Abstract
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
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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.
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