Individual cells within a population undergo apoptosis at distinct, apparently random time points. By analyzing cellular mitotic history, we identified that sibling HeLa cell pairs, in contrast to random cell pairs, underwent apoptosis synchronously. This allowed us to use high-speed cellular imaging to investigate mitochondrial outer membrane permeabilization (MOMP), a highly coordinated, rapid process during apoptosis, at a temporal resolution approximately 100 times higher than possible previously. We obtained new functional and mechanistic insight into the process of MOMP: We were able to determine the kinetics of pore formation in the outer mitochondrial membrane from the initiation phase of cytochrome-c-GFP redistribution, and showed differential pore formation kinetics in response to intrinsic or extrinsic apoptotic stimuli (staurosporine, tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL)). We also detected that the onset of mitochondrial permeabilization frequently proceeded as a wave through the cytosol, and that the frequency of wave occurrence in response to TRAIL was reduced by inhibition of protein kinase CK2. Computational analysis by a partial differential equation model suggested that the spread of permeabilization signals could sufficiently be explained by diffusion-adsorption velocities of locally generated permeabilization inducers. Taken together, our study yielded the first comprehensive analysis of clonal cell-to-cell variability in apoptosis execution and allowed to visualize and explain the dynamics of MOMP in cells undergoing apoptosis. Apoptosis is an evolutionary conserved cell death process that is fundamental to remove superfluous and damaged cells from the bodies of multicellular organisms. Impaired or excessive apoptosis has repeatedly been implicated as a major contributor to proliferative and degenerative diseases. Initiated by BH-3-only proteins of the Bcl-2 protein family, mitochondrial outer membrane permeabilization (MOMP) and the subsequent activation of effector caspases are rapid key processes crucial for the efficient execution of apoptotic cell death.Previous single-cell imaging studies using fluorescently tagged proteins showed that MOMP results in the coordinate release of mitochondrial intermembrane space proteins, including cytochrome-c (cyt-c) and Smac/Diablo, into the cytosol. The release was suggested to occur synchronously throughout the cytosol and with very fast kinetics, which seemed independent of the type of the apoptotic stimulus used, and also independent of downstream effector caspase activity. [1][2][3][4][5] Cytosolic cyt-c induces the rapid formation of the large multiprotein apoptosome complex, caspase-9 activation and subsequent activation of effector caspases-3 and -7. 6,7 In parallel, cytosolic Smac neutralizes x-linked inhibitor of apoptosis protein, the key inhibitor of caspases-9, -3 and -7. 8 As long as all key components of the execution network are present, effector caspase activation proceeds as a rapid and kinetically largely i...

TRAIL and agonistic antibodies raised against TRAIL death receptors are highly promising new anticancer agents. In this brief review, we describe the recent advances in the molecular understanding of TRAIL signaling and the progress made in using TRAIL or agonistic antibodies clinically in mono-and combination therapies. Synergies have been reported in various scenarios of TRAIL-based multidrug treatments, and these can be used to potentiate the efficacy of therapies targeting TRAIL death receptors. We pay particular attention to structure the current knowledge on the diverse molecular mechanisms that are thought to give rise to these synergies and describe how different signaling features evoking synergies can be associated with distinct classes of drugs used in TRAIL-based combination treatments. Mol Cancer Ther; 11(1); 3-13. Ó2012 AACR.

BackgroundGlioblastoma multiforme is the most common lethal brain tumor in human adults, with no major therapeutic breakthroughs in recent decades. Research is based mostly on human tumor cell lines deprived of their organotypic environment or inserted into immune-deficient animals required for graft survival. Here, we describe how glioblastoma specimens obtained from surgical biopsy material can be sectioned and transferred into cultures within minutes.MethodsSlices were kept in 6-well plates, allowing direct observation, application of temozolomide, and irradiation. At the end of experiments, slice cultures were processed for histological analysis including hematoxylin-eosin staining, detection of proliferation (Ki67), apoptosis/cell death (cleaved caspase 3, propidium iodide), DNA double-strand breaks (γH2AX), and neural subpopulations. First clinical trials employed irradiation with the heavy ion carbon for the treatment of glioblastoma patients, but the biological effects and most effective dose regimens remain to be established. Therefore, we developed an approach to expose glioblastoma slice cultures to 12C and X-rays.ResultsWe found preservation of the individual histopathology over at least 16 days. Treatments resulted in activation of caspase 3, inhibition of proliferation, and cell loss. Irradiation induced γH2AX. In line with clinical observations, individual tumors differed significantly in their susceptibility to temozolomide (0.4%–2.5% apoptosis and 1%–15% cell loss).ConclusionGlioblastoma multiforme slice cultures provide a unique tool to explore susceptibility of individual tumors for specific therapies including heavy ions, thus potentially allowing more personalized treatments plus exploration of mechanisms of (and strategies to overcome) tumor resistance.