Dynamics of caspase-3 activation and inhibition in embryonic micromasses evaluated by a photon-counting chemiluminescence approach

Chlastáková, Ivana; Lišková, Marcela; Kudelová, Jarmila; Dubská, Lenka Zdražilová; Klepárnı́k, Karel; Matalová, Eva

doi:10.1007/s11626-012-9542-8

Cited by 12 publications

(8 citation statements)

References 18 publications

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“…The medium with different treatments was changed every 2 days. The effectiveness of the individual caspase inhibitors was verified with Caspase-Glo assays (Supplement 2), and the constant inhibition effect of the FMK inhibitors in cells was confirmed previously 38 RNA isolation, real-time PCR, PCR Arrays. Cells for RNA isolation were lysed in 350 µl of RLT buffer with β-mercaptoethanol.…”

Section: Methodsmentioning

confidence: 62%

Osteogenic impact of pro-apoptotic caspase inhibitors in MC3T3-E1 cells

Kratochvílová

Veselá

Ledvina

et al. 2020

Sci Rep

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Caspases are proteases traditionally associated with inflammation and cell death. Recently, they have also been shown to modulate cell proliferation and differentiation. The aim of the current research was to search for osteogenic molecules affected by caspase inhibition and to specify the individual caspases critical for these effects with a focus on proapoptotic caspases: caspase-2,-3,-6,-7,-8 and-9. Along with osteocalcin (Ocn), general caspase inhibition significantly decreased the expression of the Phex gene in differentiated MC3T3-E1 cells. The inhibition of individual caspases indicated that caspase-8 is a major contributor to the modification of Ocn and Phex expression. Caspase-2 and-6 had effects on Ocn and caspase-6 had an effect on Phex. These data confirm and expand the current knowledge about the nonapoptotic roles of caspases and the effect of their pharmacological inhibition on the osteogenic potential of osteoblastic cells. Caspases are proteases that are currently associated with inflammation and cell death. Their use has broad implications for pathological conditions, such as cancer and degenerative disorders. Caspase inhibitors have been tested in several therapeutic approaches 1. Additionally, a much broader spectrum of caspase functions has been demonstrated 2 , particularly proapoptotic caspases, including apical caspases-8 and-9, the executive trio of caspase-3,-6 and-7 and the still enigmatic caspase-2. New functions of caspases have also been reported in osteogenesis 3,4. Bmp4-induced differentiation of osteoblastic MC3T3-E1 cells leads to the activation of caspase-2,-3 and-8 without increasing the apoptosis rate 5. Pharmaceutical inhibition of caspases reduced Alp activity in MC3T3-E1 cells and the expression levels of osteocalcin, a molecule typically found in osteoblasts 4,5. Notably, osteocalcin is also used in medical diagnoses as a biochemical marker of bone formation and metabolic risk 6. Pharmacological caspase inhibitors are considered potential tools in several therapies 1. Previous works have focused on the alteration of gene expression during MC3T3-E1 cell differentiation 7,8 , which consists of several phases, including proliferation, differentiation and matrix deposition, accompanied by the production of specific osteogenic factors 9. Since these phases were first characterized 10 , hundreds of reports have been based on experiments performed using MC3T3-E1 cells. Given their intramembranous origin, MC3T3-E1 cells are recognized as suitable in vitro models for direct osteoblastic differentiation and pleiotropic studies 11. Based on published results and our preliminary data, we hypothesized that proapoptotic caspases impact gene expression in differentiated MC3T3-E1 cells. The following investigation addresses the effects of caspase blockade on gene expression in differentiated cells. For the first time, a cell bioluminescence-based approach was used to record the activation of individual proapoptotic caspases during MC3T3-E1 cell cultivation. Therefore, the osteogenic profi...

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Section: Methodsmentioning

confidence: 62%

Osteogenic impact of pro-apoptotic caspase inhibitors in MC3T3-E1 cells

Kratochvílová

Veselá

Ledvina

et al. 2020

Sci Rep

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“…Micromass cultures (primary mesenchymal cell cultures) were prepared from E12 mouse forelimbs (as, for example, in Mello and Tuan, 15 and Chlastakova et al 16 ). The forelimbs were disintegrated mechanically in Puck's saline A buffer (PSA; 0.4 g of KCl/l, 8 g of NaCl/l, 0.35 g of NaHCO 3 /l, 1 g of glucose/l) with 10% fetal bovine serum (FBS; Invitrogen, Karlsruhe, Germany, 9:1) and enzymatically in 10 U/ml of Dispase II (Sigma-Aldrich; Darmstadt, Germany) in 10% FBS/PSA at 37C, 1-1.5 hr, 1000 rpm, and vortexed every 15 min.…”

Section: Micromass Culturesmentioning

confidence: 99%

Osteogenic Potential of Caspases Related to Endochondral Ossification

Janečková

Bíliková

Matalová

2017

J Histochem Cytochem.

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Caspases have functions particularly in apoptosis and inflammation. Increasing evidence indicates novel roles of these proteases in cell differentiation, including those involved in osteogenesis. This investigation provides a complex screening of osteogenic markers affected by pan caspase inhibition in micromass cultures derived from mouse forelimbs. PCR Array analysis showed significant alterations in expression of 49 osteogenic genes after 7 days of inhibition. The largest change was a decrease in CD36 expression, which was confirmed at organ level by caspase inhibition in cultured mouse ulnae followed by CD36 immunohistochemical analysis. So far, available data point to osteogenic potential of pro-apoptotic caspases. Therefore, the expression of pro-apoptotic caspases (-3, -6, -7, -8, -9) within the growth plate of mouse forelimbs at the stage where the individual zones are clearly apparent was studied. Caspase-9 was reported in the growth plate for the first time as well as caspase-6 and -7 in the resting zone, caspase-7 in the proliferation, and caspase-6 and -8 in the ossification zone. For all caspases, there was a gradient increase in activation toward the ossification zone. The distribution of staining varied significantly from that of apoptotic cells, and thus, the results further support non-apoptotic participation of caspases in osteogenesis.

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“…For the experimentally induced approach, camptothecin (90%, from Sigma‐Aldrich) was used for targeted caspase‐3 activation. Camptothecin was dissolved directly in micromass medium at the concentration of 5 μM and applied as described in detail in Chlastakova et al . The cells for caspase measurement were collected in the peak as determined earlier .…”

Section: Methodsmentioning

confidence: 99%

“…Camptothecin was dissolved directly in micromass medium at the concentration of 5 μM and applied as described in detail in Chlastakova et al . The cells for caspase measurement were collected in the peak as determined earlier . Cells from digital regions of the limbs and untreated cells cultured under the same conditions, respectively, were engaged as the negative controls.…”

Section: Methodsmentioning

confidence: 99%

“…The most sensitive method will be optimized and used for the detection and quantification of activated caspases at the level of a single cell. It has already been proved that single cell analyses can reveal the apoptotic pathways more accurately than analyses of cellular extracts, which average the heterogeneity of a cell population . As a result, the developed instrumentation and methodology can potentially become a valuable tool for research in biology and medicine.…”

Section: Introductionmentioning

confidence: 99%

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Bioluminescence determination of active caspase‐3 in single apoptotic cells

et al. 2013

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Caspase-3 is an executive caspase, in the central position within apoptotic machinery. Apoptosis as a way of programmed cell death is a physiological process that plays an essential role in the development and homeostasis maintenance; moreover, its deregulations are linked to tumor progression or various autoimmune disorders. Therefore, an investigation of apoptosis pathways on the level of individual cells is not only of biological but also medical importance. In this work we report on the development of a high-sensitivity instrumentation and protocol for detection of active caspase-3 in individual mammalian apoptotic cells. The technology is based on the specific cleavage of modified luciferin by caspase-3, an immediate bioluminescence reaction of free luciferin with luciferase followed by emissions of photons and their detection by photomultiplier tube working in the photon counting regime. Three different instrumental arrangements are compared for the determination of caspase-3 in free cells or tissue samples. Thus, in our best miniaturized system the mean amount as low as about 6.5 fg corresponding to 122 000 molecules of caspase-3 can be detected in individual apoptotic mouse leg cells.

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Dynamics of caspase-3 activation and inhibition in embryonic micromasses evaluated by a photon-counting chemiluminescence approach

Cited by 12 publications

References 18 publications

Osteogenic impact of pro-apoptotic caspase inhibitors in MC3T3-E1 cells

Osteogenic impact of pro-apoptotic caspase inhibitors in MC3T3-E1 cells

Osteogenic Potential of Caspases Related to Endochondral Ossification

Bioluminescence determination of active caspase‐3 in single apoptotic cells

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