Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8

Takahashi, Atsushi; Hirata, Hirokazu; Yonehara, Shin; Imai, Yuzuru; Lee, Kyung-Kwon; Moyer, Richard W.; Turner, P.W.; Mesner, Peter W.; Okazaki, Toshiro; Sawai, Hirofumi; Kishi, Shuji; Yamamoto, Kokichi; Okuma, Minoru; Sasada, Masataka

doi:10.1038/sj.onc.1201131

Cited by 110 publications

(91 citation statements)

References 43 publications

(87 reference statements)

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“…As shown in Figure 4A-I, both caspase-3 and caspase-6 activities were significantly enhanced by STS treatment in control Jurkat cells or by anti-Fas engagement, which is consistent with previously reported data about the sensitivity of Jurkat cells to apoptosis induced by both of these pathways (28,(45)(46)(47). The induction of both DEVDase and VEIDase was inhibited nearly completely in bcl-x L overexpressing cells, whereas anti-Fas-induced caspase activities were partially inhibited (Fig.…”

Section: Use Of the Dual Fret Caspase Indicator Probe In Cells In Whisupporting

confidence: 91%

Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe

Simone

Hewgill

et al. 2006

Cytometry Pt A

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Background: A number of fluorescent caspase substrates and FRET-based indicators have been developed to study the in vivo activation of caspases, a conserved family of proteases critical in inflammatory, and apoptosis signaling pathways. To date, all substrates have measured only one caspase activity. Here, we describe a FRET-based probe for simultaneously measuring two distinct caspase activities in living cells. Methods: This probe consists of a CFP-YFP-mRFP fusion protein containing a caspase-3-cleavage motif, DEVD, between CFP and YFP, and a caspase-6-cleavage site, VEID, between YFP and mRFP. DEVDase and VEIDase activities could be assessed simultaneously by monitoring diminished FRET mediated by cleavage of either or both of these protease cleavage sites using flow cytometry. Results: DEVDase and VEIDase activities were completely inhibited by the pan-caspase inhibitor z-VAD-fmk and enhanced by DNA-damaging drugs or by anti-Fas stimula-

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Section: Use Of the Dual Fret Caspase Indicator Probe In Cells In Whisupporting

confidence: 91%

Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe

Simone

Hewgill

et al. 2006

Cytometry Pt A

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“…how each drug acts to induce apoptosis on certain cancers. Staurosporine has been shown to induce apoptosis in many cell types including, cardiomyocytes (Yue et al, 1998), chang liver cells (Swe and Sit, 1997), fibroblasts (Jacobson et al, 1996) and Jurkat T cells (Takahashi et al, 1997). Here we studied its effect and mechanisms of action on breast cancer cells.…”

Section: Effect Of Staurosporine On Bcl-2 Bcl-x L Bak and Bax Proteimentioning

confidence: 99%

Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells

et al. 2002

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To investigate the mechanisms underlying apoptosis in breast cancer cells, staurosporine was used as an apoptotic stimulus in the human breast cancer cell lines MCF-7 and T47D. Staurosporine induced dose and time dependent increases in DNA fragmentation which was abrogated by z-VAD-fmk. MCF-7 cells did not express caspase-3, suggesting that DNA fragmentation occurred in the absence of caspase-3 and that other caspases may be involved. Staurosporine induced DEVDase activity in T47D cells suggesting the involvement of caspase-3 and/or caspase-7, yet there was no DEVDase activity in MCF-7 cells, probably ruling out the involvement caspase-7. However, staurosporine induced the cleavage of pro-caspase-6 in MCF-7 cells, but not in T47D cells. Caspase dependent PARP cleavage was detected in MCF-7 cells at 3 h, whereas only partial PARP cleavage was detected in T47D cells and then only after 24 h. Moreover, staurosporine led to cytochrome c release at 2 h in MCF-7 cells and 6 h in T47D cells. In addition, a time dependent and caspase-independent reduction of the mitochondrial transmembrane potential was observed; which appeared to occur after the release of cytochrome c. Translocation of Bax from the cytosol to mitochondria was observed in both cell types, and this preceded cytochrome c release in both T47D and MCF-7 cells. Apoptotic events in both cell types differ temporally, involving activation of different caspases and mitochondrial changes.

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“…One unique property of staurosporine is its ability to rapidly and completely drive virtually all mammalian cells into apoptosis. Staurosporine-induced apoptosis involves mitochondrial caspase activation that is inhibited by Bcl-2 (Bertrand et al, 1994;Jacobson et al, 1994Jacobson et al, , 1996Takahashi et al, 1997). However, despite its widespread use, the primary site of action of staurosporine in apoptosis induction is unknown.…”

Section: Introductionmentioning

confidence: 99%

Staurosporine and conventional anticancer drugs induce overlapping, yet distinct pathways of apoptosis and caspase activation

et al. 2001

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Apoptosis can be induced by various stimuli including DNA-damaging anticancer drugs and the protein kinase inhibitor staurosporine. It is generally believed that the molecular events during execution of apoptosis are shared, as both anticancer drugs and staurosporine derivatives induce mitochondrial damage, cytochrome c release and the activation of the caspase-9 proteolytic cascade. In the present study we show that overexpression of a dominant-negative caspase-9 mutant abolished the activation of endogenous caspase-9, caspase-3 and the cleavage of the caspase substrate Bid in response to anticancer drug treatment. Surprisingly, however, only marginal eects were observed during staurosporine-induced apoptosis. Furthermore, we describe a Jurkat T-cell clone that is completely resistant towards dierent anticancer drugs, but remains sensitive towards staurosporine-induced apoptosis. In these cells only staurosporine, but neither anti-CD95 nor anticancer drugs were able to trigger caspase activity and the cleavage of caspase substrates. Our results therefore suggest that the mechanism of staurosporine-induced apoptosis is more complex and at least partially diers from anticancer drug-induced caspase activation. These distinct features of staurosporine may allow to bypass chemoresistance of tumor cells and may encourage further clinical trials for the use of staurosporine derivatives in antitumor therapy. Oncogene (2001) 20, 1193 ± 1202.

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Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8

Cited by 110 publications

References 43 publications

Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe

Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe

Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells

Staurosporine and conventional anticancer drugs induce overlapping, yet distinct pathways of apoptosis and caspase activation

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