scholarly journals E2A-HLF-mediated cell transformation requires both the trans-activation domains of E2A and the leucine zipper dimerization domain of HLF.

1995 ◽  
Vol 15 (6) ◽  
pp. 3247-3255 ◽  
Author(s):  
T Yoshihara ◽  
T Inaba ◽  
L H Shapiro ◽  
J Y Kato ◽  
A T Look
Keyword(s):  
Leucine Zipper ◽  
Target Genes ◽  
Cell Transformation ◽  
Fusion Gene ◽  
Phase Cell ◽  
3T3 Cells ◽  
Dimerization Domain ◽  
Activation Domains ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13) translocation in childhood acute pro-B-cell leukemia, encodes a hybrid protein that contains the paired trans-activation domains of E2A (E12/E47) linked to the basic region/leucine zipper DNA-binding and dimerization domain of hepatic leukemia factor (HLF). To assess the transforming potential of this novel gene, we introduced it into NIH 3T3 murine fibroblasts by using an expression vector that also contained the neomycin resistance gene. Cells selected for resistance to the neomycin analog G418 formed aberrant colonies in monolayer cultures, marked by increased cell density and altered morphology. Transfected cells also grew readily in soft agar, producing colonies whose sizes correlated with E2A-HLF expression levels. Subclones expanded from colonies with high levels of the protein reproducibly formed tumors in nude mice and grew to higher plateau-phase cell densities in reduced-serum conditions than did parental NIH 3T3 cells. By contrast, NIH 3T3 cells expressing mutant E2A-HLF proteins that lacked either of the bipartite E2A trans-activation domains or the HLF leucine zipper domain failed to show oncogenic properties, including anchorage-independent cell growth. Thus, both of the E2A trans-activation motifs and the HLF leucine zipper dimerization domain are essential for the transforming potential of the chimeric E2A-HLF protein, suggesting a model in which aberrant regulation of the expression pattern of downstream target genes contributes to leukemogenesis.

scholarly journals Cell transformation mediated by homodimeric E2A-HLF transcription factors.

1997 ◽  
Vol 17 (3) ◽  
pp. 1417-1424 ◽  
Author(s):  
T Inukai ◽  
T Inaba ◽  
T Yoshihara ◽  
A T Look
Keyword(s):  
Transcription Factors ◽  
Target Genes ◽  
Fusion Gene ◽  
Dominant Negative ◽  
Fusion Product ◽  
3T3 Cells ◽  
Par Proteins ◽  
Downstream Target ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The E2A-HLF fusion gene, created by the t(17;19)(q22;p13) chromosomal translocation in pro-B lymphocytes, encodes an oncogenic protein in which the E2A trans-activation domain is linked to the DNA-binding and protein dimerization domain of hepatic leukemia factor (HLF), a member of the proline- and acidic amino acid-rich (PAR) subfamily of bZIP transcription factors. This fusion product binds to its DNA recognition site not only as a homodimer but also as a heterodimer with HLF and two other members of the PAR bZIP subfamily, thyrotroph embryonic factor (TEF) and albumin promoter D-box binding protein (DBP). Thus, E2A-HLF could transform cells by direct regulation of downstream target genes, acting through homodimeric or heterodimeric complexes, or by sequestering normal PAR proteins into nonfunctional heterocomplexes (dominant-negative interference). To distinguish among these models, we constructed mutant E2A-HLF proteins in which the leucine zipper domain of HLF was extended by one helical turn or altered in critical charged amino acids, enabling the chimera to bind to DNA as a homodimer but not as a heterodimer with HLF or other PAR proteins. When introduced into NIH 3T3 cells in a zinc-inducible vector, each of these mutants induced anchorage-independent growth as efficiently as unaltered E2A-HLF, indicating that the chimeric oncoprotein can transform cells in its homodimeric form. Transformation also depended on an intact E2A activator region, providing further support for a gain-of-function contribution to oncogenesis rather than one based on a dominant-interfering or dominant-negative mechanism. Thus, the tumorigenic effects of E2A-HLF and its mutant forms in NIH 3T3 cells favor a straightforward model in which E2A-HLF homodimers bind directly to promoter/enhancer elements of downstream target genes and alter their patterns of expression in early B-cell progenitors.

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.

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.

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.

Loss of the amino-terminal helix-loop-helix domain of the vav proto-oncogene activates its transforming potential

1991 ◽  
Vol 11 (4) ◽  
pp. 1912-1920
Author(s):  
S Katzav ◽  
J L Cleveland ◽  
H E Heslop ◽  
D Pulido
Keyword(s):  
Leucine Zipper ◽  
Ectopic Expression ◽  
Cdna Clones ◽  
3T3 Cells ◽  
Amino Terminal ◽  
Helix Loop Helix ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

vav, a novel human oncogene, was originally generated in vitro by replacement of its normal 5' coding sequences with sequences from pSV2neo DNA, cotransfected as a selectable marker (S. Katzav, D. Martin-Zanca, and M. Barbacid, EMBO J. 8:2283-2290, 1989). The vav proto-oncogene is normally expressed in cells of hematopoietic origin. To determine whether the 5' rearrangement of vav or its ectopic expression in NIH 3T3 cells contributes to its transforming potential, we isolated murine and human proto-vav cDNA clones as well as human genomic clones corresponding to the 5' end of the gene. Normal proto-vav was poorly transforming in NIH 3T3 cells, whereas truncation of its 5' end greatly enhanced its transforming activity. The relative failure of full-length proto-vav cDNA clones to transform NIH 3T3 cells indicates that the transforming activity of vav is not simply due to ectopic expression. Analysis of the predicted amino terminus of the vav proto-oncogene shows that it contains a helix-loop-helix domain and a leucine zipper motif similar to that of myc family proteins, though it lacks a basic region that is usually found adjacent to helix-loop-helix domains. Loss of the helix-loop-helix domain of proto-vav, either by truncation or by rearrangement with pSV2neo sequences, activates its oncogenic potential.

scholarly journals 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.

scholarly journals Loss of the amino-terminal helix-loop-helix domain of the vav proto-oncogene activates its transforming potential.

1991 ◽  
Vol 11 (4) ◽  
pp. 1912-1920 ◽  
Author(s):  
S Katzav ◽  
J L Cleveland ◽  
H E Heslop ◽  
D Pulido
Keyword(s):  
Leucine Zipper ◽  
Ectopic Expression ◽  
Cdna Clones ◽  
3T3 Cells ◽  
Amino Terminal ◽  
Helix Loop Helix ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

vav, a novel human oncogene, was originally generated in vitro by replacement of its normal 5' coding sequences with sequences from pSV2neo DNA, cotransfected as a selectable marker (S. Katzav, D. Martin-Zanca, and M. Barbacid, EMBO J. 8:2283-2290, 1989). The vav proto-oncogene is normally expressed in cells of hematopoietic origin. To determine whether the 5' rearrangement of vav or its ectopic expression in NIH 3T3 cells contributes to its transforming potential, we isolated murine and human proto-vav cDNA clones as well as human genomic clones corresponding to the 5' end of the gene. Normal proto-vav was poorly transforming in NIH 3T3 cells, whereas truncation of its 5' end greatly enhanced its transforming activity. The relative failure of full-length proto-vav cDNA clones to transform NIH 3T3 cells indicates that the transforming activity of vav is not simply due to ectopic expression. Analysis of the predicted amino terminus of the vav proto-oncogene shows that it contains a helix-loop-helix domain and a leucine zipper motif similar to that of myc family proteins, though it lacks a basic region that is usually found adjacent to helix-loop-helix domains. Loss of the helix-loop-helix domain of proto-vav, either by truncation or by rearrangement with pSV2neo sequences, activates its oncogenic potential.

Sign in / Sign up

Export Citation Format

Share Document