bluuebunny/crossref_metadata_2025 huggingface dataset:
PPARα activation and increased dietary lipid oppose thyroid hormone signaling and rescue impaired glucose-stimulated insulin secretion in hyperthyroidism
Authors: Holness Mark J. , Greenwood Gemma K. , Smith Nicholas D. , Sugden Mary C.
Published: October 2008

The aim of the study was to investigate the impact of hyperthyroidism on the characteristics of the islet insulin secretory response to glucose, particularly the consequences of competition between thyroid hormone and peroxisome proliferator-activated receptor (PPAR)α in the regulation of islet adaptations to starvation and dietary lipid-induced insulin resistance. Rats maintained on standard (low-fat/high-carbohydrate) diet or high-fat/low-carbohydrate diet were rendered hyperthyroid (HT) by triiodothyronine (T3) administration (1 mg·kg body wt−1·day−1sc, 3 days). The PPARα agonist WY14643 (50 mg/kg body wt ip) was administered 24 h before sampling. Glucose-stimulated insulin secretion (GSIS) was assessed during hyperglycemic clamps or after acute glucose bolus injection in vivo and with step-up and step-down islet perifusions. Hyperthyroidism decreased the glucose responsiveness of GSIS, precluding sufficient enhancement of insulin secretion for the degree of insulin resistance, in rats fed either standard diet or high-fat diet. Hyperthyroidism partially opposed the starvation-induced increase in the glucose threshold for GSIS and decrease in glucose responsiveness. WY14643 administration restored glucose tolerance by enhancing GSIS in fed HT rats and relieved the impact of hyperthyroidism to partially oppose islet starvation adaptations. Competition between thyroid hormone receptor (TR) and PPARα influences the characteristics of GSIS, such that hyperthyroidism impairs GSIS while PPARα activation (and increased dietary lipid) opposes TR signaling and restores GSIS in the fed hyperthyroid state. Increased islet PPARα signaling and decreased TR signaling during starvation facilitates appropriate modification of islet function.
Methyl-2-acetamidoacrylate, an ethyl pyruvate analog, decreases sepsis-induced acute kidney injury in mice
Authors: Leelahavanichkul Asada , Yasuda Hideo , Doi Kent , Hu Xuzhen , Zhou Hua , Yuen Peter S. T. , Star Robert A.
Published: October 2008

We tested the anti-inflammatory agent methyl-2-acetamidoacrylate (M2AA), an ethyl pyruvate analog, in a cecal ligation-and-puncture (CLP) model of sepsis in CD-1 mice. M2AA administration at the time of CLP improved survival, renal function, kidney histology, liver injury, and splenocyte apoptosis, and lowered cytokine levels (TNF-α, IL-6, IFN-γ, and IL-10). When M2AA treatment was delayed 6 h (but not 12 h), M2AA still significantly reduced kidney dysfunction, liver injury, splenocyte apoptosis, and cytokine levels. NF-κB, a M2AA target, was transiently activated in spleen, peaking at 6 h; kidney and liver NF-κB increased steadily with a plateau at 12–24 h. M2AA reduced NF-κB activation in spleen at 6 h and in kidney and liver at 24 h. Splenectomy diminished the ability of M2AA to reduce cytokines, especially IL-6, but M2AA still decreased kidney and liver dysfunction, suggesting that splenic NF-κB is not central to M2AA action. In contrast, beneficial effects of chloroquine on cytokines and organ damage were neutralized by splenectomy, demonstrating a spleen-specific chloroquine target. Because M2AA and chloroquine act differently, we tested this combination. Survival at 96 h was highest with combination therapy (57%) vs. chloroquine (38%), M2AA (47.6%), or vehicle (5%). The benefit of combination therapy over chloroquine or M2AA alone did not reach statistical significance, indicating potential mechanistic overlap. We conclude that the transient target(s) for M2AA responsible for the narrow 6-h therapeutic window is not splenic NF-κB. Identifying this new target and downstream signaling pathways could lengthen the therapeutic window and improve combination therapy with chloroquine.
LC-MS/MS analysis of differential centrifugation fractions from native inner medullary collecting duct of rat
Authors: Sachs Aaron N. , Pisitkun Trairak , Hoffert Jason D. , Yu Ming-Jiun , Knepper Mark A.
Published: October 2008

We carried out LC-MS/MS-based proteomic profiling of differential centrifugation fractions from rat inner medullary collecting duct (IMCD): 1) to provide baseline knowledge of the IMCD proteome and 2) to evaluate the utility of differential centrifugation in assessing trafficking of the water channel aquaporin-2 (AQP2). IMCD suspensions were freshly prepared from rat kidneys using standard methods. Homogenized samples were subjected to sequential centrifugations at 1,000, 4,000, 17,000, and 200,000 g. These samples, as well as the final supernatant, were subjected to LC-MS/MS analysis. Preliminary immunoblotting confirmed that the ratio of AQP2 in the 17,000- g fraction to the 200,000- g fraction underwent an increase in response to the vasopressin analog dDAVP, largely due to a reduction in the 200,000- g fraction. Immunoblotting for the major phosphorylated forms of AQP2 revealed that phosphorylated AQP2 was present in both the 17,000- and 200,000- g fractions. LC-MS/MS analysis showed that markers of “intracellular vesicles,” chiefly endosomal markers, were present in both the 17,000- and the 200,000- g fractions. In contrast, plasma membrane proteins were predominantly present in the 4,000- and 17,000- g fractions. Proteins associated with several multiprotein complexes (e.g., actin-related protein 2/3 complex and proteasome complex) were virtually exclusively present in the 200,000- g fraction. Overall, we identified 656 proteins, including 189 not previously present in the IMCD database. The data show that both the 17,000- and 200,000- g fractions are highly heterogeneous and cannot be equated with “plasma membrane” and “intracellular vesicle” fractions, respectively, leading us to propose an alternative approach for use of differential centrifugation to assess vesicular trafficking to the plasma membrane.
Double-Pulse Magnetic Brain Stem Stimulation: Mimicking Successive Descending Volleys
Authors: Matsumoto Hideyuki , Hanajima Ritsuko , Hamada Masashi , Terao Yasuo , Yugeta Akihiro , Inomata-Terada Satomi , Nakatani-Enomoto Setsu , Tsuji Shoji , Ugawa Yoshikazu
Published: October 2008

Magnetic stimulation with a double-cone-coil over the back of the head activates the motor tracts at the level of pyramidal decussation (brain stem stimulation [BST]). However, single-pulse BST (single BST) sometimes cannot elicit motor-evoked potentials (MEPs) in patients with corticospinal tract involvement. We developed a technique using double-pulse BST (double BST) to elicit MEPs even in patients whose threshold is abnormally elevated. Subjects were 11 healthy volunteers and 12 patients with corticospinal tract involvement in whom single BST evoked no discernible MEP. Double BST was performed at the intensities of resting and active motor threshold for single BST; MEPs were recorded from the first dorsal interosseous muscle. Interstimulus intervals (ISIs) between two pulses were 1.5, 2, 3, 5, and 10 ms in healthy subjects. Double BST enlarged MEPs at ISIs of 1.5–5 ms with a peak at 2 ms in the relaxed condition, but not in the active condition. At an ISI of 2 ms in the relaxed condition, the MEP amplitude was 15 times as large as that to single BST in relaxed muscles. The onset latency of the enlarged MEP from the second pulse in relaxed muscles was the same as that by single BST in active muscles. Double BST at a 2-ms interval elicited MEPs in eight patients. Double BST can enhance MEPs probably by temporal summations of excitatory postsynaptic potentials at the spinal motoneurons. Using this new technique, we can obtain more information about the central motor conduction even when single BST fails to elicit any MEP.
Localization and Targeting of Voltage-Dependent Ion Channels in Mammalian Central Neurons
Authors: Vacher Helene , Mohapatra Durga P. , Trimmer James S.
Published: October 2008

The intrinsic electrical properties and the synaptic input-output relationships of neurons are governed by the action of voltage-dependent ion channels. The localization of specific populations of ion channels with distinct functional properties at discrete sites in neurons dramatically impacts excitability and synaptic transmission. Molecular cloning studies have revealed a large family of genes encoding voltage-dependent ion channel principal and auxiliary subunits, most of which are expressed in mammalian central neurons. Much recent effort has focused on determining which of these subunits coassemble into native neuronal channel complexes, and the cellular and subcellular distributions of these complexes, as a crucial step in understanding the contribution of these channels to specific aspects of neuronal function. Here we review progress made on recent studies aimed to determine the cellular and subcellular distribution of specific ion channel subunits in mammalian brain neurons using in situ hybridization and immunohistochemistry. We also discuss the repertoire of ion channel subunits in specific neuronal compartments and implications for neuronal physiology. Finally, we discuss the emerging mechanisms for determining the discrete subcellular distributions observed for many neuronal ion channels.
Ion Channels in Microbes
Authors: Martinac Boris , Saimi Yoshiro , Kung Ching
Published: October 2008

Studies of ion channels have for long been dominated by the animalcentric, if not anthropocentric, view of physiology. The structures and activities of ion channels had, however, evolved long before the appearance of complex multicellular organisms on earth. The diversity of ion channels existing in cellular membranes of prokaryotes is a good example. Although at first it may appear as a paradox that most of what we know about the structure of eukaryotic ion channels is based on the structure of bacterial channels, this should not be surprising given the evolutionary relatedness of all living organisms and suitability of microbial cells for structural studies of biological macromolecules in a laboratory environment. Genome sequences of the human as well as various microbial, plant, and animal organisms unambiguously established the evolutionary links, whereas crystallographic studies of the structures of major types of ion channels published over the last decade clearly demonstrated the advantage of using microbes as experimental organisms. The purpose of this review is not only to provide an account of acquired knowledge on microbial ion channels but also to show that the study of microbes and their ion channels may also hold a key to solving unresolved molecular mysteries in the future.
Diffusion in Brain Extracellular Space
Authors: Syková Eva , Nicholson Charles
Published: October 2008

Diffusion in the extracellular space (ECS) of the brain is constrained by the volume fraction and the tortuosity and a modified diffusion equation represents the transport behavior of many molecules in the brain. Deviations from the equation reveal loss of molecules across the blood-brain barrier, through cellular uptake, binding, or other mechanisms. Early diffusion measurements used radiolabeled sucrose and other tracers. Presently, the real-time iontophoresis (RTI) method is employed for small ions and the integrative optical imaging (IOI) method for fluorescent macromolecules, including dextrans or proteins. Theoretical models and simulations of the ECS have explored the influence of ECS geometry, effects of dead-space microdomains, extracellular matrix, and interaction of macromolecules with ECS channels. Extensive experimental studies with the RTI method employing the cation tetramethylammonium (TMA) in normal brain tissue show that the volume fraction of the ECS typically is ∼20% and the tortuosity is ∼1.6 (i.e., free diffusion coefficient of TMA is reduced by 2.6), although there are regional variations. These parameters change during development and aging. Diffusion properties have been characterized in several interventions, including brain stimulation, osmotic challenge, and knockout of extracellular matrix components. Measurements have also been made during ischemia, in models of Alzheimer's and Parkinson's diseases, and in human gliomas. Overall, these studies improve our conception of ECS structure and the roles of glia and extracellular matrix in modulating the ECS microenvironment. Knowledge of ECS diffusion properties is valuable in contexts ranging from understanding extrasynaptic volume transmission to the development of paradigms for drug delivery to the brain.
Calcium Sparks
Authors: Cheng Heping , Lederer W. J.
Published: October 2008

The calcium ion (Ca2+) is the simplest and most versatile intracellular messenger known. The discovery of Ca2+sparks and a related family of elementary Ca2+signaling events has revealed fundamental principles of the Ca2+signaling system. A newly appreciated “digital” subsystem consisting of brief, high Ca2+concentration over short distances (nanometers to microns) comingles with an “analog” global Ca2+signaling subsystem. Over the past 15 years, much has been learned about the theoretical and practical aspects of spark formation and detection. The quest for the spark mechanisms [the activation, coordination, and termination of Ca2+release units (CRUs)] has met unexpected challenges, however, and raised vexing questions about CRU operation in situ. Ample evidence shows that Ca2+sparks catalyze many high-threshold Ca2+processes involved in cardiac and skeletal muscle excitation-contraction coupling, vascular tone regulation, membrane excitability, and neuronal secretion. Investigation of Ca2+sparks in diseases has also begun to provide novel insights into hypertension, cardiac arrhythmias, heart failure, and muscular dystrophy. An emerging view is that spatially and temporally patterned activation of the digital subsystem confers on intracellular Ca2+signaling an exquisite architecture in space, time, and intensity, which underpins signaling efficiency, stability, specificity, and diversity. These recent advances in “sparkology” thus promise to unify the simplicity and complexity of Ca2+signaling in biology.
Structural Basis of Protein Kinase C Isoform Function
Authors: Steinberg Susan F.
Published: October 2008

Protein kinase C (PKC) isoforms comprise a family of lipid-activated enzymes that have been implicated in a wide range of cellular functions. PKCs are modular enzymes comprised of a regulatory domain (that contains the membrane-targeting motifs that respond to lipid cofactors, and in the case of some PKCs calcium) and a relatively conserved catalytic domain that binds ATP and substrates. These enzymes are coexpressed and respond to similar stimulatory agonists in many cell types. However, there is growing evidence that individual PKC isoforms subserve unique (and in some cases opposing) functions in cells, at least in part as a result of isoform-specific subcellular compartmentalization patterns, protein-protein interactions, and posttranslational modifications that influence catalytic function. This review focuses on the structural basis for differences in lipid cofactor responsiveness for individual PKC isoforms, the regulatory phosphorylations that control the normal maturation, activation, signaling function, and downregulation of these enzymes, and the intra-/intermolecular interactions that control PKC isoform activation and subcellular targeting in cells. A detailed understanding of the unique molecular features that underlie isoform-specific posttranslational modification patterns, protein-protein interactions, and subcellular targeting (i.e., that impart functional specificity) should provide the basis for the design of novel PKC isoform-specific activator or inhibitor compounds that can achieve therapeutically useful changes in PKC signaling in cells.
Application of He′s Variational Iteration Method to Solve Semidifferential Equations of nth Order
Authors: Ghorbani Asghar , Alavi Abdolsaeed
Published: October 2008

He′s variational iteration method is applied to solve nth order semidifferential equations. Comparison is made between collocation spline method based on Lagrange interpolation and the present method. In this method, the solution is calculated in the form of a convergent series with an easily computable component. This approach does not need linearization, weak nonlinearity assumptions, or perturbation theory. Some examples are given to illustrate the effectiveness of the method; the results show that He′s method provides a straightforward and powerful mathematical tool for solving various semidifferential equations of the nth order.
Power Distribution and Possible Influence on Fuel Failure in WWER-1000
Authors: Mikuš Ján
Published: October 2008

The work is focused on the influence of investigation of some core heterogeneities and construction materials on the space power (fission rate) distribution in WWER-1000-type cores, especially from viewpoint of the values and gradient occurrence that could result in static loads with some consequences, for example, fuel pin (FP) or fuel assembly (FA) bowing and possible contribution to the FP failure root causes. For this purpose, experimental data and their analysis from two earlier performed measurements on light water, zero-power reactor LR-0 were used, concerning the relative radial power distribution determined by measurements in a WWER-1000-type core containing single FPs with homogeneous gadolinium admixture () and the relative radial power distribution determined by measurements in FA situated on the periphery of a WWER-1000-type core neighbouring the baffle (thermal shielding).
Kinetics and Mechanism of Oxidation of Leucine and Alanine by Ag(III) Complex in Alkaline Medium
Authors: Song Changying , Chen Lei , Shan Jinhuan
Published: October 2008

Kinetics and mechanism of oxidation of leucine and alanine by Ag(III) complex were studied spectrophotometrically in alkaline medium at constant ion strength. The reaction was in first order with respect to Ag(III) complex and amino acids (leucine, alanine). The second‐order rate constant, k−, decreased with the increasing in [OH−] and . A plausible mechanism was proposed from the kinetics study, and the rate equations derived from mechanism can explain all experimental phenomena. The activation parameters were calculated at 298.2 K.
Semaphorin 3B Inhibits the Phosphatidylinositol 3-Kinase/Akt Pathway through Neuropilin-1 in Lung and Breast Cancer Cells
Authors: Castro-Rivera Emely , Ran Sophia , Brekken Rolf A. , Minna John D.
Published: October 2008

Semaphorin 3B (SEMA3B), located at 3p21.3, is a secreted member of the semaphorin family important in axonal guidance. SEMA3B undergoes allele and expression loss in lung and breast cancer and can function as a tumor suppressor. Previously, we found that SEMA3B induces apoptosis in tumor cells either by reexpression or when applied as a soluble ligand. SEMA3B-induced apoptosis was mediated, in part, by blocking vascular endothelial growth factor autocrine activity in tumor cells. In the current study, treatment of lung and breast cancer cells with picomolar concentrations of soluble SEMA3B inhibited their growth; induced apoptosis; and was associated with decreased Akt phosphorylation, increase in cytochrome c release and caspase-3 cleavage, as well as increased phosphorylation of several proapoptotic proteins, including glycogen synthase kinase-3β, FKHR, and MDM-2. Lung and breast cancer lines resistant to SEMA3B did not show these signaling changes and a tumor-derived missense SEMA3B mutant was inactive in this regard, providing specificity. SEMA3B-mediated inhibition of proliferation and induction of apoptosis in cancer cells were blocked by expressing a constitutively active Akt mutant and are linked to tumor cell expression of neuropilin-1 (Np-1). SEMA3B-insensitive Np-1–negative tumor cells acquired sensitivity to SEMA3B after forced expression of Np-1, whereas SEMA3B-sensitive Np-1–positive tumor cells lost sensitivity to SEMA3B after knockdown of Np-1 by small interfering RNA. We conclude that SEMA3B is a potential tumor suppressor that induces apoptosis in SEMA3B-inactivated tumor cells through the Np-1 receptor by inactivating the Akt signaling pathway. [Cancer Res 2008;68(20):8295–303]
Feedback Mechanisms Promote Cooperativity for Small Molecule Inhibitors of Epidermal and Insulin-Like Growth Factor Receptors
Authors: Buck Elizabeth , Eyzaguirre Alexandra , Rosenfeld-Franklin Maryland , Thomson Stuart , Mulvihill Mark , Barr Sharon , Brown Eric , O'Connor Mathew , Yao Yan , Pachter Jonathan , Miglarese Mark , Epstein David , Iwata Kenneth K. , Haley John D. , Gibson Neil W. , Ji Qun-Sheng
Published: October 2008

Epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR) can cooperate to regulate tumor growth and survival, and synergistic growth inhibition has been reported for combined blockade of EGFR and IGF-IR. However, in preclinical models, only a subset of tumors exhibit high sensitivity to this combination, highlighting the potential need for patient selection to optimize clinical efficacy. Herein, we have characterized the molecular basis for cooperative growth inhibition upon dual EGFR and IGF-IR blockade and provide biomarkers that seem to differentiate response. We find for epithelial, but not for mesenchymal-like, tumor cells that Akt is controlled cooperatively by EGFR and IGF-IR. This correlates with synergistic apoptosis and growth inhibition in vitro and growth regression in vivo upon combined blockade of both receptors. We identified two molecular aspects contributing to synergy: (a) inhibition of EGFR or IGF-IR individually promotes activation of the reciprocal receptor; (b) inhibition of EGFR-directed mitogen-activated protein kinase (MAPK) shifts regulation of Akt from EGFR toward IGF-IR. Targeting the MAPK pathway through downstream MAPK/extracellular signal-regulated kinase kinase (MEK) antagonism similarly promoted IGF-driven pAkt and synergism with IGF-IR inhibition. Mechanistically, we find that inhibition of the MAPK pathway circumvents a negative feedback loop imposed on the IGF-IR– insulin receptor substrate 1 (IRS-1) signaling complex, a molecular scenario that parallels the negative feedback loop between mTOR-p70S6K and IRS-1 that mediates rapamycin-directed IGF-IR signaling. Collectively, these data show that resistance to inhibition of MEK, mTOR, and EGFR is associated with enhanced IGF-IR–directed Akt signaling, where all affect feedback loops converging at the level of IRS-1. [Cancer Res 2008;68(20):8322–32]
Genetic Instability Caused by Loss of MutS Homologue 3 in Human Colorectal Cancer
Authors: Haugen Astrid C. , Goel Ajay , Yamada Kanae , Marra Giancarlo , Nguyen Thuy-Phuong , Nagasaka Takeshi , Kanazawa Shinsaku , Koike Junichi , Kikuchi Yoshinori , Zhong Xiaoling , Arita Michitsune , Shibuya Kazutoshi , Oshimura Mitsuo , Hemmi Hiromichi , Boland C. Richard , Koi Minoru
Published: October 2008

Microsatellite instability (MSI) is a hallmark of mismatch repair (MMR) deficiency. High levels of MSI at mononucleotide and dinucleotide repeats in colorectal cancer (CRC) are attributed to inactivation of the MMR genes, hMLH1 and hMSH2. CRC with low levels of MSI (MSI-L) exists; however, its molecular basis is unclear. There is another type of MSI—elevated microsatellite alterations at selected tetranucleotide repeats (EMAST)—where loci containing [AAAG]n or [ATAG]n repeats are unstable. EMAST is frequent in non-CRCs; however, the incidence of EMAST and its cause in CRC is not known. Here, we report that MutS homologue 3 (MSH3) knockdown or MSH3-deficient cells exhibit the EMAST phenotype and low levels of mutations at dinucleotide repeats. About 60% of 117 sporadic CRC cases exhibit EMAST. All of the cases defined as MSI-H (16 cases) exhibited high levels of EMAST. Among 101 non–MSI-H cases, all 19 cases of MSI-L and 35 of 82 cases of MSS exhibited EMAST. Although non–MSI-H CRC tissues contained MSH3-negative tumor cells ranging from 2% to 50% of the total tumor cell population, the tissues exhibiting EMAST contained more MSH3-negative cells (average, 31.5%) than did the tissues not exhibiting EMAST (8.4%). Taken together, our results support the concept that MSH3 deficiency causes EMAST or EMAST with low levels of MSI at loci with dinucleotide repeats in CRC. [Cancer Res 2008;68(20):8465–72]
Oncolytic Semliki Forest Virus Vector as a Novel Candidate against Unresectable Osteosarcoma
Authors: Ketola Anna , Hinkkanen Ari , Yongabi Felicitas , Furu Petra , Määttä Ann-Marie , Liimatainen Timo , Pirinen Risto , Björn Marko , Hakkarainen Tanja , Mäkinen Kimmo , Wahlfors Jarmo , Pellinen Riikka
Published: October 2008

Oncolytic viruses are a promising tool for treatment of cancer. We studied an oncolytic Semliki Forest virus (SFV) vector, VA7, carrying the enhanced green fluorescent protein gene (EGFP), as a novel virotherapy candidate against unresectable osteosarcoma. The efficiency and characteristics of the VA7-EGFP treatment were compared with a widely studied oncolytic adenovirus, Ad5Δ24, both in vitro and in vivo. VA7-EGFP resulted in more rapid oncolysis and was more efficient at low multiplicities of infection (MOI) when compared with Ad5Δ24 in vitro. Yet, in MG-63 cells, a subpopulation resistant to the VA7-EGFP vector emerged. In subcutaneous human osteosarcoma xenografts in nude mice treatment with either vector reduced tumor size, whereas tumors in control mice expanded quickly. The VA7-EGFP–treated tumors were either completely abolished or regressed to pinpoint size. The efficacy of VA7-EGFP vector was studied also in an orthotopic osteosarcoma nude mouse model characterized by highly aggressive tumor growth. Treatment with oncolytic SFV extended survival of the animals significantly (P < 0.01), yet none of the animals were finally cured. Sera from SFV-treated mice contained neutralizing antibodies, and as nude mice are not able to establish IgG response, the result points out the role of IgM class antibodies in clearance of virus from peripheral tumors. Furthermore, biodistribution analysis at the survival end point verified the presence of virus in some of the brain samples, which is in line with previous studies demonstrating that IgG is required for clearance of SFV from central nervous system. [Cancer Res 2008;68(20):8342–50]
IFN-α and Bortezomib Overcome Bcl-2 and Mcl-1 Overexpression in Melanoma Cells by Stimulating the Extrinsic Pathway of Apoptosis
Authors: Lesinski Gregory B. , Raig Ene T. , Guenterberg Kristan , Brown Lloyd , Go Michael R. , Shah Nisha N. , Lewis Adrian , Quimper Megan , Hade Erinn , Young Gregory , Chaudhury Abhik Ray , Ladner Katherine J. , Guttridge Denis C. , Bouchard Page , Carson William E.
Published: October 2008

We hypothesized that IFN-α would enhance the apoptotic activity of bortezomib on melanoma cells. Combined treatment with bortezomib and IFN-α induced synergistic apoptosis in melanoma and other solid tumor cell lines. Apoptosis was associated with processing of procaspase-3, procaspase-7, procaspase-8, and procaspase-9 and with cleavage of Bid and poly(ADP-ribose) polymerase. Bortezomib plus IFN-α was effective at inducing apoptosis in melanoma cells that overexpressed Bcl-2 or Mcl-1, suggesting that this treatment combination can overcome mitochondrial pathways of cell survival and resistance to apoptosis. The proapoptotic effects of this treatment combination were abrogated by a caspase-8 inhibitor, led to increased association of Fas and FADD before the onset of cell death, and were significantly reduced in cells transfected with a dominant-negative FADD construct or small interfering RNA targeting Fas. These data suggest that bortezomib and IFN-α act through the extrinsic pathway of apoptosis via FADD-induced caspase-8 activation to initiate cell death. Finally, bortezomib and IFN-α displayed statistically significant antitumor activity compared with either agent alone in both the B16 murine model of melanoma and in athymic mice bearing human A375 xenografts. These data support the future clinical development of bortezomib and IFN-α for malignant melanoma. [Cancer Res 2008;68(20):8351–60]
Activated Gα13 Impairs Cell Invasiveness through p190RhoGAP-Mediated Inhibition of RhoA Activity
Authors: Bartolomé Rubén A. , Wright Natalia , Molina-Ortiz Isabel , Sánchez-Luque Francisco J. , Teixidó Joaquin
Published: October 2008

The GTPase RhoA is a downstream target of heterotrimeric G13 proteins and plays key roles in cell migration and invasion. Here, we show that expression in human melanoma cells of a constitutively active, GTPase-deficient Gα13 form (Gα13QL) or lysophosphatidylcholine (LPC)-promoted signaling through Gα13-coupled receptors led to a blockade of chemokine-stimulated RhoA activation and cell invasion that was rescued by active RhoA. Melanoma cells expressing Gα13QL or cells stimulated with LPC displayed an increase in p190RhoGAP activation, and defects in RhoA activation and invasion were recovered by knocking down p190RhoGAP expression, thus identifying this GTPase-activating protein (GAP) protein as a downstream Gα13 target that is responsible for these inhibitory responses. In addition, defective stress fiber assembly and reduced migration speed underlay inefficient invasion of Gα13QL melanoma cells. Importantly, Gα13QL expression in melanoma cells led to impairment in lung metastasis associated with prolonged survival in SCID mice. The data indicate that Gα13-dependent downstream effects on RhoA activation and invasion tightly depend on cell type–specific GAP activities and that Gα13-p190RhoGAP signaling might represent a potential target for intervention in melanoma metastasis. [Cancer Res 2008;68(20):8221–30]
Fetal Cell Microchimerism in Papillary Thyroid Cancer: A Possible Role in Tumor Damage and Tissue Repair
Authors: Cirello Valentina , Recalcati Maria Paola , Muzza Marina , Rossi Stefania , Perrino Michela , Vicentini Leonardo , Beck-Peccoz Paolo , Finelli Palma , Fugazzola Laura
Published: October 2008

Fetal cells enter the maternal circulation during pregnancy and can persist in the maternal blood or tissues for decades, creating a physiologic microchimerism. Because papillary thyroid cancer (PTC) is more frequent in women, the role of persisting fetal male cells in this tumor has been investigated. Tumor tissue specimens were obtained from 63 women with PTC who had a male pregnancy before the diagnosis. Male cells, identified by PCR amplification of a male-specific gene, the sex-determining region Y, was detected in 47.5% of women. By fluorescence in situ hybridization (FISH) analyses, the total number of microchimeric cells was significantly higher in neoplastic tissue than in controlateral normal sections. By combined FISH and immunohistochemistry (immuno-FISH), male cells expressing thyroglobulin were found in tumor and normal tissues, whereas male microchimeric cells stained with the CD45 antigen were detected only in tumor sections. Microchimeric cells negative for either marker were detected both in tumor and normal tissues. Moreover, both CD45+ and Tg+ fetal cells did not express MHC II antigens. In conclusion, fetal microchimerism has been documented in a high proportion of women with PTC. The immuno-FISH studies indicate that CD45+/MHC II− male cells found in neoplastic tissues might be committed to destroy tumor cells, whereas Tg+/MHC II− cells could have a repair function. Finally, microchimeric cells negative for either CD45 or Tg could have “progenitor-like” properties able to transdifferentiate in different cellular types. Although a pathogenetic mechanism cannot be excluded, the whole of the present results indicates a protective role of microchimerism in thyroid cancer. [Cancer Res 2008;68(20):8482–8]
Noninvasive Molecular Imaging of Hypoxia in Human Xenografts: Comparing Hypoxia-Induced Gene Expression with Endogenous and Exogenous Hypoxia Markers
Authors: He Fuqiu , Deng Xuelong , Wen Bixiu , Liu Yueping , Sun Xiaorong , Xing Ligang , Minami Akiko , Huang Yunhong , Chen Qing , Zanzonico Pat B. , Ling C. Clifton , Li Gloria C.
Published: October 2008

Tumor hypoxia is important in the development and treatment of human cancers. We have developed a novel xenograft model for studying and imaging of hypoxia-induced gene expression. A hypoxia-inducible dual reporter herpes simplex virus type 1 thymidine kinase and enhanced green fluorescence protein (HSV1-TKeGFP), under the control of hypoxia response element (9HRE), was stably transfected into human colorectal HT29 cancer cells. Selected clones were further enriched by repeated live cell sorting gated for hypoxia-induced eGFP expression. Fluorescent microscopy, fluorescence-activated cell sorting, and radioactive substrate trapping assays showed strong hypoxia-induced expression of eGFP and HSV1-tk enzyme in the HT29-9HRE cells in vitro. Sequential micropositron emission tomography (PET) imaging of tumor-bearing animals, using the hypoxic cell tracer 18F-FMISO and the reporter substrate 124I-FIAU, yielded similar tumor hypoxia images for the HT29-9HRE xenograft but not in the parental HT29 tumor. Using autoradiography and IHC, detailed spatial distributions in tumor sections were obtained and compared for the following hypoxia-associated biomarkers in the HT29-9HRE xenograft: 124I-FIAU, 18F-FMISO, Hoechst (perfusion), lectin-TRITC (functional blood vessels), eGFP, pimonidazole, EF5, and CA9. Intratumoral distributions of 124I-FIAU and 18F-FMISO were similar, and eGFP, pimonidazole, EF5, and CA9 colocalized in the same areas but not in well-perfused regions that were positive for Hoechst and lectin-TRITC. In enabling the detection of hypoxia-induced molecular events and mapping their distribution in vivo with serial noninvasive positron emission tomography imaging, and multiple variable analysis with immunohistochemistry and fluorescence microscopy, this human xenograft model provides a valuable tool for studying tumor hypoxia and in validating existing and future exogenous markers for tumor hypoxia. [Cancer Res 2008;68(20):8597–606]
Dual Inhibition of PI3Kα and mTOR as an Alternative Treatment for Kaposi's Sarcoma
Authors: Chaisuparat Risa , Hu Jiadi , Jham Bruno C. , Knight Zachary A. , Shokat Kevan M. , Montaner Silvia
Published: October 2008

Rapamycin (or sirolimus), the prototypical inhibitor of the mammalian target of rapamycin (mTOR) and an immunosuppressant used for the prevention of renal transplant rejection, has recently emerged as an effective treatment for Kaposi's sarcoma (KS), an enigmatic vascular tumor and a model for pathologic angiogenesis. Indeed, recent work supports a role for mTOR as a central player in the transformation of endothelial cells by the KS-associated herpesvirus–encoded G protein–coupled receptor (vGPCR), the viral oncogene believed to be responsible for causing KS. However, emerging evidence that rapamycin may transiently promote the activation of Akt may limit its use as an anti-KS therapy. Here, we show that activation of Akt in endothelial cells expressing vGPCR is augmented by treatment with rapamycin, resulting in the up-regulation of several Akt proliferative and survival pathways. However, use of a novel dual phosphatidylinositol 3-kinase α (PI3Kα)/mTOR inhibitor, PI-103, effectively and independently blocked activation of both PI3K and mTOR in vGPCR-expressing endothelial cells. This resulted in more effective inhibition of endothelial cell proliferation and survival in vitro and tumor growth in vivo. Our results suggest that PI-103 may be an effective therapeutic option for the treatment of patients with KS. Moreover, as KS may serve as a model for pathologic angiogenesis, our results further provide the basis for the early assessment of PI-103 as an antiangiogenic chemotherapeutic. [Cancer Res 2008;68(20):8361–8]
Sphingosine Kinase 1: A New Modulator of Hypoxia Inducible Factor 1α during Hypoxia in Human Cancer Cells
Authors: Ader Isabelle , Brizuela Leyre , Bouquerel Pierre , Malavaud Bernard , Cuvillier Olivier
Published: October 2008

Here, we provide the first evidence that sphingosine kinase 1 (SphK1), an oncogenic lipid kinase balancing the intracellular level of key signaling sphingolipids, modulates the transcription factor hypoxia inducible factor 1α (HIF-1α), master regulator of hypoxia. SphK1 activity is stimulated under low oxygen conditions and regulated by reactive oxygen species. The SphK1-dependent stabilization of HIF-1α levels is mediated by the Akt/glycogen synthase kinase-3β signaling pathway that prevents its von Hippel-Lindau protein–mediated degradation by the proteasome. The pharmacologic and RNA silencing inhibition of SphK1 activity prevents the accumulation of HIF-1α and its transcriptional activity in several human cancer cell lineages (prostate, brain, breast, kidney, and lung), suggesting a canonical pathway. Therefore, we propose that SphK1 can act as a master regulator for hypoxia, giving support to its inhibition as a valid strategy to control tumor hypoxia and its molecular consequences. [Cancer Res 2008;68(20):8635–42]
Hypermethylator Phenotype in Sporadic Colon Cancer: Study on a Population-Based Series of 582 Cases
Authors: Barault Ludovic , Charon-Barra Céline , Jooste Valérie , de la Vega Mathilde Funes , Martin Laurent , Roignot Patrick , Rat Patrick , Bouvier Anne-Marie , Laurent-Puig Pierre , Faivre Jean , Chapusot Caroline , Piard Francoise
Published: October 2008

The CpG island methylator phenotype (CIMP) is a distinct phenotype in colorectal cancer, associated with specific clinical, pathologic, and molecular features. However, most of the studies stratified methylation according to two subgroups (CIMP-High versus No-CIMP/CIMP-Low). In our study, we defined three different subgroups of methylation (No-CIMP, CIMP-Low, and CIMP-High) and evaluated the prognostic significance of methylation status on a population-based series of sporadic colon cancers. A total of 582 colon adenocarcinomas were evaluated using methylation-specific PCR for 5 markers (hMLH1, P16, MINT1, MINT2, and MINT31). No-CIMP status was defined as no methylated locus, CIMP-Low status as one to three methylated loci, and CIMP-High status as four or five methylated loci. Clinicopathologic and molecular characteristics were correlated to the methylation status. Crude and relative survival was compared according to methylation status. In the microsatellite-stable (MSS) group, CIMP-High was significantly associated with proximal location (P = 0.011) and BRAF mutation (P < 0.001). KRAS mutations were more associated with CIMP-High and CIMP-Low status (P = 0.008). A shorter 5-year survival was observed in MSS cancer patients with CIMP-Low or CIMP-High status. These results remained significant in multivariate analysis adjusted for age, stage, and BRAF and KRAS mutational status [CIMP-Low: hazard ratio (HR), 1.85; 95% confidence interval (95% CI), 1.37–2.51; CIMP-High, HR, 2.90; 95% CI, 1.53–5.49 compared with No-CIMP]. Within the high-level microsatellite instability group, no difference in survival was observed between the different CIMP groups. Our results show the interest of defining three subgroups of patients according to their methylation status (No-CIMP/CIMP-Low/CIMP-High). Methylation is an independent prognostic factor in MSS colon cancer. [Cancer Res 2008;68(20):8541–6]
Targeted and Nontargeted Effects of Ionizing Radiation That Impact Genomic Instability
Authors: Maxwell Christopher A. , Fleisch Markus C. , Costes Sylvain V. , Erickson Anna C. , Boissière Arnaud , Gupta Rishi , Ravani Shraddha A. , Parvin Bahram , Barcellos-Hoff Mary Helen
Published: October 2008

Radiation-induced genomic instability, in which the progeny of irradiated cells display a high frequency of nonclonal genomic damage, occurs at a frequency inconsistent with mutation. We investigated the mechanism of this nontargeted effect in human mammary epithelial cells (HMEC) exposed to low doses of radiation. We identified a centrosome-associated expression signature in irradiated HMEC and show here that centrosome deregulation occurs in the first cell cycle after irradiation, is dose dependent, and that viable daughters of these cells are genomically unstable as evidenced by spontaneous DNA damage, tetraploidy, and aneuploidy. Clonal analysis of genomic instability showed a threshold of >10 cGy. Treatment with transforming growth factor β1 (TGFβ), which is implicated in regulation of genomic stability and is activated by radiation, reduced both the centrosome expression signature and centrosome aberrations in irradiated HMEC. Furthermore, TGFβ inhibition significantly increased centrosome aberration frequency, tetraploidy, and aneuploidy in nonirradiated HMEC. Rather than preventing radiation-induced or spontaneous centrosome aberrations, TGFβ selectively deleted unstable cells via p53-dependent apoptosis. Together, these studies show that radiation deregulates centrosome stability, which underlies genomic instability in normal human epithelial cells, and that this can be opposed by radiation-induced TGFβ signaling. [Cancer Res 2008;68(20):8304–11]
Agglomerative Epigenetic Aberrations Are a Common Event in Human Breast Cancer
Authors: Novak Petr , Jensen Taylor , Oshiro Marc M. , Watts George S. , Kim Christina J. , Futscher Bernard W.
Published: October 2008

Changes in DNA methylation patterns are a common characteristic of cancer cells. Recent studies suggest that DNA methylation affects not only discrete genes, but it can also affect large chromosomal regions, potentially leading to LRES. It is unclear whether such long-range epigenetic events are relatively rare or frequent occurrences in cancer. Here, we use a high-resolution promoter tiling array approach to analyze DNA methylation in breast cancer specimens and normal breast tissue to address this question. We identified 3,506 cancer-specific differentially methylated regions (DMR) in human breast cancer with 2,033 being hypermethylation events and 1,473 hypomethylation events. Most of these DMRs are recurrent in breast cancer; 90% of the identified DMRs occurred in at least 33% of the samples. Interestingly, we found a nonrandom spatial distribution of aberrantly methylated regions across the genome that showed a tendency to concentrate in relatively small genomic regions. Such agglomerates of hypermethylated and hypomethylated DMRs spanned up to several hundred kilobases and were frequently found at gene family clusters. The hypermethylation events usually occurred in the proximity of the transcription start site in CpG island promoters, whereas hypomethylation events were frequently found in regions of segmental duplication. One example of a newly discovered agglomerate of hypermethylated DMRs associated with gene silencing in breast cancer that we examined in greater detail involved the protocadherin gene family clusters on chromosome 5 (PCDHA, PCDHB, and PCDHG). Taken together, our results suggest that agglomerative epigenetic aberrations are frequent events in human breast cancer. [Cancer Res 2008;68(20):8616–25]
Natural Killer Cell Accumulation in Tumors Is Dependent on IFN-γ and CXCR3 Ligands
Authors: Wendel Marco , Galani Ioanna E. , Suri-Payer Elisabeth , Cerwenka Adelheid
Published: October 2008

Several studies have correlated high numbers of tumor-infiltrating natural killer (NK) cells with a good prognosis for cancer patients. Our study aimed at identifying factors controlling intratumoral NK cell accumulation in s.c. injected NK cell sensitive tumor models and at studying their effect on survival time of recipient mice. We observed that fewer NK cells infiltrated the tumors in IFN-γ receptor knockout (IFN-γR−/−) mice compared with wild-type controls that correlated with decreased survival rate. Exogenous application of IFN-γ in the tumor augmented levels of ligands of the chemokine receptor CXCR3, increased NK cell accumulation, and prolonged survival. Furthermore, our data show that CD27high NK cells, which under steady-state conditions express CXCR3, preferentially accumulated in the tumor tissue. Accordingly, significantly lower numbers of tumor-infiltrating NK cells were detected in CXCR3−/− mice, and the capacity of adoptively transferred CXCR3−/− NK cells to accumulate in the tumor was severely impaired. Finally, exogenous application of the CXCR3 ligand CXCL10 in the tumor or ectopic expression of CXCL10 by tumor cells increased the numbers of NK cells in the tumors and prolonged NK cell–dependent survival. Our results identify IFN-γ and the expression of CXCR3 on NK cells as prerequisites for NK cell infiltration into tumors. Exploiting strategies to augment NK cell accumulation in the tumor might lead to the development of effective antitumor therapies. [Cancer Res 2008;68(20):8437–45]
Balancing between Antitumor Efficacy and Autoimmune Pathology in T-Cell–Mediated Targeting of Carcinoembryonic Antigen
Authors: Bos Rinke , van Duikeren Suzanne , Morreau Hans , Franken Kees , Schumacher Ton N.M. , Haanen John B. , van der Burg Sjoerd H. , Melief Cornelis J.M. , Offringa Rienk
Published: October 2008

Carcinoembryonic antigen (CEA) is intensively studied as a potential target for immunotherapy of colorectal cancers. Although overexpressed by tumors, CEA is also expressed in normal tissues, raising questions about the feasibility and safety of CEA-targeted immunotherapy. We investigated these issues in transgenic mice in which the expression of human CEA in normal tissues closely resembles that in man. Our data show that the T-cell response against CEA in these mice is blunted by both thymic and peripheral tolerance. Consequently, effective tumor targeting is only achieved by adoptive transfer of T cells from nontolerant donors in combination with interventions that eliminate peripheral immune regulatory mechanisms. However, such treatments can result in severe intestinal autoimmune pathology associated with weight loss and mortality. Interestingly, preconditioning of recipient mice by depletion of T-regulatory cells results in immune-mediated tumor control in the absence of toxicity. In this setting, CEA-specific T-cell responses are lower than those induced by toxic regimens and accompanied by additional T-cell responses against non-self antigen. These findings illustrate the importance of testing adoptive immunotherapies targeting self antigens such as CEA in preclinical in vivo models and show that the choice of immune intervention regimen critically determines the balance between therapeutic efficacy and toxicity. [Cancer Res 2008;68(20):8446–55]
Transient Nutlin-3a Treatment Promotes Endoreduplication and the Generation of Therapy-Resistant Tetraploid Cells
Authors: Shen Hong , Moran Diarmuid M. , Maki Carl G.
Published: October 2008

p53 activity is controlled in large part by MDM2, an E3 ubiquitin ligase that binds p53 and promotes its degradation. The MDM2 antagonist Nutlin-3a stabilizes p53 by blocking its interaction with MDM2. Several studies have supported the potential use of Nutlin-3a in cancer therapy. Two different p53 wild-type cancer cell lines (U2OS and HCT116) treated with Nutlin-3a for 24 hours accumulated 2N and 4N DNA content, suggestive of G1 and G2 phase cell cycle arrest. This coincided with increased p53 and p21 expression, hypophosphorylation of pRb, and depletion of Cyclin B1, Cyclin A, and CDC2. Upon removal of Nutlin-3a, 4N cells entered S phase and re-replicated their DNA without an intervening mitotic division, a process known as endoreduplication. p53-p21 pathway activation was required for the depletion of Cyclin B1, Cyclin A, and CDC2 in Nutlin-3a–treated cells and for endoreduplication after Nutlin-3a removal. Stable tetraploid clones could be isolated from Nutlin-3a treated cells, and these tetraploid clones were more resistant to ionizing radiation and cisplatin-induced apoptosis than diploid counterparts. These data indicate that transient Nutlin-3a treatment of p53 wild-type cancer cells can promote endoreduplication and the generation of therapy-resistant tetraploid cells. These findings have important implications regarding the use of Nutlin-3a in cancer therapy.[Cancer Res 2008;68(20):8260–8]