A novel protein expressed in mammalian cells undergoing apoptosis

Human and rodent cells undergoing Apoptosis were observed to express high levels of a novel 45,000 M(r) protein. The protein, which we have termed Apoptosis specific protein (ASP), was found in Burkitt lymphoma (BL) cells and in adenovirus-transformed human and rat embryo cells induced into Apoptosis by a variety of stimuli, including serum deprivation, exposure to the Ca2+ ionophore, ionomycin, treatment with inhibitors of macromolecular synthesis (cycloheximide and actinomycin D), and cold shock. In BL cells treated with apoptotic stimuli, expression of the oncoprotein Bcl-2 was found to both protect from Apoptosis and prevent expression of ASP. ASP was not detected either in viable cells or in cells dying passively by necrosis. Laser scanning confocal microscopy showed high levels of ASP in the cytoplasm of cells displaying the chromatin condensation and fragmentation patterns typical of Apoptosis. Retention of ASP was observed even when DNA was no longer detectable, and two-color immunofluorescence staining indicated that the protein primarily colocalized with, but was clearly distinct from, non-muscle actin. These findings, together with the observation that biochemical extraction of ASP was only possible under conditions which caused solubilization of the Cytoskeleton, leads us to conclude that ASP forms part of, or at least strongly associates with, a modified Cytoskeleton unique to cells undergoing Apoptosis. While elucidation of its function will require further work, ASP constitutes a powerful marker for the diagnosis and quantitation of Apoptosis in vivo and in vitro.