For questions 5 through 9: You have isolated an epithelial cell line which is unresponsive to EGF (normal epithelial derived cell lines require EGF in the culture media for growth). You suspect that EGF receptor signaling in this cell line is impaired, and you designate this a mutant cell line. As an assay for EGFR signaling, you have stably transfected this mutant cell line and a normal cell line with a luciferase-based reporter construct that is responsive to the transcription factor Elk-1. You want to determine what component of the signaling pathway is impaired.
1. Briefly describe a method you could use to transfect the luciferase- based reporter construct DNA into a cell, and then describe how you could establish (or select for) a stable cell line expressing this reporter construct. 2. The luciferase activity is measured in both the mutant cell line and the normal cell line following treatment with (+) or without(-) EGF in the culture media. Luciferase Activity (A.U. +10%) *A.U. =arbitrary units
-EGF +EGF
Mutant 9 10
Normal 8 182 a. Briefly state what the results tell you and indicate whether the impaired component in the mutant cell line lies upstream or downstream in the EGF signaling cascade, or if the component indicated is in fact the impaired component. 3. Introduction of EGF receptor that is constitutively phosphorylated at tyrosine 1068 results in prolonged Elk-1-mediated transcription in wild-type cells, but has no effect on the mutant cell line (i.e. no Elk-1-mediated transcription). a. Briefly state what the results tell you and indicate whether the impaired component in the mutant cell line lies upstream or downstream in the EGF signaling cascade, or if the component indicated is in fact the impaired component.

4. Expression of a constitutively active raf results in the following:
Luciferase Activity (A.U. + 10%)
-EGF +EGF
Mutant 192 190
Normal 186 189

a. Briefly state what the results tell you and indicate whether the impaired component in the mutant cell line lies upstream or downstream in the EGF signaling cascade, or if the component indicated is in fact the impaired component.