Bcl-2 homology domain (BH) 3-only proteins couple stress signals to evolutionarily conserved mitochondrial apoptotic pathways. Caspase 8-mediated cleavage of the BH3-only protein Bid into a truncated protein (tBid) and subsequent translocation of tBid to mitochondria has been implicated in death receptor signaling. We utilized a recombinant fluorescence resonance energy transfer (FRET) Bid probe to determine the kinetics of Bid cleavage and tBid translocation during death receptor-induced apoptosis in caspase 3-deficient MCF-7 cells. Cells treated with tumor necrosis factor-␣ (200 ng/ml) showed a rapid cleavage of the Bid-FRET probe occurring 75.4 ؎ 12.6 min after onset of the tumor necrosis factor-␣ exposure. Cleavage of the Bid-FRET probe coincided with a translocation of tBid to the mitochondria and a collapse of the mitochondrial membrane potential (⌬⌿m). We next investigated the role of Bid cleavage in a model of caspase-independent, glutamate-induced excitotoxic apoptosis. Rat cerebellar granule neurons were transfected with the Bid-FRET probe and exposed to glutamate for 5 min. In contrast to death receptor-induced apoptosis, neurons showed a translocation of full-length Bid to the mitochondria. This translocation occurred 5.6 ؎ 1.7 h after the termination of the glutamate exposure and was also paralleled with a collapse of the ⌬⌿m. Proteolytic cleavage of the FRET probe also occurred, however, only 25.2 ؎ 3.5 min after its translocation to the mitochondria. Subfractionation experiments confirmed a translocation of full-length Bid from the cytosolic to the mitochondrial fraction during excitotoxic apoptosis. Our data demonstrate that both tBid and full-length Bid have the capacity to translocate to mitochondria during apoptosis.The Bcl-2-homology domain-3 (BH3) 2 -only proteins are a subfamily of the Bcl-2 protein family involved in the initiation of apoptosis through the mitochondrial pathway (1, 2). The key event in the mitochondrial pathway is the release of proapoptotic factors from the mitochondrial intermembrane space into the cytosol (3) resulting in the downstream activation of a family of cytosolic cysteine proteases, caspases, that are required for many of the morphological changes that occur during apoptosis. The mitochondrial release of cytochrome-c and Smac/DIABLO allows for the formation of the apoptosome (4 -6), a complex that enables the activation of caspases within the cell.In many apoptotic models the proapoptotic Bcl-2 family members Bax or Bak are required for the release of caspase-activating factors from mitochondria (7). Both proteins are believed to form pores that make the outer mitochondrial membrane sufficiently permeable for the release of these intermembrane proteins (8). For this to occur, Bax and Bak must undergo conformational changes and insert into the outer mitochondrial membrane (9, 10). BH3-only proteins promote the activation of Bax and Bak either by direct protein association or through antagonizing the function of anti-apoptotic Bcl-2 family members (11).The BH3-on...

Colorectal cancer (CRC) is one of the most common cancers in the Western world. 5-Fluorouracil (5FU)-based chemotherapy (CT) remains the mainstay treatment of CRC in the advanced setting, and activates executioner caspases in target cells. Executioner caspases are key proteins involved in cell disassembly during apoptosis. Activation of executioner caspases also has a role in tissue regeneration and repopulation by stimulating signal transduction and cell proliferation in neighbouring, non-apoptotic cells as reported recently. Tissue microarrays (TMAs) consisting of tumour tissue from 93 stage II and III colon cancer patients were analysed by immunohistochemistry. Surprisingly, patients with low levels of active Caspase-3 had an increased disease-free survival time. This was particularly pronounced in patients who received 5FU-based adjuvant CT. In line with this observation, lower serum levels of active Caspase-3 were found in patients with metastasised CRC who revealed stable disease or tumour regression compared with those with disease progression. The role of Caspase-3 in treatment responses was explored further in primary human tumour explant cultures from fresh patient tumour tissue. Exposure of explant cultures to 5FU-based CT increased the percentage of cells positive for active Caspase-3 and Terminal Deoxynucleotidyl Transferase dUTP Nick end Labelling (TUNEL), but also the expression of regeneration and proliferation markers β-Catenin and Ki-67, as well as cyclooxygenase-2 (COX-2). Of note, selective inhibition of Caspase-3 with Ac-DNLD-CHO, a selective, reversible inhibitor of Caspase-3, significantly reduced the expression of proliferation markers as well as COX-2. Inhibition of COX-2 with aspirin or celecoxib did not affect Caspase-3 levels but also reduced Ki-67 and β-Catenin levels, suggesting that Caspase-3 acted via COX-2 to stimulate cell proliferation and tissue regeneration. This indicates that low levels of active Caspase-3 may represent a new predictor of CT responsiveness, and inhibition of Caspase-3, or antagonising downstream effectors of Caspase-3 paracrine signalling, such as COX-2 may improve patient outcomes following CT in advanced CRC.

Mitochondria are pivotal for cellular bioenergetics, but are also a core component of the cell death machinery. Hypothesis-driven research approaches have greatly advanced our understanding of the role of mitochondria in cell death and cell survival, but traditionally focus on a single gene or specific signalling pathway at a time. Predictions originating from these approaches become limited when signalling pathways show increased complexity and invariably include redundancies, feedback loops, anisotropies or compartmentalisation. By introducing methods from theoretical chemistry, control theory, and biophysics, computational models have provided new quantitative insights into cell decision processes and have led to an increased understanding of the key regulatory principles of apoptosis. In this review, we describe the currently applied modelling approaches, discuss the suitability of different modelling techniques, and evaluate their contribution to the understanding of the mitochondrial apoptosis pathway. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.

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Abstract-Fluorescence microscopy imaging is a constant trade off between signal to noise ratio, total observation time and spatio-temporal resolution due to photo toxicity. In this paper, we propose a method to estimate the quality of a fluorescent image acquisition, from a single image, taking into account both signal dependent and signal independent noise. We propose a method for the calculation of the signal to noise ratio globally and locally. We validated our algorithm on real experimental data and data with known simulated noise. Results allow us to conclude that this fully automatic method provides a good quantification of the image quality., where 8 is the signal corrected for background, V is the estimated noise variance, and A, P and M are the variances of the three noise components considered: additive, Poisson and multiplicative noise, respectively. In [4], the estimation of A was considered inaccurate and moreover bad results were obtained when the background value was O. In addition, the parameters fitting was done on times series of 128 images.In general, noise estimation methods follow two different approaches. The first is the smoothing based approach, where the noise is estimated by the difference between the original