Xenotransplantation research has made considerable progress in recent years, largely through the increasing availability of pigs with multiple genetic modifications. We suggest that a pig with nine genetic modifications (ie, currently available) will provide organs (initially kidneys and hearts) that would function for a clinically valuable period of time, for example, >12 months, after transplantation into patients with end‐stage organ failure. The national regulatory authorities, however, will likely require evidence, based on in vitro and/or in vivo experimental data, to justify the inclusion of each individual genetic modification in the pig. We provide data both from our own experience and that of others on the advantages of pigs in which (a) all three known carbohydrate xenoantigens have been deleted (triple‐knockout pigs), (b) two human complement‐regulatory proteins (CD46, CD55) and two human coagulation‐regulatory proteins (thrombomodulin, endothelial cell protein C receptor) are expressed, (c) the anti‐apoptotic and “anti‐inflammatory” molecule, human hemeoxygenase‐1 is expressed, and (d) human CD47 is expressed to suppress elements of the macrophage and T‐cell responses. Although many alternative genetic modifications could be made to an organ‐source pig, we suggest that the genetic manipulations we identify above will all contribute to the success of the initial clinical pig kidney or heart transplants, and that the beneficial contribution of each individual manipulation is supported by considerable experimental evidence.

The most common form of neutrophil death is apoptosis. In the present study, we report surprising differences in the molecular mechanisms used for caspase activation between FAS/CD95-stimulated and TNF receptor 1 (TNFR1)-stimulated neutrophils. Whereas FAS-induced apoptosis was followed by caspase-8 activation and required Bid to initiate the mitochondrial amplification loop, TNF-␣-induced apoptosis involved class IA PI3Ks, which were activated by MAPK p38. TNF-␣-induced PI3K activation resulted in the generation of reactive oxygen species, which activated caspase-3, a mechanism that did not operate in neutrophils without active NADPH oxidase. We conclude that in neutrophils, proapoptotic pathways after TNFR1 stimulation are initiated by p38 and PI3K, but not by caspase-8, a finding that should be considered in anti-inflammatory drug-development strategies. (Blood. 2011;117(22): 5953-5962) IntroductionNeutrophils are the most abundant leukocytes in human blood and are essential in innate immune responses against pathogens. 1 Both in vitro and in vivo, apoptosis is the most common physiologic cause of neutrophil death, preventing the release of histotoxic contents from the dying cell and therefore limiting tissue damage. 2 Moreover, neutrophil apoptosis contributes to the regulation of the duration and intensity of inflammatory responses. 3,4 Activation of neutrophils with members of the TNF cytokine family such as FAS ligand or TNF-␣ can result in apoptosis and might therefore be relevant in controlling inflammation. Whereas apoptosis induction has consistently been reported in neutrophils after stimulation of FAS/CD95, 5 the consequences of TNF-␣ stimulation on the life span of neutrophils is less clear because apoptosis, survival, and no effect have all been described. 5 The diverse outcomes of TNF-␣ stimulation of neutrophils seem to depend on culture conditions, including the concentration of TNF-␣. 6 Whereas exposure to TNF-␣ concentrations below 1 ng/mL results in neutrophil survival, stimulation with concentrations above 10 ng/mL leads to neutrophil apoptosis. 7 Under inflammatory conditions, TNF-␣ concentrations between 1 and 10 ng/mL have been measured in broncho-alveolar fluids of patients suffering from acute respiratory distress syndrome 8 and in the serum and joint fluids of subjects with rheumatoid arthritis, 9 suggesting that local tissue concentrations may even exceed these numbers.The proapoptotic signaling cascades induced by FAS ligand and TNF-␣ have been intensively studied in multiple cellular systems. Whereas at least 2 TNF-␣ receptors exist, proapoptotic TNF-␣ signaling is mediated through TNF receptor 1 (TNFR1). 10 Both FAS and TNFR1 proapoptotic signaling results in caspase-8 activation. 11 Active caspase-8 can process its own pro-caspase and effector pro-caspases such as pro-caspase-3. 12 Caspase-8 can also process the BCL-2 family member BID, which is involved in mitochondrial outer membrane permeabilization characterized by cytochrome c release and subsequent caspase-9 and caspase-3 a...

The transcription factor PU.1 is a master regulator of myeloid differentiation and function. On the other hand, only scarce information is available on PU.1-regulated genes involved in cell survival. We now identified the glycolytic enzyme hexokinase 3 (HK3), a gene with cytoprotective functions, as transcriptional target of PU.1. Interestingly, HK3 expression is highly associated with the myeloid lineage and was significantly decreased in acute myeloid leukemia patients compared with normal granulocytes. Moreover, HK3 expression was significantly lower in acute promyelocytic leukemia (APL) compared with non-APL patient samples. In line with the observations in primary APL patient samples, we observed significantly higher HK3 expression during neutrophil differentiation of APL cell lines. Moreover, knocking down PU.1 impaired HK3 induction during neutrophil differentiation. In vivo binding of PU.1 and PML-RARA to the HK3 promoter was found, and PML-RARA attenuated PU.1 activation of the HK3 promoter. Next, inhibiting HK3 in APL cell lines resulted in significantly reduced neutrophil differentiation and viability compared with control cells. Our findings strongly suggest that HK3 is: (1) directly activated by PU.1, IntroductionAcute promyelocytic leukemia (APL or AML-M3) accounts for 5% to 8% of acute myeloid leukemia (AML) subtypes. 1 APL is characterized by a balanced chromosomal translocation involving the promyelocytic leukemia (PML) gene on chromosome 15 and the retinoic acid receptor␣ (RARA) on chromosome 17 that results in expression of the oncogenic fusion gene PML-RARA. 2 PML-RARA is a gain-of-function protein that promotes leukemic transformation by impairing the formation of functional PML nuclear bodies 3 and repressing RARA target genes in a dominantnegative manner. 4 This repression interferes with gene expression programs involved in differentiation, apoptosis, and self-renewal and leads to increased cell survival and inhibition of terminal differentiation, with an accumulation of promyelocytes. The block in differentiation that is observed in APL cells can be reverted with pharmacologic doses of ATRA by triggering PML-RARA degradation and thus restoring normal myeloid differentiation (reviewed by de Thé and Chen 5 ).The Ets-family member PU.1 is a master transcriptional regulator of myeloid differentiation, 6,7 as evidenced in PU.1 null mice that die at birth because of the lack of functional myeloid cells. 7 Inhibition of PU.1-induced myeloid differentiation may represent a critical step in APL leukemogenesis. On the one hand, PU.1 is inhibited at the transcriptional level by PML-RARA binding to its promoter region 8 ; on the other hand, a recent study identified PML-RARA as a binding partner of PU.1, 4 resulting in repression of PU.1-activated genes. A majority of PU.1 target genes are directly involved in myeloid differentiation and function, such as CD11b, CD45, the granulocyte/macrophage colonystimulating factor receptor (GM-CSFR), myeloperoxidase, lysozyme, and neutrophil elastase (reviewed...

Purpose: Evasion from chemotherapy-induced apoptosis due to p53 loss strongly contributes to drug resistance. Identification of specific targets for the treatment of drug-resistant p53-null tumors would therefore increase the effectiveness of cancer therapy.Experimental Design: By using a kinase-directed short hairpin RNA library and HCT116p53KO drugresistant colon carcinoma cells, glycogen synthase kinase 3 beta (GSK3B) was identified as a target whose silencing bypasses drug resistance due to loss of p53. p53-null colon cancer cell lines with different sets of mutations were used to validate the role of GSK3B in sustaining resistance and to characterize cell death mechanisms triggered by chemotherapy when GSK3B is silenced. In vivo xenograft studies were conducted to confirm resensitization of drug-resistant cells to chemotherapy upon GSK3 inhibition. Colon cancer samples from a cohort of 50 chemotherapy-treated stage II patients were analyzed for active GSK3B expression.Results: Downregulation of GSK3B in various drug-resistant p53-null colon cancer cell lines abolished cell viability and colony growth after drug addition without affecting cell proliferation or cell cycle in untreated cells. Cell death of 5-fluorouracil (5FU)-treated p53-null GSK3B-silenced colon carcinoma cells occurred via PARP1-dependent and AIF-mediated but RIP1-independent necroptosis. In vivo studies showed that drug-resistant xenograft tumor mass was significantly reduced only when 5FU was given after GSK3B inhibition. Tissue microarray analysis of colon carcinoma samples from 5FU-treated patients revealed that GSK3B is significantly more activated in drug-resistant versus responsive patients.Conclusions: Targeting GSK3B, in combination with chemotherapy, may represent a novel strategy for the treatment of chemotherapy-resistant tumors.