Recently, we showed that the internalization of the epidermal growth factor (EGF) receptor is inhibited by hydrogen peroxide (H(2)O(2)) in human fibroblasts. In order to test the effect of various stress conditions on receptor internalization and to test a variety of antioxidants in their capacity to prevent or reduce the H(2)O(2)-induced inhibition of internalization, a screening assay was developed to measure the internalization in 96-well plates. In this assay, cells are exposed to biotin-conjugated EGF and the amount of internalized EGF is detected with horseradish peroxidase-conjugated streptavidin. We show that the results obtained by this new assay are comparable with those from internalization studies performed with radioactive labeled EGF. Therefore, the cellular internalization assay as presented here is a reliable method to measure EGF receptor internalization. Moreover, because elaborate processing of the cells is not required, the assay is a relatively fast and inexpensive method to study ligand-induced internalization in 96-well plates and thereby is suitable for large-scale screening of compounds or conditions interfering with this internalization.
This thesis described the effects of exposure of cells to oxidative stress,induced by
H 2 O 2 ,on the functioning of proteins involved in signal transduction pathways.In addition,
H 2 O 2 was chosen as oxidant in order to produce cellular screening assays to measure
antioxidant efficacy in preventing the H 2 O 2 -induced modifications of protein functioning.
Afamily of kinases that plays a key role in the transduction of extracellular signals
into intracellular events is formed by the MAP kinases.1 Moreover,MAP kinases are rapidly
activated in response to various extracellular signals,including different types of cellular
stress.2-5 Therefore,the effect of H 2 O 2 on the phosphorylation of MAP kinase was
investigated and this revealed that exposure of Rat-1 fibroblasts resulted in a transient
phosphorylation of p44/p42 MAPK .Subsequently,the H 2 O 2 -induced phosphorylation of
p44/p42 MAPK was used as a marker for oxidative stress and the availability of a phospho-
specific p44/p42 MAPK antibody provided us the ability to develop a cellular enzyme-linked
immunosorbent assay (Cell-ELISA)to measure the phosphorylation of p44/p42 MAPK (chapter
2 ).This assay was subsequently used for the screening of antioxidant efficacy in Rat-1
fibroblasts and in addition,the assay was applicable to test other stresses,such as
menadione,cumene hydroperoxide,AMVN and hypoxanthine/xanthine oxidase.
Asecond screening assay was developed to measure the internalization of the EGF
receptor in 96-well plates (chapter 4 ).Internalization and subsequent degradation of
activated EGF receptors,also referred to as receptor downregulation or receptor-mediated
endocytosis,results in a reduction of the amount of EGF receptors expressed at the plasma
membrane and therefore in a reduction of binding sites for EGF.Another cellular feedback
mechanism to attenuate receptor signaling involves the activation of phosphatases.6
Dephosphorylation of the C-terminal Tyr residues of the EGF receptor,for instance,
abrogates docking sites for downstream signaling proteins and furthermore,the enzymatic
activity of several signaling proteins can be negatively regulated by dephosphorylation.
Finally,a third mechanism to regulate EGF-induced signaling is receptor transmodulation,
which results in lowered affinity of the receptor for its ligand and in addition,receptor Tyr
kinase activity is reduced.7 We decided to investigate the effect of H 2 O 2 on receptor
downregulation,because an inhibition of EGF receptor-mediated endocytosis had been
described for different forms of cellular stress,8 suggesting that oxidative stress might
interfere with this process as well.H 2 O 2 was found to inhibit the internalization of the EGF
receptor in HER14 fibroblasts (chapter 3 )and this inhibition was subsequently considered as
a marker for oxidative stress.T easily study ligand-induced internalization,a cellular
screening assay in 96-well plates was developed,which was partly based on the cellular
MAP kinase assay as described in chapter 2 .In this assay,internalization was studied using
biotin-conjugated EGF and we showed that the results obtained with this internalization
assay were comparable with results obtained with radioactive labeled EGF (chapter 4 ).
In conclusion,the newly developed 96-well plate assays are nonradioactive,
relatively fast and reliable methods for quantitative detection of changes in phosphorylation
of MAP kinases or changes in ligand-induced internalization in 96-well plates.Furthermore,
both assays are applicable for the screening of various stress conditions on these processes
and for testing a variety of antioxidants in their capacity to prevent or reduce the H 2 O 2 -
induced changes in these cellular responses.
Mitogen-activated protein (MAP) kinases are serine/threonine kinases that are activated by phosphorylation and are involved in the cellular response to various physiologic stimuli and stress conditions. Because MAP kinases play an important role in cellular functioning, a screening assay to determine the phosphorylation of MAP kinase upon various conditions was desirable. Therefore, we have developed a cellular enzyme-linked immunosorbent assay (Cell-ELISA), in which the phosphorylated forms of p42MAPK and p 44 MAPK are detected. We show that in this Cell-ELISA, MAP kinase becomes phosphorylated in a dose- and time-dependent manner under proliferative or stress conditions. This dose- and time-dependent phosphorylation agrees with observations using classical gel-electrophoresis and Western blotting techniques. Furthermore, we show that our assay is applicable to different cell types and that serum-starvation is not required for detection of an increase in MAP kinase phosphorylation. From these experiments, it is concluded that the Cell-ELISA is a reliable and fast method for quantitative detection of the phosphorylation, and thus the activation, of MAP kinase. This assay is applicable for a large-scale screening of the effectivity of biological or chemical compounds that modulate the cellular response to physiologic stimuli or stress through phosphorylation and activation of MAP kinase.
Recently, we demonstrated that hydrogen peroxide (H2O2) inhibits the internalization of the epidermal growth factor (EGF) receptor and the EGF-induced mono-ubiquitination of EGF receptor pathway substrate clone #15 (Eps15) in fibroblasts. In addition, it was suggested that EGF receptor internalization might be inhibited by H2O2 by inhibition of ubiquitination of proteins involved in endocytosis. Here, we show that H2O2 also inhibits the poly-ubiquitination of the EGF receptor in fibroblasts. Furthermore, recovery of the cells resulted in re-establishment of ubiquitination of both the EGF receptor and Eps15 and coincided with restoration of internalization of those receptors that had bound EGF in the presence of H2O2. In addition, EGF receptor internalization was inhibited by the sulphydryl reagent N-ethylmaleimide (NEM), indicating that intact SH groups might be required for receptor-mediated endocytosis. Furthermore, H2O2 rapidly induced an increase in the cellular ratio of GSSG:GSH (oxidized glutathione:reduced glutathione) and removal of H2O2 resulted in a fast restoration of the ratio of GSSG:GSH. Therefore, these results suggest a relation between the inhibition of internalization ubiquitination and an increase in GSSG:GSH ratio, which strengthens the hypothesis that H2O2 inhibits EGF receptor internalization by an inhibition of ubiquitination of proteins involved in EGF receptor-mediated endocytosis.
