bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancement of Adhesion Strength of Electroless-Plated Ni Under Bump Metallurgy by Introduction of Inductively Coupled Plasma Enhanced Bias Sputtering Ni Seed Layer
Authors: Kim Eung-Do , Lee Won-Jong
Published: December 2005

Electroless Ni plating using zincate treatment on Al pad is the most popular under bump metallurgy (UBM) fabrication method because of its low process cost. However, it has a demerit of poor adhesion strength. In this study, instead of using conventional zincate treatment, Ni thin film was used as a seed layer for the fabrication of Ni UBM film using electroless plating. 60-nm-thick Ni seed layer was deposited by inductively coupled plasma enhanced bias sputtering (ICPBS). The bombardment of energetic ions causes the compressive stress in the Ni seed layer and the atomic mixing at the interface. The introduction of Ti buffer layer successfully keeps the Ni seed layer from swelling due to the hydrogen generated by reducing agent in the electroless plating solution. The adoption of Ni seed layer deposited by ICPBS method enables the fabrication of electroless-plated Ni UBM film with maximum of 20 times superb adhesion strength measured by scratch test compared to that fabricated by conventional technique.
Attempted and Completed Suicide in Adolescence
Authors: Spirito Anthony , Esposito-Smythers Christianne
Published: December 2005

Suicide is the third leading cause of death in adolescence, and medically serious suicide attempts occur in approximately 3% of adolescents. This review examines a number of risk factors that contribute to suicidal behavior. A prior suicide attempt is one of the best predictors of both a repeat attempt and eventual completed suicide. Depression, disruptive behavior disorders, and substance-use disorders also place adolescents at high risk for suicidal behavior, with comorbidity further increasing risk. Research on families indicates that suicidal behavior is transmitted through families. Groups at high risk for suicidal behavior include gay, lesbian, and bisexual youths, incarcerated adolescents, and homeless/runaway teens. Although abnormalities in the serotonergic system have not been consistently linked to suicidal behavior, genetic and neurobiologic studies suggest that impulsive aggression may be the mechanism through which decreased serotonergic activity is related to suicidal behavior. Findings from prevention and intervention studies are modest and indicate the need for substantially more theory-driven treatment research.
HUMAN T CELL RESPONSES AGAINST MELANOMA
Authors: Boon Thierry , Coulie Pierre G. , Eynde Benoît J. Van den , Bruggen Pierre van der
Published: December 2005

Many antigens recognized by autologous T lymphocytes have been identified on human melanoma. Melanoma patients usually mount a spontaneous T cell response against their tumor. But at some point, the responder T cells become ineffective, probably because of a local immunosuppressive process occurring at the tumor sites. Therapeutic vaccination of metastatic melanoma patients with these antigens is followed by tumor regressions only in a small minority of the patients. The T cell responses to the vaccines show correlation with the tumor regressions. The local immunosuppression may be the cause of the lack of vaccination effectiveness that is observed in most patients. In patients who do respond to the vaccine, the antivaccine T cells probably succeed in reversing focally this immunosuppression and trigger a broad activation of other antitumor T cells, which proceed to destroy the tumor.
ATMOSPHERIC FIELD MEASUREMENTS OF THE HYDROXYL RADICAL USING LASER-INDUCED FLUORESCENCE SPECTROSCOPY
Authors: Heard Dwayne E.
Published: December 2005

▪ Abstract  The hydroxyl radical, OH, is the most important cleansing agent in the Earth's atmosphere, removing the majority of trace gases by oxidation, including greenhouse gases and CFC replacements. It is intimately involved in the chemistry that generates photochemical smog, which includes many substances harmful to health, such as ozone and particulate matter. In this review, the technique of laser-induced fluorescence for the detection of OH in the atmosphere is described, using as an example the fluorescence assay by gas expansion (FAGE) instrument developed at the University of Leeds. The comparison of measured OH concentrations at a given field site with those calculated by an atmospheric model, which is a mathematical representation of the underlying chemistry, provides one of the best methods to test whether the key chemical and physical processes are understood. Examples are given for field measurements made in clean and polluted environments.
POLITICAL ISSUES AND PARTY ALIGNMENTS: Assessing the Issue Evolution Perspective
Authors: Carmines Edward G. , Wagner Michael W.
Published: December 2005

▪ Abstract  Although the study of realignment is an essential component of the rich and fruitful tradition of examining long-term partisan change, questions about the usefulness of the concept persist. We seek to redirect and reinvigorate the study of lasting political change by evaluating the critiques of classic realignment theory, examining the issue evolution perspective, and assessing whether the theory of issue evolution can be used to explain recent research on the relationship between political issues and partisan change. Our review of the theoretical and empirical literature investigating political issues and party alignments sheds light on both the utility of the issue evolution perspective and the conditions under which durable changes in party alignments are most likely to occur.
Low-amplitude trapezius activity in work and leisure and the relation to shoulder and neck pain
Authors: Mork Paul Jarle , Westgaard Rolf H.
Published: December 2005

The aim of this study is to obtain evidence supporting or negating the hypothesis that muscle pain is associated with sustained activation of low-threshold motor units. Long-term surface electromyographic (EMG) recordings of trapezius activity pattern were related to subjectively reported shoulder and neck pain in work and leisure. Recordings from 118 female subjects (73 recorded both during work and leisure) were analyzed. Computer operators, secretaries, and health care and retail workers were represented in the material. The recordings were calibrated by the root-mean-square-detected response at maximal voluntary contraction (%maximum EMG). The analysis was performed by quantifying duration and amplitude of surface EMG activity exceeding 2% maximum EMG (“EMG bursts”). Three response categories were defined by duration of the burst periods during work: low- (<50%), intermediate- (50–70%), and high-response (>70%) groups. Shoulder and neck pain was assessed by hourly visual analog score throughout work and leisure and by pain score for the last 6 mo. Shoulder and neck pain was higher at work than leisure for subjects with long-term pain in both the high- and the low-response groups. Persistent pain, defined by the 6-mo score, was more prevalent in the high- than the low- and intermediate-response groups (73 vs. 37%); relative risk was 2.0. Trapezius activity was reduced from work to leisure for the high- but not the low-response group. The activity pattern is consistent with low-threshold motor unit overexertion for the high- but not the low-response group. We speculate that different mechanisms of muscle pain causation, dependent and independent of motor activity pattern, coexist.
Processing of Vibrotactile Inputs From Hairy Skin by Neurons of the Dorsal Column Nuclei in the Cat
Authors: Sahai V. , Mahns D. A. , Robinson L. , Perkins N. M. , Coleman G. T. , Rowe M. J.
Published: December 2005

The capacity of single neurons of the dorsal column nuclei (DCN) for coding vibrotactile information from the hairy skin has been investigated in anesthetized cats to permit quantitative comparison first with the capacities of DCN neurons responding to glabrous skin vibrotactile inputs and second with those of spinocervical tract neurons responding to vibrotactile inputs from hairy skin. Dynamically sensitive tactile neurons of the DCN the input of which came from hairy skin could be divided into two classes, one associated with hair follicle afferent (HFA) input, the other with Pacinian corpuscle (PC) input. The HFA-related class was most sensitive to low-frequency (<50 Hz) vibration and had a graded response output as a function of vibrotactile intensity changes. PC-related neurons had a broader vibrotactile sensitivity, extending to ≥300 Hz and appeared to derive their input from the margins of hairy skin, near the footpads, or from deeper PC sources such as the interosseous membranes or joints. HFA-related neurons had phaselocked responses to vibration frequencies up to ∼75 Hz, whereas PC neurons retained this capacity up to frequencies of ∼300 Hz with tightest phaselocking between 50 and 200 Hz. Quantitative measures of phaselocking revealed that the HFA-related neurons provide the better signal of vibrotactile frequency up to ∼50 Hz with a switch-over to the PC-related neurons above that value. In conclusion, the functional capacities of these two classes of cuneate neuron appear to account for behavioral vibrotactile frequency discriminative performance in hairy skin, in contrast to the limited capacities of vibrotactile-sensitive neurons within the spinocervical tract system.
Time Course and Magnitude of Illusory Translation Perception During Off-Vertical Axis Rotation
Authors: Vingerhoets R.A.A. , Medendorp W. P. , Van Gisbergen J.A.M.
Published: December 2005

Human spatial orientation relies on vision, somatosensory cues, and signals from the semicircular canals and the otoliths. The canals measure rotation, whereas the otoliths are linear accelerometers, sensitive to tilt and translation. To disambiguate the otolith signal, two main hypotheses have been proposed: frequency segregation and canal–otolith interaction. So far these models were based mainly on oculomotor behavior. In this study we investigated their applicability to human self-motion perception. Six subjects were rotated in yaw about an off-vertical axis (OVAR) at various speeds and tilt angles, in darkness. During the rotation, subjects indicated at regular intervals whether a briefly presented dot moved faster or slower than their perceived self-motion. Based on such responses, we determined the time course of the self-motion percept and characterized its steady state by a psychometric function. The psychophysical results were consistent with anecdotal reports. All subjects initially sensed rotation, but then gradually developed a percept of being translated along a cone. The rotation percept could be described by a decaying exponential with a time constant of about 20 s. Translation percept magnitude typically followed a delayed increasing exponential with delays up to 50 s and a time constant of about 15 s. The asymptotic magnitude of perceived translation increased with rotation speed and tilt angle, but never exceeded 14 cm/s. These results were most consistent with predictions of the canal–otolith-interaction model, but required parameter values that differed from the original proposal. We conclude that canal–otolith interaction is an important governing principle for self-motion perception that can be deployed flexibly, dependent on stimulus conditions.
Influence of Location of a Fluorescent Zinc Probe in Brain Slices on Its Response to Synaptic Activation
Authors: Kay Alan R. , Tóth Katalin
Published: December 2005

The precise role of the high concentration of ionic zinc found in the synaptic vesicles of certain glutamatergic terminals is unknown. Fluorescent probes with their ability to detect ions at low concentrations provide a powerful approach to monitoring cellular Zn2+ levels. In the last few years, a number of fluorescent probes (indicators) have been synthesized that can be used to visualize Zn2+ in live cells. The interpretation of data gathered using such probes depends crucially on the location of the probe. Using acutely prepared hippocampal slices, we provide evidence that the Zn2+ probes, ZnAF-2 and ZP4, are membrane permeant and are able to pass into synaptic vesicles. In addition, we show that changes in fluorescence of the Zn2+ probes can be used to monitor presynaptic activity; however, these changes are inconsistent with Zn2+ release.
Local Connections to Specific Types of Layer 6 Neurons in the Rat Visual Cortex
Authors: Zarrinpar Amir , Callaway Edward M.
Published: December 2005

Because layer 6 of the cerebral cortex receives direct thalamic input and provides projections back to the thalamus, it is in a unique position to influence thalamocortical interactions. Different types of layer 6 pyramidal neurons provide output to different thalamic nuclei, and it is therefore of interest to understand the sources of functional input to these neurons. We studied the morphologies and local excitatory input to individual layer 6 neurons in rat visual cortex by combining intracellular labeling and recording with laser-scanning photostimulation. As in previous photostimulation studies, we found significant differences in the sources of local excitatory input to different cell types. Most notably, there were differences in local input to neurons that, based on analogy to barrel cortex, are likely to project only to the lateral geniculate nucleus of the thalamus versus those that are likely to also project to the lateral posterior nucleus. The more striking finding, however, was the paucity of superficial layer input to layer 6 neurons in the rat visual cortex, contrasting sharply with layer 6 neurons in the primate visual cortex. These observations provide insight into differences in function between cortical projections to first-order versus higher-order thalamic nuclei and also show that these circuits can be organized differently in different species.
Enhanced Targeting and Killing of Tumor Cells Expressing the CXC Chemokine Receptor 4 by Transducible Anticancer Peptides
Authors: Snyder Eric L. , Saenz Cheryl C. , Denicourt Catherine , Meade Bryan R. , Cui Xian-Shu , Kaplan Ian M. , Dowdy Steven F.
Published: December 2005

Protein transduction domains (PTDs), such as the TAT PTD, have been shown to deliver a wide variety of cargo in cell culture and to treat preclinical models of cancer and cerebral ischemia. The TAT PTD enters cells by a lipid raft–dependent macropinocytosis mechanism that all cells perform. Consequently, PTDs resemble small-molecule therapeutics in their lack of pharmacologic tissue specificity in vivo. However, several human malignancies overexpress specific receptors, including HER2 in breast cancer, GnRH in ovarian carcinomas, and CXC chemokine receptor 4 (CXCR4) in multiple malignancies. To target tumor cells that overexpress the CXCR4 receptor, we linked the CXCR4 DV3 ligand to two transducible anticancer peptides: a p53-activating peptide (DV3-TATp53C′) and a cyclin-dependent kinase 2 antagonist peptide (DV3-TAT-RxL). Treatment of tumor cells expressing the CXCR4 receptor with either the DV3-TATp53C′ or DV3-TAT-RxL targeted peptides resulted in an enhancement of tumor cell killing compared with treatment with nontargeted parental peptides. In contrast, there was no difference between DV3 targeted peptide and nontargeted, parental peptide treatment of non-CXCR4-expressing tumor cells. These observations show that a multidomain approach can be used to further refine and enhance the tumor selectivity of biologically active, transducible macromolecules for treating cancer.
Cancer Cells Become Susceptible to Natural Killer Cell Killing after Exposure to Histone Deacetylase Inhibitors Due to Glycogen Synthase Kinase-3–Dependent Expression of MHC Class I–Related Chain A and B
Authors: Skov Søren , Pedersen Marianne Terndrup , Andresen Lars , Thor Straten Per , Woetmann Anders , Ødum Niels
Published: December 2005

We show that histone deacetylase (HDAC) inhibitors lead to functional expression of MHC class I–related chain A and B (MICA/B) on cancer cells, making them potent targets for natural killer (NK) cell–mediated killing through a NK group 2, member D (NKG2D) restricted mechanism. Blocking either apoptosis or oxidative stress caused by HDAC inhibitor treatment did not affect MICA/B expression, suggesting involvement of a separate signal pathway not directly coupled to induction of cell death. HDAC inhibitor treatment induced glycogen synthase kinase-3 (GSK-3) activity and down-regulation of GSK-3 by small interfering RNA or by different inhibitors showed that GSK-3 activity is essential for the induced MICA/B expression. We thus present evidence that cancer cells which survive the direct induction of cell death by HDAC inhibitors become targets for NKG2D-expressing cells like NK cells, γδ T cells, and CD8 T cells.
Antitumor Activity of Human Papillomavirus Type 16 E7–Specific T Cells against Virally Infected Squamous Cell Carcinoma of the Head and Neck
Authors: Albers Andreas , Abe Koji , Hunt Jennifer , Wang Jun , Lopez-Albaitero Andres , Schaefer Carsten , Gooding William , Whiteside Theresa L. , Ferrone Soldano , DeLeo Albert , Ferris Robert L.
Published: December 2005

Human papillomavirus (HPV)–associated squamous cell carcinoma of the head and neck (SCCHN) seems to be a suitable target for cancer vaccination. HPV-encoded oncogenic proteins, such as E7, are promising tumor-specific antigens and are obligatory for tumor growth. Because few immunologic studies have analyzed the endogenous HPV-specific immune response in this subset of SCCHN patients, we studied T-cell frequencies against HPV-16 E711-20 or E786-93 in tumor-bearing, human leukocyte antigen (HLA)-A*0201+ SCCHN patients, whose tumors were either HPV-16+ or HPV-16−. In HPV-16+ SCCHN patients, frequencies of T cells against either peptide were significantly elevated (P < 0.005) compared with HPV-16− patients or healthy volunteers. Tetramer+ T cells showed evidence of terminally differentiated phenotype (CD45RA+CCR7−) and an elevated level of CD107a staining for degranulation. Despite detectable expression of the restricting HLA class I allele, HLA-A*0201-E711-20– or HLA-A*0201-E786-93–specific CTL obtained by in vitro stimulation of healthy donor peripheral blood mononuclear cells only recognize a naturally HPV-16-transformed, HLA-A*0201+ SCCHN cell line after pretreatment with IFN-γ. This cell line had little or no expression of LMP2, TAP1, and tapasin, critical components of the HLA class I antigen-processing machinery, which were up-regulated by IFN-γ treatment. Immunohistochemistry of HPV-16+ SCCHN tumors showed that these antigen-processing machinery components are down-regulated in tumors in vivo compared with adjacent normal squamous epithelium. Thus, immunity to HPV-16 E7 is associated with the presence of HPV-16 infection and presentation of E7-derived peptides on SCCHN cells, which show evidence of immune escape. These findings support further development of E7-specific immunotherapy and strategies for up-regulation of antigen-processing machinery components in HPV-associated SCCHN.
p53-Independent Negative Regulation of p21/Cyclin-Dependent Kinase–Interacting Protein 1 by the Sonic Hedgehog-Glioma-Associated Oncogene 1 Pathway in Gastric Carcinoma Cells
Authors: Ohta Miki , Tateishi Keisuke , Kanai Fumihiko , Watabe Hirotsugu , Kondo Shintaro , Guleng Bayasi , Tanaka Yasuo , Asaoka Yoshinari , Jazag Amarsanaa , Imamura Jun , Ijichi Hideaki , Ikenoue Tsuneo , Sata Masataka , Miyagishi Makoto , Taira Kazunari , Tada Minoru , Kawabe Takao , Omata Masao
Published: December 2005

The activation of Hedgehog (Hh) signaling has been implicated in the growth of various tumor types, including gastric carcinoma. However, the precise mechanisms of Hh activation and suppression of tumor growth by the blockade of Hh signaling in gastric carcinoma cells remain unknown. The aim of this study was to elucidate the mechanism of abnormal Hh signaling and the key molecules contributing to dysregulated growth of gastric carcinoma. The Sonic hedgehog (Shh) ligand and its receptor Patched were expressed in all five gastric carcinoma cell lines examined (MKN1, MKN7, MKN45, MKN74, and AGS cells). The blockade of Hh signaling with anti-Shh antibody inhibited the growth of all five gastric carcinoma cell lines. Shh was overexpressed (mean, 12.8-fold) in 8 of 14 (57.0%) cancerous tissue samples from patients with gastric carcinoma as compared with expression in the surrounding noncancerous tissues. The disruption of glioma-associated oncogene 1 (Gli1) by small interfering RNA induced an increase in p21/cyclin-dependent kinase–interacting protein 1 (CIP1), interfered with the G1-S transition, and suppressed cell proliferation. The stimulation or inhibition of Hh signaling did not affect p53 activity and the induction of p21/CIP1 expression and the G1 arrest by inhibition of Hh signaling were not affected by the p53 status. These findings suggest that the overexpression of Shh contributes to constitutive Hh activation and that this signaling pathway negatively regulates p21/CIP1 through a Gli1-dependent and p53-independent mechanism in gastric carcinoma cells.
MCT-1 Oncogene Contributes to Increased In vivo Tumorigenicity of MCF7 Cells by Promotion of Angiogenesis and Inhibition of Apoptosis
Authors: Levenson Anait S. , Thurn Kenneth E. , Simons Laura A. , Veliceasa Dorina , Jarrett Jennifer , Osipo Clodia , Jordan V. Craig , Volpert Olga V. , Satcher Robert L. , Gartenhaus Ronald B.
Published: December 2005

Overexpression of a novel oncogene MCT-1 (multiple copies in a T cell malignancy) causes malignant transformation of murine fibroblasts. To establish its role in the pathogenesis of breast cancer in humans, we generated stable transfectants of MCF7 breast cancer cells negative for endogenous MCT-1 (MCF7-MCT-1). Overexpression of MCT-1 in these cells resulted in a slight elevation of estrogen receptor-α, and higher rates of DNA synthesis and growth in response to estradiol compared with the empty vector control (MCF7-EV). The pure antiestrogen fulvestrant inhibited the estradiol-stimulated proliferation of MCF7-MCT-1 cells. The MCF7-MCT-1 clones showed increased invasiveness in the presence of 50% serum compared with the MCF7-EV. In a tumor xenograft model, MCT-1–overexpressing cells showed higher take rates and formed significantly larger tumors than MCF7-EV controls. When we examined angiogenic phenotype and molecular mediators of angiogenesis in MCF7-MCT-1 tumors in vivo, we found greater microvascular density and lower apoptosis in the MCF7-MCT-1 tumors compared with MCF7-EV controls accompanied by a dramatic decline in the levels of angiogenesis inhibitor, thrombospondin-1 (TSP1). In vitro, blocking TSP1 in the medium conditioned by MCT-1–negative cells restored its angiogenic potential to that of the MCF7-MCT-1 cells. Conversely, despite an increase in mRNA encoding vascular endothelial growth factor upon MCT-1 overexpression, vascular endothelial growth factor protein levels have not been notably altered. Taken together, our results suggest that MCT-1 may contribute to the pathogenesis and progression of human breast cancer via at least two routes: promotion of angiogenesis through the decline of TSP1 and inhibition of apoptosis.
Rapamycin-Sensitive Pathway Regulates Mitochondrial Membrane Potential, Autophagy, and Survival in Irradiated MCF-7 Cells
Authors: Paglin Shoshana , Lee Na-Young , Nakar Charles , Fitzgerald Megan , Plotkin Jason , Deuel Bethanne , Hackett Nadia , McMahill Melissa , Sphicas Eleana , Lampen Nina , Yahalom Joachim
Published: December 2005

Radiation-induced inhibition of rapamycin-sensitive pathway and its effect on the cellular response to radiation were studied in the human breast cancer cell line MCF-7. Both radiation and rapamycin shared molecular targets and induced similar physiologic responses. Each of these treatments increased immunostaining of mammalian target of rapamycin (mTOR) in the nucleus, and radiation led to decreased phosphorylation of its autophosphorylation site Ser2481. In addition to dephosphorylation of established mTOR downstream effectors 4E-binding protein 1 and p70 ribosomal S6 kinase, both treatments decreased the level of eukaryotic initiation factor 4G. Experiments with the potentiometric dye, JC-1, revealed an oligomycin-dependent increase in mitochondrial membrane potential following radiation or rapamycin treatment, suggesting that both lead to reversal of F0F1ATPase activity. Both radiation and rapamycin induced sequestration of cytoplasmic material in autophagic vacuoles. In both cases, appearance of autophagic vacuoles involved the participation of microtubule-associated protein 1 light chain 3 (LC3). Transient cotransfection of green fluorescent protein-LC3 with either wild-type or dominant-negative mTOR further showed that inactivation of mTOR pathway is sufficient to induce autophagy in these cells. Finally, administration of rapamycin in combination with radiation led to enhanced mitochondria hyperpolarization, p53 phosphorylation, and increased cell death. Taken together, these experiments show that radiation-induced inhibition of rapamycin-sensitive pathway in MCF-7 cells causes changes in mitochondria metabolism, development of autophagy, and an overall decrease in cell survival.
Point Mutation at Single Tyrosine Residue of Novel Oncogene NOK Abrogates Tumorigenesis in Nude Mice
Authors: Chen Yue , Li Ying-Hua , Chen Xi-Ping , Gong Li-Min , Zhang Shu-Ping , Chang Zhi-Jie , Zhang Xiu-Fang , Fu Xin-Yuan , Liu Li
Published: December 2005

Receptor protein-tyrosine kinases (RPTKs) are tightly regulated during normal cellular processes including cell growth, differentiation, and metabolism. Recently, a RPTK-like molecule named novel oncogene with kinase-domain (NOK) has been cloned and characterized. Overexpression of NOK caused severe cellular transformation as well as tumorigenesis and metastasis in nude mice. In the current study, we generated two tyrosine→phenylalanine (Y→F) point mutations (Y327F and Y356F) within the endodomain of NOK that are well conserved in many RPTK subfamilies and are the potential tyrosine phosphorylation sites important for major intracellular signaling. Using BaF3 cells stably expressing the ectodomain of mouse erythropoietin receptor, and the transmembrane and endodomain of NOK (BaF3-E/N), we were able to show that point mutations at either Y327 or Y356 dramatically blocked cellular transformation by NOK as examined by colony formation and cellular DNA synthesis. In addition, tumorigenesis induced by BaF3-E/N was completely abrogated upon the introduction of either single mutation. Importantly, signaling studies revealed that the activation of extracellular signal-regulated kinase was inhibited by Y356F and was significantly reduced by Y327F. Both mutations significantly impaired Akt phosphorylation. Interestingly, both mutations did not affect the kinase activity of NOK. Moreover, apoptotic analysis revealed that both mutations accelerated cell death by activating caspase-3–mediated pathways. Thus, our study shows that these potential tyrosine phosphorylation sites may play critical roles in NOK-mediated tumorigenesis both in vitro and in vivo.
Human Aromatase: Gene Resequencing and Functional Genomics
Authors: Ma Cynthia X. , Adjei Araba A. , Salavaggione Oreste E. , Coronel Josefa , Pelleymounter Linda , Wang Liewei , Eckloff Bruce W. , Schaid Daniel , Wieben Eric D. , Adjei Alex A. , Weinshilboum Richard M.
Published: December 2005

Aromatase [cytochrome P450 19 (CYP19)] is a critical enzyme for estrogen biosynthesis, and aromatase inhibitors are of increasing importance in the treatment of breast cancer. We set out to identify and characterize genetic polymorphisms in the aromatase gene, CYP19, as a step toward pharmacogenomic studies of aromatase inhibitors. Specifically, we “resequenced” all coding exons, all upstream untranslated exons plus their presumed core promoter regions, all exon-intron splice junctions, and a portion of the 3′-untranslated region of CYP19 using 240 DNA samples from four ethnic groups. Eighty-eight polymorphisms were identified, resulting in 44 haplotypes. Functional genomic studies were done with the four nonsynonymous coding single nucleotide polymorphisms (cSNP) that we observed, two of which were novel. Those cSNPs altered the following amino acids: Trp39Arg, Thr201Met, Arg264Cys, and Met364Thr. The Cys264, Thr364, and double variant Arg39Cys264 allozymes showed significant decreases in levels of activity and immunoreactive protein when compared with the wild-type (WT) enzyme after transient expression in COS-1 cells. A slight decrease in protein level was also observed for the Arg39 allozyme, whereas Met201 displayed no significant changes in either activity or protein level when compared with the WT enzyme. There was also a 4-fold increase in apparent Km value for Thr364 with androstenedione as substrate. Of the recombinant allozymes, only the double mutant (Arg39Cys264) displayed a significant change from the WT enzyme in inhibitor constant for the aromatase inhibitors exemestane and letrozole. These observations indicate that genetic variation in CYP19 might contribute to variation in the pathophysiology of estrogen-dependent disease.
Interaction between Polyamines and the Mitogen-Activated Protein Kinase Pathway in the Regulation of Cell Cycle Variables in Breast Cancer Cells
Authors: Hu Xin , Washington Sharlene , Verderame Michael F. , Manni Andrea
Published: December 2005

Inhibition of polyamine biosynthesis with α-difluoromethylornithine (DFMO) has been shown to inhibit proliferation of breast cancer cells although its mechanism of action has not been fully elucidated. To address this issue, we tested the effects of DFMO on cell cycle variables of MDA-MB-435 human breast cancer cells in culture. We also focused on the possible mediatory role of the mitogen-activated protein kinase (MAPK) pathway on the cell cycle effects of DFMO because this compound has been shown to activate MAPK signaling. We found that DFMO caused a p53-independent increase in p21 and its association with cyclin-dependent kinase (cdk)-2 and decreased cdk-2 protein as well as its phosphorylation on Thr160. In addition, DFMO markedly suppressed the expression of the full-length and low molecular weight forms of cyclin E. These effects of DFMO were reversible with exogenous putrescine, thus indicating that they are specifically mediated through polyamine depletion. Cdk-2 activity was drastically reduced in DFMO-treated breast cancer cells which exhibited a reduction in retinoblastoma (Rb) phosphorylation and protein. As a predictable consequence of these effects, DFMO caused a G1-S block. In addition, DFMO inhibited G2-M transition, most likely as a result of its induction of p21 expression. Inhibition of the MAPK pathway with PD98059 or U0126 blocked the DFMO-induced induction of p21 and the reduction of cdk-2 protein. PD98059 reversed the G2-M block induced by DFMO (probably as a result of suppression of p21) but not the G1-S arrest. MDA-MB-435 cells treated with PD98059 or U0126 in the presence and absence of DFMO exhibited a marked increase in the expression of p27 and its association with cdk-2, a decrease in phosphorylation of cdk-2 on Thr160, and a decrease in cyclin E expression. As predicted, PD98059 treatment reduced cdk-2 activity and Rb phosphorylation while reversing the decrease in Rb protein induced by DFMO. Neither DFMO nor PD98059, either alone or in combination, reduced cdk-4 activity despite a marked induction in p15 expression caused by DFMO. Our results indicate that activation of the MAPK pathway accounts for some of the effects of DFMO on cell cycle events of breast cancer cells. Inhibition of the MAPK pathway, however, does not reverse the cell cycle arrest induced by DFMO because of activation of alternative mechanisms leading to suppression of cdk-2 activity.
Antibody Microarray Profiling Reveals Individual and Combined Serum Proteins Associated with Pancreatic Cancer
Authors: Orchekowski Randal , Hamelinck Darren , Li Lin , Gliwa Ewa , VanBrocklin Matt , Marrero Jorge A. , Vande Woude George F. , Feng Ziding , Brand Randall , Haab Brian B.
Published: December 2005

We used antibody microarrays to probe the associations of multiple serum proteins with pancreatic cancer and to explore the use of combined measurements for sample classification. Serum samples from pancreatic cancer patients (n = 61), patients with benign pancreatic disease (n = 31), and healthy control subjects (n = 50) were probed in replicate experiment sets by two-color, rolling circle amplification on microarrays containing 92 antibodies and control proteins. The antibodies that had reproducibly different binding levels between the patient classes revealed different types of alterations, reflecting inflammation (high C-reactive protein, α-1-antitrypsin, and serum amyloid A), immune response (high IgA), leakage of cell breakdown products (low plasma gelsolin), and possibly altered vitamin K usage or glucose regulation (high protein-induced vitamin K antagonist-II). The accuracy of the most significant antibody microarray measurements was confirmed through immunoblot and antigen dilution experiments. A logistic-regression algorithm distinguished the cancer samples from the healthy control samples with a 90% and 93% sensitivity and a 90% and 94% specificity in duplicate experiment sets. The cancer samples were distinguished from the benign disease samples with a 95% and 92% sensitivity and an 88% and 74% specificity in duplicate experiment sets. The classification accuracies were significantly improved over those achieved using individual antibodies. This study furthered the development of antibody microarrays for molecular profiling, provided insights into the nature of serum-protein alterations in pancreatic cancer patients, and showed the potential of combined measurements to improve sample classification accuracy.
Helicobacter pylori but not High Salt Induces Gastric Intraepithelial Neoplasia in B6129 Mice
Authors: Rogers Arlin B. , Taylor Nancy S. , Whary Mark T. , Stefanich Erinn D. , Wang Timothy C. , Fox James G.
Published: December 2005

Helicobacter pylori is responsible for most human stomach cancers. Gastric cancer also is overrepresented in populations consuming high-salt diets. Attempts to test the hypothesis that high salt promotes H. pylori carcinogenesis have been hindered by the lack of a wild-type mouse model. Based on pilot observations of unexpectedly early gastric adenocarcinoma in C57BL/6 × 129S6/SvEv (B6129) mice infected with Helicobacter felis, we conducted a study to characterize H. pylori infection in these mice and to determine whether high salt promotes tumorigenesis. Male and female mice were gavaged with H. pylori Sydney strain-1 or vehicle only and divided into four groups based on infection status and maintenance on a basal (0.25%) or high (7.5%) salt diet. In uninfected mice, the high-salt diet enhanced proliferation and marginally increased parietal cell mucous metaplasia with oxyntic atrophy. Lesions in H. pylori infected mice without regard to diet or gender were of equivalent severity and characterized by progressive gastritis, oxyntic atrophy, hyperplasia, intestinal metaplasia, and dysplasia. Infected mice on the high-salt diet exhibited a shift in antimicrobial humoral immunity from a Th1 to a Th2 pattern, accompanied by significantly higher colonization and a qualitative increase in infiltrating eosinophils. No mice developed anti-parietal cell antibodies suggestive of autoimmune gastritis. At 15 months of age infected mice in both dietary cohorts exhibited high-grade dysplasia consistent with gastric intraepithelial neoplasia. In summary, we report for the first time H. pylori–induced gastric intraepithelial neoplasia in a wild-type mouse model and show no additive effect of high-salt ingestion on tumor progression.
Induction of the Hypoxia-Inducible Factor System by Low Levels of Heat Shock Protein 90 Inhibitors
Authors: Ibrahim Nadia O. , Hahn Torsten , Franke Corinna , Stiehl Daniel P. , Wirthner Renato , Wenger Roland H. , Katschinski Dörthe M.
Published: December 2005

The heterodimeric hypoxia-inducible factor-1 (HIF-1) is involved in key steps of tumor progression and therapy resistance and thus represents an attractive antitumor target. Because heat shock protein 90 (HSP90) plays an important role in HIF-1α protein stabilization and because HSP90 inhibitors are currently being tested in clinical phase I trials for anticancer treatment, we investigated their role as anti-HIF-1α agents. Surprisingly, low-dose (5-30 nmol/L) treatment of HeLa cells with three different HSP90 inhibitors (17-AAG, 17-DMAG, and geldanamycin) increased HIF-1–dependent reporter gene activity, whereas higher doses (1-3 μmol/L) resulted in a reduction of hypoxia-induced HIF-1 activity. In line with these data, low-dose treatment with HSP90 inhibitors increased and high-dose treatment reduced hypoxic HIF-1α protein levels, respectively. HIF-1α protein stabilized by HSP90 inhibitors localized to the nucleus. As a result of HSP90-modulated HIF-1 activity, the levels of the tumor-relevant HIF-1 downstream targets carbonic anhydrase IX, prolyl-4-hydroxylase domain protein 3, and vascular endothelial growth factor were increased or decreased after low-dose or high-dose treatment, respectively. Bimodal effects of 17-AAG on vessel formation were also seen in the chick chorioallantoic membrane angiogenesis assay. In summary, these results suggest that dosage will be a critical factor in the treatment of tumor patients with HSP90 inhibitors.
Melatonin-Depleted Blood from Premenopausal Women Exposed to Light at Night Stimulates Growth of Human Breast Cancer Xenografts in Nude Rats
Authors: Blask David E. , Brainard George C. , Dauchy Robert T. , Hanifin John P. , Davidson Leslie K. , Krause Jean A. , Sauer Leonard A. , Rivera-Bermudez Moises A. , Dubocovich Margarita L. , Jasser Samar A. , Lynch Darin T. , Rollag Mark D. , Zalatan Frederick
Published: December 2005

The increased breast cancer risk in female night shift workers has been postulated to result from the suppression of pineal melatonin production by exposure to light at night. Exposure of rats bearing rat hepatomas or human breast cancer xenografts to increasing intensities of white fluorescent light during each 12-hour dark phase (0-345 μW/cm2) resulted in a dose-dependent suppression of nocturnal melatonin blood levels and a stimulation of tumor growth and linoleic acid uptake/metabolism to the mitogenic molecule 13-hydroxyoctadecadienoic acid. Venous blood samples were collected from healthy, premenopausal female volunteers during either the daytime, nighttime, or nighttime following 90 minutes of ocular bright, white fluorescent light exposure at 580 μW/cm2 (i.e., 2,800 lx). Compared with tumors perfused with daytime-collected melatonin-deficient blood, human breast cancer xenografts and rat hepatomas perfused in situ, with nocturnal, physiologically melatonin-rich blood collected during the night, exhibited markedly suppressed proliferative activity and linoleic acid uptake/metabolism. Tumors perfused with melatonin-deficient blood collected following ocular exposure to light at night exhibited the daytime pattern of high tumor proliferative activity. These results are the first to show that the tumor growth response to exposure to light during darkness is intensity dependent and that the human nocturnal, circadian melatonin signal not only inhibits human breast cancer growth but that this effect is extinguished by short-term ocular exposure to bright, white light at night. These mechanistic studies are the first to provide a rational biological explanation for the increased breast cancer risk in female night shift workers.
Altered Localization of p120 Catenin During Epithelial to Mesenchymal Transition of Colon Carcinoma Is Prognostic for Aggressive Disease
Authors: Bellovin David I. , Bates Richard C. , Muzikansky Alona , Rimm David L. , Mercurio Arthur M.
Published: December 2005

We examined the expression and localization of p120 catenin (p120ctn) as a consequence of the epithelial to mesenchymal transition (EMT) of highly differentiated colon carcinoma cells (LIM1863 cells). This unique line grows in suspension as spheroids and undergoes an EMT within 24 hours following stimulation with transforming growth factor-β and tumor necrosis factor-α. Although p120ctn expression remains stable during the EMT, its localization shifts from cell-cell junctions to the cytoplasm. Interestingly, a marked decrease in RhoA activation coincident with E-cadherin loss occurs during the EMT and correlates with the formation of a p120ctn/RhoA complex. Use of RNA interference showed that p120ctn reduction results in increased RhoA activity and a significant decrease in the motility of post-EMT cells. To determine the relevance of these findings to colorectal cancer progression, we assessed p120ctn expression by immunohistochemistry in 557 primary tumors. Of note, we observed that 53% of tumors presented cytoplasmic staining for p120ctn, and statistical analysis revealed that this localization is predictive of poor patient outcome. Cytoplasmic p120ctn correlated with later-stage tumors, significantly reduced 5- and 10-year survival times and a greater propensity for metastasis to lymph nodes compared with junctional p120ctn. We also confirmed that altered localization of p120ctn corresponded with loss or cytoplasmic localization of E-cadherin. These alterations in E-cadherin are also associated with a significant reduction in patient survival time and an increase in tumor stage and lymph node metastasis. These data provide a compelling argument for the importance of both p120ctn and the EMT itself in the progression of colorectal carcinoma.
A Novel Dietary Triterpene Lupeol Induces Fas-Mediated Apoptotic Death of Androgen-Sensitive Prostate Cancer Cells and Inhibits Tumor Growth in a Xenograft Model
Authors: Saleem Mohammad , Kweon Mee-Hyang , Yun Jung-Mi , Adhami Vaqar Mustafa , Khan Naghma , Syed Deeba N. , Mukhtar Hasan
Published: December 2005

In prostate cancer, a fine balance between cell proliferation and apoptotic death is lost, resulting in increased cellular mass and tumor progression. One approach to redress this imbalance and control this malignancy is its preventive intervention through the use of dietary natural agents. Here, we investigated the growth-inhibitory effect and associated mechanisms of Lupeol, a triterpene present in fruits and vegetables, in androgen-sensitive human prostate cancer cells. Lupeol treatment resulted in significant inhibition of cell viability in a dose-dependent manner and caused apoptotic death of prostate cancer cells. Lupeol was found to induce the cleavage of poly(ADP-ribose) polymerase protein and degradation of acinus protein with a significant increase in the expression of FADD protein. Among all death receptor targets examined, Lupeol specifically caused a significant increase in the expression of Fas receptor. The small interfering RNA–mediated silencing of the Fas gene and inhibition of caspase-6, caspase-8, and caspase-9 by their specific inhibitors confirmed that Lupeol specifically activates the Fas receptor–mediated apoptotic pathway in androgen-sensitive prostate cancer cells. The treatment of cells with a combination of anti-Fas monoclonal antibody and Lupeol resulted in higher cell death compared with the additive effect of the two compounds alone, suggesting a synergistic effect. Lupeol treatment resulted in a significant inhibition in growth of tumors with concomitant reduction in prostate-specific antigen secretion in athymic nude mice implanted with CWR22Rν1 cells. Because early clinical prostate cancer growth is an androgen-dependent response, the results of the present study suggest that Lupeol may have a potential to be an effective agent against prostate cancer.
Prospective Identification of Tumorigenic Prostate Cancer Stem Cells
Authors: Collins Anne T. , Berry Paul A. , Hyde Catherine , Stower Michael J. , Maitland Norman J.
Published: December 2005

Existing therapies for prostate cancer eradicates the bulk of cells within a tumor. However, most patients go on to develop androgen-independent disease that remains incurable by current treatment strategies. There is now increasing evidence in some malignancies that the tumor cells are organized as a hierarchy originating from rare stem cells that are responsible for maintaining the tumor. We report here the identification and characterization of a cancer stem cell population from human prostate tumors, which possess a significant capacity for self-renewal. These cells are also able to regenerate the phenotypically mixed populations of nonclonogenic cells, which express differentiated cell products, such as androgen receptor and prostatic acid phosphatase. The cancer stem cells have a CD44+/α2β1hi/CD133+ phenotype, and we have exploited these markers to isolate cells from a series of prostate tumors with differing Gleason grade and metastatic states. Approximately 0.1% of cells in any tumor expressed this phenotype, and there was no correlation between the number of CD44+/α2β1hi/CD133+ cells and tumor grade. The identification of a prostate cancer stem cell provides a powerful tool to investigate the tumorigenic process and to develop therapies targeted to the stem cell.
Novel Extracellular Vesicles Mediate an ABCG2-Dependent Anticancer Drug Sequestration and Resistance
Authors: Ifergan Ilan , Scheffer George L. , Assaraf Yehuda G.
Published: December 2005

Overexpression of the multidrug efflux transporter ABCG2 in the plasma membrane of cancer cells confers resistance to various anticancer drugs, including mitoxantrone. Here, we explored the mechanism underlying drug resistance in the MCF-7 breast cancer sublines MCF-7/MR and MCF-7/FLV1000 cells in which wild-type (R482) ABCG2 overexpression is highly confined to cell-cell attachment zones. The latter comprised the membrane of novel extracellular vesicles in which mitoxantrone was rapidly and dramatically sequestered. After 12 hours of incubation with mitoxantrone, the estimated intravesicular drug concentration was ∼1,000-fold higher than in the culture medium. This drug compartmentalization was prevented by the specific and potent ABCG2 transport inhibitors Ko143 and fumitremorgin C, thereby resulting in restoration of drug sensitivity. Consistently, this intravesicular drug concentration was abrogated by energy deprivation and was restored upon provision of energy substrates. Fine-structure studies corroborated the presence of numerous large extracellular vesicles that were highly confined to cell-cell attachment zones between neighbor cells. Furthermore, high-resolution electron microscopy revealed that the membrane of these extracellular vesicles contained microvilli-like invaginations protruding into the intravesicular lumen. It is likely that these microvilli-like projections increase the vesicular membrane surface, thereby allowing for a more efficient ABCG2-dependent intravesicular anticancer drug concentration. Hence, these novel extracellular vesicles mediate the ABCG2-dependent extraction of intracellular drug, thereby serving as cytotoxic drug disposal chambers shared by multiple neighbor cancer cells. This constitutes a novel modality of anticancer drug resistance.