Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions.

Life and death fate decisions allow cells to avoid massive apoptotic death in response to genotoxic stress. Although the regulatory mechanisms and signalling pathways controlling DNA repair and apoptosis are well characterized, the precise molecular strategies that determine the ultimate choice of DNA repair and survival or apoptotic cell death remain incompletely understood. Here we report that a protein tyrosine phosphatase, EYA, is involved in promoting efficient DNA repair rather than apoptosis in response to genotoxic stress in mammalian embryonic kidney cells by executing a damage-signal-dependent dephosphorylation of an H2AX carboxy-terminal tyrosine phosphate (Y142). This post-translational modification determines the relative recruitment of either DNA repair or pro-apoptotic factors to the tail of serine phosphorylated histone H2AX (gamma-H2AX) and allows it to function as an active determinant of repair/survival versus apoptotic responses to DNA damage, revealing an additional phosphorylation-dependent mechanism that modulates survival/apoptotic decisions during mammalian organogenesis

A functional yeast survival screen of tumor-derived cDNA libraries designed to identify anti-apoptotic mammalian oncogenes

Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems

Q&A: Cellular near death experiences-what is anastasis?

Apoptosis is a form of programmed cell death that is carried out by proteolytic enzymes called caspases. Executioner caspase activity causes cells to shrink, bleb, and disintegrate into apoptotic bodies and has been considered a point of no return for apoptotic cells. However, relatively recent work has shown that cells can survive transient apoptotic stimuli, even after executioner caspase activation. This process is called anastasis. In this Q&A, we answer common questions that arise regarding anastasis, including how it is defined, the origin of the name, the potential physiological consequences, molecular mechanisms, and open questions for this new field of study

Evaluation of the cell viability of human Wharton's jelly stem cells for use in cell therapy

Human umbilical cord Wharton's jelly stem cells (HWJSCs) are gaining attention as a possible clinical source of mesenchymal stem cells for cell therapy and tissue engineering due to their high accessibility, expansion potential, and plasticity. We employed a combination of highly sensitive techniques to determine the average cell viability levels and proliferation capabilities of 10 consecutive cell passages of cultured HWJSCs and then used RNA microarrays to identify genes associated with changes in cell viability levels. We found an initial decrease in cell viability from the first to the third cell passage followed by an increase until the sixth passage and a final decrease from the sixth to tenth cell passages. The highest cell viability levels corresponded to the fifth and sixth passages. The intracellular ionic contents of potassium, sodium, and chlorine suggest that the lower cell viability levels at passages 2, 3, and 8-10 may be associated with apoptotic cell death. In fact, gene expression analysis revealed that the average cell viability was significantly associated with genes with a function in apoptotic cell death, especially pro-apoptotic FASTKD2, BNIP3L genes and anti-apoptotic TNFAIP8 and BCL2L2 genes. This correlation with both pro-apoptotic and anti-apoptotic genes suggests that there may be a complex live-death equilibrium in cultured HWJSCs kept in culture for multiple cell passages. In this study, the highest cell viability levels corresponded to the fifth and sixth HWJSC passages, suggesting that these passages should be preferentially employed in cell therapy or tissue engineering protocols using this cell type

Ginger extract (Zingiber officinale Roscoe) triggers apoptosis in hepatocarcinogenesis induced rats

Ginger extract has been reported previously by our group to exhibit anticancer and an-tioxidant effects by reducing tumour burden and lipid peroxidation respectively in he-patocarcinogenesis induced rats. The current study examined the expression of pro-apoptotic protein caspase-8 and anti-apoptotic protein Bcl-2 in hepatocarcinogenesis treated rats. Thirty normal male Wistar rats were divided into 5 groups based on the diet given: i) control (normal rat chow), ii) olive oil, iii) ginger extract (100mg/kg body weight), iv) choline deficient diet + ethionine, CDE (to induce liver cancer) and v) CDE+ ginger extract. Rats were killed at week 8, and liver tissues were excised for immuno-histochemical study to identify pro-apoptotic and anti-apoptotic proteins, caspase-8 and Bcl-2. The observation on H&E staining confirmed the CDE diet induced liver can-cer as indicated by the presence of numerous oval cells. Identification of Bcl-2 expres-sion showed that 91.6% (11/12) of the samples from the CDE group revealed positive staining while treatment with ginger extract however inhibited the expression with only 8.4% (1/12) samples showing positive staining for Bcl-2. As for caspase-8 protein, 41.7% (5/12) of the samples from CDE group showed positive staining, which in-creased to 100% (12/12) with ginger extract treatment. Our findings suggest that gin-ger extract has an anticancer effect by inducing apoptosis in liver cancer cells via up-regulation of the expression of pro-apoptotic protein, caspase-8 and down-regulation of the expression of anti-apoptotic protein Bcl-

Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death.

Apoptosis is a genetically regulated cell suicide programme mediated by activation of the effector caspases 3, 6 and 7. If apoptotic cells are not scavenged, they progress to a lytic and inflammatory phase called secondary necrosis. The mechanism by which this occurs is unknown. Here we show that caspase-3 cleaves the GSDMD-related protein DFNA5 after Asp270 to generate a necrotic DFNA5-N fragment that targets the plasma membrane to induce secondary necrosis/pyroptosis. Cells that express DFNA5 progress to secondary necrosis, when stimulated with apoptotic triggers such as etoposide or vesicular stomatitis virus infection, but disassemble into small apoptotic bodies when DFNA5 is deleted. Our findings identify DFNA5 as a central molecule that regulates apoptotic cell disassembly and progression to secondary necrosis, and provide a molecular mechanism for secondary necrosis. Because DFNA5-induced secondary necrosis and GSDMD-induced pyroptosis are dependent on caspase activation, we propose that they are forms of programmed necrosis

Spatial control of Draper receptor signaling initiates apoptotic cell engulfment.

The engulfment of apoptotic cells is essential for tissue homeostasis and recovering from damage. Engulfment is mediated by receptors that recognize ligands exposed on apoptotic cells such as phosphatidylserine (PS). In this study, we convert Drosophila melanogaster S2 cells into proficient phagocytes by transfecting the Draper engulfment receptor and replacing apoptotic cells with PS-coated beads. Similar to the T cell receptor (TCR), PS-ligated Draper forms dynamic microclusters that recruit cytosolic effector proteins and exclude a bulky transmembrane phosphatase, consistent with a kinetic segregation-based triggering mechanism. However, in contrast with the TCR, localized signaling at Draper microclusters results in time-dependent depletion of actin filaments, which facilitates engulfment. The Draper-PS extracellular module can be replaced with FRB and FKBP, respectively, resulting in a rapamycin-inducible engulfment system that can be programmed toward defined targets. Collectively, our results reveal mechanistic similarities and differences between the receptors involved in apoptotic corpse clearance and mammalian immunity and demonstrate that engulfment can be reprogrammed toward nonnative targets