Quantitative imaging of apoptosis commitment in colorectal tumor cells.
We have studied caspase-3 activation by combined DNA damage induction and EGFR kinase inhibition in order to identify potential EGFR-mediated survival signals conferring resistance to apoptosis in human colorectal tumor cells. The onset of apoptosis was microscopically imaged with a newly developed caspase-3 substrate sensor based on EGFP and tHcred1, enabling us to monitor caspase-3 activation in cells by fluorescence lifetime imaging microscopy or fluorescence correlation spectroscopy. Both optical approaches provide parameters quantitatively reporting the ratio between cleaved and uncleaved sensor, thereby facilitating the comparison of caspase-3 activation between different cells. Using these methods, 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.
Keese
Michael
M
Chirurgische Klinik, Universitätsklinikum Mannheim, Mannheim, Germany.
Offterdinger
Martin
M
Tischer
Christian
C
Girod
Andreas
A
Lommerse
Piet H M
PH
Yagublu
Vugar
V
Magdeburg
Richard
R
Bastiaens
Philippe I H
PI
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis; D053148:Caspase 3 / Q000032:analysis*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000201:enzymology*; D004249:DNA Damage; D066246:ErbB Receptors / Q000037:antagonists & inhibitors; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology
D000964:Antimetabolites, Antineoplastic; D047428:Protein Kinase Inhibitors; D066246:ErbB Receptors; D053148:Caspase 3; D005472:Fluorouracil