the c-myc promoter is a v-Abl response element and that v-Abl appears to induce c-myc by initiating a phosphorylation cascade that ultimately activates E2F-binding proteins. In this work we have investigated the signaling pathway between the v-Abl tyrosine kinase and activated E2F proteins. We show that the Ras GTPase and Raf1 serine/threonine kinase are required in this pathway. However, in contrast to other aspects of v-Abl signaling, induction of c-myc transcription is independent of the Rac GTPase. Our results also establish a requirement for activated cyclin-dependent kinases (cdks), as v-Abl-dependent induction of c-myc transcription is blocked by cdk inhibitor p21 and induction of c-myc is accompanied by activation of cdk2 and cdk4. Finally, we show that v-Abl-dependent induction of c-myc is accompanied by hyperphosphorylation of pRb, p107, and p130. On the basis of these data, we propose a model for the signaling path from v-Abl to c-myc.

A 62 kDa protein is highly phosphorylated in many cells containing activated tyrosine kinases. This protein, characterized mainly by its avid association with rasGAP, has proved elusive. Anti-phosphotyrosine antibody was used to purify p62. From peptide sequence, molecular cloning revealed a cDNA encoding a novel protein, p62dok, with little homology to others but with a prominent set of tyrosines and nearby sequences suggestive of SH2 binding sites. In cells, v-Abl tyrosine kinase binds and strongly phosphorylates p62dok, which then binds rasGAP. A monoclonal antibody, 2C4, to the rasGAP-associated p62 reacts with p62dok. Thus, p62dok appears to be the long-sought major substrate of many tyrosine kinases.

Primary cell cultures are in general resistant to the transforming effect of a single oncogene, a finding considered consistent with the multistage theory of carcinogenesis. In the present studies, we examined whether cellular age, differentiation stage, and/or tissue origin of primary cells plays a role in determining their response to v-src transformation. To study the role of cellular age, rat mammary fibroblasts were isolated from a 50-day-old female rat and infected with a recombinant retrovirus carrying a v-src gene after 2, 7, 14, 21, and 28 days of continuous growth. To determine whether cellular differentiation is important, fibroblasts were isolated from embryos at 12 and 16 days of gestation, from newborns, and from a 30-day-old rat and similarly infected. Finally, the role of primary-cell histogenesis was assessed by infecting primary cultures of fibroblasts isolated from the mammary gland, dermis, and lungs of a mature rat. When compared to 3Y1 cells, all preparations of primary cultures exhibited considerable resistance to v-src transformation. However, whereas primary cells isolated from different tissues responded similarly to the transforming effect of the oncogene, major differences were observed when cells were transduced at different stages of their in vitro life span. v-src was capable of inducing formation of foci and growth in soft agar in early-passage cells but failed to do so in primary cultures infected after 14 days of continuous passaging. Similarly, both the number of foci and the number of colonies in soft agar decreased with tissue donor age. The differential response of young and senescing cells could not be explained by mutations in v-src provirus, by differences in functional v-src expression, or by growth stimulation or suppression via paracrine mechanisms. Furthermore, v-src cooperated with an immortalizing gene, like simian virus 40 large T, polyomavirus large T, E6 and E7 of human papillomavirus, or an activated p53 mutant, to induce anchorage-independent growth of primary cultures but failed to do so with cytoplasmic transforming genes, like v-abl, v-ras, or v-raf, which did not confer indefinite division potential. These studies indicate that cellular aging is a critical determinant of primary-cell resistance to v-src transformation. It is suggested that v-src requires a nuclear auxiliary function for transformation which is present in early-passage cells, particularly when these cells are derived from embryonic tissue, but is lost as cells approach replicative senescence. This auxiliary function is provided by nuclear oncogenes but not cytoplasmic transforming genes.

Nickel compounds have been found to be carcinogenic based upon epidemiological, animal and cell culture studies. Previous studies suggest that epigenetic mechanisms play a role in Nickel-induced carcinogenesis such as DNA methylation and histone modification. In this study, we investigated the role of microRNAs (miRNAs) in nickel-induced carcinogenesis. The expression of several miRNAs which may function as tumor suppressor genes revealed a strong downregulation of miR-203 in Ni3S2-transformed 16HBE cells (NSTCs). Meanwhile, we observed hypermethylation of CpGs in miR-203 promoter and first exon area, and proved that the hyper-methylated miR-203 was involved in the Nickel-induced tumorigenesis. Moreover, we identified that miR-203 may suppress the tumorigenesis at least in part through negatively regulating its target gene ABL1. Our findings indicate that DNA methylation-associated silencing of tumor suppressor miRNAs contributes to the development of Nickel-induced cancer.

Rho family GTPases coordinate cytoskeletal rearrangements in neurons, and mutations in their regulators are associated with mental retardation and other neurodevelopmental disorders (Billuart et al., 1998; Kutsche et al., 2000; Newey et al., 2005; Benarroch, 2007). Chromosomal microdeletions encompassing p190RhoGAP or its upstream regulator, the Abl2/Arg tyrosine kinase, have been observed in cases of mental retardation associated with developmental defects (Scarbrough et al., 1988; James et al., 1996; Takano et al., 1997; Chaabouni et al., 2006; Leal et al., 2009). Genetic knockout of Arg in mice leads to synapse, dendritic spine, and dendrite arbor loss accompanied by behavioral deficits (Moresco et al., 2005; Sfakianos et al., 2007). To elucidate the cell-autonomous mechanisms by which Arg regulates neuronal stability, we knocked down Arg in mouse hippocampal neuronal cultures. We find that Arg knockdown significantly destabilizes dendrite arbors and reduces dendritic spine density by compromising dendritic spine stability. Inhibiting RhoA prevents dendrite arbor loss following Arg knockdown in neurons, but does not block spine loss. Interestingly, Arg-deficient neurons exhibit increased miniature excitatory postsynaptic current amplitudes, and their remaining spines exhibit larger heads deficient in the actin stabilizing protein cortactin. Spine destabilization in Arg knockdown neurons is prevented by blocking NMDA receptor-dependent relocalization of cortactin from spines, or by forcing cortactin into spines via fusion to an actin-binding region of Arg. Thus, Arg employs distinct mechanisms to selectively regulate spine and dendrite stability: Arg dampens activity-dependent disruption of cortactin localization to stabilize spines, and attenuates Rho activity to stabilize dendrite arbors.

The v-Abl tyrosine kinase encoded by the Abelson murine leukemia virus (A-MuLV) can either stimulate or inhibit cell proliferation, depending on the cell context. In a NIH-3T3-derived cell line, N3T3, v-Abl blocks the serum-induced entry into S phase. In these G1-arrested cells v-Abl does not interfere with the activation of cyclin D1 or cyclin E-dependent kinases. As a result, v-Abl does not block the hyperphosphorylation and inactivation of the retinoblastoma protein RB. However, activation of cyclin A-dependent kinase is inhibited due to a v-Abl-induced block in the accumulation of cyclin A mRNA and protein. Ectopic expression of cyclin A enabled the v-Abl-arrested cells to enter S phase, whereas cyclins E and D1, or E2Fs 1 and 4 could not overcome the v-Abl arrest. Taken together, these results suggest that v-Abl tyrosine kinase arrests cell cycle progression in G1 by inhibiting the expression of cyclin A.

A necessary downstream element of Abelson murine leukemia virus (Ab-MLV)-mediated transformation is Ras, which can be activated by the phosphotyrosine-dependent association of Shc with the Grb2-mSos complex. Here we show that Shc is tyrosine-phosphorylated and associates with Grb2 in v-Abl-transformed cells, whereas Shc in NIH3T3 cells is phosphorylated solely on serine and is not Grb2-associated. In addition, Shc coprecipitates with P120 v-Abl and P70 v-Abl, which lacks the carboxyl terminus. Surprisingly, a kinase-defective mutant of P120 also binds Shc, demonstrating that Shc/v-Abl association is a phosphotyrosine-independent interaction. Glutathione S-transferase fusion proteins were used to map the interacting domains and showed that Shc from both NIH3T3 and v-Abl-transformed cells binds to the Abl SH2 domain and that P120 v-Abl binds to a region in the amino terminus of Shc. Consistent with these data, a v-Abl mutant encoding only the Gag and SH2 regions was able to bind Shc in vivo. The unique non-phosphotyrosine-mediated binding of Shc may allow direct tyrosine phosphorylation of Shc by v-Abl and subsequent activation of the Ras pathway through assembly of a signaling complex with Grb2-mSos.

Abelson murine leukemia virus (Ab-MLV) mutants expressing v-Abl proteins lacking the carboxyl terminus are compromised in the ability to transform lymphoid but not NIH 3T3 cells. This feature correlates with the presence of low levels of phosphotyrosine in lymphoid cells infected with carboxyl-terminal truncation mutants. In contrast, high levels of phosphotyrosine are observed in NIH 3T3 cells infected with wild-type and mutant Ab-MLV. Two downstream targets affected in lymphoid transformants are the GTPase-activating protein and GTPase-activating protein-associated protein p62, molecules which are heavily tyrosine phosphorylated in lymphoid cells transformed by wild-type Ab-MLV but not carboxyl-terminal truncation mutants of Ab-MLV. This difference suggested that signaling mediated via the Ras pathway may be compromised in lymphoid cells expressing the carboxyl-terminal truncation mutants. Consistent with this idea, expression of v-Ha-ras complemented these mutants in primary bone marrow transformation assays and increased transformation frequencies obtained with the Ab-MLV mutants 8- to 20-fold. These data suggest that a biologically important link exists between the carboxyl terminus of v-Abl protein and the Ras pathway. Signals transmitted via this connection may enhance those mediated via other regions of the v-Abl protein and facilitate transformation of primary, nonimmortalized cells such as pre-B lymphocytes.