Autophagy supports generation of cells with high CD44 expression via modulation of oxidative stress and Parkin-mediated mitochondrial clearance.

High CD44 expression is associated with enhanced malignant potential in esophageal squamous cell carcinoma (ESCC), among the deadliest of all human carcinomas. Although alterations in autophagy and CD44 expression are associated with poor patient outcomes in various cancer types, the relationship between autophagy and cells with high CD44 expression remains incompletely understood. In transformed oesophageal keratinocytes, CD44Low-CD24High (CD44L) cells give rise to CD44High-CD24-/Low (CD44H) cells via epithelial-mesenchymal transition (EMT) in response to transforming growth factor (TGF)-ß. We couple patient samples and xenotransplantation studies with this tractable in vitro system of CD44L to CD44H cell conversion to investigate the functional role of autophagy in generation of cells with high CD44 expression. We report that high expression of the autophagy marker cleaved LC3 expression correlates with poor clinical outcome in ESCC. In ESCC xenograft tumours, pharmacological autophagy inhibition with chloroquine derivatives depletes cells with high CD44 expression while promoting oxidative stress. Autophagic flux impairment during EMT-mediated CD44L to CD44H cell conversion in vitro induces mitochondrial dysfunction, oxidative stress and cell death. During CD44H cell generation, transformed keratinocytes display evidence of mitophagy, including mitochondrial fragmentation, decreased mitochondrial content and mitochondrial translocation of Parkin, essential in mitophagy. RNA interference-mediated Parkin depletion attenuates CD44H cell generation. These data suggest that autophagy facilitates EMT-mediated CD44H generation via modulation of redox homeostasis and Parkin-dependent mitochondrial clearance. This is the first report to implicate mitophagy in regulation of tumour cells with high CD44 expression, representing a potential novel therapeutic avenue in cancers where EMT and CD44H cells have been implicated, including ESCC.

TNF-alpha modulates angiopoietin-1 expression in rheumatoid synovial fibroblasts via the NF-kappa B signalling pathway.

Angiopoietin-1 (Ang-1) is one of a family of ligands for the Tie-2 receptor which has been demonstrated to be involved in angiogenesis. Little is known about the regulation of Ang-1 gene expression. We have previously demonstrated that TNF-alpha is able to up-regulate the expression of Ang-1 mRNA in synovial fibroblasts. This present study investigated the signal transduction pathways involved in the TNF-alpha induced expression of Ang-1. TNF-alpha signals primarily through the p38, JNK, MAP kinase, and IKK pathways resulting in the activation of the transcription factors AP-1 and NF-kappa B. Experiments with inhibitors and siRNA for these various signal transduction pathways revealed that TNF-alpha stimulation of Ang-1 expression occurs via the NF-kappa B signal transduction pathway.

Potent and selective nonpeptide inhibitors of caspases 3 and 7.

5-Dialkylaminosulfonylisatins have been identified as potent, nonpeptide inhibitors of caspases 3 and 7. The most active compound within this series (34) inhibited caspases 3 and 7 in the 2-6 nM range and exhibited approximately 1000-fold selectivity for caspases 3 and 7 versus a panel of five other caspases (1, 2, 4, 6, and 8) and was at least 20-fold more selective versus caspase 9. Sequence alignments of the active site residues of the caspases strongly suggest that the basis of this selectivity is due to binding in the S2 subsite comprised of residues Tyr204, Trp206, and Phe256 which are unique to caspases 3 and 7. These compounds inhibit apoptosis in three cell-based models: human Jurkat T cells, human chondrocytes, and mouse bone marrow neutrophils.

Nebulized sildenafil is a selective pulmonary vasodilator in lambs with acute pulmonary hypertension.

OBJECTIVE: To determine whether inhalation of aerosolized sildenafil with and without inhaled nitric oxide (NO) causes selective pulmonary vasodilation in a sheep model of pulmonary hypertension. DESIGN: A controlled laboratory study in instrumented, awake, spontaneously breathing lambs. SETTING: Animal research laboratory affiliated with a university hospital. SUBJECT: Twenty Suffolk lambs. INTERVENTIONS: Lambs were instrumented with a carotid artery catheter, a pulmonary artery catheter, and a tracheostomy tube and studied awake. After baseline measurements, pulmonary hypertension was induced by the continuous infusion of U46619, a thromboxane A2 analog. After breathing three concentrations of inhaled NO (2, 5, and 20 ppm), lambs were divided into two groups. Group 1 (n = 7) breathed aerosols containing 1, 10, and 30 mg of sildenafil alone, and group 2 (n = 4) simultaneously breathed NO (2 and 5 ppm) and aerosols containing 10 mg of sildenafil. Hemodynamic measurements were obtained before and at the end of each drug administration. Venous admixture was calculated, and plasma cyclic guanosine monophosphate and sildenafil concentrations were measured. MEASUREMENTS AND MAIN RESULTS: Aerosols containing 10 mg and 30 mg of sildenafil selectively decreased the pulmonary artery pressure by 21% +/- 3% and 26% +/- 3%, respectively (p < .05 vs. baseline pulmonary hypertension). When 10 mg of sildenafil was inhaled while simultaneously breathing 2 ppm and 5 ppm NO, the pulmonary artery pressure decreased by 35% +/- 3% and 43% +/- 2% (p < .05 vs. baseline pulmonary hypertension). Inhaled sildenafil did not impair systemic oxygenation, increase right-to-left intrapulmonary shunting, or impair the ability of inhaled NO to reduce right-to-left shunting. CONCLUSIONS: Nebulized sildenafil is a selective pulmonary vasodilator that can potentiate the pulmonary vasodilating effects of inhaled NO.

Production and characterization of a Tie2 agonist monoclonal antibody.

The Tie2 receptor and its known ligands, the angiopoietins, play a critical role in endothelial cell differentiation during the process of angiogenesis. Recent experimental observations indicate that the agonistic ligand, angiopoietin-1, can stimulate endothelial cell sprouting and act as a chemo-attractant in vitro and induce increased and enhanced angiogenesis both alone and in conjunction with vascular endothelial growth factor (VEGF) in vivo. Here, we present a monoclonal antibody (MAb), which binds to the extracellular portion of the Tie2 receptor and elicits similar agonist effects. Upon MAb binding to the native Tie2 receptor of cultured human umblical vein endothelial cells (HUVEC), there is a rapid increase in receptor autophosphorylation with a concomitant enhancement in the recruitment and association of the signalling intermediates Grb2 and SH-PTP2. The antibody further demonstrates functional activity in vascular tissues. In vitro, the antibody promotes the survival of cultured HUVEC and elicits a dose dependent outgrowth and branching of microvessels from cultured explants of rat aorta. When administered in vivo, the antibody enhances the vascularization of subcutaneous Matrigel implants in mice. Together these data suggest that the antibody is capable of acting as a surrogate ligand for Tie2 and further confirms the role of Tie2 in the differentiation of endothelial cells during angiogenesis.

Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality.

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.

Caffeine abolishes the mammalian G(2)/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity.

Recent evidence indicates that arrest of mammalian cells at the G(2)/M checkpoint involves inactivation and translocation of Cdc25C, which is mediated by phosphorylation of Cdc25C on serine 216. Data obtained with a phospho-specific antibody against serine 216 suggest that activation of the DNA damage checkpoint is accompanied by an increase in serine 216 phosphorylated Cdc25C in the nucleus after exposure of cells to gamma-radiation. Prior treatment of cells with 2 mM caffeine inhibits such a change and markedly reduces radiation-induced ataxia-telangiectasia-mutated (ATM)-dependent Chk2/Cds1 activation and phosphorylation. Chk2/Cds1 is known to localize in the nucleus and to phosphorylate Cdc25C at serine 216 in vitro. Caffeine does not inhibit Chk2/Cds1 activity directly, but rather, blocks the activation of Chk2/Cds1 by inhibiting ATM kinase activity. In vitro, ATM phosphorylates Chk2/Cds1 at threonine 68 close to the N terminus, and caffeine inhibits this phosphorylation with an IC(50) of approximately 200 microM. Using a phospho-specific antibody against threonine 68, we demonstrate that radiation-induced, ATM-dependent phosphorylation of Chk2/Cds1 at this site is caffeine-sensitive. From these results, we propose a model wherein caffeine abrogates the G(2)/M checkpoint by targeting the ATM-Chk2/Cds1 pathway; by inhibiting ATM, it prevents the serine 216 phosphorylation of Cdc25C in the nucleus. Inhibition of ATM provides a molecular explanation for the increased radiosensitivity of caffeine-treated cells.

An indolocarbazole inhibitor of human checkpoint kinase (Chk1) abrogates cell cycle arrest caused by DNA damage.

Many cancer therapies cause DNA damage to effectively kill proliferating tumor cells; however, a major limitation of current therapies is the emergence of resistant tumors following initial treatment. Cell cycle checkpoints are involved in the response to DNA damage and specifically prevent cell cycle progression to allow DNA repair. Tumor cells can take advantage of the G2 checkpoint to arrest following DNA damage and avoid immediate cell death. This can contribute to acquisition of drug resistance. By abrogating the G2 checkpoint arrest, it may be possible to synergistically augment tumor cell death induced by DNA damage and circumvent resistance. This requires an understanding of the molecules involved in regulating the checkpoints. Human Chk1 is a recently identified homologue of the Schizosaccharomyces pombe checkpoint kinase gene, which is required for G2 arrest in response to DNA damage. Chk1 phosphorylates the dual specificity phosphatase cdc25C on Ser-216, and this may be involved in preventing cdc25 from activating cdc2/cyclinB and initiating mitosis. To further study the role of Chk1 in G2 checkpoint control, we identified a potent and selective indolocarbazole inhibitor (SB-218078) of Chk1 kinase activity and used this compound to assess cell cycle checkpoint responses. Limited DNA damage induced by gamma-irradiation or the topoisomerase I inhibitor topotecan was used to induce G2 arrest in HeLa cells. In the presence of the Chk1 inhibitor, the cells did not arrest following gamma-irradiation or treatment with topotecan, but continued into mitosis. Abrogation of the damage-arrest checkpoint also enhanced the cytotoxicity of topoisomerase I inhibitors. These studies suggest that Chk1 activity is required for G2 arrest following DNA damage.

Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway.

In response to DNA damage and replication blocks, cells activate pathways that arrest the cell cycle and induce the transcription of genes that facilitate repair. In mammals, ATM (ataxia telangiectasia mutated) kinase together with other checkpoint kinases are important components in this response. We have cloned the rat and human homologs of Saccharomyces cerevisiae Rad 53 and Schizosaccharomyces pombe Cds1, called checkpoint kinase 2 (chk2). Complementation studies suggest that Chk2 can partially replace the function of the defective checkpoint kinase in the Cds1 deficient yeast strain. Chk2 was phosphorylated and activated in response to DNA damage in an ATM dependent manner. Its activation in response to replication blocks by hydroxyurea (HU) treatment, however, was independent of ATM. Using mass spectrometry, we found that, similar to Chk1, Chk2 can phosphorylate serine 216 in Cdc25C, a site known to be involved in negative regulation of Cdc25C. These results suggest that Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Activation of Chk2 might not only delay mitotic entry, but also increase the capacity of cultured cells to survive after treatment with gamma-radiation or with the topoisomerase-I inhibitor topotecan.

Selective metals determination with a photoreversible spirobenzopyran.

The photoreversible metal-ion complexation behavior of nitroquinolinospiropyranindoline (NQSP) was studied in combination with the selective identification of six different transition-metal ions, Zn(2+), Co(2+), Hg(2+), Cu(2+), Cd(2+), and Ni(2+), in single- and binary-component mixtures via partial least squares discriminant (PLSD) analysis. The plasticizer, dicapryl phthalate, was chosen as the support medium for this study on the basis of (1) its enhancement of the photoreversibility and hypsochromic shifts seen in metal complexation and (2) its potential application to supported liquid membranes for eventual sensor applications. Complexation of divalent transition-metal ions by NQSP in dicapryl phthalate produced variable hypsochromic shifts in the absorption spectra (30-60 nm), requiring chemometric techniques in order to overcome the spectral overlaps. PLSD analysis was used to build classification analysis models to differentiate between the six divalent transition-metal ions. The feasibility of performing mixture analysis was studied using the concept of net analyte signal prior to experimental verification. Single- and binary-component mixtures of metals were identified with 100 and 97.4% accuracy, respectively, which included no false positives in either the training or prediction sets.

Kupffer cell-mediated lymphocyte apoptosis: a PGE2-dependent mechanism of portal venous transfusion-induced immunosuppression?

BACKGROUND: Kupffer cells, after exposure to alloantigen via the portal vein, mediate an immunosuppressive effect involving enhanced production of PGE2. We hypothesize that up-regulation of Kupffer cell CoA-independent transacylase (CoA-IT) by portal venous transfusion (PVT) is a possible mechanism of increased PGE2 production. Additionally, enhanced lymphocyte apoptosis, a process known to be macrophage dependent and facilitated by PGE2, is postulated as a possible mechanism of PVT-induced, Kupffer cell-mediated immunosuppression. METHODS: Lewis rat Kupffer cells were isolated after portal venous infusion with 1 ml of Wistar-Firth blood (PVT) or saline (PV sal). Kupffer cell PGE2 production and CoA-IT activity was assessed. Lymphocyte apoptosis after exposure to PVT or PV sal-treated Kupffer cells was also assessed by flow cytometry. RESULTS: PVT-treated Kupffer cells produced significantly more PGE2 and had increased CoA-IT activity when compared to PV sal-treated Kupffer cells. Treatment of Kupffer cells with a selective inhibitor of CoA-IT significantly decreased PVT-induced Kupffer cell PGE2 production. Increased lymphocyte apoptosis was observed after coculture with PVT-treated Kupffer cells compared to PV sal-treated cells. CONCLUSIONS: PVT increases Kupffer cell PGE2 production via increased CoA-IT activity and induces Kupffer cell-mediated lymphocyte apoptosis. Lymphocyte apoptosis facilitated by Kupffer cells within the hepatic sinusoid may be an important mechanism of PVT-induced immunosuppression in organ transplantation.

The role of platelet activating factor and other lipid mediators in inflammatory angiogenesis.

Chronic inflammatory diseases are often accompanied by intense angiogenesis. A model of inflammatory angiogenesis is the murine air pouch granuloma which has a hyperangiogenic component. Proinflammatory lipid mediator generation is also a hallmark of chronic inflammation and the role of endogenous production of these mediators in angiogenesis is not known. The 14 kDa phospholipase A2 (PLA2) deacylates phospholipid, liberating arachidonic acid, which is used for leukotriene production, and lysophospholipid, which can drive the production of platelet-activating factor (PAF). Therefore, SB 203347, an inhibitor of the 14 kDa PLA2, zileuton, an inhibitor of 5-lipoxygenase, and Ro 24-4736 a PAF receptor antagonist were evaluated for their effects in the murine air pouch granuloma. SB 203347 reduced both LTB4 and PAF, but not PGD2 levels measured in the day 6 granuloma. This correlated with a significant reduction in angiogenesis. Zileuton reduced LTB4 levels as expected, but did not significantly inhibit angiogenesis, whereas Ro 24-4736 potently reduced angiogenesis. These data support the hypothesis that PAF, and to a lesser extent leukotrienes contribute to the angiogenic phenotype in chronic inflammation.

Interactions of substrate and product with cytochrome P450: P4502B4 versus P450cam.

In the present study, two P450s (P4502B4 and P450cam) have been examined with regard to their interactions with their substrates and products utilizing the characteristic spectral perturbations as criteria for their binding. The results indicate that although there are differences between the two P450s (E) in regard to their precise interactions with their substrates (S) and products (P), the spectral titration data were consistent with the two-site model--E + S<-->ES (K1), E + P<-->EP (K2); EP + S<-->ESP (K3); ES + P<-->ESP (K4) in which S and P bind to E forming ESP. The data were inconsistent with the two-site model in which S and P compete for the same site. As required by the two-site model, the relationship K2K3 = K1K4 was maintained with both P450s at all product concentrations tested, although K3 and K4 decreased considerably when product concentration was increased. The relationship K3 >> K4 was also maintained, indicating that with both enzymes' ESP is formed predominantly by binding of S to EP rather than binding of P to ES, and that ESP dissociates predominantly to ES and P rather than EP and S. In other words, binding of S to EP facilitates the dissociation of P. This indicates that the relative parameter values are compatible for ESP to have functional significance. The possible role of ESP in controlling catalytic rate and catalytic efficiency is discussed.

Pharmacological effects of SB 220025, a selective inhibitor of P38 mitogen-activated protein kinase, in angiogenesis and chronic inflammatory disease models.

Chronic inflammatory diseases often are accompanied by intense angiogenesis, supporting the destructive proliferation of inflammatory tissues. A model of inflammatory angiogenesis is the murine air pouch granuloma, which has a hyperangiogenic component. In this model, we explored the regulation of inflammatory angiogenesis using SB 220025, a specific inhibitor of human p38 mitogen-activated protein (MAP) kinase, with an IC50 value of 60 nM and 50- to 1000-fold selectivity vs. other kinases tested. In vivo, this compound reduced the lipopolysaccharide-induced production of tumor necrosis factor at an ED50 value of 7.5 mg/kg. In the inflammatory angiogenesis model, over the course of granuloma development, we observed elevated levels of interleukin-1beta and tumor necrosis factor-alpha during the chronic inflammatory phase when intense angiogenesis occurs. SB 220025 at 30 mg/kg b.i.d. p.o. was able to greatly reduce the expression of these cytokines and inhibit angiogenesis by approximately 40%. To further study the effects of p38/CSBP MAP kinase inhibition in angiogenesis-dependent chronic inflammatory disease, SB 220025 was tested in murine collagen-induced arthritis. In this model, SB 220025 was able to prevent the progression of established arthritis. Thus, this p38/CSBP MAP kinase inhibitor, which can reduce inflammatory cytokine production and inhibit angiogenesis, is an effective treatment for chronic proliferative inflammatory disease.

Beta-lactams SB 212047 and SB 216754 are irreversible, time-dependent inhibitors of coenzyme A-independent transacylase.

The enzyme coenzyme A-independent transacylase (CoA-IT) has been demonstrated to be the key mediator of arachidonate remodeling, a process that moves arachidonate into 1-ether-containing phospholipids. Blockade of CoA-IT by reversible inhibitors has been shown to block the release of arachidonate in stimulated neutrophils and inhibit the production of eicosanoids and platelet-activating factor. We describe novel inhibitors of CoA-IT activity that contain a beta-lactam nucleus. beta-Lactams were investigated as potential mechanism-based inhibitors of CoA-IT on the basis of the expected formation of an acyl-enzyme intermediate complex. Two beta-lactams, SB 212047 and SB 216754, were shown to be specific, time-dependent inhibitors of CoA-IT activity (IC50 = 6 and 20 microM, respectively, with a 10-min pretreatment time). Extensive washing and dilution could not remove the inhibition, suggesting it was irreversible. In stimulated human monocytes, SB 216754 decreased the production of eicosanoids in a time-dependent manner. In an in vivo model of phorbol ester-induced ear inflammation, SB 216754 was able to inhibit indices of both edema and cell infiltration. Taken together, the results support two hypotheses: 1) CoA-IT activity is important for the production of inflammatory lipid mediators in stimulated cells and in vivo and 2) the mechanism by which CoA-IT acts to transfer arachidonate is through an acyl-enzyme intermediate.

Cytosolic 85-kDa phospholipase A2-mediated release of arachidonic acid is critical for proliferation of vascular smooth muscle cells.

Recent evidence suggests that arachidonic acid (AA) may be involved in regulating cellular proliferation. The predominant mechanism of AA release from cellular phospholipids is via phospholipase A2 (PLA2) hydrolysis. The purpose of this study was to examine the roles of the distinct 14-kDa and 85-kDa PLA2 enzymes in human coronary artery vascular smooth muscle cell (hCAVSMC) proliferation. Cultured hCAVSMCs proliferate in the presence of growth medium with a typical doubling time of 30-40 h, grow at a slower proliferative rate upon reaching confluency (day 8), and eventually undergo contact inhibition of growth (day 10). Neither Type II 14-kDa PLA2 activity nor mass changed over a 10-day culture period. In contrast, 85-kDa PLA2 protein activity and mRNA decreased as time in culture progressed. This reduction in 85-kDa PLA2 correlated with reductions in DNA synthesis and suggested a possible association between 85-kDa PLA2 and proliferation. To directly evaluate the role of the 85-kDa PLA2 in proliferation we examined the effects of an 85-kDa PLA2 inhibitor (AACOCF3) and 85-kDa PLA2 antisense oligonucleotides on proliferation. Both reagents dose dependently inhibited proliferation, whereas a 14-kDa PLA2 inhibitor (SB203347), a calcium-independent PLA2 inhibitor (HELSS), an 85-kDa sense oligonucleotide, and a nonrelevant scrambled control oligonucleotide had no effect. The mechanism by which 85-kDa PLA2 influences cellular proliferation remains unclear. Inhibition of 85-kDa PLA2 activity produced neither phase-specific cell cycle arrest nor apoptosis (fluorescence-activated cell sorter analysis). Addition of AA (20 mu M) attenuated the effects of both AACOCF3 and 85-kDa antisense oligonucleotides implicating AA as a key mediator in cellular proliferation. However, although prostaglandin E2 (PGE2) was present in the culture medium, it peaked early (day 3) in culture, and indomethacin had no effect on cellular proliferation indicating that hCAVSMC proliferation was not mediated through PGE2. These data provide the first direct evidence that PLA2 is involved in control of VSMC proliferation and indicate that 85-kDa PLA2-mediated liberation of AA is critical for cellular proliferation.