Correlating cell cycle with apoptosis in a cell line expressing a tandem green fluorescent protein substrate specific for group II caspases.
BACKGROUND
We describe a rapid flow cytometric assay that correlates cell cycle with apoptotic cell death in a cell line expressing a tandem green fluorescent protein (GFP).


METHODS
A Jurkat cell line was transfected with a gene construct coding for constitutive expression of a tandem GFP molecule carrying a consensus cleavage site (DEVD) for group II caspases (C-2-Y). Cells were treated with CD95 antibody (Ab), then incubated with annexin V-phycoerythrin (PE), propidium iodide (PI), and Hoechst 33342.


RESULTS
After CD95 treatment, the C-2-Y cell line had twice the number of nonapoptotic cells compared with both control cell lines. This proportion of viable, nonapoptotic cells after treatment was unaffected by the level of GFP (DEVD) expression in the cells, as confirmed by sorted populations. The early apoptotic cells in the C-2-Y cell line had an increased G0-G1 phase population compared with the control cell lines.


CONCLUSIONS
Apoptosis is delayed in the C-2-Y cell line and the early apoptotic cells have a higher G0-G1 cell cycle frequency. The artificial substrate competes with the natural substrate(s), thereby slowing the apoptotic process. The expression level of DEVD-GFP does not alter the delayed induction of apoptosis. Caspase activation occurs prior to phosphatidylserine translocation.
Donahue
C J
CJ
Discovery Technologies, Pfizer Global Research and Development, Ann Arbor, Michigan 48105, USA. christopher.donahue@pfizer.com
Santoro
M
M
Hupe
D
D
Jones
J M
JM
Pollok
B
B
Heim
R
R
Giegel
D
D
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D058846:Antibodies, Monoclonal, Murine-Derived; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D020169:Caspases / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D016193:G1 Phase / Q000187:drug effects / Q000502:physiology*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000378:metabolism; D016192:Resting Phase, Cell Cycle / Q000187:drug effects / Q000502:physiology*; D013223:Statistics as Topic; D013379:Substrate Specificity / Q000502:physiology; D020080:Tandem Repeat Sequences / Q000502:physiology; D014162:Transfection
D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D008164:Luminescent Proteins; C411023:anti-Fas monoclonal antibody; D049452:Green Fluorescent Proteins; D020169:Caspases