Mandal, Soma (2001) A study of EGF-mediated early and late signaling events in relation to epidermal growth factor receptor tyrosine kinase activity in the human breast cancer cell line, MDA 468. Doctoral (PhD) thesis, Memorial University of Newfoundland.
- Accepted Version
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The human breast cancer cell line, MDA 468 overexpresses the epidermal growth factor receptor, the EGFR to 1-2 x 10⁶ receptors per cell. Receptor phosphorylation influences the EGF-mediated signaling events initiated at the cell surface. In this study we used a protein tyrosine kinase inhibitor (PTK) to study the effects of inhibition of EGFR kinase activity on EGF-dependent effects on phosphatidylinositol (PI) turnover, cell viability, and cell proliferation. -- In the initial phase of the study, we tested some of the frequently used PTK inhibitors for their ability to inhibit the EGF-stimulated EGFR autophosphorylation. MDA 468 cells exhibited a differential sensitivity to inhibition of receptor autophosphorylation with these PTK inhibitors. Of the inhibitors tested, only the dihydroxybenzene moiety containing inhibitor lavendustin A (LA) effectively inhibited (~ 95% at 1 μM) the EGF-stimulated EGFR autophosphorylation in intact cells and in crude membrane preparations in a time and dose dependent manner. Exposure of cells to 1 μM LA beyond 10 h caused morphological changes such as nuclear condensation and membrane blebbing. Morphological changes and occurrence of a subdiploid peak at higher concentrations are consistent with the fact that LA produced cell death by apoptosis. -- Earlier experiments from this laboratory had shown that EGF stimulates an increase in the total PI turnover and that a major portion of the metabolites was being converted to a component whose elution time did not correspond to the that of the elution times of non-cyclic inositol phosphates. This is an outcome of EGF- stimulated EGFR autophosphorylation. This metabolite was acid-labile and comprised over 50% of the PI turnover components in control untreated cells. Treatment of cells with EGF increased the levels of this component, while 1 μM LA treatment decreased it. Using the technique of electrospray ionization tandem mass spectrometry, we identified this metabolite to be myo-inositol 1,2-(cyclic) monophosphate (cIP) through the specific fragmentation pattern as compared to the standard. Change in the total PI turnover by stimulation (with EGF) or inhibition (with LA) of EGFR phosphorylation could be entirely accounted for by alterations in the percentage of cIP generated by these cells. This type of PI turnover profile is atypical and unlike that generated by phospholipase C-γ. cIP is the major constituent of EGF-stimulated PI turnover and it is the metabolite which is most closely linked to changes in phosphorylation of EGFR in MDA 468 cells. -- MDA 468 cells are growth inhibited by 10⁻⁸ M EGF between 48-72 h of exposure. Previous work has shown that this growth inhibition is due to a pronounced G₁ growth arrest. Using a PTK inhibitor we hoped to see how changes in this relationship would affect the proliferative response in MDA 468 cells. At 0.2 μM, LA reduced cell proliferation (as estimated by cell number) and DNA synthesis ([³H]-thymidine uptake). Growth inhibition was accompanied by perturbations in the cell cycle. LA, EGF, and EGF+LA produced a pronounced G₁ arrest. LA and EGF+LA-induced arrest was reversed 24 h after their removal, but EGF-treated cells showed a more persistent arrest. MDA 468 cells possess the p53²⁷³·His mutant, which positively enhances cell proliferation, but it transforms into an alternate "pseudo-wild type" conformation during EGF-mediated G₁ arrest as demonstrated by previous work in this laboratory. In fact, p53 in cells treated with EGF, LA, or both, showed an altered subcellular localization at 24 h. The p53 in the nucleus lost reactivity to PAb 240 (it reacts with mutant p53), but reacted positively to PAb 1620 (it reacts with wild-type p53). This altered conformational shift of p53²⁷³·His from mutant to wild-type was also accompanied by a strong nuclear localization of p21WAF1/CIP1 and an absence of nuclear localization of cyclin dependent kinase 2 (cdk2). Thus we hypothesize a role of p53²⁷³·His dependent participation of p21WAF1/CIP1 and cdk2 in G₁ arrest in response to EGF, LA alone or both in combination. The augmented growth inhibitory response in presence of both EGF and LA is intriguing and is probably indicative of a segregated relationship between receptor phosphorylation and proliferative response. -- In this study we provide evidence of EGF-dependent changes associated with phosphorylation in the levels of an unusual PI metabolite, whose levels cause changes in total PI turnover. Apparently, this receptor phosphorylation-mediated changes in cIP during short-term exposure do not correlate with changes in proliferative response. Time and concentration dependent inhibition of receptor kinase activity by LA is reflected in later times by growth inhibition. Furthermore, the data presented suggests a function of p53²⁷³·His in growth arrest.
|Item Type:||Thesis (Doctoral (PhD))|
|Additional Information:||Bibliography: leaves 188-241.|
|Department(s):||Medicine, Faculty of|
|Library of Congress Subject Heading:||Cellular signal transduction; Protein-tyrosine kinase; Breast--Cancer|
|Medical Subject Heading:||Breast Neoplasms; Signal Transduction; Receptor, Epidermal Growth Factor|
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