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Quantitative dissociation between EGF effects on c-myc and c-fos gene expression, DNA synthesis, and epidermal growth
S Hauguel-DeMouzon1, P Csermely, G Zoppini
1Joslin Diabetes Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts.
Abstract:
The exact relationship between EGF-stimulated tyrosine phosphorylation, induction of the cellular proto-oncogenes c-myc and c-fos, and DNA synthesis remains uncertain. Madin-Darby Canine Kidney (MDCK) cells possess EGF receptor sites with high binding capacity, and in contrast to A431 cells, respond to EGF by increasing DNA synthesis. Following EGF stimulation of intact MDCK cells, there was a rapid and marked increase in the autophosphorylation of the EGF receptor. This was associated with an increase in the tyrosine phosphorylation of a 120 kDa phosphoprotein believed to be an endogenous substrate of this receptor kinase. The ED50 for stimulation of phosphorylation of pp120 was approximately 0.05 nM versus 1.0 nM for receptor autophosphorylation, consistent with amplification of signalling at this step in EGF action. Stimulation of DNA synthesis occurred after 12 to 24 hours and revealed even further amplification with an ED50 of about 0.1 nM. Intermediate between these events was a time-dependent activation of c-fos and c-myc gene expression. However, the ED50 for these processes was approximately 10 nM, indicating a relatively lower sensitivity of EGF for stimulation of proto-oncogene expression. Tyrphostin (RG 50864), a compound reported to inhibit specifically the EGF receptor kinase, completely blocked EGF stimulation of proto-oncogene induction. Interestingly, under the same experimental conditions, EGF receptor autophosphorylation was decreased only 60%. These data, along with the dose-response studies, indicate that proto-oncogene induction requires near maximal stimulation of EGF receptor autophosphorylation. They also suggest that, in MDCK cells, the EGF dependent induction of the c-fos and c-myc genes is not strictly correlated to the extent of EGF receptor autophosphorylation or EGF-stimulated DNA synthesis, and that EGF stimulation of DNA synthesis likely involves additional rate-limiting intermediate steps.
Insights
Epidermal Growth Factor (EGF) stimulates DNA synthesis and proto-oncogene expression in MDCK cells. Proto-oncogene induction requires high EGF receptor autophosphorylation, but isn't directly correlated with DNA synthesis.
Area of Science:
- Cell Biology
- Molecular Biology
- Oncology
Background:
- The precise link between EGF-stimulated tyrosine phosphorylation, proto-oncogene induction (c-myc, c-fos), and DNA synthesis is unclear.
- Madin-Darby Canine Kidney (MDCK) cells exhibit high EGF receptor capacity and respond to EGF by increasing DNA synthesis.
Purpose of the Study:
- To elucidate the relationship between EGF receptor activity, proto-oncogene expression, and DNA synthesis in MDCK cells.
- To investigate the signaling cascade initiated by EGF stimulation.
Main Methods:
- Stimulation of intact MDCK cells with Epidermal Growth Factor (EGF).
- Measurement of EGF receptor autophosphorylation and tyrosine phosphorylation of a 120 kDa protein (pp120).
- Assay of c-fos and c-myc gene expression and DNA synthesis.
- Inhibition studies using Tyrphostin (RG 50864).
Main Results:
- EGF rapidly increased EGF receptor autophosphorylation and pp120 tyrosine phosphorylation, with amplification observed at the pp120 phosphorylation step.
- Proto-oncogene induction (c-fos, c-myc) and DNA synthesis were time-dependent, with distinct dose-response curves.
- Proto-oncogene induction required near-maximal EGF receptor autophosphorylation, while DNA synthesis involved additional rate-limiting steps.
- Tyrphostin inhibited proto-oncogene induction more effectively than EGF receptor autophosphorylation.
Conclusions:
- EGF-stimulated DNA synthesis and proto-oncogene induction in MDCK cells involve complex signaling pathways with differential sensitivities.
- Proto-oncogene induction is linked to high levels of EGF receptor autophosphorylation but not strictly correlated with DNA synthesis extent.
- EGF-induced DNA synthesis likely proceeds through intermediate steps beyond receptor autophosphorylation and proto-oncogene activation.