bluuebunny/crossref_metadata_2025 huggingface dataset:
Incorporation of topsoil and burn-pile debris substantially increases early growth of lodgepole pine on landings
Authors: Campbell D. Bruce , Bulmer Chuck E. , Jones Melanie D. , Philip Leanne J. , Zwiazek Janusz J.
Published: March 2008

Rehabilitation of skid trails, temporary roads, and log landings is required for many harvested sites in British Columbia; however, more information is needed regarding practical methods to return these access areas to productive forest. Lodgepole pine ( Pinus contorta Dougl. ex Loud. var. latifolia Engelm.) seedlings (1 + 0) were planted into (i) fully rehabilitated landings (burn-pile debris and topsoil incorporated), (ii) tilled landings, and (iii) unprepared portions of the adjacent cutblock. After two seasons of growth in the field, seedlings planted on fully rehabilitated landings were 38% larger, more robust, and exhibited 44% greater absolute growth and 22% greater relative growth rates, than seedlings planted in the adjacent cutblock. Seedlings planted on tilled landings were 33% smaller, and exhibited absolute growth rates 38% less, and relative growth rates 18% less, than seedlings planted in the adjacent cutblock. Seedlings planted in the cutblock exhibited higher ectomycorrhizal colonization rates. Our results indicate that tillage alone was not sufficient to fully rehabilitate these landings, but that incorporation of recovered topsoil and burn-pile debris produced an excellent growth substrate. This method, using materials found on site, can prevent a significant loss of land regenerating to productive second-growth forest.
Characterization of an amphimull under Mediterranean evergreen oak forest (Quercus ilex): micromorphological and biodynamic descriptions
Authors: Tagger Simone , Périssol Claude , Criquet Stéven , Aubert Guy , Neville Pierre , Le Petit Jean , Toutain François
Published: March 2008

The relationships between microorganisms and microfauna were studied in an evergreen oak ( Quercus ilex L.) forest floor located in a French Mediterranean region characterized by hot and dry summers. The soil was a brown fersialitic soil with an amphimull, i.e., a mull with a thick litter. A micromorphological study of both litter and organomineral layers was used to observe relationships between white-rot fungi and fauna, such as oribatids, millipedes, enchytraeids, and earthworms. Microbiological properties of the litter and physicochemical properties of the soil were analysed. Enchytraeids comminute the faeces of other animals, whereas earthworms form aggregates. These two opposite actions probably modify aeration and water movements in deeper layers. Brown leaves and animal faeces constitute a nutritional substrate for white-rot fungi and other animals. Inside degraded cells of rootlets and bleached leaves, fungi form calcium oxalate crystals because the absorbent complex is saturated by an excess of calcium. Enzymes such as laccases, manganese peroxidases, cellulases, or xylanases were detected in the litter at significant levels. The high activity values of phosphatases in the litter could reflect a deficiency in available phosphorus. This deficiency could partly explain a low rate of litter degradation and the presence of an OH sublayer.
Multidrug Resistance-Associated Protein–Overexpressing Teniposide-Resistant Human Lymphomas Undergo Apoptosis by a Tubulin-Binding Agent
Authors: Aneja Ritu , Liu Min , Yates Clayton , Gao Jinmin , Dong Xin , Zhou Binfei , Vangapandu Surya N. , Zhou Jun , Joshi Harish C.
Published: March 2008

Several DNA- and microtubule-binding agents are used to manage hematologic malignancies in the clinic. However, drug resistance has been a challenge, perhaps due to a few surviving cancer stem cells. Toxicity is another major impediment to successful chemotherapy, leading to an impoverished quality of life. Here, we show that a semisynthetic nontoxic tubulin-binding agent, 9-bromonoscapine (EM011), effectively inhibits growth and regresses multidrug resistance-associated protein (MRP)-overexpressing teniposide-resistant T-cell lymphoma xenografts and prolongs longevity. As expected, teniposide treatment failed to regress teniposide-resistant xenografts, rather, treated mice suffered tremendous body weight loss. Mechanistically, EM011 displays significant antiproliferative activity, perturbs cell cycle progression by arresting mitosis, and induces apoptosis in teniposide-resistant lymphoblastoid T cells both in vitro and in vivo. EM011-induced apoptosis has a mitochondrially-mediated component, which was attenuated by pretreatment with cyclosporin A. We also observed alterations of apoptosis-regulatory molecules such as inactivation of Bcl2, translocation of BAX to the mitochondrial membrane, cytochrome c release, and activation of downstream apoptotic signaling. EM011 caused DNA degradation as evident by terminal deoxynucleotidyl transferase–mediated dUTP-biotin end labeling staining of the increased concentration of 3′-DNA ends. Furthermore, the apoptotic induction was caspase dependent as shown by cleavage of the caspase substrate, poly(ADP)ribose polymerase. In addition, EM011 treatment caused a suppression of natural survival pathways such as the phosphatidylinositol-3′-kinase/Akt signaling. These preclinical findings suggest that EM011 is an excellent candidate for clinical evaluation. [Cancer Res 2008;68(5):1495–503]
MicroRNA-137 Targets Microphthalmia-Associated Transcription Factor in Melanoma Cell Lines
Authors: Bemis Lynne T. , Chen Robert , Amato Carol M. , Classen Elizabeth H. , Robinson Steven E. , Coffey David G. , Erickson Paul F. , Shellman Yiqun G. , Robinson William A.
Published: March 2008

Micropthalmia-associated transcription factor (MITF) is the master regulator of melanocyte development, survival, and function. Frequent alteration in the expression of MITF is detected in melanoma, but the mechanism(s) underlying the alteration in expression have not been completely determined. In these studies, we have identified microRNA-137 (miR-137) as a regulator of MITF expression. The genomic locus of miR-137 at chromosome 1p22 places it in a region of the human genome previously determined to harbor an allele for melanoma susceptibility. Here, we show that expression of mature miR-137 in melanoma cell lines down-regulates MITF expression. Further, we have identified a 15-bp variable nucleotide tandem repeat located just 5′ to the pre-miR-137 sequence, which alters the processing and function of miR-137 in melanoma cell lines. [Cancer Res 2008;68(5):1362–8]
T-cadherin Supports Angiogenesis and Adiponectin Association with the Vasculature in a Mouse Mammary Tumor Model
Authors: Hebbard Lionel W. , Garlatti Michèle , Young Lawrence J.T. , Cardiff Robert D. , Oshima Robert G. , Ranscht Barbara
Published: March 2008

T-cadherin delineates endothelial, myoepithelial, and ductal epithelial cells in the normal mouse mammary gland, and becomes progressively restricted to the vasculature during mammary tumorigenesis. To test the function of T-cadherin in breast cancer, we inactivated the T-cadherin (Cdh13) gene in mice and evaluated tumor development and pathology after crossing the mutation into the mouse mammary tumor virus (MMTV)-polyoma virus middle T (PyV-mT) transgenic model. We report that T-cadherin deficiency limits mammary tumor vascularization and reduces tumor growth. Tumor transplantation experiments confirm the stromal role of T-cadherin in tumorigenesis. In comparison with wild-type MMTV-PyV-mT controls, T-cadherin–deficient tumors are pathologically advanced and metastasize to the lungs. T-cadherin is a suggested binding partner for high molecular weight forms of the circulating, fat-secreted hormone adiponectin. We discern adiponectin in association with the T-cadherin–positive vasculature in the normal and malignant mammary glands and report that this interaction is lost in the T-cadherin null condition. This work establishes a role for T-cadherin in promoting tumor angiogenesis and raises the possibility that vascular T-cadherin-adiponectin association may contribute to the molecular cross-talk between tumor cells and the stromal compartment in breast cancer. [Cancer Res 2008;68(5):1407–16]
Selective Induction of Expression of a Ligand for the NKG2D Receptor by Proteasome Inhibitors
Authors: Valés-Gómez Mar , Chisholm Susan E. , Cassady-Cain Robin L. , Roda-Navarro Pedro , Reyburn Hugh T.
Published: March 2008

The interaction of the activating receptor NKG2D with its ligands plays an important role in immunosurveillance of tumors and infectious pathogens, but dysregulation of this system may lead to autoimmunity. The expression of NKG2D ligands is induced by cellular “stress.” However, the regulation of expression of these molecules is not well understood. Here, we show that cells treated with proteasome inhibitors can become more susceptible to cytotoxicity mediated by natural killer cells because of the induction of expression of ligands for NKG2D, specifically ULBP2, but not down-regulation of MHC class I. Treatment with proteasome inhibitors led to up-regulation of ULBP2 expression in multiple, but not all, cell lines tested. This increase in expression of ULBP2 at the cell surface correlated with induction of transcription of the ULBP2 gene and synthesis of ULBP2 protein. In contrast, treatment with inhibitors of histone deacetylases led to increased levels of mRNA and protein, for both ULBP2 and MHC class I–related chain A/B molecules. Thus, different types of stress can trigger up-regulated expression of different sets of NKG2D ligands. Proteasome inhibitors are proving to be of significant value in the treatment of hematologic malignancies and these observations may help to better understand the biology of therapy with these compounds. [Cancer Res 2008;68(5):1546–54]
Loss of Keap1 Function Activates Nrf2 and Provides Advantages for Lung Cancer Cell Growth
Authors: Ohta Tsutomu , Iijima Kumiko , Miyamoto Mamiko , Nakahara Izumi , Tanaka Hiroshi , Ohtsuji Makiko , Suzuki Takafumi , Kobayashi Akira , Yokota Jun , Sakiyama Tokuki , Shibata Tatsuhiro , Yamamoto Masayuki , Hirohashi Setsuo
Published: March 2008

Oxidative and electrophilic stresses are sensed by Keap1, which activates Nrf2 to achieve cytoprotection by regulating the expression of drug-metabolizing and antioxidative stress enzymes/proteins. Because oxidative and electrophilic stresses cause many diseases, including cancer, we hypothesized that an abnormality in the Nrf2-Keap1 system may facilitate the growth of cancer cells. We sequenced the KEAP1 gene of 65 Japanese patients with lung cancer and identified five nonsynonymous somatic mutations at a frequency of 8%. We also identified two nonsynonymous somatic KEAP1 gene mutations and two lung cancer cell lines expressing KEAP1 at reduced levels. In lung cancer cells, low Keap1 activity (due to mutations or low-level expression) led to nuclear localization and constitutive activation of Nrf2. The latter resulted in constitutive expression of cytoprotective genes encoding multidrug resistance pumps, phase II detoxifying enzymes, and antioxidative stress enzymes/proteins. Up-regulation of these target genes in lung cancer cells led to cisplatin resistance. Nrf2 activation also stimulated growth of lung cancer–derived cell lines expressing KEAP1 at low levels and in mutant cell lines and in Keap1-null mouse embryonic fibroblasts under homeostatic conditions. Thus, inhibition of NRF2 may provide new therapeutic approaches in lung cancers with activation of Nrf2. [Cancer Res 2008;68(5):1303–9]
Discovery and Validation of Protein Abundance Differences between Follicular Thyroid Neoplasms
Authors: Netea-Maier Romana T. , Hunsucker Stephen W. , Hoevenaars Brigiet M. , Helmke Steve M. , Slootweg Pieter J. , Hermus Ad R. , Haugen Bryan R. , Duncan Mark W.
Published: March 2008

Distinguishing between benign follicular thyroid adenoma (FTA) and malignant follicular thyroid carcinoma (FTC) by cytologic features alone is not possible. Molecular markers may aid distinguishing FTA from FTC in patients with indeterminate cytology. The aim of this study is to define protein abundance differences between FTC from FTA through a discovery (proteomics) and validation (immunohistochemistry) approach. Difference gel electrophoresis (DIGE) and peptide mass fingerprinting were performed on protein extracts from five patients with FTC and compared with six patients with FTA. Individual gel comparisons (i.e., each FTC extract versus FTA pool) were also performed for the five FTC patients. Immunohistochemical validation studies were performed on three of the identified proteins. Based on DIGE images, 680 protein spots were matched on individual gels. Of these, 102 spots showed statistically significant differences in abundance between FTC and FTA in the individual gel analyses and were therefore studied further. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify 54 of these protein spots. Three candidates involved in protein folding (heat shock protein gp96, protein disulfide isomerase A3, and calreticulin) were studied by immunohistochemistry. Moderate calreticulin immunohistochemical staining was the best single marker with a high negative predictive value (88%); combining all three markers (any marker less than moderate staining) had the best positive predictive value (75%) while still retaining a good negative predictive value (68%). With DIGE, we identified 54 proteins differentially abundant between FTC and FTA. Three of these were validated by immunohistochemistry. These findings provide further insights into the diagnosis, prognosis, and pathophysiology of follicular-derived thyroid neoplasms. [Cancer Res 2008;68(5):1572–80]
An Actin-Binding Protein Girdin Regulates the Motility of Breast Cancer Cells
Authors: Jiang Ping , Enomoto Atsushi , Jijiwa Mayumi , Kato Takuya , Hasegawa Taisaku , Ishida Maki , Sato Tomoko , Asai Naoya , Murakumo Yoshiki , Takahashi Masahide
Published: March 2008

Girdin (girders of actin filaments) is a novel actin-binding Akt substrate that plays an important role in actin organization and Akt-dependent cell motility in fibroblasts. Here, we find that Girdin is expressed in a variety of cancer cell lines, including the breast cancer cell line MDA-MB-231, and is phosphorylated by the stimulation of insulin-like growth factor (IGF-I). In vitro migration and invasion assays showed that Girdin is required for the IGF-I–dependent cell movement of MDA-MB-231 cells. Short hairpin interfering RNA directed against Girdin markedly inhibited the metastasis of s.c. transplanted MDA-MB-231 cells in nude mice. In addition, Girdin is highly expressed in a variety of human malignant tissues, including breast, colon, lung, and uterine cervical carcinomas. These findings highlight the important role of Girdin in tumor progression in which the Akt signaling pathway is aberrantly activated. [Cancer Res 2008;68(5):1310–8]
Tempol Protects against Oxidative Damage and Delays Epithelial Tumor Onset in Fanconi Anemia Mice
Authors: Zhang Qing-Shuo , Eaton Laura , Snyder Eric R. , Houghtaling Scott , Mitchell James B. , Finegold Milton , Van Waes Carter , Grompe Markus
Published: March 2008

Fanconi anemia (FA) is a genetic disorder characterized by congenital abnormalities, bone marrow failure, and marked cancer susceptibility. FA patients have an elevated risk of developing hematologic malignancies and solid tumors. Using Fancd2−/− knockout mice as a model of FA, we examined the potential of tempol, a nitroxide antioxidant and a superoxide dismutase mimetic, as a tumor-delaying agent for solid tumors. Dietary tempol increased the mean tumor-free survival time of Fancd2−/− Trp53+/− mice by 27% (P < 0.01), from 308 to 390 days, without changing the overall tumor spectrum. More strikingly, tempol delayed the onset of epithelial tumors and increased the mean epithelial tumor-free survival time by 38% (P < 0.0001), from 312 to 432 days, in Fancd2−/− Trp53+/− mice. These results show that tempol can significantly delay tumor formation in Fancd2−/− Trp53+/− mice. Furthermore, tempol treatment did not adversely affect the repopulating ability of FA hematopoietic stem cells. The reduction in oxidative DNA damage in tempol-treated FA fibroblasts and mice suggests that its tumor-delaying function may be attributed to its antioxidant activity. [Cancer Res 2008;68(5):1601–8]
TGFBR1*6A Enhances the Migration and Invasion of MCF-7 Breast Cancer Cells through RhoA Activation
Authors: Rosman Diana S. , Phukan Sharbani , Huang Chiang-Ching , Pasche Boris
Published: March 2008

TGFBR1*6A is a common hypomorphic variant of the type 1 transforming growth factor β receptor (TGFBR1), which has been associated with increased cancer risk in some studies. Although TGFBR1*6A is capable of switching TGF-β growth-inhibitory signals into growth-stimulatory signals when stably transfected into MCF-7 breast cancer cells, the biological effects of TGFBR1*6A are largely unknown. To broadly explore the potential oncogenic properties of TGFBR1*6A, we assessed its effects on NIH-3T3 cells as well as its effect on the migration and invasion of MCF-7 cells. We found that TGFBR1*6A has decreased oncogenic properties compared with TGFBR1. However, TGFBR1*6A significantly enhances MCF-7 cell migration and invasion in a TGF-β signaling–independent manner. Gene expression profiling studies identified two down-regulated genes involved in cell migration and invasion: ARHGAP5, encoding ARHGAP5, and FN1, encoding fibronectin-1 (FN1). ARHGAP5 and FN1 expression was similarly down-regulated in MCF-7 cells stably transfected with a kinase-inactivated TGFBR1*6A construct. Functional assays show that TGFBR1*6A-mediated decreased ARHGAP5 expression is associated with higher RhoA activation, a crucial mediator of cell migration. Extracellular signal-regulated kinase (ERK) activation is also higher in cells that harbor the TGFBR1*6A allele. We conclude that TGFBR1*6A is not an oncogene but enhances MCF-7 cell migration and invasion through RhoA and ERK pathway activation and down-regulates two crucial mediators of this phenotype. These results provide the first evidence that TGFBR1*6A may contribute to cancer progression in a TGF-β signaling–independent manner. [Cancer Res 2008;68(5):1319–28]
Synergistic Effect of Oncogenic RET and Loss of p18 on Medullary Thyroid Carcinoma Development
Authors: van Veelen Wendy , van Gasteren Carola J.R. , Acton Dennis S. , Franklin David S. , Berger Ruud , Lips Cornelis J.M. , Höppener Jo W.M.
Published: March 2008

Activating mutations in the RET proto-oncogene are associated with both familial and sporadic medullary thyroid carcinoma (MTC) development; however, the genetic mechanisms underlying MTC tumorigenesis remain largely unknown. Recently, we have identified somatic inactivating mutations in the cell cycle inhibitor gene P18 in human MTC, which coincided with activating RET mutations, suggesting a role for loss of P18 in combination with oncogenic RET in the multistep process of MTC development. Therefore, we crossed transgenic mice expressing oncogenic RET (RET2B) with mice lacking p18 (and p27, another cell cycle inhibitor) and monitored MTC development. RET2B;p18+/− mice and RET2B;p18−/− mice developed MTC with a highly increased incidence compared with their corresponding single mutant littermates. In addition, expression of oncogenic RET causes an earlier age of onset and larger MTCs in p18−/−;p27+/− mice. In a subset of MTCs of RET2B;p18+/−(;p27+/−) mice, p18Ink4c expression was completely lost. This loss of p18Ink4c expression correlated with higher proliferation rates as well as with larger MTCs, indicating that loss of p18 in combination with oncogenic RET not only increases the risk for MTC development but also enhances MTC progression. Our data strongly indicate that oncogenic RET and loss of p18 cooperate in the multistep tumorigenesis of MTC. [Cancer Res 2008;68(5):1329–37]
Preclinical Cancer Therapy in a Mouse Model of Neurofibromatosis-1 Optic Glioma
Authors: Hegedus Balazs , Banerjee Debasish , Yeh Tu-Hsueh , Rothermich Stefan , Perry Arie , Rubin Joshua B. , Garbow Joel R. , Gutmann David H.
Published: March 2008

Mouse models of human cancers afford unique opportunities to evaluate novel therapies in preclinical trials. For this purpose, we analyzed three genetically engineered mouse (GEM) models of low-grade glioma resulting from either inactivation of the neurofibromatosis-1 (Nf1) tumor suppressor gene or constitutive activation of KRas in glial cells. Based on tumor proliferation, location, and penetrance, we selected one of these Nf1 GEM models for preclinical drug evaluation. After detection of an optic glioma by manganese-enhanced magnetic resonance imaging, we randomized mice to either treatment or control groups. We first validated the Nf1 optic glioma model using conventional single-agent chemotherapy (temozolomide) currently used for children with low-grade glioma and showed that treatment resulted in decreased proliferation and increased apoptosis of tumor cells in vivo as well as reduced tumor volume. Because neurofibromin negatively regulates mammalian target of rapamycin (mTOR) signaling, we showed that pharmacologic mTOR inhibition in vivo led to decreased tumor cell proliferation in a dose-dependent fashion associated with a decrease in tumor volume. Interestingly, no additive effect of combined rapamycin and temozolomide treatment was observed. Lastly, to determine the effect of these therapies on the normal brain, we showed that treatments that affect tumor cell proliferation or apoptosis did not have a significant effect on the proliferation of progenitor cells within brain germinal zones. Collectively, these findings suggest that this Nf1 optic glioma model may be a potential preclinical benchmark for identifying novel therapies that have a high likelihood of success in human clinical trials. [Cancer Res 2008;68(5):1520–8]
Molecular Basis of Nuclear Factor-κB Activation by Astrocyte Elevated Gene-1
Authors: Sarkar Devanand , Park Eun Sook , Emdad Luni , Lee Seok-Geun , Su Zao-zhong , Fisher Paul B.
Published: March 2008

Malignant glioma is a consistently fatal brain cancer. The tumor invades the surrounding tissue, limiting complete surgical removal and thereby initiating recurrence. Identifying molecules critical for glioma invasion is essential to develop targeted, effective therapies. The expression of astrocyte elevated gene-1 (AEG-1) increases in malignant glioma and AEG-1 regulates in vitro invasion and migration of malignant glioma cells by activating the nuclear factor-κB (NF-κB) signaling pathway. The present studies elucidate the domains of AEG-1 important for mediating its function. Serial NH2-terminal and COOH-terminal deletion mutants were constructed and functional analysis revealed that the NH2-terminal 71 amino acids were essential for invasion, migration, and NF-κB–activating properties of AEG-1. The p65-interaction domain was identified between amino acids 101 to 205, indicating that p65 interaction alone is not sufficient to mediate AEG-1 function. Coimmunoprecipitation assays revealed that AEG-1 interacts with cyclic AMP-responsive element binding protein–binding protein (CBP), indicating that it might act as a bridging factor between NF-κB, CBP, and the basal transcription machinery. Chromatin immunoprecipitation assays showed that AEG-1 is associated with the NF-κB binding element in the interleukin-8 promoter. Thus, AEG-1 might function as a coactivator for NF-κB, consequently augmenting expression of genes necessary for invasion of glioma cells. In these contexts, AEG-1 represents a viable potential target for the therapy of malignant glioma. [Cancer Res 2008;68(5):1478–84]
Enhanced Expression of RAD51 Associating Protein-1 Is Involved in the Growth of Intrahepatic Cholangiocarcinoma Cells
Authors: Obama Kazutaka , Satoh Seiji , Hamamoto Ryuji , Sakai Yoshiharu , Nakamura Yusuke , Furukawa Yoichi
Published: March 2008

Purpose: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary cancer in the liver, and its incidence is increasing in developed countries.

Experimental Design: To discover novel molecular targets for the diagnosis and treatment of ICCs, we earlier analyzed expression profiles of 25 ICCs using a cDNA microarray containing 27,648 genes. In this study, we focused on the RAD51 associating protein-1 (RAD51AP1) gene because its expression was frequently elevated in our microarray data.

Results: Quantitative PCR confirmed that RAD51AP1 expression was elevated in the great majority of the ICCs examined. Immunohistochemical analysis with anti-RAD51AP1 antibody further corroborated its accumulation in 14 of 23 ICC tissues (61%). Notably, suppression of RAD51AP1 by short interfering RNA resulted in growth suppression of cholangiocarcinoma cells, suggesting its involvement in the development and/or progression of ICC. Because RAD51AP1 interacts with RAD51, a molecule involved in DNA repair, we investigated whether RAD51AP1 is implicated in DNA strand breaks using γ-irradiation. As a result, γ-irradiation augmented RAD51AP1 protein expression and brought a focus formation in the nuclei, where accumulated RAD51AP1 colocalized with phosphorylated histone 2AX (γ-H2AX) and RAD51. These data suggest that RAD51AP1 may play a role in cell proliferation as well as DNA repair.

Conclusion: Our findings may contribute to the better understanding of cholangiocarcinogenesis and open a new avenue to the development of novel therapeutic and/or diagnostic approach to this type of tumor.
Therapeutic Nanoparticles for Drug Delivery in Cancer
Authors: Cho Kwangjae , Wang Xu , Nie Shuming , Chen Zhuo (Georgia) , Shin Dong M.
Published: March 2008

Cancer nanotherapeutics are rapidly progressing and are being implemented to solve several limitations of conventional drug delivery systems such as nonspecific biodistribution and targeting, lack of water solubility, poor oral bioavailability, and low therapeutic indices. To improve the biodistribution of cancer drugs, nanoparticles have been designed for optimal size and surface characteristics to increase their circulation time in the bloodstream. They are also able to carry their loaded active drugs to cancer cells by selectively using the unique pathophysiology of tumors, such as their enhanced permeability and retention effect and the tumor microenvironment. In addition to this passive targeting mechanism, active targeting strategies using ligands or antibodies directed against selected tumor targets amplify the specificity of these therapeutic nanoparticles. Drug resistance, another obstacle that impedes the efficacy of both molecularly targeted and conventional chemotherapeutic agents, might also be overcome, or at least reduced, using nanoparticles. Nanoparticles have the ability to accumulate in cells without being recognized by P-glycoprotein, one of the main mediators of multidrug resistance, resulting in the increased intracellular concentration of drugs. Multifunctional and multiplex nanoparticles are now being actively investigated and are on the horizon as the next generation of nanoparticles, facilitating personalized and tailored cancer treatment.
Sensitivity to Epidermal Growth Factor Receptor Inhibitor Requires E-Cadherin Expression in Urothelial Carcinoma Cells
Authors: Black Peter C. , Brown Gordon A. , Inamoto Teruo , Shrader Marissa , Arora Ameeta , Siefker-Radtke Arlene O. , Adam Liana , Theodorescu Dan , Wu Xifeng , Munsell Mark F. , Bar-Eli Menashe , McConkey David J. , Dinney Colin P.N.
Published: March 2008

Purpose: Epidermal growth factor receptor (EGFR) is an attractive target for the treatment of urothelial carcinoma, but a clinical response can be expected in only a small proportion of patients. The aim of this study was to define molecular markers of response to cetuximab therapy in a panel of urothelial carcinoma cell lines.

Experimental Design: Eleven cell lines were investigated for antiproliferative response to cetuximab based on [3H]thymidine incorporation. A variety of markers, including EGFR expression, phosphorylation, and gene amplification, as well as the expression of other growth factor receptors, their ligands, and markers of epithelial-to-mesenchymal transition were investigated. Cohen's κ statistic was used to estimate the agreement between response and expression of these markers. E-cadherin was silenced by small interfering RNA in two sensitive cell lines, and the effect on the response to cetuximab was measured.

Results: We were able to identify a panel of relevant markers pertaining especially to alternate growth factor receptor expression and epithelial-to-mesenchymal transition that predicted response to cetuximab. The data suggested that expression of intact HER-4 (κ, 1.00; P = 0.008), E-cadherin (κ, 0.81; P = 0.015), and β-catenin (κ, 0.81; P = 0.015) and loss of expression of platelet-derived growth factor receptor β (κ, 0.57; P = 0.167) were associated with response to cetuximab therapy. Silencing E-cadherin in two sensitive cell lines reduced responsiveness to cetuximab in both (P < 0.001).

Conclusions: A panel of predictive markers for cetuximab response has been established in vitro and is currently being evaluated in a prospective clinical trial of neoadjuvant EGFR-targeted therapy. Most importantly, E-cadherin seems to play a central role in modulation of EGFR response in urothelial carcinoma.
Basal-Like Breast Cancer Defined by Five Biomarkers Has Superior Prognostic Value than Triple-Negative Phenotype
Authors: Cheang Maggie C.U. , Voduc David , Bajdik Chris , Leung Samuel , McKinney Steven , Chia Stephen K. , Perou Charles M. , Nielsen Torsten O.
Published: March 2008

Purpose: Basal-like breast cancer is associated with high grade, poor prognosis, and younger patient age. Clinically, a triple-negative phenotype definition [estrogen receptor, progesterone receptor, and human epidermal growth factor receptor (HER)-2, all negative] is commonly used to identify such cases. EGFR and cytokeratin 5/6 are readily available positive markers of basal-like breast cancer applicable to standard pathology specimens. This study directly compares the prognostic significance between three- and five-biomarker surrogate panels to define intrinsic breast cancer subtypes, using a large clinically annotated series of breast tumors.

Experimental Design: Four thousand forty-six invasive breast cancers were assembled into tissue microarrays. All had staging, pathology, treatment, and outcome information; median follow-up was 12.5 years. Cox regression analyses and likelihood ratio tests compared the prognostic significance for breast cancer death-specific survival (BCSS) of the two immunohistochemical panels.

Results: Among 3,744 interpretable cases, 17% were basal using the triple-negative definition (10-year BCSS, 6 7%) and 9% were basal using the five-marker method (10-year BCSS, 62%). Likelihood ratio tests of multivariable Cox models including standard clinical variables show that the five-marker panel is significantly more prognostic than the three-marker panel. The poor prognosis of triple-negative phenotype is conferred almost entirely by those tumors positive for basal markers. Among triple-negative patients treated with adjuvant anthracycline-based chemotherapy, the additional positive basal markers identified a cohort of patients with significantly worse outcome.

Conclusions: The expanded surrogate immunopanel of estrogen receptor, progesterone receptor, human HER-2, EGFR, and cytokeratin 5/6 provides a more specific definition of basal-like breast cancer that better predicts breast cancer survival.
Transcription Factor Signal Transducer and Activator of Transcription 5 Promotes Growth of Human Prostate Cancer Cells In vivo
Authors: Dagvadorj Ayush , Kirken Robert A. , Leiby Benjamin , Karras James , Nevalainen Marja T.
Published: March 2008

Purpose: Signal transducer and activator of transcription 5a/b (Stat5a/b) is the key mediator of prolactin effects in prostate cancer cells via activation of Janus-activated kinase 2. Prolactin is a locally produced growth factor in human prostate cancer. Prolactin protein expression and constitutive activation of Stat5a/b are associated with high histologic grade of clinical prostate cancer. Moreover, activation of Stat5a/b in primary prostate cancer predicts early disease recurrence. Here, we inhibited Stat5a/b by several different methodologic approaches. Our goal was to establish a proof of principle that Stat5a/b is critical for prostate cancer cell viability in vitro and for prostate tumor growth in vivo.

Experimental Design: We inhibited Stat5a/b protein expression by antisense oligonucleotides or RNA interference and transcriptional activity of Stat5a/b by adenoviral expression of a dominant-negative mutant of Stat5a/b in prostate cancer cells in culture. Moreover, Stat5a/b activity was suppressed in human prostate cancer xenograft tumors in nude mice. Stat5a/b regulation of Bcl-XL and cyclin D1 protein levels was shown by antisense suppression of Stat5a/b protein expression followed by Western blotting.

Results and Conclusions: We show here that inhibition of Stat5a/b by antisense oligonucleotides, RNA interference, or adenoviral expression of dominant-negative Stat5a/b effectively kills prostate cancer cells. Moreover, we show that Stat5a/b is critical for human prostate cancer xenograft growth in nude mice. The effects of Stat5a/b on the viability of prostate cancer cells involve Stat5a/b regulation of Bcl-XL and cyclin D1 protein levels but not the expression or activation of Stat3. This work establishes Stat5a/b as a therapeutic target protein for prostate cancer. Pharmacologic inhibition of Stat5a/b in prostate cancer can be achieved by small-molecule inhibitors of transactivation, dimerization, or DNA binding of Stat5a/b.
Evaluation of Colon Cancer–Specific Antigen 2 as a Potential Serum Marker for Colorectal Cancer
Authors: Leman Eddy S. , Schoen Robert E. , Magheli Ahmed , Sokoll Lori J. , Chan Daniel W. , Getzenberg Robert H.
Published: March 2008

Purpose: A blood test to detect colon cancer at a preventable stage would represent a major advancement. We have previously identified colon cancer–specific markers using focused proteomics analysis of nuclear structural proteins. Two of these markers, colon cancer–specific antigen (CCSA)-3 and CCSA-4, have been developed into blood-based markers that are able to distinguish individuals with colorectal cancer from those without. CCSA-2 is a distinct novel colon cancer marker identified using focused proteomics.

Experimental Design: Using an indirect ELISA on serum samples obtained from two institutions, we evaluated CCSA-2 as a serum-based colon cancer marker. A total of 111 serum samples from individuals who underwent colonoscopy and were subsequently diagnosed as either being normal or having hyperplastic polyps, nonadvanced adenomas, advanced adenomas, and colorectal cancer were evaluated. A diverse control population that consisted of 125 serum samples was also included in this study.

Results: Receiver operating characteristic analyses were used to measure the sensitivity and specificity of CCSA-2. CCSA-2 at a cutoff of 10.8 μg/mL has overall specificity of 78.4% [95% confidence interval (95% CI), 67.3-87.1%] and sensitivity of 97.3% (95% CI, 85.8-99.5%) in separating individuals with advanced adenomas and colorectal cancer from normal, hyperplastic, and nonadvanced adenoma populations. The receiver operating characteristic curve for CCSA-2 has an area under the curve of 0.90 (95% CI, 0.83-0.95).

Conclusion: Our initial study shows that CCSA-2 is a potential serum-based marker for colon cancer detection with high sensitivity and specificity.
The Severe Cognitive Impairment Rating Scale – An Instrument for the Assessment of Cognition in Moderate to Severe Dementia Patients
Authors: Choe J.Y. , Youn J.C. , Park J.H. , Park I.S. , Jeong J.W. , Lee W.H. , Lee S.B. , Park Y.S. , Jhoo J.H. , Lee D.Y. , Kim K.W.
Published: March 2008

Background/Aims: This study aimed to develop a brief, reliable and valid test for cognitive function of severely demented patients. Methods: We constructed the Severe Cognitive Impairment Rating Scale, which consisted of 11 items covering memory, language, visuospatial function, frontal function and orientation, and investigated its reliability and validity on 267 subjects [normal: 65, very mild Alzheimer’s disease (AD): 42, mild AD: 58, moderate AD: 36, severe AD: 44, profound AD: 22]. Results: The internal consistency obtained by Cronbach’s coefficient α was 0.93. The interrater reliability and test-retest reliability in the moderately to severely impaired subjects with an MMSE score of ≤14 was 0.99 (p < 0.001) and 0.90 (p < 0.001), respectively. It showed significant correlation with Severe MMSE (r = 0.96, p < 0.01), MMSE (r = 0.86, p < 0.01) and Clinical Dementia Rating (r = –0.83, p < 0.01). It was robust to both the floor effect in the severe/profound stage of AD and the ceiling effect in the mild/moderate stage of AD. Exploratory factor analysis yielded 2 factors (automatic informational processing and controlled informational processing) accounting for 73.5% of the total variance. Conclusions: The Severe Cognitive Impairment Rating Scaleis a valid and reliable test for evaluating the cognitive function of advanced AD patients.
The “Book of Will” of Petrus Woskan (1680-1751): Some Insights into the Global Commercial Network of the Armenians in the Indian Ocean
Authors: Bhattacharya Bhaswati
Published: March 2008

This paper argues that “insider” material, preserved by the Armenians in the archives of the cathedral in New Julfa presents, by virtue of its logic, a one-sided, consensual picture. “Outsider” material contests this view and highlights the underlying conflicts of interest that even existed among the core members of the network. The picture that emerges from this study points to a more dynamic, complex structure in which the multiple interests of individual members of family firms, kin, and community members in different capacities were grafted into one. Loopholes in the system were visible as early as the 1720s, long before the center at New Julfa collapsed. Les archives arméniennes ont été conservées dans la cathédrale de la Nouvelle Julfa. Ces sources “internes” présentent, selon nous, une image partiale de consensus. L'examen minutieux des sources “externes” par contre fait ressortir des conflits d'intérêts même entre les grands négociants du réseau arménien. Ces documents-là attestent des structures complexes plus dynamiques. Dans ce cadre s'inscrivaient les multiples intérêts des membres individuels de la firma familiale, des parents, et des membres de la communauté. Et les faiblesses du système sont déjà perceptibles dans les années vingt du XVIIème siècle, bien avant l'effondrement de son centre à la Nouvelle Julfa.
Teaching Concepts Versus Facts in Developmental Biology
Authors: Wood William B.
Published: March 2008

Note from the Editor

Points of View (POV) address issues faced by many people within the life science education community. CBE—Life Sciences Education (CBE-LSE) publishes the POV Feature to present two or more opinions published side-by-side on a common topic. We consider POVs to be “Op-Ed” pieces designed to stimulate thought and dialogue on significant educational issues. They are not meant to be exhaustive treatments of a subject. In this issue, we ask the question, “What are key concepts in developmental biology?” We present three POVs. The first is by CBE-LSE Editor-in-Chief, William Wood, and it is in part based on his experience teaching developmental biology to undergraduates at the University of Colorado, Boulder, including his collaborative experiments in the classroom with Jennifer Knight, the first results of which have been published in CBE-LSE ( Knight and Wood, 2005 ). The second, a partially tongue-in-cheek list of key concepts to convey to students about embryonic development, is by Scott Gilbert (Swarthmore College), author of the leading textbook worldwide for teaching developmental biology, Developmental Biology, 8th ed. (Sinauer Associates, Inc.). The third is by Jeff Hardin (University of Wisconsin–Madison), who has produced Web-based educational materials for teaching developmental biology that are used nationally and internationally for conveying dynamic events during early development (see the WWW feature in this issue by Stark for more details), and who deals with the vexing problem of trying to convey the essential four-dimensional nature of embryonic development to introductory students.
Hypokalaemic paralysis precipitated by distal renal tubular acidosis secondary to Sjögren's syndrome
Authors: Comer D M , Droogan A G , Young I S , Maxwell A P
Published: March 2008

A 43-year-old woman presented with a sudden onset of hypokalaemic paralysis requiring intubation and ventilatory support. Subsequent biochemical and clinical assessments established a diagnosis of distal renal tubular acidosis (RTA) in association with underlying Sjögren's syndrome as the aetiology of her profound hypokalaemia. Distal RTA is rare, but Sjögren's syndrome is one of the more common causes in adults and should be considered in the differential diagnosis of patients who present with hypokalaemic muscular paralysis.
Online extraction of 5-hydroxyindole acetic acid from urine for analysis by liquid chromatography–tandem mass spectrometry
Authors: Perry Heather , Keevil Brian
Published: March 2008

Background

Urinary 5-hydroxyindole acetic acid (5-HIAA) is a useful marker for the turnover of tryptophan metabolites in the diagnosis and monitoring of carcinoid tumours and the carcinoid syndrome. We have developed a simple and cost-effective assay for urinary 5-HIAA using liquid chromatography–tandem mass spectrometry (LC–MS/MS) incorporating an online sample clean-up process to replace a liquid chromatography electrochemical (LC-EC) technique.

Methods

Acidified urine was serially diluted in ammonium acetate buffer followed by ammonium acetate buffer enriched with 5-hydroxyindole-3-acetic-2,2-D2 acid internal standard. A 2.1 × 10 mm C18 column was used for primary online clean-up and eluted with 100% methanolic mobile phase onto a second dC18 Atlantis 2.1 × 20 mm column. Analytes were detected by mass spectrometry using transitions 192.1 > 146.3 and 194.1 > 148.0 for 5-HIAA and deuterated analyte, respectively.

Results

Run time was 3 min with 5-HIAA eluting at 1.37 min. The inter and intra-assay imprecision and accuracies of the three levels of inhouse quality control (QC) (30, 300 and 600 μmol/L) were acceptable with coefficient of variations (CVs) and deviation from target values <12% ( n = 15). The average recovery of 5-HIAA spiked into urine was 93.7% with no ion suppression observed. The limit of detection was 2.8 and lower limit of quantification 4.0 μmol/L. Passing–Bablok regression of LC-EC with LC–MS/MS results showed good agreement between the methods, the relationship described as LC–MS/MS = 1.01(LC–EC)–1.22. No systematic or proportional biases were observed over the working range of the method. The assay was linear to at least 2000 μmol/L.

Conclusions

We have developed a robust method offering a more than six-fold improvement in linearity compared to the existing LC–EC method.
High-resolution melting analysis for detection of familial ligand-defective apolipoprotein B-100 mutations
Authors: Liyanage Khemanganee E , Hooper Amanda J , Defesche Joep C , Burnett John R , van Bockxmeer Frank M
Published: March 2008

Background

Familial ligand-defective apolipoprotein B-100 (FDB) is characterized by elevated plasma concentrations of LDL-cholesterol and apolipoprotein (apo) B, normal triglyceride and HDL-cholesterol levels, the presence of tendon xanthomas, and premature coronary artery disease. FDB cannot be clinically distinguished from heterozygous LDL-receptor-defective familial hypercholesterolaemia (FH) without genetic testing.

Methods

Amplicons in exon 26 and exon 29 of the APOB gene were screened for established genetic variants including mutations and polymorphisms using high-resolution melting analysis. Six novel variants associated with FDB in hypercholesterolaemic Dutch patients (S3476L, S3488G, Y3533C, T3540M, I4350T, G4368D) were also studied.

Results

All positive controls, a total of 10 mutations in exon 26 and four mutations in exon 29, were readily detectable by melting curve analysis. In addition, a patient previously not known to be heterozygous for the H3543Y mutation was identified in a screen of hypercholesterolaemic subjects. The method was validated by comparison of high-resolution melting analysis with DNA sequence data in a ‘blinded’ manner in 35 consecutive patients attending a lipid disorders clinic. These patients were classified as ‘definite FH’ by the Dutch Lipid Clinic Network criteria. Five patients were found to be heterozygous for the R3500Q and one for H3543Y.

Conclusions

We have established a novel, robust method of FDB mutation detection using high-resolution melting analysis in conjunction with DNA sequencing. Compared with existing methods it is not only more cost-effective, but is also capable of detecting new sequence changes and will have importance in cascade screening of affected subjects.
HDL-cholesterol modulation and its impact on the management of cardiovascular risk
Authors: Ferns Gordon , Keti Vuyolethu
Published: March 2008

There is strong epidemiological evidence that HDL-cholesterol (HDL-C) is an independent cardiovascular risk factor. A low HDL-C is a common finding in the general population, and is a feature of metabolic syndrome, the prevalence of which is increasing globally. The importance of HDL-C as a coronary risk factor is generally accepted, and has been incorporated into most of the commonly applied risk engines. There are several causes of a low HDL-C, including rare genetic abnormalities, and more common conditions associated with lifestyle and drug therapy. Not all of these are associated with an increased risk of coronary disease. A better understanding of HDL metabolism and the mechanisms by which it may exert its protective effects has revealed why this may be the case. New therapeutic targets have also been identified that may allow HDL-C to be raised in patients with low levels. However, good clinical endpoint studies are still required to enable the setting of absolute target values for HDL-C in patients at high cardiovascular risk.
Adolescents' reasons for using a microbicide-like product over time
Authors: Short Mary B , Succop Paul A , Rupp Richard , Rosenthal Susan L
Published: March 2008

Summary

This study examined the reasons that adolescent girls used a product over time. Adolescent girls ( n = 208; 14–21 years) participated in a six-month study in which they completed three face-to-face interviews and 24-weekly phone call interviews. Participants were given microbicide-like products (vaginal lubricants) and encouraged to use them with condoms when they had intercourse. Most of the reasons for use were endorsed by most of the adolescent girls, at some time during the study. Over time, the adolescents were significantly more likely to report the following reasons for use: did not think the product would leak out ( z = 2.49; P < 0.01), the product felt comfortable ( z = 2.41; P < 0.02) and the product was not messy to use ( z = 3.11; P < 0.01). Although it appears that adolescents are interested in microbicides, they may worry about certain characteristics of the product. It will be important to develop anticipatory guidance that focuses on the experience of using the product, making sure adolescent girls continue to use over time.