Increased phosphotyrosine content and inhibition of proliferation in EGF-treated A431 cells

http://www.nature.com/nature/journal/v293/n5830/pdf/293305a0.pdf

Nature 293, 305 - 307 (24 September 1981); doi:10.1038/293305a0

Gordon N. Gill & Cheri S. Lazar
Department of Medicine, Division of Endocrinology, University of California, San Diego, School of Medicine, La Jolla, California 92093, USA

Abstract

Epidermal growth factor (EGF), which binds to specific high-affinity cell-surface receptors, stimulates replication of a number of cell types1,2. In vitro EGF stimulates a membrane-associated protein kinase which catalyses phosphorylation of the EGF receptor at tyrosine residues3,4. The transforming proteins of several RNA tumour viruses are protein kinases which also specifically catalyse phosphorylation at tyrosine residues5−10. An elevated level of phosphotyrosine is found in cells transformed by Rous sarcoma, Fujinami sarcoma, PRCII, Y73, Snyder−Theilin and Gardner−Armstrong feline sarcoma and Abelson murine leukaemia viruses5,10−12. At least four of these viruses, which encode distinct protein kinases, catalyse phosphorylation of tyrosine residues in the same cellular substrate proteins13. In vitro EGF-stimulated protein kinase catalyses the phosphorylation of anti-p60src heavy chains, suggesting that this enzyme recognizes similar substrate determinants to p60src (refs 14,15). Here we demonstrate that EGF treatment of A431 human epidermoid carcinoma cells increases phosphotyrosine content, indicating that EGF stimulates tyro sine-specific protein kinase activity in vivo as well as in vitro. In contrast to Rous sarcoma virus (RSV) transformation, EGF inhibits replication of A431 cells. This inhibition by EGF is influenced by both cell density and tissue culture substratum.

Summary

A431 human epidermoid carcinoma cells have an unusually high density of EGF receptors. It has been known that EGF increases the phosphotyrosine content of exponentially growing A431 cells. EGF stimulates a membrane-associated protein kinase through phosphorylation. EGF increased the phosphotyrosine content of A431 cells sevenfold (0.14 and 0.02% in EGF-treated and untreated A431 cells respectively), indicating that EGF activates tyrosine-specific protein kinase activity in vivo.



Figure.1
Exponentially growing A431 cells were cultured. In control, no EGF was given while in EGF group, EGF was added. After culturing, cells were broken down and the proteins were collected. Electrophoresis was done and following Western blot was done and then overexposed to phosphoamino acids. The results shows phosphorylated tyrosine(p-tyr) only exists in EGF-treated cells.


Figure 2.
Effect of EGF on growth of A431 cells plated at different cell densities. Cells were cultured with different densities: 550(□), 2500(○) and 11000(●) cells / cm^2. The possibility that increased EGF degradation at high cell densities on tissue culture dishes contributed to the rightward shift in the dose-response curve shown in Figure 2.


Figure 3.
Effect of cell density on binding of EGF to A431 cells. A431 cells were subcultured into 3-cm tissue culture dishes and used 24-48 hr later. EGF-binding to A431 cells grown on tissue culture dishes: ●, I-EGF binding of constant specific activity. KD is the equilibrium dissociation constant. This figure shows that there is an inverse relationship between cell density and the affinity of EGF receptors for EGF.


Critique

Epithermal growth factor(EGF) is one of the molecules that cancer researchers are interested in. Many cancer cells have extraordinarily high density of EGF-receptors, and EGF is known to affect cell proliferation and apoptosis by phosphorylating EGF receptors.

This article is related to our groups project, whis is to see the effect of EGF on A431 cells in their morphology and proliferation. This research done by Gill and Lazar is one of the beginning researches of EGF on cancer cells. At the time of this research, the molecular activity of EGF on EGF receptor was figured out: in vitro, EGF was found out that it catalysed the phosphorylation of the EGF receptor at tyrosine residues. The main purpose of this research was to see the effect of EGF in vivo.

This article was published on Nature in 1981, so it does not show the current hot issues in molecular biology/bioengineering. However, it shows several interesting points that are worth taking a look. First, this research began as to see the effect of EGF in vivo, but as the experiment went on, the authors could discover a new experimental result that the effect of EGF was influenced by both cell density and tissue culture environment. This shows that even we start an experiment with a clear hypothesis and purpose, we may make unexpected but new discoveries.

Secondly, the methods used in this research are outdated and different from current methods. For example, if you look at Figure 1 which shows the protein electrophoresis result, you will easily see that the figure seems messy. The purpose of the electrophoresis was to visualize the amount of phosphorylated tyrosine in EGF-treated cells and control, so the dark background is actually a noise. Considering the fact the phosphorylated tyrosine had been studied well in vitro before this research was performed, I think there was a reason that they could not perform immunoblotting skills, which would have clearly shown the difference in amount of p-tyrosine. In my opinion, Western blot was not popular at the time of this research, because the Western blot was only invented in 1975, a few years before this research. Nevertheless, the researchers proved that there was significant difference in the amount of p-tyrosine by manually separating the proteins.

Lastly, this article does not have small titles as "introduction", "materials & methods", and "results" which can be found in most of today's articles. By reading an article like this, I realized that those small titles expedite the understanding of readers.

To sum up, this article is interesting because it is one of the first researches to see the effect of EGF in vivo. Even though it is from 1981, the skills and logic through the experiment are understandable, and it will let you have an opportunity to think about current research techniques in different way.