Investigation of a potential scintigraphic marker of apoptosis: radioiodinated Z -Val-Ala-DL-Asp( O -methyl)-fluoromethyl ketone

Two irreversible calpain inhibitors, benzyloxycarbonyl (Cbz)-Leu-Leu-Tyr-Ch2F and Cbz-Leu-Leu-Tyr-CHN2, were shown earlier [Anagli, Hagmann and Shaw (1991) Biochem. J. 274, 497-502] to penetrate intact platelets and to inactivate calpain. This permitted an evaluation of certain functions attributed to this proteinase. For example, in platelets pretreated with these inhibitors, talin and actin-binding protein were protected from subsequent degradation when the Ca2+ level was raised. On the other hand, additional properties of stimulated platelets attributed to calpain remained unaffected by this treatment, and such hypotheses may be dismissed. Radioiodinated inhibitors permitted confirmation of the labelling of calpain by the procedures used. Although Cbz-Leu-Leu-Tyr-CHN2 is more effective in vitro than the corresponding fluoromethyl ketone, we now show that the latter penetrates more readily. These two inhibitors, and two additional ones, t-butyloxycarbonyl-Val-Lys(Cbz)-Leu-Tyr- CHN2 and Cbz-Leu-Tyr-CH2F, have been radioiodinated to permit a comparison of their intracellular labelling patterns in activated platelets. Calpain is the major target of all four inhibitors. Although they are closely related peptide structures, variations with respect to the labelling of additional proteins were observed. These were minor in the case of the peptidyl diazomethyl ketones, but were major in the case of the fluoromethyl ketones. However, in contrast to calpain, this labelling was neither time-dependent nor Ca(2+)-dependent. Radiolabelling and cellular fractionation studies were used to localize active calpain during platelet activation. Calpain appears to be activated in the cytosol and translocated to the membrane or cytoskeletal sites.

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Assessment of Caspase Activities in Intact Apoptotic Thymocytes Using Cell-Permeable Fluorogenic Caspase Substrates

Journal of Experimental Medicine ◽

10.1084/jem.191.11.1819 ◽

2000 ◽

Vol 191 (11) ◽

pp. 1819-1828 ◽

Cited By ~ 117

Author(s):

Akira Komoriya ◽

Beverly Z. Packard ◽

Martin J. Brown ◽

Ming-Lei Wu ◽

Pierre A. Henkart

Keyword(s):

Apoptotic Cell ◽

Apoptotic Cells ◽

Caspase Activation ◽

Peptide Recognition ◽

Caspase Substrates ◽

Intracellular Target ◽

Recognition Motifs ◽

In Vitro Treatment ◽

Fluoromethyl Ketone

To detect caspase activities in intact apoptotic cells at the single cell level, cell-permeable fluorogenic caspase substrates were synthesized incorporating the optimal peptide recognition motifs for caspases 1, 3/7, 6, 8, and 9. Caspase activities were then assessed at various times after in vitro treatment of mouse thymocytes with dexamethasone or anti-Fas antibody. Dexamethasone induced the following order of appearance of caspase activities as judged by flow cytometry: LEHDase, WEHDase, VEIDase, IETDase, and DEVDase. Since the relative order of caspases 3 (DEVDase) and 6 (VEIDase) in the cascade has been controversial, this caspase activation order was reexamined using confocal microscopy. The VEIDase activity appeared before DEVDase in every apoptotic cell treated with dexamethasone. In contrast, anti-Fas stimulation altered this sequence: IETDase was the first measurable caspase activity and DEVDase preceded VEIDase. In an attempt to determine the intracellular target of the potent antiapoptotic agent carbobenzoxy-valyl-alanyl-aspartyl(β-methyl ester)-fluoromethyl ketone (Z-VAD[OMe]-FMK), we examined its ability to inhibit previously activated intracellular caspases. However, no significant reductions of these activities were observed. These fluorogenic caspase substrates allow direct observation of the caspase cascade in intact apoptotic cells, showing that the order of downstream caspase activation is dependent on the apoptotic stimulus.

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Structure of Recombinant Human CPP32 in Complex with the Tetrapeptide Acetyl-Asp-Val-Ala-Asp Fluoromethyl Ketone

Journal of Biological Chemistry ◽

10.1074/jbc.272.10.6539 ◽

1997 ◽

Vol 272 (10) ◽

pp. 6539-6547 ◽

Cited By ~ 161

Author(s):

Peer R. E. Mittl ◽

Stefania Di Marco ◽

Joseph F. Krebs ◽

Xu Bai ◽

Donald S. Karanewsky ◽

...

Keyword(s):

Fluoromethyl Ketone

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Caspase-3 Activity and Carbonyl Cyanide m-Chlorophenylhydrazone-induced Apoptosis in HL-60 cells

Alternatives to Laboratory Animals ◽

10.1177/026119290102900313 ◽

2001 ◽

Vol 29 (3) ◽

pp. 243-249 ◽

Cited By ~ 8

Author(s):

Petr Mlejnek

Keyword(s):

Cell Death ◽

Dna Cleavage ◽

Caspase 3 ◽

Chromatin Condensation ◽

Experimental System ◽

Apoptotic Bodies ◽

Nucleosomal Dna ◽

Carbonyl Cyanide ◽

Induced Apoptosis ◽

Fluoromethyl Ketone

The role of caspase proteases in carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced apoptosis of human promyelocytic HL-60 cells was examined. Treatment of HL-60 cells with micromolar concentrations of CCCP resulted in cell death, with typical apoptotic features such as chromatin condensation, formation of apoptotic bodies, nucleosomal fragmentation of DNA and a distinct increase in caspase-3 activity. The results, however, indicated that full caspase-3 inhibition by the selective inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone (Z-DEVD-FMK) did not prevent cell death, nor did it affect the manifestation of apoptotic hallmarks, including apoptotic bodies formation and nucleosomal DNA fragmentation. The only distinct effect that Z-DEVD-FMK exhibited was to retard the disruption of the plasma membrane. We therefore assume that caspase-3 activity itself is not essential for the manifestation of apoptotic features mentioned above. Similarly, the pan-specific caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD-FMK) did not prevent cell death. On the contrary, Z-VAD-FMK completely prevented DNA cleavage and apoptotic body formation, but it failed to completely counteract chromatin condensation. Thus, in the presence of Z-VAD-FMK, application of CCCP concentrations that otherwise induced apoptosis, resulted in the appearance of two morphologically different groups of dead cells with intact DNA. The first group included cells with necrotic-like nuclear morphology, and therefore could be taken as being “truly” necrotic in nature, because they had intact DNA. The cells of the second group formed small single-spherical nuclei with condensed chromatin. In spite of having intact DNA, they could not be taken as “truly” necrotic cells. It is evident that in the experimental system, caspase proteases play an essential role in the formation of apoptotic bodies and in the cleavage of nucleosomal DNA, but not in the condensation of chromatin. Therefore, it is likely that the choice between cell death modalities is not solely a matter of the caspase proteases present.

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Different Interleukin-1β Converting Enzyme (ICE) Family Protease Requirements for the Apoptotic Death of T Lymphocytes Triggered by Diverse Stimuli

Journal of Experimental Medicine ◽

10.1084/jem.184.6.2445 ◽

1996 ◽

Vol 184 (6) ◽

pp. 2445-2450 ◽

Cited By ~ 115

Author(s):

Apurva Sarin ◽

Ming-Lei Wu ◽

Pierre A. Henkart

Keyword(s):

T Cell ◽

T Lymphocytes ◽

Apoptotic Cell ◽

Cell Types ◽

Interleukin 1Β ◽

Converting Enzyme ◽

Apoptotic Death ◽

Cell Permeable Peptide ◽

Fluoromethyl Ketone

Two cell permeable peptide fluoromethyl ketone inhibitors of Interleukin-1β converting enzyme (ICE) family proteases were tested as inhibitors of apoptotic cell death of T lymphocytes at various stages of differentiation. The CPP-32–like protease activity in apoptotic cell lysates was blocked by both the ICE inhibitor Cbz-Val-Ala-Asp(OMe)-fluoromethyl ketone (ZVADFMK) as well as its truncated analog Boc-Asp(OMe)-fluoromethyl ketone (BD-FMK), which failed to block ICE. In vitro apoptotic death in murine thymocytes triggered by the independent agents dexamethasone, etoposide, radiation, anti-Fas, and anti-CD3 was blocked equally well by BD-FMK and ZVAD-FMK, but not by the control reagent Cbz-Phe-Ala-fluoromethyl ketone. In activated T cell blasts, while anti-CD3/ Fas-induced death was almost completely inhibited by both ZVAD-FMK and BD-FMK, death induced by dexamethasone, etoposide, or irradiation was more sensitive to inhibition by BD-FMK. In the murine T cell line CTLL-2, apoptotic death induced by IL-2 withdrawal, etoposide, or dexamethasone was inhibited by BD-FMK, while ZVAD-FMK was without effect. These data indicate that ICEfamily proteases comprise a common functional step in distinct T cell apoptotic death pathways, but suggest that different family members are likely to be critical in various differentiated T cell types, even when triggered by the same stimulus.

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Regiospecific preparation and allylation of fluoromethyl ketone enol ethers via fluoro-olefination of 1,2,3-triol derivatives

Tetrahedron Letters ◽

10.1016/s0040-4039(00)95392-9 ◽

1987 ◽

Vol 28 (15) ◽

pp. 1677-1680 ◽

Cited By ~ 5

Author(s):

Makoto Shimizu ◽

Yuko Nakahara ◽

Shigekazu Kanemoto ◽

Hirosuke Yoshioka

Keyword(s):

Enol Ethers ◽

Fluoromethyl Ketone

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Blockade of Caspases Inhibits Amebic Liver Abscess Formation in a Mouse Model of Disease

Infection and Immunity ◽

10.1128/iai.69.12.7911-7914.2001 ◽

2001 ◽

Vol 69 (12) ◽

pp. 7911-7914 ◽

Cited By ~ 34

Author(s):

Le Yan ◽

Samuel L. Stanley

Keyword(s):

Mouse Model ◽

Entamoeba Histolytica ◽

Liver Abscess ◽

Abscess Formation ◽

Control Group ◽

Amebic Liver Abscess ◽

Caspase Inhibitor ◽

The Mean ◽

Mouse Model Of Infection ◽

Fluoromethyl Ketone

ABSTRACT We looked at the effect of inhibiting caspases on amebic liver abscess in the mouse model of infection. A dose of the pan-caspase inhibitor benzyloxycarbonyl-V-A-D-O-methyl fluoromethyl ketone (Z-VAD-FMK; R & D Systems) given to SCID mice 2 h prior to direct hepatic inoculation with Entamoeba histolyticatrophozoites, and 12 h after amebic inoculation, reduced the mean liver abscess size by 70% at 24 h compared to a control group. These data indicate that apoptosis plays a significant but not an exclusive role in amebic liver abscess formation in the mouse model.

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Involvement of the ICE family of proteases in silica-induced apoptosis in human alveolar macrophages

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.273.4.l760 ◽

1997 ◽

Vol 273 (4) ◽

pp. L760-L767 ◽

Cited By ~ 7

Author(s):

Rashi Iyer ◽

Andrij Holian

Keyword(s):

Alveolar Macrophages ◽

Current Evidence ◽

Silica Dust ◽

Molecular Events ◽

Human Alveolar Macrophages ◽

Silica Treatment ◽

Induced Apoptosis ◽

Interleukin Converting Enzyme ◽

Fluoromethyl Ketone

Exposure to silica dust can result in lung inflammation that may progress to fibrosis for which there is no effective clinical treatment. The mechanisms involved in the development of pulmonary silicosis have not been well defined; however, most current evidence implicates a central role for alveolar macrophages in this process. We have previously demonstrated that fibrotic agents, such as asbestos and silica, induce apoptosis in human alveolar macrophages. The goal of this study was to identify molecular events in the silica-induced apoptotic process to better understand the mechanism by which fibrotic agents may be inducing apoptosis in human alveolar macrophages. To elucidate the possible mechanism by which silica causes apoptosis, we investigated the involvement of the interleukin-converting enzyme (ICE) family of proteases. Human alveolar macrophages were treated with silica in vitro and were examined for the involvement of ICE, Ich-1L, and cpp32β in silica-induced apoptosis. Pretreatment of cells with 10 μM of the ICE inhibitor z-Val-Ala-Asp-fluoromethyl ketone and the cpp32β inhibitor Asp-Glu-Val-Asp-fluoromethyl ketone completely blocked silica-induced apoptosis. Additionally, an increased formation of the active p20 fragments of ICE and Ich-1Las well as degradation of the inactive zymogen form of cpp32β protein were observed in silica-treated human alveolar macrophages, indicating activation of these proteases. Furthermore, degradation of the nuclear protein poly(ADP-ribose) polymerase was observed within 2 h of silica treatment. These results suggest that silica-induced apoptosis involves activation of the ICE family of proteases and is the first step in elucidating the intracellular mechanism of particulate-induced apoptosis in human alveolar macrophages.

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