bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of genetic knockout of Pept2 on the in vivo disposition of endogenous and exogenous carnosine in wild-type and Pept2 null mice
Authors: Kamal Mohamed A. , Jiang Huidi , Hu Yongjun , Keep Richard. F. , Smith David E.
Published: February 2009

Carnosine (β-alanyl-l-histidine), an endogenous dipeptide substrate of the proton-coupled oligopeptide transporter PEPT2, plays an important role in many physiological processes. This study examined the effect of PEPT2 on the disposition of endogenous and exogenous carnosine in wild-type and Pept2 null mice. After exogenous dosing of [3H]carnosine (1 nmol/g iv bolus), a marked increase was observed in its systemic clearance in Pept2 null mice (0.50 vs. 0.29 ml/min), resulting in a decreased systemic exposure of dipeptide (area under the curve = 43.7 vs. 73.0 min×μM). Carnosine uptake was substantially reduced in the kidney of Pept2 null mice, and renal clearance increased 18-fold in this genotype (206 vs. 11.5 μl/min). Fractional reabsorption of carnosine in Pept2 null mice was only one-fifth that in wild-type animals (0.20 vs. 0.94). PEPT2 also had a substantial impact in brain where the cerebrospinal fluid (CSF)-to-plasma concentration ratio of carnosine was eightfold greater in Pept2 null mice (0.70 vs. 0.08). With respect to endogenous carnosine levels, significant reductions were observed in Pept2 null compared with wild-type mice for choroid plexus (0.026 vs. 0.20 mmol/kg), olfactory bulb (1.12 vs. 1.79 mmol/kg), and spleen (0.019 vs. 0.029 mmol/kg). In contrast, carnosine levels in the skeletal muscle of Pept2 null mice were significantly increased (1.70 vs. 1.14 mmol/kg), and no differences were observed between genotypes for endogenous carnosine levels in plasma and CSF. These results demonstrate that PEPT2 significantly modulates the disposition of exogenous carnosine. However, endogenous carnosine levels may be under homeostatic control to maintain systemic and central concentrations under physiological in vivo conditions.
GABAAergic and Glycinergic Inhibition in the Phrenic Nucleus Organizes and Couples Fast Oscillations in Motor Output
Authors: Marchenko Vitaliy , Rogers Robert F.
Published: February 2009

One of the characteristics of respiratory motor output is the presence of fast synchronous oscillations, at rates far exceeding the basic breathing rhythm, within a given functional population. However, the mechanisms responsible for organizing phrenic output into two dominant bands in vivo, medium (MFO)- and high (HFO)-frequency oscillations, have yet to be elucidated. We hypothesize that GABAAergic and glycinergic inhibition within the phrenic motor nucleus underlies the specific organization of these oscillations. To test this, the phrenic nuclei (C4) of 14 unanesthetized, decerebrate adult male Sprague-Dawley rats were microinjected unilaterally with either 4 mM strychnine ( n = 7) or GABAzine ( n = 7) to block glycine or GABAA receptors, respectively. Application of GABAzine caused an increase in overall phrenic amplitude during all three phases of respiration (inspiration, postinspiration, and expiration), while the increases caused by strychnine were most pronounced during postinspiration. Neither antagonist produced changes in inspiratory duration or respiratory rate. Power spectral analysis of inspiratory phrenic bursts showed that blockade of inhibition caused significant reduction in the relative power of MFO (GABAA and glycine receptors) and HFO (GABAA receptors only). In addition, analysis of the coherence between the firing of the ipsi- and contralateral phrenic nerves revealed that HFO coupling was significantly reduced by both antagonists and that of MFO was significantly reduced only by strychnine. We conclude that both GABAA and glycine receptors play critical roles in the organization of fast oscillations into MFO and HFO bands in the phrenic nerve, as well as in their bilateral coupling.
Muscle genome-wide expression profiling during disease evolution in mdx mice
Authors: Marotta Mario , Ruiz-Roig Claudia , Sarria Yaris , Peiro Jose Luis , Nuñez Fatima , Ceron Julian , Munell Francina , Roig-Quilis Manuel
Published: February 2009

Mdx mice show a milder phenotype than Duchenne patients despite bearing an analogous genetic defect. Our aim was to sort out genes, differentially expressed during the evolution of skeletal muscle mdx mouse disease, to elucidate the mechanisms by which these animals overcome the lack of dystrophin. Genome-wide microarray-based gene expression analysis was carried out at 3 wk and 1.5 and 3 mo of life. Candidate genes were selected by comparing: 1) mdx vs. controls at each point in time, and 2) mdx mice and 3) control mice among the three points in time. The first analysis showed a strong upregulation (96%) of inflammation-related genes and in >75% of genes related to cell adhesion, muscle structure/regeneration, and extracellular matrix remodeling during mdx disease evolution. Lgals3, Postn, Ctss, and Sln genes showed the strongest variations. The analysis performed among points in time demonstrated significant changes in Ecm1, Spon1, Thbs1, Csrp3, Myo10, Pde4b, and Adamts-5 exclusively during mdx mice lifespan. RT-PCR analysis of Postn, Sln, Ctss, Thbs1, Ecm1, and Adamts-5 expression from 3 wk to 9 mo, confirmed microarray data and demonstrated variations beyond 3 mo of age. A high-confidence functional network analysis demonstrated a strong relationship between them and showed two main subnetworks, having Dmd- Utrn- Myo10 and Adamts5- Thbs1- Spon1-Postn as principal nodes, which are functionally linked to Abca1, Actn4, Crebbp, Csrp3, Lama1, Lama3, Mical2, Mical3, Myf6, Pxn, and Sparc genes. Candidate genes may participate in the decline of muscle necrosis in mdx mice and could be considered potential therapeutic targets for Duchenne patients.
Granulocyte Macrophage Colony-Stimulating Factor Inhibits Breast Cancer Growth and Metastasis by Invoking an Anti-Angiogenic Program in Tumor-Educated Macrophages
Authors: Eubank Tim D. , Roberts Ryan D. , Khan Mahmood , Curry Jennifer M. , Nuovo Gerard J. , Kuppusamy Periannan , Marsh Clay B.
Published: February 2009

Tumor-educated macrophages facilitate tumor metastasis and angiogenesis. We discovered that granulocyte macrophage colony-stimulating factor (GM-CSF) blocked macrophages vascular endothelial growth factor (VEGF) activity by producing soluble VEGF receptor-1 (sVEGFR-1) and determined the effect on tumor-associated macrophage behavior and tumor growth. We show GM-CSF treatment of murine mammary tumors slowed tumor growth and slowed metastasis. These tumors had more macrophages, fewer blood vessels, and lower oxygen concentrations. This effect was sVEGFR-1 dependent. In situ hybridization and flow cytometry identified macrophages as the primary source of sVEGFR-1. These data suggest that GM-CSF can re-educate macrophages to reduce angiogenesis and metastases in murine breast cancer. [Cancer Res 2009;69(5):2133–40]
Identification of Polo-like Kinase 1 as a Potential Therapeutic Target in Anaplastic Thyroid Carcinoma
Authors: Nappi Tito Claudio , Salerno Paolo , Zitzelsberger Horst , Carlomagno Francesca , Salvatore Giuliana , Santoro Massimo
Published: February 2009

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and chemoresistant cancers. The serine/threonine kinase Polo-like kinase 1 (PLK1), a key regulator of multiple steps during mitotic progression, is highly expressed in ATC. Here, we used the BI 2536 PLK1 inhibitor on ATC and nontransformed thyroid follicular cell lines. Our data show that ATC cells are addicted to high levels of PLK1 activity for proliferation, survival, anchorage-independent growth, and tumorigenicity. On treatment with nanomolar doses of BI 2536, ATC cells progressed normally through S phase but died thereafter, directly from mitotic arrest. Immunofluorescence microscopy, immunoblot, and flow cytometry analysis showed that, on PLK1 blockade, ATC cells arrested in prometaphase with a 4N DNA content. Treated ATC cells accumulated phosphohistone H3 and displayed characteristic mitotic (Polo) spindle aberrations. Nontransformed thyroid cells were 3.2- to 18.4-fold less susceptible to BI 2536–induced cell cycle effects compared with ATC cells. These findings identify PLK1 as a promising target for the molecular therapy of ATC. [Cancer Res 2009;69(5):1916–23]
Lysine-Specific Demethylase 1 Is Strongly Expressed in Poorly Differentiated Neuroblastoma: Implications for Therapy
Authors: Schulte Johannes H. , Lim Soyoung , Schramm Alexander , Friedrichs Nicolaus , Koster Jan , Versteeg Rogier , Ora Ingrid , Pajtler Kristian , Klein-Hitpass Ludger , Kuhfittig-Kulle Steffi , Metzger Eric , Schüle Roland , Eggert Angelika , Buettner Reinhard , Kirfel Jutta
Published: February 2009

Aberrant epigenetic changes in DNA methylation and histone acetylation are hallmarks of most cancers, whereas histone methylation was previously considered to be irreversible and less versatile. Recently, several histone demethylases were identified catalyzing the removal of methyl groups from histone H3 lysine residues and thereby influencing gene expression. Neuroblastomas continue to remain a clinical challenge despite advances in multimodal therapy. Here, we address the functional significance of the chromatin-modifying enzyme lysine-specific demethylase 1 (LSD1) in neuroblastoma. LSD1 expression correlated with adverse outcome and was inversely correlated with differentiation in neuroblastic tumors. Differentiation of neuroblastoma cells resulted in down-regulation of LSD1. Small interfering RNA–mediated knockdown of LSD1 decreased cellular growth, induced expression of differentiation-associated genes, and increased target gene–specific H3K4 methylation. Moreover, LSD1 inhibition using monoamine oxidase inhibitors resulted in an increase of global H3K4 methylation and growth inhibition of neuroblastoma cells in vitro. Finally, targeting LSD1 reduced neuroblastoma xenograft growth in vivo. Here, we provide the first evidence that a histone demethylase, LSD1, is involved in maintaining the undifferentiated, malignant phenotype of neuroblastoma cells. We show that inhibition of LSD1 reprograms the transcriptome of neuroblastoma cells and inhibits neuroblastoma xenograft growth. Our results suggest that targeting histone demethylases may provide a novel option for cancer therapy. [Cancer Res 2009;69(5):2065–71]
LY303511 Enhances TRAIL Sensitivity of SHEP-1 Neuroblastoma Cells via Hydrogen Peroxide–Mediated Mitogen-Activated Protein Kinase Activation and Up-regulation of Death Receptors
Authors: Shenoy Kirthan , Wu Yongfei , Pervaiz Shazib
Published: February 2009

We recently reported that LY294002 (LY29) and LY303511 (LY30) sensitized tumor cells to drug-induced apoptosis independent of the phosphoinositide 3-kinase/Akt pathway. Here, we investigated the mechanism of LY30-induced sensitization of human neuroblastoma cells to TRAIL-mediated apoptosis. We provide evidence that LY30-induced increase in intracellular H2O2 up-regulates the expression of TRAIL receptors (DR4 and DR5) in SHEP-1 cells by activating mitogen-activated protein kinases, resulting in a significant amplification of TRAIL-mediated caspase-8 processing and activity, cytosolic translocation of cytochrome c, and cell death. Involvement of the death receptors was further confirmed by the ability of blocking antibodies against DR4 and/or DR5 to inhibit LY30-induced TRAIL sensitization. Pharmacologic inhibition of c-Jun NH2 terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activation by SP600125 and PD98059, respectively, blocked LY30-induced increase in sensitization to TRAIL-mediated death. Finally, small interfering RNA–mediated gene silencing of JNK and ERK inhibited LY30-induced increase in surface expression of DR4 and DR5, respectively. These data show that JNK and ERK are two crucial players involved in H2O2-mediated increase in TRAIL sensitization of tumor cells upon exposure to LY30 and underscore a novel mode of action of this inactive analogue of LY29. Our findings could have implications for the use of LY30 and similar compounds for enhancing the apoptotic sensitivity of neuroblastoma cells that often become refractory to chemotherapy. [Cancer Res 2009;69(5):1941–50]
cIAP1, cIAP2, and XIAP Act Cooperatively via Nonredundant Pathways to Regulate Genotoxic Stress–Induced Nuclear Factor-κB Activation
Authors: Jin Hyung-Seung , Lee Dong-Hee , Kim Dong-Hwan , Chung Ji-Hye , Lee Seul-Ji , Lee Tae H.
Published: February 2009

Various genotoxic agents cause monoubiquitination of NEMO/IKKγ—the regulatory subunit of IκB kinase (IKK) complex—in the nucleus. Ubiquitinated NEMO exits from the nucleus and forms a complex with the IKK catalytic subunits IKKα and IKKβ, resulting in IKK activation and, ultimately, nuclear factor-κB (NF-κB) activation. Thus, NEMO ubiquitination is a prerequisite for IKK-dependent activation of NF-κB. However, the IKK activation mechanism is unknown and the NEMO-ubiquitinating E3 enzyme has not been identified. We found that inhibitors of apoptosis protein (IAP) regulate genotoxic stress–induced NF-κB activation at different levels. XIAP mediates activation of the upstream IKK kinase, TAK1, and couples activated TAK1 to the IKK complex. This XIAP-dependent event occurs in response to camptotechin or etoposide/VP16; however, XIAP is dispensable for activation of NF-κB by doxorubicin, which engages a MEK-ERK pathway to activate IKK. We also show that cIAP1 mediates NEMO ubiquitination and cIAP2 regulates an event downstream of NEMO ubiquitination. Our study highlights nonredundant cooperative contributions of IAPs to antiapoptotic NF-κB activation by genotoxic signals beyond their classic caspase inhibitory functions. [Cancer Res 2009;69(5):1782–91]
Xenobiotic Metabolizing Gene Variants, Dietary Heterocyclic Amine Intake, and Risk of Prostate Cancer
Authors: Koutros Stella , Berndt Sonja I. , Sinha Rashmi , Ma Xiaomei , Chatterjee Nilanjan , Alavanja Michael C.R. , Zheng Tongzhang , Huang Wen-Yi , Hayes Richard B. , Cross Amanda J.
Published: February 2009

We recently reported that heterocyclic amines (HCA) are associated with prostate cancer risk in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. We now use extensive genetic data from this resource to determine if risks associated with dietary HCAs {2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP); 2-amino-3,8-dimethylimidazo[4,5-b]quinoxaline (MeIQx); and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx)} from cooked meat are modified by single nucleotide polymorphisms (SNP) in genes involved in HCA metabolism (CYP1A1, CYP1A2, CYP1B1, GSTA1, GSTM1, GSTM3, GSTP1, NAT1, NAT2, SULT1A1, SULT1A2, and UGT1A locus). We conducted a nested case-control study that included 1,126 prostate cancer cases and 1,127 controls selected for a genome-wide association study for prostate cancer. Unconditional logistic regression was used to estimate odds ratios (OR), 95% confidence intervals (95% CI), and P values for the interaction between SNPs, HCA intake, and risk of prostate cancer. The strongest evidence for an interaction was noted between DiMeIQx and MeIQx and the polymorphism rs11102001 downstream of the GSTM3 locus (Pinteraction = 0.001 for both HCAs; statistically significant after correction for multiple testing). Among men carrying the A variant, the risk of prostate cancer associated with high DiMeIQx intake was 2-fold greater than that with low intake (OR, 2.3; 95% CI, 1.2–4.7). The SNP rs11102001, which encodes a nonsynonymous amino acid change P356S in EPS8L3, is a potential candidate modifier of the effect of HCAs on prostate cancer risk. The observed effect provides evidence to support the hypothesis that HCAs may act as promoters of malignant transformation by altering mitogenic signaling. [Cancer Res 2009;69(5):1877–84]
E-Cadherin Deficiency Initiates Gastric Signet-Ring Cell Carcinoma in Mice and Man
Authors: Humar Bostjan , Blair Vanessa , Charlton Amanda , More Helen , Martin Iain , Guilford Parry
Published: February 2009

The importance of loss of the cell-cell adhesion molecule E-cadherin (encoded by CDH1) to tumor progression is well established. However, CDH1 germ-line mutations predispose to the cancer susceptibility syndrome hereditary diffuse gastric cancer (HDGC), suggesting a role for E-cadherin in tumor initiation. The earliest indications of cancer in the stomachs of CDH1 mutation carriers are microscopic foci of intramucosal signet-ring cell carcinoma (SRCC; designated “eHDGC”). Here, we used N-methyl-N-nitrosourea (MNU) to promote gastric carcinogenesis in wild-type (wt) and cdh1+/− mice. MNU induced a variety of gastric tumors; however, intramucosal SRCC developed with an 11 times higher incidence in cdh1+/− mice compared with wt mice. The murine SRCC resembled the human eHDGCs in that they were hypoproliferative, lacked nuclear β-catenin accumulation, and had reduced membrane localization of E-cadherin and its interacting junctional proteins. The down-regulation of E-cadherin in the murine SRCCs confirmed the importance of the second CDH1 hit to the initiation of diffuse gastric cancer. CDH1 promoter hypermethylation has been proposed to be a major second hit in advanced HDGC; however, its contribution to eHDGC was unknown. We thus examined a series of human eHDGC and detected CDH1 promoter methylation in 50% of foci. Promoter methylation was accompanied by reduced wt CDH1 mRNA levels in the foci and had a monoclonal pattern, consistent with an epigenetic initiation of disease. Together, these findings provide compelling evidence for a deficiency in cell-to-cell adhesion being sufficient to initiate diffuse gastric cancer in the absence of hyperproliferation and β-catenin activation. [Cancer Res 2009;69(5):2050–6]
Kinetic Preservation of Dual Specificity of Coprogrammed Minor Histocompatibility Antigen-Reactive Virus-Specific T Cells
Authors: van Loenen Marleen M. , Hagedoorn Renate S. , Kester Michel G.D. , Hoogeboom Manja , Willemze Roel , Falkenburg J.H. Frederik , Heemskerk Mirjam H.M.
Published: February 2009

Adoptive transfer of antigen-specific T cells is an attractive strategy for the treatment of hematologic malignancies. It has been shown that T cells recognizing minor histocompatibility antigens (mHag) selectively expressed on hematopoietic cells mediate antileukemic reactivity after allogeneic stem cell transplantation. However, large numbers of T cells with defined specificity are difficult to attain. An attractive strategy to obtain large numbers of leukemia-reactive T cells is retroviral transfer of mHag-specific T-cell receptors (TCR). TCR transfer into T cells specific for persistent viruses may enable these T cells to proliferate both after encountering with viral antigens as well as mHags, increasing the possibility of in vivo survival. We analyzed whether the dual specificity of the TCR-transferred T cells after repetitive stimulation via either the introduced antileukemic HA-2-TCR or the endogenous cytomegalovirus (CMV) specific CMV-TCR was preserved. We show that after repetitive stimulation, T cells skew to a population predominantly expressing the triggered TCR. However, HA-2-TCR–transferred CMV-specific T cells with high antileukemic HA-2-TCR expression but low CMV-TCR expression were able to persist and proliferate after repetitive stimulation with pp65. Moreover, HA-2-TCR–transferred CMV-specific T cells remained dual specific after repetitive stimulation and TCR expression could be reverted after additional stimulation via the previously nonstimulated TCR, restoring high-avidity interactions. These data imply persistence of TCR-transferred virus-specific T cells with both antileukemic and antivirus reactivity in vivo. [Cancer Res 2009;69(5):2034–41]
Sustained Src Inhibition Results in Signal Transducer and Activator of Transcription 3 (STAT3) Activation and Cancer Cell Survival via Altered Janus-Activated Kinase–STAT3 Binding
Authors: Sen Banibrata , Saigal Babita , Parikh Nila , Gallick Gary , Johnson Faye M.
Published: February 2009

Locoregional and distant recurrence remains common and usually fatal for patients with advanced head and neck squamous cell carcinoma (HNSCC). One promising molecular target in HNSCC is the Src family kinases (SFK). SFKs can affect cellular proliferation and survival by activating the signal transducer and activator of transcription (STAT) family of transcription factors, especially STAT3. Surprisingly, sustained SFK inhibition resulted in only transient inhibition of STAT3. We investigated the mechanism underlying STAT3 activation and its biological importance. Specific c-Src knockdown with small interfering RNA (siRNA) resulted in STAT3 activation showing specificity, which was inhibited by Janus-activated kinase (JAK; TYK2 and JAK2) depletion with siRNA. Sustained SFK inhibition also resulted in recovered JAK-STAT3 binding and JAK kinase activity after an initial reduction, although JAK phosphorylation paradoxically decreased. To determine the biological significance of STAT3 activation, we combined specific STAT3 depletion with a pharmacologic SFK inhibitor and observed increased cell cycle arrest and apoptosis. Likewise, the addition of STAT3- or JAK-specific siRNA to c-Src–depleted cells enhanced cytotoxicity relative to cells incubated with c-Src siRNA alone. These results show that reactivation of STAT3 after sustained, specific c-Src inhibition is mediated through altered JAK-STAT3 binding and JAK kinase activity and that this compensatory pathway allows for cancer cell survival and proliferation despite durable c-Src inhibition. To our knowledge, this novel feedback pathway has never been described previously. Given that pharmacologic SFK inhibitors are currently being evaluated in clinical trials, these results have potential clinical implications for cancer therapy. [Cancer Res 2009;69(5):1958–65]
Neuronal Guidance Protein Netrin-1 Induces Differentiation in Human Embryonal Carcinoma Cells
Authors: Mancino Mario , Esposito Claudia , Watanabe Kazuhide , Nagaoka Tadahiro , Gonzales Monica , Bianco Caterina , Normanno Nicola , Salomon David S. , Strizzi Luigi
Published: February 2009

Pluripotent cells within embryonal carcinoma (EC) can differentiate in vivo or in vitro on treatment with specific agents. Differentiating EC cells express lower levels of stem cell–related genes, such as Cripto-1. We show that migration of human EC cells (NTERA/2 and NCCIT) can be reduced following treatment with the guidance molecule Netrin-1. Moreover, Netrin-1 treatment increased the levels of β-III tubulin, glial filament acidic protein, Nestin, and γ-aminobutyric acid and reduced the expressions of Cripto-1, Nanog, and Oct4 in EC cells. These Netrin-1–induced effects in the EC cells were mediated via binding of Netrin-1 to the Neogenin receptor and activation of SHP-2, resulting in increased levels of inactive phosphorylated c-src(Y527). These results suggest that Netrin-1 can induce neuroectodermal-like differentiation of human EC cells by affecting c-src signaling via SHP-2 activation and regulation of Nanog, Oct4, and Cripto-1 expressions. [Cancer Res 2009;69(5):1717–21]
Activation of Ribosomal RNA Transcription by Hepatitis C Virus Involves Upstream Binding Factor Phosphorylation via Induction of Cyclin D1
Authors: Raychaudhuri Santanu , Fontanes Vanessa , Barat Bhaswati , Dasgupta Asim
Published: February 2009

Hepatitis C virus (HCV) causes chronic infection in humans leading to liver cirrhosis and hepatocellular carcinoma. rRNA transcription, catalyzed by RNA polymerase I (Pol I), plays a critical role in ribosome biogenesis, and changes in Pol I transcription rate are associated with profound alterations in the growth rate of the cell. Because rRNA synthesis is intimately linked to cell growth and frequently up-regulated in many cancers, we hypothesized that HCV might have the ability to activate rRNA synthesis in infected cells. We show here that rRNA promoter–mediated transcription is significantly (10- to 12-fold) activated in human liver–derived cells following infection with type 2 JFH-1 HCV or transfection with the subgenomic type 1 HCV replicon. Further analysis revealed that HCV nonstructural protein 5A (NS5A) was responsible for activation of rRNA transcription. Both the NH2-terminal amphipathic helix and the polyproline motifs of NS5A seem to be essential for rRNA transcription activation. The NS5A-dependent activation of rRNA transcription seems to be due to hyperphosphorylation and consequent activation of upstream binding factor (UBF), a Pol I DNA binding transcription factor. We further show that hyperphosphorylation of UBF occurs as a result of up-regulation of both cyclin D1 and cyclin-dependent kinase 4 by the HCV NS5A polypeptide. These results suggest that the endoplasmic reticulum–associated NS5A is able to transduce signals into the nucleoplasm via UBF hyperphosphorylation leading to rRNA transcription activation. These results could, at least in part, explain a mechanism by which HCV contributes to transformation of liver cells. [Cancer Res 2009;69(5):2057–64]
Enhanced Immune Recognition of Cryptic Glycan Markers in Human Tumors
Authors: Newsom-Davis Thomas E. , Wang Denong , Steinman Lawrence , Chen Paul F-T. , Wang Lai-Xi , Simon A. Katharina , Screaton Gavin R.
Published: February 2009

Abnormal glycosylation is one of the hallmarks of the cancer cell and is associated with tumor invasion and metastasis. The development of tumor-associated carbohydrate antigen (TACA) vaccines has been problematic due to poor immunogenicity. However, when appropriate targets can be identified, passive immunization with monoclonal antibodies (mAbs) directed against TACAs has been shown to have antitumor activity. Fas ligand (FasL) is a transmembrane protein that induces apoptosis in cells expressing its receptor, Fas. When grafted into mice, FasL-expressing tumor cells break immunologic tolerance to self-antigens and induce antibody-mediated tumor immunity. Here, five IgM mAbs were produced from mice vaccinated with FasL-expressing B16F10 mouse melanoma cells. They recognize various syngeneic and allogeneic murine tumor cell lines. One mAb, TM10, recognizes a range of human tumor cell lines, including melanoma, prostate, and ovarian cancer. It does not bind to untransformed cells. The epitopes recognized by all the mAbs were carbohydrates expressed on proteins. Using carbohydrate microarrays, the antigenic targets of TM10 were found to be high-mannose core structures of N-linked glycans. In normal cells, high-mannose clusters are hidden by extensive saccharide branching but they become exposed in cancer cells as a result of abnormal glycosylation pathways. Vaccination with FasL-expressing tumors therefore enables the immune system to break tolerance to self-antigens, allowing identification of novel TACAs that can form the basis of future humoral anticancer therapy. [Cancer Res 2009;69(5):2018–25]
Introducing Nanochemoprevention as a Novel Approach for Cancer Control: Proof of Principle with Green Tea Polyphenol Epigallocatechin-3-Gallate
Authors: Siddiqui Imtiaz A. , Adhami Vaqar M. , Bharali Dhruba J. , Hafeez Bilal B. , Asim Mohammad , Khwaja Sabih I. , Ahmad Nihal , Cui Huadong , Mousa Shaker A. , Mukhtar Hasan
Published: February 2009

Chemoprevention, especially through the use of naturally occurring phytochemicals capable of impeding the process of one or more steps of carcinogenesis process, is a promising approach for cancer management. Despite promising results in preclinical settings, its applicability to humans has met with limited success largely due to inefficient systemic delivery and bioavailability of promising chemopreventive agents. Here, we introduce the concept of nanochemoprevention, which uses nanotechnology for enhancing the outcome of chemoprevention. We encapsulated green tea polyphenol epigallocatechin-3-gallate (EGCG) in polylactic acid–polyethylene glycol nanoparticles and observed that encapsulated EGCG retains its biological effectiveness with over 10-fold dose advantage for exerting its proapoptotic and angiogenesis inhibitory effects, critically important determinants of chemopreventive effects of EGCG in both in vitro and in vivo systems. Thus, this study could serve as a basis for the use of nanoparticle-mediated delivery to enhance bioavailability and limit any unwanted toxicity of chemopreventive agents, such as EGCG. [Cancer Res 2009;69(5):1712–6]
HMGA Proteins Up-regulate CCNB2 Gene in Mouse and Human Pituitary Adenomas
Authors: De Martino Ivana , Visone Rosa , Wierinckx Anne , Palmieri Dario , Ferraro Angelo , Cappabianca Paolo , Chiappetta Gennaro , Forzati Floriana , Lombardi Gaetano , Colao Annamaria , Trouillas Jacqueline , Fedele Monica , Fusco Alfredo
Published: February 2009

The high mobility group As (HMGAs) belong to a family of nonhistone nuclear proteins that orchestrate the assembly of nucleoprotein complexes. Through a complex network of protein-DNA and protein-protein interaction, they play important roles in gene transcription, recombination, and chromatin structure. This protein family is involved, through different mechanisms, in both benign and malignant neoplasias. We have recently reported that transgenic mice carrying the Hmga1 or Hmga2 genes under transcriptional control of the cytomegalovirus promoter develop pituitary adenomas secreting prolactin and growth hormone. We have shown that the mechanism of the HMGA2-induced pituitary adenoma is based on the increased E2F1 activity. The expression profile of mouse normal pituitary glands and adenomas induced in HMGA transgenic mice revealed an increased expression of the ccnb2 gene, coding for the cyclin B2 protein, in the neoplastic tissues compared with the normal pituitary gland. Here, we show, by electrophoretic mobility shift assay and chromatin immunoprecipitation, a direct binding of HMGA proteins to the promoter of ccnb2 gene, whereas luciferase assays showed that HMGAs are able to up-regulate ccnb2 promoter activity. Finally, we report an increased CCNB2 expression in human pituitary adenomas of different histotypes that is directly correlated with HMGA1 and HMGA2 expression. Because cyclin B2 is involved in the regulation of the cell cycle, these results taken together indicate that HMGA-induced cyclin B2 overexpression gives an important contribution to experimental and human pituitary tumorigenesis. [Cancer Res 2009;69(5):1844–50]
Detection of Hepatocellular Carcinoma Using Glycomic Analysis
Authors: Goldman Radoslav , Ressom Habtom W. , Varghese Rency S. , Goldman Lenka , Bascug Gregory , Loffredo Christopher A. , Abdel-Hamid Mohamed , Gouda Iman , Ezzat Sameera , Kyselova Zuzana , Mechref Yehia , Novotny Milos V.
Published: February 2009

Purpose: Hepatocellular carcinoma (HCC) represents an increasing health problem in the United States. Serum α-fetoprotein, the currently used clinical marker, is elevated in only ∼60% of HCC patients; therefore, the identification of additional markers is expected to have significant public health impact. The objective of our study was to quantitatively assess N-glycans originating from serum glycoproteins as alternative markers for the detection of HCC.

Experimental Design: We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for quantitative comparison of 83 N-glycans in serum samples of 202 participants (73 HCC cases, 77 age- and gender-matched cancer-free controls, and 52 patients with chronic liver disease). N-glycans were enzymatically released from serum glycoproteins and permethylated before mass spectrometric quantification.

Results: The abundance of 57 N-glycans was significantly altered in HCC patients compared with controls. The sensitivity of six individual glycans evaluated for separation of HCC cases from population controls ranged from 73% to 90%, and the specificity ranged from 36% to 91%. A combination of three selected N-glycans was sufficient to classify HCC with 90% sensitivity and 89% specificity in an independent validation set of patients with chronic liver disease. The three N-glycans remained associated with HCC after adjustment for chronic viral infection and other known covariates, whereas the other glycans increased significantly at earlier stages of the progression of chronic viral infection to HCC.

Conclusion: A set of three identified N-glycans is sufficient for the detection of HCC with 90% prediction accuracy in a population with high rates of hepatitis C viral infection. Further evaluation of a wider clinical utility of these candidate markers is warranted.
MicroRNA-mediated Regulation of Ubc9 Expression in Cancer Cells
Authors: Wu Fangting , Zhu Shuomin , Ding Yanna , Beck William T. , Mo Yin-Yuan
Published: February 2009

Purpose: As an E2-conjugating enzyme for sumoylation, Ubc9 plays a critical role in sumoylation-mediated cellular pathways, ultimately impacting cell growth and cancer development. The aim of this study was to investigate the regulation of Ubc9 in cancer cells.

Experimental Design: Immunohistochemistry and Western blot were used to determine Ubc9 expression in paraffin-embedded tumor tissue and frozen specimens of the matched tumors from the same patient, respectively. To establish the causal relationship between miR-30e and Ubc9 expression, we overexpressed miR-30e and then determined the resultant effects on Ubc9 expression. To determine whether miR-30e directly targets Ubc9, we did luciferase assays using luciferase reporters carrying the 3′-untranslated region (3′-UTR) of the Ubc9 gene.

Results: We found that Ubc9 is up-regulated in breast, head and neck, and lung cancer specimens. In addition, an examination of eight pairs of matched breast tumor specimens by Western blot analysis revealed that, on average, the level of Ubc9 is 5.7-fold higher in tumor than in the matched normal breast tissue. Of interest, we present evidence that Ubc9 is subjected to posttranscriptional regulation by microRNA, and the miR-30 family, such as miR-30e, negatively regulates Ubc9 expression. In contrast to Ubc9, miR-30e is underexpressed in tumors. Moreover, ectopic expression of miR-30e suppresses cell growth, which can be partially reversed by Ubc9. Finally, using luciferase-Ubc9-3′-UTR reporters, we show that Ubc9 is a direct target for miR-30e by interactions with the putative miR-30e binding sites.

Conclusion: These results provide new insight into regulation of Ubc9 in cancer cells.
Tumor Suppressor BRCA1 Is Expressed in Prostate Cancer and Controls Insulin-like Growth Factor I Receptor (IGF-IR) Gene Transcription in an Androgen Receptor–Dependent Manner
Authors: Schayek Hagit , Haugk Kathy , Sun Shihua , True Lawrence D. , Plymate Stephen R. , Werner Haim
Published: February 2009

Purpose: The insulin-like growth factor (IGF) system plays an important role in prostate cancer. The BRCA1 gene encodes a transcription factor with tumor suppressor activity. The involvement of BRCA1 in prostate cancer, however, has not yet been elucidated. The purpose of the present study was to examine the functional correlations between BRCA1 and the IGF system in prostate cancer.

Experimental Design: An immunohistochemical analysis of BRCA1 was done on tissue microarrays comprising 203 primary prostate cancer specimens. In addition, BRCA1 levels were measured in prostate cancer xenografts and in cell lines representing early stages (P69 cells) and advanced stages (M12 cells) of the disease. The ability of BRCA1 to regulate IGF-I receptor (IGF-IR) expression was studied by coexpression experiments using a BRCA1 expression vector along with an IGF-IR promoter-luciferase reporter.

Results: We found significantly elevated BRCA1 levels in prostate cancer in comparison with histologically normal prostate tissue (P < 0.001). In addition, an inverse correlation between BRCA1 and IGF-IR levels was observed in the androgen receptor (AR)–negative prostate cancer–derived P69 and M12 cell lines. Coexpression experiments in M12 cells revealed that BRCA1 was able to suppress IGF-IR promoter activity and endogenous IGF-IR levels. On the other hand, BRCA1 enhanced IGF-IR levels in LNCaP C4-2 cells expressing an endogenous AR.

Conclusions: We provide evidence that BRCA1 differentially regulates IGF-IR expression in AR-positive and AR-negative prostate cancer cells. The mechanism of action of BRCA1 involves modulation of IGF-IR gene transcription. In addition, immunohistochemical data are consistent with a potential survival role of BRCA1 in prostate cancer.
Utility of Incorporating Genetic Variants for the Early Detection of Prostate Cancer
Authors: Nam Robert K. , Zhang William W. , Trachtenberg John , Seth Arun , Klotz Laurence H. , Stanimirovic Aleksandra , Punnen Sanoj , Venkateswaran Vasundara , Toi Ants , Loblaw D. Andrew , Sugar Linda , Siminovitch Katherine A. , Narod Steven A.
Published: February 2009

Purpose: Several single nucleotide polymorphisms (SNP) have been associated with the risk of prostate cancer. The clinical utility of using SNPs in the early detection of prostate cancer has not been evaluated.

Experimental Design: We examined a panel of 25 SNPs from candidate genes and chromosomal regions in 3,004 unselected men who were screened for prostate cancer using serum prostate-specific antigen (PSA) and digital rectal examination. All underwent a prostate biopsy. We evaluated the ability of these SNPs to help predict the presence of prostate cancer at biopsy.

Results: Of the 3,004 patients, 1,389 (46.2%) were found to have prostate cancer. Fifteen of the 25 SNPs studied were significantly associated with prostate cancer (P = 0.02-7 × 10−8). We selected a combination of 4 SNPs with the best predictive value for further study. After adjusting for other predictive factors, the odds ratio for patients with all four of the variant genotypes compared with men with no variant genotype was 5.1 (95% confidence interval, 1.6-16.5; P = 0.006). When incorporated into a nomogram, genotype status contributed more significantly than PSA, family history, ethnicity, urinary symptoms, and digital rectal examination (area under the curve = 0.74). The positive predictive value of the PSA test ranged from 42% to 94% depending on the number of variant genotypes carried (P = 1 × 10−15).

Conclusions: SNP genotyping can be used in a clinical setting for the early detection of prostate cancer in a nomogram approach and by improving the positive predictive value of the PSA test.
Dynamic Spectral Imaging: Improving Colposcopy
Authors: Soutter William P. , Diakomanolis Emmanuel , Lyons Deirdre , Ghaem-Maghami Sadaf , Ajala Tosin , Haidopoulos Dimitrios , Doumplis Dimitrios , Kalpaktsoglou Costas , Sakellaropoulos Gerasimos , Soliman Suzan , Perryman Karen , Hird Vicky , Buckley C. Hilary , Pavlakis Kitty , Markaki Sofia , Dina Roberto , Healy Vourneen , Balas Costas
Published: February 2009

Purpose: Colposcopy occupies a key role in the prevention of cervical cancer by identifying preinvasive or invasive lesions. However, colposcopy is subjective and is responsible for 52% of screening failures. Dynamic spectral imaging (DSI) is based on the objective, quantitative assessment of the acetowhitening effect. This study compared DSI with colposcopy.

Experimental Design: Women referred for colposcopy were examined simultaneously with colposcopy and DSI using a precommercial DySIS model (FPC-03) in an international, multicenter trial. The colposcopy impression and DySIS values were compared with consensus histology reports of biopsies. Subjects were recruited to a training group and subsequently to a test group. Measures were taken to avoid verification bias.

Results: The training and test groups comprised 82 and 308 eligible women, respectively. A cutoff value to identify high-grade disease was selected from the results of the training group and data from previous work. Receiver operator curve analysis of the test data showed an area under the curve of 0.844. DySIS detected 62.9% more high-grade cases than colposcopy (57 versus 35, P = 0.0001). DySIS exceeded end points approved by the Food and Drug Administration for similar studies, with increments in the true positive rate of 22/308 (7.1%; lower 95% CL, 4.5% versus 2%) and in the false positive rate of 32/308 (10.4%; upper 95% CL, 14.7% versus 15%).

Conclusions: DySIS is more sensitive than colposcopy in detecting high-grade lesions and can provide improved guidance for biopsy. The results are obtained in a user-independent fashion, making it suitable for use by nursing personnel.
Unique Methylation Pattern of Oncostatin M Receptor Gene in Cancers of Colorectum and Other Digestive Organs
Authors: Deng Guoren , Kakar Sanjay , Okudiara Keisuke , Choi Esther , Sleisenger Marvin H. , Kim Young S.
Published: February 2009

Purpose: Oncostatin M (OSM) is an interleukin-6 cytokine family member, which inhibits cell proliferation and induces cell differentiation and apoptosis in cancers. In melanoma cells, epigenetic silencing of OSM receptor (OSMR) by histone deacetylation contributes to escape of cell growth control by OSM. However, the silencing of OSMR by DNA methylation in any cancer has not been examined.

Experimental Design: Methylation status of OSMR was determined by sequencing or methylation-specific PCR in primary tumors and cell lines. Cell lines were treated with DNA methyltransferase inhibitors 5-aza-2-deoxycytidine or DNA methyltransferase 1 small interfering RNA or a histone deacetylase inhibitor trichostatin A. OSMR mRNA level was determined by reverse transcription-PCR. The acetylation of histone H3 was analyzed by chromatin immunoprecipitation assay.

Results: We observed methylation of OSMR in 88 of 98 (90%) colorectal cancers, 34 of 38 (89%) colorectal polyps, 17 of 31 (55%) normal-appearing mucosa adjacent to colorectal cancers, 13 of 40 (33%) gastric cancers, and 2 of 10 (20%) pancreatic cancers. OSMR methylation was absent or rarely detected in normal colonic mucosa from noncancer patients or in cancers of nondigestive organs, including breast, lung, liver, prostate, kidney, and melanoma. We observed a significant correlation between OSMR methylation and loss of mRNA expression in 39 cancer cell lines. Following the treatment of colorectal cancer cell lines with 5-aza-2-deoxycytidine, DNA methyltransferase 1 small interfering RNA, or trichostatin A, the induction of OSMR mRNA and the enrichment in the level of histone acetylation were observed.

Conclusions: The epigenetic silencing and DNA methylation of OSMR occur frequently in colorectal cancers and rarely in cancers of nondigestive organs. OSMR methylation is an early event in the colorectal carcinogenesis.
NRH:Quinone Oxidoreductase 2-Deficient Mice Are Highly Susceptible to Radiation-Induced B-Cell Lymphomas
Authors: Iskander Karim , Barrios Roberto J. , Jaiswal Anil K.
Published: February 2009

Purpose: NRH:quinone oxidoreductase 2 (NQO2) is known to protect against myelogenous hyperplasia. However, the role of NQO2 in prevention of hematologic malignancies remains unknown. Present studies investigated in vivo role of NQO2 in prevention of myeloproliferative disease and lymphomas.

Experimental Design: Wild-type and NQO2-null mice were exposed to 0, 1, and 3 Gy γ-radiation. One year later, the mice were analyzed for the development of myeloproliferative disease and lymphomas. Immunohistochemistry analysis determined the B- and T-cell origin of lymphomas. The mice were also sacrificed at 6 and 48 h after radiation exposure and bone marrow was collected and analyzed for p53, Bax, and B-cell apoptosis. Bone marrow cells were cultured and the rate of degradation of p53 was analyzed.

Results: Seventy-two percent NQO2-null mice showed development of B-cell lymphomas in multiple tissues compared with 11% in wild-type mice exposed to 3 Gy γ-radiation. In contrast, only 22% NQO2-null mice showed myeloproliferation compared with none in wild-type mice. Further analysis revealed that bone marrow from NQO2-null mice contained lower levels of p53 compared with wild-type mice due to rapid degradation of p53. In addition, the exposure to radiation resulted in lower induction of p53 and Bax and decreased B-cell apoptosis in NQO2-null mice.

Conclusion: NQO2-null mice are highly susceptible to develop radiation-induced B-cell lymphomas. The lack of significant induction of p53 and Bax and decrease in B-cell apoptosis presumably contributed to the development of lymphomas. NQO2 functions as endogenous factor in prevention against radiation-induced B-cell lymphomas.
Short Pigment Epithelial-Derived Factor-Derived Peptide Inhibits Angiogenesis and Tumor Growth
Authors: Mirochnik Yelena , Aurora Arin , Schulze-Hoepfner Frank T. , Deabes Ahmed , Shifrin Victor , Beckmann Richard , Polsky Charles , Volpert Olga V.
Published: February 2009

Purpose: Pigment epithelial-derived factor (PEDF) is a potent angiogenesis inhibitor with multiple other functions, some of which enhance tumor growth. Our previous studies mapped PEDF antiangiogenic and prosurvival activities to distinct epitopes. This study was aimed to determine the minimal fragment of PEDF, which maintains antiangiogenic and antitumor efficacy.

Experimental Design: We analyzed antigenicity, hydrophilicity, and charge distribution of the angioinhibitory epitope (the 34-mer) and designed three peptides covering its COOH terminus, P14, P18, and P23. We analyzed their ability to block endothelial cell chemotaxis and induce apoptosis in vitro and their antiangiogenic activity in vivo. The selected peptide was tested for the antitumor activity against mildly aggressive xenografted prostate carcinoma and highly aggressive renal cell carcinoma. To verify that P18 acts in the same manner as PEDF, we used immunohistochemistry to measure PEDF targets, vascular endothelial growth factor receptor 2, and CD95 ligand expression in P18-treated vasculature.

Results: P14 and P18 blocked endothelial cell chemotaxis; P18 and P23 induced apoptosis. P18 showed the highest IC50 and blocked angiogenesis in vivo: P23 was inactive and P14 was proangiogenic. P18 increased the production of CD95 ligand and reduced the expression of vascular endothelial growth factor receptor 2 by the endothelial cells in vivo. In tumor studies, P18 was more effective in blocking the angiogenesis and growth of the prostate cancer than parental 34-mer; in the renal cell carcinoma, P18 strongly decreased angiogenesis and halted the progression of established tumors.

Conclusions: P18 is a novel and potent antiangiogenic biotherapeutic agent that has potential to be developed for the treatment of prostate and renal cancer.
Overexpression of Phosphorylated Mammalian Target of Rapamycin Predicts Lymph Node Metastasis and Prognosis of Chinese Patients with Gastric Cancer
Authors: Yu Guanzhen , Wang Jiejun , Chen Ying , Wang Xi , Pan Jun , Li Gang , Jia Zhiliang , Li Qiang , Yao James C. , Xie Keping
Published: February 2009

Purpose: We determined the expression of mammalian target of rapamycin (mTOR) and its activated form, p-mTOR, in Chinese patients with gastric cancer and its clinical effects and underlying mechanisms.

Experimental Design: Tissue microarray blocks containing gastric cancer tissue and matched noncancer gastric tissue specimens obtained from 1,072 patients were constructed. Expression of total mTOR and p-mTOR in these specimens was analyzed using immunohistochemical studies and confirmed by Western blotting.

Results: The overall rates of total mTOR and p-mTOR overexpression were 50.8% (545 of 1,072) and 46.5% (499 of 1,072), respectively. The p-mTOR overexpression was significantly correlated with total mTOR overexpression. Overexpression of total mTOR protein was significantly correlated with tumor differentiation, T1/T2 tumors, and stage I/II/III disease, whereas p-mTOR overexpression was significantly correlated with lymph node metastasis and all stage disease. The Cox proportional hazards model revealed that the overexpression of p-mTOR, but not total mTOR, was an independent prognostic factor for gastric cancer. The overexpression of p-mTOR also predicted the angiogenic phenotype of human gastric cancer and regulated angiogenesis of gastric cancer cells.

Conclusions: Increased activation of mTOR is frequent in human gastric cancer and overexpression of p-mTOR is an independent prognostic factor, suggesting that mTOR pathway could be a potential target for therapy of this malignancy.