Single-cell fluorescence resonance energy transfer analysis demonstrates that caspase activation during apoptosis is a rapid process. Role of caspase-3.
Activation of effector caspases is considered to be the final step in many apoptosis pathways. We transfected HeLa cells with a recombinant caspase substrate composed of cyan and yellow fluorescent protein and a linker peptide containing the caspase cleavage sequence DEVD, and we examined the cleavage kinetics at the single-cell level by fluorescence resonance energy transfer (FRET) analysis. Caspase activation in response to tumor necrosis factor-alpha, staurosporine, or etoposide resulted in cleavage of the linker peptide and subsequent disruption of the FRET signal. The time to caspase activation varied among individual cells, depending on the type of treatment and concentration used. However, once initiated, disruption of the FRET signal was always rapid (<or=15 min) and largely independent of these parameters. In contrast, FRET probe cleavage was significantly slower in the caspase-3-deficient MCF-7 cells, particularly at low concentrations of the pro-apoptotic agents. Under these conditions, MCF-7 cells required up to 90 min for the FRET probe cleavage, whereas MCF-7/Casp-3 cells displayed rapid cleavage kinetics. Interestingly, we could still observe comparable cell death rates in MCF-7 and MCF-7/Casp-3 cells. Our results suggest that caspase activation during apoptosis occurs in an "all or nothing" fashion. Caspase-3 is required for rapid cleavage kinetics when the onset of apoptosis is slow, suggesting the existence of caspase-3-dependent feedback loops.
Rehm
Markus
M
Interdisciplinary Center for Clinical Research (IZKF), Research Group "Apoptosis and Cell Death," the Department of Experimental Dermatology, Division of Immunology and Cell Biology, Westphalian Wilhelms University, D-48149 Münster, Germany.
Dussmann
Heiko
H
Janicke
Reiner U
RU
Tavare
Jeremy M
JM
Kogel
Donat
D
Prehn
Jochen H M
JH
D017209:Apoptosis / Q000502:physiology*; D001943:Breast Neoplasms; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D007700:Kinetics; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases