Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis.

Lazebnik, Yuri; Cole, Susan P. C.; Cooke, Carol; Nelson, William G.; Earnshaw, William C.

doi:10.1083/jcb.123.1.7

Cited by 433 publications

(334 citation statements)

References 54 publications

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“…Strikingly, the nuclear lamina in Ad-Bin1-infected cells remained essentially intact. This feature supported the lack of caspase involvement because lamins are subjected to caspase-mediated proteolysis during PCD (Lazebnik et al, 1993;Rao et al, 1996). In addition, lamin cleavage is separable from chromatin collapse (Lazebnik et al, 1995) but is important for complete nuclear degeneration (Rao et al, 1996), so the more limited nuclear degeneration in Ad-Bin1-infected cells was also consistent with a caspase-independent process.…”

Section: Bin1 Does Not Activate Caspasesmentioning

confidence: 65%

The c-Myc-interacting adaptor protein Bin1 activates a caspase-independent cell death program

et al. 2000

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Cell death processes are progressively inactivated during malignant development, in part by loss of tumor suppressors that can promote cell death. The Bin1 gene encodes a nucleocytosolic adaptor protein with tumor suppressor properties, initially identi®ed through its ability to interact with and inhibit malignant transformation by c-Myc and other oncogenes. Bin1 is frequently missing or functionally inactivated in breast and prostate cancers and in melanoma. In this study, we show that Bin1 engages a caspase-independent cell death process similar to type II apoptosis, characterized by cell shrinkage, substratum detachment, vacuolated cytoplasm, and DNA degradation. Cell death induction was relieved by mutation of the BAR domain, a putative e ector domain, or by a missplicing event that occurs in melanoma and inactivates suppressor activity. Cells in all phases of the cell cycle were susceptible to death and p53 and Rb were dispensable. Notably, Bin1 did not activate caspases and the broad spectrum caspase inhibitor ZVAD.fmk did not block cell death. Consistent with the lack of caspase involvement, dying cells lacked nucleosomal DNA cleavage and nuclear lamina degradation. Moreover, neither Bcl-2 or dominant inhibition of the Fas pathway had any e ect. In previous work, we showed that Bin1 could not suppress cell transformation by SV40 large T antigen. Consistent with this ®nding, we observed that T antigen suppressed the death program engaged by Bin1. This observation was interesting in light of emerging evidence that T antigen has roles in cell immortalization and human cell transformation beyond Rb and p53 inactivation. In support of a link to c-Mycinduced death processes, AEBSF, a serine protease inhibitor that inhibits apoptosis by c-Myc, potently suppressed DNA degradation by Bin1. Our ®ndings suggest that the tumor suppressor activity of Bin1 re¯ects engagement of a unique cell death program. We propose that loss of Bin1 may promote malignancy by blunting death penalties associated with oncogene activation. Oncogene (2000) 19, 4669 ± 4684.

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Section: Bin1 Does Not Activate Caspasesmentioning

confidence: 65%

The c-Myc-interacting adaptor protein Bin1 activates a caspase-independent cell death program

et al. 2000

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“…Nuclei from HeLa cells (puri®ed on a sucrose gradient, as described (Lazebnik et al, 1993) were cultured in the presence of mitochondrial supernatants for 90 min at 378C. Nuclei were stained with propidium iodide (PI; 10 mg/ml; Sigma), followed by cyto¯uorometric analysis in an EPICS Pro®le II Analyzer (Coulter, Hialeh, FL) (Susin et al, 1997b).…”

Section: Cell-free System Of Apoptosismentioning

confidence: 99%

The thiol crosslinking agent diamide overcomes the apoptosis-inhibitory effect of Bcl-2 by enforcing mitochondrial permeability transition

et al. 1998

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In several di erent cell lines, Bcl-2 prevents the induction of apoptosis (DNA fragmentation, PARP cleavage, phosphatidylserine exposure) by the pro-oxidant terbutylhydroperoxide (t-BHP) but has no cytoprotective e ect when apoptosis is induced by the thiol crosslinking agent diazenedicarboxylic acid bis 5N,N-dimethylamide (diamide). Both t-BHP and diamide cause a disruption of the mitochondrial transmembrane potential DC m that is not inhibited by the broad spectrum caspase inhibitor Z-VAD.fmk, although Z-VAD.fmk does prevent nuclear DNA fragmentation and poly(ADP-ribose) polymerase cleavage in these models. Bcl-2 stabilizes the DC m of t-BHP-treated cells but has no inhibitory e ect on the DC m collapse induced by diamide. As compared to normal controls, isolated mitochondria from Bcl-2 overexpressing cells are relatively resistant to the induction of DC m disruption by t-BHP in vitro. Such Bcl-2 overexpressing mitochondria also fail to release apoptosis-inducing factor (AIF) and cytochrome c from the intermembrane space, whereas control mitochondria not overexpressing Bcl-2 do liberate AIF and cytochrome c in response to t-BHP. In contrast, Bcl-2 does not confer protection against diamidetriggered DC m collapse and the release of AIF and cytochrome c. This indicates that Bcl-2 suppresses the permeability transition (PT) and the associated release of intermembrane proteins induced by t-BHP but not by diamide. To further investigate the mode of action of Bcl-2, semi-puri®ed PT pore complexes were reconstituted in liposomes in a cell-free, organelle-free system. Recombinant Bcl-2 or Bcl-X L proteins augment the resistance of reconstituted PT pore complexes to pore opening induced by t-BHP. In contrast, mutated Bcl-2 proteins which have lost their cytoprotective potential also lose their PTmodulatory capacity. Again, Bcl-2 fails to confer protection against diamide in this experimental system. The reconstituted PT pore complex itself cannot release cytochrome c encapsulated into liposomes. Altogether these data suggest that pro-oxidants, thiol-reactive agents, and Bcl-2 can regulate the PT pore complex in a direct fashion, independently from their e ects on cytochrome c. Furthermore, our results suggest a strategy for inducing apoptosis in cells overexpressing apoptosis-inhibitory Bcl-2 analogs.

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“…Nuclei from mock and Ad2 (moi=100) infected HeLa cells were prepared by the method described by Lazebnik et al (1993). 10 6 nuclei were mixed with 10 ml of extract for each time point with the addition of 10 mM HEPES (7.0), 50 mM NaCl, 2 mM MgCl 2 , 0.1 mM CaCl 2 , 40 mM b-glycerolphosphate and 1 mM DTT.…”

Section: Lamin Degradation Assaymentioning

confidence: 99%

The E1B 19K protein associates with lamins in vivo and its proper localization is required for inhibition of apoptosis

1997

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Expression of the E1B 19K protein is required to inhibit apoptosis induced by E1A during adenovirus infection and transformation. E1B 19K is homologous to Bcl-2 in function and the two proteins also share limited amino acid sequence homology. Consequently, the E1B 19K and Bcl-2 proteins bind to and inhibit the cellular deathinducing proteins Bax, Bak and Nbk/Bik. Both E1B 19K and Bcl-2 localize to membranes of the nucleus and the endoplasmic reticulum. In addition to membrane association, and unlike Bcl-2, the E1B 19K protein is found associated with intermediate ®lament proteins in the cytoplasm and the nuclear lamina and copuri®es with the lamins both during infection and transformation. While a membrane targeting domain at the C-terminus of Bcl-2 ensures its proper localization, the mechanism by which the E1B 19K protein localizes is unknown. Not surprisingly, lamin A fragments were cloned from a yeast two-hybrid screen for E1B 19K-interacting proteins. The interaction was demonstrated in yeast and mammalian cells in vivo and in vitro and was unique and speci®c to E1B 19K, with no interaction evident between Bcl-2 and lamin A. Mutants of lamin A/C which localized inappropriately in the cytoplasm or nucleus but retained E1B 19K binding, interfered with the nuclear envelope and cytoplasmic membrane targeting of the E1B 19K protein. Improper localization impaired the ability of the E1B 19K protein to inhibit apoptosis. Thus, proper localization of the E1B 19K protein is required for its function and the interaction of the E1B 19K protein with lamin A/C may represent a means for nuclear envelope localization.

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Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis.

Cited by 433 publications

References 54 publications

The c-Myc-interacting adaptor protein Bin1 activates a caspase-independent cell death program

The c-Myc-interacting adaptor protein Bin1 activates a caspase-independent cell death program

The thiol crosslinking agent diamide overcomes the apoptosis-inhibitory effect of Bcl-2 by enforcing mitochondrial permeability transition

The E1B 19K protein associates with lamins in vivo and its proper localization is required for inhibition of apoptosis

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