Evolution Of Cell Death Research Articles

Electroporation Parameters for Human Cardiomyocyte Ablation In Vitro.

Cardiac ablation with irreversible electroporation (IRE) is quickly being established as a modality of choice for atrial fibrillation treatment. While it has not yet been optimised, IRE has the potential to significantly limit collateral damage and improve cell-specific targeting associated with other energy sources. However, more tissue and cell-specific evidence is required to demonstrate the selective threshold parameters for human cells. The aim here is to determine the optimal ablation threshold parameters related to lesion size for human cardiomyocytes in 2D culture. Conventional biphasic pulses of different field strengths and on-times were delivered in a monolayer culture system of human AC16 cardiomyocytes. The dynamics of cell death and lesion dimensions were examined at different time points. Human cardiomyocytes are susceptible to significant electroporation and cell death at a field strength of 750 V/cm or higher with 100 μs pulses. Increasing the IRE on-time from 3 ms to 60 ms reduces the effective field threshold to 250 V/cm. Using very short pulses of 2 μs and 5 μs also causes significant cell death, but only at fields higher than 1000 V/cm. A longer on-time results in more cell death and induced greater lesion area in 2D models. In addition, different forms of cell death are predicted based on the evolution of cell death over time. This study presents important findings on the ability of different IRE parameters to induce human cardiomyocyte cell death. Lesion size can be tuned by appropriate choice of IRE parameters and cardiomyocytes display an upregulation of delayed cell death 24 h after electroporation, which is an important consideration for clinical practice.

Transcriptional and epigenetic control of regulated cell death in yeast.

Unicellular organisms like yeast can undergo controlled demise in a manner that is partly reminiscent of mammalian cell death. This is true at the levels of both mechanistic and functional conservation. Yeast offers the combination of unparalleled genetic amenability and a comparatively simple biology to understand both the regulation and evolution of cell death. In this minireview, we address the capacity of the nucleus as a regulatory hub during yeast regulated cell death (RCD), which is becoming an increasingly central question in yeast RCD research. In particular, we explore and critically discuss the available data on stressors and signals that specifically impinge on the nucleus. Moreover, we also analyze the current knowledge on nuclear factors as well as on transcriptional control and epigenetic events that orchestrate yeast RCD. Altogether we conclude that the functional significance of the nucleus for yeast RCD in undisputable, but that further exploration beyond correlative work is necessary to disentangle the role of nuclear events in the regulatory network.

Abstract 5337: An in silico platform for characterizing ADC bystander effects

Abstract Introduction The “bystander effect” is the hypothesized process by which neighboring cancer cells are killed when free drug is released into the tumor environment by dying cells. The extent to which an antibody drug conjugate (ADC) elicits the bystander effect can have significant impact on the magnitude and dynamics of efficacy. Understanding the bystander effect though experimental methods (e.g. video-microscopy) are challenging; however, when supplemented with in silico modeling, mechanistic details can be elucidated. The purpose of this work is to develop a spatial-temporal in silico platform describing tumor cell progression in the presence of an ADC in order to quantify the bystander effect. Methods The model employs spatial-stochastic methods to simulate the time evolution of cell death resulting from ADCs and freely diffusing payload based on following parameters: drug to antibody ratio (DAR), linker stability, antigen concentration, ADC cell membrane permeability, ADC-antigen internalization, payload permeability, and payload potency. Specifically, a 2-D rectangular grid of tumor cells, ADCs, and payload are numerically implemented through binary pixel occupation of the grid. Governing rules are then assigned for: mobility for freely diffusing ADCs and payload, reaction rate for ADC binding to antigen-expressing cells, rate of cell death dependent on payload concentration, and internalization rate of payload into non-antigen expressing cells. The simulation is run until stopping criteria are met, e.g. all cells are killed. Baseline parameters can be optimized by fitting the model to in vitro experimental video-microscopy data. Results Experimental conditions were simulated with the model to characterize the influence of the aforementioned ADC and biological parameters on the bystander effect. Conclusions By incorporating the spatial aspect of ADC mechanics, spatially dependent metrics can be formulated to quantify the bystander effect. The model allows for wide adjustability of the ADC design parameters as well as relevant biological parameters. Thus, by understanding the relative parameter sensitivities, the model can explore design optimization of ADCs that maximize the bystander effect. Through such optimization, the magnitude and dynamics of drug exposure at the site of action can be controlled to maximize efficacy and improve the therapeutic window. Citation Format: Jackson Burton, Shu-Wen Teng, Christopher J. Zopf, Ryan Nolan, Arijit Chakravarty. An in silico platform for characterizing ADC bystander effects. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5337. doi:10.1158/1538-7445.AM2015-5337

Multiple Modes of Cell Death Discovered in a Prokaryotic (Cyanobacterial) Endosymbiont

Programmed cell death (PCD) is a genetically-based cell death mechanism with vital roles in eukaryotes. Although there is limited consensus on similar death mode programs in prokaryotes, emerging evidence suggest that PCD events are operative. Here we present cell death events in a cyanobacterium living endophytically in the fern Azolla microphylla, suggestive of PCD. This symbiosis is characterized by some unique traits such as a synchronized development, a vertical transfer of the cyanobacterium between plant generations, and a highly eroding cyanobacterial genome. A combination of methods was used to identify cell death modes in the cyanobacterium. Light- and electron microscopy analyses showed that the proportion of cells undergoing cell death peaked at 53.6% (average 20%) of the total cell population, depending on the cell type and host developmental stage. Biochemical markers used for early and late programmed cell death events related to apoptosis (Annexin V-EGFP and TUNEL staining assays), together with visualization of cytoskeleton alterations (FITC-phalloidin staining), showed that all cyanobacterial cell categories were affected by cell death. Transmission electron microscopy revealed four modes of cell death: apoptotic-like, autophagic-like, necrotic-like and autolytic-like. Abiotic stresses further enhanced cell death in a dose and time dependent manner. The data also suggest that dynamic changes in the peptidoglycan cell wall layer and in the cytoskeleton distribution patterns may act as markers for the various cell death modes. The presence of a metacaspase homolog (domain p20) further suggests that the death modes are genetically programmed. It is therefore concluded that multiple, likely genetically programmed, cell death modes exist in cyanobacteria, a finding that may be connected with the evolution of cell death in the plant kingdom.

When is a cell not a cell? The evolution of cell death as a developmental strategy

When is a cell not a cell? The evolution of cell death as a developmental strategy

Choice, methodology, and characterization of focal ischemic stroke models: the search for clinical relevance.

To develop novel neuroprotective or neurorestorative agents for clinical application, the appropriate selection and characterization of preclinical focal stroke models is required to provide confidence in predicting therapeutic efficacy. Compelling evidence for novel therapies derived from the pathological and functional consequences of models of cerebral ischemia in the rat (and higher species) is an essential prerequisite before large expensive clinical trials are begun. This chapter provides an overview of focal ischemic models, with an emphasis on objective functional assessment of pathological mechanisms and efficacy of novel therapeutic strategies. The ability to predict functional consequences from structural abnormalities is a critical theme that can be extrapolated from the preclinical to the clinical setting, in that certain brain regions are inextricably linked to specific behavioral functions. This underlying approach is highly relevant, as monitoring the dynamic pathological and functional changes attributed to focal stroke will reveal new insights into novel mechanisms and targets that play a role in the evolution of cell death and impaired function. The utility of novel genomic technologies that are aligned with methods to determine structure-function relationships in preclinical models will facilitate a greater understanding of the pathophysiological process and potentially generate new targets that may ultimately be used to predict or offer clinical benefit.

Reduction of DNA Fragmentation and Hydroxyl Radical Production by Hyaluronic Acid and Chondroitin-4-sulphate in Iron Plus Ascorbate-induced Oxidative Stress in Fibroblast Cultures

Glycosaminoglycans (GAGs), components of extracellular matrix, are thought to play important roles in cell proliferation and differentiation in the repair process of injured tissue. Oxidative stress is one of the most frequent causes of tissue and cell injury and the consequent lipid peroxidation is the main manifestation of free radical damage. It has been found to play an important role in the evolution of cell death. Since several reports have shown that hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S) are able to inhibit lipid peroxidation during oxidative stress, We investigated the antioxidant capacity of these GAGs in reducing oxidative damage in fibroblast cultures.Free radicals production was induced by the oxidizing system employing iron (Fe2+) plus ascorbate. We evaluated cell death, membrane lipid peroxidation, DNA damage, protein oxidation, hydroxyl radical (OH•) generation and endogenous antioxidant depletion in human skin fibroblast cultures.The exposition of fibroblasts to FeSO4 and ascorbate caused inhibition of cell growth and cell death, increased OH• production determined by the aromatic trap method; furthermore it caused DNA strand breaks and protein oxidation as shown by the DNA fragments analysis and protein carbonyl content, respectively. Moreover, it enhanced lipid peroxidation evaluated by the analysis of conjugated dienes (CD) and decreased antioxidant defenses assayed by means of measurement of superoxide dismutase (SOD) and catalase (CAT) activities.When fibroblasts were treated with two different doses of HYA or C4S a protective effect, following oxidative stress induction, was shown. In fact these GAGs were able to limit cell death, reduced DNA fragmentation and protein oxidation, decreased OH• generation, inhibited lipid peroxidation and improved antioxidant defenses.Our results confirm the antioxidant activity of HYA and C4S and this could represent a useful step in the understanding of the exact role played by GAGs in living organisms.

Immunohistochemical and biochemical assessment of caspase-3 activation and DNA fragmentation following transient focal ischemia in the rat

In the present study, we evaluated the time-course of caspase-3 activation, and the evolution of cell death following focal cerebral ischemia produced by transient middle cerebral artery occlusion in rats. Ischemia-induced active caspase-3 immunoreactivity in the striatum but not the cortex at 3 and 6 h time points post-reperfusion. Furthermore, using a novel approach to visualize enzymatic activity, ΔC-APP, a C-terminal cleavage product of APP generated by caspase-3, was found to immunolocalize to the same areas as active caspase-3. Double-labeling studies demonstrated co-localization of these two proteins at the cellular level. Further double-labeling experiments revealed that active caspase-3 was confined to neuronal cells which were still viable and thus immunoreactive for NeuN. DNA fragmentation, assessed histologically by terminal dUTP nick-end labeling (TUNEL), was observed in a small number of cells in the striatum as early as 3 h, but only began to appear in the cortex by 6 h. DNA fragmentation was progressive, and by 24 h post-reperfusion, large portions of both the striatum and cortex showed TUNEL positive cells. However, double-labeling of active caspase-3 with TUNEL showed only minimal co-localization at all time-points. Thus, caspase-3 activation is an event that appears to occur prior to DNA fragmentation. As a confirmation of the histological TUNEL data, 24 h ischemia also induced the generation of nucleosome fragments, evidenced by cell death enzyme-linked immunosorbent assay. Using a novel ischemia-induced substrate cleavage biochemical approach, spectrin P120 fragment, a caspase-specific cleavage product of alpha II spectrin, a cytoskeletal protein, was shown to be elevated by western blotting. Brain concentrations of both nucleosomes and spectrin P120 correlate with the degree of injury previously assessed by triphenyltetrazolium chloride staining and infarct volume calculation. Together, our findings suggest a possible association between caspase-3 activation and ischemic cell death following middle cerebral artery occlusion brain injury.