| Programmed cell death is characterized by
a progressive series of morphological and
biochemical changes ultimately resulting in
disassembly of the cell proper. Caspases are
a family of proteolytic enzymes that mediate
key aspects of this process from initiating
early signaling events to directing cellular
breakdown through cleavage of structural
proteins. We have combined the detection
of mitotic state with several parameters of
apoptosis to investigate the action of several
test compounds. This multiplex assay system
detects: (1) the enzymatic activity of Caspases
3/7, and 8; (2) changes in mitochondrial
membrane potential; (3) translocation of
phosphatidylserine residues to the cell surface;
(4) membrane permeability; and (5) cell cycle
analysis. The simultaneous interrogation of
both caspase activity and cell cycle progression,
along with the information regarding the
relative frequencies of treated cells in the
early, mid-or late stages of apoptosis offers a
powerful approach to endpoint analysis for
characterizing compounds. In addition, this
strategy will provide a more complete and
detailed understanding of the mechanism of
action both at the level of the individual cell
and the overall cell population. |
