The K-fgf/hst oncogene induces transformation through an autocrine mechanism that requires extracellular stimulation of the mitogenic pathway

1991 ◽  
Vol 11 (2) ◽  
pp. 1138-1145
Author(s):  
D Talarico ◽  
C Basilico
Keyword(s):  
Growth Factor ◽  
Receptor Activation ◽  
Soft Agar ◽  
Serum Free Medium ◽  
Free Medium ◽  
3T3 Cells ◽  
Serum Free ◽  
Nih 3T3 ◽  
Autocrine Mechanism ◽  
Nih 3T3 Cells

The K-fgf/hst oncogene encodes a secreted growth factor of the fibroblast growth factor (FGF) family. The ability of K-fgf-transformed cells to grow in soft agar and in serum-free medium is inhibited by anti-K-FGF neutralizing antibodies, consistent with an autocrine mechanism of transformation. The transformed properties of clones that express high levels of K-FGF are, however, only partially affected. To better define the autocrine mechanism of transformation by K-fgf and to determine whether receptor activation could occur intracellularly, we constructed two mutants of the K-fgf cDNA. Deletion of the sequences encoding the signal peptide suppressed K-fgf ability to induce foci in NIH 3T3 cells. A few morphologically transformed colonies were observed in cotransfection experiments, and they were found to express high levels of cytoplasmic K-FGF. However, their ability to grow in serum-free medium and in soft agar was inhibited by anti-K-FGF antibodies. Addition of a sequence encoding the KDEL endoplasmic reticulum and Golgi retention signal to the K-fgf cDNA led to accumulation of the growth factor in intracellular compartments. The ability of the KDEL mutant to induce foci in NIH 3T3 cells was much lower than that of the wild-type cDNA, and also in this case the transformed phenotype was reverted by anti-K-FGF antibodies. These and other findings indicate that the transformed phenotype of cells expressing a nonsecretory K-FGF is due to the extracellular activation of the receptor by the small amounts of growth factor that these cells still release. Thus, transformation by K-fgf appears to be due to an autocrine growth mechanisms that requires activation of the mitogenic pathway at the cell surface.

scholarly journals The K-fgf/hst oncogene induces transformation through an autocrine mechanism that requires extracellular stimulation of the mitogenic pathway.

1991 ◽  
Vol 11 (2) ◽  
pp. 1138-1145 ◽  
Author(s):  
D Talarico ◽  
C Basilico
Keyword(s):  
Growth Factor ◽  
Receptor Activation ◽  
Soft Agar ◽  
Serum Free Medium ◽  
Free Medium ◽  
3T3 Cells ◽  
Serum Free ◽  
Nih 3T3 ◽  
Autocrine Mechanism ◽  
Nih 3T3 Cells

The K-fgf/hst oncogene encodes a secreted growth factor of the fibroblast growth factor (FGF) family. The ability of K-fgf-transformed cells to grow in soft agar and in serum-free medium is inhibited by anti-K-FGF neutralizing antibodies, consistent with an autocrine mechanism of transformation. The transformed properties of clones that express high levels of K-FGF are, however, only partially affected. To better define the autocrine mechanism of transformation by K-fgf and to determine whether receptor activation could occur intracellularly, we constructed two mutants of the K-fgf cDNA. Deletion of the sequences encoding the signal peptide suppressed K-fgf ability to induce foci in NIH 3T3 cells. A few morphologically transformed colonies were observed in cotransfection experiments, and they were found to express high levels of cytoplasmic K-FGF. However, their ability to grow in serum-free medium and in soft agar was inhibited by anti-K-FGF antibodies. Addition of a sequence encoding the KDEL endoplasmic reticulum and Golgi retention signal to the K-fgf cDNA led to accumulation of the growth factor in intracellular compartments. The ability of the KDEL mutant to induce foci in NIH 3T3 cells was much lower than that of the wild-type cDNA, and also in this case the transformed phenotype was reverted by anti-K-FGF antibodies. These and other findings indicate that the transformed phenotype of cells expressing a nonsecretory K-FGF is due to the extracellular activation of the receptor by the small amounts of growth factor that these cells still release. Thus, transformation by K-fgf appears to be due to an autocrine growth mechanisms that requires activation of the mitogenic pathway at the cell surface.

Processing, secretion, and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential

1988 ◽  
Vol 8 (7) ◽  
pp. 2933-2941
Author(s):  
P Delli-Bovi ◽  
A M Curatola ◽  
K M Newman ◽  
Y Sato ◽  
D Moscatelli ◽  
...  
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Kaposi Sarcoma ◽  
Biological Properties ◽  
Fibroblast Growth ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
The Stability ◽  
Nih 3T3

We recently reported that the protein encoded in a novel human oncogene isolated from Kaposi sarcoma DNA was a growth factor with significant homology to basic and acidic fibroblast growth factors (FGFs). To study the properties of this growth factor (referred to as K-FGF) and the mechanism by which the K-fgf oncogene transforms cells, we have studied the production and processing of K-FGF in COS-1 cells transfected with a plasmid encoding the K-fgf cDNA. The results show that, unlike basic and acidic FGFs, the K-FGF protein is cleaved after a signal peptide, glycosylated, and efficiently secreted as a mature protein of 176 or 175 amino acids. Inhibition of glycosylation impaired secretion, and the stability of the secreted K-FGF was greatly enhanced by the presence of heparin in the cultured medium. We have used the conditioned medium from transfected COS-1 cells to test K-FGF biological activity. Similar to basic FGF, the K-FGF protein was mitogenic for fibroblasts and endothelial cells and induced the growth of NIH 3T3 mouse cells in serum-free medium. Accordingly, K-fgf-transformed NIH 3T3 cells grew in serum-free medium, consistent with an autocrine mechanism of growth. We have also expressed the protein encoded in the K-fgf protooncogene in COS-1 cells, and it was indistinguishable in its molecular weight, glycosylation, secretion, and biological activity from K-FGF. Taken together, these results suggest that the mechanism of activation of this oncogene is due to overexpression rather than to mutations in the coding sequences.

Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells

1992 ◽  
Vol 12 (9) ◽  
pp. 3883-3889
Author(s):  
Z Pietrzkowski ◽  
C Sell ◽  
R Lammers ◽  
A Ullrich ◽  
R Baserga
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
Epidermal Growth Factor ◽  
Platelet Derived Growth Factor ◽  
Serum Free Medium ◽  
Free Medium ◽  
3T3 Cells ◽  
Serum Free ◽  
Insulinlike Growth Factor ◽  
Epidermal Growth

BALB/c3T3 cells are exquisitely growth regulated and require platelet-derived growth factor, epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1) for growth. When BALB/c3T3 cells are transfected with plasmids constitutively expressing both EGF and the human IGF-1 receptor mRNAs, the cells are capable of growing in serum-free medium without the addition of any exogenous growth factor. These cells, called p5 cells, can grow for prolonged periods in serum-free medium. BALB/c3T3 cells transfected with only the IGF-1 receptor expression plasmid (p6 cells) do not grow in serum-free medium but do grow if IGF-1 (or insulin in supraphysiological concentrations) is added. p6 cells also grow in response to EGF, confirming that the combination of EGF and an overexpressed IGF-1 receptor is sufficient for the growth of 3T3 cells. We have found that in EGF-stimulated p6 cells there is an increase in the expression of IGF-1 mRNA, that IGF-1 is secreted into the medium, and that the growth of p5 cells and EGF-stimulated p6 cells is inhibited by exposure to antisense oligodeoxynucleotides to IGF-1 receptor RNA. Finally, while cells constitutively expressing both EGF and EGF receptor RNAs grow, albeit modestly, in serum-free medium, their growth is also inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA. In contrast, in cells overexpressing the IGF-1 receptor, IGF-1-mediated cell growth occurs independently of the platelet-derived growth factor and EGF receptors (Z. Pietrzkowski, R. Lammers, G. Carpenter, A. M. Soderquist, M. Limardo, P. D. Phillips, A. Ullrich, and R. Baserga, Cell Growth Differ. 3:199-205, 1992, and this paper). These data indicate that an important role for EGF is participation in the activation of an autocrine loop based on the IGF-1-IGF-1 receptor interaction, which is obligatory for the proliferation of 3T3 cells.

The growth-stimulatory effect of simian virus 40 T antigen requires the interaction of insulinlike growth factor 1 with its receptor

1992 ◽  
Vol 12 (11) ◽  
pp. 5069-5077
Author(s):  
P Porcu ◽  
A Ferber ◽  
Z Pietrzkowski ◽  
C T Roberts ◽  
M Adamo ◽  
...  
Keyword(s):  
Growth Factor ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Serum Free Medium ◽  
Free Medium ◽  
3T3 Cells ◽  
Sv40 T Antigen ◽  
Serum Free ◽  
Insulinlike Growth Factor

We have used a plasmid expressing a temperature-sensitive (ts) mutant of simian virus 40 (SV40) T antigen, stably transfected into 3T3 cells, to study the role of insulinlike growth factor 1 (IGF-1) and its receptor in T-antigen-mediated growth. While 3T3 cells do not grow in serum-free medium, in 1% serum, or with the sole addition of either platelet-derived growth factor (PDGF) or IGF-1, cells expressing the tsA T antigen (BALB 58 cells) grow at 34 degrees C in either PDGF or 1% serum but not in IGF-1. At the restrictive temperature (39.6 degrees C), these cells can only grow in 10% serum. We show that BALB 58 cells, at 34 degrees C, have a markedly increased expression of IGF-1 and IGF-1 mRNA and that their growth in 1% serum (at 34 degrees C) is inhibited by an antisense oligodeoxynucleotide to the IGF-1 receptor RNA. When this tsA plasmid is stably transfected into cells constitutively overexpressing the human IGF-1 receptor cDNA, the resulting cell lines show a constitutively phosphorylated IGF-1 receptor and grow in serum-free medium at 34 degrees C (but not at 39.6 degrees C). A functional SV40 T antigen also increases the expression of a plasmid in which the reporter luciferase gene is under the control of a rat IGF-1 promoter. We conclude (i) that the SV40 T antigen induces the expression of IGF-1 and IGF-1 mRNA, at least in part by a transcriptional mechanism, thus altering the growth factors requirements, and (ii) that, in BALB/c3t3 cells, the SV40 T antigen necessitates a functional IGF-1 receptor for its growth-stimulating effect in low serum (or PDGF).

scholarly journals Processing, secretion, and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential.

1988 ◽  
Vol 8 (7) ◽  
pp. 2933-2941 ◽  
Author(s):  
P Delli-Bovi ◽  
A M Curatola ◽  
K M Newman ◽  
Y Sato ◽  
D Moscatelli ◽  
...  
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Kaposi Sarcoma ◽  
Biological Properties ◽  
Fibroblast Growth ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
The Stability ◽  
Nih 3T3

We recently reported that the protein encoded in a novel human oncogene isolated from Kaposi sarcoma DNA was a growth factor with significant homology to basic and acidic fibroblast growth factors (FGFs). To study the properties of this growth factor (referred to as K-FGF) and the mechanism by which the K-fgf oncogene transforms cells, we have studied the production and processing of K-FGF in COS-1 cells transfected with a plasmid encoding the K-fgf cDNA. The results show that, unlike basic and acidic FGFs, the K-FGF protein is cleaved after a signal peptide, glycosylated, and efficiently secreted as a mature protein of 176 or 175 amino acids. Inhibition of glycosylation impaired secretion, and the stability of the secreted K-FGF was greatly enhanced by the presence of heparin in the cultured medium. We have used the conditioned medium from transfected COS-1 cells to test K-FGF biological activity. Similar to basic FGF, the K-FGF protein was mitogenic for fibroblasts and endothelial cells and induced the growth of NIH 3T3 mouse cells in serum-free medium. Accordingly, K-fgf-transformed NIH 3T3 cells grew in serum-free medium, consistent with an autocrine mechanism of growth. We have also expressed the protein encoded in the K-fgf protooncogene in COS-1 cells, and it was indistinguishable in its molecular weight, glycosylation, secretion, and biological activity from K-FGF. Taken together, these results suggest that the mechanism of activation of this oncogene is due to overexpression rather than to mutations in the coding sequences.

scholarly journals Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells.

1992 ◽  
Vol 12 (9) ◽  
pp. 3883-3889 ◽  
Author(s):  
Z Pietrzkowski ◽  
C Sell ◽  
R Lammers ◽  
A Ullrich ◽  
R Baserga
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
Epidermal Growth Factor ◽  
Platelet Derived Growth Factor ◽  
Serum Free Medium ◽  
Free Medium ◽  
3T3 Cells ◽  
Serum Free ◽  
Insulinlike Growth Factor ◽  
Epidermal Growth

BALB/c3T3 cells are exquisitely growth regulated and require platelet-derived growth factor, epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1) for growth. When BALB/c3T3 cells are transfected with plasmids constitutively expressing both EGF and the human IGF-1 receptor mRNAs, the cells are capable of growing in serum-free medium without the addition of any exogenous growth factor. These cells, called p5 cells, can grow for prolonged periods in serum-free medium. BALB/c3T3 cells transfected with only the IGF-1 receptor expression plasmid (p6 cells) do not grow in serum-free medium but do grow if IGF-1 (or insulin in supraphysiological concentrations) is added. p6 cells also grow in response to EGF, confirming that the combination of EGF and an overexpressed IGF-1 receptor is sufficient for the growth of 3T3 cells. We have found that in EGF-stimulated p6 cells there is an increase in the expression of IGF-1 mRNA, that IGF-1 is secreted into the medium, and that the growth of p5 cells and EGF-stimulated p6 cells is inhibited by exposure to antisense oligodeoxynucleotides to IGF-1 receptor RNA. Finally, while cells constitutively expressing both EGF and EGF receptor RNAs grow, albeit modestly, in serum-free medium, their growth is also inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA. In contrast, in cells overexpressing the IGF-1 receptor, IGF-1-mediated cell growth occurs independently of the platelet-derived growth factor and EGF receptors (Z. Pietrzkowski, R. Lammers, G. Carpenter, A. M. Soderquist, M. Limardo, P. D. Phillips, A. Ullrich, and R. Baserga, Cell Growth Differ. 3:199-205, 1992, and this paper). These data indicate that an important role for EGF is participation in the activation of an autocrine loop based on the IGF-1-IGF-1 receptor interaction, which is obligatory for the proliferation of 3T3 cells.

scholarly journals Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts.

1994 ◽  
Vol 14 (6) ◽  
pp. 3604-3612 ◽  
Author(s):  
C Sell ◽  
G Dumenil ◽  
C Deveaud ◽  
M Miura ◽  
D Coppola ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Soft Agar ◽  
T Antigen ◽  
Insulin Like Growth Factor ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Factor I ◽  
Growth Factor I

Fibroblast cell lines, designated R- and W cells, were generated, respectively, from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 insulin-like growth factor receptor, and from their wild-type littermates. W cells grow normally in serum-free medium supplemented with various combinations of purified growth factors, while pre- and postcrisis R- cells cannot grow, as they are arrested before entering the S phase. R- cells are able to grow in 10% serum, albeit more slowly than W cells, and with all phases of the cell cycle being elongated. An activated Ha-ras expressed from a stably transfected plasmid is unable to overcome the inability of R- cells to grow in serum-free medium supplemented with purified clones. Nevertheless, even in the presence of serum, R- cells stably transfected with Ha-ras, alone or in combination with simian virus 40 large T antigen, fail to form colonies in soft agar. Reintroduction into R- cells (or their derivatives) of a plasmid expressing the human insulin-like growth factor I receptor RNA and protein restores their ability to grow with purified growth factors or in soft agar. The signaling pathways participating in cell growth and transformation are discussed on the basis of these results.

Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts

1994 ◽  
Vol 14 (6) ◽  
pp. 3604-3612
Author(s):  
C Sell ◽  
G Dumenil ◽  
C Deveaud ◽  
M Miura ◽  
D Coppola ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Soft Agar ◽  
T Antigen ◽  
Insulin Like Growth Factor ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Factor I ◽  
Growth Factor I

Fibroblast cell lines, designated R- and W cells, were generated, respectively, from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 insulin-like growth factor receptor, and from their wild-type littermates. W cells grow normally in serum-free medium supplemented with various combinations of purified growth factors, while pre- and postcrisis R- cells cannot grow, as they are arrested before entering the S phase. R- cells are able to grow in 10% serum, albeit more slowly than W cells, and with all phases of the cell cycle being elongated. An activated Ha-ras expressed from a stably transfected plasmid is unable to overcome the inability of R- cells to grow in serum-free medium supplemented with purified clones. Nevertheless, even in the presence of serum, R- cells stably transfected with Ha-ras, alone or in combination with simian virus 40 large T antigen, fail to form colonies in soft agar. Reintroduction into R- cells (or their derivatives) of a plasmid expressing the human insulin-like growth factor I receptor RNA and protein restores their ability to grow with purified growth factors or in soft agar. The signaling pathways participating in cell growth and transformation are discussed on the basis of these results.

scholarly journals The growth-stimulatory effect of simian virus 40 T antigen requires the interaction of insulinlike growth factor 1 with its receptor.

1992 ◽  
Vol 12 (11) ◽  
pp. 5069-5077 ◽  
Author(s):  
P Porcu ◽  
A Ferber ◽  
Z Pietrzkowski ◽  
C T Roberts ◽  
M Adamo ◽  
...  
Keyword(s):  
Growth Factor ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Serum Free Medium ◽  
Free Medium ◽  
3T3 Cells ◽  
Sv40 T Antigen ◽  
Serum Free ◽  
Insulinlike Growth Factor

We have used a plasmid expressing a temperature-sensitive (ts) mutant of simian virus 40 (SV40) T antigen, stably transfected into 3T3 cells, to study the role of insulinlike growth factor 1 (IGF-1) and its receptor in T-antigen-mediated growth. While 3T3 cells do not grow in serum-free medium, in 1% serum, or with the sole addition of either platelet-derived growth factor (PDGF) or IGF-1, cells expressing the tsA T antigen (BALB 58 cells) grow at 34 degrees C in either PDGF or 1% serum but not in IGF-1. At the restrictive temperature (39.6 degrees C), these cells can only grow in 10% serum. We show that BALB 58 cells, at 34 degrees C, have a markedly increased expression of IGF-1 and IGF-1 mRNA and that their growth in 1% serum (at 34 degrees C) is inhibited by an antisense oligodeoxynucleotide to the IGF-1 receptor RNA. When this tsA plasmid is stably transfected into cells constitutively overexpressing the human IGF-1 receptor cDNA, the resulting cell lines show a constitutively phosphorylated IGF-1 receptor and grow in serum-free medium at 34 degrees C (but not at 39.6 degrees C). A functional SV40 T antigen also increases the expression of a plasmid in which the reporter luciferase gene is under the control of a rat IGF-1 promoter. We conclude (i) that the SV40 T antigen induces the expression of IGF-1 and IGF-1 mRNA, at least in part by a transcriptional mechanism, thus altering the growth factors requirements, and (ii) that, in BALB/c3t3 cells, the SV40 T antigen necessitates a functional IGF-1 receptor for its growth-stimulating effect in low serum (or PDGF).

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