Tyrosine Kinase Oncoproteins Research Articles

Identification and Characterization of Ovarian Cancer-Initiating Cells from Primary Human Tumors

The objective of this study was to identify and characterize a self-renewing subpopulation of human ovarian tumor cells (ovarian cancer-initiating cells, OCICs) fully capable of serial propagation of their original tumor phenotype in animals. Ovarian serous adenocarcinomas were disaggregated and subjected to growth conditions selective for self-renewing, nonadherent spheroids previously shown to derive from tissue stem cells. To affirm the existence of OCICs, xenoengraftment of as few as 100 dissociated spheroid cells allowed full recapitulation of the original tumor (grade 2/grade 3 serous adenocarcinoma), whereas >10(5) unselected cells remained nontumorigenic. Stemness properties of OCICs (under stem cell-selective conditions) were further established by cell proliferation assays and reverse transcription-PCR, demonstrating enhanced chemoresistance to the ovarian cancer chemotherapeutics cisplatin or paclitaxel and up-regulation of stem cell markers (Bmi-1, stem cell factor, Notch-1, Nanog, nestin, ABCG2, and Oct-4) compared with parental tumor cells or OCICs under differentiating conditions. To identify an OCIC cell surface phenotype, spheroid immunostaining showed significant up-regulation of the hyaluronate receptor CD44 and stem cell factor receptor CD117 (c-kit), a tyrosine kinase oncoprotein. Similar to sphere-forming OCICs, injection of only 100 CD44(+)CD117(+) cells could also serially propagate their original tumors, whereas 10(5) CD44(-)CD117(-) cells remained nontumorigenic. Based on these findings, we assert that epithelial ovarian cancers derive from a subpopulation of CD44(+)CD117(+) cells, thus representing a possible therapeutic target for this devastating disease.

Coexpression of c-kit and bcl-2 in Small Cell Carcinoma and Large Cell Neuroendocrine Carcinoma of the Lung

It has been shown that tyrosine kinase oncoprotein c-kit and antiapoptotic molecule bcl-2 are overexpressed in several types of malignancy, including small cell carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC) of the lung. Whether these 2 molecules are coexpressed in lung neuroendocrine tumors has not been investigated. Here, we analyzed immunohistochemical results to determine expression and coexpression patterns of c-kit and bcl-2 in the spectrum of lung neuroendocrine tumors. Using a polyclonal antibody against c-kit and a monoclonal antibody against bcl-2, our data demonstrated that all 7 cases (100%) of SCLC included in this study were positive for both c-kit and bcl-2. Among 14 LCNECs, 7 (50%) stained positive for c-kit and 9 (64%) for bcl-2. All cases of high grade neuroendocrine carcinomas (SCLCs and LCNECs) that showed positive staining for c-kit coexpressed bcl-2. In contrast, all typical and atypical carcinoids (TC and AC) were negative for c-kit, and only 1 of 16 (6.3%) TCs and 1 of 6 (16.7%) ACs stained positive for bcl-2. These results indicate a progressive increase in the frequency of c-kit and bcl-2 expression and coexpression, from carcinoid tumors (TC and AC) to LCNEC and to SCLC. High grade neuroendocrine carcinomas are more likely to coexpress c-kit and bcl-2 when compared with carcinoid tumors. The high frequency of coexpression of these 2 molecules in high grade neuroendocrine carcinomas of the lung suggests that they may be involved in the carcinogenic pathway, given their important roles in carcinogenesis. Therapeutic targeting on both c-kit and bcl-2 molecules might be beneficial in the management of patients with high grade neuroendocrine carcinomas of the lung in the future.

Construction and Validation of a RET TK Catalytic Domain by Homology Modeling

RET tyrosine kinase (TK) oncoproteins are potential targets for anticancer therapy. However, the search for novel RET inhibitors has been hampered by the lack of a 3D structure of the receptor. In this study, the "open" and the "closed" structure of the RET TK catalytic domain have been built by homology modeling techniques. The structures were validated by extensive docking studies with practically all the inhibitors reported in the literature and through molecular dynamics simulations of the resulting complexes. All the expected major interactions between the active domain amino acids and the inhibitors have been reproduced in their details. Furthermore, the proposed 3D models are in agreement with the results of available mutation studies. Therefore, these models could be profitably used to filter off from large libraries new potential hit compounds able to target this enzyme.

Roles of Phosphorylated p210bcr/abl and Hsp90 Protein in Apoptosis Induced by Berbamine in K562 Cells.

Chronic myeloid leukemia (CML) is a pluripotent hematopoietic stem cell disorder characterized by accumulation of mature and immature granulocytes in peripheral blood and bone marrow due to uncontrolled growth and resistance to apoptosis. The dysregulated activity of the bcr/abl oncoprotein tyrosine kinase, which is encoded by the bcr-abl fusion gene generated from the chromosomal translocation of t(9; 22) and present in approximately 95% of CML patients, has been shown to be responsible for these malignant phenotypes. Numerous studies have demonstrated that only being phosphorylated that p210bcr/abl oncoprotein can promote cell proliferation and survival, and block apoptosis of tumor cells. Bcr/abl tyrosine kinase has chaperone association with heat shock protein 90 (Hsp90), which plays an essential role in stabilization of bcr/abl protein. Here we describe the activity of a natural small molecular compound, berbamine from plant Berberis amurensis that can selectively induce cell death of both Gleevecsensitive and -resistant Ph+ CML cells, but little is known about its exact mechanisms. We investigated the the expression levels of phosphorylated p210bcr/abl protein and Hsp90 protein in apoptosis induced by berbamine in K562 cells by means of various technologies, including cell culture system, flow cytomerty, western blot and immunoprecipitation (IP).Methods: Human Ph+ CML leukemia K562 cells, which express endogenous p210bcr/abl protein, were cultured in RPMI 1640 and treated with berbamine as indicated time and dose. Flow cytometry (FCM) and Annexin V-FLUOS/PI staining kit were used to evaluate apoptosis of leukemic cells; FCM and cytoperm/cytofix plus Caspase-3-McAb-PE were employed to measure leukemic cells with activated Caspase-3. Phosphorylation of p210bcr/abl protein in leukemic cells were assessed by a combination of immunoprecipitation (IP) with c-abl antibody and Western blot with p-Tyr(pY99)antibody. The protein levels of P210bcr/abl, Hsp90 and Hsp70 in leukemic cells were determined by Western blot with antibodies to c-abl, Hsp90 and Hsp70, respectively.Results:After treatment with berbamine at 8 μg/ml for 48 h, the percentages of leukemic cells expressing activated caspase-3 and apoptotic cells were 45.69% and 48.43%, respectively.IP and WB results showed that berbamine at low concentration markedly inhibited phosphorylation of p210bcr/abl protein in leukemia cells, and the amount of phosphorylated p210bcr/abl in leukemia cells exposured to berbamine at 8 μg/ml for 6 h were only 8.41% of that of untreated leukemia cells without the protein levels of P210bcr/abl down-regulated.Berbamine also down-regulated chaperone Hsp90 protein, and the amount of Hsp90 protein in leukemia cells treated with berbamine at 8 μg/ml for 48 h accounted for 18.37% of that of untreated leukemia cells. Interestingly, berbamine at 8 μg/ml had no obvious effects on chaperone Hsp70 protein expression associated with the resistance of leukemia cells to apoptosis.Conclusions:Berbamine could induce caspase-3-mediated apoptosis of Ph+ leukemia cells through inhibiting phosphorylation of p210bcr/abl protein and down-regulating its chaperone Hsp90 protein.Unlike Hsp90 inhibitor GA that could upregulate Hsp70, berbamine had no obvious effects on chaperone Hsp70 protein expression in leukemia cells, suggesting that berbamine may be a novel class of Hsp90 inhibitor, and further study is required.

STAT3: A multifaceted oncogene

Signal transducers and activators of transcription (STAT) proteins are a family of transcription factors first characterized for their role in cytokine signaling. These versatile proteins contain a site for specific tyrosine phosphorylation, a modification that results in a conformational rearrangement and dimerization through phosphotyrosine–SH2 domain interactions (1). Once activated in this manner, phosphorylated STAT protein dimers accumulate in the cell nucleus, bound to enhancer elements of target genes. STAT proteins were first characterized as intermediaries in IFN signaling, a growth inhibitory cytokine pathway involved in antiviral and innate immune responses. However, as additional members of this protein family were discovered, they caught the eyes of cancer biologists as potential mediators of growth control and possibly oncogenic events. In particular, it became recognized that STAT3 and, to a lesser extent, STAT5 were stimulated by classic growth-promoting signals, such as activated growth factor receptors. Even more compelling were the seminal observations that STAT3 was a substrate for the catalytic activity of the tyrosine kinase oncoprotein v-Src (2) and that phosphorylated STAT3 accumulated in many human cancers, leading to the hypothesis that activated STAT3 is an oncogene (3). A considerable amount of experimental and clinical observations have now confirmed such a role for STAT3, and a remarkable degree of diversity has been uncovered for the molecular mechanisms at the basis of STAT3 action. Much early work has focused on direct growth-control and cell survival …

Increased Proliferation and Altered Growth Factor Dependence of Human Mammary Epithelial Cells Overexpressing the Gab2 Docking Protein

The docking protein Gab2 is a proto-oncogene product that is overexpressed in primary breast cancers. To determine the functional consequences of Gab2 overexpression, we utilized the immortalized human mammary epithelial cell line MCF-10A. In monolayer culture, expression of Gab2 at levels comparable with those detected in human breast cancer cells accelerated epidermal growth factor (EGF)-induced cell cycle progression and was associated with increased basal Stat5 tyrosine phosphorylation and enhanced and/or more sustained EGF-induced Erk and Akt activation. Three-dimensional Matrigel culture of MCF-10A cells resulted in the formation of polarized, growth-arrested acini with hollow lumina. Under these conditions, Gab2 increased cell proliferation during morphogenesis, leading to significantly larger acini, an effect dependent on Gab2 binding to Grb2 and Shp2 and enhanced by recruitment of the p85 subunit of phosphatidylinositol 3-kinase. Pharmacological inhibition of MEK revealed that, in addition to direct activation of phosphatidylinositol 3-kinase, increased Erk signaling also contributed to Gab2-mediated enhancement of acinar size. In addition, Gab2 overcame the proliferative suppression that normally occurs in late stage cultures and conferred independence of the morphogenetic program from exogenous EGF. Finally, higher levels of Gab2 expression led to the formation of large disorganized structures with defective luminal clearance. These findings support a role for Gab2 in mammary tumorigenesis.

Meis1 programs transcription of FLT3 and cancer stem cell character, using a mechanism that requires interaction with Pbx and a novel function of the Meis1 C-terminus

Meis1 is a homeodomain transcription factor coexpressed with Hoxa9 in most human acute myeloid leukemias (AMLs). In mouse models of leukemia produced by Hoxa9, Meis1 accelerates leukemogenesis. Because Hoxa9 immortalizes myeloid progenitors in the absence of Meis1 expression, the contribution of Meis1 toward leukemia remains unclear. Here, we describe a cultured progenitor model in which Meis1 programs leukemogenicity. Progenitors immortalized by Hoxa9 in culture are myeloid-lineage restricted and only infrequently caused leukemia after more than 250 days. Coexpressed Meis1 programmed rapid AML-initiating character, maintained multipotent progenitor potential, and induced expression of genes associated with short-term hematopoietic stem cells (HSCs), such as FLT3 and CD34, whose expression also characterizes the leukemia-initiating stem cells of human AML. Meis1 leukemogenesis functions required binding to Pbx, binding to DNA, and a conserved function of its C-terminal tail. We hypothesize that Meis1 is required for the homing and survival of leukemic progenitors within their hematopoietic niches, functions mediated by HSC-specific genes such as CD34 and Fms-like tyrosine kinase 3 (FLT3), respectively. This is the first example of a transcription factor oncoprotein (Meis1) that establishes expression of a tyrosine kinase oncoprotein (FLT3), and explains their coexpression in human leukemia. This cultured progenitor model will be useful to define the genetic basis of leukemogenesis involving Hoxa9 and Meis1.

How thyroid tumors start and why it matters: kinase mutants as targets for solid cancer pharmacotherapy

Treatment of patients with thyroid cancer is usually successful, and most patients are cured of the disease. However, we do not have effective therapies for patients with invasive or metastatic thyroid cancer if the disease is not surgically resectable and does not concentrate radio-iodine. Conventional external beam radiotherapy and chemotherapy are of marginal benefit. In other types of cancer, new therapies are being developed that take advantage of our knowledge of cancer pathogenesis to interfere with the activity of specific oncoproteins believed to be important in disease causation. Because these approaches are being considered for thyroid cancer, I will briefly describe in this review examples of recent breakthroughs in medical therapy of certain hematological malignancies and some solid tumors using drugs that work in this fashion, focusing in particular on compounds that block the enzymatic activity of specific tyrosine kinase oncoproteins. It should be noted, however, that cancers commonly harbor mutations or other disruptions of many genes, each of which could conceivably play a role in disease pathogenesis. This makes the choice of molecular target a difficult and critical decision if these approaches are to succeed. Here I will argue that priority should be given to blocking the function of oncoproteins activated early in tumor development. We have a fairly good understanding of the genetic changes involved in thyroid cancer initiation, and hence these cancers may prove to be particularly well suited for oncoprotein-specific therapies.

Cd117 overexpression in neuroendocrine carcinomas

9595 BACKGROUND: Overexpression of receptor-type tyrosine kinase is found in various malignant tumors and may be associated with aggressive biologic potential. The CD117 (c-kit) tyrosine kinase oncoprotein is overexpressed in many tumors including neuroendocrine carcinomas (NEC) of the lung. Our hypothesis is that CD117 overexpression in NEC influences disease outcome. METHODS: Lesions from patients with low, intermediate and high-grade NEC were biopsied or resected from several anatomic sites (lung, GI tract, skin). The tumors were studied for CD117 overexpression. Archival formalin fixed, paraffin-embedded tissue was immunostained with antibodies against CD117, p53 and Ki-67 using the avidin-biotin-peroxidase method. Staining was assessed quantitatively by four observers using a four-tiered (quartile) scoring method. Cases scoring 2+ or greater were considered positive (overexpressed). Survival of patients with high-grade NEC was estimated using the Kaplan-Meier method. RESULTS: Tissue was analyzed from 106 subjects diagnosed between 1997 and 2003 (M:F=42:64, median age 66 years). NE tumors were distributed as follows: 20 low grade (typical carcinoid), 4 intermediate grade (atypical carcinoid) and 82 high grade (76 small cell, 1 large cell NEC, 5 Merkel cell). CD117 overexpression by tumor grade was distributed as follows: low-grade: 0% (0/20), intermediate-grade: 0% (0/4), high-grade: 66% (54/82). Survival analysis was performed on the subjects with high-grade NEC. High-grade NEC were classified as either positive for CD117 overexpression (n=54) or negative (n=28). There were Median survival time showed no significant survival difference between the two groups (P=0.25). CONCLUSION: Our data suggests that CD117 does not overexpress in low and intermediate-grade NE tumors and does overexpress in a high percentage of high-grade NEC. Given our small sample size, the overexpression or lack of expression in high-grade NEC does not appear to affect the aggressiveness of this disease in terms of survival. Also in our study, a high percentage of the high-grade NEC did overexpress CD117 and it is likely that imatinib (Gleevec) may be an effective form of therapy in this patient population. No significant financial relationships to disclose.

Tyrosine kinase oncoprotein, RET/PTC3, induces the secretion of myeloid growth and chemotactic factors.

Differentiated thyroid carcinomas are the most frequent endocrine neoplasms, but account for few cancer-related deaths. Although the indolent growth of these cancers correlates well with longevity, the biological basis for this good prognosis is not known. In contrast, two of the most frequent autoimmune diseases involve the thyroid suggesting a high propensity for this organ to invoke destructive immunity. Unfortunately, the mechanism linking malignancy and autoimmunity is not clear, although the expression of the oncogenic fusion protein RET/PTC3 (RP3) in both of these disorders may provide a clue. Interestingly, the signaling caused by activated RET kinase involves overlapping pathways and some common to the inflammatory response. Accordingly, we analyzed the function of RP3 and a mutant RP3 molecule to induce proinflammatory pathways in thyroid epithelial cells. Indeed, we find that RP3 alone causes increases in nuclear NF-kappaB activity and secretion of MCP-1 and GM-CSF. Finally, transfer of RP3-expressing thyrocytes into mice in vivo attracted dense macrophage infiltrates, which lead to rapid thyroid cell death. Further, cytokine synthesis and inflammation was largely abrogated by mutation of RP3 Tyr588; an important protein-binding site for downstream signaling. Together, these studies implicate oncogene-induced cytokine-signaling pathways in a new mechanism linking inflammation with cancer.

Involvement of Akt kinase in the action of STI571 on chronic myelogenous leukemia cells

To elucidate the role of mitogen-activated protein kinases (MAPKs) and Akt kinase in leukemogenesis caused by the breakpoint cluster region (BCR)–Abelson (ABL) tyrosine kinase oncoprotein, we examined the activities of MAPKs and Akt kinase and their roles in the action of STI571, a specific inhibitor of BCR-ABL tyrosine kinase, in chronic myelogenous leukemia (CML) cells. We found that extracellular signal-regulated kinase (ERK) 1/2 and Akt kinase are constitutively active in the chronic phase of CML, blast crisis of CML, and the CML-derived K562 cell line. Both interferon-α and STI571 suppressed ERK1/2 activity in K562 cells. In contrast, Akt kinase activity was inhibited only by STI571. K562 cell proliferation was markedly suppressed by LY294002, a specific inhibitor of PI3K/Akt kinase, and STI571 but not by PD98059, a specific inhibitor of MEK1/2. In addition, caspase-3 was activated by treatment of cells with STI571 and LY294002 but not with PD98059. These data indicate that Akt kinase may play a role in the proliferation of CML leukemia cells and the action of STI571. Primary leukemia cells from patients with CML blast crisis did not show inhibition of ERK1/2 or Akt kinase activity and were resistant to caspase-3-associated apoptosis after treatment with STI571. These findings suggest that STI571 does not effectively block signaling molecules downstream of the BCR–ABL tyrosine kinase in some cases of CML blast crisis.

Cell proliferation, nuclear ploidy, and EGFr and HER2/neu tyrosine kinase oncoproteins in infiltrating ductal breast carcinoma

The purpose of this work was to study if the variations in the nuclear DNA content and the expression of EGFr and HER2/neu transmembrane oncoproteins were related and if this influences the rate of cell proliferation modifying, in each case, the potential aggressivity of the neoplasia. Thirty-four ductal breast carcinoma tissue samples of tumors of up to 2 cm in diameter were analyzed by flow cytometry. HER2/neu and EGFr were measured by immunohistochemical methods. Twenty cases were diploid (DNA index of 1) and 14 cases were aneuploid (DNA index other than 1). The expression of EGFr and HER2/neu was significantly higher in aneuploid tumors compared with diploid tumors. The cell proliferation rate was significantly higher in tumors with an aneuploid pattern. The expression of EGFr was associated with a higher rate of cell proliferation. The higher expression of EGFr and HER 2/neu oncoproteins in aneuploid tumors suggests that the increased proliferative activity of aneuploid carcinomas is influenced by the activity of such oncoproteins, which favors a more aggressive biological behavior.

Use of signal specific receptor tyrosine kinase oncoproteins reveals that pathways downstream from Grb2 or Shc are sufficient for cell transformation and metastasis.

Many human cancers have been associated with the deregulation of receptor tyrosine kinases (RTK). However, the individual contribution of receptor-associated signaling proteins in cellular transformation and metastasis is poorly understood. To examine the role of RTK activated signal transduction pathways to processes involved in cell transformation, we have exploited the oncogenic derivative of the Met RTK (Tpr-Met). Unlike other RTKs, twin tyrosine residues in the carboxy-terminal tail of the Met oncoprotein and receptor are required for all biological and transforming activities, and a mutant lacking these tyrosines is catalytically active but non transforming. Using this mutant we have inserted oligonucleotide cassettes, each encoding a binding site for a specific signaling protein derived from other RTKs. We have generated variant forms of the Tpr-Met oncoprotein with the ability to bind individually to the p85 subunit of PI3'K, PLCgamma, or to the Grb2 or Shc adaptor proteins. Variants that recruit the Shc or Grb2 adaptor proteins generated foci of morphologically transformed fibroblast cells and induced anchorage-independent growth, scattering of epithelial cells and experimental metastasis. In contrast, variants that bind and activate PI3'K or PLCgamma failed to generate readily detectable foci. Although cell lines expressing the PI3'K variant grew in soft-agar, these cells were non metastatic. Using this unique RTK oncoprotein model, we have established that Grb2 or Shc dependent signaling pathways are sufficient for cell transformation and metastatic spread.

Dissecting the Molecular Mechanism of Chronic Myelogenous Leukemia Using Murine Models

Chronic myelogenous leukemia (CML) is a complex disease that impinges on stem cell biology, the regulation of blood lineage determination and/or selection, as well as the overall regulation of hematopoietic cell proliferation, survival, adhesion and migration. Establishment of murine models for CML in recent years has enabled experimental analyses of molecular mechanisms in the pathogenesis of CML at the organismal level. This review summarizes the approaches used to develop murine models for CML and the analyses of the roles of functional domains and downstream signaling pathways of BCR-ABL (an oncoprotein generated by the t(9;22)(q34;q11) translocation found in CML patients) and the roles of related tyrosine kinase oncoproteins, altered cytokine production and oncogene cooperation in the pathogenesis of CML-like disease using murine models. These in vivo studies of leukemogenesis will help to advance therapies for CML, as well as to understand fundamental rules of leukemogenesis and hematopoiesis, which should contribute in turn to the development of therapies for other related diseases.

The mechanism of cell cycle regulation by v-Src.

The tyrosine kinase oncoprotein v-Src can overcome the requirements for serum growth factors and anchorage which restrain normal cell growth. Here we investigated the biochemical mechanisms whereby v-Src induces quiescent cells to enter S phase in the absence of serum mitogens. Activating a temperature sensitive v-Src in quiescent cells sequentially induced cyclins D1, E and A and also down regulated p27. We addressed whether p27 down regulation was required to activate cyclin D1/CDK4/6 or cyclin E/CDK2 by engineering cells with inducible p27. Both S phase entry and activation of cyclin/CDKs were inhibited by over expression of p27. Using cells engineered with inducible p16 we showed that Cyclin D/CDK4/6 activity was required for v-Src to increase expression of cyclin A but not cyclin E. To determine which downstream kinases mediated these effects of v-Src we added pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3-K), LY294002 or mitogen activated protein kinase kinase (MEK), U0126. PI3-K was required for v-Src to activate MEK and MEK was required for v-Src to increase expression of cyclins D1 and E. However, the MEK inhibitor prevented p27 protein down regulation whereas the PI3-K inhibitor did not. This was because reduced PI3-K activity lead to proteolytic degradation of p27.

The Reciprocal Role of Egr-1 and Sp Family Proteins in Regulation of the PTP1B Promoter in Response to the p210 Bcr-Abl Oncoprotein-tyrosine Kinase

Protein-tyrosine phosphatase 1B (PTP1B) is an important regulator of protein-tyrosine kinase-dependent signaling pathways. Changes in expression and activity of PTP1B have been associated with various human diseases; however, the mechanisms by which PTP1B expression is regulated have yet to be characterized. Previously, we have shown that the expression of PTP1B is enhanced by p210 Bcr-Abl and that PTP1B is a specific antagonist of transformation induced by this oncoprotein protein-tyrosine kinase. Here we have characterized the PTP1B promoter and demonstrate that a motif with features of a stress-response element acts as a p210 Bcr-Abl-responsive sequence, termed PRS. We have shown that three C(2)H(2) zinc finger proteins, namely Sp1, Sp3, and Egr-1, bind to PRS. Whereas binding of either Sp1 or Sp3 induced promoter function, Egr-1 repressed Sp3-mediated PTP1B promoter activation. The binding of Egr-1 to PRS is suppressed by p210 Bcr-Abl due to the inhibition of Egr-1 expression, resulting in the enhancement of PTP1B promoter activity. Our data indicate that Egr-1 and Sp family proteins play a reciprocal role in the control of expression from the PTP1B promoter.

Full Oncogenic Activities of v-Src Are Mediated by Multiple Signaling Pathways: Ras AS AN ESSENTIAL MEDIATOR FOR CELL SURVIVAL

Tyrosine kinase oncoproteins cause simultaneous activation of multiple intracellular signaling pathways. However, the precise mechanisms by which individual pathways induce oncogenesis are not well understood. We have investigated the roles of individual signaling pathways in v-Src-dependent cell growth and survival by inhibiting one particular pathway. v-Src induced constitutive activation of signal transducers and activators of transcription 3 (STAT3), phosphatidylinositol 3-kinase, and Ras in murine Ba/F3 cells and led to factor-independent proliferation. Dominant-negative mutants of STAT3 (STAT3D) and phosphatidylinositol 3-kinase (Deltap85) inhibited v-Src-dependent growth by approximately 60 and approximately 40%, respectively. Moreover, dominant-negative Ras (N17) induced severe apoptosis, which was accompanied by down-regulation of Bcl-2 and activation of caspase-3. Although cells overexpressing Bcl-2 or caspase-3 inhibitors remained viable even when N17 was expressed, the growth was reduced by approximately 85%. During N17- and STAT3D-induced growth suppression, expression of cyclin D2, cyclin D3, c-myc, and c-fos was suppressed by N17, whereas that of cyclin D2, cyclin E, and c-myc was suppressed by STAT3D. Thus, v-Src-activated Ras and STAT3 are involved in distinct but partly overlapping transcriptional regulation of cell cycle regulatory molecules. These results suggest that the full oncogenic activity of v-Src requires simultaneous activation of multiple signalings, in which Ras is particularly required for survival.

BCR-ABL and v-SRC tyrosine kinase oncoproteins support normal erythroid development in erythropoietin receptor-deficient progenitor cells.

Erythropoietin (Epo)-independent differentiation of erythroid progenitors is a major characteristic of myeloproliferative disorders, including chronic myeloid leukemia. Epo receptor (EpoR) signaling is crucial for normal erythroid development, as evidenced by the properties of Epo(-/-) and EpoR(-/-) mice, which contain a normal number of fetal liver erythroid progenitors but die in utero from a severe anemia attributable to the absence of red cell maturation. Here we show that two constitutively active cytoplasmic protein tyrosine kinases, P210(BCR-ABL) and v-SRC, can functionally replace the EpoR and support full proliferation, differentiation, and maturation of fetal liver erythroid progenitors from EpoR(-/-) mice. These protein tyrosine kinases can also partially complement the myeloid growth factors IL-3, IL-6, and Steel factor, which are normally required in addition to Epo for erythroid development. Additionally, BCR-ABL mutants that lack residues necessary for transformation of fibroblasts or bone marrow cells can fully support normal erythroid development. These results demonstrate that activated tyrosine kinase oncoproteins implicated in tumorigenesis and human leukemia can functionally complement for cytokine receptor signaling pathways to support normal erythropoiesis in EpoR-deficient cells. Moreover, terminal differentiation of erythroid cells requires generic signals provided by activated protein tyrosine kinases and does not require a specific signal unique to a cytokine receptor.

Mechanism of transformation by v-ErbA: substitution for steroid hormone receptor function in self renewal induction.

V-ErbA, a mutated thyroid hormone receptor (TR) alpha cooperates with tyrosine kinase oncoproteins to induce fatal erythroleukemia in chicks. In vitro, v-ErbA employs a similar cooperation to induce sustained proliferation and arrest differentiation of committed erythroid progenitors. V-ErbA has been proposed to function as a dominant-negative c-ErbA/TR alpha, since it lacks an AF-2 transactivation domain and cannot be activated by hormone but retains the capacity to bind corepressors. However, v-ErbA fails to heterodimerize with the coreceptor RXR, exhibits an altered DNA binding specificity and fails to suppress the action of coexpressed TR alpha/c-ErbA in erythroblasts. In this paper, we identify a novel mechanism by which v-ErbA contributes to leukemogenesis. Recently, the glucocorticoid receptor (GR) was identified as a key regulator of proliferation and differentiation in normal erythroid progenitors. For this, the GR required to cooperate with endogenous receptor tyrosine kinases (c-Kit) and with the estrogen receptor (ER). Here, we demonstrate that v-ErbA can substitute for the ligand-activated GR and ER, inducing proliferation and arresting differentiation in the presence of specific GR and ER antagonists. Like the GR, v-ErbA required to cooperate with c-Kit for both proliferation induction and differentiation arrest, being devoid of biological activity in the absence of an active c-Kit. In self-renewing erythroblasts, v-ErbA not only repressed known v-ErbA target genes but also maintained high expression of c-myb. These biological activities of v-ErbA depended on distinct mutations in the DNA-binding domain. Additionally, v-ErbA acted as a partial, weak repressor of c-ErbA/TR alpha function in normal erythroblasts. It could be converted into a truly dominant-negative receptor by restoring its ability to heterodimerize with RXR.

Efficient Cellular Transformation by the Met Oncoprotein Requires a Functional Grb2 Binding Site and Correlates with Phosphorylation of the Grb2-associated Proteins, Cbl and Gab1

The Tpr-Met oncoprotein consists of the catalytic kinase domain of the hepatocyte growth factor/scatter factor receptor tyrosine kinase (Met) fused downstream from sequences encoded by the tpr gene. Tpr-Met is a member of a family of tyrosine kinase oncoproteins generated following genomic rearrangement and has constitutive kinase activity. We have previously demonstrated that a single carboxyl-terminal tyrosine residue, Tyr489, is essential for efficient transformation of Fr3T3 fibroblasts by Tpr-Met and forms a multisubstrate binding site for Grb2, phosphatidylinositol 3' kinase, phospholipase Cgamma, SHP2, and an unknown protein of 110 kDa. A mutant Tpr-Met protein that selectively fails to bind Grb2 has reduced transforming activity, implicating pathways downstream of Grb2 in Tpr-Met mediated cell transformation. We show here that the 110-kDa Tpr-Met substrate corresponds to the recently identified Grb2-associated protein, Gab1. Moreover, we show that tyrosine phosphorylation of the Cbl protooncogene product as well as Gab1 required Tyr489 and correlated with the ability of Tpr-Met to associate with Grb2 and to transform cells, providing evidence that pathways downstream of Gab1 and/or Cbl may play a role in Tpr-Met-mediated cell transformation.