bluuebunny/crossref_metadata_2025 huggingface dataset:
Simian Virus 40 Large T Antigen Can Specifically Unwind the Central Palindrome at the Origin of DNA Replication
Authors: Wang Weiping , Simmons Daniel T.
Published: January 2009

The hydrophilic channels between helicase domains of simian virus 40 (SV40) large T antigen play a critical role in DNA replication. Previous mutagenesis of residues in the channels identified one class of mutants (class A: D429A, N449S, and N515S) with normal DNA binding and ATPase and helicase activities but with a severely reduced ability to unwind origin DNA and to support SV40 DNA replication in vitro. Here, we further studied these mutants to gain insights into how T antigen unwinds the origin. We found that the mutants were compromised in melting the imperfect palindrome (EP) but normal in untwisting the AT-rich track. However, the mutants' defect in EP melting was not the major reason they failed to unwind the origin because supplying an EP region as a mismatched bubble, or deleting the EP region altogether, did not rescue their unwinding deficiency. These results suggested that specific separation of the central palindrome of the origin (site II) is an essential step in unwinding origin DNA by T antigen. In support of this, wild-type T antigen was able to specifically unwind a 31-bp DNA containing only site II in an ATPase-dependent reaction, whereas D429A and N515S failed to do so. By performing a systematic mutagenesis of 31-bp site II DNA, we identified discrete regions in each pentanucleotide necessary for normal origin unwinding. These data indicate that T antigen has a mechanism to specifically unwind the central palindrome. Various models are proposed to illustrate how T antigen could separate the central origin.
5′
cis
Elements Direct Nodavirus RNA1 Recruitment to Mitochondrial Sites of Replication Complex Formation
Authors: Van Wynsberghe Priscilla M. , Ahlquist Paul
Published: January 2009

Positive-strand RNA viruses replicate their genomes on intracellular membranes, usually in conjunction with virus-induced membrane rearrangements. For the nodavirus flock house virus (FHV), we recently showed that multifunctional FHV replicase protein A induces viral RNA template recruitment to a membrane-associated state, but the site(s) and function of this recruitment were not determined. By tagging viral RNA with green fluorescent protein, we show here in
Drosophila
cells that protein A recruits FHV RNA specifically to the outer mitochondrial membrane sites of RNA replication complex formation. Using
Drosophila
cells and yeast cells, which also support FHV replication, we also defined the
cis
-acting regions that direct replication and template recruitment for FHV genomic RNA1. RNA1 nucleotides 68 to 205 were required for RNA replication and directed efficient protein A-mediated RNA recruitment in both cell types. RNA secondary structure prediction, structure probing, and phylogenetic comparisons in this region identified two stable, conserved stem-loops with nearly identical loop sequences. Further mutational analysis showed that both stem-loops and certain flanking sequences were required for RNA1 recruitment, negative-strand synthesis, and subsequent positive-strand amplification in yeast and
Drosophila
cells. Thus, we have shown that protein A recruits RNA1 templates to mitochondria, as expected for RNA replication, and identified a new RNA1
cis
element that is necessary and sufficient for RNA1 template recognition and recruitment to these mitochondrial membranes for negative-strand RNA1 synthesis. These results establish RNA recruitment to the sites of replication complex formation as an essential, distinct, and selective early step in nodavirus replication.
Discovery of Herpes B Virus-Encoded MicroRNAs
Authors: Besecker Michael I. , Harden Mallory E. , Li Guanglin , Wang Xiu-Jie , Griffiths Anthony
Published: January 2009

Herpes B virus (BV) naturally infects macaque monkeys and is a close relative of herpes simplex virus. BV can zoonotically infect humans to cause a rapidly ascending encephalitis with ∼80% mortality. Therefore, BV is a serious danger to those who come into contact with these monkeys or their tissues and cells. MicroRNAs are regulators of gene expression, and there have been reports of virus-encoded microRNAs. We hypothesize that BV-encoded microRNAs are important for the regulation of viral and cellular genes. Herein, we report the discovery of three herpes B virus-encoded microRNAs.
INFINITE ORDER PARAMETRIC NORMAL FORM OF HOPF SINGULARITY
Authors: GAZOR MAJID , YU PEI
Published: January 2009

In this paper, we introduce a suitable algebraic structure for efficient computation of the parametric normal form of Hopf singularity based on a notion of formal decompositions. Our parametric state and time spaces are respectively graded parametric Lie algebra and graded ring. As a consequence, the parametric state space is also a graded module. Parameter space is observed as an integral domain as well as a vector space, while the near-identity parameter map acts on the parametric state space. The method of multiple Lie bracket is used to obtain an infinite order parametric normal form of codimension-one Hopf singularity. Filtration topology is revisited and proved that state, parameter and time (near-identity) maps are continuous. Furthermore, parametric normal form is a convergent process with respect to filtration topology. All the results presented in this paper are verified by using Maple.
PERIOD-DOUBLING AND SCROLL-DOUBLING BIFURCATIONS IN A CLASS OF NONSYMMETRIC CELLULAR NEURAL NETWORKS
Authors: TANG CHANGBING , ZENG KAIJUN , CHEN FANGYUE
Published: January 2009

Two kinds of bifurcation behaviors in a simple nonsymmetric CNN system are discovered with the help of computer simulation. The first kind of bifurcation exhibits a cascade of period doubling bifurcations which lead to the birth of two coexisting Chua's single-scroll attractors. The second one presents a scroll doubling bifurcation which gives rise from Chua's single-scroll attractor to a Chua's double-scroll attractor.
GLOBAL SYNCHRONIZATION IN TRANSLATION INVARIANT COUPLED MAP LATTICES
Authors: FERNANDEZ BASTIEN
Published: January 2009

A sufficient condition for global synchronization in coupled map lattices (CML) with translation invariant coupling and arbitrary individual map is proved. As in [Jost & Joy, 2001] where CML with reflection invariant couplings are considered, the condition only involves the linearized dynamics in the diagonal, namely for all points in the diagonal, the derivative must be contractive in all transverse directions. In addition to this result, a (weaker) condition that ensures the CML attractor to be composed of either 2-periodic or constant configurations, is also obtained.
SYNCHRONIZATION OF A CLASS OF SECOND-ORDER NONLINEAR SYSTEMS
Authors: MIJOLARO A. P. , ABERTO L. F. C. , BRETAS N. G.
Published: January 2009

The asymptotic behavior of a class of coupled second-order nonlinear dynamical systems is studied in this paper. Using very mild assumptions on the vector-field, conditions on the coupling parameters that guarantee synchronization are provided. The proposed result does not require solutions to be ultimately bounded in order to prove synchronization, therefore it can be used to study coupled systems that do not globally synchronize, including synchronization of unbounded solutions. In this case, estimates of the synchronization region are obtained. Synchronization of two-coupled nonlinear pendulums and two-coupled Duffing systems are studied to illustrate the application of the proposed theory.
FLIPPING COINS, FOLDING PAPER, AND FINDING FAMILIAR FRACTALS IN THE TOWER OF HANOI
Authors: CAMP DANE R.
Published: January 2009

This manuscript describes three activities connecting the Tower of Hanoi puzzle to three familiar fractal forms. The first connects coin flipping, paper folding, and the Tower of Hanoi to the Dragon Curve. The second illustrates mathematician Ian Stewart's method for showing how the relationships between possible states of the Tower of Hanoi are related to stages in Sierpinski's Gasket. The final activity compares right-left moves of Tower of Hanoi disks to iterations of the Von Koch Curve.
CONTROLLING WAVE PACKET INTERFERENCE OF DISSOCIATING MOLECULES BY SHAPING LASER PULSES IN FREQUENCY DOMAIN
Authors: HAN YONG-CHANG , YUAN KAI-JUN , CONG SHU-LIN
Published: January 2009

The interference of dissociating wave packets for the Br 2 molecule in femtosecond laser field is studied theoretically using time-dependent quantum wave packet method. The interference of dissociating wave packets can be determined by the spectrum of laser field. By shaping laser pulses in frequency domain, the corresponding R- and v-dependent density functions can be effectively controlled. Compared with the 2-pulse excitation scheme, the resolution of the interference patterns can be improved by using 3- and 4-pulse excitation schemes. The dissociating velocity can be steered by varying laser parameters.
STUDY OF THEORETICAL VIBRATIONAL SPECTRA AND THEIR ASSIGNMENTS FOR VINYLPHOSPHONIC AND VINYLTHIOPHOSPHONIC ACIDS
Authors: FÖRNER WOLFGANG , BADAWI HASSAN M.
Published: January 2009

The structures and conformational stability of vinylphosphonic and vinylthiophosphonic acids were investigated using calculations mostly at DFT/6-311G** level and ab initio ones at MP2/6-311G** level. From the calculations the molecules were predicted to exist in a nonplanar near-cis (nc) to trans -gauche (tg) conformational equilibrium with nc (phosphonic oxygen or sulfur nearly eclipses the vinyl group) being the predominant conformer at ambient temperature, at least on the basis of Gibbs free energy (in the case of the thio compound the potential energy of tg is slightly lower than that of nc). The antisymmetric potential function for the internal rotation was determined for each one of the molecules. However, when starting optimizations from a structure too close to the full cis symmetry, the cis form results as an extremum on the potential hyper-surface. Consequently cis was reported in the literature several times as minimum. However, when we calculated the vibrational frequencies the cis form turned out to have an imaginary frequency, and thus cis is a local maximum along one of the normal coordinates. The true minimum, with only real frequencies, actually is an nc one with a very low barrier to rotation. The rotational angle out of cis is rather small; however, HOPX dihedral angles are quite away from real cis. This holds true not only with DFT but also with MP2. The vibrational frequencies were computed and the spectra were plotted as a prediction. Normal coordinate calculations were carried out and potential energy distributions were calculated for the molecules in the nc and the tg conformations. From our results and their analysis, we conclude, in agreement with results from the literature based on localized orbitals, that conjugation effects are absent – or at least negligible – as compared with electrostatic or steric ones in determining the structures of the stable conformers in the vinyl derivatives.
MOLECULAR DYNAMICS STUDIES ON Fe2+, Co2+, AND Ni2+ AQUEOUS SOLUTIONS BASED ON ABEEM/MM FLUCTUATING CHARGE MODEL
Authors: GUAN QING-MEI , HAN YAN , YANG ZHONG-ZHI
Published: January 2009

Based on atom-bond electronegativity equalization method fused into molecular mechanics (ABEEM/MM), the intermolecular potential for transition metal ion Fe 2+, Co 2+, and Ni 2+ cations in water has been derived. Parameters for the effective interaction between a cation and a water molecule were determined by reproducing the ab initio results. Many structural and dynamic properties of Fe 2+(aq.), Co 2+(aq.), and Ni 2+(aq.) were studied using these potential parameters. Strong influences of the twofold charged cations on the structures of the hydration shells and some other properties of aqueous ionic solutions are discussed and compared with the results of a previous study of alkali metal and alkaline-earth metal cations in water. At the same time, comparative study of the hydration properties of each cation is also discussed. This work demonstrates that ABEEM/MM provides a useful tool in the exploration of the hydration of transition metal cations in water.
Statistical Methods for Risk-Outcome Research: Being Sensitive to Longitudinal Structure
Authors: Cole David A. , Maxwell Scott E.
Published: January 2009

The relation between risk and outcome consists of myriad, complex, longitudinal processes. To study these relations requires research designs and statistical methods that are sensitive to the longitudinal structure of the risk, the outcome, and the risk-outcome relation. This review presents four longitudinal characteristics that can complicate psychopathology risk-outcome research. We represent each complication with an example data set. We demonstrate how conventional statistical approaches can yield highly misleading results. Finally, we review alternative statistical approaches that can handle these complications quite well.
Diffusion Theory and Knowledge Dissemination, Utilization, and Integration in Public Health
Authors: Green Lawrence W. , Ottoson Judith M. , García César , Hiatt Robert A.
Published: January 2009

Legislators and their scientific beneficiaries express growing concerns that the fruits of their investment in health research are not reaching the public, policy makers, and practitioners with evidence-based practices. Practitioners and the public lament the lack of relevance and fit of evidence that reaches them and barriers to their implementation of it. Much has been written about this gap in medicine, much less in public health. We review the concepts that have guided or misguided public health in their attempts to bridge science and practice through dissemination and implementation. Beginning with diffusion theory, which inspired much of public health's work on dissemination, we compare diffusion, dissemination, and implementation with related notions that have served other fields in bridging science and practice. Finally, we suggest ways to blend diffusion with other theory and evidence in guiding a more decentralized approach to dissemination and implementation in public health, including changes in the ways we produce the science itself.
Effect of pH on l- and d-methionine uptake across the apical membrane of Caco-2 cells
Authors: Martín-Venegas Raquel , Rodríguez-Lagunas M. José , Mercier Yves , Geraert Pierre-André , Ferrer Ruth
Published: January 2009

The transport systems involved in intestinal methionine (Met) absorption are described as Na+-dependent and Na+-independent mechanisms. However, since recent studies have suggested the importance of the H+ gradient as a driving force for intestinal nutrient absorption, the aim of the present work was to test whether Met transport across the apical membrane of Caco-2 cells is affected by extracellular pH. The results show that l- and d-Met uptake was increased by lowering extracellular pH from 7.4 to 5.5, in both the presence and absence of Na+. Cis-inhibition experiments revealed that inhibition of l-Met transport by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) or l-lysine (l-Lys) was higher at a pH of 5.5. Moreover, the BCH-insensitive component was not affected by pH, whereas the l-Lys-insensitive component was increased by lowering extracellular pH, thus suggesting the participation of system L. The contribution of another mechanism, sensitive to both BCH and l-Lys, was also considered. The inhibition obtained with taurine (Tau) was also higher at a pH of 5.5, thus suggesting the involvement of system B0,+ on pH-stimulated component. As for d-Met uptake, the results showed higher inhibition with l-Lys and Tau at a pH of 5.5 and no effect on the l-Lys- or Tau-insensitive component. In conclusion, Met transport across the apical membrane of Caco-2 cells is increased by low extracellular pH as the result of the stimulation of two transport systems functionally identified with systems L and B0,+ for l-Met and with system B0,+ for d-Met.
Vascular nitric oxide and superoxide anion contribute to sex-specific programmed cardiovascular physiology in mice
Authors: Roghair Robert D. , Segar Jeffrey L. , Volk Kenneth A. , Chapleau Mark W. , Dallas Lindsay M. , Sorenson Anna R. , Scholz Thomas D. , Lamb Fred S.
Published: January 2009

Intrauterine environmental pertubations have been linked to the development of adult hypertension. We sought to evaluate the interrelated roles of sex, nitric oxide, and reactive oxygen species (ROS) in programmed cardiovascular disease. Programming was induced in mice by maternal dietary intervention (DI; partial substitution of protein with carbohydrates and fat) or carbenoxolone administration (CX, to increase fetal glucocorticoid exposure). Adult blood pressure and locomotor activity were recorded by radiotelemetry at baseline, after a week of high salt, and after a week of high salt plus nitric oxide synthase inhibition (by l-NAME). In male offspring, DI or CX programmed an elevation in blood pressure that was exacerbated by Nω-nitro-l-arginine methyl ester administration, but not high salt alone. Mesenteric resistance vessels from DI male offspring displayed impaired vasorelaxation to ACh and nitroprusside, which was blocked by catalase and superoxide dismutase. CX-exposed females were normotensive, while DI females had nitric oxide synthase-dependent hypotension and enhanced mesenteric dilation. Despite the disparate cardiovascular phenotypes, both male and female DI offspring displayed increases in locomotor activity and aortic superoxide production. Despite dissimilar blood pressures, DI and CX-exposed females had reductions in cardiac baroreflex sensitivity. In conclusion, both maternal malnutrition and fetal glucocorticoid exposure program increases in arterial pressure in male but not female offspring. While maternal DI increased both superoxide-mediated vasoconstriction and nitric oxide mediated vasodilation, the balance of these factors favored the development of hypertension in males and hypotension in females.
Calpain 10 is required for cell viability and is decreased in the aging kidney
Authors: Covington Marisa D. , Arrington David D. , Schnellmann Rick G.
Published: January 2009

Aging is associated with abnormalities in kidney function, but the exact mechanisms are unknown. We examined calpains 1, 2, and 10 protein levels in kidneys from rats, mice, and humans of various ages and determined whether calpain 10 is required for cell viability. Calpain 10 protein expression decreased in the kidney, but not in the liver, of aging Fischer 344 rats, and this decrease was attenuated with caloric restriction. There was no change in calpains 1 or 2 levels in the kidney or liver in control and caloric-restricted aging rats. Aging mice also exhibited decreased calpain 10 protein levels. Calpain 10 protein and mRNA levels decreased linearly in human kidney samples with age in the absence of changes in calpains 1 or 2. Our laboratory previously found calpain 10 to be expressed in both the cytosol and mitochondria of rabbit renal proximal tubular cells (RPTC). Adenoviral-delivered shRNA to rabbit RPTC decreased mitochondrial calpain 10 expression below detectable levels by 3 days while cytosolic calpain 10 levels remained unchanged at 3 days and decreased to ∼20% of control by 5 days. Knockdown of mitochondrial calpain 10 resulted in nuclear condensation and cleaved procaspase 3, markers of apoptosis. In summary, mitochondrial calpain 10 is required for cell viability and calpain 10 levels specifically decrease in aging rat, mice, and human kidney tissues when renal function decreases, suggesting that calpain 10 is required for renal function and is a biomarker of the aging kidney.
The copper transporter Ctr1 contributes to cisplatin uptake by renal tubular cells during cisplatin nephrotoxicity
Authors: Pabla Navjotsingh , Murphy Robert F. , Liu Kebin , Dong Zheng
Published: January 2009

The usefulness and efficacy of cisplatin, a chemotherapeutic drug, are limited by its toxicity to normal tissues and organs, including the kidneys. The uptake of cisplatin in renal tubular cells is high, leading to cisplatin accumulation and tubular cell injury and death, culminating in acute renal failure. While extensive investigations have been focused on the signaling pathways of cisplatin nephrotoxicity, much less is known about the mechanism of cisplatin uptake by renal cells and tissues. In this regard, evidence has been shown for the involvement of organic cation transporters (OCT), specifically OCT2. The copper transporter Ctr1 is highly expressed in the renal tubular cells; however, its role in cisplatin nephrotoxicity is not known. In this study, we demonstrate that Ctr1 is mainly expressed in both proximal and distal tubular cells in mouse kidneys. We further show that Ctr1 is mainly localized on the basolateral side of these cells, a proposed site for cisplatin uptake. Importantly, downregulation of Ctr1 by small interfering RNA or copper pretreatment results in decreased cisplatin uptake. Consistently, downregulation of Ctr1 suppresses cisplatin toxicity, including cell death by both apoptosis and necrosis. Cimetidine, a pharmacological inhibitor of OCT2, can also partially attenuate cisplatin uptake. Notably, cimetidine can further reduce cisplatin uptake and cisplatin toxicity in Ctr1-downregulated cells. The results have demonstrated the first evidence for a role of Ctr1 in cisplatin uptake and nephrotoxicity.
Strength training the free limb attenuates strength loss during unilateral immobilization
Authors: Farthing Jonathan P. , Krentz Joel R. , Magnus Charlene R. A.
Published: January 2009

The objective was to determine if strength training the free limb during a 3-wk period of unilateral immobilization attenuates strength loss in the immobilized limb through cross-education. Thirty right-handed participants were assigned to three groups. One group ( n = 10) wore a cast and trained the free arm (Cast-Train). A second group ( n = 10) wore a cast and did not train (Cast). A third group ( n = 10) received no treatment (control). Casts were applied to the nondominant (left) wrist and hand by a physician. Strength training was maximal isometric ulnar deviation (right hand) 5 days/wk. Peak torque (dynamometer), electromyography (EMG), and muscle thickness (ultrasound) were assessed in both arms before and after the intervention. Cast-Train improved right arm strength [14.3 (SD 5.0) to 17.7 (SD 4.8) N·m; P < 0.05] with no significant muscle hypertrophy [3.73 (SD 0.43) to 3.84 (SD 0.52) cm; P = 0.09]. The immobilized arm of Cast-Train did not change in strength [13.9 (SD 4.3) to 14.2 (SD 4.6) N·m] or muscle thickness [3.61 (SD 0.51) to 3.57 (SD 0.43) cm]. The immobilized arm of Cast decreased in strength [12.2 (SD 3.8) to 10.4 (SD 2.5) N·m; P < 0.05] and muscle thickness [3.47 (SD 0.59) to 3.32 (SD 0.55) cm; P < 0.05]. Control showed no changes in the right arm [strength: 15.3 (SD 6.1) to 14.3 (SD 5.8) N·m; muscle thickness: 3.57 (SD 0.68) to 3.52 (SD 0.75) cm] or left arm [strength: 14.5 (SD 5.3) to 13.7 (SD 6.1) N·m; muscle thickness: 3.55 (SD 0.77) to 3.51 (SD 0.70) cm]. Agonist muscle activation remained unchanged after the intervention for both arms [right: 302 (SD 188) to 314 (SD 176) μV; left: 261 (SD 139) to 288 (SD 151) μV] with no group differences. Strength training of the free limb attenuated strength loss in the immobilized limb during unilateral immobilization. Strength training may have prevented muscle atrophy in the immobilized limb.
Estimation of critical torque using intermittent isometric maximal voluntary contractions of the quadriceps in humans
Authors: Burnley Mark
Published: January 2009

To determine whether the asymptote of the torque-duration relationship (critical torque) could be estimated from the torque measured at the end of a series of maximal voluntary contractions (MVCs) of the quadriceps, eight healthy men performed eight laboratory tests. Following familiarization, subjects performed two tests in which they were required to perform 60 isometric MVCs over a period of 5 min (3 s contraction, 2 s rest), and five tests involving intermittent isometric contractions at ∼35–60% MVC, each performed to task failure. Critical torque was determined using linear regression of the torque impulse and contraction time during the submaximal tests, and the end-test torque during the MVCs was calculated from the mean of the last six contractions of the test. During the MVCs voluntary torque declined from 263.9 ± 44.6 to 77.8 ± 17.8 N·m. The end-test torque was not different from the critical torque (77.9 ± 15.9 N·m; 95% paired-sample confidence interval, −6.5 to 6.2 N·m). The root mean squared error of the estimation of critical torque from the end-test torque was 7.1 N·m. Twitch interpolation showed that voluntary activation declined from 90.9 ± 6.5% to 66.9 ± 13.1% ( P < 0.001), and the potentiated doublet response declined from 97.7 ± 23.0 to 46.9 ± 6.7 N·m ( P < 0.001) during the MVCs, indicating the development of both central and peripheral fatigue. These data indicate that fatigue during 5 min of intermittent isometric MVCs of the quadriceps leads to an end-test torque that closely approximates the critical torque.
Helicobacter pyloriCytotoxin-Associated Gene A Activates the Signal Transducer and Activator of Transcription 3 PathwayIn vitroandIn vivo
Authors: Bronte-Tinkew Dana M. , Terebiznik Mauricio , Franco Aime , Ang Michelle , Ahn Diane , Mimuro Hitomi , Sasakawa Chihiro , Ropeleski Mark J. , Peek Richard M. , Jones Nicola L.
Published: January 2009

Persistent infection with Helicobacter pylori confers an increased risk for the development of gastric cancer. However, the exact mechanisms whereby this bacterium causes carcinogenesis have not been completely elucidated. Recent evidence indicates that aberrant activation of the signal transducers and activators of transcription 3 (STAT3) signaling pathway may play a role in gastric carcinogenesis. Therefore, we hypothesized that H. pylori infection modulates STAT3 signaling, favoring gastric cancer development. In epithelial cells infected with H. pylori, STAT3 was activated, as assessed by immunoblotting for phosphorylated STAT3, immunofluorescence of translocated STAT3, fluorescence recovery after photobleaching, and luciferase activation in transfected cells. Activation was dependent on translocation but not phosphorylation of cytotoxin-associated gene A (CagA) in host cells. Activation seemed to be receptor-mediated because preincubation of cells with the interleukin-6 (IL-6) receptor superantagonist sant7 or inhibition of gp130 by a monoclonal antibody prevented H. pylori–mediated STAT3 activation. However, activation was not related to autocrine activation by IL-6 or IL-11. CagA+ wild-type H. pylori, but not the noncarcinogenic cagA− mutant, activated STAT3 in gastric epithelial cells in vivo in the gerbil model of H. pylori–mediated gastric carcinogenesis. Collectively, these results indicate that H. pylori CagA activates the STAT3 signaling pathway in vitro and in vivo, providing a potential mechanism by which chronic H. pylori infection promotes the development of gastric cancer. [Cancer Res 2009;69(2):632–9]
High Frequency of TP53 Mutation in BRCA1 and Sporadic Basal-like Carcinomas but not in BRCA1 Luminal Breast Tumors
Authors: Manié Elodie , Vincent-Salomon Anne , Lehmann-Che Jacqueline , Pierron Gaelle , Turpin Elisabeth , Warcoin Mathilde , Gruel Nadège , Lebigot Ingrid , Sastre-Garau Xavier , Lidereau Rosette , Remenieras Audrey , Feunteun Jean , Delattre Olivier , de Thé Hugues , Stoppa-Lyonnet Dominique , Stern Marc-Henri
Published: January 2009

Breast tumors with a germ-line mutation of BRCA1 (BRCA1 tumors) and basal-like carcinoma (BLC) are associated with a high rate of TP53 mutation. Because BRCA1 tumors frequently display a basal-like phenotype, this study was designed to determine whether TP53 mutations are correlated with the hereditary BRCA1 mutated status or the particular phenotype of these tumors. The TP53 gene status was first investigated in a series of 35 BRCA1 BLCs using immunohistochemistry, direct sequencing of the coding sequence, and functional analysis of separated alleles in yeast, and compared with the TP53 status in a series of 38 sporadic (nonhereditary) BLCs. Using this sensitive approach, TP53 was found to be frequently mutated in both BRCA1 (34 of 35, 97%) and sporadic (35 of 38, 92%) BLCs. However, the spectrum of mutation was different, particularly with a higher rate of complex mutations, such as insertion/deletion, in BRCA1 BLCs than in the sporadic group [14 of 33 (42%) and 13 of 34 (9%), respectively; P = 0.002]. Secondly, the incidence of TP53 mutations was analyzed in 19 BRCA1 luminal tumors using the same strategy. Interestingly, only 10 of these 19 tumors were mutated (53%), a frequency similar to that found in grade-matched sporadic luminal tumors. In conclusion, TP53 mutation is highly recurrent in BLCs independently of BRCA1 status, but not a common feature of BRCA1 luminal tumors. [Cancer Res 2009;69(2):663–71]
Down-regulation of Rap1GAP via Promoter Hypermethylation Promotes Melanoma Cell Proliferation, Survival, and Migration
Authors: Zheng Hong , Gao Ling , Feng Yunfeng , Yuan Liya , Zhao Haibo , Cornelius Lynn A.
Published: January 2009

Melanoma is the most serious, highly aggressive form of skin cancer with recent dramatic increases in incidence. Current therapies are relatively ineffective, highlighting the need for a better understanding of the molecular mechanisms contributing to the disease. We have previously shown that activation of Rap1 promotes melanoma cell proliferation and migration through the mitogen-activated protein kinase pathway and integrin activation. In the present study, we show that expression of Rap1GAP, a specific negative regulator of Rap1, is decreased in human melanoma tumors and cell lines. Overexpression of Rap1GAP in melanoma cells blocks Rap1 activation and extracellular signal-regulated kinase (ERK) phosphorylation and inhibits melanoma cell proliferation and survival. In addition, overexpression of Rap1GAP also inhibits focal adhesion formation and decreases melanoma cell migration. Rap1GAP down-regulation is due to its promoter methylation, a mechanism of gene silencing in tumors. Furthermore, treatment of melanoma cells with the demethylating agent 5-aza-2'-deoxycytidine reinduces Rap1GAP expression, followed by decreased Rap1 activity, ERK phosphorylation, and cell proliferation and survival—changes that are significantly blunted in cells transfected by small interfering RNA–mediated Rap1GAP knockdown. Taken together, our findings indicate that down-regulation of Rap1GAP via promoter hypermethylation promotes melanoma cell proliferation, survival, and migration. [Cancer Res 2009;69(2):449–57]
Epigallocatechin-3-Gallate, a Histone Acetyltransferase Inhibitor, Inhibits EBV-Induced B Lymphocyte Transformation via Suppression of RelA Acetylation
Authors: Choi Kyung-Chul , Jung Myung Gu , Lee Yoo-Hyun , Yoon Joo Chun , Kwon Seung Hyun , Kang Hee-Bum , Kim Mi-Jeong , Cha Jeong-Heon , Kim Young Jun , Jun Woo Jin , Lee Jae Myun , Yoon Ho-Geun
Published: January 2009

Because the p300/CBP-mediated hyperacetylation of RelA (p65) is critical for nuclear factor-κB (NF-κB) activation, the attenuation of p65 acetylation is a potential molecular target for the prevention of chronic inflammation. During our ongoing screening study to identify natural compounds with histone acetyltransferase inhibitor (HATi) activity, we identified epigallocatechin-3-gallate (EGCG) as a novel HATi with global specificity for the majority of HAT enzymes but with no activity toward epigenetic enzymes including HDAC, SIRT1, and HMTase. At a dose of 100 μmol/L, EGCG abrogates p300-induced p65 acetylation in vitro and in vivo, increases the level of cytosolic IκBα, and suppresses tumor necrosis factor α (TNFα)–induced NF-κB activation. We also showed that EGCG prevents TNFα-induced p65 translocation to the nucleus, confirming that hyperacetylation is critical for NF-κB translocation as well as activity. Furthermore, EGCG treatment inhibited the acetylation of p65 and the expression of NF-κB target genes in response to diverse stimuli. Finally, EGCG reduced the binding of p300 to the promoter region of interleukin-6 gene with an increased recruitment of HDAC3, which highlights the importance of the balance between HATs and histone deacetylases in the NF-κB–mediated inflammatory signaling pathway. Importantly, EGCG at 50 μmol/L dose completely blocks EBV infection-induced cytokine expression and subsequently the EBV-induced B lymphocyte transformation. These results show the crucial role of acetylation in the development of inflammatory-related diseases. [Cancer Res 2009;69(2):583–92]
MicroRNA Silencing in Primates: Towards Development of Novel Therapeutics
Authors: Petri Andreas , Lindow Morten , Kauppinen Sakari
Published: January 2009

MicroRNAs (miRNA) comprise an abundant class of small noncoding RNAs that act as important posttranscriptional regulators of gene expression. Accumulating evidence showing that aberrantly expressed miRNAs play important roles in human cancers underscores them as potential targets for therapeutic intervention. Recent reports on efficient miRNA silencing in rodents and nonhuman primates using high-affinity targeting by chemically modified antisense oligonucleotides highlight the utility of such compounds in the development of miRNA-based cancer therapeutics. [Cancer Res 2009;69(2):393–5]
Modulation of G Protein Signaling Normalizes Tumor Vessels
Authors: Manzur Mitali , Hamzah Juliana , Ganss Ruth
Published: January 2009

G protein–coupled biological processes are important for an ever-increasing number of human diseases and require fine-tuning through accessory molecules such as the regulators of G protein signaling (RGS). RGS5, a marker for tumor-resident pericytes, was recently established as playing a pivotal role in vascular maturation and vessel remodeling during carcinogenesis. Remarkably, tumors arising in a RGS5-deficient background display vessels with normalized morphology and an overall improved blood flow. Furthermore, these morphologic changes also lead to dramatic improvements in lymphocyte access to tumors and success of antitumor immunotherapy. Here, we consider the implications of these findings, and how they contribute to enhancing our understanding of remodeling angiogenic vessels as means for improving anticancer therapies. [Cancer Res 2009;69(2):396–9]
Protein Kinase C βII and PKCι/λ: Collaborating Partners in Colon Cancer Promotion and Progression
Authors: Murray Nicole R. , Weems Justin , Braun Ursula , Leitges Michael , Fields Alan P.
Published: January 2009

We previously showed that elevated expression of either protein kinase CβII (PKCβII) or PKCι/λ enhances colon carcinogenesis in mice. Here, we use novel bitransgenic mice to determine the relative importance of PKCβII and PKCι/λ in colon carcinogenesis in two complimentary models of colon cancer in vivo. Bitransgenic mice overexpressing PKCβII and constitutively active PKCι (PKCβII/caPKCι) or kinase-deficient, dominant-negative PKCι (PKCβII/kdPKCι) in the colon exhibit a similar increase in colon tumor incidence, tumor size, and tumor burden in response to azoxymethane (AOM) when compared with nontransgenic littermates. However, PKCβII/kdPKCι mice develop predominantly benign colonic adenomas, whereas PKCβII/caPKCι mice develop malignant carcinomas. In contrast, PKCβ-deficient (PKCβ−/−) mice fail to develop tumors even in the presence of caPKCι. Our previous data indicated that PKCβII drives tumorigenesis and proliferation by activating β-catenin/Apc signaling. Consistent with this conclusion, genetic deletion of PKCβ has no effect on spontaneous tumorigenesis in Apcmin/+ mice. In contrast, tissue-specific knockout of PKCλ significantly suppresses intestinal tumor formation in Apcmin/+ mice. Our data show that PKCβII and PKCι/λ serve distinct, nonoverlapping functions in colon carcinogenesis. PKCβII is required for AOM-induced tumorigenesis but is dispensable for tumor formation in ApcMin/+ mice. PKCι/λ promotes tumor progression in both AOM- and Apcmin/+-induced tumorigenesis. Thus, PKCβII and PKCι, whose expression is elevated in both rodent and human colon tumors, collaborate to drive colon tumor formation and progression, respectively. [Cancer Res 2009;69(2):656–62]
Potentiating Endogenous Antitumor Immunity to Prostate Cancer through Combination Immunotherapy with CTLA4 Blockade and GM-CSF
Authors: Fong Lawrence , Kwek Serena S. , O'Brien Shaun , Kavanagh Brian , McNeel Douglas G. , Weinberg Vivian , Lin Amy M. , Rosenberg Jonathan , Ryan Charles J. , Rini Brian I. , Small Eric J.
Published: January 2009

CTL-associated antigen 4 (CTLA4) is a costimulatory molecule expressed on activated T cells that delivers an inhibitory signal to these T cells. CTLA4 blockade with antibody treatment has been shown to augment antitumor immunity in animal models and is being developed as a treatment for cancer patients. As has been seen in preclinical models, combining CTLA4 blockade and granulocyte macrophage colony-stimulating factor (GM-CSF)–based immunotherapies can enhance the antitumor efficacy of this approach. We therefore examined whether CTLA4 blockade could be combined with GM-CSF administration. We treated 24 patients with metastatic, castration-resistant prostate cancer in a phase I trial where sequential cohorts were treated with increasing doses of ipilimumab, a fully human anti-CTLA4 antibody. Study subjects also received s.c. injections of GM-CSF at a fixed dose. Of the six patients treated at the highest dose level, three had confirmed PSA declines of >50%, including one patient that had a partial response in visceral metastases. Expansion of activated, circulating CD25+ CD69+ CD8+ T cells occurred more frequently at higher doses of treatment and was greater in magnitude than was seen in patients who received the same doses of either ipilimumab or GM-CSF alone. By screening sera with protein arrays, we showed that our treatment can induce antibody responses to NY-ESO-1. These results show that this combination immunotherapy can induce the expansion not only of activated effector CD8 T cells in vivo but also of T cells that are specific for known tumor-associated antigens from the endogenous immune repertoire. [Cancer Res 2009;69(2):609–15]
Targeting Inflammatory Pathways for Prevention and Therapy of Cancer: Short-Term Friend, Long-Term Foe
Authors: Aggarwal Bharat B. , Vijayalekshmi R.V. , Sung Bokyung
Published: January 2009

Chronic infections, obesity, alcohol, tobacco, radiation, environmental pollutants, and high-calorie diet have been recognized as major risk factors for the most common types of cancer. All these risk factors are linked to cancer through inflammation. Although acute inflammation that persists for short-term mediates host defense against infections, chronic inflammation that lasts for long term can predispose the host to various chronic illnesses, including cancer. Linkage between cancer and inflammation is indicated by numerous lines of evidence; first, transcription factors nuclear factor-κB (NF-κB) and signal transducers and activators of transcription 3 (STAT3), two major pathways for inflammation, are activated by most cancer risk factors; second, an inflammatory condition precedes most cancers; third, NF-κB and STAT3 are constitutively active in most cancers; fourth, hypoxia and acidic conditions found in solid tumors activate NF-κB; fifth, chemotherapeutic agents and γ-irradiation activate NF-κB and lead to chemoresistance and radioresistance; sixth, most gene products linked to inflammation, survival, proliferation, invasion, angiogenesis, and metastasis are regulated by NF-κB and STAT3; seventh, suppression of NF-κB and STAT3 inhibits the proliferation and invasion of tumors; and eighth, most chemopreventive agents mediate their effects through inhibition of NF-κB and STAT3 activation pathways. Thus, suppression of these proinflammatory pathways may provide opportunities for both prevention and treatment of cancer.
The Expression of Phospho-AKT, Phospho-mTOR, and PTEN in Extrahepatic Cholangiocarcinoma
Authors: Chung Joon-Yong , Hong Seung-Mo , Choi Byeong Yeob , Cho HyungJun , Yu Eunsil , Hewitt Stephen M.
Published: January 2009

Purpose: The protein kinase B (AKT) pathway plays a key role in the regulation of cellular survival, apoptosis, and protein translation, and has been shown to have prognostic significance in a number of cancers. We sought to define its role in extrahepatic cholangiocarcinoma.

Experimental Design: Two hundred twenty-one extrahepatic cholangiocarcinoma patients with clinicopathologic data, including survival, were arrayed into tissue microarrays. Phosphorylated AKT (p-AKT), phosphorylated mammalian target of rapamycin (p-mTOR), and total phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein expressions were studied with multiplex tissue immunoblotting assay.

Results: Expressions of p-AKT and p-mTOR were significantly increased in extrahepatic cholangiocarcinoma cases compared with normal and dysplastic bile duct epithelium (P < 0.05 both). Decreased PTEN expression was observed in patients with increasing depth of invasion (P < 0.05), T classification (P < 0.05), and stage grouping (P < 0.05), and the presence of invasion of the pancreas (P < 0.05) and duodenum (P < 0.05). Decreased PTEN expression (P = 0.004) as well as decreased PTEN/p-AKT (P = 0.003) and PTEN/p-mTOR (P = 0.009) expression showed shorter survival by univariate but not by multivariate analysis.

Conclusions: The AKT pathway is activated in a subset of extrahepatic cholangiocarcinoma. Elevated PTEN expression correlates with longer survival. Quantitative data obtained by multiplex tissue immunoblotting may provide additional information than assessment of immunohistochemistry alone. Quantitative analysis of PTEN, PTEN/p-AKT and PTEN/p-mTOR shows differences in survival by univariate analysis.
Improvement of Gemcitabine-Based Therapy of Pancreatic Carcinoma by Means of Oncolytic Parvovirus H-1PV
Authors: Angelova Assia L. , Aprahamian Marc , Grekova Svitlana P. , Hajri Amor , Leuchs Barbara , Giese Nathalia A. , Dinsart Christiane , Herrmann Alexia , Balboni Ginette , Rommelaere Jean , Raykov Zahari
Published: January 2009

Pancreatic carcinoma is a gastrointestinal malignancy with poor prognosis. Treatment with gemcitabine, the most potent chemotherapeutic against this cancer up to date, is not curative, and resistance may appear. Complementary treatment with an oncolytic virus, such as the rat parvovirus H-1PV, which is infectious but nonpathogenic in humans, emerges as an innovative option.

Purpose: To prove that combining gemcitabine and H-1PV in a model of pancreatic carcinoma may reduce the dosage of the toxic drug and/or improve the overall anticancer effect.

Experimental Design: Pancreatic tumors were implanted orthotopically in Lewis rats or subcutaneously in nude mice and treated with gemcitabine, H-1PV, or both according to different regimens. Tumor size was monitored by micro-computed tomography, whereas bone marrow, liver, and kidney functions were monitored by measuring clinically relevant markers. Human pancreatic cell lines and gemcitabine-resistant derivatives were tested in vitro for sensitivity to H-1PV infection with or without gemcitabine.

Results: In vitro studies proved that combining gemcitabine with H-1PV resulted in synergistic cytotoxic effects and achieved an up to 15-fold reduction in the 50% effective concentration of the drug, with drug-resistant cells remaining sensitive to virus killing. Toxicologic screening showed that H-1PV had an excellent safety profile when applied alone or in combination with gemcitabine. The benefits of applying H-1PV as a second-line treatment after gemcitabine included reduction of tumor growth, prolonged survival of the animals, and absence of metastases on CT-scans.

Conclusion: In addition to their potential use as monotherapy for pancreatic cancer, parvoviruses can be best combined with gemcitabine in a two-step protocol.
Phase II Trial of OGX-011 in Combination with Docetaxel in Metastatic Breast Cancer
Authors: Chia Stephen , Dent Susan , Ellard Susan , Ellis Peter M. , Vandenberg Ted , Gelmon Karen , Powers Jean , Walsh Wendy , Seymour Lesley , Eisenhauer Elizabeth A.
Published: January 2009

Purpose: Clusterin is an antiapoptotic protein activated in response to cellular stress. OGX-011 is a second-generation antisense oligonucleotide that inhibits clusterin expression. The primary objective of this phase II trial was to assess the safety and efficacy of the combination of OGX-011 and docetaxel for metastatic breast cancer.

Experimental Design: Women with measurable metastatic breast cancer and ≤1 chemotherapy regimen were eligible. Three loading doses of OGX-011 640 mg i.v. followed by weekly OGX-011 and docetaxel 75 mg/m2 (every 3 weeks) were given. A two-stage design was used with a hypothesis of H0 ≤35% and Ha ≥55%. Objective response in ≥6 of the first 14 patients was required for the trial to continue to the second stage.

Results: Fifteen patients were enrolled. A median of six cycles were delivered (range, 2-10). Five partial responses were confirmed for a 33% response rate (95% confidence interval, 11.8-61.6%) with a further 9 (60%) patients showing stable disease. The median duration of stable disease was 9.3 months. The median time to progression was 8 months (95% confidence interval, 5.62-9.43 months). Toxic effects were similar to those with single agent docetaxel. Although serum clusterin decreased on treatment, there was no relationship observed between the magnitude of decrease and response.

Conclusion: The combination of OGX-011 and docetaxel at 75 mg/m2 is well tolerated and clinical activity was seen in these patients with metastatic breast cancer, but there was an insufficient number of responses to meet the criteria for proceeding to the second stage of accrual.
Enhancement of Docetaxel Anticancer Activity by a Novel Diindolylmethane Compound in Human Non–Small Cell Lung Cancer
Authors: Ichite Nkechi , Chougule Mahavir B. , Jackson Tanise , Fulzele Suniket V. , Safe Stephen , Singh Mandip
Published: January 2009

Purpose: This study was conducted to examine the cytotoxic effects of a peroxisome proliferator-activated receptor γ (PPARγ) agonist, 1,1-bis (3′-indolyl)-1-(p-biphenyl) methane (DIM-C-pPhC6H5), alone and in combination with docetaxel in vitro in A549 lung cancer cells and in vivo in nude mice bearing A549 orthotopic lung tumors.

Experimental Design: Isobolographic method was used to calculate combination index values from cell viability data. Apoptosis was evaluated in A549 cells by terminal deoxynucleotidyl transferase-mediated nick end labeling assay and measurement of cleaved poly(ADP-ribose) polymerase level. Expression of proteins was studied by Western blotting. A549 cells were implanted to induce orthotopic lung tumors in nude mice and the efficacy of docetaxel, DIM-C-pPhC6H5, or combination was determined. Apoptosis and cleaved caspase-3 expression in the harvested tissues were studied by terminal deoxynucleotidyl transferase-mediated nick end labeling and immunohistochemistry, respectively.

Results: The combination index values (0.36-0.9) suggested synergistic to additive effects of docetaxel + DIM-C-pPhC6H5 and resulted in the highest increase in percentage of apoptotic cells and expression of cleaved poly(ADP-ribose) polymerase, Bax, and N-cadherin compared with treatment with either agent. The combination also enhanced procaspase-3 and -9 cleavage. In vivo, docetaxel + DIM-C-pPhC6H5 reduced lung weights by 57% compared with 39% by docetaxel or 22% by DIM-C-pPhC6H5 alone, induced apoptosis in 43% of the tumor cells compared with 29% and 22% in tumors treated with docetaxel and DIM-C-pPhC6H5, respectively, and increased procaspase-3 cleavage compared with either agent alone.

Conclusions: These findings suggest potential benefit for use of docetaxel and DIM-C-pPhC6H5 combination in lung cancer treatment.