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R Jove

Publications and source records attributed to R Jove.

At least 37 records · Page 2Linked to original sources
PubMed1999

Inhibition of pp60c-Src reduces Bcl-XL expression and reverses the transformed phenotype of cells overexpressing EGF and HER-2 receptors.

Tumors that overexpress HER-2/neu receptor or exhibit enhanced EGFR signaling have been reported to possess constitutively activated Src family kinases, especially pp60c-Src. High levels of pp60c-Src activity have also been reported for cell lines that overexpress the EGFR or the chimeric EGFR-HER-2 receptor. It has therefore been suggested that Src kinases may contribute significantly to the oncogenic phenotype of these cells and to the degree of malignancy of tumors that overexpress EGFR family receptors. In this study we show that the induced expression of c-SRC antisense RNA or the application of a selective Src kinase inhibitor induces growth arrest, programmed cell death and reverses the transformed properties of cells that overexpress EGFR or HER-2 receptors. We show that inhibition of Src kinase expression or activity results in the reduction of Stat3 tyrosine phosphorylation, decline of Bcl-XL expression, and induction of cell death. Using a construct in which the promoter of Bcl-X, which possesses putative Stat3 sites, is tethered to the luciferase reporter gene, we show that inhibition of Src activity or expression induces a decline in Bcl-X expression. We also show that the expression of activated Src induces activation of the Bcl-X promoter. This activation is inhibited by the expression of kinase dead Src or of Stat3beta, the dominant-negative form of Stat3. Taken together, these results support the hypothesis that Src positively regulates the transformed phenotype of cells overexpressing EGFR family kinases. Furthermore, these results also suggest that Src positively regulates Bcl-XL expression via Stat3 activation and thus acts not only as a potent mitogenic signaling element, but also as an anti-apoptotic signaling protein. The combination of both activities probably confers upon activated Src its oncogenic activity. Since Src kinase is activated in many tumors, pp60c-Src kinase inhibitors may prove useful as anti-cancer agents for many types of cancer.

Cell Transformation, Neoplastic↗
PubMed1999

STAT5 activation by BCR-Abl contributes to transformation of K562 leukemia cells.

Signal transducers and activators of transcription (STATs) belong to a family of transcription factors that were originally identified as mediators of cytokine-induced gene expression. Recent evidence, however, has shown that certain members of the STAT family, including STAT3, are also involved in cellular transformation. Here we show that STAT5 also plays a role in cellular transformation by the BCR-Abl oncogene. In BCR-Abl transformed K562 cells, STAT5A and 5B are constitutively phosphorylated on tyrosine and are transcriptionally active. Moreover, expression of a dominant negative form of STAT5 shows that active STAT5 is necessary for the growth in soft agar of these cells. These results show that besides STAT3, STAT5 can also be involved in cellular transformation.

Cell Transformation, Neoplastic↗
PubMed1999

Constitutive RelB activation in v-Src-transformed fibroblasts: requirement for IkappaB degradation.

RelB, an NF-kappaB/Rel-related transacting factor, was initially identified as an immediate-early gene product in fibroblasts and subsequently shown to exhibit constitutive DNA binding activity in lymphoid cells. The data presented in this report show that RelB is also constitutively active, as monitored by electrophoretic mobility shift assay, in the v-Src-transformed fibroblast cell line, SR1. By contrast, nontransformed parental (3Y1) cells displayed inducible NF-kappaB activity; RelB activity was also observed, although to a lesser extent, in two additional v-Src-transformed fibroblast lines. RelB activation in SR1 cells did not require an increase in RelB expression or result from a decrease in the levels of IkappaB alpha or p105, proteins previously shown to bind to and inhibit the activity of the Rel proteins. Numerous studies have shown that stimulus-dependent Rel activation requires degradation of IkappaB alpha, p105 or other member of the IkappaB family, and that this process is precluded by agents that inhibit proteasome activity. We show that treatment of SR1 cells with proteasome inhibitors abolishes RelB activity and thus suggest that RelB in these cells is associated with IkappaB and that v-Src transformation activates RelB by accelerating IkappaB proteolysis. Additional data show that serum and tumor necrosis factor-alpha (TNF-alpha) increase RelB protein levels in 3Y1 cells and that this process is blocked by proteasome inhibitors.

3T3 Cells↗
PubMed1999

Constitutive activation of the JAK2/STAT5 signal transduction pathway correlates with growth factor independence of megakaryocytic leukemic cell lines.

The factor-independent Dami/HEL and Meg-01 and factor-dependent Mo7e leukemic cell lines were used as models to investigate JAK/STAT signal transduction pathways in leukemic cell proliferation. Although Dami/HEL and Meg-01 cell proliferation in vitro was independent of and unresponsive to exogenous cytokines including granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, thrombopoietin (TPO), and tumor necrosis factor-alpha (TNF-alpha), the growth of Mo7e cells was dependent on hematopoietic growth factors. When these cell lines were cultured in medium without cytokines, a constitutively activated STAT-like DNA-binding factor was detected in nuclear extracts from both Dami/HEL and Meg-01 cells. However, the STAT-like factor was not detectable in untreated Mo7e cells, but was activated transiently in Mo7e cells in response to cytokine treatments. The constitutively activated and cytokine-induced STAT-like DNA-binding factor in these three cell lines was identified as STAT5 by oligonucleotide competition gel mobility assays and by specific anti-STAT antibody gel supershift assays. Constitutive activation of JAK2 also was detected in the factor-independent cell lines, but not in Mo7e cells without cytokine exposure. Meg-01 cells express a p185 BCR/ABL oncogene, which may be responsible for the constitutive activation of STAT5. Dami/HEL cells do not express the BCR/ABL oncogene, but increased constitutive phosphorylation of Raf-1 oncoprotein was detected. In cytokine bioassays using growth factor-dependent Mo7e and TF-1 cells as targets, conditioned media from Dami/HEL and Meg-01 cells did not show stimulatory effects on cell proliferation. Our results indicate that the constitutive activation of JAK2/STAT5 correlates with the factor-independent growth of Dami/HEL and Meg-01 cells. The constitutive activation of JAK2/STAT5 in Dami/HEL cells is triggered by a mechanism other than autocrine cytokines or the BCR/ABL oncoprotein.

Cell Division↗
PubMed1999

Inhibition of neurofibromin and p120 GTPase activating protein (GAP) by dietary fatty acids.

Neurofibromin and p120 GTPase activating protein (p120 GAP) down-regulate the activity of cellular Ras proteins. How the activity of these two proteins is controlled is not yet clear. In this study, we analyzed the effects of eight nutritionally relevant fatty acids on GTPase stimulatory activity of full-length neurofibromin and p120 GAP. The fatty acids tested were: saturated stearic acid, monounsaturated oleic acid, three omega-6 and three omega-3 polyunsaturated fatty acids. The analysis was performed by Ras immunoprecipitation GTPase assay. The full-length p120 GAP expressed in insect Sf9 cells and the immunoaffinity purified full-length neurofibromin were used. Neurofibromin was readily inhibited by stearic and oleic acid, but p120 GAP was not inhibited even at high concentrations (> 80 microM). Neurofibromin was also inhibited by low concentrations of all the polyunsaturated fatty acids tested (IC50 of 6 to 16 microM). p120 GAP was 2-3 fold less sensitive to inhibition by these fatty acids. The GTPase stimulatory activity of neurofibromin was also inhibited by arachidonic and oleic acid in the presence of a lipid mixture representing the major lipid components of the cell membrane. Chimeric proteins of neurofibromin and p120 GAP were used to determine that differential sensitivity to fatty acid inhibition maps to the catalytic domain of the proteins. These results indicated that fatty acids can modulate the GTPase function of the c-Ha-Ras protein by inhibiting the GTPase stimulatory activity of two Ras regulators, full-length neurofibromin and p120 GAP, at physiologically relevant concentrations in vitro.

Animals↗
PubMed1999

The Src-family kinase Lck can induce STAT3 phosphorylation and DNA binding activity.

Constitutive activation of the Src-family kinase Lck has been shown to lead to transformation. Constitutive activation of the STAT pathway of transcription factors has also been shown to be involved in transformation. An oncogenic form of the prototypical member of the Src-family, v-Src, has been shown to activate STAT3, and this activation is required for v-Src's transforming ability. To investigate whether Lck could directly activate STAT3, a baculovirus expression system was utilised. When Lck and STAT3 were coexpressed, STAT3 was found to have enhanced tyrosine phosphorylation and DNA binding activity. This finding was confirmed with experiments where exogenous Lck was added to baculovirus produced STAT3. Moreover, the activation of STAT3 by exogenous Lck could be attenuated by the Lck-specific inhibitor PP1. In addition, mammalian cells stably expressing a constitutively activated form of Lck were shown to have activated STAT3. These data provide strong evidence that, like v-Src, Lck can also directly activate STAT3, which contributes to the transformation process.

Animals↗
PubMed1999

Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells.

Interleukin 6 (IL-6) is the major survival factor for myeloma tumor cells and induces signaling through the STAT proteins. We report that one STAT family member, Stat3, is constitutively activated in bone marrow mononuclear cells from patients with multiple myeloma and in the IL-6-dependent human myeloma cell line U266. Moreover, U266 cells are inherently resistant to Fas-mediated apoptosis and express high levels of the antiapoptotic protein Bcl-xL. Blocking IL-6 receptor signaling from Janus kinases to the Stat3 protein inhibits Bcl-xL expression and induces apoptosis, demonstrating that Stat3 signaling is essential for the survival of myeloma tumor cells. These findings provide evidence that constitutively activated Stat3 signaling contributes to the pathogenesis of multiple myeloma by preventing apoptosis.

3T3 Cells↗
PubMed1999

Activating SRC mutation in a subset of advanced human colon cancers.

The discovery of Rous sarcoma virus (RSV) led to the identification of cellular Src (c-Src), a non-receptor tyrosine kinase, which has since been implicated in the development of numerous human cancers. c-Src has been found to be highly activated in colon cancers, particularly in those metastatic to the liver. Studies of the mechanism of c-Src regulation have suggested that c-Src kinase activity is downregulated by phosphorylation of a critical carboxy-terminal tyrosine (Tyr 530 in human c-Src, equivalent to Tyr 527 in chicken Src) and have implied the existence of activating mutations in this C-terminal regulatory region. We report here the identification of a truncating mutation in SRC at codon 531 in 12% of cases of advanced human colon cancer tested and demonstrate that the mutation is activating, transforming, tumorigenic and promotes metastasis. These results provide, for the first time, genetic evidence that activating SRC mutations may have a role in the malignant progression of human colon cancer.

3T3 Cells↗
PubMed1999

STAT proteins as novel targets for cancer therapy. Signal transducer an activator of transcription.

Although the signal transducer and activator of transcription (STAT) proteins were originally discovered through the study of interferon-induced responses, a large number of cytokines and growth factors have been found to activate STATs. In addition to the fundamental role of STAT pathways in normal cell signaling, accumulating evidence is defining a critical role for STATs in oncogenesis. STAT family members are constitutively activated by various oncoproteins in transformed cells and are found activated in a wide variety of human tumors, including breast cancer and diverse blood malignancies. This review discusses recent progress in understanding how aberrant activation of STAT signaling pathways participates in malignant progression of human cancers. Current evidence indicates that one mechanism by which STATs contribute to oncogenesis involves prevention of programmed cell death, or apoptosis, thereby conferring a survival advantage and, potentially, resistance to chemotherapy. These advances identify STATs as novel molecular targets for development of promising therapeutics against human cancers that harbor activated STAT proteins.

Animals↗
PubMed1999

Requirement for Ras/Rac1-mediated p38 and c-Jun N-terminal kinase signaling in Stat3 transcriptional activity induced by the Src oncoprotein.

Signal transducers and activators of transcription (STATs) are transcription factors that mediate normal biologic responses to cytokines and growth factors. However, abnormal activation of certain STAT family members, including Stat3, is increasingly associated with oncogenesis. In fibroblasts expressing the Src oncoprotein, activation of Stat3 induces specific gene expression and is required for cell transformation. Although the Src tyrosine kinase induces constitutive Stat3 phosphorylation on tyrosine, activation of Stat3-mediated gene regulation requires both tyrosine and serine phosphorylation of Stat3. We investigated the signaling pathways underlying the constitutive Stat3 activation in Src oncogenesis. Expression of Ras or Rac1 dominant negative protein blocks Stat3-mediated gene regulation induced by Src in a manner consistent with dependence on p38 and c-Jun N-terminal kinase (JNK). Both of these serine/threonine kinases and Stat3 serine phosphorylation are constitutively induced in Src-transformed fibroblasts. Furthermore, inhibition of p38 and JNK activities suppresses constitutive Stat3 serine phosphorylation and Stat3-mediated gene regulation. In vitro kinase assays with purified full-length Stat3 as the substrate show that both JNK and p38 can phosphorylate Stat3 on serine. Moreover, inhibition of p38 activity and thus of Stat3 serine phosphorylation results in suppression of transformation by v-Src but not v-Ras, consistent with a requirement for Stat3 serine phosphorylation in Src transformation. Our results demonstrate that Ras- and Rac1-mediated p38 and JNK signals are required for Stat3 transcriptional activity induced by the Src oncoprotein. These findings delineate a network of tyrosine and serine/threonine kinase signaling pathways that converge on Stat3 in the context of oncogenesis.

3T3 Cells↗
PubMed1999

Drug resistance in multiple myeloma: approaches to circumvention.

Resistance mechanisms to chemotherapy in multiple myeloma include (1) reduced drug concentrations at the target site of action, (2) alterations in the drug target, and (3) inhibition or prevention of drug-induced apoptosis. Recent advances in understanding resistance mechanisms have resulted in the investigation of novel therapies for the treatment of patients with multiple myeloma. P-glycoprotein is a drug transport protein that decreases intracellular drug concentrations at the target site. Valspodar, a third-generation cyclosporine analog, is an inhibitor of P-glycoprotein that currently is being evaluated to potentially overcome this mechanism of drug resistance. P-glycoprotein inhibitors (also known as chemosensitizers) are being investigated for use in combination with chemotherapeutic agents to enhance the apoptotic effect and prevent resistance at the target site. Other novel approaches involve blocking pathways that result in the expression of antiapoptosis factors. Interleukin-6 is an important growth factor in myeloma and has been implicated in drug resistance via an antiapoptosis effect. In vitro blocking of an interleukin-6-dependent pathway with either a JAK inhibitor (tyrphostin, AG490) or STAT3 dominant negative (STAT3-DN) reduced expression of Bcl-xL (an antiapoptosis protein), increased spontaneous apoptosis, and enhanced sensitivity to Fas-mediated apoptosis. In conclusion, several cellular mechanisms reduce the response to drug therapy in multiple myeloma. Future treatment approaches for this condition most likely will involve combinations of agents to enhance response or prevent resistance.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

Signal Transducers and Activators of Transcription: Novel Targets for Anticancer Therapeutics.

BACKGROUND: Through specific activation of gene expression, the family of proteins known as signal transducers and activators of transcription (STATs) converts extracellular stimuli into diverse biological responses. Beyond the normal signaling functions of STATs, recent evidence indicates that aberrant activation of STATs contributes to neoplastic transformation. METHODS: Current literature pertaining to the role of STAT proteins in oncogenesis is presented. Also, the rationale for developing novel approaches to disrupt STAT signaling is discussed, and the potential of STATs as anticancer targets in treating human cancer is reviewed. RESULTS: The discovery that certain oncoproteins constitutively activate specific STATs, coupled with observations that elevated STAT activity occurs frequently in a spectrum of human tumors, establishes a direct link between STAT activation and neoplastic transformation. Significantly, abrogation of STAT signaling blocks oncogenesis in model in vitro and in vivo systems. These results make STATs attractive targets for rational design of small molecule inhibitors and gene therapy approaches to disrupt STAT signaling. CONCLUSIONS: As a result of genetic, biochemical, and crystallographic analyses, the functional domains of STAT proteins have been well characterized. Based on these data, selective inhibitors of STAT function can be designed. Because disrupting STAT signaling has proven effective in blocking neoplastic transformation, it is proposed that STAT proteins represent promising targets for development of novel molecular therapeutics to treat human cancer.

Journal Article↗
PubMed1998

AKT2, a member of the protein kinase B family, is activated by growth factors, v-Ha-ras, and v-src through phosphatidylinositol 3-kinase in human ovarian epithelial cancer cells.

Three members have been identified in the protein kinase B (PKB) family, i.e., Akt/PKB alpha, AKT2/PKB beta, and AKT3/PKB gamma. Previous studies have demonstrated that only AKT2 is predominantly involved in human malignancies and has oncogenic activity. However, the mechanism of transforming activity of AKT2 is still not well understood. Here, we demonstrate the activation of AKT2 with several growth factors, including epidermal growth factor, insulin-like growth factor 1, insulin-like growth factor II, basic fibroblast growth factor, platelet-derived growth factor, and insulin, in human ovarian epithelial cancer cells. The kinase activity and the phosphorylation of AKT2 were induced by the growth factors and blocked by the phosphatidylinositol (PI) 3-kinase inhibitor, wortmannin, and dominant-negative Ras (N17Ras). Moreover, the activated Ras and v-Src, two proteins that transduce growth factor-generated signals, also activated AKT2, and this activation was not significantly enhanced by growth factor stimulation but was abrogated by wortmannin. These results indicate that AKT2 is a downstream target of PI 3-kinase and that Ras and Src function upstream of PI 3-kinase and mediate the activation of AKT2 by growth factors. The findings also provide further evidence that AKT2, in cooperation with Ras and Src, is important in the development of some human malignancies.

Enzyme Activation↗
PubMed1998

Role of tyrosine kinase activity of epidermal growth factor receptor in the lysophosphatidic acid-stimulated mitogen-activated protein kinase pathway.

Recent evidence indicates that the epidermal growth factor (EGF) receptor mediates a branch of lysophosphatidic acid (LPA)-induced signal transduction pathways that activate mitogen-activated protein (MAP) kinase. However, it is unclear whether the intrinsic tyrosine kinase activity of EGF receptor is involved. We previously showed that reactive oxygen species (ROS) were involved in the LPA-stimulated MAP kinase pathway. Here, we identify tyrosine phosphorylation of EGF receptor as an LPA signaling step that requires ROS. To evaluate the role of the tyrosine kinase activity of EGF receptor in the LPA-stimulated MAP kinase pathway, we examined the effects of an EGF receptor-specific tyrosine kinase inhibitor, PD158780. PD158780 potently inhibited the LPA-stimulated MAP kinase kinase 1/2 (MKK1/2) activation and EGF receptor tyrosine phosphorylation in HeLa cells, while it had no detectable effect on c-Src kinase activity. PD158780 also inhibited LPA-induced MKK1/2 activation and DNA synthesis in NIH 3T3 cells. Furthermore, we compared LPA-stimulated MKK1/2 and MAP kinase activation, transcriptional activity of the c-fos promoter, and DNA synthesis in B82L cells, which lack endogenous EGF receptor, and B82L cells expressing kinase-defective or wild-type human EGF receptor. Results obtained from analysis of these cell lines suggest that the EGF receptor tyrosine kinase contributes to the LPA-stimulated MAP kinase activation, c-fos transcription, and mitogenesis.

Animals↗
PubMed1998

Activation of STAT3 by the c-Fes protein-tyrosine kinase.

STATs (signal transducers and activators of transcription) are transcription factors that contain SH2 domains and are activated by tyrosine phosphorylation, often in response to cytokine stimulation. Recent evidence indicates that the transforming tyrosine kinases encoded by the v-Src, v-Abl, and v-Fps oncogenes can induce STAT activation, suggesting that their normal cellular homologs may contribute to STAT activation under physiological conditions. In this report, we provide direct evidence that c-Fes, the normal human homolog of v-Fps, potently activates STAT3. Transient transfection of human 293T cells with STAT3 and Fes resulted in strong stimulation of STAT3 DNA binding activity. In contrast, only modest activation of STAT5 by Fes was observed in this system, indicative of possible selectivity. To determine whether Fes-induced STAT3 activation is dependent upon endogenous mammalian kinases, co-expression studies were also performed in Sf-9 insect cells. Fes also induced a dramatic increase in STAT3 DNA binding activity in this system, whereas no activation of STAT5 was observed. As a positive control, both STAT3 and STAT5 were shown to be activated by the Bcr-Abl tyrosine kinase in Sf-9 cells. Fes induced strong tyrosine phosphorylation of STAT3 in both expression systems, consistent with the gel-shift results. Fes and STAT3 have been independently linked to myeloid differentiation. Results presented here suggest that these proteins may cooperate to promote differentiation signaling in response to hematopoietic cytokines.

Animals↗
PubMed1998

Activation of STAT transcription factors in oncogenic tyrosine kinase signaling.

Signal transducers and activators of transcription (STATs) are cytoplasmic transcription factors that translocate to the nucleus and regulate gene expression in response to cytokine and growth factor stimulation. Emerging evidence indicates that STAT signaling is also frequently activated by oncogenes and in tumor cells. Constitutive activation of STAT proteins has been reported in cell lines stably transformed by diverse oncoproteins that directly or indirectly activate specific tyrosine kinase signaling pathways. In addition, STAT activation has been detected in a variety of human tumors and tumor cell lines, many of which are known to harbor activated tyrosine kinases. Recent findings support a model in which activation of STAT signaling in the context of oncogenesis induces gene expression that participates in malignant transformation.

Animals↗
PubMed1998

Differential regulation of neurofibromin and p120 GTPase-activating protein by nutritionally relevant fatty acids.

Arachidonic acid, phosphatidic acid, and other lipids inhibit the catalytic fragment of neurofibromin more potently than that of p120 guanosine triphosphatase-activating protein (GAP). The effects of fatty acids other than arachidonic acid on full-length neurofibromin and p120 GAP, to our knowledge, have not been studied. In this study, we analyzed the effects of eight nutritionally relevant fatty acids on guanosine triphosphatase (GTPase) stimulatory activity of full-length neurofibromin and p120 GAP. The fatty acids tested were saturated stearic acid, monounsaturated oleic acid, and three n-6 and three n-3 polyunsaturated fatty acids. Analysis was performed by Ras immunoprecipitation GTPase assay. The full-length p120 GAP expressed in insect Sf9 cells and immunoaffinity-purified full-length neurofibromin were used. In contrast to neurofibromin, which was readily inhibited by stearic and oleic acid, p120 GAP was only weakly inhibited even at high concentrations (> 80 microM). Neurofibromin was also two- to threefold more sensitive to inhibition by other fatty acids tested. A chimeric protein in which the neurofibromin catalytic domain was fused to the NH2-terminal sequences of p120 GAP was used to determine that differential sensitivity to fatty acid inhibition maps to the catalytic domain of the proteins. These results indicate that nutritionally relevant fatty acids can modulate the GTPase function of c-Ha-Ras protein by inhibiting GTPase stimulatory activity of two Ras regulators, full-length neurofibromin and p120 GAP, at physiologically relevant concentrations in vitro.

Animals↗