You have isolated an epithelial cell line which is unrespons

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.

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.

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.

Luciferase Activity A.U. +10%) A.U. arbitrary units EGF EGF Mutant 9 10 Norma 8 182

Solution

Here, the luciferase reporter construct must have been made in such a way that the luciferase gene is downstream of a promoter sequence that has the binding site of transcription factor Elk-1.

Once the EGF signalling cascade gets activated, Elk-1 gets phosphorylated and this phosphorylated state binds to the DNA at its appropriate binding site. So, in this luciferase assay, the Elk-1-P will also bind to the upstream of the transfected luciferase gene and trigger its expression. Hence the activity of luciferase is proportional to the expression of luciferase gene i.e. binding of Elk-1-P to DNA and is an indirect read-out of the EGF signalling cascade.

According to the results shown here, in presence of EGF, luciferase activity is high in normal cells but non-existent in the mutant cell lines. Hence it can be inferred that the EGF signalling cascade is impaired in the mutant line. By this experiment, it can only be inferred that EGF signalling cascade is impaired in the mutant line and the impairment has happened upstream of Elk-1, but can happen at any point of the EGF signaling cascade- may be at the receptor level or at any intermediate level.

(To pinpoint the component of the EGF signalling cascade that is impaired, one needs to do a rescue experiment i.e. by transfecting the mutant cell line in presence of EGF, one by one, the wild type genes of the components of the EGF signalling pathway and see where the luciferase activity has been attained. The component whose transfection rescues the luciferase activity in the mutant line will be the mutated component in the mutant cell line.)