All that glitters is not gold: All that FLICA binds is not caspase. A caution in data interpretation—and new opportunities

Darzynkiewicz, Zbigniew; Pożarowski, Piotr

doi:10.1002/cyto.a.20425

Cited by 22 publications

(26 citation statements)

References 10 publications

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“…Our data suggests a potential low-level activation of the NLRP3 inflammasome by mtDNA in ARPE-19 cells as evidenced by secretion of low levels of mature IL-1β to the culture medium and increased FAM-YVAD-FMK signal. It should be noted however that concerns about the specificity of the latter probe have been raised 82 . More importantly, because of differences and serious deficiencies in AMD experimental models, there is still no consensus as to whether NLRP3 activity is detrimental or protective in the first place.…”

Section: Discussionmentioning

confidence: 99%

Mitochondrial DNA has a pro-inflammatory role in AMD

Dib

Lin

Maidana

et al. 2015

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly of industrialized nations, and there is increasing evidence to support a role for chronic inflammation in its pathogenesis. Mitochondrial DNA (mtDNA) has been recently reported to be pro-inflammatory in various diseases such as Alzheimer’s and heart failure. Here, we report that intracellular mtDNA induces ARPE-19 cells to secrete IL-6 and IL-8, inflammatory cytokines that have consistently been associated with AMD onset and progression. The induction was dependent on the size of mtDNA, but not on specific sequence. Oxidative stress plays a major role in the development of AMD, and our findings indicate that mtDNA induces IL-6 and IL-8 more potently when oxidized. Cytokine induction was mediated by STING (Stimulator of Interferon Genes) and NF-κB as evidenced by abrogation of the cytokine response with use of specific inhibitors (siRNA and Bay 11–7082, respectively). Finally, mtDNA primed the NLRP3 inflammasome and weakly activated it as shown by caspase-1 activation and mature IL-1β secretion. This study contributes to our understanding of the potential pro-inflammatory role of mtDNA in the pathogenesis of AMD.

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Section: Discussionmentioning

confidence: 99%

Mitochondrial DNA has a pro-inflammatory role in AMD

Dib

Lin

Maidana

et al. 2015

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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“…Recently published data suggest also their superior applicability in a plethora of multiparametric applications. Nevertheless, in light of recent reports one should be aware that staining with FLICA apparently does not represent affinity labeling of individual caspase active centers (Darzynkiewicz and Pozarowski, 2007; Pozarowski et al , 2003). …”

Section: Cytometric Methods To Detect Apoptosismentioning

confidence: 92%

“…As FLICA reagents withstand fixation, this strongly suggests covalent interactions with the intracellular targets becoming accessible in the course of apoptosis (Darzynkiewicz and Pozarowski, 2007; Kuzelova et al , 2007; Pozarowski et al , 2003). The reactivity of FMK moiety with intracellular thiols may provide some explanation of these interactions.…”

Section: Cytometric Methods To Detect Apoptosismentioning

confidence: 99%

“…The reactivity of FMK moiety with intracellular thiols may provide some explanation of these interactions. In this context, opening of the disulfide cysteine bridges (inter- and/or intramolecular) may provide as yet unidentified affinity sites (Darzynkiewicz and Pozarowski, 2007). Noncovalent hydrophobic interactions between fluorochrome domain and intracellular targets have also been postulated to play a role in FLICA retention (Pozarowski et al , 2003).…”

Section: Cytometric Methods To Detect Apoptosismentioning

confidence: 99%

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Apoptosis and Beyond: Cytometry in Studies of Programmed Cell Death

Wlodkowic

Telford

Skommer

et al. 2011

Methods in Cell Biology

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385

340

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A cell undergoing apoptosis demonstrates multitude of characteristic morphological and biochemical features, which vary depending on the inducer of apoptosis, cell type and the “time window” at which the process of apoptosis is observed. Because the gross majority of apoptotic hallmarks can be revealed by flow and image cytometry, the cytometric methods become a technology of choice in diverse studies of cellular demise. Variety of cytometric methods designed to identify apoptotic cells, detect particular events of apoptosis and probe mechanisms associated with this mode of cell death have been developed during the past two decades. In the present review, we outline commonly used methods that are based on the assessment of mitochondrial transmembrane potential, activation of caspases, DNA fragmentation, and plasma membrane alterations. We also present novel developments in the field such as the use of cyanine SYTO and TO-PRO family of probes. Strategies of selecting the optimal multiparameter approaches, as well as potential difficulties in the experimental procedures, are thoroughly summarized.

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“…The increase in fluorescence at late stages of apoptosis in chick cochlea may reflect the involvement of lysosomal proteases in cell death [43]. This cross-reactivity could also explain the results of studies that show that FLICA signal in flow cytometry could not be decreased by pre-treating cells with concentrations of untagged Z-DEVD-FMK or Z-VAD-FMK sufficient to block caspase activity [44, 45]. Interestingly, there have been no reported biochemical data regarding the selectivity of the FLICA reagents when used in cells.…”

Section: Applications Of Substrate- and Activity-based Probesmentioning

confidence: 99%

Functional imaging of proteases: recent advances in the design and application of substrate-based and activity-based probes

Edgington

Verdoes

Bogyo

2011

Current Opinion in Chemical Biology

155

184

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Proteases are enzymes that cleave peptide bonds in protein substrates. This process can be important for regulated turnover of a target protein but it can also produce protein fragments that then perform other functions. Because the last few decades of protease research have confirmed that proteolysis is an essential regulatory process in both normal physiology and in multiple disease-associated conditions, there has been an increasing interest in developing methods to image protease activity. Proteases are also considered to be one of the few druggable classes of proteins and therefore a large number of small molecule based inhibitors of proteases have been reported. These compounds serve as a starting point for the design of probes that can be used to target active proteases for imaging applications. Currently, several classes of fluorescent probes have been developed to visualize protease activity in live cells and even whole organisms. The two primary classes of protease probes make use of either peptide/protein substrates or covalent inhibitors that produce a fluorescent signal when bound to an active protease target. This review outlines some of the most recent advances in the design of imaging probes for proteases. In particular, it highlights the strengths and weaknesses of both substrate- and activity- based probes and their applications for imaging cysteine proteases that are important biomarkers for multiple human diseases.

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All that glitters is not gold: All that FLICA binds is not caspase. A caution in data interpretation—and new opportunities

Cited by 22 publications

References 10 publications

Mitochondrial DNA has a pro-inflammatory role in AMD

Mitochondrial DNA has a pro-inflammatory role in AMD

Apoptosis and Beyond: Cytometry in Studies of Programmed Cell Death

Functional imaging of proteases: recent advances in the design and application of substrate-based and activity-based probes

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