REGULATION OF MEMBRANE RELEASE IN APOPTOSIS

Apoptosis is a fundamental process of cell regulation whereby cells ex ecute one or more biochemical programs leading to cell death. Several mechanisms have been evaluated and suggested to Flay roles in the regu lation of apoptosis, including the activation of phospholipase A(2) (P LA(2)), usually measured as release of H-3-labelled arachidonic acid ( AA) from prelabelled cells. The current study was aimed at examining t he role of PLA(2) in regulating apoptosis in response to several induc ers (such as vincristine and etoposide) in lymphoid cell lines. Cells were labelled with [H-3]fatty acids and the released radioactivity was characterized. These studies indicated that the AA release assay did not reflect release of non-esterified fatty acid via activation of the PLA(2) pathway. Rather, studies using TLC and electron microscopy sho wed that AA release reflected a previously unsuspected shedding of a h eterogeneous population of membrane vesicles and fragments, probably a s components of apoptotic bodies. Further studies demonstrated that th is process is an integral part of apoptosis. Overexpression of Bcl-2 o r the addition of caspase peptide inhibitor benzyloxycarbonyl-Asp-Glu- Val-Asp-fluoromethane prevented the characteristic morphological chang es of cell death, and completely inhibited the release of membrane ves icles and fragments. On the other hand, release of membrane vesicles a nd fragments was caused by various inducers of apoptosis, as measured by release of either H-3-labelled AA or palmitic acid. Thus the presen t study demonstrates that the release of membrane lipids during apopto sis defines a new assay for apoptosis and has allowed the investigatio n of the mechanisms regulating formation of apoptotic bodies.