Changes of caspase activities involved in apoptosis of a macrophage-like cell line J774.1/JA-4 treated with lipopolysaccharide (LPS) and cycloheximide

The addition of lipopolysaccharide (LPS) together with cycloheximide (CHX) induced apoptosis in a subline of a J774.1 macrophage-like cell line, JA-4, as judged by terminal deoxynucleotidyl transferase (TdT)-mediatcd deoxyuri dine triphosphate (dUTP) nick end labeling (TUNEL)-staining and poly(adenos ine 5'-diphosphate (ADP)-ribose) polymerase (PARP)-cleavage. Caspase activi ties were examined in these macrophages in vitro using fluorogenic substrat es such as acetyl-DEVD-aminomethyl coumarine (Ac-DEVD-AMC, caspase-3-like), acetyl-YVAD-aminomethyl coumarine (Ac-YVAD-AMC, caspase-1-like), acetyl-VE ID-aminomethyl coumarine (Ac-VEID-AMC, caspase-6-like), and carbobenzoxy-IE TD-aminofluoro coumarine (Z-IETD-AFC; caspase-8-like). Kinetic studies reve aled these caspase activities with different K-m and V-max values in extrac ts of apoptotic macrophages. In the course of apoptosis, caspase-3-like act ivity increased first at 75 min, simultaneously with the appearance of TUNE L staining and prior to PARP cleavage, and then caspase-6 and 8-like activi ties increased at 90 and 105 min, respectively. However, caspase-1-like act ivity did not change throughout the experiment. Furthermore. removal of LPS and CHX by extensive washing of the cells for 60 min completely abolished the apoptosis and the subsequent release of lactate dehydrogenase (LDH) dur ing additional incubation until 4 h after LPS addition. However, washing of the cells after 75 min or later resulted in the progress of apoptosis and LDH release, which was coordinated with the elevation of caspase-3-like act ivity at 60 min and that of caspase-6 or 8-like activity at 90 min, but not with that of caspase-1-like activity. These results suggest that caspase-3 -like activity represents the most apical caspase among these caspases in t erms of the intiation of apoptosis in macrophages treated with LPS and CHX. In the present study, we also provide evidence on the relatively low; speci ficities of a series of caspase inhibitors other than acetyl-DEVD-aldehyde (Ac-DEVD-CHO) which specifically inhibited the caspasc-3-like activity.