KSHV-GPCR and CXCR2 Transforming Capacity and Angiogenic Responses Are Mediated through a JAK2-STAT3 Dependent Pathway.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4347-4347
Author(s):  
Meike Burger ◽  
Tanja Hartmann ◽  
Jan A. Burger ◽  
Ingrid U. Schraufstatter
Keyword(s):  
Kaposi's Sarcoma ◽  
Cell Transformation ◽  
Kaposi’S Sarcoma ◽  
Fiber Formation ◽  
Actin Stress Fiber ◽  
3T3 Cells ◽  
Focus Formation ◽  
Nih 3T3 ◽  
Dependent Pathway ◽  
Nih 3T3 Cells

Abstract The Kaposi’s sarcoma herpesvirus encodes a G-protein-coupled chemokine receptor termed KSHV-GPCR. Expression of this constitutively active GPCR leads to cell transformation and vascular overgrowth characteristic of Kaposi’s sarcoma. Previously, we have shown that CXCR2, the closest human homologue, is similarly able to transform cells if continously stimulated or constitutively activated by amino acid exchange D138V of the DRY sequence. Here, we demonstrate that STAT3 activation is a prerequisite for transformation in KSHV-GPCR and CXCR2 transfected NIH 3T3 cells. In KSHV-GPCR and D138V transfected cells, STAT-3 is constitutively phosphorylated on Tyr705. In CXCR2 transfected NIH 3T3 cells and human microvascular endothelial cells (HMEC), which express the CXCR2 constitutively, STAT3 is phosphorylated upon stimulation with IL-8 (CXCL8). Focus formation in NIH 3T3 cells transfected with the KSHV-GPCR, CXCR2, or the D138V mutant, was blocked by the specific JAK2 inhibitor AG490. Typical functions of the CXCR2 including actin stress fiber formation, haptotaxis, and the angiogenic response in HMEC shown by tube formation in Matrigel were blocked by AG490. These data suggest that the transforming capacity and migratory responses that are involved in tumor development, metastasis and angiogenesis in KSHV or CXCR2-expressing cells is at least partially mediated through a JAK2-STAT3 dependent pathway.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Prolongs the Life Span of Primary Human Umbilical Vein Endothelial Cells

2003 ◽  
Vol 77 (11) ◽  
pp. 6188-6196 ◽  
Author(s):  
Takahiro Watanabe ◽  
Makoto Sugaya ◽  
April M. Atkins ◽  
Elisabeth A. Aquilino ◽  
Aparche Yang ◽  
...  
Keyword(s):  
Life Span ◽  
Kaposi's Sarcoma ◽  
Umbilical Vein ◽  
Kaposi’S Sarcoma ◽  
Nuclear Antigen ◽  
3T3 Cells ◽  
Spindle Cells ◽  
Umbilical Vein Endothelial Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT Tumor spindle cells in all clinical types of Kaposi's sarcoma (KS) are infected with Kaposi's sarcoma-associated herpesvirus (KSHV). Although KSHV contains more than 80 genes, only a few are expressed in tumor spindle cells, including latency-associated nuclear antigen (LANA) and k-cyclin (kCYC). To assess the oncogenic potential of LANA and kCYC, primary human umbilical vein endothelial cells (HUVEC) and murine NIH 3T3 cells were stably transduced by using recombinant retroviruses expressing these genes or the known viral oncogene simian virus 40 large T antigen (LTAg). Interestingly, LANA-transduced HUVEC proliferated faster and demonstrated a greatly prolonged life span (mean ± standard deviation, 38.3 ± 11.0 passages) than untransduced cells and vector-transduced cells (<20 passages). By contrast, kCYC-transduced HUVEC did not proliferate faster or live longer than control cells. LANA- and kCYC-transduced HUVEC, but not LTAg-transduced HUVEC, retained the ability to form normal vessel-like structures in an in vitro model of angiogenesis. In cellular assays of transformation, LANA- and kCYC-transduced NIH 3T3 cells demonstrated minimal or no anchorage-independent growth in soft agar and no tumorigenicity when injected into nude mice, unlike LTAg-transduced NIH 3T3 cells. Lastly, gene expression profiling revealed down-regulation, or silencing, of a number of genes within LANA-transduced HUVEC. Taken together, these results suggest that KSHV LANA is capable of inducing prolonged life span, but not transformation, in primary human cells. These findings may explain why LANA-expressing spindle cells proliferate within KS tumors, yet most often do not demonstrate biologic characteristics of transformation or true malignant conversion.

scholarly journals Stat3 Activation by Src Induces Specific Gene Regulation and Is Required for Cell Transformation

1998 ◽  
Vol 18 (5) ◽  
pp. 2545-2552 ◽  
Author(s):  
James Turkson ◽  
Tammy Bowman ◽  
Roy Garcia ◽  
Eric Caldenhoven ◽  
Rolf P. De Groot ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Signaling Pathways ◽  
Cell Transformation ◽  
Specific Gene ◽  
3T3 Cells ◽  
Focus Formation ◽  
Stat3 Signaling ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT While signal transducers and activators of transcription (STATs) were originally discovered as intracellular effectors of normal signaling by cytokines, increasing evidence also points to a role for STAT transcription factors in oncogenesis. Previous studies have demonstrated that one STAT family member, Stat3, possesses constitutively elevated tyrosine phosphorylation and DNA-binding activity in fibroblasts stably transformed by the Src oncoprotein. To determine if this Stat3 activation by Src could induce Stat3-mediated gene expression, luciferase reporter constructs based on synthetic and authentic promoters were transfected into NIH 3T3 cells. Activation of endogenous cellular Stat3 by the Src oncoprotein induced gene expression through a Stat3-specific binding element (TTCCCGAA) of the C-reactive protein gene promoter. A naturally occurring splice variant of human Stat3 protein, Stat3β, with a deletion in the C-terminal transactivation domain abolished this gene induction in a dominant negative manner. Expression of Stat3β did not have any effect on a reporter construct based on the c-fos serum response element, which is not dependent on Stat3 signaling, indicating that Stat3β does not nonspecifically inhibit other signaling pathways or Src function. Transfection of vectors expressing Stat3β together with Src blocked cell transformation by Src as measured in a quantitative focus formation assay using NIH 3T3 cells. By contrast, Stat3β had a much less pronounced effect on focus formation induced by the Ras oncoprotein, which does not activate Stat3 signaling. In addition, three independent clones of NIH 3T3 cells stably overexpressing Stat3β were generated and characterized, demonstrating that Stat3β overexpression does not have a toxic effect on cell viability. These Stat3β-overexpressing clones were shown to be deficient in Stat3-mediated signaling and refractory to Src-induced cell transformation. We conclude that Stat3 activation by the Src oncoprotein leads to specific gene regulation and that Stat3 is one of the critical signaling pathways involved in Src oncogenesis. Our findings provide evidence that oncogenesis-associated activation of Stat3 signaling is part of the process of malignant transformation.

scholarly journals Transformation of NIH 3T3 cells by cotransfection with c-src and nuclear oncogenes.

1987 ◽  
Vol 7 (10) ◽  
pp. 3582-3590 ◽  
Author(s):  
D Shalloway ◽  
P J Johnson ◽  
E O Freed ◽  
D Coulter ◽  
W A Flood
Keyword(s):  
3T3 Cells ◽  
Focus Formation ◽  
Adenovirus E1a ◽  
Rous Sarcoma ◽  
Cellular Homolog ◽  
Sarcoma Virus ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

pp60c-src, the cellular homolog of the Rous sarcoma virus transforming protein, does not completely transform cells even when present at high levels, but has been shown to be involved in polyomavirus-induced transformation when activated by polyomavirus middle T (pmt)-antigen binding. Here we show that cotransfection, but not solo transfection, of expression plasmids for c-src and either adenovirus E1A, v-myc, c-myc, or the 5' half of polyomavirus large T (pltN) antigen into NIH 3T3 cells induces anchorage-independent growth, enhanced focus formation, and, for pltN cotransfection, tumorigenicity in adult NFS mice. Enhancement of transformation was not observed with polyomavirus small t (pst) antigen. Cotransfection of c-src with pltN induced modification of pp60c-src that altered its electrophoretic mobility and in vivo phosphorylation state and stimulated its in vitro kinase activity. Similar alterations were not seen after c-src-E1A cotransfection, suggesting that at least two different mechanisms of enhancement are involved.

Interactions of phosphatidylinositol kinase, GTPase-activating protein (GAP), and GAP-associated proteins with the colony-stimulating factor 1 receptor

1990 ◽  
Vol 10 (11) ◽  
pp. 5601-5608
Author(s):  
M Reedijk ◽  
X Q Liu ◽  
T Pawson
Keyword(s):  
Cell Transformation ◽  
Cellular Transformation ◽  
Colony Stimulating Factor ◽  
Gtpase Activating Protein ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Fibroblast Transformation ◽  
Insert Region ◽  
Stimulating Factor ◽  
Nih 3T3 Cells

The interactions of the macrophage colony-stimulating factor 1 (CSF-1) receptor with potential targets were investigated after ligand stimulation either of mouse macrophages or of fibroblasts that ectopically express mouse CSF-1 receptors. In Rat-2 cells expressing the mouse CSF-1 receptor, full activation of the receptor and cellular transformation require exogenous CSF-1, whereas NIH 3T3 cells expressing mouse c-fms are transformed by autocrine stimulation. Activated CSF-1 receptors physically associate with a phosphatidylinositol (PI) 3'-kinase. A mutant CSF-1 receptor with a deletion of the kinase insert region was deficient in its ability to bind functional PI 3'-kinase and to induce PI 3'-kinase activity precipitable with antiphosphotyrosine antibodies. In fibroblasts, CSF-1 stimulation also induced the phosphorylation of the GTPase-activating protein (GAP)-associated protein p62 on tyrosine, although GAP itself was a relatively poor substrate. In contrast to PI 3'-kinase association, phosphorylation of p62 and GAP was not markedly affected by deletion of the kinase insert region. These results indicate that the kinase insert region selectively enhances the CSF-1-dependent association of the CSF-1 receptor with active PI 3'-kinase. The insert deletion mutant retains considerable transforming activity in NIH 3T3 cells (G. Taylor, M. Reedijk, V. Rothwell, L. Rohrschneider, and T. Pawson, EMBO J. 8:2029-2037, 1989). This mutant was more seriously impaired in Rat-2 cell transformation, although mutant-expressing Rat-2 cells still formed small colonies in soft agar in the presence of CSF-1. Therefore, phosphorylation of GAP and p62 through activation of the CSF-1 receptor does not result in full fibroblast transformation. The interaction between the CSF-1 receptor and PI 3'-kinase may contribute to c-fms fibroblast transformation and play a role in CSF-1-stimulated macrophages.

Overexpressed pp60c-src can induce focus formation without complete transformation of NIH 3T3 cells

1985 ◽  
Vol 5 (5) ◽  
pp. 1073-1083
Author(s):  
P J Johnson ◽  
P M Coussens ◽  
A V Danko ◽  
D Shalloway
Keyword(s):  
Cancer Cells ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
3T3 Cells ◽  
Focus Formation ◽  
Molecular Control ◽  
Nih 3T3 ◽  
Murine Leukemia ◽  
Nih 3T3 Cells

NIH 3T3 cells were transfected with plasmids containing Moloney murine leukemia virus long terminal repeats and either chicken c-src or v-src genes. In contrast with the effects observed after transfection with plasmids containing c-src and avian retrovirus or simian virus 40 promoter-enhancers (H. Hanafusa, H. Iba, T. Takeya, and F. R. Cross, p. 1-8, in G. F. Vande Woude, A. J. Levine, W. C. Topp, and J. D. Watson, ed., Cancer Cells, vol. 2, 1984; H. Iba, T. Takeya, F. R. Cross, T. Hanafusa, and H. Hanafusa, Proc. Natl. Acad. Sci. U.S.A. 81:4424-4428, 1984; R. C. Parker, R. Swanstrom, H. E. Varmus, and J. M. Bishop, p. 19-26, in G. F. Vande Woude et al., ed., Cancer Cells, vol. 2, 1984; R. C. Parker, H. E. Varmus, and J. M. Bishop, Cell 37:131-139, 1984; D. Shalloway, P. M. Coussens, and P. Yaciuk, p. 9-17, in G. F. Vande Woude et al., ed., Cancer Cells, vol. 2, 1984; D. Shalloway, P. M. Coussens, and P. Yaciuk, Proc. Natl. Acad. Sci. U.S.A. 81:7071-7075; and K. C. Wilhelmsen, W. G. Tarpley, and H. M. Temin, p. 303-308, in G. F. Vande Woude et al., ed., Cancer Cells, vol. 2, 1984), we found that both types of Moloney murine leukemia virus long terminal repeat-src expression plasmids induced focus formation, although c-src induced only 1% as many foci as v-src. The focus-selected c-src overexpressed cells had altered morphology and limited growth in soft agarose but were not tumorigenic in vivo. Cleveland digests, comparative in vitro kinase assays, secondary transfections, and immunoprecipitations indicated that focus formation was caused by rare transfection events that resulted in very high-level pp60c-src expression rather than by mutations of the transfected c-src genes. These results suggest that pp60v-src induced transformation is not a completely spurious activity which is unrelated to the function of pp60c-src but that it represents a perturbation of already existent molecular control processes involving pp60c-src.

Muscarinic receptors transform NIH 3T3 cells through a Ras-dependent signalling pathway inhibited by the Ras-GTPase-activating protein SH3 domain

1994 ◽  
Vol 14 (12) ◽  
pp. 7943-7952
Author(s):  
R R Mattingly ◽  
A Sorisky ◽  
M R Brann ◽  
I G Macara
Keyword(s):  
Muscarinic Receptor ◽  
Muscarinic Receptors ◽  
Cellular Transformation ◽  
Sh3 Domain ◽  
Gtpase Activating Protein ◽  
3T3 Cells ◽  
Focus Formation ◽  
Ras Gtpase ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Expression of certain subtypes of human muscarinic receptors in NIH 3T3 cells provides an agonist-dependent model of cellular transformation by formation of foci in response to carbachol. Although focus formation correlates with the ability of the muscarinic receptors to activate phospholipase C, the actual mitogenic signal transduction pathway is unknown. Through cotransfection experiments and measurement of the activation state of native and epitope-tagged Ras proteins, the contributions of Ras and Ras GTPase-activating protein (Ras-GAP) to muscarinic receptor-dependent transformation were defined. Transforming muscarinic receptors were able to activate Ras, and such activation was required for transformation because focus formation was inhibited by coexpression of either Ras with a dominant-negative mutation or constructs of Ras-GAP that include the catalytic domain. Coexpression of the N-terminal region of GAP or of its isolated SH3 (Src homology 3) domain, but not its SH2 domain, was also sufficient to suppress muscarinic receptor-dependent focus formation. Point mutations at conserved residues in the Ras-GAP SH3 domain reversed its action, leading to an increase in carbachol-dependent transformation. The inhibitory effect of expression of the Ras-GAP SH3 domain occurs proximal to Ras activation and is selective for the mitogenic pathway activated by carbachol, as cellular transformation by either v-Ras or trkA/nerve growth factor is unaffected.

scholarly journals Muscarinic receptors transform NIH 3T3 cells through a Ras-dependent signalling pathway inhibited by the Ras-GTPase-activating protein SH3 domain.

1994 ◽  
Vol 14 (12) ◽  
pp. 7943-7952 ◽  
Author(s):  
R R Mattingly ◽  
A Sorisky ◽  
M R Brann ◽  
I G Macara
Keyword(s):  
Muscarinic Receptor ◽  
Muscarinic Receptors ◽  
Cellular Transformation ◽  
Sh3 Domain ◽  
Gtpase Activating Protein ◽  
3T3 Cells ◽  
Focus Formation ◽  
Ras Gtpase ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Expression of certain subtypes of human muscarinic receptors in NIH 3T3 cells provides an agonist-dependent model of cellular transformation by formation of foci in response to carbachol. Although focus formation correlates with the ability of the muscarinic receptors to activate phospholipase C, the actual mitogenic signal transduction pathway is unknown. Through cotransfection experiments and measurement of the activation state of native and epitope-tagged Ras proteins, the contributions of Ras and Ras GTPase-activating protein (Ras-GAP) to muscarinic receptor-dependent transformation were defined. Transforming muscarinic receptors were able to activate Ras, and such activation was required for transformation because focus formation was inhibited by coexpression of either Ras with a dominant-negative mutation or constructs of Ras-GAP that include the catalytic domain. Coexpression of the N-terminal region of GAP or of its isolated SH3 (Src homology 3) domain, but not its SH2 domain, was also sufficient to suppress muscarinic receptor-dependent focus formation. Point mutations at conserved residues in the Ras-GAP SH3 domain reversed its action, leading to an increase in carbachol-dependent transformation. The inhibitory effect of expression of the Ras-GAP SH3 domain occurs proximal to Ras activation and is selective for the mitogenic pathway activated by carbachol, as cellular transformation by either v-Ras or trkA/nerve growth factor is unaffected.

scholarly journals Human c-fgr induces a monocyte-specific enzyme in NIH 3T3 cells.

1991 ◽  
Vol 11 (12) ◽  
pp. 6279-6285 ◽  
Author(s):  
K Inoue ◽  
B Wongsasant ◽  
T Akiyama ◽  
K Toyoshima
Keyword(s):  
Tyrosine Kinases ◽  
Cell Transformation ◽  
Peritoneal Macrophages ◽  
Marker Enzyme ◽  
3T3 Cells ◽  
Dihydroxyvitamin D3 ◽  
Tyrosine Residues ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The mutant c-fgr protein (p58c-fgr/F523) containing Phe-523 instead of Tyr-523 exhibited transforming activity in NIH 3T3 cells like other protein-tyrosine kinases of the src family, but normal p58c-fgr (p58c-fgr/wt) did not. The mutant protein showed tyrosine kinase activity threefold higher than that of the normal protein in vitro. Surprisingly, transfection of the normal c-fgr gene into NIH 3T3 cells resulted in induction of sodium fluoride (NaF)-sensitive alpha-naphthyl butyrate esterase (alpha-NBE), a marker enzyme of cells of monocytic origin, which was not induced in v-src-, v-fgr-, or lyn-transfected NIH 3T3 cells. The NaF-sensitive alpha-NBE induced in c-fgr transfectants was shown by isoelectric focusing to have a pI of 5.2 to 5.4, a range which was the same as those for thioglycolate-induced murine peritoneal macrophages and 1 alpha,25-dihydroxyvitamin D3-treated WEHI-3B cells. Immunoblotting studies with antiphosphotyrosine antibodies revealed that 58-, 62-, 75-, 120-, 200-, and 230-kDa proteins were commonly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in cells transfected with the mutated c-fgr. These findings suggest that tyrosine phosphorylation of specific cellular substrate proteins is important in induction of NaF-sensitive alpha-NBE and cell transformation by p58c-fgr.

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