Hubbard, Christopher J.
M.S. (Master of Science)
Department of Biological Sciences
Epidermal growth factor; Carcinogenesis
Past studies indicate that regulation of epidermal growth factor receptor (EGF-R) mRNA stability plays a major role in EGF-R gene expression. In particular, stimulation of the EGF-R by EGF stabilizes the EGF-R message by a currently undetermined mechanism. The present study was conducted to further evaluate the role that EGF plays in regulating EGF-R stability. KB cells, an epidermal carcinoma cell line, were incubated in serum-free medium for 48 hours in 100 mm culture dishes prior to experimental treatment. Cells were then incubated with various additives after which the transcription inhibitor actinomyin D (Act D) was added and the cells were incubated for an additional period. At the end of each incubation cells were lysed in guanidine lysis buffer or catrimox-14 surfactant and EGF-R mRNA concentrations were measured by RNase protection assay. When cells were stimulated for 5 hours with EGF prior to adding Act D, EGF stabilized the EGF-R message (control half-life = 2.8 hr; +EGF half-life = 4.5 hr). This effect was augmented by cyclohexamide and inhibited by the tyrosine kinase inhibitor lavendustin A. Activating other tyrosine kinase systems showed PDGF to stabilize the message, whereas IGF-I had no effect on EGF-R message decay. The cells were pulsed with EGF (EGF was washed from the medium prior to Act D) for times shorter than 5 hours to determine the length of time required for EGF mediated stabilization of EGF-R message. An exposure time between 1 and 2 hours was required before EGF increased EGF-R mRNA stability beyond that of the controls. These results suggest that EGF stabilizes EGF receptor message through a mechanism involving the EGF receptor tyrosine kinase and protein synthesis. The length of time required for EGF to mediate this event indicates that additional steps beyond tyrosine phosphorylation are required. It is likely that transcription and translation of other proteins which stabilizes the transcript are involved.
Delbrook, Kathy E., "The role of the epidermal growth factor receptor in carcinogenesis" (1994). Graduate Research Theses & Dissertations. 6265.
Northern Illinois University
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