Rescue Pathway Research Articles

Inhibition of nicotinamide phosphoribosyltransferase modifies LPS-induced inflammatory responses of human monocytes

Recent studies have identified enzymes that use NAD as a substrate, thus contributing to its net consumption. To maintain the intracellular pool, NAD is re-synthesized by a salvage pathway using nicotinamide, the by-product generated by the enzymatic cleavage of NAD. Enzymes involved in NAD re-synthesis include nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase. Our studies show, that NAMPT was substantially up-regulated by LPS in primary human monocytes, suggesting that it may be especially required during the process of monocyte activation. To evaluate the contribution of the NAD rescue pathway to LPS-induced biological responses in human monocytes, we used APO866, a well-characterized inhibitor of NAMPT. Concomitant with the inhibition of NAMPT, LPS-induced TNF-α protein synthesis declined, while TNF-α mRNA levels were minimally affected. Moreover, APO866 strongly decreased the production of reactive oxygen species (ROS), increased surface expression of the NAD-consuming enzyme CD38, and modified the production of selective eicosanoids. We further demonstrate that protein ADP-ribosylation was strongly reduced, indicating a possible link between this post-translational protein modification and human monocyte inflammatory responses. Despite a substantial reduction in intracellular NAD levels, activated monocytes were resistant to apoptosis, while resting monocytes were not. Taken together, our data suggest that activated monocytes strongly depend on the NAD salvage pathway to mount an appropriate inflammatory response. Their survival is not affected by NAD-depletion, probably as a result of LPS-mediated anti-apoptotic signals.

Wheat germ cell-free expression system as a pathway to improve protein yield and solubility for the SSGCID pipeline.

Recombinant expression of proteins of interest in Escherichia coli is an important tool in the determination of protein structure. However, lack of expression and insolubility remain significant challenges to the expression and crystallization of these proteins. The SSGCID program uses a wheat germ cell-free expression system as a rescue pathway for proteins that are either not expressed or insoluble when produced in E. coli. Testing indicates that the system is a valuable tool for these protein targets. Further increases in solubility were obtained by the addition of the NVoy polymer reagent to the reaction mixture. These data indicate that this eukaryotic cell-free expression system has a high success rate and that the addition of specific reagents can increase the yield of soluble protein.

Activation of PDGFr-β Signaling Pathway after Imatinib and Radioimmunotherapy Treatment in Experimental Pancreatic Cancer

Pancreatic cancer does not respond to a single-agent imatinib therapy. Consequently, multimodality treatments are contemplated. Published data indicate that in colorectal cancer, imatinib and radioimmunotherapy synergize to delay tumor growth. In pancreatic cancer, the tumor response is additive. This disparity of outcomes merited further studies because interactions between these modalities depend on the imatinib-induced reduction of the tumor interstitial fluid pressure. The examination of human and murine PDGFr-β/PDGF-B pathways in SW1990 pancreatic cancer xenografts revealed that the human branch is practically dormant in untreated tumors but the insult on the stromal component produces massive responses of human cancer cells. Inhibition of the stromal PDGFr-β with imatinib activates human PDGFr-β/PDGF-B signaling loop, silent in untreated xenografts, via an apparent paracrine rescue pathway. Responses are treatment-and time-dependent. Soon after treatment, levels of human PDGFr-β, compared to untreated tumors, are 3.4×, 12.4×, and 5.7× higher in imatinib-, radioimmunotherapy + imatinib-, and radioimmunotherapy-treated tumors, respectively. A continuous 14-day irradiation of imatinib-treated xenografts reduces levels of PDGFr-β and phosphorylated PDGFr-β by 5.3× and 4×, compared to earlier times. Human PDGF-B is upregulated suggesting that the survival signaling via the autocrine pathway is also triggered after stromal injury. These findings indicate that therapies targeting pancreatic cancer stromal components may have unintended mitogenic effects and that these effects can be reversed when imatinib is used in conjunction with radioimmunotherapy.

Chemical rescue of enzymes: proton transfer in mutants of human carbonic anhydrase II.

In human carbonic anhydrase II (HCA II), the mutation of position 64 from histidine to alanine (H64A) disrupts the rate limiting proton transfer (PT) event, resulting in a reduction of the catalytic activity of the enzyme as compared to the wild-type. Potential of mean force (PMF) calculations utilizing the multistate empirical valence bond (MS-EVB) methodology for H64A HCA II yields a PT free energy barrier significantly higher than that found in the wild-type enzyme. This high barrier, determined in the absence of exogenous buffer and assuming no additional ionizable residues in the PT pathway, indicates the likelihood of alternate enzyme pathways that utilize either ionizable enzyme residues (self-rescue) and/or exogenous buffers (chemical rescue). It has been shown experimentally that the catalytic activity of H64A HCA II can be chemically rescued to near wild-type levels by the addition of the exogenous buffer 4-methylimidazole (4MI). Crystallographic studies have identified two 4MI binding sites, yet site-specific mutations intended to disrupt 4MI binding have demonstrated these sites to be nonproductive. In the present work, MS-EVB simulations show that binding of 4MI near Thr199 in the H64A HCA II mutant, a binding site determined by NMR spectroscopy, results in a viable chemical rescue pathway. Additional viable rescue pathways are also identified where 4MI acts as a proton transport intermediary from the active site to ionizable residues on the rim of the active site, revealing a probable mode of action for the chemical rescue pathway.

Hypochlorite-modified high-density lipoprotein promotes induction of HO-1 in endothelial cells via activation of p42/44 MAPK and zinc finger transcription factor Egr-1

Modification/chlorination of high-density lipoprotein (HDL) by hypochlorous acid (HOCl), formed by the myeloperoxidase–H2O2–chloride system of activated phagocytes, converts an anti-atherogenic lipoprotein into a pro-inflammatory lipoprotein particle. Chlorinated HDL is present in human lesion material, binds to and is internalized by endothelial cells and impairs expression and activity of endothelial nitric oxide synthase. The present study aimed at clarifying whether exposure of endothelial cells to pro-inflammatory HOCl–HDL impacts on expression of heme oxygenase-1, a potential rescue pathway against endothelial dysfunction. Our findings revealed that HDL modified by HOCl, added as reagent or generated enzymatically, induced phosphorylation of p42/44 mitogen-activated protein kinase, expression of transcription factor early growth response-1 (Egr-1) and enhanced expression of heme oxygenase-1 in human endothelial cells. Upregulation of heme oxygenase-1 could be blocked by an inhibitor upstream of p42/44 mitogen-activated protein kinase and/or knockdown of Egr-1 by RNA-interference. Electrophoretic mobility shift assays demonstrated HOCl–HDL-mediated induction of the Egr-1 DNA binding activity. Immunocytochemical and immunoblotting experiments demonstrated HOCl–HDL-induced translocation of Egr-1 to the nucleus. The present study demonstrates a novel compensatory pathway against adverse effects of HOCl–HDL, providing cytoprotection in a number of pathological conditions including cardiovascular disease.

Fatty acid omega‐oxidation as a rescue pathway for fatty acid oxidation disorders in humans

Fatty acids (FAs) can be degraded via different mechanisms including α-, β- and ω-oxidation. In humans, a range of different genetic diseases has been identified in which either mitochondrial FA β-oxidation, peroxisomal FA β-oxidation or FA α-oxidation is impaired. Treatment options for most of these disorders are limited. This has prompted us to study FA ω-oxidation as a rescue pathway for these disorders, based on the notion that if the ω-oxidation of specific FAs could be upregulated one could reduce the accumulation of these FAs and the subsequent detrimental effects in the different groups of disorders. In this minireview, we describe our current state of knowledge in this area with special emphasis on Refsum disease and X-linked adrenoleukodystrophy.

Inhibition of Nicotinamide Phosphoribosyltransferase: CELLULAR BIOENERGETICS REVEALS A MITOCHONDRIAL INSENSITIVE NAD POOL

The NAD rescue pathway consists of two enzymatic steps operated by nicotinamide phosphoribosyltransferase (Nampt) and nicotinamide mononucleotide adenylyltransferases. Recently, the potent Nampt inhibitor FK866 has been identified and evaluated in clinical trials against cancer. Yet, how Nampt inhibition affects NAD contents and bioenergetics is in part obscure. It is also unknown whether NAD rescue takes place in mitochondria, and FK866 alters NAD homeostasis within the organelle. Here, we show that FK866-dependent reduction of the NAD contents is paralleled by a concomitant increase of ATP in various cell types, in keeping with ATP utilization for NAD resynthesis. We also show that poly- and mono(ADP-ribose) transferases rather than Sirt-1 are responsible for NAD depletion in HeLa cells exposed to FK866. Mass spectrometry reveals that the drug distributes in the cytosolic and mitochondrial compartment. However, the cytoplasmic but not the mitochondrial NAD pool is reduced upon acute or chronic exposure to the drug. Accordingly, Nampt does not localize within the organelles and their bioenergetics is not affected by the drug. In the mouse, FK866-dependent reduction of NAD contents in various organs is prevented by inhibitors of poly(ADP-ribose) polymerases or the NAD precursor kynurenine. For the first time, our data indicate that mitochondria lack the canonical NAD rescue pathway, broadening current understanding of cellular bioenergetics.

Serum albumin concentration: a predictive factor of infliximab pharmacokinetics and clinical response in patients with ulcerative colitis

Infliximab, an IgG1 monoclonal antibody (mab), has large inter-individual serum concentration variability. The objective was to determine the extent of the association of baseline albumin concentration and infliximab disposition in patient with ulcerative colitis. Data from 728 patients with ulcerative colitis from two clinical trials were analyzed to evaluate trends between infliximab pharmacokinetics and serum albumin, or liver or kidney function. Response in the placebo and treated groups were compared by baseline serum albumin concentrations (SAC) groups. Patients with higher SAC maintained higher infliximab concentrations, lower clearance, and longer half-life than patients with lower SAC. When analyzed by SAC quartiles, patients in the highest quartile had several-fold greater trough infliximab concentrations when compared with those in the lowest quartile. These observations were consistent in both studies and at different dose levels. Generally, clinical response in patients did not vary with SAC when the SAC was within the normal range, apparently because serum infliximab concentrations remained at therapeutic levels. However, patients with SAC lower than the normal laboratory reference range had much lower median serum infliximab concentrations and lower response rates compared with patients within normal SAC. Infliximab pharmacokinetics did not correlate with SGOT or creatinine clearance. It is hypothesized that the common rescue pathway for both albumin and IgG involving the neonatal Fc receptor may be responsible for the relationship between serum albumin and serum infliximab levels. Baseline albumin level may serve as a valuable and convenient measure of mab pharmacokinetic expectations in these patients.

Dicentric breakage at telomere fusions

Nonhomologous end-joining (NHEJ) inhibition at telomeres ensures that native chromosome ends do not fuse together. But the occurrence and consequences of rare telomere fusions are not well understood. It is notably unclear whether a telomere fusion could be processed to restore telomere ends. Here we address the behavior of individual dicentrics formed by telomere fusion in the yeast Saccharomyces cerevisiae. Our approach was to first stabilize and amplify fusions between two chromosomes by temporarily inactivating one centromere. Next we analyzed dicentric breakage following centromere reactivation. Unexpectedly, dicentrics often break at the telomere fusions during progression through mitosis, a process that restores the parental chromosomes. This unforeseen result suggests a rescue pathway able to process telomere fusions and to back up NHEJ inhibition at telomeres.

Poly(ADP-Ribose) Polymerase Inhibitors in Triple-Negative Breast Cancer

Poly(ADP-ribose) polymerases (PARPs) are involved in many aspects of the cellular response to various forms of damage. PARP-1 and PARP-2, the most abundant PARPs, are central to the response to specific types of DNA damage, especially single-strand breaks. Inhibition of PARP activity may sensitize the cell to exogenous agents such as chemotherapy and radiation. In circumstances where rescue pathways are deficient, particularly the homologous recombination (HR)-directed DNA repair pathway, inhibition of PARP may result in "synthetic lethality." BRCA mutation-associated breast cancers are a paradigm of HR-directed repair deficient tumors. Early clinical trials have demonstrated significant activity of single-agent PARP inhibitors in BRCA-deficient breast and ovarian cancer. Because of phenotypic similarities between some "triple-negative" breast cancers (TNBC) and the most prevalent type of breast cancer seen in BRCA1 mutation carriers, some have hypothesized that TNBC might also be specifically sensitive to PARP inhibition. The activity of single-agent PARP inhibitors in TNBC has not been reported. One trial did suggest significant enhancement of the activity of platinum-based combination chemotherapy, without incremental toxicity. These studies indicate that PARP inhibition is an exciting new approach to the treatment of breast cancers in women with underlying BRCA mutations and possibly in sporadic cancers with defects in HR-directed repair. Future studies will be necessary to determine whether the effectiveness of PARP inhibitors in nonhereditary cancer requires an underlying HR defect or whether these agents may improve the activity of conventional chemotherapy by other means. In addition, studies will be required to determine whether PARP inhibitors may induce synthetic lethality in tumors with defects in pathways other than the BRCA-dependent DNA repair pathway. If either or both of these prove to be the case, then PARP inhibition may benefit a wide spectrum of cancer patients.

Kinetics of Paused Ribosome Recycling in Escherichia coli

The bacterial tmRNA·SmpB system recycles stalled translation complexes in a process termed ‘ribosome rescue.’ tmRNA·SmpB specifically recognizes ribosomes that are paused at or near the 3′ end of truncated mRNA; therefore, nucleolytic mRNA processing is required before paused ribosomes can be rescued from full-length transcripts. Here, we examine the recycling of ribosomes paused on both full-length and truncated mRNAs. Peptidyl-tRNAs corresponding to each paused translation complex were identified, and their turnover kinetics was used to estimate the half-lives of paused ribosomes in vivo. Ribosomes were paused at stop codons on full-length mRNA using a nascent peptide motif that interferes with translation termination and elicits tmRNA·SmpB activity. Peptidyl-tRNA turnover from these termination-paused ribosomes was slightly more rapid in tmRNA+ cells (T1/2=22±2.2 s) than in ΔtmRNA cells (T1/2=32±1.6 s). Overexpression of release factor (RF) 1 greatly accelerated peptidyl-tRNA turnover from termination-paused ribosomes in both tmRNA+ and ΔtmRNA cells, whereas other termination factors had little or no effect on recycling. In contrast to inefficient translation termination, ribosome recycling from truncated transcripts lacking in-frame stop codons was dramatically accelerated by tmRNA·SmpB. However, peptidyl-tRNA still turned over from nonstop-paused ribosomes at a significant rate (t1/2=61±7.3 s) in ΔtmRNA cells. Overexpression of RF-1, RF-3, and ribosome recycling factor in ΔtmRNA cells failed to accelerate ribosome recycling from nonstop mRNA. These results indicate that tmRNA·SmpB activity is rate limited by mRNA cleavage, and that RF-3 and ribosome recycling factor do not constitute a tmRNA-independent rescue pathway, as previously suggested. Peptidyl-tRNA turnover from nonstop-paused ribosomes in ΔtmRNA cells suggests the existence of another uncharacterized ribosome rescue pathway.

Endogenous leukemia inhibitory factor protects photoreceptor cells against light-induced degeneration.

Expression of leukemia inhibitory factor (LIF) by a subset of Müller glia cells has recently been implicated in an endogenous survival response to photoreceptor injury in a model of inherited retinal degeneration. To investigate whether such a LIF-controlled survival pathway might be commonly induced upon photoreceptor injury independently of the nature of the toxic stimulus, we analyzed the role of LIF during light-induced retinal degeneration. Lif(+/-) and Lif(-/-) mice were exposed to 15,000 lx of white light for 2 h. Retinal morphology and rhodopsin content were analyzed nine days after light exposure. Gene expression studies were done using real-time PCR. Protein levels were determined by western blotting using specific antibodies. A lack of LIF reduced survival of photoreceptor cells after light exposure. In the absence of LIF several genes encoding molecules involved in the Janus kinase/signal transducer and activator of transcription (Jak/STAT) signaling pathway were not activated after light exposure. Presence or absence of LIF did not affect AKT (also known as protein kinase B, PKB) signaling and had only a mild effect on extracellular regulated kinase (ERK) phosphorylation. Stress-induced glial fibrillary acidic protein (GFAP) induction was minimal in the absence of LIF. Our results suggest that increased retinal expression of LIF is a general response to photoreceptor injury. Independent of the nature of the toxic insult (gene mutation, light), LIF may activate an endogenous rescue pathway that protects viable photoreceptor cells, leading to an increased photoreceptor survival in the stressed retina. This defense system may depend on the Jak/STAT pathway and may involve endothelin 2 (EDN2) but not (or only minimally) AKT and ERK1,2 signaling.

Detection and pharmacological modulation of nicotinamide mononucleotide (NMN) in vitro and in vivo

The emerging key role of NAD-consuming enzymes in cell biology has renewed the interest in NAD resynthesis through the rescue pathways. The first step of the nicotinamide-dependent NAD-rescue pathway is operated by nicotinamide phosphoribosyl transferase (NaPRT) forming nicotinamide mononucleotide (NMN). Because of the difficulties in measuring NMN, numerous open questions exist about the pathophysiological relevance of NaPRT and NMN itself. Here, we describe a new method of fluorimetric NMN detection upon derivatization of its alkylpyridinium group with acetophenone. By adopting this method, we analyzed the kinetics of nicotinamide-dependent NAD recycling in HeLa and U937 cells. Measurement of NMN contents in subcellular fractions revealed that the nucleotide is highly enriched in mitochondria, suggesting intramitochondrial NAD synthesis. NMN increases in cells undergoing hyperactivation of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1, or exposed to gallotannin, a putative inhibitor of NMN-adenylyl transferases. Evidence that the inhibitor of NAD resynthesis FK866 selectively inhibits NaPRT having no effect on NMNAT activity is also provided. Importantly, NMN reduces NAD and ATP depletion in cells undergoing PARP-1 hyperactivation, significantly delaying cell death. Finally, we show that a single injection of FK866 in the mouse induces long-lasting (up to 16 h) but mild (∼20%) reduction of NMN contents in different organs, suggesting slow rate of basal NAD consumption in vivo. Data provide new information on the biochemistry and pharmacology of NAD biosynthesis, allowing a better understanding of pyridine nucleotide metabolism.

Activation of the Unfolded Protein Response (UPR) Induces CEBPA mRNA Decay in AML Cells.

The myeloid key transcription factor CEBPA (CCAAT-enhancer binding protein alpha) is crucial for normal neutrophil differentiation, and cebpa-deficient mice lack mature granulocytes. In addition, deregulation of CEBPA function is a frequent event in AML patients. The endoplasmatic reticulum (ER) is involved in proper protein folding. Alterations in calcium levels or insufficient protein folding capacity leads to ER stress, thereby inducing rescue pathways that are commonly summarized as the unfolded protein response (UPR). The UPR aims either to re-establish ER homeostasis or to induce cell death. So far, induction of ER stress has been associated with a number of non-hematopoietic diseases. Here, we analyzed whether activated ER stress in leukemic cell lines and in AML patients affects the myeloid key regulator CEBPA. We induced ER stress in the leukemic HL60 cell line by treatment with 0.4mg/ml of Calcimycin (deregulation of calcium homeostasis) or with 3mg/ml of Tunicamycin (block of protein glycosylation). Consistently, we observed a rapid decrease of CEBPA mRNA to 10% after 6 hours of treatment. In accordance, CEBPA protein was no more detectable after 8 hours. Experiments in HL60 cells with Actinomycin D and Cycloheximide as well as competition experiments with various constructs of wild-type CEBPA sequences suggest a decay mechanism involving one or several elements in the 3'UTR of the CEBPA mRNA. We excluded microRNA-124a, a previously identified CEBPA regulator acting through the 3'UTR, to be involved. The precise elements mediating ER stress induced CEBPA mRNA decay remain to be elucidated. We also determined the expression of CEBPA mRNA and mediators of UPR - such as CHOP and GRP78 as well as alternative splicing of the XBP1 mRNA - in leukemic cells from 76 AML patients of all FAB subtypes. We found that AML patients with induced ER stress (13%) tended to have two-fold lower CEBPA mRNA expression, thus underlining the in vitro results reported above. In conclusion, our experiments indicate activated ER stress to be involved in a significant subgroup of AML patients. Moreover, it suggests that activation of the UPR in myeloid leukemic cells efficiently induces 3'UTR mediated decay of CEBPA mRNA expression, thereby contributing to the block in myeloid differentiation in these leukemias.

Targeting the Nuclear Factor-κB Rescue Pathway Has Promising Future in Human Renal Cell Carcinoma Therapy

Metastatic renal cell carcinoma (RCC) remains refractory to therapies. The nuclear factor-kappaB (NF-kappaB) transcription factor is involved in cell growth, cell motility, and vascularization. We evaluated whether targeting NF-kappaB could be of therapeutic and prognostic values in human RCC. The activation of the NF-kappaB pathway in human RCC cells and tumors was investigated by Western blot. In vitro, the effects of BAY 11-7085 and sulfasalazine, two NF-kappaB inhibitors, on tumor cell growth were investigated by cell counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and fluorescence-activated cell sorting. Their specificity toward NF-kappaB was analyzed by Western blot, confocal microscopy, NF-kappaB small interfering RNA, and NF-kappaB transcription assay. In vivo, the effects of BAY 11-7085 on the growth of human RCC tumors were investigated in nude mice. A tissue microarray (TMA) containing 241 cases of human RCC with 12 to 22 years of clinical follow-up and corresponding normal tissues was built up to assess prognostic significance of activated NF-kappaB. NF-kappaB is constitutively activated in cultured cells expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene as a consequence of Akt kinase activation and in tumors. In vitro and in vivo NF-kappaB inhibition blocked tumor cell growth by inducing cell apoptosis. On the TMA, NF-kappaB activation was correlated with tumor dimension but was not found to be an independent prognostic factor for patient survival. This report provides strong evidence that the mechanisms responsible for the intrinsic resistance of RCC cells to apoptosis converge on NF-kappaB independently of VHL expression and that targeting this pathway has great anticancer potential.

Oncogenic c-H-ras deregulates survivin expression: An improvement for survival

Survivin protein accomplishes two basic functions: cell cycle regulation and control of apoptosis. It is only expressed in G2/M phase and it influences rescue pathways in apoptosis-induced cells. Overexpression of constitutive active c-H-ras in HeLa, or induction of c-H-ras in a stable HeLaDiR cell line, led to sustained survivin expression in all cell cycle phases and even protected cells from drug induced apoptosis. siRNA-mediated silencing of survivin reversed this protection. Here we link the anti-apoptotic property of survivin to its cell cycle (in)dependent regulation via the activity of oncogenic c-H-ras.

A novel function for alternative polyadenylation as a rescue pathway from NMD surveillance

Premature termination codon (PTC) containing transcripts are subjected to a rapid degradation via nonsense-mediated decay (NMD) surveillance mechanism. By and large degradation is desired in order to prevent the translation of truncated, most likely deleterious, protein. Nevertheless, several dissimilar NMD-rescue events, capable of turning NMD-candidates into NMD-immune, are described. Yet, the extent and nature of this phenomenon is unknown. We screened the human genome for NMD-candidates transcripts. Among which we sub-grouped “pseudo-NMD” genes, which all their annotated transcripts contain PTCs, and therefore allegedly are transcribed but never translated. Here we show that alternative polyadenylation can rescue prematurely terminated transcripts, by truncating the pre-mRNA so that the PTC is now “legally” positioned. ESTs-based analysis shows that NMD-rescued genes are indeed expressed in human tissues. Furthermore, predicted NMD-rescue variants’ existence is computationally verified. Hence, we suggest a novel role for the exon-truncated class of alternative polyadenylation as an NMD-rescue regulatory mechanism.

Drosophila homologs of mammalian TNF/TNFR-related molecules regulate segregation of Miranda/Prospero in neuroblasts

During neuroblast (NB) divisions, cell fate determinants Prospero (Pros) and Numb, together with their adaptor proteins Miranda (Mira) and Partner of Numb, localize to the basal cell cortex at metaphase and segregate exclusively to the future ganglion mother cells (GMCs) at telophase. In inscuteable mutant NBs, these basal proteins are mislocalized during metaphase. However, during anaphase/telophase, these mutant NBs can partially correct these earlier localization defects and redistribute cell fate determinants as crescents to the region where the future GMC "buds" off. This compensatory mechanism has been referred to as "telophase rescue". We demonstrate that the Drosophila homolog of the mammalian tumor-necrosis factor (TNF) receptor-associated factor (DTRAF1) and Eiger (Egr), the homolog of the mammalian TNF, are required for telophase rescue of Mira/Pros. DTRAF1 localizes as an apical crescent in metaphase NBs and this apical localization requires Bazooka (Baz) and Egr. The Mira/Pros telophase rescue seen in inscuteable mutant NBs requires DTRAF1. Our data suggest that DTRAF1 binds to Baz and acts downstream of Egr in the Mira/Pros telophase rescue pathway.

PI3-Kinase Inhibition Decreases the Proliferation of ALL Cells, Potentiates mTOR Inhibition, and Abrogates Growth Factor-Mediated Reversal of mTOR Inhibition.

Biologically targeted cancer agents, including signal transduction inhibitors, have shown great promise in treating hematologic malignancies. However, used as single agents, these drugs may not be curative secondary to innate or acquired cellular resistance. Thus, acute lymphoblastic leukemia (ALL) and other cancer cells may become resistant to rapamycin, an mTOR inhibitor (MTI), following extended exposure to the drug. A strategy to overcome such resistance is to combine targeted agents, and thereby inhibit multiple targets simultaneously. Previously, we have shown activity of MTI in models of both human and murine ALL. In mouse models, treatment of ALL with MTI prolongs survival but may not cure disease. IL-7, a lymphoid growth factor important in the regulation of progenitor B cell development and proliferation, can reverse the inhibitory effects of MTI on human and murine pre-B ALL cells. We wished to further explore the mechanisms by which IL-7-mediated signaling protects ALL cells from the inhibitory effects of MTI, through the investigation of modulators of growth factor signaling in ALL. Thus, we have evaluated the impact of LY294002, an inhibitor of phosphatidyl inositol-3 kinase (PI3K). PI3K is a critical signaling molecule in cell survival and proliferation, with one of its central roles being signal transduction from growth factor receptors to the activation of AKT (an upstream regulator of mTOR). PI3K/AKT pathway over-activation has been implicated in many different cancers. Treatment of ALL cell lines with the PI3K inhibitor LY294002 markedly decreased cell proliferation in a dose-dependent manner. More importantly, the inhibitory effects of LY294002 were additive or synergistic with the inhibitory effects of MTI, and prevented the ability of IL-7 to reverse the inhibitory effects of rapamycin. Treatment of pre-B ALL cell lines with 2.5 μM LY294002 resulted in decreased proliferation to 20–45% of baseline as compared to untreated cells, whereas treatment with a higher dose (5 μM) reduced cell proliferation to 10–20%. Combinations of LY294002 and rapamycin, even at low doses, inhibited cell proliferation to a greater degree than each drug individually. Co-treatment with 2.5 μM LY294002 and low dose rapamycin (1 ng/ml) resulted in profound inhibition of proliferation to <=5%, compared to 20–30% with rapamycin alone. Furthermore, co-treatment with low-dose LY294002 and low-dose rapamycin resulted in greater inhibition than even higher doses of each of these agents individually. While the addition of IL-7 (1 U/ml) to rapamycin-treated cells resulted in the reversal of rapamycin-mediated cell inhibition, the further addition of 2.5 μM LY294002 significantly antagonized this growth factor rescue of MTI-treated ALL cells. The blockade by LY294002 of the IL-7 effect was most apparent in ALL cell lines that were IL-7 dependent, with cell proliferation reduced to <20%. However, the effects were still significant in IL-7 independent cell lines, with proliferation reduced to 20–60%. Similar results were seen using human ALL cell lines. These data suggest that the PI3K signaling pathway serves as a potential rescue pathway from mTOR inhibition, mediating the ability of growth factors to rescue cells from rapamycin;PI3K itself is a therapeutic target for ALL; andcombination therapy with MTI and PI3K inhibitors may be more active than either agent alone.

Survival signalling in Alzheimer's disease

Significant advancements in our understanding of cell-survival signalling in AD (Alzheimer's disease) stem from recent investigations into the metabolism, trafficking and fate of the essential omega-3 fatty acid DHA (docosahexaenoic acid) (C(22:6), n=3). Brain synaptic terminals and neuronal plasma membranes are highly enriched in DHA, and deficiencies in this polyunsaturated fatty acid are characteristic of AD-affected brain. Oxidative stress, targeting phospholipids containing DHA, and age-related DHA depletion are associated with the progressive erosion of normal cognitive function in AD. Current studies support the idea that DHA itself and novel DHA-derived neural synapse- and membrane-derived lipid messengers have considerable potential to modulate cell survival signalling in stressed cultured neural cell models in vitro and in mammalian models of learning, memory and AD in vivo. Key players in this intrinsic rescue system include the alpha-secretase-processed neurotrophin sAPPalpha [soluble APPalpha (amyloid precursor protein alpha)] peptide, the DHA-derived 10,17S-docosatriene NPD1 (neuroprotectin D1), a tandem brain cytosolic phospholipase A(2) and 15-lipoxygenase enzymatic system that biosynthesizes NPD1, and a small family of anti-apoptotic neuroprotective genes that encode Bcl-2, Bcl-X(L) and Bfl-1 (A1). This paper reviews current ideas regarding DHA and the oxygenated DHA derivative NPD1, intrinsically triggered biolipid neuroprotectants that along with their associated rescue pathways, contribute to life-or-death decisions of brain cells during homoeostasis, aging and neurodegenerative disease.