Advances in cytochemical methods for detection of apoptosis

In an earlier article from this laboratory, the current methods developed t o detect apoptosis in cells and tissues were highlighted, along with the ch allenges in their interpretation. Recent discoveries concerning the underly ing biochemical mechanisms of apoptotic effector pathways have made possibl e further assays that allow a more direct measure of the activation of the apoptotic machinery in cells. This article summarizes some of these newer m ethods and extends the interpretation of the more classical assays of apopt osis in a defined cell system. We present data in KB and PC3 cell model cul ture systems induced to undergo apoptosis by the plant toxin ricin. Using a modified in situ nick translation assay (ISNT) with either Bodipy or BUdR labeling, we confirm that most cells showing altered nuclear morphology do not show reactivity with this assay until very late in the apoptotic proces s. We also show that only a minority of cells label with fluorescent annexi n V during apoptosis but that apoptotic cells continue to internalize mater ial from the cell surface through endocytosis after becoming reactive with annexin V. In addition, we describe the utility of a prototype of new assay s for caspase substrate cleavage products, the detection of cleaved cytoker atin 18. It is these newer cleavage product assays that perhaps hold the gr eatest promise for specific detection of apoptosis in cells either in cell culture or in intact tissues.