bluuebunny/crossref_metadata_2025 huggingface dataset:
Coupling EPIC to LS-DYNA for Simulation of Blast-Structure-Fragmentation Interaction
Authors: Littlefield David L. , Walls Kenneth C. , Danielson Kent T.
Published: January 2011

In this work we have coupled the EPIC code to the LS-DYNA code to provide a high-fidelity simulation framework for simulation of blast-structure-fragmentation interaction. The coupled code exploits the strengths of the two original codes: EPIC, which has special algorithms and models for weapons effects analysis, and LS-DYNA, which is a general purpose finite element code for modeling large-scale structural deformation. Example problems are shown which illustrate the advantages of this approach.
High-corrosion-resistant Al2O3 passivation-film formation by selective oxidation on austenitic stainless steel containing Al
Authors: Kitano Masafumi , Ishii Hidekazu , Shirai Yasuyuki , Ohmi Tadahiro
Published: January 2011

We have developed Al2O3 passivation film having very high anticorrosion resistance on the surface of austenitic stainless steel containing 3 wt % aluminum. Al2O3 passivation film is formed by selective oxidation of aluminum in the austenitic stainless steel in the Ar and H2 ambient including a small amount of H2O at predetermined temperatures. Al2O3 film is obtained at temperatures higher than 750 °C in the Ar and H2 ambient, where the partial pressure ratio of H2 and H2O is set higher than 2×103. Al2O3 films have been confirmed to exhibit very high anticorrosion resistance for various halogen gases and various plasma ambients (Cl2, H2, and O2) with ion-bombardment energies less than 100 eV at temperatures less than 150 °C. In the case of fluorine-gas plasma, the Al2O3 film surface has been converted to AlF3 with a depth of 15 nm, where AlF3 film is thermodynamically stable, as well as Al2O3, resulting in an excellent passivation film exhibiting very high anticorrosion capability. Moreover, the Al2O3 film surface has been confirmed to exhibit no catalytic activity for various specialty gases at temperatures less than 150 °C.
Identification of a
Saccharomyces cerevisiae
Glucosidase That Hydrolyzes Flavonoid Glucosides
Authors: Schmidt Sabine , Rainieri Sandra , Witte Simone , Matern Ulrich , Martens Stefan
Published: January 2011

Baker's yeast (
Saccharomyces cerevisiae
) whole-cell bioconversions of naringenin 7-
O
-β-glucoside revealed considerable β-glucosidase activity, which impairs any strategy to generate or modify flavonoid glucosides in yeast transformants. Up to 10 putative glycoside hydrolases annotated in the
S. cerevisiae
genome database were overexpressed with His tags in yeast cells. Examination of these recombinant, partially purified polypeptides for hydrolytic activity with synthetic chromogenic α- or β-glucosides identified three efficient β-glucosidases (EXG1, SPR1, and YIR007W), which were further assayed with natural flavonoid β-glucoside substrates and product verification by thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Preferential hydrolysis of 7- or 4′-
O
-glucosides of isoflavones, flavonols, flavones, and flavanones was observed
in vitro
with all three glucosidases, while anthocyanins were also accepted as substrates. The glucosidase activities of EXG1 and SPR1 were completely abolished by Val168Tyr mutation, which confirmed the relevance of this residue, as reported for other glucosidases. Most importantly, biotransformation experiments with knockout yeast strains revealed that only EXG1 knockout strains lost the capability to hydrolyze flavonoid glucosides.
Identification of Cold-Temperature-Regulated Genes in
Flavobacterium psychrophilum
Authors: Hesami Shohreh , Metcalf Devon S. , Lumsden John S. , MacInnes Janet I.
Published: January 2011

Flavobacterium psychrophilum
is the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS). It causes disease primarily in fresh water-reared salmonids, but other fish species can also be affected. A diverse array of clinical conditions is associated with BCWD, including tail rot (peduncle disease), necrotic myositis, and cephalic osteochondritis. Degradation of connective and muscular tissues by extracellular proteases is common to all of these presentations. There are no effective vaccines to prevent BCWD or RTFS, and antibiotics are often used to prevent and control disease. To identify virulence factors that might permit development of an efficacious vaccine, cDNA suppression subtractive hybridization (SSH) was used to identify cold-regulated genes in a virulent strain of
F. psychrophilum.
Genes predicted to encode a two-component system sensor histidine kinase (LytS), an ATP-dependent RNA helicase, a multidrug ABC transporter permease/ATPase, an outer membrane protein/protective antigen OMA87, an M43 cytophagalysin zinc-dependent metalloprotease, a hypothetical protein, and four housekeeping genes were upregulated at 8°C versus the level of expression at 20°C. Because no
F. psychrophilum
gene was known to be suitable as an internal standard in reverse transcription-quantitative real-time PCR (RT-qPCR) experiments, the expression stability of nine commonly used reference genes was evaluated at 8°C and 20°C. Expression of the 16S rRNA was equivalent at both temperatures, and this gene was used in RT-qPCR experiments to verify the SSH findings. With the exception of the ATCC 49513 strain, similar patterns of gene expression were obtained with 11 other representative strains of
F. psychrophilum.
Formic Acid Triggers the “Acid Crash” of Acetone-Butanol-Ethanol Fermentation by
Clostridium acetobutylicum
Authors: Wang Shaohua , Zhang Yanping , Dong Hongjun , Mao Shaoming , Zhu Yan , Wang Runjiang , Luan Guodong , Li Yin
Published: January 2011

Solvent production by
Clostridium acetobutylicum
collapses when cells are grown in pH-uncontrolled glucose medium, the so-called “acid crash” phenomenon. It is generally accepted that the fast accumulation of acetic acid and butyric acid triggers the acid crash. We found that addition of 1 mM formic acid into corn mash medium could trigger acid crash, suggesting that formic acid might be related to acid crash. When it was grown in pH-uncontrolled glucose medium or glucose-rich medium,
C. acetobutylicum
DSM 1731 containing the empty plasmid pIMP1 failed to produce solvents and was found to accumulate 0.5 to 1.24 mM formic acid intracellularly. In contrast, recombinant strain DSM 1731 with formate dehydrogenase activity did not accumulate formic acid intracellularly and could produce solvent as usual. We therefore conclude that the accumulation of formic acid, rather than acetic acid and butyric acid, is responsible for the acid crash of acetone-butanol-ethanol fermentation.
The Airway Epithelium: Soldier in the Fight against Respiratory Viruses
Authors: Vareille Marjolaine , Kieninger Elisabeth , Edwards Michael R. , Regamey Nicolas
Published: January 2011

The airway epithelium acts as a frontline defense against respiratory viruses, not only as a physical barrier and through the mucociliary apparatus but also through its immunological functions. It initiates multiple innate and adaptive immune mechanisms which are crucial for efficient antiviral responses. The interaction between respiratory viruses and airway epithelial cells results in production of antiviral substances, including type I and III interferons, lactoferrin, β-defensins, and nitric oxide, and also in production of cytokines and chemokines, which recruit inflammatory cells and influence adaptive immunity. These defense mechanisms usually result in rapid virus clearance. However, respiratory viruses elaborate strategies to evade antiviral mechanisms and immune responses. They may disrupt epithelial integrity through cytotoxic effects, increasing paracellular permeability and damaging epithelial repair mechanisms. In addition, they can interfere with immune responses by blocking interferon pathways and by subverting protective inflammatory responses toward detrimental ones. Finally, by inducing overt mucus secretion and mucostasis and by paving the way for bacterial infections, they favor lung damage and further impair host antiviral mechanisms.
Clinical Significance of Microbial Infection and Adaptation in Cystic Fibrosis
Authors: Hauser Alan R. , Jain Manu , Bar-Meir Maskit , McColley Susanna A.
Published: January 2011

A select group of microorganisms inhabit the airways of individuals with cystic fibrosis. Once established within the pulmonary environment in these patients, many of these microbes adapt by altering aspects of their structure and physiology. Some of these microbes and adaptations are associated with more rapid deterioration in lung function and overall clinical status, whereas others appear to have little effect. Here we review current evidence supporting or refuting a role for the different microbes and their adaptations in contributing to poor clinical outcomes in cystic fibrosis.
Fig1 Facilitates Calcium Influx and Localizes to Membranes Destined To Undergo Fusion during Mating in Candida albicans
Authors: Yang Meng , Brand Alexandra , Srikantha Thyagarajan , Daniels Karla J. , Soll David R. , Gow Neil A. R.
Published: January 2011

Few mating-regulated genes have been characterized in
Candida albicans. C. albicans FIG1
(Ca
FIG1
) is a fungus-specific and mating-induced gene encoding a putative 4-transmembrane domain protein that shares sequence similarities with members of the claudin superfamily. In
Saccharomyces cerevisiae
, Fig1 is required for shmoo fusion and is upregulated in response to mating pheromones. Expression of Ca
FIG1
was also strongly activated in the presence of cells of the opposite mating type. CaFig1-green fluorescent protein (GFP) was visible only during the mating response, when it localized predominantly to the plasma membrane and perinuclear zone in mating projections and daughter cells. At the plasma membrane, CaFig1-GFP was visualized as discontinuous zones, but the distribution of perinuclear CaFig1-GFP was homogeneous. Exposure to pheromone induced a 5-fold increase in Ca
2+
uptake in mating-competent opaque cells. Uptake was reduced substantially in the
fig1
Δ null mutant. CaFig1 is therefore involved in Ca
2+
influx and localizes to membranes that are destined to undergo fusion during mating.
Plasmids with a Chromosome-Like Role in Rhizobia
Authors: Landeta Cristina , Dávalos Araceli , Cevallos Miguel Ángel , Geiger Otto , Brom Susana , Romero David
Published: January 2011

Replicon architecture in bacteria is commonly comprised of one indispensable chromosome and several dispensable plasmids. This view has been enriched by the discovery of additional chromosomes, identified mainly by localization of rRNA and/or tRNA genes, and also by experimental demonstration of their requirement for cell growth. The genome ofRhizobium etliCFN42 is constituted by one chromosome and six large plasmids, ranging in size from 184 to 642 kb. Five of the six plasmids are dispensable for cell viability, but plasmid p42e is unusually stable. One possibility to explain this stability would be that genes on p42e carry out essential functions, thus making it a candidate for a secondary chromosome. To ascertain this, we made an in-depth functional analysis of p42e, employing bioinformatic tools, insertional mutagenesis, and programmed deletions. Nearly 11% of the genes in p42e participate in primary metabolism, involving biosynthetic functions (cobalamin, cardiolipin, cytochromeo, NAD, and thiamine), degradation (asparagine and melibiose), and septum formation (minCDE). Synteny analysis and incompatibility studies revealed highly stable replicons equivalent to p42e in content and gene order in otherRhizobiumspecies. A systematic deletion analysis of p42e allowed the identification of two genes (RHE_PE00001 and RHE_PE00024), encoding, respectively, a hypothetical protein with a probable winged helix-turn-helix motif and a probable two-component sensor histidine kinase/response regulator hybrid protein, which are essential for growth in rich medium. These data support the proposal that p42e and its homologous replicons (pA, pRL11, pRLG202, and pR132502) merit the status of secondary chromosomes.
Identification and Characterization of ZapC, a Stabilizer of the FtsZ Ring in
Escherichia coli
Authors: Durand-Heredia Jorge M. , Yu Helen H. , De Carlo Sacha , Lesser Cammie F. , Janakiraman Anuradha
Published: January 2011

In
Escherichia coli
, spatiotemporal control of cell division occurs at the level of the assembly/disassembly process of the essential cytoskeletal protein FtsZ. A number of regulators interact with FtsZ and modulate the dynamics of the assembled FtsZ ring at the midcell division site. In this article, we report the identification of an FtsZ stabilizer, ZapC (Z-associated protein C), in a protein localization screen conducted with
E. coli
. ZapC colocalizes with FtsZ at midcell and interacts directly with FtsZ, as determined by a protein-protein interaction assay in yeast. Cells lacking or overexpressing ZapC are slightly elongated and have aberrant FtsZ ring morphologies indicative of a role for ZapC in FtsZ regulation. We also demonstrate the ability of purified ZapC to promote lateral bundling of FtsZ in a sedimentation reaction visualized by transmission electron microscopy. While ZapC lacks sequence similarity with other nonessential FtsZ regulators, ZapA and ZapB, all three Zap proteins appear to play an important role in FtsZ regulation during rapid growth. Taken together, our results suggest a key role for lateral bundling of the midcell FtsZ polymers in maintaining FtsZ ring stability during division.
Complete Genome Sequences for the Anaerobic, Extremely Thermophilic Plant Biomass-Degrading Bacteria
Caldicellulosiruptor hydrothermalis
,
Caldicellulosiruptor kristjanssonii
,
Caldicellulosiruptor kronotskyensis
,
Caldicellulosiruptor owensensis
, and
Caldicellulosiruptor lactoaceticus
Authors: Blumer-Schuette Sara E. , Ozdemir Inci , Mistry Dhaval , Lucas Susan , Lapidus Alla , Cheng Jan-Fang , Goodwin Lynne A. , Pitluck Samuel , Land Miriam L. , Hauser Loren J. , Woyke Tanja , Mikhailova Natalia , Pati Amrita , Kyrpides Nikos C. , Ivanova Natalia , Detter John C. , Walston-Davenport Karen , Han Shunsheng , Adams Michael W. W. , Kelly Robert M.
Published: January 2011

The genus
Caldicellulosiruptor
contains the most thermophilic, plant biomass-degrading bacteria isolated to date. Previously, genome sequences from three cellulolytic members of this genus were reported (
C. saccharolyticus
,
C. bescii
, and
C. obsidiansis
). To further explore the physiological and biochemical basis for polysaccharide degradation within this genus, five additional genomes were sequenced:
C. hydrothermalis
,
C. kristjanssonii
,
C. kronotskyensis
,
C. lactoaceticus
, and
C. owensensis
. Taken together, the seven completed and one draft-phase
Caldicellulosiruptor
genomes suggest that, while central metabolism is highly conserved, significant differences in glycoside hydrolase inventories and numbers of carbohydrate transporters exist, a finding which likely relates to variability observed in plant biomass degradation capacity.
IL-1β and ADAM17 are central regulators of β-defensin expression in Candida esophagitis
Authors: Pahl Rene , Brunke Gabriele , Steubesand Nadine , Schubert Sabine , Böttner Martina , Wedel Thilo , Jürgensen Christian , Hampe Jochen , Schäfer Heiner , Zeissig Sebastian , Schreiber Stefan , Rosenstiel Philip , Reiss Karina , Arlt Alexander
Published: January 2011

Candida albicans resides on epithelial surfaces as part of the physiological microflora. However, under certain conditions, it may cause life-threatening infections, including Candida sepsis. We have recently shown that human β-defensins (hBDs) hBD-2 and hBD-3 are upregulated in Candida esophagitis and that this antifungal host response is distinctly regulated by NF-κB and MAPK/activator protein-1 (AP-1) pathways. Here, we show that C. albicans induces hBD-2 through an autocrine IL-1β loop and that activation of the epidermal growth factor receptor (EGFR) by endogenous transforming growth factor-α (TGF-α) is a crucial event in the induction of hBD-3. To further dissect upstream signaling events, we investigated expression of the central sheddases for EGFR ligands ADAM10 and ADAM17 in the healthy and infected esophagus. Next, we used pharmaceutical inhibitors and small-interfering RNA-mediated knock down of ADAM10 and ADAM17 to reveal that ADAM17-induced shedding of TGF-α is a crucial step in the induction of hBD-3 expression in response to Candida infection. In conclusion, we describe for the first time an autocrine IL-1β loop responsible for the induction of hBD-2 expression and an ADAM17-TGF-α-EGFR-MAPK/AP-1 pathway leading to hBD-3 upregulation in the course of a Candida infection of the esophagus.
Endothelial NOS activity and myocardial oxygen metabolism define the salvageable ischemic time window for ischemic postconditioning
Authors: Cai Ming , Li Yuanjing , Xu Yi , Swartz Harold M. , Chen Chwen-Lih , Chen Yeong-Renn , He Guanglong
Published: January 2011

Ischemic postconditioning (IPOC) could be ineffective or even detrimental if the index ischemic duration is either too short or too long. The present study is to demonstrate that oxygen supply and metabolism defines a salvageable ischemic time window of IPOC in mice. C57BL/6 mice underwent coronary artery occlusion followed by reperfusion (I/R), with or without IPOC by three cycles of 10 s/10 s R/I. In vivo myocardial tissue oxygenation was monitored with electron paramagnetic resonance oximetry. Regional blood flow (RBF) was measured with a laser Doppler monitor. At the end of 60 min reperfusion, tissue from the risk area was collected, and mitochondrial enzyme activities were assayed. Tissue oximetry demonstrated that I/R induced a reperfusion hyperoxygenation state in the 30- and 45-min but not 15- and 60-min ischemia groups. IPOC attenuated the hyperoxygenation with 45 but not 30 min ischemia. RBF, eNOS phosphorylation, and mitochondrial enzyme activities were suppressed after I/R with different ischemic time, and IPOC afforded protection with 30 and 45 but not 60 min ischemia. Infarct size measurement indicated that IPOC reduced infarction with 30 and 45 min but not 60 min ischemia. Clearly, IPOC protected mouse heart with a defined ischemic time window between 30 and 45 min. This salvageable time window was accompanied by the improvement of RBF due to increased phosphorylated eNOS and the preservation of mitochondrial oxygen consumption due to conserved mitochondrial enzyme activities. Interestingly, this salvageable ischemic time window was mirrored by tissue hyperoxygenation status in the postischemic heart.
Pulmonary and systemic vasodilator responses to the soluble guanylyl cyclase activator, BAY 60–2770, are not dependent on endogenous nitric oxide or reduced heme
Authors: Pankey Edward A. , Bhartiya Manish , Badejo Adeleke M. , Haider Umair , Stasch Johannes-Peter , Murthy Subramanyam N. , Nossaman Bobby D. , Kadowitz Philip J.
Published: January 2011

4-({(4-Carboxybutyl)[2-(5-fluoro-2-{[4′-(trifluoromethyl)biphenyl-4-yl]methoxy}phenyl)ethyl]amino}methyl)benzoic acid (BAY 60–2770) is a nitric oxide (NO)-independent activator of soluble guanylyl cyclase (sGC) that increases the catalytic activity of the heme-oxidized or heme-free form of the enzyme. In this study, responses to intravenous injections of the sGC activator BAY 60–2770 were investigated under baseline and elevated tone conditions induced by the thromboxane mimic U-46619 when NO synthesis was inhibited by Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), when sGC activity was inhibited by 1H-[1,2,4]-oxadizaolo[4,3]quinoxaline-1-one (ODQ), an agent that oxidizes sGC, and in animals with monocrotaline-induced pulmonary hypertension. The intravenous injections of BAY 60–2770 under baseline conditions caused small decreases in pulmonary arterial pressure, larger decreases in systemic arterial pressure, and no change or small increases in cardiac output. Under elevated tone conditions during infusion of U-46619, intravenous injections of BAY 60–2770 caused larger decreases in pulmonary arterial pressure, smaller decreases in systemic arterial pressure, and increases in cardiac output. Pulmonary vasodilator responses to BAY 60–2770 were enhanced by l-NAME or by ODQ in a dose that attenuated responses to the NO donor sodium nitroprusside. ODQ had no significant effect on baseline pressures and attenuated pulmonary and systemic vasodilator responses to the sGC stimulator BAY 41–8543 2-{1-[2-(fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}-5(4-morpholinyl)-4,6-pyrimidinediamine. BAY 60–2770 and sodium nitroprusside decreased pulmonary and systemic arterial pressures in monocrotaline-treated rats in a nonselective manner. The present data show that BAY 60–2770 has vasodilator activity in the pulmonary and systemic vascular beds that is enhanced by ODQ and NOS inhibition, suggesting that the heme-oxidized form of sGC can be activated in vivo in an NO-independent manner to promote vasodilation . These results show that BAY 60–2770 and sodium nitroprusside decreased pulmonary and systemic arterial pressures in monocrotaline-treated rats, suggesting that BAY 60–2770 does not have selective pulmonary vasodilator activity in animals with monocrotaline-induced pulmonary hypertension.
Antigen Spreading Contributes to MAGE Vaccination-Induced Regression of Melanoma Metastases
Authors: Corbière Véronique , Chapiro Jacques , Stroobant Vincent , Ma Wenbin , Lurquin Christophe , Lethé Bernard , van Baren Nicolas , Van den Eynde Benoît J. , Boon Thierry , Coulie Pierre G.
Published: January 2011

A core challenge in cancer immunotherapy is to understand the basis for efficacious vaccine responses in human patients. In previous work we identified a melanoma patient who displayed a low-level antivaccine cytolytic T-cell (CTL) response in blood with tumor regression after vaccination with melanoma antigens (MAGE). Using a genetic approach including T-cell receptor β (TCRβ) cDNA libraries, we found very few antivaccine CTLs in regressing metastases. However, a far greater number of TCRβ sequences were found with several of these corresponding to CTL clones specific for nonvaccine tumor antigens, suggesting that antigen spreading was occurring in regressing metastases. In this study, we found another TCR belonging to tumor-specific CTL enriched in regressing metastases and detectable in blood only after vaccination. We used the TCRβ sequence to detect and clone the desired T cells from tumor-infiltrating lymphocytes isolated from the patient. This CD8 clone specifically lysed autologous melanoma cells and displayed HLA-A2 restriction. Its target antigen was identified as the mitochondrial enzyme caseinolytic protease. The target antigen gene was mutated in the tumor, resulting in production of a neoantigen. Melanoma cell lysis by the CTL was increased by IFN-γ treatment due to preferential processing of the antigenic peptide by the immunoproteasome. These results argue that tumor rejection effectors in the patient were indeed CTL responding to nonvaccine tumor-specific antigens, further supporting our hypothesis. Among such antigens, the mutated antigen we found is the only antigen against which no T cells could be detected before vaccination. We propose that antigen spreading of an antitumor T-cell response to truly tumor-specific antigens contributes decisively to tumor regression. Cancer Res; 71(4); 1253–62. ©2011 AACR.
LOXL2-Mediated Matrix Remodeling in Metastasis and Mammary Gland Involution
Authors: Barker Holly E. , Chang Joan , Cox Thomas R. , Lang Georgina , Bird Demelza , Nicolau Monica , Evans Holly R. , Gartland Alison , Erler Janine T.
Published: January 2011

More than 90% of cancer patient mortality is attributed to metastasis. In this study, we investigated a role for the lysyl oxidase-related enzyme lysyl oxidase-like 2 (LOXL2) in breast cancer metastasis, in both patient samples and in vivo models. Analysis of a published microarray data set revealed that LOXL2 expression is correlated with metastasis and decreased survival in patients with aggressive breast cancer. In immunocompetent or immunocompromised orthotopic and transgenic breast cancer models we showed that genetic, chemical or antibody-mediated inhibition of LOXL2 resulted in decreased metastasis. Mechanistic investigations revealed that LOXL2 promotes invasion by regulating the expression and activity of the extracellular proteins tissue inhibitor of metalloproteinase-1 (TIMP1) and matrix metalloproteinase-9 (MMP9). We found that LOXL2, TIMP1, and MMP9 are coexpressed during mammary gland involution, suggesting they function together in glandular remodeling after weaning. Finally, we found that LOXL2 is highly expressed in the basal/myoepithelial mammary cell lineage, like many other genes that are upregulated in basal-like breast cancers. Our findings highlight the importance of LOXL2 in breast cancer progression and support the development of anti-LOXL2 therapeutics for the treatment of metastatic breast cancer. Cancer Res; 71(5); 1561–72. ©2011 AACR.
Epigenetic Silencing Mediated through Activated PI3K/AKT Signaling in Breast Cancer
Authors: Zuo Tao , Liu Ta-Ming , Lan Xun , Weng Yu-I , Shen Rulong , Gu Fei , Huang Yi-Wen , Liyanarachchi Sandya , Deatherage Daniel E. , Hsu Pei-Yin , Taslim Cenny , Ramaswamy Bhuvaneswari , Shapiro Charles L. , Lin Huey-Jen L. , Cheng Alfred S. L. , Jin Victor X. , Huang Tim H.-M.
Published: January 2011

Trimethylation of histone 3 lysine 27 (H3K27me3) is a critical epigenetic mark for the maintenance of gene silencing. Additional accumulation of DNA methylation in target loci is thought to cooperatively support this epigenetic silencing during tumorigenesis. However, molecular mechanisms underlying the complex interplay between the two marks remain to be explored. Here we show that activation of PI3K/AKT signaling can be a trigger of this epigenetic processing at many downstream target genes. We also find that DNA methylation can be acquired at the same loci in cancer cells, thereby reinforcing permanent repression in those losing the H3K27me3 mark. Because of a link between PI3K/AKT signaling and epigenetic alterations, we conducted epigenetic therapies in conjunction with the signaling-targeted treatment. These combined treatments synergistically relieve gene silencing and suppress cancer cell growth in vitro and in xenografts. The new finding has important implications for improving targeted cancer therapies in the future. Cancer Res; 71(5); 1752–62. ©2011 AACR.
Pretreatment EGFR T790M Mutation and BRCA1 mRNA Expression in Erlotinib-Treated Advanced Non–Small-Cell Lung Cancer Patients with EGFR Mutations
Authors: Rosell Rafael , Molina Miguel Angel , Costa Carlota , Simonetti Sara , Gimenez-Capitan Ana , Bertran-Alamillo Jordi , Mayo Clara , Moran Teresa , Mendez Pedro , Cardenal Felipe , Isla Dolores , Provencio Mariano , Cobo Manuel , Insa Amelia , Garcia-Campelo Rosario , Reguart Noemi , Majem Margarita , Viteri Santiago , Carcereny Enric , Porta Ruth , Massuti Bartomeu , Queralt Cristina , de Aguirre Itziar , Sanchez Jose Miguel , Sanchez-Ronco Maria , Mate Jose Luis , Ariza Aurelio , Benlloch Susana , Sanchez Jose Javier , Bivona Trever G , Sawyers Charles L , Taron Miquel
Published: January 2011

Purpose: Advanced non–small-cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR) mutations (deletion in exon 19 or L858R) show an impressive progression-free survival of 14 months when treated with erlotinib. However, the presence of EGFR mutations can only imperfectly predict outcome. We hypothesized that progression-free survival could be influenced both by the pretreatment EGFR T790M mutation and by components of DNA repair pathways.

Experimental Design: We assessed the T790M mutation in pretreatment diagnostic specimens from 129 erlotinib-treated advanced NSCLC patients with EGFR mutations. The expression of eight genes and two proteins involved in DNA repair and four receptor tyrosine kinases was also examined.

Results: The EGFR T790M mutation was observed in 45 of 129 patients (35%). Progression-free survival was 12 months in patients with and 18 months in patients without the T790M mutation (P = 0.05). Progression-free survival was 27 months in patients with low BRCA1 mRNA levels, 18 months in those with intermediate levels, and 10 months in those with high levels (P = 0.02). In the multivariate analysis, the presence of the T790M mutation (HR, 4.35; P = 0.001), intermediate BRCA1 levels (HR, 8.19; P < 0.0001), and high BRCA1 levels (HR, 8.46; P < 0.0001) emerged as markers of shorter progression-free survival.

Conclusions: Low BRCA1 levels neutralized the negative effect of the T790M mutation and were associated with longer progression-free survival to erlotinib. We advocate baseline assessment of the T790M mutation and BRCA1 expression to predict outcome and provide alternative individualized treatment to patients based on T790M mutations and BRCA1 expression. Clin Cancer Res; 17(5); 1–9. ©2011 AACR.
Specifically Targeting Angiopoietin-2 Inhibits Angiogenesis, Tie2-Expressing Monocyte Infiltration, and Tumor Growth
Authors: Huang Hanhua , Lai Jing-Yu , Do Janet , Liu Dingguo , Li Lingna , Del Rosario Joselyn , Doppalapudi Venkata R. , Pirie-Shepherd Steven , Levin Nancy , Bradshaw Curt , Woodnutt Gary , Lappe Rodney , Bhat Abhijit
Published: January 2011

Purpose: Angiopoietin-1 (Ang1) plays a key role in maintaining stable vasculature, whereas in a tumor Ang2 antagonizes Ang1's function and promotes the initiation of the angiogenic switch. Specifically targeting Ang2 is a promising anticancer strategy. Here we describe the development and characterization of a new class of biotherapeutics referred to as CovX-Bodies, which are created by chemical fusion of a peptide and a carrier antibody scaffold.

Experimental Design: Various linker tethering sites on peptides were examined for their effect on CovX-Body in vitro potency and pharmacokinetics. Ang2 CovX-Bodies with low nmol/L IC50s and significantly improved pharmacokinetics were tested in tumor xenograft studies alone or in combination with standard of care agents. Tumor samples were analyzed for target engagement, via Ang2 protein level, CD31-positive tumor vasculature, and Tie2 expressing monocyte penetration.

Results: Bivalent Ang2 CovX-Bodies selectively block the Ang2–Tie2 interaction (IC50 < 1 nmol/L) with dramatically improved pharmacokinetics (T½ > 100 hours). Using a staged Colo-205 xenograft model, significant tumor growth inhibition (TGI) was observed (40%–63%, P < 0.01). Ang2 protein levels were reduced by approximately 50% inside tumors (P < 0.01), whereas tumor microvessel density (P < 0.01) and intratumor proangiogenic Tie2+CD11b+ cells (P < 0.05) were significantly reduced. When combined with sunitinib, sorafenib, bevacizumab, irinotecan, or docetaxel, Ang2 CovX-Bodies produced even greater efficacy (∼80% TGI, P < 0.01).

Conclusion: CovX-Bodies provide an elegant solution to overcome the pharmacokinetic–pharmacodynamic problems of peptides. Long-acting Ang2 specific CovX-Bodies will be useful as single agents and in combination with standard-of-care agents. Clin Cancer Res; 17(5); 1001–11. ©2011 AACR.
JAK1 Activates STAT3 Activity in Non-Small–Cell Lung Cancer Cells and IL-6 Neutralizing Antibodies Can Suppress JAK1-STAT3 Signaling
Authors: Song Lanxi , Rawal Bhupendra , Nemeth Jeffrey A. , Haura Eric B.
Published: January 2011

Members of the signal transducer and activator of transcription (STAT) family of transcription factors are potential targets for the treatment and prevention of cancers including non-small–cell lung cancer. STAT proteins can be phosphorylated and activated by diverse upstream kinases including cytokine receptors and tyrosine kinases. We examined STAT protein activation in lung cancer cell lines including those with activating mutations in the EGFR and examined upstream kinases responsible for STAT3 phosphorylation and activation using small molecules, antibodies, and RNA interference. We found more pronounced STAT3 activation in cells with activating EGFR mutations, yet inhibition of EGFR activity had no effect on STAT3 activation. Inhibition of JAK1 with small molecules or RNA interference resulted in loss of STAT3 tyrosine phosphorylation and inhibition of cell growth. An interleukin-6 neutralizing antibody, siltuximab (CNTO 328) could inhibit STAT3 tyrosine phosphorylation in a cell-dependent manner. Siltuximab could completely inhibit STAT3 tyrosine phosphorylation in H1650 cells, and this resulted in inhibition of lung cancer cell growth in vivo. Combined EGFR inhibition with erlotinib and siltuximab resulted in dual inhibition of both tyrosine and serine STAT3 phosphorylation, more pronounced inhibition of STAT3 transcriptional activity, and translated into combined effects on lung cancer growth in a mouse model. Our results suggest that JAK1 is responsible for STAT3 activation in lung cancer cells and that indirect attacks on JAK1-STAT3 using an IL-6 neutralizing antibody with or without EGFR inhibition can inhibit lung cancer growth in lung cancer subsets. Mol Cancer Ther; 10(3); 481–94. ©2011 AACR.
Silencing of Tubulin Binding Cofactor C Modifies Microtubule Dynamics and Cell Cycle Distribution and Enhances Sensitivity to Gemcitabine in Breast Cancer Cells
Authors: Hage-Sleiman Rouba , Herveau Stéphanie , Matera Eva-Laure , Laurier Jean-Fabien , Dumontet Charles
Published: January 2011

Tubulin binding cofactor C (TBCC) is essential for the proper folding of α- and β-tubulins into microtubule polymerizable heterodimers. Because microtubules are considered major targets in the treatment of breast cancer, we investigated the influence of TBCC silencing on tubulin pools, microtubule dynamics, and cell cycle distribution of breast cancer cells by developing a variant MCF7 cells with reduced content of TBCC (MC−). MC− cells displayed decreased content in nonpolymerizable tubulins and increased content of polymerizable/microtubule tubulins when compared with control MP6 cells. Microtubules in MC− cells showed stronger dynamics than those of MP6 cells. MC− cells proliferated faster than MP6 cells and showed an altered cell cycle distribution, with a higher percentage in S-phase of the cell cycle. Consequently, MC− cells presented higher sensitivity to the S-phase–targeting agent gemcitabine than MP6 cells in vitro. Although the complete duration of mitosis was shorter in MC− cells and their microtubule dynamics was enhanced, the percentage of cells in G2-M phase was not altered nor was there any difference in sensitivity to antimicrotubule-targeting agents when compared with MP6 cells. Xenografts derived from TBCC variants displayed significantly enhanced tumor growth in vivo and increased sensitivity to gemcitabine in comparison to controls. These results are the first to suggest that proteins involved in the proper folding of cytoskeletal components may have an important influence on the cell cycle distribution, proliferation, and chemosensitivity of tumor cells. Mol Cancer Ther; 10(2); 303–12. ©2011 AACR.
Interpreting Mammalian Target of Rapamycin and Cell Growth Inhibition in a Genetically Engineered Mouse Model of Nf1-Deficient Astrocytes
Authors: Banerjee Sutapa , Gianino Scott M. , Gao Feng , Christians Uwe , Gutmann David H.
Published: January 2011

The identification of mammalian target of rapamycin (mTOR) as a major mediator of neurofibromatosis-1 (NF1) tumor growth has led to the initiation of clinical trials using rapamycin analogs. Previous studies from our laboratory have shown that durable responses to rapamycin treatment in a genetically engineered mouse model of Nf1 optic glioma require 20 mg/kg/day, whereas only transient tumor growth suppression was observed with 5 mg/kg/day rapamycin despite complete silencing of ribosomal S6 activity. To gain clinically relevant insights into the mechanism underlying this dose-dependent effect, we used Nf1-deficient glial cells in vitro and in vivo. First, there was an exponential relationship between blood and brain rapamycin levels. Second, we show that currently used biomarkers of mTOR pathway inhibition (phospho-S6, phospho-4EBP1, phospho-STAT3, and Jagged-1 levels) and tumor proliferation (Ki67) do not accurately reflect mTOR target inhibition or Nf1-deficient glial growth suppression. Third, the incomplete suppression of Nf1-deficient glial cell proliferation in vivo following 5 mg/kg/day rapamycin treatment reflects mTOR-mediated AKT activation, such that combined 5 mg/kg/day rapamycin and PI3-kinase (PI3K) inhibition or dual PI3K/mTOR inhibition recapitulates the growth suppressive effects of 20 mg/kg/day rapamycin. These new findings argue for the identification of more accurate biomarkers for rapamycin treatment response and provide reference preclinical data for comparing human rapamycin levels with target effects in the brain. Mol Cancer Ther; 10(2); 279–91. ©2011 AACR.
p53R2 Inhibits the Proliferation of Human Cancer Cells in Association with Cell-Cycle Arrest
Authors: Zhang Keqiang , Wu Jun , Wu Xiwei , Wang Xiaochen , Wang Yan , Zhou Ning , Kuo Mei-ling , Liu Xiyong , Zhou Bingsen , Chang Lufen , Ann David , Yen Yun
Published: January 2011

Deregulation of the expression of p53R2, a p53-inducible homologue of the R2 subunit of ribonucleotide reductase, has been found in various human cancer tissues; however, the roles p53R2 plays in cancer progression and malignancy remain controversial. In the present study, we examined changes in gene expression profiles associated with p53R2 in cancer cells, using the analysis of cDNA microarray. Gene set enrichment analysis identified that the gene set regulating cell-cycle progression was significantly enriched in p53R2-silencing human oropharyngeal carcinoma KB cells. Attenuation of p53R2 expression significantly reduced p21 expression and moderately increased cyclin D1 expression in both wild-type p53 cancer cells (KB and MCF-7) and mutant p53 cancer cells (PC3 and MDA-MB-231). Conversely, overexpression of p53R2-GFP resulted in an increase in the expression of p21 and decrease in the expression of cyclin D1, which correlated with reduced cell population in S-phase in vitro and suppressed growth in vivo. Furthermore, the MAP/ERK kinase inhibitor PD98059 partially abolished modulation of p21 and cyclin D1 expression by p53R2. Moreover, under the conditions of nonstress and adriamycin-induced genotoxic stress, attenuation of p53R2 in KB cells significantly increased phosphorylated H2AX, which indicates that attenuation of p53R2 may enhance DNA damage induced by adriamycin. Overall, our study shows that p53R2 may suppress cancer cell proliferation partially by upregulation of p21 and downregulation of cyclin D1; p53R2 plays critical roles not only in DNA damage repair but also in proliferation of cancer cells. Mol Cancer Ther; 10(2); 269–78. ©2011 AACR.
Antisense Inhibition of Survivin Expression as a Cancer Therapeutic
Authors: Carrasco Rosa A. , Stamm Nancy B. , Marcusson Eric , Sandusky George , Iversen Philip , Patel Bharvin K.R.
Published: January 2011

Survivin, a family member of the inhibitor of apoptosis proteins that is expressed during mitosis in a cell cycle–dependent manner and localized to different components of the mitotic apparatus, plays an important role in both cell division and inhibition of apoptosis. Survivin is expressed in a vast majority of human cancers, but not in normal adult tissues. Survivin expression is often correlated with poor prognosis in a wide variety of cancer patients. These features make survivin an attractive target against which cancer therapeutics could be developed. We have identified a survivin antisense oligonucleotide (ASO) that potently downregulated survivin expression in human cancer cells derived from lung, colon, pancreas, liver, breast, prostate, ovary, cervix, skin, and brain as measured by quantitative RT-PCR and immunoblotting analysis. Specific inhibition of survivin expression in multiple cancer cell lines by this ASO (LY2181308) induced caspase-3–dependent apoptosis, cell cycle arrest in the G2-M phase, and multinucleated cells. We also showed that inhibition of survivin expression by LY2181308 sensitized tumor cells to chemotherapeutic-induced apoptosis. Most importantly, in an in vivo human xenograft tumor model, LY2181308 produced significant antitumor activity as compared with saline or its sequence-specific control oligonucleotide and sensitized to gemcitabine, paclitaxel, and docetaxel. Furthermore, we showed that this antitumor activity was associated with significant inhibition of survivin expression in these xenograft tumors. On the basis of these, LY2181308 is being evaluated in a clinical setting (Phase II) in combination with docetaxel for the treatment of prostate cancer. Mol Cancer Ther; 10(2); 221–32. ©2011 AACR.
Ovarian Cancer Stem Cell–Like Side Populations Are Enriched Following Chemotherapy and Overexpress EZH2
Authors: Rizzo Siân , Hersey Jenny M. , Mellor Paul , Dai Wei , Santos-Silva Alessandra , Liber Daniel , Luk Louisa , Titley Ian , Carden Craig P , Box Garry , Hudson David L. , Kaye Stanley B. , Brown Robert
Published: January 2011

Platinum-based chemotherapy, with cytoreductive surgery, is the cornerstone of treatment of advanced ovarian cancer; however, acquired drug resistance is a major clinical obstacle. It has been proposed that subpopulations of tumor cells with stem cell–like properties, such as so-called side populations (SP) that overexpress ABC drug transporters, can sustain the growth of drug-resistant tumor cells, leading to tumor recurrence following chemotherapy. The histone methyltransferase EZH2 is a key component of the polycomb-repressive complex 2 required for maintenance of a stem cell state, and overexpression has been implicated in drug resistance and shorter survival of ovarian cancer patients. We observed higher percentage SP in ascites from patients that have relapsed following chemotherapy compared with chemonaive patients, consistent with selection for this subpopulation during platinum-based chemotherapy. Furthermore, ABCB1 (P-glycoprotein) and EZH2 are consistently overexpressed in SP compared with non-SP from patients' tumor cells. The siRNA knockdown of EZH2 leads to loss of SP in ovarian tumor models, reduced anchorage-independent growth, and reduced tumor growth in vivo. Together, these data support a key role for EZH2 in the maintenance of a drug-resistant, tumor-sustaining subpopulation of cells in ovarian cancers undergoing chemotherapy. As such, EZH2 is an important target for anticancer drug development. Mol Cancer Ther; 10(2); 325–35. ©2010 AACR.
Pushing the Envelope in the mTOR Pathway: The Second Generation of Inhibitors
Authors: Vilar Eduardo , Perez-Garcia Jose , Tabernero Josep
Published: January 2011

The phosphatidylinositol-3-kinase (PI3K)/mTOR pathway has been a major focus of attention for cancer researchers in the past decade. A preliminary and incomplete understanding of the molecular biology of this complex network has importantly conditioned not only the development of the first generation of mTOR inhibitors, but also the biomarker studies designed to identify the best responders to these agents. Most recently, research in this pathway has focused on the dual nature of mTOR that is integrated by the mTOR complex 1 and complex 2. These two complexes are formed and regulated by different proteins and are also driven by multiple different compensatory feedback loops. This deeper understanding has allowed the development of a promising second generation of inhibitors, which are able to block simultaneously both complexes due to their catalytic activity over mTOR. Moreover, some of them also exert an inhibitory effect over PI3K that is a key player in the feedback loops. This article reviews the newest insights in the signaling of the mTOR pathway and then focuses on the development of the new wave of mTOR inhibitors. Mol Cancer Ther; 10(3); 395–403. ©2011 AACR.
Inhibition of Intestinal Polyp Formation by Pitavastatin, a HMG-CoA Reductase Inhibitor
Authors: Teraoka Naoya , Mutoh Michihiro , Takasu Shinji , Ueno Toshiya , Yamamoto Masafumi , Sugimura Takashi , Wakabayashi Keiji
Published: January 2011

It has been suggested that hyperlipidemia is positively associated with colon carcinogenesis. Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors, reduce serum lipid levels. In this study, we clarified the effects of a novel chemically synthesized statin, pitavastatin, on intestinal polyp formation in Min mice, and further examined serum lipid and adipocytokine levels, and proinflammatory and adipocytokine gene levels in intestinal mucosa of Min mice. Treatment with pitavastatin at doses of 20 and 40 ppm decreased the total number of polyps dose-dependently to 85.2% and 65.8% (P < 0.05) of the untreated value, respectively. Serum levels of total cholesterol and triglyceride were slightly reduced and those of IL-6, leptin, and MCP-1 were decreased by 40-ppm pitavastatin treatment. mRNA expression levels of cyclooxygenase-2, IL-6, inducible nitric oxide (iNOS), MCP-1, and Pai-1 were significantly reduced in intestinal nonpolyp parts by pitavastatin treatment. Among them, iNOS mRNA levels were also reduced in the intestinal polyps. Moreover, oxidative stress represented by 8-nitroguanosine in the small intestinal epithelial cells was reduced by pitavastatin treatment. Related to these proinflammatory genes, PPARγ activity was activated in the intestinal nonpolyp parts and in the liver of Min mice with pitavastatin treatment. These results indicated that pitavastatin has potential benefit for the suppression of intestinal polyp development. Cancer Prev Res; 4(3); 445–53. ©2011 AACR.
Flanking Recipient Vasculature, Not Circulating Progenitor Cells, Contributes to Endothelium and Smooth Muscle in Murine Allograft Vasculopathy
Authors: Hagensen Mette K. , Shim Jeong , Falk Erling , Bentzon Jacob F.
Published: January 2011

Objective—

The prevailing view assumes that circulating endothelial and smooth muscle progenitor cells participate in allograft vasculopathy (AV), although the seminal studies in the field were not designed to distinguish between circulating and migrating cells of recipient origin. We developed a double-transplantation technique to overcome this problem and reinvestigated the origin of endothelial cells (ECs) and smooth muscle cells (SMCs) in murine AV.

Methods and Results—

Carotid artery segments from BALB/c mice were allografted to apolipoprotein E
−/−
B6 mice with or without a “flanking” isograft interpositioned between the allograft and the recipient artery. Either recipient mice or interpositioned isografts expressed enhanced green fluorescent protein, and consequently, cells migrating into the allograft from the flanking vasculature could easily be tracked and distinguished from recruited circulating cells. Without immunosuppression, allograft donor cells vanished as expected, and AV developed by replacement and accumulation of ECs and SMCs of recipient origin. The double transplantation models revealed that all ECs and SMCs in AV had migrated into the allograft from the flanking vasculature without any contribution from putative progenitor cells in the blood.

Conclusion—

Migrating cells from the flanking vasculature, not circulating progenitor cells, are the source of recipient-derived ECs and SMCs in murine AV.