The Role of S-Palmitoylation of the Human Glucocorticoid Receptor (hGR) in Mediating the Nongenomic Glucocorticoid Actions.

BACKGROUND: Many rapid nongenomic glucocorticoid actions are mediated by membrane-bound glucocorticoid receptors (GRs). S-palmitoylation is a lipid post-translational modification that mediates the membrane localization of some steroid receptors. A highly homologous amino acid sequence (663YLCM KTLLL671) is present in the ligand-binding domain of hGRα, suggesting that hGRα might also undergo S-palmitoylation. AIM: To investigate the role of the motif 663YLCMKTLLL671 in membrane localization of the hGRα and in mediating rapid nongenomic glucocorticoid signaling. METHODS AND RESULTS: We showed that the mutant receptors hGRαY663A, hGRαC665A and hGRαLL670/671AA, and the addition of the palmitoylation inhibitor 2-bromopalmitate did not prevent membrane localization of hGRα and co-localization with caveolin-1, and did not influence the biphasic activation of mitogen-activated protein kinase (MAPK) signaling pathway in the early time points. Finally, the hGRα was not shown to undergo S-palmitoylation. CONCLUSIONS: The motif 663YLCMKTLLL671 does not play a role in membrane localization of hGRα and does not mediate the nongenomic glucocorticoid actions.

Bioinformatically Informed Design of Synthetic Mammalian Promoters.

Synthetic promoters have been developed in a number of different organisms and are capable of mediating specific and enhanced levels of gene expression. Typically, cis-regulatory regions from a few genes are randomly combined to generate a synthetic promoter library, and the sequences with the highest activity are selected for in target cell lines. Here we describe a novel approach that can be employed in the construction of synthetic promoters . Specifically, we use gene expression profiles obtained from microarray datasets to select the cis-regulatory elements that comprise the synthetic promoter library. By adopting this approach, we were able to construct several promoters that could specifically mediate gene expression in colorectal cancer cells. We develop a new selection criteria based on the observed transcriptome of target cells, the frequency that identified cis-regulatory sequences occur in identified gene modules, and the length of identified cis-regulatory regions. Our method allows for the generation of synthetic promoter libraries with increased level of specificity and facilitates the selection of promoters that are highly active only under predefined gene expression profiles.

A novel mutation of the hGR gene causing Chrousos syndrome.

BACKGROUND: Natural mutations in the human glucocorticoid receptor (hGR, NR3C1) gene cause Chrousos syndrome, a rare condition characterized by generalized, partial, target-tissue insensitivity to glucocorticoids. OBJECTIVE: To present a new case of Chrousos syndrome caused by a novel mutation in the hGR gene, and to elucidate the molecular mechanisms through which the natural mutant receptor affects glucocorticoid signal transduction. DESIGN AND RESULTS: The index case presented with hirsutism, acne, alopecia, anxiety, fatigue and irregular menstrual cycles, but no clinical manifestations suggestive of Cushing's syndrome. Endocrinologic evaluation revealed elevated 08:00 h plasma adrenocorticotropic hormone, serum cortisol and androstenedione concentrations and increased urinary free cortisol excretion. The patient harbored a novel A > G transition at nucleotide position 2177, which resulted in histidine (H) to arginine (R) substitution at amino acid position 726 of the receptor (c.2177A > G, p.H726R). Compared with the wild-type receptor, the mutant receptor hGRαH726R demonstrated decreased ability to transactivate glucocorticoid-responsive genes and to transrepress the nuclear factor-κB signalling pathway, displayed 55% lower affinity for the ligand and a four-fold delay in nuclear translocation, and interacted with the glucocorticoid receptor-interacting protein 1 coactivator mostly through its activation function-1 domain. Finally, a 3-dimensional molecular modelling study of the H726R mutation revealed a significant structural shift in the rigidity of helix 10 of the receptor, which resulted in reduced flexibility and decreased affinity of the mutant receptor for binding to the ligand. CONCLUSIONS: The natural mutant receptor hGRαH726R impairs multiple steps of glucocorticoid signal transduction, thereby decreasing tissue sensitivity to glucocorticoids.

A novel point mutation of the human glucocorticoid receptor gene causes primary generalized glucocorticoid resistance through impaired interaction with the LXXLL motif of the p160 coactivators: dissociation of the transactivating and transreppressive activities.

CONTEXT: Primary generalized glucocorticoid resistance is a rare genetic disorder characterized by generalized, partial, target-tissue insensitivity to glucocorticoids. The molecular basis of the condition has been ascribed to inactivating mutations in the human glucocorticoid receptor (hGR) gene. OBJECTIVE: The objective of the study was to present three new cases caused by a novel mutation in the hGR gene and to delineate the molecular mechanisms through which the mutant receptor impairs glucocorticoid signal transduction. DESIGN AND RESULTS: The index case (father) and his two daughters presented with increased urinary free cortisol excretion and resistance of the hypothalamic-pituitary-adrenal axis to dexamethasone suppression in the absence of clinical manifestations suggestive of Cushing syndrome. All subjects harbored a novel, heterozygous, point mutation (T→G) at nucleotide position 1724 of the hGR gene, which resulted in substitution of valine by glycine at amino acid 575 of the receptor. Compared with the wild-type receptor, the hGRαV575G demonstrated a significant (33%) reduction in its ability to transactivate the mouse mammary tumor virus promoter in response to dexamethasone, a 50% decrease in its affinity for the ligand, and a 2.5-fold delay in nuclear translocation. Although it did not exert a dominant negative effect on the wild-type receptor and preserved its ability to bind to DNA, hGRαV575G displayed significantly enhanced (∼80%) ability to transrepress the nuclear factor-κΒ signaling pathway. Finally, the mutant receptor hGRαV575G demonstrated impaired interaction with the LXXLL motif of the glucocorticoid receptor-interacting protein 1 coactivator in vitro and in computer-based structural simulation via its defective activation function-2 (AF-2) domain. CONCLUSIONS: The natural mutant receptor hGRαV575G causes primary generalized glucocorticoid resistance by affecting multiple steps in the glucocorticoid signaling cascade, including the affinity for the ligand, the time required for nuclear translocation, and the interaction with the glucocorticoid-interacting protein-1 coactivator.

A novel point mutation in the DNA-binding domain (DBD) of the human glucocorticoid receptor causes primary generalized glucocorticoid resistance by disrupting the hydrophobic structure of its DBD.

CONTEXT: Primary generalized glucocorticoid resistance is a rare genetic condition characterized by partial end-organ insensitivity to glucocorticoids. Most affected subjects present with clinical manifestations of mineralocorticoid and androgen excess. The condition has been associated with inactivating mutations in the human glucocorticoid receptor (hGR) gene, which impair the molecular mechanisms of hGRα action, thereby reducing tissue sensitivity to glucocorticoids. OBJECTIVE: ΤHE aim of our study was to investigate the molecular mechanisms through which one previously described natural heterozygous V423A mutation, the second mutation detected in the DNA-binding domain (DBD) of the hGRα, affects glucocorticoid signal transduction. DESIGN AND RESULTS: Compared with the wild-type receptor, hGRαV423A demonstrated a 72% reduction in its ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter in response to dexamethasone. The hGRαV423A receptor showed a significant reduction in its ability to bind to glucocorticoid-response elements of glucocorticoid-responsive genes, owing to structural alterations of the DBD confirmed by computer-based structural analysis. In addition, hGRαV423A demonstrated a 2.6-fold delay in nuclear translocation following exposure to the ligand, although it did not exert a dominant negative effect on the wild-type hGRα, had a similar affinity to the ligand with the wild-type receptor, and displayed a normal interaction with the GRIP1 coactivator in vitro. CONCLUSIONS: The natural mutant receptor hGRαV423A causes primary generalized glucocorticoid resistance by affecting multiple steps in the cascade of glucocorticoid receptor action, which primarily involve decreased ability to bind to target glucocorticoid response elements and delayed translocation into the nucleus.

Mitochondrial uncoupler FCCP activates proton conductance but does not block store-operated Ca(2+) current in liver cells.

Uncouplers of mitochondrial oxidative phosphorylation, including carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) and carbonilcyanide m-cholorophenylhydrazone (CCCP), are widely used in experimental research to investigate the role of mitochondria in cellular function. Unfortunately, it is very difficult to interpret the results obtained in intact cells using FCCP and CCCP, as these agents not only inhibit mitochondrial potential, but may also affect membrane potential and cell volume. Here we show by whole-cell patch clamping that in primary rat hepatocytes and H4IIE liver cells, FCCP induced large proton currents across the plasma membrane, but did not activate any other observable conductance. In intact hepatocytes FCCP inhibits thapsigargin-activated store-operated Ca(2+) entry, but in patch clamping under the conditions of strong Ca(2+) buffering it has no effect on store-operated Ca(2+) current (I(SOC)). These results indicate that there is no direct connection between mitochondria and activation of I(SOC) in liver cells and support the notion of indirect regulation of I(SOC) by mitochondrial Ca(2+) buffering.

Gene expression profile associated with oncogenic ras-induced senescence, cell death, and transforming properties in human cells.

We developed inducible and constitutive expression systems of Ha-RasV12 in HEK 293 cells to examine early oncogenic RasV12 signaling. Inducible expression of oncogenic Ras-triggered growth arrest, early senescence, and later apoptosis. Gene expression profile analysis revealed early Ras proliferation and cell cycle genes like c-fos, cyclin E, cdk2, cell-cell contact, and signaling like integrin a6, MEK5, and free radical signaling genes, like proline oxidase. Therefore, Ras-mediated signaling is a fine regulated process both positively and negatively influencing cell cycle, senescence, and apoptosis pathways. Novel early RAS-target genes could be potentially exploited in cancer diagnostics and therapeutics.

Efficient liver-directed gene transfer by in situ generation of retroviral vector from adenoviral templates.

UNLABELLED: To improve liver-directed retroviral-mediated gene transfer, we injected C57/BL10 mice intravenously with three adenoviral vectors encoding retroviral vector genome and structural components: AdGagPol expressing the respective structural genes of Moloney murine leukaemia virus, Ad10A1Env expressing the 10A1 envelope protein of 10A1-MuLV, and AdLEIN, encoding the LEIN retrovirus genome, expressing green fluorescence protein (eGFP) and the neomycin resistance gene. MATERIALS AND METHODS: The extent of eGFP expression was determined after 1 and 15 weeks by fluorescence microscopy and FACS analysis. Proviral integration was determined by a novel PCR-based technique. RESULTS: Hepatocytes infected with all three Ad vectors generated LEIN retrovirus after one week and in situ transduction of neighbouring cells resulted in stable proviral integration associated with eGFP expression ranging from 4.3% to 20.5% in different liver cell populations 15 weeks post-infection. CONCLUSION: Hybrid adeno-retroviral vectors can be efficiently used to improve the efficiency of retroviral-mediated gene transfer to the liver.

The contribution of voltage-gated Ca2+ currents to K+ channel activation during ovine adrenal chromaffin cell development.

Prior to the development of adrenal innervation, the adrenal medulla is capable of responding to low blood oxygen directly. However, this response is lost once adrenal innervation is established. Previous work by our group has outlined mechanisms involved in this direct hypoxic response and the means by which innervation causes the loss of the direct hypoxic response in the ovine adrenal. The current study further investigates mechanisms which may underlie the developmental loss of the direct hypoxic response by concentrating on two aspects of cell function which regulate catecholamine secretion: the contribution of different types of Ca2+ channels to the total Ca2+ current and the contribution of each Ca2+ channel type to K+ channel activation. We identified that Ca2+ current size at -40 to -10 mV is increased in amplitude in fetal chromaffin cells. This is not due to the increased prevalence or size of T-type Ca2+ currents present at these voltages. The relative contribution of L-, N- or P/Q-type Ca2+ channels to total Ca2+ current and to activation of the K+ current is unchanged during chromaffin cell development, however K+ current density increases with age. Our results indicate that there is a developmental shift in relative expression of T-type, but not L-, N- or P/Q-type, Ca2+ channels in ovine chromaffin cells. The increased K+ current density in adult cells may result in an altered response to an equal stimulus, while larger Ca2+ current at negative voltages in fetal cells may facilitate Ca2+ entry and catecholamine secretion in response to small depolarisations such as those induced by hypoxia.

In vivo investigation of tolerance and antitumor activity of cisplatin-loaded PLGA-mPEG nanoparticles.

The tolerance of BALB/c mice to different doses of blank and cisplatin-loaded PLGA-mPEG nanoparticles and the in vivo anticancer activity of these nanoparticles on SCID mice xenografted with colorectal adenocarcinoma HT 29 cells were investigated. Nanoparticles with an average size of 150-160 nm and approximately 2% w/w cisplatin content were prepared by a modified emulsification and solvent evaporation method. Normal BALB/c mice tolerated three weekly intravenous injections of a relatively high dose of blank PLGA-mPEG nanoparticles (500 mg/kg, equivalent to about 10mg nanoparticles/mouse) and three weekly intravenous injections of a high dose of nanoparticle-entrapped cisplatin (10 mg/kg). Also, histopathology examination indicated that there were no differences in the kidneys or spleens from animals treated with cisplatin-loaded nanoparticles or blank nanoparticles compared to the untreated control group. A moderate granulation of protoplasm of hepatic cells was observed in the livers from mice treated with cisplatin-loaded nanoparticles and blank nanoparticles, however, both the hepatic lobe and the portal hepatis maintained their normal architecture. The cisplatin-loaded PLGA-mPEG nanoparticles appeared to be effective at delaying tumor growth in HT 29 tumor-bearing SCID mice. The group of mice treated with cisplatin-loaded nanoparticles exhibited higher survival rate compared to the free cisplatin group. The results justify further evaluation of the in vivo antitumor efficacy of the PLGA-mPEG/cisplatin nanoparticles.

Adenovirus-based targeting in myoblasts is hampered by nonhomologous vector integration.

Chromosomal correction of dystrophin gene mutations is a most desirable therapeutic solution for Duchenne muscular dystrophy, as it allows production of the full-length dystrophin under the control of locus-specific promoters. Here we explored gene targeting in conditionally immortal mouse dystrophin-deficient myoblasts. We constructed an adenoviral vector for the correction of the mdx mutation, containing 6.0 kb of sequence homologous to the target locus (partial intron 21 through to exon 24 with the normal sequence of exon 23) and a neomycin expression cassette inserted in intron 23. Adenovirus-based gene targeting was previously reported to be beneficial in mouse embryonic stem cells, resulting in one targeted integration per three integration events. However, we found no targeted integration events among 144 stably transduced G418-resistant myoblast clones, reflecting efficient random integration of the adenoviral vector in myogenic cells. We found that mouse myoblasts are capable of integrating recombinant adenoviral DNA with an efficiency approaching 1%. Interestingly, dermal fibroblasts integrate adenoviral DNA up to 100 times less efficiently than myoblasts from the same mice. We also show that the efficiency of recombinant adenoviral DNA integration is influenced by preinfection cell density, possibly indicating the importance of cellular DNA replication for adenoviral integration.

Synthesis, characterization, and remarkable biological properties of cyclodextrins bearing guanidinoalkylamino and aminoalkylamino groups on their primary side.

The introduction of aminoalkylamino and guanidinoalkylamino substituents on the primary side of beta- and gamma-cyclodextrin (CDs) resulted in a series of novel compounds that were extensively characterized by NMR spectroscopy and mass spectrometry. Bromination of the primary side of beta- and gamma-CD, and reaction with neat alkylene diamines at a pressure of 7 atm afforded aminoalkylamino derivatives that were then guanylated at the primary amino group to give the corresponding guanidinoalkylamino-CDs. These compounds are water soluble and display pK(a) values that allow them to be mostly protonated at neutral pH; for example, pK(a(1)) approximately 6.4 and pK(a(2)) approximately 9.5 for the aminoethylamino-beta-CD and pK(a(1)) approximately 7.8 and pK(a(2)) approximately 11.0 for the guanidinoethylamino-beta-CD. The title CDs are rigid, cyclic alpha-D-glucopyranose oligomers (heptamers or octamers) with branches that resemble lysine and arginine side chains that enable multiple interactions with suitable substrates. Thus, they bear similarities to known cell-penetrating peptides. Indeed, the compounds were found to cross the membranes of HeLa cells and penetrate inside the cytoplasm quickly, the guadinylated ones within 15 min, as shown by fluorescence microscopy using fluorescein-labeled derivatives. The toxicity of the compounds, measured by performing MTT tests, ranged from 50 to 300 microM. Furthermore, some of the aminated CDs could facilitate the transfection of DNA expressing the green fluorescent protein (GFP) in HEK 293T cells, with effectiveness comparable to the commercial agent Lipofectamine 2000. Circular dichroism, atomic force microscopy and electrophoresis experiments confirmed the strong interaction of the compounds with DNA. Because of their carbohydrate, non-peptide nature the title compounds are not anticipated to be enzymatically labile or immunogenic, and thus they fulfill many of the criteria for non-hazardous transport vectors in biological and pharmaceutical applications.

TNF receptor I sensitizes neurons to erythropoietin- and VEGF-mediated neuroprotection after ischemic and excitotoxic injury.

CNS neurons use robust cytoprotective mechanisms to ensure survival and functioning under conditions of injury. These involve pathways induced by endogenous neuroprotective cytokines such as erythropoietin (EPO). Recently, in contrast to its well known deleterious roles, TNF has also been shown to exhibit neuroprotective properties. In the present study, we investigated the molecular mechanisms by which TNF receptor (TNFR)I mediates neuroprotection by comparing the gene expression profiles of lesioned cortex from WT and TNFRI KO mice after permanent middle cerebral artery occlusion. Several known neuroprotective molecules were identified as TNFRI targets, notably members of the Bcl-2 family, DNA repair machinery and cell cycle, developmental, and differentiation factors, neurotransmitters and growth factors, as well as their receptors, including EPO receptor (EPOR), VEGF, colony-stimulating factor receptor 1, insulin-like growth factor (IGF), and nerve growth factor (NGF). Further analysis showed that induction of EPOR and VEGF expression in primary cortical neurons after glucose deprivation (GD) largely depended on TNFRI and was further up-regulated by TNF. Also, EPO- and VEGF-induced neuroprotection against GD, oxygen-glucose deprivation, and NMDA excitotoxicity depended significantly on TNFRI presence. Finally, EPO prevented neuronal damage induced by kainic acid in WT but not TNFRI KO mice. Our results identify cross-talk between tissue protective cytokines, specifically that TNFRI is necessary for constitutive and GD-induced expression of EPOR and VEGF and for EPO-mediated neuroprotection.

Interpreting microarray data: towards the complete bioinformatics toolkit for cancer.

Functional genomics has been applied in the study of human malignancies since the inception of this field nearly a decade ago. Microarray analysis has been specifically used in an attempt to re-classify carcinomas at the molecular level, to aid in diagnosis/prognosis and to predict how various types of tumour respond to different therapeutic agents. Bioinformatics is now at the forefront of the post-genomics era and is providing a number of tools with which to mine the large datasets produced by genome-wide analysis. Of particular importance is the emergence of techniques that give the ability to reveal the transcription regulatory networks that are active in the cell in response to environmental stimuli or disease states. Deciphering the transcription networks that function in malignant cells not only will provide the knowledge to understand how carcinomas progress, but would also allow the construction of useful therapeutic tools for their effective treatment. In this review the recent advances that have been made in functional genomics that allow microarray data to be more fully interpreted and reveal the transcription networks that have gone awry in transformed cells are described.

FLIP(L) protects neurons against in vivo ischemia and in vitro glucose deprivation-induced cell death.

Knowledge of the molecular mechanisms that underlie neuron death after stroke is important to allow the development of effective neuroprotective strategies. In this study, we investigated the contribution of death receptor signaling pathways to neuronal death after ischemia using in vitro and in vivo models of ischemic injury and transgenic mice that are deficient in tumor necrosis factor receptor I (TNFRI KO) or show neuron-specific overexpression of the long isoform of cellular Fas-associated death domain-like interleukin-1-beta-converting enzyme-inhibitory protein (FLIP(L)). Caspase 8 was activated in brain lesions after permanent middle cerebral artery occlusion (pMCAO) and in cortical neurons subjected to glucose deprivation (GD) and was necessary for GD-induced neuron death. Thus, neurons treated with zIETD-FMK peptide or overexpressing a dominant-negative caspase 8 mutant were fully protected against GD-induced death. The presence of the neuroprotective TNFRI was necessary for selectively sustaining p50/p65NF-kappaB activity and the expression of the p43 cleavage form of FLIP(L), FLIP(p43), an endogenous inhibitor of caspase 8, in pMCAO lesions and GD-treated neurons. Moreover, TNF pretreatment further upregulated p50/p65NF-kappaB activity and FLIP(p43) expression in neurons after GD. The knock-down of FLIP in wild-type (WT) neurons using a short hairpin RNA revealed that FLIP(L) is essential for TNF/TNFRI-mediated neuroprotection after GD. Furthermore, the overexpression of FLIP(L) was sufficient to rescue TNFRI KO neurons from GD-induced death and to enhance TNF neuroprotection in WT neurons, and neuron-specific expression of FLIP(L) in transgenic mice significantly reduced lesion volume after pMCAO. Our results identify a novel role for the TNFRI-NF-kappaB-FLIP(L) pathway in neuroprotection after ischemia and identify potential new targets for stroke therapy.

Quercetin mediates preferential degradation of oncogenic Ras and causes autophagy in Ha-RAS-transformed human colon cells.

Several food polyphenols act as chemopreventers by reducing the incidence of many types of cancer, especially in colon epithelia. In this study, we have investigated whether the flavonoid quercetin can modulate cell proliferation and survival by targeting key molecules and/or biological processes responsible for tumor cell properties. The effect of quercetin on the expression of Ras oncoproteins was specifically studied using systems of either constitutive or conditional expression of oncogenic RAS in human epithelial cells. Our findings suggest that quercetin inhibits cell viability as well as cancer cell properties like anchorage-independent growth. These findings were further supported at the molecular level, since quercetin treatment resulted in a preferential reduction of Ras protein levels in cell lines expressing oncogenic Ras proteins. Notably, in cells that only express wild-type Ras or in those where the oncogenic Ras allele was knocked out, quercetin had no evident effects upon Ras levels. We have shown that quercetin drastically reduces half-life of oncogenic Ras but has no effect when the cells are treated with a proteasome inhibitor. Moreover, in Ha-RAS-transformed cells, quercetin induces autophagic processes. Since quercetin downregulates the levels of oncogenic Ras in cancer cells, we propose that this flavonoid could act as a chemopreventive agent for cancers with frequent mutations of RAS genes.

Glucagon activates Ca2+ and Cl- channels in rat hepatocytes.

Glucagon is one of the major hormonal regulators of glucose metabolism, counteracting the hepatic effects of insulin when the concentration of glucose in the bloodstream falls below a certain level. Glucagon also regulates bile flow, hepatocellular volume and membrane potential of hepatocytes. It is clear that changes in cell volume and membrane potential cannot occur without significant ion fluxes across the plasma membrane. The effects of glucagon on membrane currents in hepatocytes, however, are not well understood. Here we show, by patch-clamping of rat hepatocytes, that glucagon activates two types of currents: a small inwardly rectifying Ca2+ current with characteristics similar to those of the store-operated Ca2+ current and a larger outwardly rectifying Cl- current similar to that activated by cell swelling. We show that the mechanism of glucagon action on membrane conductance involves phospholipase C and adenylyl cyclase. Contribution of the adenylyl cyclase-dependent pathway to activation of the currents depended on Epac (exchange protein directly activated by cAMP), but not on protein kinase A. The activation of Ca2+ and Cl- channels is likely to play a key role in the mechanisms by which glucagon regulates hepatocyte metabolism and volume.

Microarray analysis of the differential transformation mediated by Kirsten and Harvey Ras oncogenes in a human colorectal adenocarcinoma cell line.

Colorectal cancer arises after a series of mutational events in the colon epithelia and is often used as a model of the multistep progression of tumorigenesis. Mutations in Ki-Ras have been detected in some 50% of cases and are thought to occur at an early stage. Almost never do mutations arise in the loci of other Ras isoforms (Ha- and N-), leading to the assumption that Ki-Ras plays a unique role in tumorigenesis. In order to examine the distinctive function that Ki-Ras plays in cancer development in the colon, we introduced constitutively active mutant Ki- and Ha-Ras genes into an intermediate-stage colon adenoma cell line (Caco-2). We found that mutant active Ha-RasV12 was more efficient at transforming these colon epithelial cells as assessed by anchorage-independent growth, tumor formation in SCID mice and the development of mesenchymal morphology compared to transformation by Ki-RasV12. We conducted microarray analysis in an attempt to reveal the genes whose aberrant expression is a direct result of overexpression of either Ki-RasV12 or Ha-RasV12. We used Clontech's Atlas cancer cDNA (588 genes) and RZPD's Onco Set 1 (1,544 genes) arrays. We identified fewer genes that were commonly regulated than were differentially expressed between Ki- and Ha-RasV12 isoforms. Specifically, we found that Ki-RasV12 regulated genes involved in cytokine signaling, cell adhesion and colon development, whereas Ha-RasV12 mainly regulated genes involved in controlling cell morphology, correlating to an epithelial-mesenchymal transition only observed in these cells. Our results demonstrate how 2 Ras isoforms regulate disparate biologic processes, revealing a number of genes whose deregulated expression may influence colon carcinogenesis (supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html).

Oxygen-sensing pathway for SK channels in the ovine adrenal medulla.

1. The intracellular pathways that modulate the opening of oxygen-sensitive ion channels during periods of hypoxia are poorly understood. Different tissues appear to use either NADPH oxidase or a rotenone-sensitive mechanism as an oxygen sensor. The aim of the present study was to identify the oxygen-sensing pathway in the oxygen-sensitive sheep adrenal medullary chromaffin cell (AMCC). 2. The whole-cell patch-clamp technique was used to measure K+ currents in dissociated adult ovine chromaffin cells as well as SK channel currents expressed in the H4IIE cell line. 3. Diphenyliodonium, an inhibitor of NADPH oxidase, had no effect on the hypoxia-evoked closure of K+ channels in primary AMCC, whereas the mitochondrial inhibitor rotenone abolished the hypoxia-evoked response. Both these compounds significantly reduced K+ current amplitude under normoxic conditions. 4. One possible mechanism through which the oxygen sensor may modulate K+ channel activity is by altering the redox state of the cell. In sheep AMCC, altering the redox state by the addition of H2O2 to the extracellular solution increased K+ conductance. 5. The oxygen-sensitive K+ (Ko2) channels in sheep chromaffin cells are from the SK family and the whole-cell conductance of cells expressing mouse SK2 or SK3, but not human SK1, was increased by H2O2 and decreased by the reducing agent dithiothreitol. 6. These studies show that, in sheep AMCC, Ko2 channels are modulated via a rotenone-sensitive mechanism and that alteration of the cellular redox state mimics the change produced by alterations in Po2. In a heterologous expression system, SK2 and SK3 channels, the channels that initiate hypoxia-evoked changes in AMCC function, are modulated appropriately by changes in cellular redox state.

Zinc inhibits human ClC-1 muscle chloride channel by interacting with its common gating mechanism.

Transition metals block the muscle Cl- channel ClC-1, which belongs to a large family of double-barreled Cl- channels and transporters. In the Torpedo Cl- channel ClC-0, Zn2+ block is closely related to the common gating mechanism that opens and closes both pores of the channel simultaneously, and the mutation C212S, which locks the common gate open, also eliminates the block. In ClC-1, however, previous results suggested that Zn2+ block is independent of gating, and that the cysteine residues involved in Zn2+ binding are in different positions to those that confer Zn2+ sensitivity on ClC-0. In this work, we show that Zn2+ block of ClC-1 is faster at hyperpolarized potentials where the channel is more likely to be in the closed state. Mutation C277S, equivalent to C212S in ClC-0, which locks the common gate in ClC-1 open, virtually eliminates Zn2+ block. A mutation, V321A, which reduces open probability of the common gate, facilitated Zn2+ block. These results demonstrate that Zn2+ block is state dependent, acting on the common gate. The extent of the block, however, is not a simple function of the open probability of the common gate. The Q10 of approximately 13 of the time course of Zn2+ block, which is significantly higher than the Q10 of common gating transitions in WT ClC-1, suggests that Zn2+ binds to a very high temperature-dependent low-probability closed substate of the common gate, which has not yet been characterized in this channel.