bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross-Linked Forms of the Isolated N-Terminal Domain of the Lethal Factor Are Potent Inhibitors of Anthrax Toxin
Authors: Juris Stephen J. , Melnyk Roman A. , Bolcome Robert E. , Chan Joanne , Collier R. John
Published: July 2007

The proteins that comprise anthrax toxin self-assemble at the mammalian cell surface into a series of toxic complexes, each containing a heptameric form of protective antigen (PA) plus up to a total of three molecules of the enzymatic moieties of the toxin (lethal factor [LF] and edema factor [EF]). These complexes are trafficked to the endosome, where the PA heptamer forms a pore in the membrane under the influence of low pH, and bound LF and EF unfold and translocate through the pore to the cytosol. To explore the hypothesis that the PA pore can translocate multiple, cross-linked polypeptides simultaneously, we cross-linked LF
N
, the N-terminal domain of LF, via an introduced cysteine at its N or C terminus and characterized the products. Both dimers and trimers of LF
N
retained the ability to bind to PA pores and block ion conductance, but they were unable to translocate across the membrane, even at high voltages or with a transmembrane pH gradient. The multimers were remarkably potent inhibitors of toxin action in mammalian cells (20- to 50-fold more potent than monomeric LF
N
) and in a zebrafish model system. These findings show that the PA pore cannot translocate multimeric, cross-linked polypeptides and demonstrate a new approach to generating potent inhibitors of anthrax toxin.
Human Defensin α-1 CausesTrypanosoma cruziMembrane Pore Formation and Induces DNA Fragmentation, Which Leads to Trypanosome Destruction
Authors: Madison M. Nia , Kleshchenko Yuliya Y. , Nde Pius N. , Simmons Kaneatra J. , Lima Maria F. , Villalta Fernando
Published: July 2007

Human defensins play a fundamental role in the initiation of innate immune responses to some microbial pathogens. Here we show that human defensin α-1 displays a trypanocidal role againstTrypanosoma cruzi, the causative agent of Chagas' disease. The toxicity of human defensin α-1 againstT. cruziis mediated by membrane pore formation and the induction of nuclear and mitochondrial DNA fragmentation, leading to trypanosome destruction. Exposure of trypomastigote and amastigote forms ofT. cruzito defensin α-1 significantly reduced parasite viability in a peptide concentration-dependent and saturable manner. The toxicity of defensin α-1 againstT. cruziis blocked by anti-defensin α-1 immunoglobulin G. Electron microscopic analysis of trypomastigotes exposed to defensin α-1 revealed pore formation in the cellular and flagellar membranes, membrane disorganization, and blebbing as well as cytoplasmic vacuolization. Furthermore, human defensin α-1 enters the trypanosome when membrane pores are present and is associated with later intracellular damage. Trypanosome membrane depolarization abolished the toxicity of defensin α-1 against the parasite. Preincubation of trypomastigotes with defensin α-1 followed by exposure to human epithelial cells significantly reducedT. cruziinfection in these cells. Thus, human defensin α-1 is an innate immune molecule that causes severe toxicity toT. cruziand plays an important role in reducing cellular infection. This is the first report showing that human defensin α-1 causes membrane pore formation in a human parasite, leading to trypanosome destruction.
Translocation of ExsE into Chinese Hamster Ovary Cells Is Required for Transcriptional Induction of the
Pseudomonas aeruginosa
Type III Secretion System
Authors: Urbanowski Mark L. , Brutinel Evan D. , Yahr Timothy L.
Published: July 2007

Transcription of the
Pseudomonas aeruginosa
type III secretion system (T3SS) is induced under Ca
2+
-limiting growth conditions or following the contact of the bacteria with host cells. The regulatory response to low Ca
2+
levels is initiated by the T3SS-mediated secretion of ExsE, a negative regulatory protein that prevents T3SS gene transcription. In the present study, we demonstrated that ExsE plays an analogous role in transcriptional induction following host cell contact. By using a flow cytometry assay, the host contact-dependent induction of T3SS gene expression was found to be dependent upon the presence of functional type III translocation machinery. Using three independent assays, we demonstrated that ExsE was translocated into Chinese hamster ovary cells in a T3SS-dependent manner. Deletion mapping experiments indicated that the amino terminus of ExsE is required both for secretion under Ca
2+
-limiting growth conditions and for translocation into host cells. A
P. aeruginosa
mutant expressing an
exsE
allele lacking codons 3 through 20 was deficient in ExsE secretion and translocation and showed constitutive repression of T3SS gene expression under Ca
2+
-limiting growth conditions. The mutant also failed to induce T3SS gene expression following host cell contact and demonstrated a significant reduction in T3SS-dependent cytotoxicity towards Chinese hamster ovary cells, indicating that the translocation of ExsE is required for the host contact-dependent induction of T3SS gene expression.
Influence of Host Immunity on Parasite Diversity inTheileria parva
Authors: Katzer Frank , Ngugi Daniel , Schnier Christian , Walker Alan R. , McKeever Declan J.
Published: July 2007

We examined the influence of host immunity on the genotypic diversity of the intracellular transforming cattle parasiteTheileria parva. By tracking the emergence of discrete parasite genotypes in an animal challenged with a bulk stabilate following immunization with its major component clone, we observed a profound modulation of genotypic frequencies in the breakthrough schizont population. In particular, no incidences of the immunizing clone were observed and a progressive decline was apparent in the relatedness of breakthrough genotypes to it. These observations were reflected in the genotypic profile of transmissible parasite stages that emerged in the erythrocyte fraction of the animal and in parasite progeny generated by tick pickup. In a separate experiment, genotypic profiles of breakthrough parasite populations were observed to vary between unrelated immune animals selected on the basis of the major histocompatibility complex (MHC) class I phenotype, a known determinant of the specificity of the immune response. Furthermore, immunization and challenge of calves with molecularly distinct but cross-protective parasite populations revealed that infection results in transmissible erythrocyte forms in spite of a protective immune response. These observations suggest that immunity does not prevent transmission of challenge parasites and that its impact on the parasite at a population level is influenced by herd MHC diversity.
Role for
sagA
and
siaA
in Quorum Sensing and Iron Regulation in
Streptococcus pyogenes
Authors: Salim Kowthar Y. , de Azavedo Joyce C. , Bast Darrin J. , Cvitkovitch Dennis G.
Published: July 2007

Streptococcus pyogenes
is a ubiquitous and versatile pathogen that causes a variety of infections with a wide range of severity. The versatility of this organism is due in part to its capacity to regulate virulence gene expression in response to the many environments that it encounters during an infection. We analyzed the expression of two potential virulence factors,
sagA
and
siaA
(also referred to as
pel
and
htsA
, respectively), in response to conditions of varying cell densities and iron concentrations. The
sagA
gene was up-regulated in conditioned medium from a wild-type strain but not from
sagA
-deficient mutants, and the gene was also up-regulated in the presence of streptolysin S (SLS), the gene product of
sagA
, thus indicating that this gene or its product is involved in density-dependent regulation of
S. pyogenes
. By comparison,
siaA
responded in a manner consistent with a role in iron acquisition since it was up-regulated under iron-restricted conditions. Although
siaA
expression was also up-regulated in the presence of SLS and in conditioned media from both wild-type and
sagA
-deficient mutants, this up-regulation was not growth phase dependent. We conclude that
sagA
encodes a quorum-sensing signaling molecule, likely SLS, and further support the notion that
siaA
is likely involved in iron acquisition.
MEASURING THE DIRECTIONALITY OF COUPLING: PHASE VERSUS STATE SPACE DYNAMICS AND APPLICATION TO EEG TIME SERIES
Authors: OSTERHAGE HANNES , MORMANN FLORIAN , WAGNER TOBIAS , LEHNERTZ KLAUS
Published: July 2007

Measuring the directionality of coupling between dynamical systems is one of the challenging problems in nonlinear time series analysis. We investigate the relative merit of two approaches to assess directionality, one based on phase dynamics modeling and one based on state space topography. We analyze unidirectionally coupled model systems to investigate the ability of the two approaches to detect driver-responder relationships and discuss certain problems and pitfalls. In addition we apply both approaches to the intracranial electroencephalogram (EEG) recorded from one epilepsy patient during the seizure-free interval to demonstrate the general suitability of directionality measures to reflect the pathological interaction of the epileptic focus with other brain areas.
A FLEXIBLE ANNEALING CHAOTIC NEURAL NETWORK TO MAXIMUM CLIQUE PROBLEM
Authors: YANG GANG , TANG ZHENG , ZHANG ZHIQIANG , ZHU YUNYI
Published: July 2007

Based on the analysis and comparison of several annealing strategies, we present a flexible annealing chaotic neural network which has flexible controlling ability and quick convergence rate to optimization problem. The proposed network has rich and adjustable chaotic dynamics at the beginning, and then can converge quickly to stable states. We test the network on the maximum clique problem by some graphs of the DIMACS clique instances, p-random and k random graphs. The simulations show that the flexible annealing chaotic neural network can get satisfactory solutions at very little time and few steps. The comparison between our proposed network and other chaotic neural networks denotes that the proposed network has superior executive efficiency and better ability to get optimal or near-optimal solution.
AVERAGING EINSTEIN'S EQUATIONS: THE LINEARIZED CASE
Authors: STOEGER WILLIAM R. , HELMI AMINA , TORRES DIEGO F.
Published: July 2007

We introduce a simple and straightforward averaging procedure, which is a generalization of one which is commonly used in electrodynamics, and show that it possesses all the characteristics we require for linearized averaging in general relativity and cosmology — for weak-field and perturbed FLRW situations. In particular, we demonstrate that it yields quantities which are approximately tensorial in these situations, and that its application to an exact FLRW metric yields another FLRW metric, to first-order in integrals over the local coordinates. Finally, we indicate some important limits of any linearized averaging procedure with respect to cosmological perturbations which are the result of averages over large amplitude small and intermediate scale inhomogeneities, and show our averaging procedure can be approximately implemented by that of Zotov and Stoeger in these cases.
ARE RADIO SOURCES AND GAMMA RAY BURSTS LUMINAL BOOMS?
Authors: THULASIDAS MANOJ
Published: July 2007

The softening of the gamma ray burst (GRB) afterglow bears remarkable similarities to the frequency evolution in a sonic boom. At the front end of the sonic boom cone, the frequency is infinite, much like a GRB. Inside the cone, the frequency rapidly decreases to infrasonic ranges and the sound source appears at two places at the same time, mimicking the double-lobed radio sources. Although a "luminal" boom violates the Lorentz invariance and is therefore forbidden, it is tempting to work out the details and compare them with existing data. This temptation is further enhanced by the observed superluminality in the celestial objects associated with radio sources and some GRBs. In this article, we calculate the temporal and spatial variation of observed frequencies from a hypothetical luminal boom and show remarkable similarity between our calculations and current observations.
ON INFLATION AND VARIATION OF THE STRONG COUPLING CONSTANT
Authors: CHAMOUN N. , LANDAU S. J. , VUCETICH H.
Published: July 2007

Variation of constants in the very early universe can generate inflation. We consider a scenario where the strong coupling constant was changing in time and where the gluon condensate underwent a phase transition ending the inflation.
DE SITTER STABILITY IN QUADRATIC GRAVITY
Authors: TOPORENSKY A. V. , TRETYAKOV P. V.
Published: July 2007

Quadratic curvature corrections to the Einstein–Hilbert action lead in general to higher-order equations of motion, which can induce instability of some unperturbed solutions of General Relativity. We study the conditions for the stability of the de Sitter cosmological solution. We argue that the simple form of this condition known for the FRW background in (3+1) dimensions changes seriously if at least one of these two assumptions is violated. In the present paper, the stability conditions for the de Sitter solution are found for the multidimensional FRW background and for Bianchi I metrics in (3 + 1) dimensions.
REALIZATIONS OF OBSERVABLES IN HAMILTONIAN SYSTEMS WITH FIRST CLASS CONSTRAINTS
Authors: BRATCHIKOV A. V.
Published: July 2007

In a Hamiltonian system with first class constraints, observables can be defined as elements of a quotient Poisson bracket algebra. In the gauge fixing method, observables form a quotient Dirac bracket algebra. We show that these two algebras are isomorphic. A new realization of the observable algebras through the original Poisson bracket is found. Generators, brackets and pointwise products of the algebras under consideration are calculated.
TOPOLOGY CHANGE AND SIGNATURE CHANGE IN NON-LINEAR FIRST-ORDER GRAVITY
Authors: BOROWIEC ANDRZEJ , FRANCAVIGLIA MAURO , VOLOVICH IGOR
Published: July 2007

We show that different topologies of a space-time manifold and different signatures of its metric can be encompassed into a single Lagrangian formalism, provided one adopts the first-order (Palatini) formulation and relies on nonlinear Lagrangians, that were earlier shown to produce, in the generic case, universality of Einstein field equations and of Komar's energy-momentum complex as well. An example in Relativistic Cosmology is provided.
AFM and Acoustics: Fast, Quantitative Nanomechanical Mapping
Authors: Huey Bryan D.
Published: July 2007

Combining atomic force microscopy and ultrasonic methods allows near-field detection of acoustic signals and thereby otherwise inaccessible nanoscale mechanical characterization. The two predominant variations, ultrasonic force microscopy and atomic force acoustic microscopy, are reviewed in detail. Applications of each to ceramics, polymers, metals, biological materials, and even subsurface structures are discussed, with a particular emphasis on image contrast mechanisms, data analysis, and experimental challenges. Finally, recent advances of these concepts into high-speed surface property mapping are presented, demonstrating 100-fold enhancements in full-frame imaging speeds.
The E2F-Regulated Gene Chk1 Is Highly Expressed in Triple-Negative Estrogen Receptor−/Progesterone Receptor−/HER-2− Breast Carcinomas
Authors: Verlinden Lieve , Vanden Bempt Isabelle , Eelen Guy , Drijkoningen Maria , Verlinden Ilse , Marchal Kathleen , De Wolf-Peeters Christiane , Christiaens Marie-Rose , Michiels Luc , Bouillon Roger , Verstuyf Annemieke
Published: July 2007

We previously showed that checkpoint kinase 1 (Chk1) and Claspin, two DNA-damage checkpoint proteins, were down-regulated by 1,25-dihydroxyvitamin D3, a known inhibitor of cell proliferation. In the present study, we aimed to investigate the transcriptional regulation of Chk1 and Claspin and to study their expression levels in human breast cancer tissue. Transient transfection experiments in MCF-7 breast cancer cells showed that promoter activities of Chk1 and Claspin were regulated by the E2F family of transcription factors. Subsequently, transcript levels of Chk1, Claspin, and E2F1 were determined by quantitative reverse transcriptase-PCR analysis in 103 primary invasive breast carcinomas and were compared with several clinicopathologic variables in breast cancer. A strong correlation was found between Chk1 and Claspin transcript levels. Transcript levels of Chk1, Claspin, and E2F1 were highest in histologic grade 3 tumors and in tumors in which the expression of estrogen receptor (ER) and progesterone receptor (PR) was lost. Moreover, Chk1 expression was significantly elevated in grade 3 breast carcinomas showing a triple-negative ER−/PR−/HER-2− phenotype compared with other grade 3 tumors. Further research is warranted to validate the use of Chk1 inhibitors in triple-negative breast carcinomas for which treatment strategies are limited at present. [Cancer Res 2007;67(14):6574–81]
The Vascular Disrupting Agent, DMXAA, Directly Activates Dendritic Cells through a MyD88-Independent Mechanism and Generates Antitumor Cytotoxic T Lymphocytes
Authors: Wallace Africa , LaRosa David F. , Kapoor Veena , Sun Jing , Cheng Guanjun , Jassar Arminder , Blouin Aaron , Ching Lai-Ming , Albelda Steven M.
Published: July 2007

5,6-Di-methylxanthenone-4-acetic acid (DMXAA) is a small molecule in the flavanoid class that has antitumor activity. Although classified as a “vascular disrupting agent,” we have recently conducted studies showing that DMXAA has remarkable efficacy in a range of tumors, working primarily as an immune modulator that activates tumor-associated macrophages and induces a subsequent CD8+ T-cell–mediated response. To more completely analyze the effect of DMXAA on CD8+ T-cell generation, we treated mice bearing tumors derived from EG7 thymoma cells that express the well-characterized chicken ovalbumin neotumor antigen. Treatment with DMXAA led to cytokine release, tumor cell necrosis, and ultimately reduction in tumor size that was lymphocyte dependent. Within 24 h of administration, we observed dendritic cell activation in tumor-draining lymph nodes (TDLN). This was followed by a rapid and marked increase in the number of tetramer-specific CD8+ T cells in the spleens of treated animals. In contrast, the vascular disrupting agent combretastatin A4-phosphate, which caused a similar amount of immediate tumor necrosis, did not activate dendritic cells, nor induce an effective antitumor response. Using in vitro systems, we made the observation that DMXAA has the ability to directly activate mouse dendritic cells, as measured by increased expression of costimulatory molecules and proinflammatory cytokine release via a pathway that does not require the Toll-like receptor adaptor molecule MyD88. DMXAA thus has the ability to activate tumor-specific CD8+ T cells through multiple pathways that include induction of tumor cell death, release of stimulatory cytokines, and direct activation of dendritic cells. [Cancer Res 2007;67(14):7011–9]
Subcellular Localization of Sigma-2 Receptors in Breast Cancer Cells Using Two-Photon and Confocal Microscopy
Authors: Zeng Chenbo , Vangveravong Suwanna , Xu Jinbin , Chang Katherine C. , Hotchkiss Richard S. , Wheeler Kenneth T. , Shen Duanwen , Zhuang Zhi-Ping , Kung Hank F. , Mach Robert H.
Published: July 2007

Sigma-2 receptor agonists have been shown to induce cell death via caspase-dependent and caspase-independent pathways. Unfortunately, there is little information regarding the molecular function of sigma-2 receptors that can explain these results. In this study, two fluorescent probes, SW107 and K05-138, were used to study the subcellular localization of sigma-2 receptors by two-photon and confocal microscopy. The results indicate that sigma-2 receptors colocalize with fluorescent markers of mitochondria, lysosomes, endoplasmic reticulum, and the plasma membrane in both EMT-6 mouse and MDA-MB-435 human breast cancer cells. The fluorescent probe, K05-138, was internalized rapidly, reaching a plateau of fluorescent intensity at 5 min. The internalization of K05-138 was reduced ∼40% by phenylarsine oxide, an inhibitor of endocytosis. These data suggest that sigma-2 ligands are internalized, in part, by an endocytotic pathway. The localization of sigma-2 receptors in several organelles known to have a role in both caspase-dependent and caspase-independent pathways of cell death supports the conclusions of previous studies suggesting that sigma-2 receptor ligands should be evaluated as potential cancer chemotherapeutic agents. [Cancer Res 2007;67(14):6708–16]
Tumor Paint: A Chlorotoxin:Cy5.5 Bioconjugate for Intraoperative Visualization of Cancer Foci
Authors: Veiseh Mandana , Gabikian Patrik , Bahrami S-Bahram , Veiseh Omid , Zhang Miqin , Hackman Robert C. , Ravanpay Ali C. , Stroud Mark R. , Kusuma Yumiko , Hansen Stacey J. , Kwok Deborah , Munoz Nina M. , Sze Raymond W. , Grady William M. , Greenberg Norman M. , Ellenbogen Richard G. , Olson James M.
Published: July 2007

Toward the goal of developing an optical imaging contrast agent that will enable surgeons to intraoperatively distinguish cancer foci from adjacent normal tissue, we developed a chlorotoxin:Cy5.5 (CTX:Cy5.5) bioconjugate that emits near-IR fluorescent signal. The probe delineates malignant glioma, medulloblastoma, prostate cancer, intestinal cancer, and sarcoma from adjacent non-neoplastic tissue in mouse models. Metastatic cancer foci as small as a few hundred cells were detected in lymph channels. Specific binding to cancer cells is facilitated by matrix metalloproteinase-2 (MMP-2) as evidenced by reduction of CTX:Cy5.5 binding in vitro and in vivo by a pharmacologic blocker of MMP-2 and induction of CTX:Cy5.5 binding in MCF-7 cells following transfection with a plasmid encoding MMP-2. Mouse studies revealed that CTX:Cy5.5 has favorable biodistribution and toxicity profiles. These studies show that CTX:Cy5.5 has the potential to fundamentally improve intraoperative detection and resection of malignancies. [Cancer Res 2007;67(14):6882–8]
Effect of Dominant Negative Transforming Growth Factor-β Receptor Type II on Cytotoxic Activity of RAW 264.7, a Murine Macrophage Cell Line
Authors: Lee Geun Taek , Hong Jun Hyuk , Kwak Cheol , Woo Jaesung , Liu Victoria , Lee Chung , Kim Isaac Yi
Published: July 2007

Transforming growth factor-β (TGF-β) is a potent suppressor of the immune system. In the present study, we investigated the effect of TGF-β resistance on a murine macrophage cell line, RAW 264.7, by overexpressing a dominant negative TGF-β receptor type II (TβRIIDN) construct. As expected, TβRIIDN-expressing RAW cells, designated as RAW-TβRIIDN, were resistant to TGF-β signaling. When these cells were cocultured with the murine renal cell carcinoma cell line, Renca, a dramatic increase in apoptosis of Renca cells was observed. Simultaneously, elevated levels of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α) in association with IFN-γ were detected in RAW-TβRIIDN cells. When the effects of TNF-α and iNOS were neutralized through the use of neutralizing antibody and NG-methyl-l-arginine, respectively, the enhanced cytotoxicity of TβRIIDN-RAW cells was partially reversed. Taken together, these results show that TGF-β–resistant RAW 264.7 murine macrophage cells have increased cytotoxic activity that is in part mediated by iNOS and TNF-α. [Cancer Res 2007;67(14):6717–24]
Inhibition of Nuclear Factor-κB Activity by Temozolomide Involves O6-Methylguanine–Induced Inhibition of p65 DNA Binding
Authors: Yamini Bakhtiar , Yu Xiaohong , Dolan M. Eileen , Wu Michael H. , Kufe Donald W. , Weichselbaum Ralph R.
Published: July 2007

The alkylating agent temozolomide, commonly used in the treatment of malignant glioma, causes cellular cytotoxicity by forming O6-methylguanine adducts. In this report, we investigated whether temozolomide alters the activity of the transcription factor nuclear factor-κB (NF-κB). Temozolomide inhibits basal and tumor necrosis factor α (TNFα)–induced NF-κB transcriptional activity without altering phosphorylation or degradation of inhibitor of κB-α. Inhibition of NF-κB is secondary to attenuation of p65 DNA binding, not nuclear translocation. Inhibition of DNA binding is shown both in vitro, with gel shift studies and DNA binding assays, and in vivo at κB sites. Consistent with inhibition of NF-κB activity, temozolomide reduces basal and TNFα-induced κB-dependent gene expression. Temozolomide also inhibits NF-κB activated by inducers other than TNFα, including lipopolysaccharide, doxorubicin, and phorbol 12-myristate 13-acetate. The inhibitory action of temozolomide on NF-κB is observed to be maximal following pretreatment of cells with temozolomide for 16 h and is also seen with the SN1-type methylating agent methylnitrosourea. The ability of temozolomide to form O6-methylguanine adducts is important for inhibition of NF-κB as is the presence of a functioning mismatch repair system. Activation of NF-κB with TNFα before administration of temozolomide reduces the cytotoxicity of temozolomide, whereas 16-h pretreatment with temozolomide resensitizes cells to killing. This work shows a mechanism whereby O6-methylguanine adducts formed by temozolomide lead to inhibition of NF-κB activity and illustrates a link between mismatch repair processing of alkylator-induced DNA damage and cell death. [Cancer Res 2007;67(14):6889–98]
Transforming Growth Factor α–Dependent Cancer Progression Is Modulated by Muc1
Authors: Pochampalli Mamata R. , Bitler Benjamin G. , Schroeder Joyce A.
Published: July 2007

Transforming growth factor α (TGFα) is a potent inducer of cellular transformation, through its binding and activation of the epidermal growth factor receptor (EGFR). Previous studies in our laboratory showed that EGFR could also be affected by the glycoprotein MUC1, which inhibits ligand-stimulated degradation of EGFR in breast epithelial cell lines. To determine the effect of Muc1 expression on TGFα/EGFR-dependent breast transformation, we crossed the WAP-TGFα transgenic mouse model of breast cancer onto a Muc1-null background. We found that the loss of Muc1 expression dramatically affects mammary gland transformation and progression. Although 100% of WAP-TGFα/Muc1+/+ mice form mammary gland tumors by 1 year, only 37% of WAP-TGFα/Muc1−/− form tumors by this time. This difference is also associated with a delay in onset, with a doubling of onset time observed in the WAP-TGFα/Muc1−/− compared with the WAP-TGFα/Muc1+/+ mice. Analysis of signal transduction pathways revealed that activation of cyclin D1 expression is significantly suppressed in tumors derived from WAP-TGFα/Muc1−/− animals compared with those expressing Muc1. The loss of Muc1 expression also results in a significant inhibition in the formation of hyperplastic lesions during tumor progression. On the C57Bl/6 inbred background, pulmonary lesions were observed in 28 of 29 WAP-TGFα/Muc1+/+ animals (including one metastatic pulmonary adenocarcinoma and multiple perivascular lymphomas), although none were detected in the WAP-TGFα/Muc1−/− animals. Together, these data indicate that Muc1 is an important modulator of TGFα-dependent tumor progression. [Cancer Res 2007;67(14):6591–8]
The RET Kinase Inhibitor NVP-AST487 Blocks Growth and Calcitonin Gene Expression through Distinct Mechanisms in Medullary Thyroid Cancer Cells
Authors: Akeno-Stuart Nagako , Croyle Michelle , Knauf Jeffrey A. , Malaguarnera Roberta , Vitagliano Donata , Santoro Massimo , Stephan Christine , Grosios Konstantina , Wartmann Markus , Cozens Robert , Caravatti Giorgio , Fabbro Doriano , Lane Heidi A. , Fagin James A.
Published: July 2007

The RET kinase has emerged as a promising target for the therapy of medullary thyroid cancers (MTC) and of a subset of papillary thyroid cancers. NVP-AST487, a N,N′-diphenyl urea with an IC50 of 0.88 μmol/L on RET kinase, inhibited RET autophosphorylation and activation of downstream effectors, and potently inhibited the growth of human thyroid cancer cell lines with activating mutations of RET but not of lines without RET mutations. NVP-AST487 induced a dose-dependent growth inhibition of xenografts of NIH3T3 cells expressing oncogenic RET, and of the MTC cell line TT in nude mice. MTCs secrete calcitonin, a useful indicator of tumor burden. Human plasma calcitonin levels derived from the TT cell xenografts were inhibited shortly after treatment, when tumor volume was still unchanged, indicating that the effects of RET kinase inhibition on calcitonin secretion were temporally dissociated from its tumor-inhibitory properties. Accordingly, NVP-AST487 inhibited calcitonin gene expression in vitro in TT cells, in part, through decreased gene transcription. These data point to a previously unknown physiologic role of RET signaling on calcitonin gene expression. Indeed, the RET ligands persephin and GDNF robustly stimulated calcitonin mRNA, which was blocked by pretreatment with NVP-AST487. Antagonists of RET kinase activity in patients with MTC may result in effects on plasma calcitonin that are either disproportionate or dissociated from the effects on tumor burden, because RET kinase mediates a physiologic pathway controlling calcitonin secretion. The role of traditional tumor biomarkers may need to be reassessed as targeted therapies designed against oncoproteins with key roles in pathogenesis are implemented. [Cancer Res 2007;67(14):6956–64]
Substrate Overlap between Mrp4 and Abcg2/Bcrp Affects Purine Analogue Drug Cytotoxicity and Tissue Distribution
Authors: Takenaka Kazumasa , Morgan Jessica A. , Scheffer George L. , Adachi Masashi , Stewart Clinton F. , Sun Daxi , Leggas Markos , Ejendal Karin F.K. , Hrycyna Christine A. , Schuetz John D.
Published: July 2007

The use of probe substrates and combinations of ATP-binding cassette (ABC) transporter knockout (KO) animals may facilitate the identification of common substrates between apparently unrelated ABC transporters. An unexpectedly low concentration of the purine nucleotide analogue, 9-(2-(phosphonomethoxy)ethyl)-adenine (PMEA), and up-regulation of Abcg2 in some tissues of the Mrp4 KO mouse prompted us to evaluate the possibility that Abcg2 might transport purine-derived drugs. Abcg2 transported and conferred resistance to PMEA. Moreover, a specific Abcg2 inhibitor, fumitremorgin C, both increased PMEA accumulation and reversed Abcg2-mediated PMEA resistance. We developed Mrp4 and Abcg2 double KO mice and used both single KOs of Abcg2 and Mrp4 mice to assess the role of these transporters in vivo. Abcg2 contributed to PMEA accumulation in a variety of tissues, but in some tissues, this contribution was only revealed by the concurrent absence of Mrp4. Abcg2 also transported and conferred resistance to additional purine analogues, such as the antineoplastic, 2-chloro-2′-deoxyadenosine (cladribine) and puromycin, a protein synthesis inhibitor that is often used as a dominant selectable marker. Purine analogues interact with ABCG2 by a site distinct from the prazosin binding site as shown by their inability to displace the substrate analogue and photoaffinity tag [125I]iodoarylazidoprazosin. These studies show that Abcg2, like Mrp4, transports and confers resistance to purine nucleoside analogues and suggest that these two transporters work in parallel to affect drug cytotoxicity and tissue distribution. This new knowledge will facilitate an understanding of how Abcg2 and Mrp4, separately and in combination, protect against purine analogue host toxicity as well as resistance to chemotherapy. [Cancer Res 2007;67(14):6965–72]
Hemizygous Disruption of Cdc25A Inhibits Cellular Transformation and Mammary Tumorigenesis in Mice
Authors: Ray Dipankar , Terao Yasuhisa , Nimbalkar Dipali , Hirai Hiroyuki , Osmundson Evan C. , Zou Xianghong , Franks Roberta , Christov Konstantin , Kiyokawa Hiroaki
Published: July 2007

CDC25A phosphatase activates multiple cyclin-dependent kinases (CDK) during cell cycle progression. Inactivation of CDC25A by ubiquitin-mediated degradation is a major mechanism of DNA damage-induced S-G2 checkpoint. Although increased CDC25A expression has been reported in various human cancer tissues, it remains unclear whether CDC25A activation is a critical rate-limiting step of carcinogenesis. To assess the role for CDC25A in cell cycle control and carcinogenesis, we used a Cdc25A-null mouse strain we recently generated. Whereas Cdc25A−/− mice exhibit early embryonic lethality, Cdc25A+/− mice show no appreciable developmental defect. Cdc25A+/− mouse embryonic fibroblasts (MEF) exhibit normal kinetics of cell cycle progression at early passages, modestly enhanced G2 checkpoint response to DNA damage, and shortened proliferative life span, compared with wild-type MEFs. Importantly, Cdc25A+/− MEFs are significantly resistant to malignant transformation induced by coexpression of H-rasV12 and a dominant negative p53 mutant. The rate-limiting role for CDC25A in transformation is further supported by decreased transformation efficiency in MCF-10A human mammary epithelial cells stably expressing CDC25A small interfering RNA. Consistently, Cdc25A+/− mice show substantially prolonged latency in mammary tumorigenesis induced by MMTV-H-ras or MMTV-neu transgene, whereas MMTV-myc–induced tumorigenesis is not significantly affected by Cdc25A heterozygosity. Mammary tissues of Cdc25A+/−;MMTV-neu mice before tumor development display less proliferative response to the oncogene with increased tyrosine phosphorylation of CDK1/2, but show no significant change in apoptosis. These results suggest that Cdc25A plays a rate-limiting role in transformation and tumor initiation mediated by ras activation. [Cancer Res 2007;67(14):6605–11]
SIRT1 Is Significantly Elevated in Mouse and Human Prostate Cancer
Authors: Huffman Derek M. , Grizzle William E. , Bamman Marcas M. , Kim Jeong-su , Eltoum Isam A. , Elgavish Ada , Nagy Tim R.
Published: July 2007

Evidence suggests that the histone deacetylase, SIRT1, is a mediator of life span extension by calorie restriction; however, SIRT1 may paradoxically increase the risk of cancer. To better understand the relationship among SIRT1, energy balance, and cancer, two experiments were done. First, a transgenic mouse model of prostate cancer (transgenic adenocarcinoma of mouse prostate; TRAMP) was used to determine the role of energy balance on SIRT1 expression and the effect of cancer stage on SIRT1 and hypermethylated in cancer-1 (HIC-1). Second, immunohistochemistry was done on human prostate tumors to determine if SIRT1 was differentially expressed in tumor cells versus uninvolved cells. Results show that SIRT1 is not increased in the dorsolateral prostate (DLP) of calorie-restricted mice during carcinogenesis. In contrast, when examined in the DLP as a function of pathologic score, SIRT1 was significantly elevated in mice with poorly differentiated adenocarcinomas compared with those with less-advanced disease. HIC-1, which has been shown to regulate SIRT1 levels, was markedly reduced in the same tumors, suggesting that a reduction in HIC-1 may be in part responsible for the increased expression of SIRT1 in prostatic adenocarcinomas. Furthermore, immunostaining of human prostate tumors showed that cancer cells had greater SIRT1 expression than uninvolved cells. In conclusion, DLP SIRT1 expression from calorie-restricted mice was not altered during carcinogenesis. However, SIRT1 expression was increased in mice with poorly differentiated adenocarcinomas and in human prostate cancer cells. Because SIRT1 may function as a tumor promoter, these results suggest that SIRT1 should be considered as a potential therapeutic target for prostate cancer. [Cancer Res 2007;67(14):6612–8]
Genetic Ablation of CCAAT/Enhancer Binding Protein α in Epidermis Reveals Its Role in Suppression of Epithelial Tumorigenesis
Authors: Loomis Kari D. , Zhu Songyun , Yoon Kyungsil , Johnson Peter F. , Smart Robert C.
Published: July 2007

CCAAT/enhancer binding protein α (C/EBPα) is a basic leucine zipper transcription factor that inhibits cell cycle progression and regulates differentiation in various cell types. C/EBPα is inactivated by mutation in acute myeloid leukemia (AML) and is considered a human tumor suppressor in AML. Although C/EBPα mutations have not been observed in malignancies other than AML, greatly diminished expression of C/EBPα occurs in numerous human epithelial cancers including lung, liver, endometrial, skin, and breast, suggesting a possible tumor suppressor function. However, direct evidence for C/EBPα as an epithelial tumor suppressor is lacking due to the absence of C/EBPα mutations in epithelial tumors and the lethal effect of C/EBPα deletion in mouse model systems. To examine the function of C/EBPα in epithelial tumor development, an epidermal-specific C/EBPα knockout mouse was generated. The epidermal-specific C/EBPα knockout mice survived and displayed no detectable abnormalities in epidermal keratinocyte proliferation, differentiation, or apoptosis, showing that C/EBPα is dispensable for normal epidermal homeostasis. In spite of this, the epidermal-specific C/EBPα knockout mice were highly susceptible to skin tumor development involving oncogenic Ras. These mice displayed decreased tumor latency and striking increases in tumor incidence, multiplicity, growth rate, and the rate of malignant progression. Mice hemizygous for C/EBPα displayed an intermediate-enhanced tumor phenotype. Our results suggest that decreased expression of C/EBPα contributes to deregulation of tumor cell proliferation. C/EBPα had been proposed to block cell cycle progression through inhibition of E2F activity. We observed that C/EBPα blocked Ras-induced and epidermal growth factor–induced E2F activity in keratinocytes and also blocked Ras-induced cell transformation and cell cycle progression. Our study shows that C/EBPα is dispensable for epidermal homeostasis and provides genetic evidence that C/EBPα is a suppressor of epithelial tumorigenesis. [Cancer Res 2007;67(14):6768–76]
Doxorubicin Affects Testicular Lipids with Long-Chain (C18-C22) and Very Long-Chain (C24-C32) Polyunsaturated Fatty Acids
Authors: Zanetti Samanta R. , Maldonado Eduardo N. , Aveldaño Marta I.
Published: July 2007

Doxorubicin disrupts spermatogenesis by causing apoptosis of spermatogonia and primary spermatocytes. The aim of this study was to examine the effect of this agent on adult rat testicular lipids and their fatty acids. A single dose (7.5 mg/kg) and a multidose regime (3 mg/kg once a week for 4 weeks) were evaluated. Both treatments resulted in the gradual loss of spermatogenic cells and determined a marked reduction in testicular size and weight 9 weeks after their start. Germ cell loss was accompanied by a decrease in phospholipids, including glycerophospholipids and sphingomyelin. Concomitantly, glycerophospholipids lost selectively their major polyunsaturated fatty acid (PUFA), 22:5n-6, and sphingomyelin lost its major very long-chain PUFA (VLCPUFA), 28:4n-6 and 30:5n-6. The molecular species from which the lost polyenes originated were thus a trait of germ cells. A transient peak of 16:0-ceramide was observed 48 h after the single dose. In both doxorubicin regimes, sphingomyelin and ceramide with reduced amounts of VLCPUFA after about 4 weeks and with no VLCPUFA after 9 weeks resulted. By contrast, triglycerides and especially cholesterol esters (CE) tended to accumulate in the testes undergoing germ cell death, probably in the surviving Sertoli cells, their fatty acid patterns suggesting that initially, these lipids retained part of the PUFA coming from, or no longer used for, the synthesis of germ cell glycerophospholipids. As the latter decreased, CE accumulated massively 9 weeks after starting doxorubicin treatment, 20:4n-6 becoming their major PUFA. Part of these CEs may derive from surviving steroidogenic cells. [Cancer Res 2007;67(14):6973–80]
Human Polynucleotide Kinase Participates in Repair of DNA Double-Strand Breaks by Nonhomologous End Joining but not Homologous Recombination
Authors: Karimi-Busheri Feridoun , Rasouli-Nia Aghdass , Allalunis-Turner Joan , Weinfeld Michael
Published: July 2007

Human polynucleotide kinase (hPNK) is a bifunctional enzyme possessing a 5′-DNA kinase activity and a 3′-phosphatase activity. Studies based on cell extracts and purified proteins have indicated that hPNK can act on single-strand breaks and double-strand breaks (DSB) to restore the termini to the chemical form required for further action by DNA repair polymerases and ligases (i.e., 5′-phosphate and 3′-hydroxyl termini). These studies have revealed that hPNK can bind to XRCC4, and as a result, hPNK has been implicated as a participant in the nonhomologous end joining (NHEJ) pathway for DSB repair. We sought to confirm the role of hPNK in NHEJ in the cellular setting using a genetic approach. hPNK was stably down-regulated by RNA interference expression in M059K glioblastoma cells, which are NHEJ positive, and M059J cells, which are NHEJ deficient due to a lack of DNA-PK catalytic subunit (DNA-PKcs). Whereas depletion of hPNK significantly sensitized M059K cells to ionizing radiation, no additional sensitization was conferred to M059J cells, clearly implying that hPNK operates in the same DNA repair pathway as DNA-PKcs. On the other hand, depletion of hPNK did not increase the level of sister chromatid exchanges, indicating that hPNK is not involved in the homologous recombination DSB repair pathway. We also provide evidence that the action of hPNK in the repair of camptothecin-induced topoisomerase 1 “dead-end” complexes is independent of DNA-PKcs and that hPNK is not involved in the nucleotide excision repair pathway. [Cancer Res 2007;67(14):6619–25]
Hierarchical Organization of Prostate Cancer Cells in Xenograft Tumors: The CD44+α2β1+ Cell Population Is Enriched in Tumor-Initiating Cells
Authors: Patrawala Lubna , Calhoun-Davis Tammy , Schneider-Broussard Robin , Tang Dean G.
Published: July 2007

Prostate cancer cells are heterogeneous in their tumorigenicity. For example, the side population cells isolated from LAPC9 xenografts are 100 to 1,000 times more tumorigenic than the corresponding non–side population cells. Highly purified CD44+ prostate cancer cells from several xenografts are also enriched in prostate cancer stem/progenitor cells. Because the CD44+ prostate cancer cell population is still heterogeneous, we wonder whether we could further enrich for tumorigenic prostate cancer cells in this population using other markers. Integrin α2β1 has been proposed to mark a population of normal human prostate stem cells. Therefore, we first asked whether the α2β1+/hi cells in prostate tumors might also represent prostate cancer stem cells. Highly purified (≥98%) α2β1+/hi cells from three human xenograft tumors, Du145, LAPC4, and LAPC9, show higher clonal and clonogenic potential than the α2β1−/lo cells in vitro. However, when injected into the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse prostate or s.c., the α2β1+/hi prostate cancer cells are no more tumorigenic than the α2β1−/lo cells. Immunofluorescence studies reveal that CD44 and α2β1 identify an overlapping and inclusive population of prostate cancer cells in that ∼70% of α2β1+/hi cells are CD44+ and 20% to 30% of CD44+ cells are distributed in the α2β1−/lo cell population. Subsequently, we sorted out CD44+α2β1+/hi, CD44+α2β1−/lo, CD44−α2β1+/hi, and CD44−α2β1−/lo cells from LAPC9 tumors and carried out tumorigenicity experiments. The results revealed a hierarchy in tumorigenic potential in the order of CD44+α2β1+/hi ≈ CD44+α2β1−/lo > CD44−α2β1+/hi ≫ CD44−α2β1−/lo. These observations together suggest that prostate cancer cells are organized as a hierarchy. [Cancer Res 2007;67(14):6796–805]
The Putative Tumor Suppressor Gene PTPN13/PTPL1 Induces Apoptosis through Insulin Receptor Substrate-1 Dephosphorylation
Authors: Dromard Mathilde , Bompard Guillaume , Glondu-Lassis Murielle , Puech Carole , Chalbos Dany , Freiss Gilles
Published: July 2007

The protein tyrosine phosphatase (PTP) PTPL1/PTPN13 is a candidate tumor suppressor gene. Indeed, PTPL1 activity has been reported recently to be decreased through somatic mutations, allelic loss, or promoter methylation in some tumors. We showed previously that its expression was necessary for inhibition of Akt activation and induction of apoptosis by antiestrogens in breast cancer cells. Implications of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in cancer progression are now well established, and our study was therefore designed to define whether PTPL1 is sufficient to inhibit this pathway and, if so, to identify a direct substrate of this PTP, which may trigger a proapoptotic effect. We first show by complementary approaches that PTPL1 specifically dephosphorylates insulin receptor substrate-1 (IRS-1) in vitro and in cellulo. Next, our experiments using a dominant-negative mutant and RNA interference confirm the crucial role of PTPL1 in IRS-1 dephosphorylation. Finally, we report that PTPL1 expression is sufficient to block the IRS-1/PI3K/Akt signaling pathway, to inhibit the insulin-like growth factor-I effect on cell survival, and to induce apoptosis. Altogether, these data provide the first evidence for a direct positive role of the putative tumor suppressor gene PTPL1/PTPN13 on apoptosis and identify its target in the IRS-1/PI3K/Akt signaling pathway. [Cancer Res 2007;67(14):6806–13]
Eukaryotic Translation Initiation Factor 4E–Induced Progression of Primary Human Mammary Epithelial Cells along the Cancer Pathway Is Associated with Targeted Translational Deregulation of Oncogenic Drivers and Inhibitors
Authors: Larsson Ola , Li Shunan , Issaenko Olga A. , Avdulov Svetlana , Peterson Mark , Smith Karen , Bitterman Peter B. , Polunovsky Vitaly A.
Published: July 2007

Pathologic redirection of translational control by constitutive activation of eukaryotic translation initiation factor 4F (eIF4F), the cap-dependent translation initiation apparatus, is an obligatory step in oncogenesis; however, its mechanism remains undefined. Here, we simulate this pro-oncogenic state by overexpressing eIF4E, the rate-limiting component of eIF4F, in primary human mammary epithelial cells (HMECs) and examine the resultant changes in cell biology and gene expression profiles of total and polyribosome-bound mRNA genome wide. Overexpressed eIF4E rescues primary HMECs from telomere-independent growth arrest and disables checkpoints governing S-phase entry as well as apoptosis in HMECs immortalized by telomerase, imparting cells with proliferative and survival autonomy. Although the transcriptional response to increased eIF4E was modest, the translational response was large, selective, and bidirectional. In addition to translational activation of known and novel eIF4E-responsive oncogenic drivers regulating cell growth and survival, our data unveil previously unrecognized cellular defenses including translational activation of tumor suppressors, translational repression of transcripts enriched with miRNA target sites, and translational modulation of genes governing translation itself. These findings provide insight into the proneoplastic and compensatory mechanisms embedded in the oncogenic translational program. They support a model whereby deregulated eIF4E moves human epithelial cells along the cancer pathway by profoundly altering ribosomal recruitment to cancer-related transcripts, and eIF4E-modified cells counter these potentially oncogenic alterations with a compensatory translational mechanism that mitigates acquisition of malignancy. [Cancer Res 2007;67(14):6814–24]