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Following the establishment of histone deacetylases (HDACs) as promising therapeutic targets for the reversal of aberrant epigenetic states associated with cancer, the development of HDAC inhibitors (HDACi) and their underlying mechanisms of action has been a significant area of scientific interest. HDACi induce diverse biological responses including the inhibition of cell proliferation by blocking progression through the G1 or G2/M phases of the cell cycle. As a putative tumor-suppressor protein, p21waf1/cip1 influences cell proliferation by inhibiting the activity of cyclin-cyclin-dependent kinase (CDK) complexes at the G1/S and G2/M cell cycle checkpoints. HDACi transcriptionally activate CDKN1A, and it has been proposed that induction of p21waf1/cip1 can determine if a cell undergoes apoptosis or cell cycle arrest following HDACi treatment. In the Eμ-myc transgenic mouse model of B-cell lymphoma, knockout of cdkn1a had no effect on disease latency, indicating that p21waf1/cip1 did not function as a tumor suppressor in this system. Although HDACi robustly induced expression of p21waf1/cip1 in wild-type Eμ-myc lymphomas, deletion of cdkn1a did not sensitize the lymphoma cells to HDACi-induced apoptosis and HDACi-induced cell cycle arrest still occurred. However, knockdown of cdkn1b in cdkn1a knockout lymphomas resulted in defective vorinostat-mediated arrest at G1/S indicating an essential role of p27Kip1 in mediating this biological response to vorinostat. These data demonstrate that induction of cdkn1a does not regulate HDACi-mediated tumor cell apoptosis and refute the notion that p21waf1/cip1 is an obligate mediator of HDACi-induced cell cycle arrest. [ABSTRACT FROM AUTHOR]

The human lymphocyte toxins granzyme B (hGrzB) and perforin cooperatively induce apoptosis of virus-infected or transformed cells: perforin pores enable entry of the serine protease hGrzB into the cytosol, where it processes Bid to selectively activate the intrinsic apoptosis pathway. Truncated Bid (tBid) induces Bax/Bak-dependent mitochondrial outer membrane permeability and the release of cytochrome c and Smac/Diablo. To identify cellular proteins that regulate perforin/hGrzB-mediated Bid cleavage and subsequent apoptosis, we performed a gene-knockdown (KD) screen using a lentiviral pool of short hairpin RNAs embedded within a miR30 backbone (shRNAmiR). We transduced HeLa cells with a lentiviral pool expressing shRNAmiRs that target 1213 genes known to be involved in cell death signaling and selected cells with acquired resistance to perforin/hGrzB-mediated apoptosis. Twenty-two shRNAmiRs were identified in the positive-selection screen including two, PCAF and ADA3, whose gene products are known to reside in the same epigenetic regulatory complexes. Small interfering (si)RNA-mediated gene-KD of PCAF or ADA3 also conferred resistance to perforin/hGrzB-mediated apoptosis providing independent validation of the screen results. Mechanistically, PCAF and ADA3 exerted their pro-apoptotic effect upstream of mitochondrial membrane permeabilization, as indicated by reduced cytochrome c release in PCAF-KD cells exposed to perforin/hGrzB. While overall levels of Bid were unaltered, perforin/hGrzB-mediated cleavage of Bid was reduced in PCAF-KD or ADA3-KD cells. We discovered that PCAF-KD or ADA3-KD resulted in reduced expression of PACS2, a protein implicated in Bid trafficking to mitochondria and importantly, targeted PACS2-KD phenocopied the effect of PCAF-KD or ADA3-KD. We conclude that PCAF and ADA3 regulate Bid processing via PACS2, to modulate the mitochondrial cell death pathway in response to hGrzB. [ABSTRACT FROM AUTHOR]

Human GraB (hGraB) preferentially induces apoptosis via Bcl-2-regulated mitochondrial damage but can also directly cleave caspases and caspase substrates in cell-free systems. How hGraB kills cells when it is delivered by cytotoxic lymphocytes (CL) and the contribution of hGraB to CL-induced death is still not clear. We show that primary human natural killer (hNK) cells, which specifically used hGraB to induce target cell death, were able to induce apoptosis of cells whose mitochondria were protected by Bcl-2. Purified hGraB also induced apoptosis of Bcl-2-overexpressing targets but only when delivered at 5- to 10-fold the concentration required to kill cells expressing endogenous Bcl-2. Caspases were critical in this process as inhibition of caspase activity permitted clonogenic survival of Bcl-2-overexpressing cells treated with hGraB or hNK cells but did not protect cells that only expressed endogenous Bcl-2. Our data therefore show that hGraB triggers caspase activation via mitochondria-dependent and mitochondria-independent mechanisms that are activated in a hierarchical manner, and that the combined effects of Bcl-2 and direct caspase inhibition can block cell death induced by hGraB and primary hNK cells.Cell Death and Differentiation (2008) 15, 708–717; doi:10.1038/sj.cdd.4402300; published online 18 January 2008 [ABSTRACT FROM AUTHOR]

Modelling the risk factors driving an environmental problem can be problematic when published data describing variables and their interactions are sparse. In such cases, expert opinion forms a vital source of information. Here we demonstrate the utility of a Bayesian Net (BN) model to integrate available information in a risk analysis setting. As an example, we use this methodology to explore the major factors influencing initiation of Lyngbya majuscula blooms in Deception Bay, Queensland, Australia. Over the past decade Lyngbya blooms have increased in both frequency and extent on seagrass beds in Deception Bay, with a range of adverse effects. This model was used to identify the main factors that could trigger a Lyngbya bloom. The five factors found to have the greatest effect on Lyngbya bloom initiation were: the available nutrient pool, water temperature, redox state of the sediments, current velocity, and light. Scenario analysis was also conducted to determine the sensitivity of the model to different combinations of variable states. The model has been used to identify knowledge gaps and therefore to direct additional research efforts in Deception Bay. With minor changes the model can be used to better understand the factors triggering Lyngbya blooms in other coastal regions. [ABSTRACT FROM AUTHOR]

Presents an analysis of 2-D finite elements using ANSYS modeling software. Simulation of electrothermal actuators; Mechanical and electrothermal properties of polysilicon; Presentation of the steps necessary to perform a sequential electrothermal-mechanical analysis using ANSYS.

Previous studies by our laboratory have shown that the drug transporter protein P-glycoprotein, P-gp, can specifically inhibit Fas-induced caspase-3 activation and apoptosis. Importantly, inhibition of both caspase-3 activation and cell death could be reversed by pharmacological and antibody inhibitors of P-gp function. However, the molecular mechanisms underpinning P-gp-mediated resistance to Fas-induced cell death and caspase activation remained unknown. We therefore sought to identify the point(s) within the death receptor pathway at which P-gp exerted its inhibitory effect and to determine whether the ATPase activity of P-gp was required. Structure-function analysis determined that ATP hydrolysis was necessary for P-gp to confer resistance to Fas-induced caspase activation and cell death. Importantly, although both FADD and caspase-8 were recruited to the Death Inducing Signal Complex (DISC) in wild-type P-gp expressing cells following Fas ligation, subsequent activation of caspase-8 at the DISC was inhibited. The ability of P-gp to inhibit caspase-8 activation was also ATP dependent. These studies demonstrate that P-gp inhibits Fas-induced caspase-8 activation but not formation of the DISC and that this activity of P-gp is dependent on ATP hydrolysis.Cell Death and Differentiation (2002) 9, 1266–1272. doi:10.1038/sj.cdd.4401081 [ABSTRACT FROM AUTHOR]