gms | German Medical Science

Introduction: Upregulation and overexpression of the epidermal growth factor (EGFR) has been correlated to many processes related to cancer, including uncontrolled cellular proliferation and autocrine stimulation of tumors producing their own growth factors. Many epithelial tumors express high EGFR densities, which are associated with advanced disease and poor clinical prognosis. We here questioned whether EGFR activity conveys resistance to apoptosis.

Materials and methods: In contrast to former assays, which were restricted to the quantitation of protein or RNA expression, we established a new approach to detect the functional activity of EGFR using fluorescence resonance energy transfer: FRET. Labelling of the F4-antibody (specific antibody against an intracellular epitope of EGFR) with Cy 3 and of the phosphotyrosin-antibody Py72 with Cy 5 allows measure FRET which is present in the activated EGFR. We determined EGFR activity in colorectal cell lines. Additionally we measured the amount of EGFR-expression. Apoptosis commitment in colorectal cell lines was microscopically imaged with a newly developed caspase-3 substrate sensor based on EGFP and tHcred1 enabling to monitor caspase-3 activation in cells by fluorescence lifetime imaging microscopy/FLIM) in transfected cells. This approach provides parameters reporting quantitatively the ratio between cleaved versus uncleaved sensor thereby facilitating the comparison of apoptosis commitment between different cells.

Results: We show that the phosphorylation state of endogenous EGFR can be quantitatively imaged in tumor cells and tissues by detecting fluorescence resonance energy transfer between fluorophores conjugated to antibodies. Five different human colorectal cell lines were analyzed for activity and expression of EGFR. All cell lines exhibited basal EGFR phosphorylation under serum starvation conditions. Phosphorylation levels increased after stimulation dependent on the level of basal EGFR phosphorylation. The basal activity correlated inversely with receptor expression. Apoptosis commitment was microscopically imaged with a novel caspase-3 substrate sensor based on EGFP and tHcred1 enabling to monitor caspase-3 activation in cells by fluorescence lifetime imaging microscopy. This approach provides parameters reporting quantitatively the ratio between cleaved versus uncleaved sensor thereby facilitating the comparison of apoptosis commitment between different cells. We show that EGFR kinase inhibitors sensitize colorectal SW-480 tumor cells for 5-fluorouracil induced apoptosis indicating that EGFR-mediated survival signaling contributes to apoptosis resistance via its intrinsic kinase activity.

Discussion: Although fluorescent biosensors are now widely used in cell biology, few studies have attempted to use them in the context of drug screening procedures. We have therefore developed FRET-FLIM assays for quantitative analysis of cell biological processes such as RTK-activity or protease activation. Using these optical approaches, we show that colorectal tumor cells display basal EGFR phosphorylation in the absence of exogenous growth factors. This basal EGFR signaling contributes to cell survival upon chemotherapy-induced DNA damage.