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Regulation of membrane release in apoptosis.

ABSTRACT: Apoptosis is a fundamental process of cell regulation whereby cells execute one or more biochemical programs leading to cell death. Several mechanisms have been evaluated and suggested to play roles in the regulation of apoptosis, including the activation of phospholipase A2 (PLA2), usually measured as release of 3H-labelled arachidonic acid (AA) from prelabelled cells. The current study was aimed at examining the role of PLA2 in regulating apoptosis in response to several inducers (such as vincristine and etoposide) in lymphoid cell lines. Cells were labelled with [3H]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 PLA2 pathway. Rather, studies using TLC and electron microscopy showed that AA release reflected a previously unsuspected shedding of a heterogeneous population of membrane vesicles and fragments, probably as components of apoptotic bodies. Further studies demonstrated that this process is an integral part of apoptosis. Overexpression of Bcl-2 or the addition of caspase peptide inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethane prevented the characteristic morphological changes of cell death, and completely inhibited the release of membrane vesicles and fragments. On the other hand, release of membrane vesicles and fragments was caused by various inducers of apoptosis, as measured by release of either 3H-labelled AA or palmitic acid. Thus the present study demonstrates that the release of membrane lipids during apoptosis defines a new assay for apoptosis and has allowed the investigation of the mechanisms regulating formation of apoptotic bodies.

SUBMITTER: Zhang J

PROVIDER: S-EPMC1219712 | biostudies-other | 1998 Sep

REPOSITORIES: biostudies-other

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Project description:Hantaviruses, zoonotic RNA viruses belonging to the order Bunyavirales, cause two severe acute diseases in humans, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Hantavirus-infected patients show strong cytotoxic lymphocyte responses and hyperinflammation; however, infected cells remain mostly intact. Hantaviruses were recently shown to inhibit apoptosis in infected cells. By inhibiting granzyme B- and TRAIL-mediated apoptosis, hantaviruses specifically and efficiently inhibit cytotoxic lymphocyte-mediated killing of infected cells. Hantaviruses also strongly inhibit apoptosis triggered intrinsically; i.e., initiated through intracellular activation pathways different from those used by cytotoxic lymphocytes. However, insights into the latter mechanisms are currently largely unknown. Here, we dissected the mechanism behind how hantavirus infection, represented by the HFRS-causing Hantaan virus and the HPS-causing Andes virus, results in resistance to staurosporine-induced apoptosis. Less active caspase-8 and caspase-9, and consequently less active caspase-3, was observed in infected compared to uninfected staurosporine-exposed cells. While staurosporine-exposed uninfected cells showed massive release of pro-apoptotic cytochrome C into the cytosol, this was not observed in infected cells. Further, hantaviruses prevented activation of BAX and mitochondrial outer membrane permeabilization (MOMP). In parallel, a significant increase in levels of the pro-survival factor BCL-2 was observed in hantavirus-infected cells. Importantly, direct inhibition of BCL-2 by the inhibitor ABT-737, as well as silencing of BCL-2 by siRNA, resulted in apoptosis in staurosporine-exposed hantavirus-infected cells. Overall, we here provide a tentative mechanism by which hantaviruses protect infected cells from intrinsic apoptosis at the mitochondrial level by inducing an increased expression of the pro-survival factor BCL-2, thereby preventing MOMPs and subsequent activation of caspases. The variety of mechanisms used by hantaviruses to ensure survival of infected cells likely contribute to the persistent infection in natural hosts and may play a role in immunopathogenesis of HFRS and HPS in humans.

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Regulation of membrane release in apoptosis.

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