Nociceptin/orphanin FQ-induced nociceptive responses through substance P release from peripheral nerve endings in mice

We have studied the in vivo signaling mechanisms involved in nociceptin/orphanin FQ (Noci)-induced pain responses by using a flexor-reflex paradigm. Noci was 10,000 times more potent than substance P (SP) in eliciting flexor responses after intraplantar injection into the hind limb of mice, but the action of Noci seems to be mediated by SP. Mice pretreated with an NK1 tachykinin receptor antagonist or capsaicin, or mice with a targeted disruption of the tachykinin 1 gene no longer respond to Noci. The action of Noci appears to be mediated by the Noci receptor, a pertussis toxin-sensitive G protein–coupled receptor that stimulates inositol trisphosphate receptor and Ca2+ influx. These findings suggest that Noci indirectly stimulates nerve endings of nociceptive primary afferent neurons through a local SP release.

Targeted disruption of the Kcnq1 gene produces a mouse model of Jervell and Lange– Nielsen Syndrome

KCNQ1 encodes KCNQ1, which belongs to a family of voltage-dependent K+ ion channel proteins. KCNQ1 associates with a regulatory subunit, KCNE1, to produce the cardiac repolarizing current, IKs. Loss-of-function mutations in the human KCNQ1 gene have been linked to Jervell and Lange–Nielsen Syndrome (JLNS), a disorder characterized by profound bilateral deafness and a cardiac phenotype. To generate a mouse model for JLNS, we created a line of transgenic mice that have a targeted disruption in the Kcnq1 gene. Behavioral analysis revealed that the Kcnq1−/− mice are deaf and exhibit a shaker/waltzer phenotype. Histological analysis of the inner ear structures of Kcnq1−/− mice revealed gross morphological anomalies because of the drastic reduction in the volume of endolymph. ECGs recorded from Kcnq1−/− mice demonstrated abnormal T- and P-wave morphologies and prolongation of the QT and JT intervals when measured in vivo, but not in isolated hearts. These changes are indicative of cardiac repolarization defects that appear to be induced by extracardiac signals. Together, these data suggest that Kcnq1−/− mice are a potentially valuable animal model of JLNS.

Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor

Aberrant blood vessel growth in the retina that underlies the pathology of proliferative diabetic retinopathy and retinopathy of prematurity is the result of the ischemia-driven disruption of the normally antiangiogenic environment of the retina. In this study, we show that a potent inhibitor of angiogenesis found naturally in the normal eye, pigment epithelium-derived growth factor (PEDF), inhibits such aberrant blood vessel growth in a murine model of ischemia-induced retinopathy. Inhibition was proportional to dose and systemic delivery of recombinant protein at daily doses as low as 2.2 mg/kg could prevent aberrant endothelial cells from crossing the inner limiting membrane. PEDF appeared to inhibit angiogenesis by causing apoptosis of activated endothelial cells, because it induced apoptosis in cultured endothelial cells and an 8-fold increase in apoptotic endothelial cells could be detected in situ when the ischemic retinas of PEDF-treated animals were compared with vehicle-treated controls. The ability of low doses of PEDF to curtail aberrant growth of ocular endothelial cells without overt harm to retinal morphology suggests that this natural protein may be beneficial in the treatment of a variety of retinal vasculopathies.

Deletion of cytosolic phospholipase A2 suppresses ApcMin-induced tumorigenesis

Although nonsteroidal antiinflammatory drugs (NSAIDs) show great promise as therapies for colon cancer, a dispute remains regarding their mechanism of action. NSAIDs are known to inhibit cyclooxygenase (COX) enzymes, which convert arachidonic acid (AA) to prostaglandins (PGs). Therefore, NSAIDs may suppress tumorigenesis by inhibiting PG synthesis. However, various experimental studies have suggested the possibility of PG-independent mechanisms. Notably, disruption of the mouse group IIA secretory phospholipase A2 locus (Pla2g2a), a potential source of AA for COX-2, increases tumor number despite the fact that the mutation has been predicted to decrease PG production. Some authors have attempted to reconcile the results by suggesting that the level of the precursor (AA), not the products (PGs), is the critical factor. To clarify the role of AA in tumorigenesis, we have examined the effect of deleting the group IV cytosolic phospholipase A2 (cPLA2) locus (Pla2g4). We report that ApcMin/+, cPLA2−/− mice show an 83% reduction in tumor number in the small intestine compared with littermates with genotypes ApcMin/+, cPLA2+/− and ApcMin/+, cPLA2+/+. This tumor phenotype parallels that of COX-2 knockout mice, suggesting that cPLA2 is the predominant source of AA for COX-2 in the intestine. The protective effect of cPLA2 deletion is thus most likely attributed to a decrease in the AA supply to COX-2 and a resultant decrease in PG synthesis. The tumorigenic effect of sPLA2 mutations is likely to be through a completely different pathway.

Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis

Signal transducer and activator of transcription (STAT) proteins perform key roles in mediating signaling by cytokines and growth factors, including platelet-derived growth factor (PDGF). In addition, Src family kinases activate STAT signaling and are required for PDGF-induced mitogenesis in normal cells. One STAT family member, Stat3, has been shown to have an essential role in cell transformation by the Src oncoprotein. However, the mechanisms by which STAT-signaling pathways contribute to mitogenesis and transformation are not fully defined. We show here that disruption of Stat3 signaling by using dominant-negative Stat3β protein in NIH 3T3 fibroblasts suppresses c-Myc expression concomitant with inhibition of v-Src-induced transformation. Ectopic expression of c-Myc is able to partially reverse this inhibition, suggesting that c-Myc is a downstream effector of Stat3 signaling in v-Src transformation. Furthermore, c-myc gene knockout fibroblasts are refractory to transformation by v-Src, consistent with a requirement for c-Myc protein in v-Src transformation. In normal NIH 3T3 cells, disruption of Stat3 signaling with dominant-negative Stat3β protein inhibits PDGF-induced mitogenesis in a manner that is reversed by ectopic c-Myc expression. Moreover, inhibition of Src family kinases with the pharmacologic agent, SU6656, blocks Stat3 activation by PDGF. These findings, combined together, delineate the signaling pathway, PDGF → Src → Stat3 → Myc, that is important in normal PDGF-induced mitogenesis and subverted in Src transformation.

Evidence for aminoacylation-induced conformational changes in human mitochondrial tRNAs.

Analysis by acid polyacrylamide/urea gel electrophoresis of 14 individual mitochondrial tRNAs (mt-tRNAs) from human cells has revealed a variable decrease in mobility of the aminoacylated relative to the nonacylated form, with the degree of separation of the two forms not being correlated with the mass, polar character, or charge of the amino acid. Separation of the charged and uncharged species has been found to be independent of tRNA denaturation, being observed also in the absence of urea. In another approach, electrophoresis through a perpendicular denaturing gradient gel of several individual mt-tRNAs has shown a progressive unfolding of the tRNA with increasing denaturant concentration, which is consistent with an initial disruption of tertiary interactions, followed by the sequential melting of the four stems of the cloverleaf structure. A detailed analysis of the unfolding process of charged and uncharged tRNALys and tRNALeu(UUR) has revealed that the separation of the two forms of these tRNAs persisted throughout the almost entire range of denaturant concentrations used and was lost upon denaturation of the last helical domain(s), which most likely included the amino acid acceptor stem. These observations strongly suggest that the electrophoretic retardation of the charged species reflects an aminoacylation-induced conformational change of the 3'-end of these mt-tRNAs, with possible significant implications in connection with the known role of the acceptor end in tRNA interactions with the ribosomal peptidyl transferase center and the elongation factor Tu.

Ultrafast thermally induced unfolding of RNase A.

A temperature jump (T-jump) method capable of initiating thermally induced processes on the picosecond time scale in aqueous solutions is introduced. Protein solutions are heated by energy from a laser pulse that is absorbed by homogeneously dispersed molecules of the dye crystal violet. These act as transducers by releasing the energy as heat to cause a T-jump of up to 10 K with a time resolution of 70 ps. The method was applied to the unfolding of RNase A. At pH 5.7 and 59 degrees C, a T-jump of 3-6 K induced unfolding which was detected by picosecond transient infrared spectroscopy of the amide I region between 1600 and 1700 cm-1. The difference spectral profile at 3.5 ns closely resembled that found for the equilibrium (native-unfolded) states. The signal at 1633 cm-1, corresponding to the beta-sheet structure, achieved 15 +/- 2% of the decrease found at equilibrium, within 5.5 ns. However, no decrease in absorbance was detected until 1 ns after the T-ump. The disruption of beta-sheet therefore appears to be subject to a delay of approximately 1 ns. Prior to 1 ns after the T-jump, water might be accessing the intact hydrophobic regions.

Disruption of cellular signaling pathways by daunomycin through destabilization of nonlamellar membrane structures.

Albeit anthracyclines are widely used in the treatment of solid tumors and leukemias, their mechanism of action has not been elucidated. The present study gives relevant information about the role of nonlamellar membrane structures in signaling pathways, which could explain how anthracyclines can exert their cytocidal action without entering the cell [Tritton, T. R. & Yee, G. (1982) Science 217, 248-250]. The anthracycline daunomycin reduced the formation of the nonlamellar hexagonal (HII) phase (i.e., the hexagonal phase propensity), stabilizing the bilayer structure of the plasma membrane by a direct interaction with membrane phospholipids. As a consequence, various cellular events involved in signal transduction, such as membrane fusion and membrane association of peripheral proteins [e.g., guanine nucleotide-binding regulatory proteins (G proteins and protein kinase C-alpha beta)], where nonlamellar structures (negative intrinsic monolayer curvature strain) are required, were altered by the presence of daunomycin. Functionally, daunomycin also impaired the expression of the high-affinity state of a G protein-coupled receptor (ternary complex for the alpha 2-adrenergic receptor) due to G-protein dissociation from the plasma membrane. In vivo, daunomycin also decreased the levels of membrane-associated G proteins and protein kinase C-alpha beta in the heart. The occurrence of such nonlamellar structures favors the association of these peripheral proteins with the plasma membrane and prevents daunomycin-induced dissociation. These results reveal an important role of the lipid component of the cell membrane in signal transduction and its alteration by anthracyclines.

Disruption of the gene for hsp30, an alpha-crystallin-related heat shock protein of Neurospora crassa, causes defects in thermotolerance.

The alpha-crystallin-related heat shock proteins are produced by all eukaryotes, but the role of these proteins in thermoprotection remains unclear. To investigate the function of one of these proteins, we disrupted expression of the single-copy hsp30 gene of Neurospora crassa, using repeat-induced point mutagenesis, and we generated and characterized mutant strains that were deficient in hsp30 synthesis. These strains could grow at high temperature and they acquired thermotolerance from a heat shock. However, the hsp30-defective strains proved to be extremely sensitive to the combined stresses of high temperature and carbohydrate limitation, enforced by the addition of a nonmetabolizable glucose analogue. Under these conditions, their survival was reduced by 90% compared with wild-type cells. This sensitive phenotype was reversed by reintroduction of a functional hsp30 gene into the mutant strains. The mutant cells contained mitochondria from which a 22-kDa protein was readily extracted with detergents, in contrast to its retention by the mitochondria of wild-type cells. Antibodies against hsp30 coimmunoprecipitated a protein also of approximately 22 kDa from wild-type cells. Results of this study suggest that hsp30 may be important for efficient carbohydrate utilization during high temperature stress and that it may interact with other mitochondrial membrane proteins and function as a protein chaperone.

Antitumor activity of 3-ingenyl angelate: Plasma membrane and mitochondrial disruption and necrotic cell death

Options for skin cancer treatment currently include surgery, radiotherapy, topical chemotherapy, cryosurgery, curettage, and electrodes-sication. Although effective, surgery is costly and unsuitable for certain patients. Radiotherapy can leave a poor cosmetic effect, and current chemotherapy is limited by low cure rates and extended treatment schedules. Here, we describe the preclinical activity of a novel topical chemotherapeutic agent for the treatment of skin cancer, 3-ingenyl angelate (PEP005), a hydrophobic diterpene ester isolated from the plant Euphorbia peplus. Three daily topical applications of 42 nmol (18 mug) of PEP005 cured a series of s.c. mouse tumors (B16 melanoma, LK2 UV-induced squamous cell carcinoma, and Lewis lung carcinoma; it = >14 tumors/group) and human tumors (DO4 melanoma, HeLa cervical carcinoma, and PC3 and DU145 prostate carcinoma; it = >4 tumors/group) previously established (5-10 mm(3)) on C57BL/6 or Fox1(nu) mice. The treatment produced a mild, short-term erythema and eschar formation but, ultimately, resulted in excellent skin cosmesis. The LD90 for PEP005 for a panel of tumor cell lines was 180-220 muM. Electron microscopy showed that treatment with PEP005 both ill vitro (230 tot) and ill vivo (42 nmol) rapidly caused swelling of mitochondria and cell death by primary necrosis. Cr-51 release, uptake of propidium iodide, and staining with the mitochondria dye JC1, revealed that PEP005 (230 muM) treatment of tumor cells ill vitro resulted in a rapid plasma membrane perturbation and loss of mitochondrial membrane potential. PEP005 thus emerges as a new topical anti-skin cancer agent that has a novel mode of action involving plasma membrane and mitochondrial disruption and primary necrosis, ultimately resulting in an excellent cosmetic outcome.

Phosphatidylserine-containing liposomes:modulators of rhinovirus induced inflammatory responses

Background: Human rhinoviral infections are major contributors to the healthcare burden associated with acute exacerbations of asthma. We, and others have recently demonstrated that rhinovirus (RV)-induced inflammatory responses are mediated by multiple signalling mechanisms, such as IL-1/MyD88 (1) and TLR3/RIGI (2). We have also previously published work showing that TLR signalling is effectively inhibited by phosphatidylserine-containing liposomes (SAPS), through the disruption of membrane microdomains (3). Evidence has also suggested that membrane microdomains may influence infections with RV. In this study, we explored the ability of SAPS to modulate responses to the natural viral pathogens, RV-1B and RV-16. Method: The immortalized bronchial epithelial cell line, BEAS-2B or primary bronchial epithelial cells were infected with RV-1B or RV-16 at a TCID50/ml of 19107 for 1 h. Immediately following infection, various concentrations of SAPS were added and changes in cytokine release were measured at 24 h. SAPS remained present throughout. Type I and III interferon (IFN) expression and rates of viral replication were measured by quantitative PCR. Virus quantification was also performed using a viral CPE assay, and IFN signalling was measured by western blot. Liposome stability was characterised and intracellular trafficking of fluorescently labelled SAPS in BEAS-2B cells was investigated using confocal microscopy. For in vivo studies, female wt Balb/c mice were pre-treated with SAPS for 2 h prior to infection with RV as previously described and changes in BAL cell number, BAL cytokine production and viral replication were quantified (4). Results: Characterisation of SAPS liposomes by mass spectrometry showed no obvious signs of oxidation over the time period tested, and liposome size remained constant. Preliminary confocal studies revealed that SAPS was rapidly internalised within the cell and was found to associate with intracellular compartments such as the early endosome and golgi. Viral infected BEAS-2B cells co-incubated with SAPS, showed notably impaired responses to RV as assessed by release of CXCL8 and CCL5. SAPS also reduced RV-induced IFNb production and STAT-1 phosphorylation, without significantly influencing viral replication rates. Modest increases in viral particle production were only observed at 48 and 72 h time points. Suppression of viral-induced cytokine production was also observed in primary bronchial epithelial cells and pilot in vivo studies showed that SAPS results in reduced KC production at 24 h post viral infection, and this was associated with reduced neutrophil numbers within the BAL fluid. Conclusion: Our data demonstrates a potential means of modulating inflammatory responses induced by human rhinovirus.

A novel role of plasma membrane calcium atpase 4 as a negative-regulator of VEGF-induced angiogenesis

Current anti-angiogenic treatments involve the attenuation of signalling via the pro-angiogenic vascular endothelial growth factor/receptor (VEGF/VEGFR) axis. Stimulation of angiogenesis by VEGF requires the activation of the calcineurin/nuclear factor of activated T-cells (NFAT) signal transduction pathway which is inhibited by Plasma Membrane Calcium ATPase 4 (PMCA4), an endogenous calcium extrusion pump. However, PMCA4s role in calcineurin/NFAT-dependent angiogenesis is unknown. Using “gain of function” studies, we show here that adenoviral overexpression of PMCA4 in human umbilical vein endothelial cells (HUVEC) inhibited NFAT activity, decreased the expression of NFAT-dependent pro-angiogenic proteins (regulator of calcineurin 1.4 (RCAN1.4) and cyclooxygenase-2) and diminished in vitro cell migration and tube formation in response to VEGF-stimulation. Furthermore, in vivo blood vessel formation was attenuated in a matrigel plug assay by ectopic expression of PMCA4. Conversely, “loss of function” experiments by si-RNA-mediated knockdown of PMCA4 in HUVEC or isolation of mouse lung endothelial cells from PMCA4−/− mice showed increased VEGF-induced NFAT activity, RCAN1.4 expression, in vitro endothelial cell migration, tube formation and in vivo blood vessel formation. Additionally, in an in vivo pathological angiogenesis model of limb ischemia, the reperfusion of the ischemic limb of PMCA4−/− mice was augmented compared to wild-type. Disruption of the interaction between endogenous PMCA4 and calcineurin by adenoviral overexpression of the region of PMCA4 that interacts with calcineurin (residues 428–651) increased NFAT activity, RCAN1.4 protein expression and in vitro tube formation. These results identify PMCA4 as an inhibitor of VEGF-induced angiogenesis, highlighting its potential as a new therapeutic target for anti-angiogenic treatments.

UV-induced melanoma mouse model dependent on endothelin 3 over-expression

Melanoma is one of the most aggressive types of cancer. It originates from the transformation of melanocytes present in the epidermal/dermal junction of the human skin. It is commonly accepted that melanomagenesis is influenced by the interaction of environmental factors, genetic factors, as well as tumor-host interactions. DNA photoproducts induced by UV radiation are, in normal cells, repaired by the nucleotide excision repair (NER) pathway. The prominent role of NER in cancer resistance is well exemplified by patients with Xeroderma Pigmentosum (XP). This disease results from mutations in the components of the NER pathway, such as XPA and XPC proteins. In humans, NER pathway disruption leads to the development of skin cancers, including melanoma. Similar to humans afflicted with XP, Xpa and Xpc deficient mice show high sensibility to UV light, leading to skin cancer development, except melanoma. The Endothelin 3 (Edn3) signaling pathway is essential for proliferation, survival and migration of melanocyte precursor cells. Excessive production of Edn3 leads to the accumulation of large numbers of melanocytes in the mouse skin, where they are not normally found. In humans, Edn3 signaling pathway has also been implicated in melanoma progression and its metastatic potential. The goal of this study was the development of the first UV-induced melanoma mouse model dependent on the over-expression of Edn3 in the skin. The UV-induced melanoma mouse model reported here is distinguishable from all previous published models by two features: melanocytes are not transformed a priori and melanomagenesis arises only upon neonatal UV exposure. In this model, melanomagenesis depends on the presence of Edn3 in the skin. Disruption of the NER pathway due to the lack of Xpa or Xpc proteins was not essential for melanomagenesis; however, it enhanced melanoma penetrance and decreased melanoma latency after one single neonatal erythemal UV dose. Exposure to a second dose of UV at six weeks of age did not change time of appearance or penetrance of melanomas in this mouse model. Thus, a combination of neonatal UV exposure with excessive Edn3 in the tumor microenvironment is sufficient for melanomagenesis in mice; furthermore, NER deficiency exacerbates this process.^

UV-Induced Melanoma Mouse Model Dependent on Endothelin 3 Over-Expression

Melanoma is one of the most aggressive types of cancer. It originates from the transformation of melanocytes present in the epidermal/dermal junction of the human skin. It is commonly accepted that melanomagenesis is influenced by the interaction of environmental factors, genetic factors, as well as tumor-host interactions. DNA photoproducts induced by UV radiation are, in normal cells, repaired by the nucleotide excision repair (NER) pathway. The prominent role of NER in cancer resistance is well exemplified by patients with Xeroderma Pigmentosum (XP). This disease results from mutations in the components of the NER pathway, such as XPA and XPC proteins. In humans, NER pathway disruption leads to the development of skin cancers, including melanoma. Similar to humans afflicted with XP, Xpa and Xpc deficient mice show high sensibility to UV light, leading to skin cancer development, except melanoma. The Endothelin 3 (Edn3) signaling pathway is essential for proliferation, survival and migration of melanocyte precursor cells. Excessive production of Edn3 leads to the accumulation of large numbers of melanocytes in the mouse skin, where they are not normally found. In humans, Edn3 signaling pathway has also been implicated in melanoma progression and its metastatic potential. The goal of this study was the development of the first UV-induced melanoma mouse model dependent on the over-expression of Edn3 in the skin. The UV-induced melanoma mouse model reported here is distinguishable from all previous published models by two features: melanocytes are not transformed a priori and melanomagenesis arises only upon neonatal UV exposure. In this model, melanomagenesis depends on the presence of Edn3 in the skin. Disruption of the NER pathway due to the lack of Xpa or Xpc proteins was not essential for melanomagenesis; however, it enhanced melanoma penetrance and decreased melanoma latency after one single neonatal erythemal UV dose. Exposure to a second dose of UV at six weeks of age did not change time of appearance or penetrance of melanomas in this mouse model. Thus, a combination of neonatal UV exposure with excessive Edn3 in the tumor microenvironment is sufficient for melanomagenesis in mice; furthermore, NER deficiency exacerbates this process.

Gold nanoparticle induced vasculature damage in radiotherapy: Comparing protons, megavoltage photons, and kilovoltage photons

Purpose: The purpose of this work is to investigate the radiosensitizing effect of gold nanoparticle (GNP) induced vasculature damage for proton, megavoltage (MV) photon, and kilovoltage (kV) photon irradiation. Methods: Monte Carlo simulations were carried out using tool for particle simulation (TOPAS) to obtain the spatial dose distribution in close proximity up to 20 µm from the GNPs. The spatial dose distribution from GNPs was used as an input to calculate the dose deposited to the blood vessels. GNP induced vasculature damage was evaluated for three particle sources (a clinical spread out Bragg peak proton beam, a 6 MV photon beam, and two kV photon beams). For each particle source, various depths in tissue, GNP sizes (2, 10, and 20 nm diameter), and vessel diameters (8, 14, and 20 µm) were investigated. Two GNP distributions in lumen were considered, either homogeneously distributed in the vessel or attached to the inner wall of the vessel. Doses of 30 Gy and 2 Gy were considered, representing typical in vivo enhancement studies and conventional clinical fractionation, respectively. Results: These simulations showed that for 20 Au-mg/g GNP blood concentration homogeneously distributed in the vessel, the additional dose at the inner vascular wall encircling the lumen was 43% of the prescribed dose at the depth of treatment for the 250 kVp photon source, 1% for the 6 MV photon source, and 0.1% for the proton beam. For kV photons, GNPs caused 15% more dose in the vascular wall for 150 kVp source than for 250 kVp. For 6 MV photons, GNPs caused 0.2% more dose in the vascular wall at 20 cm depth in water as compared to at depth of maximum dose (Dmax). For proton therapy, GNPs caused the same dose in the vascular wall for all depths across the spread out Bragg peak with 12.7 cm range and 7 cm modulation. For the same weight of GNPs in the vessel, 2 nm diameter GNPs caused three times more damage to the vessel than 20 nm diameter GNPs. When the GNPs were attached to the inner vascular wall, the damage to the inner vascular wall can be up to 207% of the prescribed dose for the 250 kVp photon source, 4% for the 6 MV photon source, and 2% for the proton beam. Even though the average dose increase from the proton beam and MV photon beam was not large, there were high dose spikes that elevate the local dose of the parts of the blood vessel to be higher than 15 Gy even for 2 Gy prescribed dose, especially when the GNPs can be actively targeted to the endothelial cells. Conclusions: GNPs can potentially be used to enhance radiation therapy by causing vasculature damage through high dose spikes caused by the addition of GNPs especially for hypofractionated treatment. If GNPs are designed to actively accumulate at the tumor vasculature walls, vasculature damage can be increased significantly. The largest enhancement is seen using kilovoltage photons due to the photoelectric effect. Although no significant average dose enhancement was observed for the whole vasculature structure for both MV photons and protons, they can cause high local dose escalation (>15 Gy) to areas of the blood vessel that can potentially contribute to the disruption of the functionality of the blood vessels in the tumor.