scholarly journals Loss of epidermal growth factor receptors and release of transforming growth factors do not correlate with sarcoma virus-transformation in clonally-related NIH/3T3-derived cell lines

1985 ◽  
Vol 229 (1) ◽  
pp. 119-125 ◽  
Author(s):  
K D Brown ◽  
D M Blakeley ◽  
P Roberts ◽  
R J Avery
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Line ◽  
Conditioned Medium ◽  
Cell Lines ◽  
Egf Receptors ◽  
3T3 Cells ◽  
Sarcoma Virus ◽  
Epidermal Growth ◽  
Nih 3T3

Transformation of NIH/3T3 cells by Kirsten murine sarcoma virus (MSV) caused a dramatic reduction in the number of cell-surface receptors for epidermal growth factor (EGF). However, the number of EGF receptors remained at a very low level in a non-tumourigenic revertant cell line isolated from the virus-transformed cells, indicating that an increase in EGF receptors is not a requirement for the phenotypic reversion of Kirsten MSV-transformed 3T3 cells. Serum-free conditioned medium from normal and virus-transformed cell lines contained similar amounts of cell growth-promoting activity as assayed by the ability to stimulate DNA synthesis in quiescent Swiss 3T3 cell cultures. However, the concentrated conditioned medium from these cell lines showed no evidence of beta-transforming growth factor (TGF) activity as assayed by promotion of anchorage-independent growth of untransformed normal rat kidney (NRK) fibroblasts in agarose. The cellular release of alpha-TGF activity was assayed by measuring the ability of concentrated conditioned medium to inhibit the binding of 125I-EGF to Swiss 3T3 cells. Conditioned medium protein from the virus-transformed cell line inhibited 125I-EGF binding but only to the same extent as conditioned medium protein prepared from the untransformed cell line. The alpha-TGF secretion by these cell lines was estimated to be 30-45-fold lower than the level of alpha-TGF released by a well-characterized alpha-TGF-producing cell line (3B11). These results suggest that the induction of TGF release is not a necessary event in the transformation of NIH/3T3 cells by Kirsten MSV.

Regulation of PG Synthase by EGF and PDGF in Human Oral, Breast, Stomach, and Fibrosarcoma Cancer Cell Lines

1994 ◽  
Vol 73 (8) ◽  
pp. 1407-1415 ◽  
Author(s):  
C.Y. Yang ◽  
C.L. Meng
Keyword(s):  
Enzyme Activity ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Platelet Derived Growth Factor ◽  
Moderate Degree ◽  
Epidermal Growth

Prostaglandins may inhibit or promote tumor cell replication, depending on the cell system that is investigated. In our laboratory, we have established and characterized four different specific human cancer cell lines. The objectives of this study were to examine and compare the prostaglandin endoperoxide synthase (PG synthase, EC 1.14.99.1) activity of these cell lines by measuring the conversion of arachidonate to 3H-PGE2 and 3H-PGF2α. We found that the oral epidermal carcinoma cell line (OEC-M1) had a moderate degree of PG synthase activity. Enzyme activity could be partially blocked (statistically significant) by the addition of epidermal growth factor (EGF) at 20 ng/mL and almost completely inhibited by platelet-derived growth factor at (PDGF) 20 mU/mL. By contrast, we discovered that the human breast adenocarcinama cell line (BC-M1) did not contain significant PG synthase, and enzyme activity could be significantly activated by the addition of epidermal growth factor at 20 ng/mL and platelet-derived growth factor at 20 mU/mL. We also found that the human stomach adenocarcinoma cell line (SCM-1) had a significant amount of PG synthase activity, and these PG synthase activities were not activated or inhibited by EGF at 20 ng/mL or PDGF at 20 mU/mL. Furthermore, the human fibrosarcoma (FS-Ml) cell line also contained a moderate degree of PG synthase activity, which could be significantly inhibited by PDGF at 20 mU/mL but was not inhibited by EGF at 20 ng/mL. The results suggest that EGF and PDGF may be involved in the regulation of the PG synthase activities of human oral, breast, stomach, and fibrosarcoma cancer cells.

scholarly journals Anti-oncogenic activity of signalling-defective epidermal growth factor receptor mutants.

1992 ◽  
Vol 12 (2) ◽  
pp. 491-498 ◽  
Author(s):  
N Redemann ◽  
B Holzmann ◽  
T von Rüden ◽  
E F Wagner ◽  
J Schlessinger ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinase ◽  
Growth Factor Receptor ◽  
Wild Type ◽  
3T3 Cells ◽  
Epidermal Growth ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Overexpression and autocrine activation of the epidermal growth factor receptor (EGF-R) cause transformation of cultured cells and correlate with tumor progression in cancer patients. Dimerization and transphosphorylation are crucial events in the process by which receptors with tyrosine kinase activity generate normal and transforming cellular signals. Interruption of this process by inactive receptor mutants offers the potential to inhibit ligand-induced cellular responses. Using recombinant retroviruses, we have examined the effects of signalling-incompetent EGF-R mutants on the growth-promoting and transforming potential of ligand-activated, overexpressed wild-type EGF-R and the v-erbB oncogene product. Expression of a soluble extracellular EGF-R domain had little if any effect on the growth and transformation of NIH 3T3 cells by either tyrosine kinase. However, both a kinase-negative EGF-R point mutant (HERK721A) and an EGF-R lacking 533 C-terminal amino acids efficiently inhibited wild-type EGF-R-mediated, de novo DNA synthesis and cell transformation in a dose-dependent manner. Furthermore, coexpression with the v-erbBES4 oncogene product in NIH 3T3 cells resulted in transphosphorylation of the HERK721A mutant receptor and reduced soft-agar colony growth but had no effect in a focus formation assay. These results demonstrate that signalling-defective receptor tyrosine kinase mutants differentially interfere with oncogenic signals generated by either overexpressed EGF-R or the retroviral v-erbBES4 oncogene product.

scholarly journals Expression and biosynthetic variation of the epidermal growth factor receptor in human hepatocellular carcinoma-derived cell lines.

1988 ◽  
Vol 8 (1) ◽  
pp. 25-34 ◽  
Author(s):  
C R Carlin ◽  
D Simon ◽  
J Mattison ◽  
B B Knowles
Keyword(s):  
Hepatocellular Carcinoma ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Line ◽  
Cell Lines ◽  
Gene Amplification ◽  
Egf Receptor ◽  
Hepatoma Cell ◽  
Receptor Gene ◽  
Epidermal Growth

Expression of the epidermal growth factor (EGF) was analyzed in six human hepatocellular carcinoma-derived and one human hepatoblastoma-derived cell line, each of which retained the differentiated phenotype and functions of the parenchymal hepatocyte. The level of receptor expression of each hepatoma cell line was similar to that of the normal human fibroblast, approximately 10(5) molecules per cell. However, NPLC/PRF/5, a subline of the PLC/PRF/5 cell line obtained following reestablishment of a xenograft tumor in vitro, was found to express 4 x 10(6) high-affinity EGF receptor molecules per cell. Proliferation of the NPLC/PRF/5 cell line was inhibited in the presence of nanomolar quantities of ligand. Receptor overexpression was found to result from EGF receptor gene amplification without apparent rearrangement of the EGF receptor coding sequences. Although cell-specific variability in posttranslational processing of EGF receptor N-linked oligosaccharides in the hepatoma cell lines was found, no difference between the receptors in PLC/PRF/5 and NPLC/PRF/5 was observed and no aberrant receptor-related species were detected. EGF receptor gene amplification in the NPLC/PRF/5 cell line is probably a reflection of genome instability and selection of variants with augmented growth potential in limiting concentrations of EGF in vivo. When viewed in this light, EGF receptor overexpression could represent a manifestation of tumor progression in the EGF-responsive hepatocyte.

scholarly journals Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha.

1988 ◽  
Vol 8 (5) ◽  
pp. 1970-1978 ◽  
Author(s):  
I Lax ◽  
A Johnson ◽  
R Howk ◽  
J Sap ◽  
F Bellot ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Alpha ◽  
3T3 Cells ◽  
Epidermal Growth ◽  
Factor Alpha ◽  
Differential Binding ◽  
Nih 3T3

The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors.

Expression and biosynthetic variation of the epidermal growth factor receptor in human hepatocellular carcinoma-derived cell lines

1988 ◽  
Vol 8 (1) ◽  
pp. 25-34
Author(s):  
C R Carlin ◽  
D Simon ◽  
J Mattison ◽  
B B Knowles
Keyword(s):  
Hepatocellular Carcinoma ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Line ◽  
Cell Lines ◽  
Gene Amplification ◽  
Egf Receptor ◽  
Hepatoma Cell ◽  
Receptor Gene ◽  
Epidermal Growth

Expression of the epidermal growth factor (EGF) was analyzed in six human hepatocellular carcinoma-derived and one human hepatoblastoma-derived cell line, each of which retained the differentiated phenotype and functions of the parenchymal hepatocyte. The level of receptor expression of each hepatoma cell line was similar to that of the normal human fibroblast, approximately 10(5) molecules per cell. However, NPLC/PRF/5, a subline of the PLC/PRF/5 cell line obtained following reestablishment of a xenograft tumor in vitro, was found to express 4 x 10(6) high-affinity EGF receptor molecules per cell. Proliferation of the NPLC/PRF/5 cell line was inhibited in the presence of nanomolar quantities of ligand. Receptor overexpression was found to result from EGF receptor gene amplification without apparent rearrangement of the EGF receptor coding sequences. Although cell-specific variability in posttranslational processing of EGF receptor N-linked oligosaccharides in the hepatoma cell lines was found, no difference between the receptors in PLC/PRF/5 and NPLC/PRF/5 was observed and no aberrant receptor-related species were detected. EGF receptor gene amplification in the NPLC/PRF/5 cell line is probably a reflection of genome instability and selection of variants with augmented growth potential in limiting concentrations of EGF in vivo. When viewed in this light, EGF receptor overexpression could represent a manifestation of tumor progression in the EGF-responsive hepatocyte.

Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor

1988 ◽  
Vol 8 (6) ◽  
pp. 2302-2308
Author(s):  
E Livneh ◽  
T J Dull ◽  
E Berent ◽  
R Prywes ◽  
A Ullrich ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Ligand Binding ◽  
Binding Affinity ◽  
Egf Receptor ◽  
Wild Type ◽  
3T3 Cells ◽  
Kinase C ◽  
Epidermal Growth ◽  
Nih 3T3

The tumor promoter phorbol ester (TPA) modulates the binding affinity and the mitogenic capacity of the epidermal growth factor (EGF) receptor. Moreover, TPA-induced kinase C phosphorylation occurs mainly on Thr-654 of the EGF receptor, suggesting that the phosphorylation state of this residue regulates ligand-binding affinity and kinase activity of the EGF receptor. To examine the role of this residue, we prepared a Tyr-654 EGF receptor cDNA construct by in vitro site-directed mutagenesis. Like the wild-type receptor, the mutant receptor exhibited typical high- and low-affinity binding sites when expressed on the surface of NIH 3T3 cells. Moreover, TPA regulated the affinity of both wild-type and mutant receptors and stimulated receptor phosphorylation of serine and threonine residues other than Thr-654. The addition of TPA to NIH 3T3 cells expressing a wild-type human EGF receptor blocked the mitogenic capacity of EGF. However, this inhibition did not occur in cells expressing the Tyr-654 EGF receptor mutant. In the latter cells, EGF was able to stimulate DNA synthesis even in the presence of inhibitory concentrations of TPA. While phosphorylation of sites other than Thr-654 may regulate ligand-binding affinity, the phosphorylation of Thr-654 by kinase C appears to provide a negative control mechanism for EGF-induced mitogenesis in mouse NIH 3T3 fibroblasts.

scholarly journals Epidermal growth factor-induced proliferation of collecting duct cells from Oak Ridge polycystic kidney mice involves activation of Na+/H+ exchanger

2014 ◽  
Vol 307 (6) ◽  
pp. C554-C560 ◽  
Author(s):  
Sonya D. Coaxum ◽  
Mary G. Blanton ◽  
Alisha Joyner ◽  
Tanjina Akter ◽  
P. Darwin Bell ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Line ◽  
Cell Lines ◽  
Egf Receptor ◽  
Collecting Duct ◽  
Polycystic Kidney ◽  
Oak Ridge ◽  
Erk Phosphorylation ◽  
Epidermal Growth

Epidermal growth factor (EGF) is linked to the pathogenesis of polycystic kidney disease (PKD). We explored signaling pathways activated by EGF in orpk cilia (−) collecting duct cell line derived from a mouse model of PKD (hypomorph of the Tg737/ Ift88 gene) with severely stunted cilia, and in a control orpk cilia (+) cell line with normal cilia. RT-PCR demonstrated mRNAs for EGF receptor subunits ErbB1, ErbB2, ErbB3, ErbB4, and mRNAs for Na+/H+ exchangers (NHE), NHE-1, NHE-2, NHE-3, NHE-4, and NHE-5 in both cell lines. EGF stimulated proton efflux in both cell lines. This effect was significantly attenuated by MIA, 5-( n-methyl- N-isobutyl) amiloride, a selective inhibitor of NHE-1 and NHE-2, and orpk cilia (−) cells were more sensitive to MIA than control cells ( P < 0.01). EGF significantly induced extracellular signal-regulated kinase (ERK) phosphorylation in both cilia (+) and cilia (−) cells (63.3 and 123.6%, respectively), but the effect was more pronounced in orpk cilia (−) cells ( P < 0.01). MIA significantly attenuated EGF-induced ERK phosphorylation only in orpk cilia (−) cells ( P < 0.01). EGF increased proliferation of orpk cilia (+) cells and orpk cilia (−) cells, respectively, and MIA at 1–5 μM attenuated EGF-induced proliferation in orpk cilia (−) cells without affecting proliferation of orpk cilia (+) cells. EGF-induced proliferation of both cell lines was significantly decreased by the EGFR tyrosine kinase inhibitor AG1478 and MEK inhibitor PD98059. These results suggest that EGF exerts mitogenic effects in the orpk cilia (−) cells via activation of growth-associated amiloride-sensitive NHEs and ERK.

scholarly journals Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor.

1988 ◽  
Vol 8 (6) ◽  
pp. 2302-2308 ◽  
Author(s):  
E Livneh ◽  
T J Dull ◽  
E Berent ◽  
R Prywes ◽  
A Ullrich ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Ligand Binding ◽  
Binding Affinity ◽  
Egf Receptor ◽  
Wild Type ◽  
3T3 Cells ◽  
Kinase C ◽  
Epidermal Growth ◽  
Nih 3T3

The tumor promoter phorbol ester (TPA) modulates the binding affinity and the mitogenic capacity of the epidermal growth factor (EGF) receptor. Moreover, TPA-induced kinase C phosphorylation occurs mainly on Thr-654 of the EGF receptor, suggesting that the phosphorylation state of this residue regulates ligand-binding affinity and kinase activity of the EGF receptor. To examine the role of this residue, we prepared a Tyr-654 EGF receptor cDNA construct by in vitro site-directed mutagenesis. Like the wild-type receptor, the mutant receptor exhibited typical high- and low-affinity binding sites when expressed on the surface of NIH 3T3 cells. Moreover, TPA regulated the affinity of both wild-type and mutant receptors and stimulated receptor phosphorylation of serine and threonine residues other than Thr-654. The addition of TPA to NIH 3T3 cells expressing a wild-type human EGF receptor blocked the mitogenic capacity of EGF. However, this inhibition did not occur in cells expressing the Tyr-654 EGF receptor mutant. In the latter cells, EGF was able to stimulate DNA synthesis even in the presence of inhibitory concentrations of TPA. While phosphorylation of sites other than Thr-654 may regulate ligand-binding affinity, the phosphorylation of Thr-654 by kinase C appears to provide a negative control mechanism for EGF-induced mitogenesis in mouse NIH 3T3 fibroblasts.

scholarly journals Epidermal growth factor receptor gene-amplified MDA-468 breast cancer cell line and its nonamplified variants.

1987 ◽  
Vol 7 (1) ◽  
pp. 251-257 ◽  
Author(s):  
J Filmus ◽  
J M Trent ◽  
M N Pollak ◽  
R N Buick
Keyword(s):  
Breast Cancer ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Line ◽  
Breast Cancer Cell Line ◽  
Egf Receptor ◽  
Receptor Gene ◽  
Cancer Cell Line ◽  
Egf Receptors ◽  
Epidermal Growth

We have recently reported (J. Filmus, M. N. Pollak, R. Cailleau, and R. N. Buick, Biochem. Biophys. Res. Commun. 128:898-905, 1985) that MDA-468, a human breast cancer cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited in vitro pharmacological doses of EGF. We have derived several MDA-468 clonal variants which are resistant to EGF-induced growth inhibition. These clones had a number of EGF receptors, similar to normal human fibroblasts, and had lost the EGF receptor gene amplification. Karyotype analysis showed that MDA-468 cells had an abnormally banded region (ABR) in chromosome 7p which was not present in the variants. It was shown by in situ hybridization that the amplified EGF receptor sequences were located in that chromosome, 7pABR. Five of the six variants studied were able to generate tumors in nude mice, but their growth rate was significantly lower than that of tumors derived from the parental cell line. The variant that was unable to produce tumors was found to be uniquely dependent on EGF for growth in soft agar.

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