Vol. 4 No. 3 November 2005

The BioTECH Quarterly

Student Research Spotlight

Quantitative Effects of ErbB-Targeted Therapeutics on Apoptosis and Proliferation in Human Cancer Cell Lines

By Danielle Carpenter '08

   Epidermal growth factor receptors (EGFRs) are a family of receptor tyrosine kinases involved in many cellular processes such as cell growth, cell death, migration, and differentiation. Given their intimate involvement in a wide variety of cellular processes, it may come as no surprise that dysregulation of EGF receptors can lead to a variety of cancers. Consequently, ErbB receptors are prime targets for cancer therapy, and pharmaceutical companies have put great effort toward the development of inhibitors (small molecules and antibodies) designed to interrupt the various steps in EGFR activation.

   Epidermal growth factor receptors (EGFRs) are a family of receptor tyrosine kinases involved in many cellular processes such as cell growth, cell death, migration, and differentiation. Given their intimate involvement in a wide variety of cellular processes, it may come as no surprise that dysregulation of EGF receptors can lead to a variety of cancers. Consequently, ErbB receptors are prime targets for cancer therapy, and pharmaceutical companies have put great effort toward the development of inhibitors (small molecules and antibodies) designed to interrupt the various steps in EGFR activation.

   However, how these EGFR mutations affect normal receptor function, and the reason for their extreme sensitivity to Iressa remains largely unknown. The specific focus of my work is to investigate how the mutant EGFR genes alter cellular proliferation and death (apoptosis), with and without EGFR inhibitors such as Iressa.

   My project employs several basic protocols for quantifying cell proliferation and apoptosis. The assays involve incubating various cell lines (with and without EGFR mutations) with varying concentrations of inhibitor, and then determining the levels of proliferation or apoptosis.

   To quantify levels of proliferation, I use a spectrophotometry-based assay to measure color change of a dye that correlates with the number of live cells in a tissue culture dish. To quantify cell death, I fix the cells subsequent to inhibitor treatment and use fluorescent antibodies to detect levels of cleaved caspase-3 and cleaved PARP (two protein markers that are indicative of cell death), and then quantify fluorescence using a flow cytometer. An example of the raw data obtained from such an experiment is shown in figure 1.

   In figure 2, we are comparing cell death of two different cell lines, one of which has a mutant EGFR gene and the other of which is wild-type. Following a three-day treatment with 50 µL of Iressa, we see 91% of H3255 cells are positive for cleaved caspase 3 and PARP, as compared to only 13% of H1666 cells, suggesting that H3255 cells are 7-fold more sensitive to Iressa than H1666 cells.

   By performing these experiments on a multitude of cell lines, containing either the mutant or wild-type EGFR gene, we will gain a quantitative understanding of the extent to which the EGFR mutation contributes to proliferation and apoptosis.

   These data are part of an even larger study of the mutant EGFR genes, in which the receptor trafficking, degradation, and signaling are also being quantified. When combined, these data will be mathematically modeled in the hopes of gaining additional insight into the development of more effective therapeutic strategies for the next generation of inhibitors.