scholarly journals Phorbol 12-myristate 13-acetate inhibits epidermal growth factor signalling in human keratinocytes, leading to decreased ornithine decarboxylase activity

1996 ◽  
Vol 319 (2) ◽  
pp. 641-648 ◽  
Author(s):  
Guo Z XUE ◽  
Zai S ZHENG ◽  
Rong Z CHEN ◽  
Mary B. LLOYD ◽  
Janet H. PRYSTOWSKY
Keyword(s):  
Biological Activity ◽  
Steady State ◽  
Growth Factor ◽  
Baseline Level ◽  
Human Keratinocytes ◽  
Enzyme Synthesis ◽  
Enzyme Protein ◽  
Epidermal Growth ◽  
Pkc Activation

Several studies have suggested that murine and human keratinocytes respond differently to phorbol 12-myristate 13-acetate (PMA). Using an in vitro assay, we found that in contrast to its effect on murine skin, PMA did not induce ornithine decarboxylase (ODC) activity in human skin biopsies. To explore the signalling induced by PMA and to determine whether an in vitro culture system could be used to predict biological activity of retinoids in human keratinocytes, we studied a simian virus 40 (SV40)-transformed human keratinocyte cell line. Epidermal growth factor (EGF) stimulates ODC activity and increases the steady-state level of ODC mRNA in a dose- and time-dependent manner in these cells [Prystowsky, Clevenger and Zheng (1993) Exp. Dermatol. 2, 125–132]. In this report, 10-10 M–10-7 M PMA induced ODC mRNA and enzyme synthesis at 7 h, but did not significantly induce ODC activity and inhibited the EGF induction of ODC activity. To explore the mechanism whereby PMA interferred with EGF signalling, the effect of PMA on EGF binding to its cell-surface receptor was studied; acute treatment with PMA (within 7 h) decreased EGF binding to 41–57% of the baseline level. In contrast, chronic treatment with PMA (24 h) increased EGF binding to 156% of the baseline level and was associated with an increase in quantity of EGF receptor protein. Protein kinase C (PKC) activation correlated with the acute decrease in EGF binding following PMA treatment. In summary, PMA induced ODC mRNA and ODC enzyme synthesis, while steady-state levels of immunoprecipitable ODC enzyme protein and ODC activity were not increased, demonstrating possible increased turnover of ODC enzyme protein. Additionally, PMA inhibited the induction of ODC by EGF through decreased EGF binding, possibly mediated by PKC activation. Finally treatment of the keratinocytes with retinoids including etretinate, Ro13-7410, etarotene, Ro40-8757, 13-cis-retinoic acid, and acitretin blocked the PMA induction of ODC mRNA, suggesting this in vitro model could be a valuable screening assay for predicting biological activity in humans.

Biological Activity In Vitro of Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies with Different Affinities

Hybridoma ◽  
2007 ◽  
Vol 26 (6) ◽  
pp. 423-432 ◽  
Author(s):  
Arlhee Diaz Miqueli ◽  
Rances Blanco ◽  
Beatriz Garcia ◽  
Teresita Badia ◽  
Ana E. Batista ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Biological Activity ◽  
Monoclonal Antibodies ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Epidermal Growth

Steady-state level of epidermal growth factor (EGF) mRNA and effect of EGF on in vitro culture of caprine preantral follicles

2011 ◽  
Vol 344 (3) ◽  
pp. 539-550 ◽  
Author(s):  
Juliana Jales H. Celestino ◽  
Jamily B. Bruno ◽  
Márcia Viviane A. Saraiva ◽  
Rebeca M. P. Rocha ◽  
Ivina R. Brito ◽  
...  
Keyword(s):  
Steady State ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
In Vitro Culture ◽  
State Level ◽  
Steady State Level ◽  
Preantral Follicles ◽  
Epidermal Growth

Regulation of steady-state follistatin mRNA levels in rat granulosa cells in vitro

1992 ◽  
Vol 9 (2) ◽  
pp. 147-156 ◽  
Author(s):  
U. Michel ◽  
J. W. McMaster ◽  
J. K. Findlay
Keyword(s):  
Steady State ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Cultures ◽  
Granulosa Cells ◽  
Internal Standard ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Epidermal Growth

ABSTRACT The regulation of steady-state follistatin mRNA levels by different pituitary hormones and peptide factors was examined in granulosa cell cultures derived from diethylstilboestrol-treated immature rats. Cytosolic RNA from cell cultures was prepared by lysis and equal amounts of RNA from all samples were analysed with a solution—hybridization assay using a 32P-labelled antisense probe corresponding to a part of exon 5 together with a part of the 5′ end of exon 6 of the rat follistatin gene. In addition, a specific 35S-labelled probe for cyclophilin was used as an internal standard. The results show that 5 μg FSH/1 for 24 to 72 h stimulated steady-state follistatin mRNA levels, reaching levels 18·5-fold higher than controls. LH (0·2-100 μg/l) had only minor effects on follistatin mRNA levels in FSH-primed granulosa cells and prolactin, GH and IGF-I did not show any significant effects. Activin raised basal as well as FSH-stimulated steady-state follistatin mRNA levels up to ten- and twofold above controls respectively, whereas epidermal growth factor was found to inhibit FSH-stimulated follistatin mRNA levels in a dose-dependent manner. It is concluded that follistatin mRNA levels in granulosa cells are regulated by FSH rather than LH, and that the stimulation by FSH can be inhibited by epidermal growth factor but enhanced by activin. Activin alone was also capable of stimulating follistatin mRNA.

17β-Estradiol enhances heparin-binding epidermal growth factor-like growth factor production in human keratinocytes

2005 ◽  
Vol 288 (4) ◽  
pp. C813-C823 ◽  
Author(s):  
Naoko Kanda ◽  
Shinichi Watanabe
Keyword(s):  
Estrogen Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
G Protein ◽  
Transcriptional Activity ◽  
Human Keratinocytes ◽  
Heparin Binding ◽  
17Β Estradiol ◽  
Epidermal Growth

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) enhances reepithelialization in wounds. Estrogen is known to promote cutaneous wound repair. We examined the in vitro effects of 17β-estradiol (E2) on HB-EGF production by human keratinocytes. E2 or membrane-impermeable BSA-conjugated E2 (E2-BSA) increased HB-EGF secretion, mRNA level, and promoter activity in keratinocytes. E2 or E2-BSA enhanced in vitro wound closure in keratinocytes, and the closure was suppressed by anti-HB-EGF antibody. Activator protein-1 (AP-1) and specificity protein 1 (Sp1) sites on HB-EGF promoter were responsible for the E2- or E2-BSA-induced transactivation. Antisense oligonucleotides against c-Fos, c-Jun, and Sp1 blocked E2- or E2-BSA-induced HB-EGF transactivation. E2 or E2-BSA enhanced DNA binding and transcriptional activity of AP-1 and generated c-Fos/c-Jun heterodimers by inducing c-Fos expression. E2 or E2-BSA enhanced DNA binding and transcriptional activity of Sp1 in parallel with the enhancement of Sp1 phosphorylation. These effects of E2 or E2-BSA were not blocked by the nuclear estrogen receptor antagonist ICI-182,780 or anti-estrogen receptor-α or -β antibodies but were blocked by inhibitors of G protein, phosphatidylinositol-specific PLC, PKC-α, and MEK1. These results suggest that E2 or E2-BSA may enhance HB-EGF production via activation of AP-1 and Sp1. These effects of E2 or E2-BSA may be dependent on membrane G protein-coupled receptors different from nuclear estrogen receptors and on the receptor-mediated activities of phosphatidylinositol-specific PLC, PKC-α, and MEK1. E2 may enhance wound reepithelialization by promoting HB-EGF production in keratinocytes.

Functional heterogeneity of proto-oncogene tyrosine kinases: the C terminus of the human epidermal growth factor receptor facilitates cell proliferation

1989 ◽  
Vol 9 (4) ◽  
pp. 1772-1778
Author(s):  
T J Velu ◽  
W C Vass ◽  
D R Lowy ◽  
L Beguinot
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Growth Factor ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Full Length ◽  
Tyrosine Residue ◽  
Gene Encoding ◽  
Epidermal Growth

Previous reports have indicated that the C termini of the membrane-associated tyrosine kinases encoded by c-src and c-fms proto-oncogenes have a negative effect on their biological activity and that this effect is mediated by their C-terminal tyrosine residue. To determine whether this was true for the human epidermal growth factor (EGF) receptor, which is also a membrane-associated tyrosine kinase proto-oncogene, we have constructed two premature termination mutants, dc19 and dc63, that delete the C-terminal 19 and 63 amino acids, respectively, from the human full-length receptor (hEGFR). The smaller deletion removes the C-terminal tyrosine residue, while the larger deletion removes the two most C-terminal tyrosines; similar deletions are found in v-erbB. As previously shown for the gene encoding the full-length EGF receptor, the two C-terminal mutants induced EGF-dependent focal transformation and anchorage-independent growth of NIH 3T3 cells. However, both dc19 and dc63 were quantitatively less efficient than the gene encoding the full-length receptor, with dc63 being less active than dc19. Although the C-terminal mutants displayed lower biological activity than the gene encoding the full-length receptor, the mutant receptors were found to be similar in several respects to the full-length receptor. These parameters included receptor localization, stability in the absence of EGF, receptor half-life in the presence of EGF, EGF binding, extent of EGF-dependent autophosphorylation in vitro, and EGF-dependent phosphorylation of an exogenous substrate in vitro. Therefore, the C-terminal 63 amino acids of the human receptor have no detectable influence on EGF-dependent early events. We conclude that in contrast

scholarly journals Functional heterogeneity of proto-oncogene tyrosine kinases: the C terminus of the human epidermal growth factor receptor facilitates cell proliferation.

1989 ◽  
Vol 9 (4) ◽  
pp. 1772-1778 ◽  
Author(s):  
T J Velu ◽  
W C Vass ◽  
D R Lowy ◽  
L Beguinot
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Growth Factor ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Full Length ◽  
Tyrosine Residue ◽  
Gene Encoding ◽  
Epidermal Growth

Previous reports have indicated that the C termini of the membrane-associated tyrosine kinases encoded by c-src and c-fms proto-oncogenes have a negative effect on their biological activity and that this effect is mediated by their C-terminal tyrosine residue. To determine whether this was true for the human epidermal growth factor (EGF) receptor, which is also a membrane-associated tyrosine kinase proto-oncogene, we have constructed two premature termination mutants, dc19 and dc63, that delete the C-terminal 19 and 63 amino acids, respectively, from the human full-length receptor (hEGFR). The smaller deletion removes the C-terminal tyrosine residue, while the larger deletion removes the two most C-terminal tyrosines; similar deletions are found in v-erbB. As previously shown for the gene encoding the full-length EGF receptor, the two C-terminal mutants induced EGF-dependent focal transformation and anchorage-independent growth of NIH 3T3 cells. However, both dc19 and dc63 were quantitatively less efficient than the gene encoding the full-length receptor, with dc63 being less active than dc19. Although the C-terminal mutants displayed lower biological activity than the gene encoding the full-length receptor, the mutant receptors were found to be similar in several respects to the full-length receptor. These parameters included receptor localization, stability in the absence of EGF, receptor half-life in the presence of EGF, EGF binding, extent of EGF-dependent autophosphorylation in vitro, and EGF-dependent phosphorylation of an exogenous substrate in vitro. Therefore, the C-terminal 63 amino acids of the human receptor have no detectable influence on EGF-dependent early events. We conclude that in contrast

Computational Evaluation and In Vitro Validation of New Epidermal Growth Factor Receptor Inhibitors

2020 ◽  
Vol 20 (18) ◽  
pp. 1628-1639
Author(s):  
Sergi Gómez-Ganau ◽  
Josefa Castillo ◽  
Andrés Cervantes ◽  
Jesus Vicente de Julián-Ortiz ◽  
Rafael Gozalbes
Keyword(s):  
Cell Proliferation ◽  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Binding Affinity ◽  
Cell Lines ◽  
Growth Factor Receptor ◽  
Significant Activity ◽  
Epidermal Growth

Background: The Epidermal Growth Factor Receptor (EGFR) is a transmembrane protein that acts as a receptor of extracellular protein ligands of the epidermal growth factor (EGF/ErbB) family. It has been shown that EGFR is overexpressed by many tumours and correlates with poor prognosis. Therefore, EGFR can be considered as a very interesting therapeutic target for the treatment of a large variety of cancers such as lung, ovarian, endometrial, gastric, bladder and breast cancers, cervical adenocarcinoma, malignant melanoma and glioblastoma. Methods: We have followed a structure-based virtual screening (SBVS) procedure with a library composed of several commercial collections of chemicals (615,462 compounds in total) and the 3D structure of EGFR obtained from the Protein Data Bank (PDB code: 1M17). The docking results from this campaign were then ranked according to the theoretical binding affinity of these molecules to EGFR, and compared with the binding affinity of erlotinib, a well-known EGFR inhibitor. A total of 23 top-rated commercial compounds displaying potential binding affinities similar or even better than erlotinib were selected for experimental evaluation. In vitro assays in different cell lines were performed. A preliminary test was carried out with a simple and standard quick cell proliferation assay kit, and six compounds showed significant activity when compared to positive control. Then, viability and cell proliferation of these compounds were further tested using a protocol based on propidium iodide (PI) and flow cytometry in HCT116, Caco-2 and H358 cell lines. Results: The whole six compounds displayed good effects when compared with erlotinib at 30 μM. When reducing the concentration to 10μM, the activity of the 6 compounds depends on the cell line used: the six compounds showed inhibitory activity with HCT116, two compounds showed inhibition with Caco-2, and three compounds showed inhibitory effects with H358. At 2 μM, one compound showed inhibiting effects close to those from erlotinib. Conclusion: Therefore, these compounds could be considered as potential primary hits, acting as promising starting points to expand the therapeutic options against a wide range of cancers.

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