bluuebunny/crossref_metadata_2025 huggingface dataset:
RESILIENCE OF SINGLET-STATE EXTRACTION AGAINST NON-OPTIMAL RESONANCE CONDITIONS
Authors: CICCARELLO FRANCESCO , PATERNOSTRO M. , KIM M. S. , PALMA G. M.
Published: August 2008

We have recently presented a protocol for extracting the singlet state of two non-interacting high-dimensional spins through post-selection of the internal state of interaction mediators sent in succession [F. Ciccarello et al., arXiv:0710.3855v1]. The scheme requires each mediator's wavevector to obey appropriate resonance conditions. Here we show the robustness of the scheme in the realistic case where such conditions are not sharply fulfilled.
SYMMETRIC COLLECTIVE ATTACKS FOR THE EAVESDROPPING OF SYMMETRIC QUANTUM KEY DISTRIBUTION
Authors: PIRANDOLA STEFANO
Published: August 2008

We consider the collective eavesdropping of the BB84 and six-state protocols. Since these protocols are symmetric in the eigenstates of conjugate bases, we consider collective attacks having the same kind of symmetry. We then show how these symmetric collective attacks are sufficiently strong in order to minimize the Devetak–Winter rates. In fact, it is quite easy to construct simple examples able to reach the unconditionally secure key rates of these protocols.
A COMPLEXITY-BASED APPROACH IN THERMODYNAMIC SIMULATION OF UNSTABLE EVOLUTIONS IN BIOSYSTEMS
Authors: SIENIUTYCZ STANISLAW
Published: August 2008

Evolution of animals with multiple organs is considered. We report computer-aided modeling and simulation of evolution in biological systems with living organisms as effect of extremum properties of classical statistical entropy of Gibbs–Boltzmann type or its associates, e.g. Tsallis q-entropy. A variational problem searches for the maximum entropy subject to the geometric constraint of constant thermodynamic distance in a non-Euclidean space of independent probabilities pi, plus possibly other constraints. Tensor form of dynamics is obtained. Some developmental processes progress in a relatively undisturbed way, whereas others may terminate rapidly due to inherent instabilities. The results show that a discrete gradient dynamics (governed by the entropy) can be predicted from variational principles for shortest paths and suitable transversality conditions.
Terrestrial Vegetation in the Coupled Human-Earth System: Contributions of Remote Sensing
Authors: DeFries Ruth
Published: August 2008

The Earth system and society's use of ecological resources are tightly coupled through exchanges of water, energy, and nutrients. Terrestrial vegetation transfers materials between the atmosphere, biosphere, and water bodies in the coupled system. Vegetation is also a primary interface between human society and the Earth system through land-cover conversion, cultivation of favorable species, and transfer of organisms between locations. Remote sensing aids analyses of these interactions and, ultimately, contributes information to decision makers for improved management. Multidecadal records from multispectral sensors have been the mainstay for studying terrestrial vegetation at regional and global scales. Representation of vegetation and carbon dynamics is now routine in Earth system models. Challenges remain to incorporate realistic ecological disturbances and human land-use activities based on remote sensing observations. Hyperspectral, LIDAR, and radar systems contribute new capabilities for observing nitrogen fluxes, vegetation stress, canopy structure, and in some cases individual species.
Forage Fish: From Ecosystems to Markets
Authors: Alder Jacqueline , Campbell Brooke , Karpouzi Vasiliki , Kaschner Kristin , Pauly Daniel
Published: August 2008

Fisheries targeting small-to-medium pelagic, so-called forage fish, impact on human food security and marine ecosystems. Because their operations are shrouded by the myth that forage fish are unsuitable for human consumption, the role of these fisheries in intensive food production is not well understood or appreciated. Thus, although they account for over 30% of global landings of marine fish annually, our knowledge of how these levels of removal impact on marine ecosystems is limited. Nevertheless, there is considerable scope for policy makers to change the current management of these fisheries and to enhance their contribution to food security and economic development. Industry and consumers also have an important role in finding the balance between these fisheries contributing to human food security and poverty alleviation on the one hand, and sustaining intensive animal food production systems, especially aquaculture, on the other.
Inhibition of ADRP prevents diet-induced insulin resistance
Authors: Varela Gladys M. , Antwi Daniel A. , Dhir Ravindra , Yin Xiaoyan , Singhal Neel S. , Graham Mark J. , Crooke Roseanne M. , Ahima Rexford S.
Published: August 2008

Diets with high fat content induce steatosis, insulin resistance, and type 2 diabetes. The lipid droplet protein adipose differentiation-related protein (ADRP) mediates hepatic steatosis, but whether this affects insulin action in the liver or peripheral organs in diet-induced obesity is uncertain. We fed C57BL/6J mice a high-fat diet and simultaneously treated them with an antisense oligonucleotide (ASO) against ADRP for 4 wk. Glucose homeostasis was assessed with clamp and tracer techniques. ADRP ASO decreased the levels of triglycerides and diacylglycerol in the liver, but fatty acids, long-chain fatty acyl CoAs, ceramides, and cholesterol were unchanged. Insulin action in the liver was enhanced after ADRP ASO treatment, whereas muscle and adipose tissue were not affected. ADRP ASO increased the phosphorylation of insulin receptor substrate (IRS)1, IRS2, and Akt, and decreased gluconeogenic enzymes and PKCε, consistent with its insulin-sensitizing action. These results demonstrate an important role for ADRP in the pathogenesis of diet-induced insulin resistance.
Heterogeneity of acid secretion induced by carbachol and histamine along the gastric gland axis and its relationship to [Ca2+]i
Authors: Perez-Zoghbi Jose Francisco , Mayora Adriana , Ruiz Marie Christine , Michelangeli Fabian
Published: August 2008

The gastric glands of the mammalian fundic mucosa are constituted by different cell types. Gastric fluid is a mixture of acid, alkali, ions, enzymes, and mucins secreted by parietal, chief, and mucous cells. We studied activation of acid secretion using LysoSensor Yellow/Blue in conjunction with fluo 3 to measure changes in pH and Ca2+ in isolated rabbit gastric glands. We evidenced a spatial heterogeneity in the amplitude of acid response along the gland axis under histamine and cholinergic stimulation. Carbachol induced a transitory pH increase before acidification. This relative alkalinization may be related to granule release from other cell types. Omeprazole inhibited the acid component but not the rise in pH. Histamine stimulated acid secretion without increase of lumen pH. We studied the relationship between Ca2+ release and/or entry and H+ secretion in glands stimulated by carbachol. Ca2+ release was associated with a fast and transient components of H+ secretion. We found a linear relationship between Ca2+ release and H+ secretion. Ca2+ entry was associated with a second slow and larger component of acid secretion. The fast component may be the result of activation of Cl− and K+ channels and hence H+/K+ pumps already present in the membrane, whereas the slow component might be associated with translocation of H+/K+ pumps to the canaliculi. In conclusion, with cholinergic stimulation, gastric glands secrete a mixture of acid and other product(s) with a pH above 4.2, both triggered by Ca2+ release. Maintenance of acid secretion depends on Ca2+ entry and perhaps membrane fusion.
Altered expression and distribution of aquaporin-9 in the liver of rat with obstructive extrahepatic cholestasis
Authors: Calamita Giuseppe , Ferri Domenico , Gena Patrizia , Carreras Flavia I. , Liquori Giuseppa E. , Portincasa Piero , Marinelli Raúl A. , Svelto Maria
Published: August 2008

Rat hepatocytes express aquaporin-9 (AQP9), a basolateral channel permeable to water, glycerol, and other small neutral solutes. Although liver AQP9 is known for mediating the uptake of sinusoidal blood glycerol, its relevance in bile secretion physiology and pathophysiology remains elusive. Here, we evaluated whether defective expression of AQP9 is associated to secretory dysfunction of rat hepatocytes following bile duct ligation (BDL). By immunoblotting, 1-day BDL resulted in a slight decrease of AQP9 protein in basolateral membranes and a simultaneous increase of AQP9 in intracellular membranes. This pattern was steadily accentuated in the subsequent days of BDL since at 7 days BDL basolateral membrane AQP9 decreased by 85% whereas intracellular AQP9 increased by 115%. However, the AQP9 immunoreactivity of the total liver membranes from day 7 of BDL rats was reduced by 49% compared with the sham counterpart. Results were confirmed by immunofluorescence and immunogold electron microscopy and consistent with biophysical studies showing considerable decrease of the basolateral membrane water and glycerol permeabilities of cholestatic hepatocytes. The AQP9 mRNA was slightly reduced only at day 7 of BDL, indicating that the dysregulation was mainly occurring at a posttranslational level. The altered expression of liver AQP9 during BDL was not dependent on insulin, a hormone known to negatively regulate AQP9 at a transcriptional level, since insulinemia was unchanged in 7-day BDL rats. Overall, these results suggest that extrahepatic cholestasis leads to downregulation of AQP9 in the hepatocyte basolateral plasma membrane and dysregulated aquaporin channels contribute to bile flow dysfunction of cholestatic hepatocyte.
Serotonin modifies cytoskeleton and brush-border membrane architecture in human intestinal epithelial cells
Authors: Gill Ravinder K. , Shen Le , Turner Jerrold R. , Saksena Seema , Alrefai Waddah A. , Pant Nitika , Esmaili Ali , Dwivedi Alka , Ramaswamy Krishnamurthy , Dudeja Pradeep K.
Published: August 2008

Serotonin or 5-hydroxytryptamine (5-HT) influences numerous functions in the gastrointestinal tract. We previously demonstrated that 5-HT treatment of Caco-2 cells inhibited Na+/H+exchangers (NHE) and Cl−/OH−exchange activities via distinct signaling mechanisms. Since regulation of several ion transporters such as NHE3 is influenced by intact cytoskeleton, we hypothesized that 5-HT modifies actin cytoskeleton and/or brush-border membrane architecture via involvement of signaling pathways. Ultrastructural analysis showed that 5-HT (0.1 μM, 1 h) treatment of Caco-2 cells caused the apical membrane to assume a convex dome shape that was associated with shortening of microvilli. To examine whether these cellular architecture changes are cytoskeleton driven, we analyzed actin cytoskeleton by fluorescence microscopy. 5-HT induced basal stress fibers with prominent cortical actin filaments via 5-HT3 and 5-HT4 receptor subtypes. This induction was partially attenuated by chelation of intracellular Ca2+and PKCα inhibition (Go6976). In vitro assays revealed that PKCα interacted with actin and this association was increased by 5-HT. Our data provide novel evidence that 5-HT-induced signaling via 5-HT3/4 receptor subtypes to cause Ca2+and PKCα-dependent regulation of actin cytoskeleton may play an important role in modulation of ion transporters that contribute to pathophysiology of diarrheal conditions associated with elevated levels of 5-HT.
Effects of cholecystokinin-58 on type 1 cholecystokinin receptor function and regulation
Authors: Wu S. Vincent , Harikumar Kaleeckal G. , Burgess Rebecca J. , Reeve Joseph R. , Miller Laurence J.
Published: August 2008

Cholecystokinin, like many peptide hormones, is present as multiple molecular forms. CCK-58 has been identified as the dominant form in the circulation, whereas most of the studies of CCK-receptor interactions have been performed with CCK-8. Despite both sharing the pharmacophoric region of CCK, representing its carboxy terminal heptapeptide amide, studies in vivo have demonstrated biological diversity of action of the two peptides, with CCK-58, but not CCK-8, stimulating pancreatic fluid secretion and lengthening the interval between meals. Here, we have directly studied the ability of these two CCK peptides to bind to the type 1 CCK receptor and to stimulate it to elicit an intracellular calcium response. The calcium response relative to receptor occupation was identical for CCK-58 and CCK-8, with the longer peptide binding with approximately fivefold lower affinity. We also examined the ability of the two peptides to elicit receptor internalization using morphological techniques and to disrupt the constitutive oligomerization of the CCK receptor using receptor bioluminescence resonance energy transfer. Here, both full agonist peptides had similar effects on these regulatory processes. These data suggest that both molecular forms of CCK act at the CCK1receptor quite similarly and elicit similar regulatory processes for that receptor, suggesting that the differences in biological activity observed in vivo most likely reflect differences in the clearance and/or metabolism of these long and short forms of CCK peptides.
Virtual electrodes and the induction of fibrillation in Langendorff-perfused rabbit ventricles: the role of intracellular calcium
Authors: Hayashi Hideki , Lin Shien-Fong , Joung Boyoung , Karagueuzian Hrayr S. , Weiss James N. , Chen Peng-Sheng
Published: August 2008

A strong premature electrical stimulus (S2) induces both virtual anodes and virtual cathodes. The effects of virtual electrodes on intracellular Ca2+ concentration ([Ca2+]i) transients and ventricular fibrillation thresholds (VFTs) are unclear. We studied 16 isolated, Langendorff-perfused rabbit hearts with simultaneous voltage and [Ca2+]i optical mapping and for vulnerable window determination. After baseline pacing (S1), a monophasic (10 ms anodal or cathodal) or biphasic (5 ms-5 ms) S2 was applied to the left ventricular epicardium. Virtual electrode polarizations and [Ca2+]i varied depending on the S2 polarity. Relative to the level of [Ca2+]i during the S1 beat, the [Ca2+]i level 40 ms after the onset of monophasic S2 increased by 36 ± 8% at virtual anodes and 20 ± 5% at virtual cathodes ( P < 0.01), compared with 25 ± 5% at both virtual cathode-anode and anode-cathode sites for biphasic S2. The VFT was significantly higher and the vulnerable window significantly narrower for biphasic S2 than for either anodal or cathodal S2 ( n = 7, P < 0.01). Treatment with thapsigargin and ryanodine ( n = 6) significantly prolonged the action potential duration compared with control (255 ± 22 vs. 189 ± 6 ms, P < 0.05) and eliminated the difference in VFT between monophasic and biphasic S2, although VFT was lower for both cases. We conclude that virtual anodes caused a greater increase in [Ca2+]i than virtual cathodes. Monophasic S2 is associated with lower VFT than biphasic S2, but this difference was eliminated by the inhibition of the sarcoplasmic reticulum function and the prolongation of the action potential duration. However, the inhibition of the sarcoplasmic reticulum function also reduced VFT, indicating that the [Ca2+]i dynamics modulate, but are not essential, to ventricular vulnerability.
A Pivotal Role for Heat Shock Protein 90 in Ewing Sarcoma Resistance to Anti-Insulin-like Growth Factor 1 Receptor Treatment:In vitroandIn vivoStudy
Authors: Martins Ana Sofia , Ordoñez José Luis , García-Sánchez Alfredo , Herrero David , Sevillano Victoria , Osuna Daniel , Mackintosh Carlos , Caballero Gemma , Otero Ana Pastora , Poremba Christopher , Madoz-Gúrpide Juan , de Alava Enrique
Published: August 2008

Ewing Sarcoma (ES) shows several deregulated autocrine loops mediating cell survival and proliferation. Therefore, their blockade is a promising therapeutic approach. We previously reported the in vitro effect of insulin-like growth factor 1 receptor (IGF1R)/KIT pathway blockade on ES cell lines, and we now extend our observations to changes induced by this treatment in interacting proteins/networks. A proteomic analysis revealed that Heat Shock Protein (HSP)90 was differentially expressed between ES cell lines sensitive and resistant to specific IGF1R/KIT inhibitors. We therefore inhibited HSP90 with 17-allylamino-17-demethoxygeldanamycin (17-AAG) and siRNA, and observed that ES cell line growth and survival were reduced, especially in the resistant cell lines. Conversely, HSP90 induced–expression conferred resistance to anti-IGF1R/KIT treatment in the sensitive cell lines. 17-AAG treatment induced HSP90 client protein degradation, including AKT, KIT, or IGF1R, by inhibiting their physical interaction with HSP90. Xenograft models developed with A673 ES cell line confirmed that HSP90 inhibition, alone or combined with IGF1R inhibition, significantly reduced tumor growth and expression of client proteins. Remarkably, using two independent clinical sample sets, we have found that nearly half of IGF1R-positive tumors also show HSP90 overexpression. This delineates a subset of patients that could benefit from combination of anti-HSP90 agents when considering IGF1R-targeting therapies. Importantly, sensitivity to drugs such as ADW/IMA depends not only on the levels of expression and basal activation of IGF1R/KIT, but also, and for the first time reported in ES, on the development of the stress response mechanism. Accordingly, HSP90 expression could be a predictive factor of response to IGF1R-targeting therapies. [Cancer Res 2008;68(15):6260–70]
Cyclic AMP Response Element-Binding Protein Overexpression: A Feature Associated with Negative Prognosis in Never Smokers with Non–Small Cell Lung Cancer
Authors: Seo Hye-Sook , Liu Diane D. , Bekele B. Nebiyou , Kim Mi-Kyoung , Pisters Katherine , Lippman Scott M. , Wistuba Ignacio I. , Koo Ja Seok
Published: August 2008

Lung cancer is the leading cause of cancer deaths worldwide. Recent advances in targeted therapies hold promise for the development of new treatments for certain subsets of cancer patients by targeting specific signaling molecule. Based on the identification of the transcription factor cyclic AMP response element-binding protein (CREB) as an important regulator of growth of several types of cancers and our recent findings of its importance in normal differentiation of bronchial epithelial cells, we hypothesized that CREB plays an important pathobiologic role in lung carcinogenesis. We conducted this initial study to determine whether the expression and activation status of CREB are altered in non–small cell lung cancer (NSCLC) and of any prognostic importance in NSCLC patients. We found that the expression levels of mRNA and protein of CREB and phosphorylated CREB (p-CREB) were significantly higher in most of the NSCLC cell lines and tumor specimens than in the normal human tracheobronchial epithelial cells and adjacent normal lung tissue, respectively. Analysis of CREB mRNA expression and the CREB gene copy number showed that CREB overexpression occurred mainly at the transcriptional level. Immunohistochemical analysis of tissue microarray slides containing sections of NSCLC specimens obtained from 310 patients showed that a decreased survival duration was significantly associated with overexpression of CREB or p-CREB in never smokers but not in current or former smokers with NSCLC. These are the first reported results illustrating the potential of CREB as a molecular target for the prevention and treatment of NSCLC, especially in never smokers. [Cancer Res 2008;68(15):6065–73]
Androgen Levels Increase by Intratumoral De novo Steroidogenesis during Progression of Castration-Resistant Prostate Cancer
Authors: Locke Jennifer A. , Guns Emma S. , Lubik Amy A. , Adomat Hans H. , Hendy Stephen C. , Wood Catherine A. , Ettinger Susan L. , Gleave Martin E. , Nelson Colleen C.
Published: August 2008

Although systemic androgen deprivation prolongs life in advanced prostate cancer, remissions are temporary because patients almost uniformly progress to a state of a castration-resistant prostate cancer (CRPC) as indicated by recurring PSA. This complex process of progression does not seem to be stochastic as the timing and phenotype are highly predictable, including the observation that most androgen-regulated genes are reactivated despite castrate levels of serum androgens. Recent evidence indicates that intraprostatic levels of androgens remain moderately high following systemic androgen deprivation therapy, whereas the androgen receptor (AR) remains functional, and silencing the AR expression following castration suppresses tumor growth and blocks the expression of genes known to be regulated by androgens. From these observations, we hypothesized that CRPC progression is not independent of androgen-driven activity and that androgens may be synthesized de novo in CRPC tumors leading to AR activation. Using the LNCaP xenograft model, we showed that tumor androgens increase during CRPC progression in correlation to PSA up-regulation. We show here that all enzymes necessary for androgen synthesis are expressed in prostate cancer tumors and some seem to be up-regulated during CRPC progression. Using an ex vivo radiotracing assays coupled to high-performance liquid chromatography-radiometric/mass spectrometry detection, we show that tumor explants isolated from CRPC progression are capable of de novo conversion of [14C]acetic acid to dihydrotestosterone and uptake of [3H]progesterone allows detection of the production of six other steroids upstream of dihydrotestosterone. This evidence suggests that de novo androgen synthesis may be a driving mechanism leading to CRPC progression following castration. [Cancer Res 2008;68(15):6407–15]
Loss of MLL PHD Finger 3 Is Necessary for MLL-ENL–Induced Hematopoietic Stem Cell Immortalization
Authors: Chen Jing , Santillan Donna A. , Koonce Mark , Wei Wei , Luo Roger , Thirman Michael J. , Zeleznik-Le Nancy J. , Diaz Manuel O.
Published: August 2008

Reciprocal chromosomal translocations at the MLL gene locus result in expression of novel fusion proteins, such as MLL-ENL, associated with leukemia. The three PHD finger cassette, one of the highly conserved domains in MLL, is absent in all fusion proteins. This domain has been shown to interact with Cyp33, a cyclophilin which enhances the recruitment of histone deacetylases (HDAC) to the MLL repression domain and mediates HOX gene repression. Insertion of the third PHD finger of MLL into MLL-ENL allows the recruitment of Cyp33 and, subsequently, HDAC1 to the fusion protein. Furthermore, expression of the fusion protein with the PHD finger insertion mediates the down-regulation of the HOXC8 gene expression in a Cyp33-dependent manner. Finally, the addition of the PHD finger domain or the third PHD finger alone into MLL-ENL blocks the hematopoietic stem cell immortalization potential of the fusion protein in serial plating colony assays. Insertion of only the first and second PHD fingers has no such effect. These data support the hypothesis that the binding of Cyp33 to the MLL third PHD finger switches the MLL function from transactivation to repression. In the immortalizing MLL fusion protein, the loss of the PHD fingers, in combination with the gain of the activation domain of ENL or of other partner proteins, makes the fusion protein a constitutive transactivator. This leads to constitutive overexpression of MLL target genes that block stem cell commitment and promote stem cell renewal, probably the first step in MLL-related leukemogenesis. [Cancer Res 2008;68(15):6199–207]
Oxidative Stress Plays a Critical Role in Inactivating Mutant BRAF by Geldanamycin Derivatives
Authors: Fukuyo Yayoi , Inoue Masahiro , Nakajima Takuma , Higashikubo Ryuji , Horikoshi Nobuko T. , Hunt Clayton , Usheva Anny , Freeman Michael L. , Horikoshi Nobuo
Published: August 2008

The geldanamycin derivatives 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) are promising chemotherapeutic drugs that inhibit heat shock protein 90 (HSP90) function. Previous studies have shown that 17-AAG/DMAG treatment induces the degradation of mutant BRAF (V600E) and inhibits the activation of mitogen-activated protein/extracellular signal-regulated kinase 1/2 (MEK1/2). We have found, however, that HSP90 inhibition alone is not sufficient for efficient BRAF(V600E) degradation in some cells. HSP90 inhibitors structurally unrelated to geldanamycin, radicicol and novobiocin, while inducing the degradation of the HSP90 client protein RAF-1 fail to induce BRAF(V600E) degradation or inhibit MEK1/2 activation in HT29 human colon cancer cells. Moreover, after treatment with 17-DMAG, the kinase activity of residual, undegraded BRAF(V600E) was also lost. Incubation of cells with a reactive oxygen species (ROS) scavenger, N-acetyl cysteine, partially restored kinase activity and also partially prevented BRAF(V600E) degradation due to 17-DMAG treatment. Conversely, treatment with the ROS producing drug menadione clearly inhibited MEK1/2 and reduced BRAF(V600E). These results suggest that in addition to direct inhibition of HSP90, the antitumor effect of geldanamycin and its derivatives is also mediated though the production of ROS, which may directly inactivate tumorigenic mutant BRAF(V600E). [Cancer Res 2008;68(15):6324–30]
Defective Transcription/Repair Factor IIH Recruitment to Specific UV Lesions in Trichothiodystrophy Syndrome
Authors: Chiganças Vanessa , Lima-Bessa Keronninn M. , Stary Anne , Menck Carlos F.M. , Sarasin Alain
Published: August 2008

Most trichothiodystrophy (TTD) patients present mutations in the xeroderma pigmentosum D (XPD) gene, coding for a subunit of the transcription/repair factor IIH (TFIIH) complex involved in nucleotide excision repair (NER) and transcription. After UV irradiation, most TTD/XPD patients are more severely affected in the NER of cyclobutane pyrimidine dimers (CPD) than of 6-4-photoproducts (6-4PP). The reasons for this differential DNA repair defect are unknown. Here we report the first study of NER in response to CPDs or 6-4PPs separately analyzed in primary fibroblasts. This was done by using heterologous photorepair; recombinant adenovirus vectors carrying photolyases enzymes that repair CPD or 6-4PP specifically by using the energy of light were introduced in different cell lines. The data presented here reveal that some TTD/XPD mutations affect the recruitment of TFIIH specifically to CPDs, but not to 6-4PPs. This deficiency is further confirmed by the inability of TTD/XPD cells to recruit, specifically for CPDs, NER factors that arrive in a TFIIH-dependent manner later in the NER pathway. For 6-4PPs, we show that TFIIH complexes carrying an NH2-terminal XPD mutated protein are also deficient in recruitment of NER proteins downstream of TFIIH. Treatment with the histone deacetylase inhibitor trichostatin A allows the recovery of TFIIH recruitment to CPDs in the studied TTD cells and, for COOH-terminal XPD mutations, increases the repair synthesis and survival after UV, suggesting that this defect can be partially related with accessibility of DNA damage in closed chromatin regions. [Cancer Res 2008;68(15):6074–83]
Tumor-Associated MICA Is Shed by ADAM Proteases
Authors: Waldhauer Inja , Goehlsdorf Dennis , Gieseke Friederike , Weinschenk Toni , Wittenbrink Mareike , Ludwig Andreas , Stevanovic Stefan , Rammensee Hans-Georg , Steinle Alexander
Published: August 2008

The immunoreceptor NKG2D promotes immunosurveillance of malignant cells and protects the host from tumor initiation by activating natural killer cells and costimulating CD8 T cells. NKG2D-mediated recognition of malignant cells by cytotoxic lymphocytes is enabled through the tumor-associated expression of NKG2D ligands (NKG2DL) resulting from cellular or genotoxic stress. Shedding of NKG2DL is thought to constitute a major countermechanism of tumor cells to subvert NKG2D-mediated immunosurveillance. Here, we report that the prototypical NKG2DL MICA is released by proteolytic cleavage in the stalk of the MICA ectodomain, where deletions, but not alanine substitutions, impede MICA shedding. Small compound-mediated stimulation and inhibition of MICA shedding adduced characteristics that indicated an involvement of members of the “a disintegrin and metalloproteinase” (ADAM) family. Accordingly, MICA shedding by tumor cells was inhibited by silencing of the related ADAM10 and ADAM17 proteases, which are known to promote tumor growth by releasing epidermal growth factor receptor ligands. Collectively, our data show that ADAM10 and ADAM17 are critically involved in the tumor-associated proteolytic release of soluble MICA facilitating tumor immune escape. Hence, therapeutic blockade of ADAM10 and ADAM17 seems promising for cancer treatment by targeting both growth and immune escape of tumors. [Cancer Res 2008;68(15):6368–76]
A Small-Molecule E2F Inhibitor Blocks Growth in a Melanoma Culture Model
Authors: Ma Yihong , Kurtyka Courtney A. , Boyapalle Sandhya , Sung Shen-Shu , Lawrence Harshani , Guida Wayne , Cress W. Douglas
Published: August 2008

HLM006474 was identified using a computer-based virtual screen and the known crystal structure of the DNA-bound E2F4/DP2 heterodimer. Treatment of multiple cell lines with HLM006474 resulted in the loss of intracellular E2F4 DNA-binding activity as measured by electrophoretic mobility shift assay within hours. Overnight exposure to HLM006474 resulted in down-regulation of total E2F4 protein as well as known E2F targets. The effects of HLM006474 treatment on different cell lines varied but included a reduction in cell proliferation and an increase in apoptosis. HLM006474 induced apoptosis in a manner distinct from cisplatin and doxorubicin. E2F4-null mouse embryonic fibroblasts were less sensitive than wild-type counterparts to the apoptosis-inducing activity of the compound, revealing its biological specificity. A375 cells were extremely sensitive to the apoptosis-inducing activity of the compound in two-dimensional culture, and HLM006474 was a potent inhibitor of melanocytes proliferation and subsequent invasion in a three-dimensional tissue culture model system. Together, these results suggest that interference with E2F activity using small molecules may have clinical application in cancer therapy. [Cancer Res 2008;68(15):6292–9]
A Six-Gene Signature Predicting Breast Cancer Lung Metastasis
Authors: Landemaine Thomas , Jackson Amanda , Bellahcène Akeila , Rucci Nadia , Sin Soraya , Abad Berta Martin , Sierra Angels , Boudinet Alain , Guinebretière Jean-Marc , Ricevuto Enrico , Noguès Catherine , Briffod Marianne , Bièche Ivan , Cherel Pascal , Garcia Teresa , Castronovo Vincent , Teti Anna , Lidereau Rosette , Driouch Keltouma
Published: August 2008

The lungs are a frequent target of metastatic breast cancer cells, but the underlying molecular mechanisms are unclear. All existing data were obtained either using statistical association between gene expression measurements found in primary tumors and clinical outcome, or using experimentally derived signatures from mouse tumor models. Here, we describe a distinct approach that consists of using tissue surgically resected from lung metastatic lesions and comparing their gene expression profiles with those from nonpulmonary sites, all coming from breast cancer patients. We show that the gene expression profiles of organ-specific metastatic lesions can be used to predict lung metastasis in breast cancer. We identified a set of 21 lung metastasis–associated genes. Using a cohort of 72 lymph node–negative breast cancer patients, we developed a 6-gene prognostic classifier that discriminated breast primary cancers with a significantly higher risk of lung metastasis. We then validated the predictive ability of the 6-gene signature in 3 independent cohorts of breast cancers consisting of a total of 721 patients. Finally, we show that the signature improves risk stratification independently of known standard clinical variables and a previously established lung metastasis signature based on an experimental breast cancer metastasis model. [Cancer Res 2008;68(15):6092–9]
Differential Cellular Internalization of Anti-CD19 and -CD22 Immunotoxins Results in Different Cytotoxic Activity
Authors: Du Xing , Beers Richard , FitzGerald David J. , Pastan Ira
Published: August 2008

B-cell malignancies routinely express surface antigens CD19 and CD22. Immunotoxins against both antigens have been evaluated, and the immunotoxins targeting CD22 are more active. To understand this disparity in cytotoxicity and guide the screening of therapeutic targets, we compared two immunotoxins, FMC63(Fv)-PE38–targeting CD19 and RFB4(Fv)-PE38 (BL22)–targeting CD22. Six lymphoma cell lines have 4- to 9-fold more binding sites per cell for CD19 than for CD22, but BL22 is 4- to 140-fold more active than FMC63(Fv)-PE38, although they have a similar cell binding affinity (Kd, ∼7 nmol/L). In 1 hour, large amounts of BL22 are internalized (2- to 3-fold more than the number of CD22 molecules on the cell surface), whereas only 5.2% to 16.6% of surface-bound FMC63(Fv)-PE38 is internalized. The intracellular reservoir of CD22 decreases greatly after immunotoxin internalization, indicating that it contributes to the uptake of BL22. Treatment of cells with cycloheximide does not reduce the internalization of BL22. Both internalized immunotoxins are located in the same vesicles. Our results show that the rapid internalization of large amounts of BL22 bound to CD22 makes CD22 a better therapeutic target than CD19 for immunotoxins and probably for other immunoconjugates that act inside cells. [Cancer Res 2008;68(15):6300–5]
Transforming Growth Factor-β (TGF-β) and TGF-β–Associated Kinase 1 Are Required for R-Ras–Mediated Transformation of Mammary Epithelial Cells
Authors: Erdogan Mete , Pozzi Ambra , Bhowmick Neil , Moses Harold L. , Zent Roy
Published: August 2008

Transforming growth factor-β (TGF-β) cooperates with oncogenic members of the Ras superfamily to promote cellular transformation and tumor progression. Apart from the classic (H-, K-, and N-) Ras GTPases, only the R-Ras subfamily (R-Ras, R-Ras2/TC21, and R-Ras3/M-Ras) has significant oncogenic potential. In this study, we show that oncogenic R-Ras transformation of EpH4 cells requires TGF-β signaling. When murine EpH4 cells were stably transfected with a constitutively active R-Ras(G38V) mutant, they were no longer sensitive to TGF-β–mediated growth inhibition and showed increased proliferation and transformation in response to exogenous TGF-β. R-Ras/EpH4 cells require TGF-β signaling for transformation to occur and they produce significantly elevated levels of endogenous TGF-β, which signals in an autocrine fashion. The effects of TGF-β are independent of Smad2/3 activity and require activation of TGF-β–associated kinase 1 (TAK1) and its downstream effectors c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase as well as the phosphoinositide 3-kinase/Akt and mammalian target of rapamycin pathways. Thus, TAK1 is a novel link between TGF-β signaling and oncogenic R-Ras in the promotion of tumorigenesis. [Cancer Res 2008;68(15):6224–31]
Neutrophil Gelatinase–Associated Lipocalin: A Novel Suppressor of Invasion and Angiogenesis in Pancreatic Cancer
Authors: Tong Zhimin , Kunnumakkara Ajaikumar B. , Wang Huamin , Matsuo Yoichi , Diagaradjane Parmeswaran , Harikumar Kuzhuvelil B. , Ramachandran Vijaya , Sung Bokyung , Chakraborty Arup , Bresalier Robert S. , Logsdon Craig , Aggarwal Bharat B. , Krishnan Sunil , Guha Sushovan
Published: August 2008

Neutrophil gelatinase–associated lipocalin (NGAL) is a 25-kDa secreted acute phase protein, which is also up-regulated in multiple cancers, including breast, lung, and pancreas. Recently, NGAL has been proposed as an early biomarker in pancreatic cancer (PaCa). However, its biological role in PaCa is unknown. In this study, we examined in vitro and in vivo the functional role of NGAL in PaCa. Well- to moderately differentiated PaCa cells (AsPC-1, BxPC-3, and Capan-2) expressed high levels of NGAL but moderately to poorly differentiated PaCa cells (PANC-1 and MIAPaCa-2) expressed undetectable NGAL levels. Immunohistochemistry of untreated tissue microarray showed specific NGAL staining in resected PaCa specimens (P = 0.0167). Stable NGAL overexpression (MIAPaCa-2 and PANC-1) significantly blocked PaCa cell adhesion and invasion in vitro and vice versa with stable PaCa clones (BxPC-3 and AsPC-1). Moreover, NGAL overexpression reduced focal adhesion kinase (FAK) tyrosine-397 phosphorylation in PaCa cells. Furthermore, NGAL overexpression potently decreased angiogenesis in vitro partly through reduced vascular endothelial growth factor (VEGF) production and vice versa. Stable NGAL overexpression or underexpression had no effect on PaCa cell survival, viability, and response to chemotherapeutic drugs. Finally, MIAPaCa-2 cells overexpressing NGAL reduced tumor volume (P = 0.012), local and distant metastasis (P = 0.002), and angiogenesis (P = 0.05) with no effect on K-67 proliferation index (P > 0.1) in an orthotopic nude mouse PaCa model. Collectively, our results suggest that NGAL reduces adhesion/invasion partly by suppressing FAK activation and inhibits angiogenesis partly by blocking VEGF production in PaCa cells. Thus, NGAL is a potential suppressor of invasion and angiogenesis in advanced PaCa. [Cancer Res 2008;68(15):6100–8]
Kinetic Stabilization of Microtubule Dynamics by Estramustine Is Associated with Tubulin Acetylation, Spindle Abnormalities, and Mitotic Arrest
Authors: Mohan Renu , Panda Dulal
Published: August 2008

Estramustine (EM) alone or in combination with other anticancer agents is clinically used for the treatment of hormone refractory prostate cancer. Furthermore, EM has been shown to potently inhibit the proliferation of different types of cancer cells in culture apparently by targeting microtubules; however, the antiproliferative mechanism of action of EM is not clear. In this work, we have shown that EM strongly suppressed the dynamic instability of individual microtubules in MCF-7 cells by reducing the rates of growing and shortening excursions and increasing the time microtubule spent in the pause state. At its half maximal proliferation inhibitory concentration (IC50), EM exerted strong suppressive effects on the dynamics of microtubules in MCF-7 cells without detectably affecting either the organization or the polymerized mass of microtubules. At relatively high concentrations (5 × IC50), EM significantly depolymerized microtubules in the cells. Furthermore, the microtubules were found highly acetylated, supporting the conclusion that they were stabilized by the drug. EM treatment induced spindle abnormalities in MCF-7 cells, and a major population of the arrested mitotic cells was multipolar. EM also perturbed the microtubule-kinetochore interaction, thereby activating the spindle assembly checkpoint and leading to apoptotic cell death. [Cancer Res 2008;68(15):6181–9]
Par-4 Binds to Topoisomerase 1 and Attenuates Its DNA Relaxation Activity
Authors: Goswami Anindya , Qiu Shirley , Dexheimer Thomas S. , Ranganathan Padhma , Burikhanov Ravshan , Pommier Yves , Rangnekar Vivek M.
Published: August 2008

The regulation of DNA relaxation by topoisomerase 1 (TOP1) is essential for DNA replication, transcription, and recombination events. TOP1 activity is elevated in cancer cells, yet the regulatory mechanism restraining its activity is not understood. We present evidence that the tumor suppressor protein prostate apoptosis response-4 (Par-4) directly binds to TOP1 and attenuates its DNA relaxation activity. Unlike camptothecin, which binds at the TOP1-DNA interface to form cleavage complexes, Par-4 interacts with TOP1 via its leucine zipper domain and sequesters TOP1 from the DNA. Par-4 knockdown by RNA interference enhances DNA relaxation and gene transcription activities and promotes cellular transformation in a TOP1-dependent manner. Conversely, attenuation of TOP1 activity either by RNA interference or Par-4 overexpression impedes DNA relaxation, cell cycle progression, and gene transcription activities and inhibits transformation. Collectively, our findings suggest that Par-4 serves as an intracellular repressor of TOP1 catalytic activity and regulates DNA topology to suppress cellular transformation. [Cancer Res 2008;68(15):6190–8]
Loss of Cannabinoid Receptor 1 Accelerates Intestinal Tumor Growth
Authors: Wang Dingzhi , Wang Haibin , Ning Wei , Backlund Michael G. , Dey Sudhansu K. , DuBois Raymond N.
Published: August 2008

Although endocannabinoid signaling is important for certain aspects of gastrointestinal homeostasis, the role of the cannabinoid receptors (CB) in colorectal cancer has not been defined. Here we show that CB1 expression was silenced in human colorectal cancer due to methylation of the CB1 promoter. Our genetic and pharmacologic studies reveal that loss or inhibition of CB1 accelerated intestinal adenoma growth in ApcMin/+ mice whereas activation of CB1 attenuated intestinal tumor growth by inducing cell death via down-regulation of the antiapoptotic factor survivin. This down-regulation of survivin by CB1 is mediated by a cyclic AMP–dependent protein kinase A signaling pathway. These results indicate that the endogenous cannabinoid system may represent a potential therapeutic target for prevention or treatment of colorectal cancer. [Cancer Res 2008;68(15):6468–76]
Antitumor Activity of Immunotoxins with T-Cell Receptor–like Specificity against Human Melanoma Xenografts
Authors: Klechevsky Eynav , Gallegos Michael , Denkberg Galit , Palucka Karolina , Banchereau Jacques , Cohen Cyril , Reiter Yoram
Published: August 2008

In this study, we have explored the use of Fab-toxin proteins (immunotoxin) to target antigen-specific MHC-peptide complexes of in vitro and in vivo cancer cells. A human phage display library was used to screen for T-cell receptor (TCR)–like antibodies that are highly specific for the peptide melanoma-associated antigen MART-126-35 presented by HLA-A201. We also used previously selected TCR-like antibodies specific for the peptide melanoma-associated antigen gp100280-288 presented by HLA-A201. The recombinant immunotoxin constructs were generated by fusing the targeting Fab fragment to a truncated form of Pseudomonas exotoxin, PE38KDEL. These immunotoxins bound with high affinity to the EBV-transformed JY cell line pulsed with the aforementioned peptides and internalized within 30 min. A significant inhibition of protein synthesis, which resulted in cell death, was detected at 24 h. MART-1–specific and gp100-specific immunotoxins bound and killed HLA-A201 melanoma MART-1+ and gp100+ cell lines that were presented at natural levels but do not bind to HLA-A201− or to HLA-A201+ MART-1− and gp100− cell lines. In severe combined immunodeficient mice, MART-1 and gp100 immunotoxins significantly and discriminately inhibited human melanoma growth. These results show that MHC class I/peptide complexes can serve as a specific target for passive immunotherapy of cancer. [Cancer Res 2008;68(15):6360–7]
Bobel-24 and Derivatives Induce Caspase-Independent Death in Pancreatic Cancer Regardless of Apoptotic Resistance
Authors: Parreño Matilde , Casanova Isolda , Céspedes María Virtudes , Vaqué Jose Pedro , Pavón Miguel Angel , Leon Javier , Mangues Ramon
Published: August 2008

The poor prognosis of pancreatic cancer and poor sensitivity to current therapeutics, associated with resistance to apoptosis, urge the search for new drugs. We previously described the induction of caspase-independent mithochondrial death in leukemia cells by Bobel-24 (AM-24) and derivatives. Here, we explored whether these compounds induce a similar cytotoxicity in human pancreatic carcinoma cell lines (NP18, NP9, NP31, and NP29). Bobel-24 or Bobel-16 induced cytotoxicity and DNA synthesis inhibition in all cell lines and apoptosis in all lines, except for NP9. Caspase and/or poly(ADP-ribose) polymerase-1 (PARP-1) activity inhibition experiments showed that cytotoxicity was mainly induced through apoptosis in NP18 and through a caspase-independent process in NP9. Moreover, in NP29 or NP31 cell lines, both caspase-dependent and caspase-independent cell death mechanisms coexisted. Cell death was associated with reactive oxygen species (ROS) production, mitochondrial depolarization, cytochrome c and apoptosis-inducing factor (AIF) release, AIF nuclear translocation, and lysosomal cathepsin release. Inhibition of ROS production, mitochondrial pore permeability, PARP-1, or phospholipase A2 partially prevented cell death. Moreover, cathepsin B inhibition or down-regulation by small interfering RNA partially blocked cell death. In conclusion, Bobel-24 and derivatives trigger caspase-independent lysosomal and mitochondrial death in all tested human pancreatic cancer lines, irrespective of their degree of apoptotic sensitivity, becoming the only active cytotoxic mechanism in the apoptosis-resistant NP9 line. This mechanism may overcome the resistance to apoptosis observed in pancreatic carcinoma when treated with current genotoxic drugs. [Cancer Res 2008;68(15):6313–23]
Implications of Apurinic/Apyrimidinic Endonuclease in Reactive Oxygen Signaling Response after Cisplatin Treatment of Dorsal Root Ganglion Neurons
Authors: Jiang Yanlin , Guo Chunlu , Vasko Michael R. , Kelley Mark R.
Published: August 2008

Peripheral neuropathy is one of the major side effects of the anticancer drug cisplatin. Although previous work suggests that this neuropathy correlates with formation of DNA adducts in sensory neurons, growing evidence suggests that cisplatin also increases the generation of reactive oxygen species (ROS), which could cause DNA damage. Apurinic/apyrimidinic endonuclease/redox factor-1 (Ape1/Ref-1) is a multifunctional protein involved in DNA base excision repair of oxidative DNA damage and in redox regulation of a number of transcription factors. Therefore, we asked whether altering Ape1 functions would influence cisplatin-induced neurotoxicity. Sensory neurons in culture were exposed to cisplatin for 24 hours and several end points of toxicity were measured, including production of ROS, cell death, apoptosis, and release of the immunoreactive calcitonin gene–related peptide (iCGRP). Reducing expression of Ape1 in neuronal cultures using small interfering RNA (siRNA) enhances cisplatin-induced cell killing, apoptosis, ROS generation, and cisplatin-induced reduction in iCGRP release. Overexpressing wild-type Ape1 attenuates all the toxic effects of cisplatin in cells containing normal endogenous levels of Ape1 and in cells with reduced Ape1 levels after Ape1siRNA treatment. Overexpressing the redox deficient/repair competent C65-Ape1 provides partial rescue, whereas the repair-deficient Ape1 (N226A + R177A) does not protect neurons from cisplatin toxicity. We also observe an increase in phosphorylation of p53 after a decrease in Ape1 levels in sensory neuronal cultures. These results strongly support the notion that Ape1 is a potential translational target such that protecting Ape1 levels and particularly its DNA repair function could reduce peripheral neuropathy in patients undergoing cisplatin treatment. [Cancer Res 2008;68(15):6425–34]