Identification of factors that regulate epidermal growth factor receptor mRNA stability
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Previous studies have shown that epidermal growth factor (EGF) stabilizes epidermal growth factor receptor (EGF-R) mRNA. Specifically, the addition of EGF to a cancer cell line causes an increase in the stability of the EGF-R mRNA. This study attempts to evaluate the role EGF has on EGF-R mRNA in vitro, the location o f the regulatory factor(s) in the cell, and what some characteristics of the regulatory factor(s) may be. KB cells, an epidermal carcinoma cell line, were incubated in serum-free medium for 48 hours in cell culture flasks prior to harvesting. Cells were then treated with or without EGF for an additional five hours. At the end of the five hours, the ells were harvested. The control and EGF-treated polysomes were then isolated and assayed for EGF-R mRNA concentrations by the RNase protection assay. When stimulated with the EGF, there was an increase in the half-life o f EGF-R mRNA, as a result o f stabilization of EGF-R mRNA. The polysomes were then subjected to a high salt wash in order to remove the proteins associated with the polysomes. The salt washed polysomes were then assayed by the RNase protection assay. Again with the washed polysomes the halflife of the EGF-treated polysomes was greater than that of control polysomes. The regulatory factor associated with the polysomes appears to be more easily washed off of the EGF-treated washed polysomes. The ribosomal salt wash (RSW) from EGF-treated cells was added back to both treatment groups and caused a decrease in the half-life of EGF-R mRNA. This result suggests that there is nuclease activity associated with the RSW from EGFtreated cells. Again, when the RSW from EGF-treated cells was added to an in vitro decay assay with deproteinized cytoplasmic RNA, the half-life of EGF-R mRNA decreased. Further studies were conducted with the radiolabeled EGF-R transcript to verify the results obtained with the washed polysomes. These results have shown that EGF stabilizes EGF-R mRNA in vitro, the regulatory factor or nuclease is associated with the ribosome and the nuclease is heat labile.