Measuring Luciferase Activity Research Articles

Three dominant‐negative mutations in factor XI‐deficient patients

Factor XI (FXI) deficiency results from genetic defects of the F11 gene and is generally considered to be inherited in an autosomal recessive manner. However, the homodimeric structure of FXI allows, in some cases, the dominant-negative transmission of the disease. The aim of this study was to characterize novel missense mutations in three unrelated patients and verify the dominant-negative effects of these mutations on the secretion of wild-type FXI protein by expression studies. The F11 gene was PCR amplified, from genomic DNA extracted from peripheral blood, and sequenced on an ABI 3100 Genetic Analyzer. Human wild-type FXI and FXI mutants were expressed in BHK570 cells using Lipofectamin transfection reagents. Conditioned media and cell lysates were collected for the measurement of luciferase activity, FXI antigen and Western blot analysis. DNA sequencing revealed three novel missense F11 mutations; c.127G>A in exon 3 (Ala43Thr), c.723C>G in exon 7 (Phe241Leu) and c.1207G>A in exon 11 (Val403Met). In vitro expression studies showed that the mutation Ala43Thr, Phe241Leu or Val403Met remarkably decreased the extracellular secretion of mutant FXI, rather than reducing synthesis of the mutant proteins. Cotransfection of wild-type FXI with mutant FXI constructs indicated that the mutation Ala43Thr, Phe241Leu or Val403Met reduced the secretion of wild-type FXI by 75.9%, 68.6% or 71.4%, respectively. Our study suggests that dominant-negative mutations in FXI-deficient patients of non-Ashkenazi Jewish origin may be more prevalent than thought, resulting from FXI's unique dimeric structure.

Minocycline Suppresses Activation of Nuclear Factor of Activated T Cells 1 (NFAT1) in Human CD4+ T Cells

Minocycline is a tetracycline family antibiotic that has anti-inflammatory and immunomodulatory properties. These properties have shown promise in the treatment of conditions such as rheumatoid arthritis, Huntington disease, and multiple sclerosis. As lymphocyte activation is involved in the pathogenesis of many of these diseases, T cells are postulated to be a primary target in minocycline therapy. Previous studies have demonstrated attenuation of CD4(+) T cell activation by minocycline, but a specific mechanism has not been elucidated. In this study, we investigated the effect of minocycline on the activity of three key transcription factors regulating CD4(+) T cell activation: NF-κB, AP-1 (activator protein 1), and NFAT (nuclear factor of activated T) cells. Our data demonstrate that minocycline selectively impairs NFAT-mediated transcriptional activation, a result of increased phosphorylation and reduced nuclear translocation of the isoform NFAT1. Minocycline increased the activity of the NFAT kinase GSK3 and decreased intracellular Ca(2+) flux, both of which facilitate NFAT1 phosphorylation. These findings provide a novel mechanism for minocycline induced suppression of CD4(+) T cell activation and may better inform the application of minocycline as an immunomodulatory agent.

1578 REGULATION OF PROSTATE STROMAL CELL GENE EXPRESSION BY THE ANDROGEN RECEPTOR AND ITS EFFECTS ON PARACRINE SIGNALING TO PROSTATE CANCER CELLS

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 20111578 REGULATION OF PROSTATE STROMAL CELL GENE EXPRESSION BY THE ANDROGEN RECEPTOR AND ITS EFFECTS ON PARACRINE SIGNALING TO PROSTATE CANCER CELLS Charles Welliver, Matthew Tanner, Mengqian Chen, Badar Mian, Alejandro Godoy, Gary Smith, and Ralph Buttyan Charles WelliverCharles Welliver Albany, NY More articles by this author , Matthew TannerMatthew Tanner Albany, NY More articles by this author , Mengqian ChenMengqian Chen Albany, NY More articles by this author , Badar MianBadar Mian Albany, NY More articles by this author , Alejandro GodoyAlejandro Godoy Buffalo, NY More articles by this author , Gary SmithGary Smith Buffalo, NY More articles by this author , and Ralph ButtyanRalph Buttyan Albany, NY More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1614AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The androgen receptor (AR) is expressed in a subset of prostate stromal cells and functional prostate stromal AR is required for normal prostate developmental. Although we are broadly aware of the specifics of genomic action of AR in prostate cancer cells, we know relatively little regarding the gene targets of functional AR in prostate stromal cells. Here, we describe a novel human prostate stromal cell model that can be used to study gene targets of AR action in stromal cells. METHODS Immortalized human WPMY1 prostate stromal cells were transduced with an AR expression lentivirus and selected for stable AR expression. Control cells (WPMY-Vec) were transduced with empty lentivirus and also selected. Conditional responsiveness to DHT was assessed by measurement of luciferase activity after transfection with a synthetic androgen-responsive promoter reporter vector. RNAs were extracted from the cells (biological duplicate cultures) with or without 10 nM dihydrotestosterone (DHT) treatment. The RNAs were labeled and hybridized to Affymetrix ST1.0 human gene chips and gene expression patterns were compared with Genespring software. Changes in gene expression induced by DHT were confirmed by quantitative RT-PCR (qPCR). The functional ontologies of genes changed by androgen were categorized according to KEGG pathway rules. RESULTS Whereas parental WPMY1 or WPMY-Vec cells express little AR and were unresponsive to DHT, WPMY-AR cells express AR at a level commensurate with LNCaP cells and were highly responsive to DHT. WPMY-AR cells were similar in morphology and growth to WPMY-Vec cells, regardless of the presence of DHT. Treatment of WPMY-AR, but not WPMY-Vec cells, with DHT significantly changed the expression of 141 genes by 2-fold or greater. qPCR testing confirmed these changes for all genes assayed. The predominant functions of the genes affected by androgen include regulation of cancer and cell signaling pathways (TGF-beta, Wnt, Hedgehog and MAP-Kinase). Most gene changes (∼60%) involved gene downregulation by DHT. CONCLUSIONS WPMY-AR provides a cell model to study androgen regulated genes in prostate stromal cells. While DHT did not affect the behavior of these cells in culture, it regulated expression of a number of genes involved in cancer and other cell signaling pathways associated with development. Remarkably, androgens downregulated more genes in these cells than were upregulated. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e633-e634 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Charles Welliver Albany, NY More articles by this author Matthew Tanner Albany, NY More articles by this author Mengqian Chen Albany, NY More articles by this author Badar Mian Albany, NY More articles by this author Alejandro Godoy Buffalo, NY More articles by this author Gary Smith Buffalo, NY More articles by this author Ralph Buttyan Albany, NY More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Poly(oligo-D-arginine) With Internal Disulfide Linkages as a Cytoplasm-sensitive Carrier for siRNA Delivery

Small interfering RNA (siRNA) has emerged as a therapeutic strategy for various diseases due to its target-specific gene silencing; however, its relatively high molecular weight, negative charge, and low stability hamper in vitro and in vivo applications. Approaches to overcome those drawbacks have relied on nonviral siRNA carriers based on cationic polymers or peptides. Nevertheless, cationic polymer-based siRNA carriers have yet to resolve intrinsic problems such as cytotoxicity and immunogenicity. An environment-sensitive carrier was recently proposed to enhance siRNA bioactivity and to reduce the carrier safety issues. Only a few studies, however, have shown cytoplasm-sensitive dissociation of the polyplex. In the present study, we clearly demonstrated decondensation of siRNA/poly(oligo-D-arginine) polyplex in the cytoplasm in response to intracellular glutathione (GSH) and the enhanced bioactivity of siRNA against VEGF (siVEGF) used as a model both in vitro and in an animal model. Reducible poly(oligo-D-arginine) (rPOA) rapidly dissociated in the cytoplasm, resulting in fast siRNA release to its target location while maintaining siRNA bioactivity both in vitro and in vivo.

Grape Seed Extract Regulates Androgen Receptor-Mediated Transcription in Prostate Cancer Cells Through Potent Anti–Histone Acetyltransferase Activity

Histone acetylation, which is regulated by histone acetyltransferases (HATs) and deacetylases, is an epigenetic mechanism that influences eukaryotic transcription. Significant changes in histone acetylation are associated with cancer; therefore, manipulating the acetylation status of key gene targets is likely crucial for effective cancer therapy. Grape seed extract (GSE) has a known protective effect against prostate cancer. Here, we showed that GSE significantly inhibited HAT activity by 30-80% in vitro (P < .05). Furthermore, we demonstrated significant repression of androgen receptor (AR)-mediated transcription by GSE in prostate cancer cells by measuring luciferase activity using a pGL3-PSA construct bearing the AR element in the human prostate cancer cell line LNCaP (P < .05). GSE treatment also decreased the mRNA level of the AR-regulated genes PSA and NKX 3.1. Finally, GSE inhibited growth of LNCaP cells. These results indicate that GSE potently inhibits HAT, leading to decreased AR-mediated transcription and cancer cell growth, and implicate GSE as a novel candidate for therapeutic activity against prostate cancer.

Cut-like Homeobox 1 (CUX1) Regulates Expression of the Fat Mass and Obesity-associated and Retinitis Pigmentosa GTPase Regulator-interacting Protein-1-like (RPGRIP1L) Genes and Coordinates Leptin Receptor Signaling

The first intron of FTO contains common single nucleotide polymorphisms associated with body weight and adiposity in humans. In an effort to identify the molecular basis for this association, we discovered that FTO and RPGRIP1L (a ciliary gene located in close proximity to the transcriptional start site of FTO) are regulated by isoforms P200 and P110 of the transcription factor, CUX1. This regulation occurs via a single AATAAATA regulatory site (conserved in the mouse) within the FTO intronic region associated with adiposity in humans. Single nucleotide polymorphism rs8050136 (located in this regulatory site) affects binding affinities of P200 and P110. Promoter-probe analysis revealed that binding of P200 to this site represses FTO, whereas binding of P110 increases transcriptional activity from the FTO as well as RPGRIP1L minimal promoters. Reduced expression of Fto or Rpgrip1l affects leptin receptor isoform b trafficking and leptin signaling in N41 mouse hypothalamic or N2a neuroblastoma cells in vitro. Leptin receptor clusters in the vicinity of the cilium of arcuate hypothalamic neurons in C57BL/6J mice treated with leptin, but not in fasted mice, suggesting a potentially important role of the cilium in leptin signaling that is, in part, regulated by FTO and RPGRIP1L. Decreased Fto/Rpgrip1l expression in the arcuate hypothalamus coincides with decreased nuclear enzymatic activity of a protease (cathepsin L) that has been shown to cleave full-length CUX1 (P200) to P110. P200 disrupts (whereas P110 promotes) leptin receptor isoform b clustering in the vicinity of the cilium in vitro. Clustering of the receptor coincides with increased leptin signaling as reflected in protein levels of phosphorylated Stat3 (p-Stat3). Association of the FTO locus with adiposity in humans may reflect functional consequences of A/C alleles at rs8050136. The obesity-risk (A) allele shows reduced affinity for the FTO and RPGRIP1L transcriptional activator P110, leading to the following: 1) decreased FTO and RPGRIP1L mRNA levels; 2) reduced LEPR trafficking to the cilium; and, as a consequence, 3) a diminished cellular response to leptin.

Peroxisome Proliferator-activated Receptor α Is Responsible for the Up-regulation of Hepatic Glucose-6-phosphatase Gene Expression in Fasting and db/db Mice

Glucose-6-phosphatase (G6Pase) is a key enzyme that is responsible for the production of glucose in the liver during fasting or in type 2 diabetes mellitus (T2DM). During fasting or in T2DM, peroxisome proliferator-activated receptor α (PPARα) is activated, which may contribute to increased hepatic glucose output. However, the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in these states is not well understood. We evaluated the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in fasting and T2DM states. In PPARα-null mice, both hepatic G6Pase and phosphoenolpyruvate carboxykinase levels were not increased in the fasting state. Moreover, treatment of primary cultured hepatocytes with Wy14,643 or fenofibrate increased the G6Pase mRNA level. In addition, we have localized and characterized a PPAR-responsive element in the promoter region of the G6Pase gene. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα binding to the putative PPAR-responsive element of the G6Pase promoter was increased in fasted wild-type mice and db/db mice. These results indicate that PPARα is responsible for glucose production through the up-regulation of hepatic G6Pase gene expression during fasting or T2DM animal models.

Abstract P1-03-09: Combination of nab-Paclitaxel and Bevacizumab Inhibited Tumor Growth and Metastasis of New Inflammatory Breast Cancer Models

Abstract Background: Inflammatory breast cancer (IBC) is a highly lethal form of breast cancer which is characterized by skin redness, irritation, swelling, pain, as well as extensive lymph nodes (LN) and hematogenous metastasis. Animal models of IBC are highly desirable for studying its pathology and for designing effective therapies. In this study, we established two new luciferase-and fluorescently-labeled IBC models and tested the efficacy of the novel drug combination nab-paclitaxel and bevacizumab. Methods: Inflammatory SUM149 breast cancer cells were stably transfected with Red Fluorescent Protein (RFP) and Renilla luciferase to establish SUM149-RR, or infected with lentivirus encoding Green Fluorescent Protein (GFP) and Firefly luciferase to establish SUM149- GL. The new cell lines were characterized both in vitro and in vivo. Immunodeficient mice bearing SUM149-RR tumors of 150mm3 in size were treated with saline (control), bevacizumab (4 mg/kg, i.p., twice a week, for 10 weeks), nab-paclitaxel (10 mg/kg, i.v., qdx5), or the combination. Metastasis was analyzed by measuring luciferase activity in the lymph nodes (LN) and lungs. Results: Luciferase measurements and in vivo imaging showed that both SUM149-RR and-GL clones were highly metastatic to LN, lungs, liver, brain, and spleen. SUM149-RR tumors in control mice displayed ulcerations, edema and redness similar to the clinical disease, while tumors in mice treated with bevacizumab or combination therapy showed no signs of inflammation. Bevacizumab alone decreased tumor growth at later but not early stages of tumor growth, whereas nab-paclitaxel alone inhibited tumor growth by 73%. Combination therapy increased inhibition to 96%, and resulted in 22% (2/9) complete responses. Histologically, tumors from bevacizumab treated groups were more morphologically intact with reduced vascular abnormalities than tumors from control or nab-paclitaxel treated mice. LN and lung metastasis was significantly reduced in all treated groups as compared with control. Conclusions: The SUM149-RR and SUM149-GL lines are new double-tagged models of human IBC that allow organ visualization and accurate quantification of metastasis. These models can be used to study the biology and treatment of IBC. Combination of nab-paclitaxel with bevacizumab was highly effective against SUM149-RR, suggesting the potential usefulness of this regimen for treatment of IBC patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-03-09.

Dynamic changes in the expression of MicroRNA-31 during inflammatory bowel disease-associated neoplastic transformation

Patients with inflammatory bowel disease (IBD) are at increased risk of developing colorectal cancer. Aberrant microRNA (miR) expression has been linked to carcinogenesis; however, no reports document a relationship between IBD-related neoplasia (IBDN) and altered miR expression. In the current study we sought to identify specific miR dysregulation along the normal-inflammation-cancer axis. miR microarrays and quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) were used to detect dysregulated miRs. Receiver operating characteristic curve analysis was employed to test for potential usefulness of miR-31 as a disease marker of IBDNs. In silico prediction analysis, Western blot, and luciferase activity measurement were employed for target identification. Several dysregulated miRs were identified between chronically inflamed mucosae and dysplasia arising in IBD. MiR-31 expression increases in a stepwise fashion during progression from normal to IBD to IBDN and accurately discriminated IBDNs from normal or chronically inflamed tissues in IBD patients. Finally, we identified factor inhibiting hypoxia inducible factor 1 as a direct target of miR-31. Our study reveals specific miR dysregulation as chronic inflammation progresses to dysplasia. MiR-31 expression levels increase with disease progression and accurately discriminates between distinct pathological entities that coexist in IBD patients. The novel effect of miR-31 on regulating factor inhibiting hypoxia inducible factor 1 expression provides a new insight on the pathogenesis of IBDN.

Claudin Association with CD81 Defines Hepatitis C Virus Entry

Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.

A convenient spectrometric assay system for intracellular quantitative measurement of DNA glycosylase activity

Cytosine methylation is a vital biology event. However, it is also the source of genomic instability due to deamination of 5'-methylcytosine by spontaneous hydrolysis, which produces thymine and results in G:T mismatches. Thymine DNA glycosylase and methyl-CpG-binding protein 4 are major DNA glycosylases involved in the mismatch repair progress, and their activities have been measured in many related researches. In this study, we developed a convenient spectrometric assay system for specific and quantitative measurement of intracellular DNA glycosylase activity. A G:T mismatch was introduced into the upstream region of firefly luciferase-coding sequence in the pGL3-control plasmid. Only if the G:T mismatches were repaired to G:C, will luciferase be expressed in transfected cells. By measuring luciferase activity, which is simple and convenient, the intracellular DNA glycosylase activity can be determined.

Secretory Mechanisms and Intercellular Transfer of MicroRNAs in Living Cells

The existence of circulating microRNAs (miRNAs) in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic marker. However, the secretory mechanism and biological function of extracellular miRNAs remain unclear. Here, we show that miRNAs are released through a ceramide-dependent secretory machinery and that the secretory miRNAs are transferable and functional in the recipient cells. Ceramide, whose biosynthesis is regulated by neutral sphingomyelinase 2 (nSMase2), triggers secretion of small membrane vesicles called exosomes. The decreased activity of nSMase2 with a chemical inhibitor, GW4869, and a specific small interfering RNA resulted in the reduced secretion of miRNAs. Complementarily, overexpression of nSMase2 increased extracellular amounts of miRNAs. We also revealed that the endosomal sorting complex required for transport system is unnecessary for the release of miRNAs. Furthermore, a tumor-suppressive miRNA secreted via this pathway was transported between cells and exerted gene silencing in the recipient cells, thereby leading to cell growth inhibition. Our findings shed a ray of light on the physiological relevance of secretory miRNAs.

Targeting of mesenchymal stem cells to ovarian tumors via an artificial receptor

BackgroundMesenchymal Progenitor/Stem Cells (MSC) respond to homing cues providing an important mechanism to deliver therapeutics to sites of injury and tumors. This property has been confirmed by many investigators, however, the efficiency of tumor homing needs to be improved for effective therapeutic delivery. We investigated the feasibility of enhancing MSC tumor targeting by expressing an artificial tumor-binding receptor on the MSC surface.MethodsHuman MSC expressing an artificial receptor that binds to erbB2, a tumor cell marker, were obtained by transduction with genetically modified adenoviral vectors encoding an artificial receptor (MSC-AR). MSC-AR properties were tested in vitro in cell binding assays and in vivo using two model systems: transient transgenic mice that express human erbB2 in the lungs and ovarian xenograft tumor model. The levels of luciferase-labeled MSCs in erbB2-expressing targeted sites were evaluated by measuring luciferase activity using luciferase assay and imaging.ResultsThe expression of AR enhanced binding of MSC-AR to erbB2-expressing cells in vitro, compared to unmodified MSCs. Furthermore, we have tested the properties of erbB2-targeted MSCs in vivo and demonstrated an increased retention of MSC-AR in lungs expressing erbB2. We have also confirmed increased numbers of erbB2-targeted MSCs in ovarian tumors, compared to unmodified MSC. The kinetic of tumor targeting by ip injected MSC was also investigated.ConclusionThese data demonstrate that targeting abilities of MSCs can be enhanced via introduction of artificial receptors. The application of this strategy for tumor cell-based delivery could increase a number of cell carriers in tumors and enhance efficacy of cell-based therapy.

Long Chain Acyl-CoA Synthetase-3 Is a Molecular Target for Peroxisome Proliferator-activated Receptor δ in HepG2 Hepatoma Cells

ACSL3 is a member of the long chain acyl-CoA synthetase (ACSL) family that plays key roles in fatty acid metabolism in various tissues in an isozyme-specific manner. Our previous studies showed that ACSL3 was transcriptionally up-regulated by the cytokine oncostatin M (OSM) in HepG2 cells, accompanied by reduced cellular triglyceride content and enhanced beta-oxidation. In this study, we investigated the molecular mechanism underlying the OSM-induced activation of ACSL3 gene transcription in HepG2 cells. We showed that OSM treatment resulted in a coordinated elevation of mRNA levels of ACSL3 and peroxisome proliferator-activated receptor delta (PPARdelta). The effect of OSM on ACSL3 mRNA expression was inhibited by cellular depletion of PPARdelta. By utilizing a PPARdelta agonist, L165041, we demonstrated that activation of PPARdelta led to increases in ACSL3 promoter activity, mRNA level, and protein level in HepG2 cells. Analysis of the ACSL3 promoter sequence identified two imperfect PPAR-responsive elements (PPRE) located in the ACSL3 promoter region -944 to -915, relative to the transcription start site. The up-regulation of ACSL3 promoter activity by PPARdelta was abolished by deletion of this PPRE-containing region or mutation to disrupt the binding sites. Direct interactions of PPARdelta with ACSL3-PPRE sequences were demonstrated by gel mobility shift and chromatin immunoprecipitation assays. Finally, we provided in vivo evidence showing that activation of PPARdelta by L165041 in hamsters increased ACSL3 mRNA and protein levels in the liver. These new findings define ACSL3 as a novel molecular target of PPARdelta in HepG2 cells and provide a regulatory mechanism for ACSL3 transcription in liver tissue.

Enhanced growth inhibition of metastatic lung tumors by intravenous injection of ATRA-cationic liposome/IL-12 pDNA complexes in mice

Interleukin 12 (IL-12) is a proinflammatory cytokine with antitumor activity. All-trans-retinoic acid (ATRA) exerts antitumor effects by regulating a variety of gene expressions, including tumor necrosis factor receptor 1 (TNFR1), increases the number of TNFR1 and potentiates TNF-alpha-induced apoptosis in cancer cells. In this study, ATRA-incorporated cationic liposome (ATRA-cationic liposome)/IL-12 plasmid DNA (pDNA) complexes were prepared to improve therapeutic efficacy of cationic liposome/IL-12 pDNA complexes in a mouse model of metastatic lung tumor after intravenous injection. IL-12 production in lungs by ATRA-cationic liposome/IL-12 pDNA complexes was comparable with that by cationic liposome/IL-12 pDNA complexes. The number of metastatic tumor cells (colon26/Luc) was quantitatively evaluated by measuring luciferase activity. ATRA-cationic liposome/IL-12 pDNA complexes reduced the number of colon26/Luc cells and tumor nodules in lungs. ATRA-cationic liposome/IL-12 pDNA complexes significantly prolonged the survival time of mice, whereas cationic liposome/IL-12 pDNA only slightly prolonged it. ATRA-cationic liposome/IL-12 pDNA complexes increased the TNFR1 mRNA upregulation and the number of apoptotic cells in the lung. Moreover, reduced serum alanine transaminase (ALT) and aspartate transaminase (AST) activities were observed in mice treated with ATRA-cationic liposome/IL-12 pDNA complexes. These results suggest that intravenous injection of ATRA-cationic liposome/IL-12 pDNA complexes is an effective method for the treatment of lung metastasis in mice.

Abstract 2354: Angiopoietin-2 role in breast cancer metastasis

Abstract Background: Angiopoietin-2 (Ang-2) is an angiogenic factor whose overexpression is associated with increased tumor vascularity, metastasis, and decreased patient survival. The pro-angiogenic effect of Ang-2 is mediated through activation of the Tie-2 receptor that leads to vessel destabilization and dismantling. The latter is an essential step for both angiogenesis and tumor entry into the lumen. We assessed the effects of Ang-2 on tumor vasculature in orthotopic breast carcinoma model MDA-MB-231 that either lacked or overexpressed this factor. Methods: Luciferase-tagged MDA-MB-231 cells were engineered to overexpress human Ang-2. The control line expressed Ang-2 in reverse orientation. Stable production of Ang-2 was confirmed by RT-PCR, qRT-PCR, Western blot, and ELISA. Ang-2 functionality was assessed by migration and proliferation assays using tumor and endothelial cells. Ang2-overexpressing and control MDA-MB-231 cells were orthotopically implanted into mice. Metastasis to lymph node and lung were determined by measuring luciferase activity in tissue extracts. Tumor blood vessel morphology was analyzed by immunohistochemistry using antibodies against MECA-32, smooth muscle actin (SMA-α), VEGFR-3 and Notch-1. Results: Ang-2-overexpressing tumor line produced 11±0.4 ng/ml of Ang-2 while expression in control line was undetectable. Tumor-produced Ang2 was functional as demonstrated by a dose-dependent migration of lymphatic endothelial cells. Soluble Tie-2 inhibited Ang-2 induced migration by 70%. Ang-2 had not effect on proliferation of MDA-MB-231 cells, tumor growth or blood vessel density. However, Ang-2 overexpressing and control tumors substantially differed in vascular morphology. The differences included 170% increase in blood vascular area, 280% increase in number of vessels with open lumen, and 600% increase in lumens’ cross-sectional area. Pericyte coverage shown by SMA-α staining decreased &amp;gt;10-fold (p&amp;gt;0.001) in Ang-2 tumors, demonstrating vessel destabilization. Tumor vascular invasion and pulmonary metastasis increased by 500% and 1100% in Ang-2 tumors compared with controls. Also, blood vessels in Ang-2 tumors displayed 2.0-2.2 fold upregulated Notch-1 and VEGFR-3 expression. Conclusions: These data suggest that Ang-2 increases metastasis via suppression of pericytes’ recruitment, which destabilizes tumor vessels and facilitates entry of tumor cells into the lumen. The increased expression of Notch-1 and VEGFR-3 on blood vessels in Ang-2 overexpressing tumors suggests that signaling of these proteins underlie the vascular morphologic changes in Ang-2-rich environment. This is consistent with prior data demonstrating up-regulation of VEGFR-3 on tumor blood vessels and association of both proteins with increased metastasis. This study demonstrates a key role for Ang-2 in hematogenous metastasis and suggests that suppression of Ang-2 and VEGFR-3 could inhibit breast cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2354.

Abstract 3279: Nab-paclitaxel and bevacizumab treatment in new models of inflammatory breast cancer, SUM149-RR and SUM149-GL

Abstract Background: Inflammatory breast cancer (IBC) is a one of the most lethal forms of breast cancer with a 5-year survival of 40%. The hallmarks of IBC include skin redness, irritation, swelling, pain, as well as extensive lymph nodes (LN) and hematogenous metastasis. Animal models are highly desirable for studying the IBC pathology and for designing therapies that increase patient survival. Here we established and characterized two luciferase- and fluorescently-labeled lines derived from the human IBC line, SUM149. We characterized the growth and metastasis of these lines in vivo as well as their sensitivity to the novel drug combination, nab-paclitaxel and bevacizumab. Methods: SUM149 cells were stably transfected with Red Fluorescent Protein (RFP) and Renilla luciferase to establish dual reporter termed RR (RFP and Renilla luciferase), or infected with lentivirus encoding Green Fluorescent Protein (GFP) and Firefly luciferase termed GL (GFP and Firefly Luciferase). The new cell lines (SUM149-RR and SUM149-GL) were characterized in vitro and in vivo after orthotopic implantation into mammary fat pads of immunodeficient mice. SUM149-RR was also examined for sensitivity to nab-paclitaxel alone or in combination with bevacizumab. Mice bearing tumors of 150mm3 in size were treated with saline (control), bevacizumab (4mg/kg i.p., twice a week, for 10 weeks), nab-paclitaxel (10mg/kg, i.v., qdx5), or the combination. Tumor growth was monitored by calipers. Metastasis was analyzed by measuring luciferase activity in the lymph nodes (LN) and lungs. Results: Luciferase measurements and in vivo imaging showed that both SUM149-RR and -GL clones were highly metastatic to LN, lungs, liver, brain, and spleen. Bevacizumab alone decreased tumor progression at later but not early stages of tumor growth. Nab-paclitaxel alone inhibited tumor growth by 73%. Combination therapy increased inhibition to 96%, and resulted in 22% (2/9) complete responses. SUM149-RR tumors in control mice displayed ulcerations, edema and redness much like the clinical disease. Tumors in mice treated with bevacizumab or combination therapy showed no signs of inflammation. Tumors from bevacizumab treated groups were more morphologically intact with reduced vascular abnormalities than tumors from control or nab-paclitaxel treated mice. LN and lung metastasis was significantly reduced in all treated groups as compared with control. Conclusions: The SUM149-RR and SUM149-GL lines are new double-tagged models for human IBC that allow organ visualization and accurate quantification of metastasis. These models can be used for better understanding of the IBC biology and for developing new therapies tailored to specific pathology of this cancer. Preliminary studies demonstrated high sensitivity of SUM149-RR to nab-paclitaxel/bevacizumab therapy suggesting the potential usefulness for treatment of IBC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3279.

Abstract 3852: Combination treatment of nab-paclitaxel and bevacizumab in a new model of triple-negative breast cancer

Abstract Background: Tumors that lack estrogen, progesterone, and Her2/nu (triple-negative) are one of the most aggressive, therapy-resistant and highly metastatic subtypes of breast cancer (BC). Most currently available models do not recapitulate expression profile of triple-negative BC, thus making it difficult to devise new treatments that target this tumor type. Here, we established and characterized a new model of triple-negative BC. We also tested the novel combination of nab-paclitaxel with bevacizumab, which has been successful in treating other metastatic cancer types. Methods: HCC1806 cells were obtained stably transfected with Red Fluorescent Protein (RFP) and Renilla luciferase to establish dual reporter termed RR (RFP and Renilla luciferase). The new derivative was designated as HCC1806-RR and characterized in proliferation and cytotoxicity assays. HCC1806-RR was also orthotopically implanted into mammary fat pads of immunodeficient mice to determine tumor cell sensitivity to chemotherapy alone or in combination with anti-VEGF-A antibody. Mice bearing orthotopic HCC1806-RR tumors of 150mm3 in size were treated with saline (control), bevacizumab (4mg/kg. i.p., twice a week, for 10 weeks), nab-paclitaxel (10mg/kg, i.v., qdx5), or with combination of nab-paclitaxel and bevacizumab. Tumor growth rate was monitored by using calipers. Metastasis was analyzed by measuring luciferase activity in the lymph nodes (LN) and lungs. Results: The parental HCC1806 and its derivative HCC1806-RR had identical morphology, proliferation rates and sensitivity to nab-paclitaxel. Luciferase measurements and imaging in vivo showed that HCC1806-RR tumors have predominant LN metastasis with lungs being a second metastatic site. Bevacizumab and nab-paclitaxel inhibited tumor growth by 0% and 90%, respectively. Combination therapy inhibited tumor growth by 100%. This result was highly significant compared with either control (P &amp;lt; 0.001) or nab-paclitaxel (P = 0.024) group. Importantly, only combination therapy reduced incidence of LN and lung metastases by 50% and 87%. This result was statistically significant as compared with all other groups, with P-values of 0.007 and 0.001 for LN and pulmonary metastases, respectively. Half of the mice in the combination therapy group (n =10) had complete regressions of primary tumors and metastases at both regional and distant sites. Conclusions: The HCC1806-RR is a new model double-tagged with RFP and Renilla luciferase. These tags allow for quantitative assessment of metastatic spread. This model can be used to study the biology of triple-negative breast cancer and for designing new approaches to treat this type of cancer. Preliminary studies demonstrated high sensitivity of HCC1806-RR to nab-paclitaxel combined with bevacizumab suggesting that this therapy could significantly improve the health outcome for patients with triple-negative breast cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3852.

Use of a New-Generation Reverse Tetracycline Transactivator System for Quantitative Control of Conditional Gene Expression in the Murine Lung

Conditional regulation of gene expression by the combined use of a lung-specific promoter and the tetracycline-regulated system provides a powerful tool for studying gene function in lung biology and disease pathogenesis in a development-independent fashion. However, the original version of the reverse tetracycline-dependent transactivator (rtTA) exhibited limited doxycycline sensitivity and residual affinity to its promoter (P(tet)), producing leaky transgene expression in the absence of doxycycline. These limitations impeded the use of this system in studying gene dosage effects in pulmonary pathogenesis and repair mechanisms in the diseased lung. Therefore, we used a new-generation rtTA, rtTA2(s)-M2, with no basal activity and increased doxycycline sensitivity, and the rat Clara cell secretory protein (CCSP) promoter to target its expression to pulmonary epithelia in mice. Novel CCSP-rtTA2(s)-M2 founder lines were crossed, with bi-transgenic reporter mice expressing luciferase and Cre recombinase. Background activity, doxycycline sensitivity, tissue and cell-type specificity, inducibility, and reversibility of doxycycline-dependent gene expression were determined by luciferase activity, immunohistochemistry, morphometry, and bioluminescence measurements in neonatal and adult lungs. We generated two distinct novel CCSP-rtTA2(s)-M2 activator mouse lines that confer tight and doxycycline dose-dependent regulation of transgene expression, with high inducibility, complete reversibility, and no background activity, in airway and alveolar epithelia. We conclude that rtTA2(s)-M2 enables quantitative control of conditional gene expression in respiratory epithelia of the murine lung, and that the new CCSP-rtTA2(s)-M2 activator mouse lines will be useful in the further elucidation of the pathogenesis of complex lung diseases and in studies of lung repair.

Abstract 2: Naked CanScript, an 18-base pair sequence, has tumor cell-specific promoter activity in vivo: Implications for targeted gene therapy

Abstract The MSLN gene encodes mesothelin and is highly active in several human malignancies including ovarian, pancreatic, bronchial, gastroesophageal, cervical, endometrial, and biliary carcinomas. The high cancer cell-specific activity of MSLN is attributable to an 18-bp upstream enhancer, CanScript, which selectively elevates transcription upon binding of TEF-1, a transcription enhancer factor, to a MCAT motif. It has previously been shown that three concatemerized copies of CanScript produce a 30-fold enhancement over a SV40 minimal promoter in MSLN-expressing cancer cells (Cancer Res. 2007 Oct 1;67(19):9055-65). To determine whether incorporation of CanScript into gene constructs may have application in enhancing activity of promoters used in cancer-targeting gene therapy strategies, we generated several luciferase reporter gene constructs having different cell type-specific promoters (MSLN, HE-4, PSA) and different copy numbers of CanScript (1X, 2X, 3X, 6X). We transfected multiple cell lines, including MSLN+ (ovarian and pancreatic) and MSLN− (liver, lung, and prostate) tumor cells, with these DNA constructs, and measured luciferase activity 48 hrs later. To replicate an in vivo therapeutic setting, we used nanoparticles to deliver the constructs to the peritoneal space of healthy and ovarian-tumor bearing mice, and measured gene expression in tumors and multiple organs by optical imaging. Surprisingly, we determined that an 18-bp CanScript sequence activates gene expression in a cell-specific manner in the absence of any other promoter sequence, and that amplification of CanScript in combination with either the MSLN or HE4 promoter sequences, i.e., two gene promoters that are highly active in ovarian cancer cells, significantly enhances their activity proportional to the number of copies of CanScript, in MSLN+ cells, including MSLN+ HE4− pancreatic cells, but has little effect in MSLN− cells. These results support the use of CanScript when increased, specific gene expression is desired to improve therapeutic efficacy in tumor cells in which MSLN is highly active. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2.