Epigenetics and cell death: DNA hypermethylation in programmed retinal cell death.

BackgroundVertebrate genomes undergo epigenetic reprogramming during development and disease. Emerging evidence suggests that DNA methylation plays a key role in cell fate determination in the retina. Despite extensive studies of the programmed cell death that occurs during retinal development and degeneration, little is known about how DNA methylation might regulate neuronal cell death in the retina.MethodsThe developing chicken retina and the rd1 and rhodopsin-GFP mouse models of retinal degeneration were used to investigate programmed cell death during retinal development and degeneration. Changes in DNA methylation were determined by immunohistochemistry using antibodies against 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC).ResultsPunctate patterns of hypermethylation paralleled patterns of caspase3-dependent apoptotic cell death previously reported to occur during development in the chicken retina. Degenerating rd1 mouse retinas, at time points corresponding to the peak of rod cell death, showed elevated signals for 5mC and 5hmC in photoreceptors throughout the retina, with the most intense staining observed in the peripheral retina. Hypermethylation of photoreceptors in rd1 mice was associated with TUNEL and PAR staining and appeared to be cCaspase3-independent. After peak rod degeneration, during the period of cone death, occasional hypermethylation was observed in the outer nuclear layer.ConclusionThe finding that cell-specific increases of 5mC and 5hmC immunostaining are associated with the death of retinal neurons during both development and degeneration suggests that changes in DNA methylation may play a role in modulating gene expression during the process of retinal degeneration. During retinal development, hypermethylation of retinal neurons associates with classical caspase-dependent apoptosis as well as caspase-3 independent cell death, while hypermethylation in the rd1 mouse photoreceptors is primarily associated with caspase-3 independent programmed cell death. These findings suggest a previously unrecognized role for epigenetic mechanisms in the onset and/or progression of programed cell death in the retina

A 3D cell death assay to quantitatively determine ferroptosis in spheroids

The failure of drug efficacy in clinical trials remains a big issue in cancer research. This is largely due to the limitations of two-dimensional (2D) cell cultures, the most used tool in drug screening. Nowadays, three-dimensional (3D) cultures, including spheroids, are acknowledged to be a better model of the in vivo environment, but detailed cell death assays for 3D cultures (including those for ferroptosis) are scarce. In this work, we show that a new cell death analysis method, named 3D Cell Death Assay (3DELTA), can efficiently determine different cell death types including ferroptosis and quantitatively assess cell death in tumour spheroids. Our method uses Sytox dyes as a cell death marker and Triton X-100, which efficiently permeabilizes all cells in spheroids, was used to establish 100% cell death. After optimization of Sytox concentration, Triton X-100 concentration and timing, we showed that the 3DELTA method was able to detect signals from all cells without the need to disaggregate spheroids. Moreover, in this work we demonstrated that 2D experiments cannot be extrapolated to 3D cultures as 3D cultures are less sensitive to cell death induction. In conclusion, 3DELTA is a more cost-effective way to identify and measure cell death type in 3D cultures, including spheroids.</jats:p

The Drosophila Caspase DRONC Cleaves following Glutamate or Aspartate and Is Regulated by DIAP1, HID, and GRIM

The caspase family of cysteine proteases plays important roles in bringing about apoptotic cell death. All caspases studied to date cleave substrates COOH-terminal to an aspartate. Here we show that the Drosophila caspase DRONC cleaves COOH-terminal to glutamate as well as aspartate. DRONC autoprocesses itself following a glutamate residue, but processes a second caspase, drICE, following an aspartate. DRONC prefers tetrapeptide substrates in which aliphatic amino acids are present at the P2 position, and the P1 residue can be either aspartate or glutamate. Expression of a dominant negative form of DRONC blocks cell death induced by the Drosophila cell death activators reaper, hid, and grim, and DRONC overexpression in flies promotes cell death. Furthermore, the Drosophila cell death inhibitor DIAP1 inhibits DRONC activity in yeast, and DIAP1's ability to inhibit DRONC-dependent yeast cell death is suppressed by HID and GRIM. These observations suggest that DRONC acts to promote cell death. However, DRONC activity is not suppressed by the caspase inhibitor and cell death suppressor baculovirus p35. We discuss possible models for DRONC function as a cell death inhibitor

Alternative cell death pathways and cell metabolism

While necroptosis has for long been viewed as an accidental mode of cell death triggered by physical or chemical damage, it has become clear over the last years that necroptosis can also represent a programmed form of cell death in mammalian cells. Key discoveries in the field of cell death research, including the identification of critical components of the necroptotic machinery, led to a revised concept of cell death signaling programs. Several regulatory check and balances are in place in order to ensure that necroptosis is tightly controlled according to environmental cues and cellular needs. This network of regulatory mechanisms includes metabolic pathways, especially those linked to mitochondrial signaling events. A better understanding of these signal transduction mechanisms will likely contribute to open new avenues to exploit our knowledge on the regulation of necroptosis signaling for therapeutic application in the treatment of human diseases

Expression of baculovirus P35 prevents cell death in Drosophila

The baculovirus P35 protein functions to prevent apoptotic death of infected cells. We have expressed P35 in the developing embryo and eye of the fly Drosophila melanogaster. P35 eliminates most, if not all, normally occurring cell death in these tissues, as well as X-irradiation-induced death. Excess pupal eye cells that are normally eliminated by apoptosis develop into pigment cells when their death is prevented by P35 expression. Our results suggest that one mechanism by which viruses prevent the death of the host cell is to block a cell death pathway that mediates normally occurring cell death. Identification of molecules that interact biochemically or genetically with P35 in Drosophila should provide important insights into how cell death is regulated

The convergence of radiation and immunogenic cell death signaling pathways.

Ionizing radiation (IR) triggers programmed cell death in tumor cells through a variety of highly regulated processes. Radiation-induced tumor cell death has been studied extensively in vitro and is widely attributed to multiple distinct mechanisms, including apoptosis, necrosis, mitotic catastrophe (MC), autophagy, and senescence, which may occur concurrently. When considering tumor cell death in the context of an organism, an emerging body of evidence suggests there is a reciprocal relationship in which radiation stimulates the immune system, which in turn contributes to tumor cell kill. As a result, traditional measurements of radiation-induced tumor cell death, in vitro, fail to represent the extent of clinically observed responses, including reductions in loco-regional failure rates and improvements in metastases free and overall survival. Hence, understanding the immunological responses to the type of radiation-induced cell death is critical. In this review, the mechanisms of radiation-induced tumor cell death are described, with particular focus on immunogenic cell death (ICD). Strategies combining radiotherapy with specific chemotherapies or immunotherapies capable of inducing a repertoire of cancer specific immunogens might potentiate tumor control not only by enhancing cell kill but also through the induction of a successful anti-tumor vaccination that improves patient survival

Deletion of AIF1 but not of YCA1/MCA1 protects Saccharomyces cerevisiae and Candida albicans cells from caspofungin-induced programmed cell death

Caspofungin was the first member of a new class of antifungals called echinocandins to be approved by a drug regulatory authority. Like the other echinocandins, caspofungin blocks the synthesis of β(1,3)-D-glucan of the fungal cell wall by inhibiting the enzyme, β(1,3)-D-glucan synthase. Loss of β(1,3)-D-glucan leads to osmotic instability and cell death. However, the precise mechanism of cell death associated with the cytotoxicity of caspofungin was unclear. We now provide evidence that Saccharomyces cerevisiae cells cultured in media containing caspofungin manifest the classical hallmarks of programmed cell death (PCD) in yeast, including the generation of reactive oxygen species (ROS), the fragmentation of mitochondria, and the production of DNA strand breaks. Our data also suggests that deleting AIF1 but not YCA1/MCA1 protects S. cerevisiae and Candida albicans from caspofungin-induced cell death. This is not only the first time that AIF1 has been specifically tied to cell death in Candida but also the first time that caspofungin resistance has been linked to the cell death machinery in yeast

Regulation of cell death in cancer - possible implications for immunotherapy

Since most anticancer therapies including immunotherapy trigger programmed cell death in cancer cells, defective cell death programs can lead to treatment resistance and tumor immune escape. Therefore, evasion of programmed cell death may provide one possible explanation as to why cancer immunotherapy has so far only shown modest clinical benefits for children with cancer. A better understanding of the molecular mechanisms that regulate sensitivity and resistance to programmed cell death is expected to open new perspectives for the development of novel experimental treatment strategies to enhance the efficacy of cancer immunotherapy in the future

Compensatory Proliferation Induced by Cell Death in the Drosophila Wing Disc Requires Activity of the Apical Cell Death Caspase Dronc in a Nonapoptotic Role

Achieving proper organ size requires a balance between proliferation and cell death. For example, at least 40%–60% of cells in the Drosophila wing disc can be lost, yet these discs go on to give rise to normal-looking adult wings as a result of compensatory proliferation 1, 2, 3. The signals that drive this proliferation are unknown. One intriguing possibility is that they derive, at least in part, from the dying cells. To explore this hypothesis, we activated cell death signaling in specific populations of cells in the developing wing but prevented these cells from dying through expression of the baculovirus p35 protein, which inhibits the activity of effector caspases that mediate apoptosis [4]. This allowed us to uncouple the activation steps of apoptosis from death itself. Here we report that stimulation of cell death signaling in the wing disc—in the absence of cell death—results in increased proliferation and ectopic expression of Wingless, a known mitogen in the wing. Activation of the apical cell death caspase Dronc is necessary and sufficient to drive both of these processes. Our results demonstrate an unanticipated function, the nonautonomous induction of proliferation, of an apical cell death caspase. This activity is likely to contribute to tissue homeostasis by promoting local compensatory proliferation in response to cell death. We speculate that dying cells may communicate cell fate or behavior instructions to their neighbors in other contexts as well