TY - GEN
T1 - Number and brightness analysis to quantify the kinetics of EGFR and its mutants in living cells before and after γ-irradiation
AU - Abdisalaam, Salim
AU - Chen, David J.
AU - Alexandrakis, George
PY - 2012
Y1 - 2012
N2 - The epidermal growth factor receptor (EGFR), which is over-expressed in tumors cells of epithelial origin is one of the determinants of tumor responses to ionizing radiation. Recently, it has been shown that higher EGFR expression levels lead to higher tumor resistance to radiation therapy through the activation of cell proliferation and survival pathways. In this study, a raster-scan imaging technique known as Number and Brightness (N&B) analysis has been employed to demonstrate the nuclear translocation of EGFR in living cells under a variety of experimental conditions. About 80% of wild type (WT) EGFR translocated to the nucleus after γ-irradiation while the L858R and ΔE746-E750 mutant EGFR did not. Subsequently, the effects of γ-irradiation together with an EGFR-blocking antibody (cetuximab) were monitored simultaneously in the same cell lines expressing EGFR and its mutants. In the combined radiation and cetuximab treatment, about 26 % of WT were translocated to the nucleus, while the L858R and ΔE746-E750 mutant EGFR did not. These results are consistent with findings attained by standard molecular techniques and support the hypothesis that a cytosolic pool of EGFR exists that cannot be accessed by cetuximab and can therefore contribute to treatment resistance.
AB - The epidermal growth factor receptor (EGFR), which is over-expressed in tumors cells of epithelial origin is one of the determinants of tumor responses to ionizing radiation. Recently, it has been shown that higher EGFR expression levels lead to higher tumor resistance to radiation therapy through the activation of cell proliferation and survival pathways. In this study, a raster-scan imaging technique known as Number and Brightness (N&B) analysis has been employed to demonstrate the nuclear translocation of EGFR in living cells under a variety of experimental conditions. About 80% of wild type (WT) EGFR translocated to the nucleus after γ-irradiation while the L858R and ΔE746-E750 mutant EGFR did not. Subsequently, the effects of γ-irradiation together with an EGFR-blocking antibody (cetuximab) were monitored simultaneously in the same cell lines expressing EGFR and its mutants. In the combined radiation and cetuximab treatment, about 26 % of WT were translocated to the nucleus, while the L858R and ΔE746-E750 mutant EGFR did not. These results are consistent with findings attained by standard molecular techniques and support the hypothesis that a cytosolic pool of EGFR exists that cannot be accessed by cetuximab and can therefore contribute to treatment resistance.
KW - DNA double strand breaks (DNA-DSBs)
KW - epidermal growth factor receptor (EGFR)
KW - fluorescence correlation spectroscopy (FCS)
KW - ionizing radiation (IR)
KW - number and brightness (N&B) analysis
KW - reactive oxygen species (ROS)
UR - http://www.scopus.com/inward/record.url?scp=84863264390&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84863264390&partnerID=8YFLogxK
U2 - 10.1117/12.909738
DO - 10.1117/12.909738
M3 - Conference contribution
AN - SCOPUS:84863264390
SN - 9780819488718
T3 - Progress in Biomedical Optics and Imaging - Proceedings of SPIE
BT - Single Molecule Spectroscopy and Superresolution Imaging V
T2 - Single Molecule Spectroscopy and Superresolution Imaging V
Y2 - 21 January 2012 through 22 January 2012
ER -