Novel oxime derivatives of radicicol induce erythroid differentiation associated with preferential G(1) phase accumulation against chronic myelogenous leukemia cells through destabilization of Bcr-Abl with Hsp90 complex

Chronic myelogenous leukemia (CML) is a clonal disorder of a pluripotent hematopoietic stem cells characterized by a chimeric bcr-abl gene giving rise to a p210(Bcr-Abl) protein with dysregulated tyrosine kinase activity. Radicicol, a macrocyclic antifungal antibiotic, binds to the N-terminal of heat shock protein 90 (Hsp90) and destabilizes Hsp90-associated proteins such as Raf-1. This study investigated the effect of radicicol, novel oxime derivatives of radicicol (KF25706 and KF58333), and herbimycin A (HA), a benzoquinoid ansamycin antibiotic, on the growth and differentiation of human K562 CML cells. Although KF25706 and KF58333 induced the expression of glycophorin A in K562 cells, radicicol and HA caused erythroid differentiation transiently. Cell cycle analysis showed that G(1) phase accumulation was observed in K562 cells treated with KF58333. KF58333 treatment depleted p210(Bcr-Abl), Raf-1, and cellular tyrosine phosphorylated proteins in K562 cells, whereas radicicol and HA showed transient depletion of these proteins. KF58333 also down-regulated the level of cell cycle-dependent kinases 4 and 6 and up-regulated cell cycle-dependent kinase inhibitor p27(Kip1) protein without an effect on the level of Erk and Hsp90 proteins. Immunoprecipitation analysis showed that p210(Bcr-Abl) formed multiple complexes with Hsp90, some containing p23 and others Hsp70; KF58333 treatment dissociated p210(Bcr-Abl) from Hsp90/p23 chaperone complexes. Furthermore, KF58333 induced apoptosis in K562 cells and administration of KF58333 prolonged the survival time of SCID mice inoculated with K562 cells. These results suggest that KF58333 may have therapeutic potential for the treatment of CML that involves abnormal cellular proliferation induced by p210(Bcr-Abl).     

Effect of herbimycin A, an antagonist of tyrosine kinase, on bcr/abl oncoprotein-associated cell proliferations: abrogative effect on the transformation of murine hematopoietic cells by transfection of a retroviral vector expressing oncoprotein P210bcr/abl and preferential inhibition on Ph1-positive leukemia cell growth.

Herbimycin A, a benzoquinoid ansamycin antibiotic, was demonstrated to decrease intracellular phosphorylation by protein tyrosine kinase (PTK). In Philadelphia chromosome (Ph1)-positive leukemias such as chronic myelogenous leukemia (CML) and Ph1-positive acute lymphoblastic leukemia (ALL), both of which express bcr-abl fused gene products (P210bcr-abl or P190bcr-abl protein kinase) with augmented tyrosine kinase activities, herbimycin A markedly inhibited the in vitro growth of the Ph1-positive ALL cells and the leukemic cells derived from CML blast crisis. However, the same dose of herbimycin A did not inhibit in vitro growth of a broad spectrum of Ph1-negative human leukemia cells, and several other protein kinase antagonists also displayed no preferential inhibition. Furthermore, we demonstrated that herbimycin A has an antagonizing effect on the growth of transformed cells by a transfection of retroviral amphotrophic vector expressing P210bcr/abl into a murine interleukin (IL)-3-dependent myeloid FDC-P2 cell line. This inhibition was abrogated by the addition of sulfhydryl compounds, similar to the reaction previously described for Rous sarcoma virus transformation. The inhibitory effect of herbimycin A on the growth of Ph1-positive cells was associated with decreased bcr/abl tyrosine kinase activity, but no decrease of bcr-abl mRNA and protein, suggesting that the inactivation of bcr-abl tyrosine kinase activity by herbimycin A may be induced by its binding to the bcr-abl protein portion that is rich with sulfhydryl groups. The present study indicates that herbimycin A is a beneficial agent for the investigation of the role of the bcr-abl gene in Ph1-positive leukemias and further suggests that the development of agents inhibiting the bcr-abl gene product may offer a new therapeutic potential for Ph1-positive leukemias.     

Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells

The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth- factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13-259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G- protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces proliferative signals, in part, through downstream activation of p21 RAS. Furthermore, p21 RAS activity is linked to pathways that regulate c-jun and c-fos expression.     

T cell protein tyrosine phosphatase (TCPTP) deficiency in muscle does not alter insulin signalling and glucose homeostasis in mice

Aims/hypothesis  

Insulin activates insulin receptor protein tyrosine kinase and downstream phosphatidylinositol-3-kinase (PI3K)/Akt signalling in muscle to promote glucose uptake. The insulin receptor can serve as a substrate for the protein tyrosine phosphatase (PTP) 1B and T cell protein tyrosine phosphatase (TCPTP), which share a striking 74% sequence identity in their catalytic domains. PTP1B is a validated therapeutic target for the alleviation of insulin resistance in type 2 diabetes. PTP1B dephosphorylates the insulin receptor in liver and muscle to regulate glucose homeostasis, whereas TCPTP regulates insulin receptor signalling and gluconeogenesis in the liver. In this study we assessed for the first time the role of TCPTP in the regulation of insulin receptor signalling in muscle.     

Crkl is constitutively tyrosine phosphorylated in platelets from chronic myelogenous leukemia patients and inducibly phosphorylated in normal platelets stimulated by thrombopoietin

Platelet functions such as aggregation and clot retraction are often abnormal in chronic mylogenous leukemia (CML) patients. However, the molecular mechanisms of these altered functions are unknown. As expression of the p210bcr-abl oncogene product, a constitutively active tyrosine kinase, is known to have an essential role in the pathogenesis of CML and tyrosine phosphorylation is intimately involved in various aspects of platelet activation, we examined the pattern of protein tyrosine phosphorylation in platelets from 15 CML patients by immunoblotting with a monoclonal antiphosphotyrosine antibody (4G10). Before and after stimulation with thrombin, the only consistent difference between normal and CML platelets was the presence of a tyrosine phosphorylated protein with a relative molecular weight of 39 kD. This tyrosine phosphorylated protein was identified as crid, an SH2, SH3 containing adapter protein. Thus, as previously demonstrated for neutrophils from CML patients, tyrosine phosphorylation of p39crkl persists in mature platelets. No tyrosine phosphorylation of crid was detected following stimulation with thrombin in normal platelets. However, crkl became incorporated into the Triton X-100 insoluble residue following thrombin stimulation in a manner dependent on platelet aggregation. Further, we found that crkl is an endogenous substrate for calpain, a protease that may be involved in postaggregation signaling processes. This suggests that crkl may be involved in the reorganization of the cytoskeleton during normal platelet aggregation and its tyrosine phosphorylation in CML platelets may contribute to the abnormal platelet function in CML patients. Finally, we found that thrombopoietin induces tyrosine phosphorylation of crk1 in normal platelets and FDCP cells genetically engineered to express human c-Mpl. This suggests that crk1 can be phosphorylated by a kinase other than p210bcr-abl and that crk1 may have a role in signaling by thrombopoietin.     

CIS1, a cytokine-inducible SH2 protein, suppresses BCR/ABL-mediated transformation. Involvement of the ubiquitin proteasome pathway

OBJECTIVE: BCR/ABL is a chimeric oncoprotein that exhibits deregulated tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph)-positive leukemia. A general understanding of BCR/ABL signaling events is emerging, but little is known about the endogenous inhibitors of p210 BCR/ABL. The present study focused attention on CIS1, a cytokine-inducible SH2 protein, as a potential physiologic antagonist for BCR/ABL. MATERIALS AND METHODS: The murine hematopoietic cell line NSF/N1.H7 stably transfected with BCR/ABL was compared to the parental counterparts for induction of CIS1 by immunoblotting and immunoprecipitation. Cells were treated with a proteasome inhibitor to examine the effect of a proteasome inhibitor on CIS1 protein expression. To determine the effect of CIS1 on BCR/ABL-mediated transformation, we generated Rat-1 fibroblasts transfected with either a control vector, CIS1, BCR/ABL p210, or CIS1 plus BCR/ABL p210. RESULTS: Three forms of CIS1 with molecular masses of 32, 37, and 47 kDa were detected in BCR/ABL-transformed cells. The 47-kDa protein was a ubiquitinated protein. The proteasome inhibitor increased the formation of complexes between CIS1 and BCR/ABL. Transformation of p210 BCR/ABL was significantly suppressed in cells overexpressing CIS1. CONCLUSION: The results suggest that CIS1 is an endogenous inhibitor of p210 BCR/ABL and is likely to be important in the pathogenesis of Ph-positive leukemia.     

Down-regulation of interleukin-3/granulocyte-macrophage colony-stimulating factor receptor beta-chain in BCR-ABL(+) human leukemic cells: association with loss of cytokine-mediated Stat-5 activation and protection from apoptosis after BCR-ABL inhibition

Several signaling cascades are engaged by expression of the p210 bcr-abl tyrosine kinase, and evidence suggests that these signals drive leukemogenesis. In this report, signaling pathways were examined and compared between cells derived from leukemic patients and cells expressing a bcr-abl construct (MBA). The effects of acute inhibition of bcr-abl with STI-571 on these signals and the survival of bcr-abl-expressing cells were also evaluated. Expression of bcr-abl in interleukin-3 (IL-3)/granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent Mo7e cells (MBA) resulted in growth factor independence, constitutive activation of Stat-5 phosphorylation, engagement of mitogen-activated protein (MAP) kinase signals, and increased expression of PTP1B and bcl-x(L). STI-571 inhibited cell growth and induced apoptosis in bcr-abl-expressing cells (MBA, K562, BV-173, KBM5) but not in bcr-abl(-) tumor cells (Mo7e, KG-1, ME-180, Daudi). STI-571-mediated apoptosis correlated with the inhibition of Stat-5 and MAP kinase activation and a reduction in overexpressed bcl-x(L) but not in PTP1B. Inhibitor had no effect on IL-3/GM-CSF-dependent Mo7e cell signaling and did not prevent activation of the other Jak/Stat pathways (interferon alpha, IL-3/GM-CSF). However, neither IL-3 nor GM-CSF could reactivate Stat-5 after the STI-571-mediated inhibition of bcr-abl. Expression of the common beta-chain of the IL-3/GM-CSF receptor was down-regulated in Stat-5-activated myeloid leukemic cells, suppressing IL-3/GM-CSF signal transduction and the ability of these cytokines to provide apoptotic protection. These studies suggest that bcr-abl activates cytokine-independent mechanisms of survival while inactivating intrinsic cytokine signaling cascades, making bcr-abl(+) myeloid cells vulnerable to apoptosis after bcr-abl inactivation.     

Pharmacological profiles of a novel protein tyrosine phosphatase 1B inhibitor,JTT‐551

Aim: Protein tyrosine phosphatase 1B (PTP1B), a negative regulator of insulin signalling, is a novel therapeutic target for type 2 diabetes mellitus. We evaluated in vitro and in vivo the pharmacological profiles of a new PTP1B inhibitor, JTT‐551: monosodium ({[5‐(1,1‐dimethylethyl)thiazol‐2‐yl]methyl} {[(4‐{4‐[4‐(1‐propylbutyl)phenoxy]methyl}phenyl)thiazol‐2‐yl]methyl}amino)acetate. Methods: PTP1B inhibitory activity and the inhibition mode were assayed with p‐nitrophenyl phosphate as a substrate, and the selectivity of JTT‐551 against other PTPs, including T‐cell protein tyrosine phosphatase (TCPTP), CD45 protein tyrosine phosphatase (CD45) and leucocyte common antigen‐related protein tyrosine phosphatase (LAR), was evaluated. Glucose uptake with JTT‐551 treatment was evaluated in L6 rat skeletal myoblasts (L6 cells). In the in vivo study, we investigated the effects on insulin receptor (IR) phosphorylation and blood chemical parameters with JTT‐551 administration in ob/ob mice and db/db mice. Results: JTT‐551 showed an inhibitory effect on PTP1B with a Ki value of 0.22 µM, and a mixed‐type inhibition mode. Ki values of TCPTP, CD45 and LAR were 9.3, 30 or higher and 30 or higher µM, respectively, and JTT‐551 exhibited clear selectivity against the other PTPs. Moreover, JTT‐551 increased the insulin‐stimulated glucose uptake in L6 cells. A single administration of JTT‐551 in ob/ob mice enhanced the IR phosphorylation of liver and reduced the glucose level. In db/db mice, chronic administration showed a hypoglycaemic effect without an acceleration of body weight gain. Conclusions: JTT‐551, a newly developed PTP1B inhibitor, improves glucose metabolism by enhancement of insulin signalling and could be useful in the treatment of type 2 diabetes mellitus.     

Comparison of effects of the tyrosine kinase inhibitors AG957, AG490, and STI571 on BCR-ABL--expressing cells, demonstrating synergy between AG490 and STI571

STI571 (formerly CGP57148) and AG957 are small molecule inhibitors of the protein tyrosine kinase (PTK) p145(abl) and its oncogenic derivative p210(bcr-abl). AG490 is an inhibitor of the PTK Janus kinase 2 (JAK2). No direct comparison of these inhibitors has previously been reported, so this study compared their effects on factor-dependent FDC-P1, 32D, and MO7e cells and their p210(bcr-abl)-expressing factor-independent derivatives. STI571 was a more potent inhibitor of (3)H-thymidine incorporation in p210(bcr-abl)-expressing cells than was AG957, and it showed superior discrimination between inhibitory effects on parental cell lines and effects on their p210(bcr-abl)-expressing derivatives. Assays performed with and without growth factor demonstrated that STI571 but not AG957 reversed the p210(bcr-abl)-driven factor independence of cell lines. p210(bcr-abl)-expressing cells were less sensitive to AG490 than to AG957 or STI571. However, for p210(bcr-abl)-expressing clones from all 3 cell lines, synergistic inhibition was demonstrated between STI571 and concentrations of AG490 with no independent inhibitory effect. Inhibition of nucleic acid synthesis with AG957 treatment was associated with reduced cell numbers, reduced viability, and small pyknotic apoptotic cells. At concentrations of STI571 that reversed the p210(bcr-abl) factor-independent phenotype, STI571 treatment and growth factor deprivation together were sufficient to induce apoptosis. This study concludes that, for the cell lines studied, (1) STI571 is a more potent and more selective inhibitor of a p210(bcr-abl)-dependent phenotype than AG957; (2) AG490 synergizes with STI571 to enhance its inhibitory effect on p210(bcr-abl)-driven proliferation; and (3) the combination of p210(bcr-abl)-tyrosine kinase inhibition and growth factor signal withdrawal can be sufficient to induce apoptotic death of transformed cells. (Blood. 2001;97:2008-2015)     

Liposomal delivery of methylphosphonate antisense oligodeoxynucleotides in chronic myelogenous leukemia [see comments]

Chronic myelogenous leukemia (CML) is a hematologic malignancy characterized by the presence of the Philadelphia (Ph) chromosome. Bcr- abl, the fusion gene associated with the Ph chromosome, expresses a p210bcr-abl protein that promotes a selective expansion of mature myeloid progenitor cells. Methylphosphonate (MP) oligodeoxynucleotides complementary to specific regions of the bcr-abl mRNA were incorporated in liposomes. We studied the effects of liposomal MP (L-MP) on the growth inhibition of CML-like cell lines. L-MP targeted to the breakpoint junctions of the bcr-abl mRNA inhibited the growth of CML cells. Fifty percent inhibition was achieved at approximately 1 mumol/L of L-MP oligonucleotide concentrations. The inhibitory effect was selective because growth inhibition was observed only with CML but not with control cell lines. Moreover, CML cell growth inhibition was dependent on the sequence of the MP oligodeoxynucleotides incorporated in the liposomes. The growth inhibition of CML cells by L-MP resulted from selective inhibition of the expression of the p210bcr-abl protein.     

Chk2 drives late G1/early S phase arrest of clonal myeloid progenitors expressing the p210 BCR-ABL tyrosine kinase in response to STI571

STI571 is the most innovative drug for the cure of Chronic Myeloid Leukemia. It inhibits, in fact, the disease causative event, the p210 bcr-abl tyrosine kinase, and addresses clonal myeloid progenitors to apoptotic death. Here, we demonstrated that STI571 also induces growth arrest by activating the Chk2-Cdc25A-Cdk2 axis, a pathway complementary to p53 in the activation of G(1)/S cell cycle checkpoint. In vitro exposure to STI571 of 32D murine myeloid progenitor cell clones transducing a temperature-sensitive p210 bcr-abl construct was associated with Chk2 phosphorylation and activation, Cdc25A degradation and persistent Cdk2 inhibitory phosphorylation, preventing, in turn, cell transition to and progression throughout the S phase of cell cycle. Chk2 and Cdc25A are both components of a complex network that integrates signals involved in regulated cell cycle progression, DNA repair and cell decision between life or death. Chk2 gene mutations or decreased expression, leading to its protein loss of function on Cdc25A target, and Cdc25A overexpression have been linked to poor prognosis of human cancers. In CML, they might further enhance the proliferative advantage and genomic instability of clonal myeloid progenitors featuring a class of poor prognosis patients eventually resistant to STI571.     

Dendritic cells from CML patients have altered actin organization,reduced antigen processing,and impaired migration

Chronic myeloid leukemia (CML) is characterized by expression of the BCR-ABL fusion gene that encodes a 210-kDa protein, which is a constitutively active tyrosine kinase. At least 70% of the oncoprotein is localized to the cytoskeleton, and several of the most prominent tyrosine kinase substrates for p210(BCR-ABL) are cytoskeletal proteins. Dendritic cells (DCs) are bone marrow-derived antigen-presenting cells responsible for the initiation of immune responses. In CML patients, up to 98% of myeloid DCs generated from peripheral blood mononuclear cells are BCR-ABL positive. In this study we have compared the morphology and behavior of myeloid DCs derived from CML patients with control DCs from healthy individuals. We show that the actin cytoskeleton and shape of CML-DCs of myeloid origin adherent to fibronectin differ significantly from those of normal DCs. CML-DCs are also defective in processing and presentation of exogenous antigens such as tetanus toxoid. The antigen-processing defect may be a consequence of the reduced capacity of CML-DCs to capture antigen via macropinocytosis or via mannose receptors when compared with DCs generated from healthy individuals. Furthermore, chemokine-induced migration of CML-DCs in vitro was significantly reduced. These observations cannot be explained by a difference in the maturation status of CML and normal DCs, because phenotypic analysis by flow cytometry showed a similar surface expression of maturation makers. Taken together, these results suggest that the defects in antigen processing and migration we have observed in CML-DCs may be related to underlying cytoskeletal changes induced by the p210(BCR-ABL) fusion protein.     

Absence of alternative splicing in bcr-abl mRNA in chronic myeloid leukemia cell lines

The major consequence of the Philadelphia (Ph) translocation in chronic myeloid leukemia (CML) is the formation of a bcr-abl hybrid oncogene encoding a tumor cell-specific protein P210bcr-abl. In contrast to this, in Ph chromosome-positive acute lymphoblastic leukemia (Ph + ALL), a P190bcr-abl can be observed. This P190bcr-abl has been implicated in acute rather than chronic leukemogenesis. Therefore, it can be hypothesized that the transition from chronic to blast phase in CML is accompanied by an alternative splice in the bcr-abl mRNA, which results in a switch of the production of P210bcr-abl into P190bcr-abl. Initial S1 nuclease protection mapping supported this theory. However, this result appears to be based on an artifact in the S1 analysis. By using the polymerase chain reaction we provide evidence for the absence of alternative splicing in bcr-abl mRNA in two CML blast crisis cell lines.     

SRC homology-2-containing protein tyrosine phosphatase-1 restrains cell proliferation in human medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) is a rare tumor originating from thyroid parafollicular C cells, where, in the inherited form, constitutive activation of the RET protooncogene is responsible for unrestrained cell proliferation. We previously demonstrated that somatostatin (SRIF) reduces cell growth in the human MTC cell line TT, which expresses all SRIF receptor (SSTR) subtypes and responds differently to selective SSTR agonists. The antiproliferative mechanism of SRIF and its analogs in MTC is still unclear. Src homology-2-containing protein tyrosine phosphatase-1 (SHP-1), a cytoplasmic protein tyrosine phosphatase (PTP), is activated by somatotropin release-inhibiting factor and reduces mutated RET autophosphorylation in a heterologous system. In this study, we explore the role of PTP activation, in particular of SHP-1, in TT cells, where RET is constitutively activated. In TT cells, SRIF stimulated the PTP activity of SHP-1, which was associated with proliferation inhibition and with reduction in the MAPK pathway activation. Blockade of PTP activity with sodium orthovanadate induced cell proliferation and MAPK phosphorylation and blunted the inhibitory effects of SRIF. Moreover, SHP-1 associates with SSTR2 depending on its activation. By using a MAPK kinase inhibitor, we demonstrated that TT cell growth depends on MAPK pathway activation. Furthermore, in TT cells overexpressing SHP-1, cell proliferation and MAPK signaling were strongly down-regulated, whereas in TT cells transfected with a dominant negative form of SHP-1, cell proliferation and MAPK signaling were markedly induced. Our data demonstrate that SRIF inhibitory effects on TT cell proliferation are mediated, at least in part, by SHP-1, which acts through a MAPK-dependent mechanism.     

BCR-ABL1 tyrosine kinase sustained MECOM expression in chronic myeloid leukaemia

MECOM oncogene expression correlates with chronic myeloid leukaemia (CML) progression. Here we show that the knockdown of MECOM (E) and MECOM (ME) isoforms reduces cell division at low cell density, inhibits colony-forming cells by 34% and moderately reduces BCR-ABL1 mRNA and protein expression but not tyrosine kinase catalytic activity in K562 cells. We also show that both E and ME are expressed in CD34(+) selected cells of both CML chronic phase (CML-CP), and non-CML (normal) origin. Furthermore, MECOM mRNA and protein expression were repressed by imatinib mesylate treatment of CML-CP CD34(+) cells, K562 and KY01 cell lines whereas imatinib had no effect in non-CML BCR-ABL1 -ve CD34(+) cells. Together these results suggest that BCR-ABL1 tyrosine kinase catalytic activity regulates MECOM gene expression in CML-CP progenitor cells and that the BCR-ABL1 oncoprotein partially mediates its biological activity through MECOM. MECOM gene expression in CML-CP progenitor cells would provide an in vivo selective advantage, contributing to CML pathogenesis.     

Bcr-Abl Efficiently Induces a Myeloproliferative Disease and Production of Excess Interleukin-3 and Granulocyte-Macrophage Colony-Stimulating Factor in Mice: A Novel Model for Chronic Myelogenous Leukemia

The bcr-abl oncogene plays a critical role in causingchronic myelogenous leukemia (CML). Effective laboratory animal models of CML are needed to study the molecular mechanisms by which the bcr-abl oncogene acts in the disease progression of CML. Weused a murine stem cell retroviral vector (MSCV) to transduce thebcr-abl/p210 oncogene into mouse bone marrow cells and foundthat expression of Bcr-Abl/p210 induced a myeloproliferative disorderthat resembled the chronic phase of human CML in 100% of bone marrowtransplanted mice in about 3 weeks. This CML-like disease was readilytransplanted to secondary recipient mice. Multiple clones of infectedcells were expanded in the primary recipients, but the leukemia was primarily monoclonal in the secondary recipient mice. Mutation analysisdemonstrated that the protein tyrosine kinase activity of Bcr-Abl/p210was essential for its leukemogenic potential in vivo. Interestingly, wefound that the leukemic cells expressed excess interleukin-3 (IL-3) andgranulocyte-macrophage colony-stimulating factor (GM-CSF) in thediseased mice. These studies demonstrate that expression of Bcr-Abl caninduce a CML-like leukemia in mice much more efficiently andreproducibly than in previously reported mouse CML models, probably dueto efficient expression in the correct target cell(s). Our first use ofthis model for analysis of the molecular mechanisms involved in CMLraises the possibility that excess expression of hematopoietic growthfactors such as IL-3 and GM-CSF may contribute to the clinicalphenotype of CML.     

Cell lines and clinical isolates derived from Ph1-positive chronic myelogenous leukemia patients express c-abl proteins with a common structural alteration.

The Philadelphia chromosome (Ph1), observed in greater than 90% of chronic myelogenous leukemia (CML) patients, results from a specific chromosomal translocation involving the c-abl gene. The translocation breakpoint occurs near c-abl and correlates with the production of an altered c-abl mRNA. In the CML-derived cell line K562, Ph1 is accompanied by a structurally altered c-abl protein (P210c-abl) with in vitro tyrosine kinase activity not detected with the normal c-abl protein (P145c-abl). We have examined c-abl proteins in other Ph1-positive CML cell lines and found that they all express P210c-abl. P210c-abl was also detected in bone marrow cells from CML patients with Ph1 in the accelerated and blast crisis phases of the disease. Comparison of the [35S]methionine-labeled tryptic peptides generated from the normal P145c-abl and P210c-abl showed that they have closely related structures, but additional polypeptide sequences are present in P210c-abl. Based on these results we propose that translocation of c-abl in Ph1-positive CML results in the creation of a chimeric gene leading to the production of a structurally altered c-abl protein with activated tyrosine kinase activity. The altered P210 c-abl protein is strongly implicated in the pathogenesis of CML.     

The role of protein tyrosine phosphatase 1B in Ras signaling

Protein tyrosine phosphatase (PTP) 1B has been implicated as a negative regulator of multiple signaling pathways downstream of receptor tyrosine kinases. Inhibition of this enzyme was initially thought to potentially lead to increased oncogenic signaling and tumorigenesis. Surprisingly, we show that platelet-derived growth factor-stimulated extracellular-regulated kinase signaling in PTP1B-deficient cells is not significantly hyperactivated. Moreover, these cells exhibit decreased Ras activity and reduced proliferation by way of previously uncharacterized pathways. On immortalization, PTP1B-deficient fibroblasts display increased expression of Ras GTPase-activating protein (p120RasGAP). Furthermore, we demonstrate that p62Dok (downstream of tyrosine kinase) is a putative substrate of PTP1B and that tyrosine phosphorylation of p62Dok is indeed increased in PTP1B-deficient cells. Consistent with the decreased Ras activity in cells lacking PTP1B, introduction of constitutively activated Ras restored extracellular-regulated kinase signaling and their proliferative potential to those of WT cells. These results indicate that loss of PTP1B can lead to decreased Ras signaling, despite enhanced signaling of other pathways. This finding may in part explain the absence of increased tumor incidence in PTP1B-deficient mice. Thus, PTP1B can positively regulate Ras activity by acting on pathways distal to those of receptor tyrosine kinases.