Inhibition of caspases inhibits the release of apoptotic bodies: Bcl-2 inhibits the initiation of formation of apoptotic bodies in chemotherapeutic agent-induced apoptosis.
During apoptosis, the cell actively dismantles itself and reduces cell size by the formation and pinching off of portions of cytoplasm and nucleus as "apoptotic bodies." We have combined our previously established quantitative assay relating the amount of release of [3H]-membrane lipid to the degree of apoptosis with electron microscopy (EM) at a series of timepoints to study apoptosis of lymphoid cells exposed to vincristine or etoposide. We find that the [3H]-membrane lipid release assay correlates well with EM studies showing the formation and release of apoptotic bodies and cell death, and both processes are regulated in parallel by inducers or inhibitors of apoptosis. Overexpression of Bcl-2 or inhibition of caspases by DEVD inhibited equally well the activation of caspases as indicated by PARP cleavage. They also inhibited [3H]-membrane lipid release and release of apoptotic bodies. EM showed that cells overexpressing Bcl-2 displayed near-normal morphology and viability in response to vincristine or etoposide. In contrast, DEVD did not prevent cell death. Although DEVD inhibited the chromatin condensation, PARP cleavage, release of apoptotic bodies, and release of labeled lipid, DEVD-treated cells showed accumulation of heterogeneous vesicles trapped in the condensed cytoplasm. These results suggest that inhibition of caspases arrested the maturation and release of apoptotic bodies. Our results also imply that Bcl-2 regulates processes in addition to caspase activation.
Zhang
J
J
Veterans Administration Geriatrics Research Education and Clinical Center and the Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Reedy
M C
MC
Hannun
Y A
YA
Obeid
L M
LM
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D020170:Caspase 1 / Q000502:physiology; D053148:Caspase 3; D061945:Caspase Inhibitors; D048429:Cell Size; D002843:Chromatin / Q000648:ultrastructure; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D005047:Etoposide / Q000494:pharmacology; D015458:Leukemia, T-Cell / Q000473:pathology; D008563:Membrane Lipids / Q000378:metabolism*; D008854:Microscopy, Electron; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000201:enzymology / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000473:pathology
D000972:Antineoplastic Agents, Phytogenic; D061945:Caspase Inhibitors; D002843:Chromatin; D015853:Cysteine Proteinase Inhibitors; D008563:Membrane Lipids; D009363:Neoplasm Proteins; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C118632:tyrosyl-valyl-alanyl-aspartal; D005047:Etoposide; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3; D020170:Caspase 1