Sequential Activation of Caspases and Serine Proteases (Serpases) During Apoptosis

Grabarek, Jerzy; Du, Litong; Johnson, Gary L.; Lee, Brian W.; David, Joel

doi:10.4161/cc.1.2.113

Cited by 29 publications

(31 citation statements)

References 44 publications

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“…As mentioned in the introductory section, these reagents have found utility as either markers of apoptotic cells or as probes of caspase activation (18 -26). In the early studies, we observed that cell pretreatment with the unlabeled inhibitors (e.g., z-VAD-FMK) at the time of induction of apoptosis prevented subsequent labeling with FLICA to a large degree (41). It was also observed that FLICA by itself was sufficient to arrest the progression of apoptosis (24).…”

Section: Discussionmentioning

confidence: 99%

“…However, when z-VAD-FMK was added along with the inducer of apoptosis, it prevented subsequent binding of FLICA (41), biotinylated inhibitor (14), or substrate cleavage (17). Of course, when z-VAD-FMK was added at the point of induction, all other events of apoptosis were prevented as well (14,41). Thus, it appears that only during the induction of apoptosis can the unlabeled inhibitor interact with the caspase binding sites, preventing subsequent binding of labeled inhibitors or cleavage of the substrate.…”

Section: Discussionmentioning

confidence: 99%

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Interactions of fluorochrome‐labeled caspase inhibitors with apoptotic cells: A caution in data interpretation

et al. 2003

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BackgroundFluorochrome‐labeled inhibitors of caspases (FLICA, e.g., FAM‐VAD‐FMK, FITC‐VAD‐FMK) have been designed as affinity labels of the enzyme active center of caspases Their binding by apoptotic cells was interpreted as reflecting activation of caspases. We have recently observed, however, that their binding is more complex and may involve additional mechanisms. Our goal in this study was to clarify the ongoing utility of these probes.MethodsApoptosis of HL‐60, Jurkat, MCF‐7 and T‐24 cells was induced by the DNA topoisomerase I inhibitor, topotecan, or by oxidative stress (H2O2). Lymphocytes were induced by their mitogenic activation. Using multiparameter laser scanning and flow cytometry analysis, the correlation between FLICA binding and the number of known apoptotic indicators was examined. These included: collapse of the mitochondrial transmembrane potential; activation of caspase‐3 (detected immunocytochemically); binding of annexin V; chromatin condensation; the presence of DNA strand breaks; and loss of plasma membrane capability to exclude propidium iodide (PI). FLICA binding specificity was tested by pretreatment with z‐VAD‐FMK or z‐DEVD‐FMK.ResultsFLICA binding was subsequent to the collapse of mitochondrial transmembrane potential, nearly concurrent with caspase‐3 activation, and preceded annexin V binding, chromatin condensation, DNA fragmentation and loss of plasma membrane integrity. The predominant portion of FAM‐VAD‐FMK, FITC‐VAD‐FMK or FAM‐DEVD‐FMK binding to apoptotic cells could not be inhibited by z‐VAD‐FMK or z‐DEVD‐FMK, respectively, when the unlabeled inhibitors were added post‐induction of apoptosis.ConclusionsFLICA are specific and convenient to use markers of apoptotic cells and they detect very early events of apoptosis associated with caspases activation. Assays that combine their binding with either the loss of mitochondrial potential or with exclusion of PI as a probe of plasma membrane integrity, distinguish sequential stages of apoptosis and are particularly useful to differentiate between apoptosis and necrosis. Our results conform with the published data that unlabeled caspase inhibitors, when added after induction of apoptosis, cannot prevent activation of caspases detected by binding of biotinylated inhibitors or by cleavage of fluorogenic substrates. While FLICA binding by apoptotic cells most likely is a consequence of caspase activation, these binding events may also involve other or additional mechanisms than simply their specific attachment to the active enzyme centers of caspases. Cytometry Part A 55A:50–60, 2003. © 2003 Wiley‐Liss, Inc.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Interactions of fluorochrome‐labeled caspase inhibitors with apoptotic cells: A caution in data interpretation

et al. 2003

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“…34 Specifically, activation of serine proteases may occur downstream from the activation of caspases during apoptosis. 35 However, the link between p53 and serine proteases may not be direct, considering the data showing that DNA damage upregulates serine proteases even in p53-null HL60 cells, human leukemia cells. 36 Interestingly, serine proteases on cell membranes are dysregulated during tumor growth and progression.…”

Section: Discussionmentioning

confidence: 99%

P53-dependent induction of serine proteases in irradiated mouse colon

Lin

Cook²,

Finnberg³

et al. 2004

Cancer Biology & Therapy

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Tumor suppressor p53 induces apoptosis through the transactivation of target genes. Previous work has shown that p53-dependent gene expression changes in response to ionizing radiation are tissue specific. To determine critical p53 target genes in the colon, we irradiated wild-type and p53-null mice and examined the global p53 gene expression patterns in response to ionizing radiation. Microarray analysis using the Affymetrix MOE430A genechip showed that many of the genes that increased in a p53-dependent manner are serine proteases (e.g., elastase, trypsin) and other proteases (e.g., carboxypeptidases). Reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blots were used to validate microarray results on trypsin 4, carboxypeptidase A1, and elastase-2, three genes that have potential p53-binding elements. Further study of tissue specific mediators of p53-dependent responses such as serine proteases will greatly increase understanding of in vivo p53-dependent pathways within the colon triggered by ionizing radiation.

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“…The cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics, as described previously (17)(18)(19). All media, supplements, and antibiotics were obtained from Life Technologies (Grand Island, NY).…”

Section: Materials and Methods Cellsmentioning

confidence: 99%

“…The DNA frequency histograms were deconvoluted with MultiCycle software (Phoenix Flow Systems, San Diego, CA). The green and red fluorescences of cells stained with F-CDV and PI was measured by flow cytometry or LSC (CompuCyte, Cambridge, MA) as described previously (17)(18)(19). Briefly, the green (F-CDV) and red (PI) fluorescence emissions were excited at 488 nm with an argon ion laser and measured by using the standard bandpass (530 Ϯ 20 nm) and long pass (Ͼ570 nm) filters.…”

Section: Fluorescence Measurementmentioning

confidence: 99%

Detection of in situ activation of transglutaminase during apoptosis: Correlation with the cell cycle phase by multiparameter flow and laser scanning cytometry

et al. 2002

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Background:One of the hallmarks of apoptosis is activation of tissue transglutaminase (Tgase; also called transglutaminase type 2 [TGase 2]). Its activation causes cross-linking of cytoplasmic proteins, making them insoluble and presumably less immunogenic. Several biochemical and cytochemical methods to detect activity of TGase 2 exist, but none has been adapted for multiparameter flow or image cytometry. Methods: Apoptosis of HL-60 or U-937 leukemic cells was induced by camptothecin, tumor necrosis factor ␣, hyperthermia, or the cytotoxic RNase onconase. Two different approaches to detect TGase 2 activation were developed: (a) the unfixed cells were treated with 4Ј,6Ј-diamidino-2-phenylindole, and sulforhodamine 101 in solutions of nonionic detergents; (b) the TGase 2 substrate fluoresceinated polyamine cadaverine (F-CDV) was administered into the cultures for several hours before cell harvesting. The cells were then fixed and their DNA counterstained with propidium. Cellular fluorescence was measured by flow or laser scanning cytometry. Results: (a) Exposure of nonapoptotic cells to detergents caused their full lysis, resulting in preparation of isolated nuclei devoid of cytoplasm. Conversely, the cross-linking of cytoplasmic protein by activated TGase 2 in apoptotic cells provided resistance to detergents: the nuclei or nuclear (chromatin) fragments of apoptotic cells remained

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Sequential Activation of Caspases and Serine Proteases (Serpases) During Apoptosis

Cited by 29 publications

References 44 publications

Interactions of fluorochrome‐labeled caspase inhibitors with apoptotic cells: A caution in data interpretation

Interactions of fluorochrome‐labeled caspase inhibitors with apoptotic cells: A caution in data interpretation

P53-dependent induction of serine proteases in irradiated mouse colon

Detection of in situ activation of transglutaminase during apoptosis: Correlation with the cell cycle phase by multiparameter flow and laser scanning cytometry

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