Flow cytometry based assays for the measurement of apoptosis-associated mitochondrial membrane depolarisation and cytochrome c release.

Mitochondria play a pivotal role in life and death of the cell because they produce the majority of energy required for survival and also regulate the intrinsic pathway to apoptosis. The involvement of mitochondria in cell death is generally measured by following mitochondrial membrane depolarisation or mitochondrial outer membrane permeabilisation (MOMP). These events can be assayed using cationic dyes that are attracted to the negative charge across the inner membrane of healthy mitochondria or by following translocation of cytochrome c from the mitochondria to the cytoplasm respectively. These events progress rapidly in individual cells but are observed as bi-phasic peaks in flow cytometry assays because cell death generally occurs asynchronously in a population. This allows researchers to use flow cytometry to easily distinguish healthy cells with intact mitochondria healthy from dying cells with permeabilised mitochondria. This article will therefore review methods using flow cytometry to follow mitochondrial membrane depolarisation and cytochrome c release during apoptosis, and will highlight some studies that resulted in development of these assays.

Maximal killing of lymphoma cells by DNA damage-inducing therapy requires not only the p53 targets Puma and Noxa, but also Bim.

DNA-damaging chemotherapy is the backbone of cancer treatment, although it is not clear how such treatments kill tumor cells. In nontransformed lymphoid cells, the combined loss of 2 proapoptotic p53 target genes, Puma and Noxa, induces as much resistance to DNA damage as loss of p53 itself. In Eµ-Myc lymphomas, however, lack of both Puma and Noxa resulted in no greater drug resistance than lack of Puma alone. A third B-cell lymphoma-2 homology domain (BH)3-only gene, Bim, although not a direct p53 target, was up-regulated in Eµ-Myc lymphomas incurring DNA damage, and knockdown of Bim levels markedly increased the drug resistance of Eµ-Myc/Puma(-/-)Noxa(-/-) lymphomas both in vitro and in vivo. Remarkably, c-MYC-driven lymphoma cell lines from Noxa(-/-)Puma(-/-)Bim(-/-) mice were as resistant as those lacking p53. Thus, the combinatorial action of Puma, Noxa, and Bim is critical for optimal apoptotic responses of lymphoma cells to 2 commonly used DNA-damaging chemotherapeutic agents, identifying Bim as an additional biomarker for treatment outcome in the clinic.

c-Cbl promotes T cell receptor-induced thymocyte apoptosis by activating the phosphatidylinositol 3-kinase/Akt pathway.

The ability of thymocytes to assess T cell receptor (TCR) signaling strength and initiate the appropriate downstream response is crucial for determining their fate. We have previously shown that a c-Cbl RING finger mutant knock-in mouse, in which the E3 ubiquitin ligase activity of c-Cbl is inactivated, is highly sensitive to TCR-induced death signals that cause thymic deletion. This high intensity signal involves the enhanced tyrosine phosphorylation of the mutant c-Cbl protein promoting a marked increase in the activation of Akt. Here we show that this high intensity signal in c-Cbl RING finger mutant thymocytes also promotes the enhanced induction of two mediators of TCR-directed thymocyte apoptosis, Nur77 and the pro-apoptotic Bcl-2 family member, Bim. In contrast, a knock-in mouse harboring a mutation at Tyr-737, the site in c-Cbl that activates phosphatidylinositol 3-kinase, shows reduced TCR-mediated responses including suppression of Akt activation, a reduced induction of Nur77 and Bim, and greater resistance to thymocyte death. These findings identify tyrosine-phosphorylated c-Cbl as a critical sensor of TCR signal strength that regulates the engagement of death-promoting signals.

Treatment of B-RAF mutant human tumor cells with a MEK inhibitor requires Bim and is enhanced by a BH3 mimetic.

B-RAF is frequently mutated in solid tumors, resulting in activation of the MEK/ERK signaling pathway and ultimately tumor cell growth and survival. MEK inhibition in these cells results in cell cycle arrest and cytostasis. Here, we have shown that MEK inhibition also triggers limited apoptosis of human tumor cell lines with B-RAF mutations and that this effect was dependent on upregulation and dephosphorylation of the proapoptotic, Bcl-2 homology 3-only (BH3-only) Bcl-2 family member Bim. However, upregulation of Bim was insufficient for extensive apoptosis and was countered by overexpression of Bcl-2. To overcome apoptotic resistance, we treated the B-RAF mutant cells both with MEK inhibitors and with the BH3 mimetic ABT-737, resulting in profound synergism and extensive tumor cell death. This treatment was successful because of both efficient antagonism of the prosurvival Bcl-2 family member Mcl-1 by Bim and inhibition of Bcl-2 and Bcl-x(L) by ABT-737. Critically, addition of ABT-737 converted the predominantly cytostatic effect of MEK inhibition to a cytotoxic effect, causing long-term tumor regression in mice xenografted with human tumor cell lines. Thus, the therapeutic efficacy of MEK inhibition requires concurrent unleashing of apoptosis by a BH3 mimetic and represents a potent combination treatment for tumors harboring B-RAF mutations.

Protection of pigs against 'in contact' challenge with classical swine fever following oral or subcutaneous vaccination with a recombinant porcine adenovirus.

A recombinant porcine adenovirus expressing the classical swine fever virus (CSFV) gp55 gene (rPAdV-gp55) was administered to commercially available outbred pigs via the subcutaneous or oral route and their susceptibility to 'in contact' challenge with classical swine fever determined. Animals vaccinated subcutaneously with a single dose of recombinant vaccine and challenged by 'in contact' exposure were protected from disease, whereas pigs given an equivalent single oral dose did not survive challenge. However, pigs given two oral doses of rPAdV-gp55, 22 days apart, were completely protected from disease. In addition, two doses of rPAdV-gp55 given subcutaneously was shown to boost CSFV neutralising antibody compared with a single dose, but neither a single dose nor two doses given orally induced detectable neutralising antibody responses.

Type I interferon drives dendritic cell apoptosis via multiple BH3-only proteins following activation by PolyIC in vivo.

BACKGROUND: DC are activated by pathogen-associated molecular patterns (PAMPs), and this is pivotal for the induction of adaptive immune responses. Thereafter, the clearance of activated DC is crucial to prevent immune pathology. While PAMPs are of major interest for vaccine science due to their adjuvant potential, it is unclear whether and how PAMPs may affect DC viability. We aimed to elucidate the possible apoptotic mechanisms that control activated DC lifespan in response to PAMPs, particularly in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We report that polyinosinic:polycytidylic acid (PolyIC, synthetic analogue of dsRNA) induces dramatic apoptosis of mouse splenic conventional DC (cDC) in vivo, predominantly affecting the CD8α subset, as shown by flow cytometry-based analysis of splenic DC subsets. Importantly, while Bim deficiency conferred only minor protection, cDC depletion was prevented in mice lacking Bim plus one of three other BH3-only proteins, either Puma, Noxa or Bid. Furthermore, we show that Type I Interferon (IFN) is necessary and sufficient for DC death both in vitro and in vivo, and that TLR3 and MAVS co-operate in IFNß production in vivo to induce DC death in response to PolyIC. CONCLUSIONS/SIGNIFICANCE: These results demonstrate for the first time in vivo that apoptosis restricts DC lifespan following activation by PolyIC, particularly affecting the CD8α cDC subset. Such DC apoptosis is mediated by the overlapping action of pro-apoptotic BH3-only proteins, including but not solely involving Bim, and is driven by Type I IFN. While Type I IFNs are important anti-viral factors, CD8α cDC are major cross-presenting cells and critical inducers of CTL. We discuss such paradoxical finding on DC death with PolyIC/Type I IFN. These results could contribute to understand immunosuppression associated with chronic infection, and to the optimization of DC-based therapies and the clinical use of PAMPs and Type I IFNs.

Glucose induces pancreatic islet cell apoptosis that requires the BH3-only proteins Bim and Puma and multi-BH domain protein Bax.

OBJECTIVE: High concentrations of circulating glucose are believed to contribute to defective insulin secretion and beta-cell function in diabetes and at least some of this effect appears to be caused by glucose-induced beta-cell apoptosis. In mammalian cells, apoptotic cell death is controlled by the interplay of proapoptotic and antiapoptotic members of the Bcl-2 family. We investigated the apoptotic pathway induced in mouse pancreatic islet cells after exposure to high concentrations of the reducing sugars ribose and glucose as a model of beta-cell death due to long-term metabolic stress. RESEARCH DESIGN AND METHODS: Islets isolated from mice lacking molecules implicated in cell death pathways were exposed to high concentrations of glucose or ribose. Apoptosis was measured by analysis of DNA fragmentation and release of mitochondrial cytochrome c. RESULTS: Deficiency of interleukin-1 receptors or Fas did not diminish apoptosis, making involvement of inflammatory cytokine receptor or death receptor signaling in glucose-induced apoptosis unlikely. In contrast, overexpression of the prosurvival protein Bcl-2 or deficiency of the apoptosis initiating BH3-only proteins Bim or Puma, or the downstream apoptosis effector Bax, markedly reduced glucose- or ribose-induced killing of islets. Loss of other BH3-only proteins Bid or Noxa, or the Bax-related effector Bak, had no impact on glucose-induced apoptosis. CONCLUSIONS: These results implicate the Bcl-2 regulated apoptotic pathway in glucose-induced islet cell killing and indicate points in the pathway at which interventional strategies can be designed.