Testing transgenic regulatory elements through live mouse imaging

AbstractTo overcome positional and methylation effects on transgene expression, we developed a universal cloning cassette for in vivo assessment of regulatory elements using the luciferase reporter gene and the CCCD camera. Monitoring luciferase expression pattern in live mice enables screening of large numbers of transgenic founders quickly and inexpensively. We demonstrate that in the engineered transgenic mice, the chicken β-globin 5′HS4 insulator did not always provide the desirable expression pattern, and the Island Element, responsible for the demethylation of the surrounding DNA region, was not beneficial. Both tested liver-specific and developmentally regulated promoters exhibited the expected expression pattern in most transgenic founders

Increased MicroRNA Activity in Human Cancers

MicroRNAs (miRNAs) are small regulatory RNAs that act by blocking the translation and increasing the degradation of target transcripts. MiRNAs play a critical role in many biological processes including development and differentiation and many studies have shown that major changes in miRNA levels occur in cancer. Since miRNAs degrade target messages, we used this property to develop a novel computational method aimed at determining the actual biological activity of miRNAs using variations in gene expression. Using the method described here, we quantified miRNA activity in papillary thyroid carcinoma and breast cancer, and found a strong and distinctive signal of increased global miRNA activity, embedded in the pertaining gene expression measurements. Interestingly, we found that in these two cancers, miRNA activity is globally increased, and is associated with a global downregulation of miRNA target genes. This downreguation of miRNA regulated genes is particularly noticeable for genes carrying multiple target sites for miRNAs. Among the miRNA-repressed genes, we found a significant enrichment of known tumor suppressors, thereby suggesting that the increased miRNA activity was indeed tumorigenic

Galectin-1 is essential for efficient liver regeneration following hepatectomy

Galectin-1 (Gal1) is a known immune/inflammatory regulator which actsboth extracellularly and intracellularly, modulating innate and adaptive immuneresponses. Here, we explored the role of Gal1 in liver regeneration using 70% partial hepatectomy (PHx) of C57BL/6 wild type and Gal1-knockout (Gal1-KO, Lgals1-/-) mice. Gene or protein expression, in liver samples collected at time intervals from 2 to 168 hours post-operation, was tested by either RT-PCR or by immunoblotting and immunohistochemistry, respectively. We demonstrated that Gal1 transcript and protein expression was induced in the liver tissue of wild type mice upon PHx. Liver regeneration following PHx was significantly delayed in the Gal1-KO compared to the control liver. This delay was accompanied by a decreased Akt phosphorylation, and accumulation of the hepatocyte nuclear p21 protein in the Gal1-KO versus control livers at 24 and 48 hours following PHx. Transcripts of several known regulators of inflammation, cell cycle and cell signaling, including some known PHx-induced genes, were aberrantly expressed (mainly down-regulated) in Gal1-KO compared to control livers at 2, 6 and 24 hours post-PHx. Transient steatosis, which is imperative for liver regeneration following PHx, was significantly delayed and decreased in the Gal1- KO compared to the control liver and was accompanied by a significantly decreased expression in the mutant liver of several genes encoding lipid metabolism regulators.Our results demonstrate that Gal1 protein is essential for efficient liver regeneration following PHx through the regulation of liver inflammation, hepatic cell proliferation, and the control of lipid storage in the regenerating liver.Fil: Potikha, Tamara. Hadassah Hebrew University Medical Center; IsraelFil: Ella, Ezra. Hadassah Hebrew University Medical Center; IsraelFil: Cerliani, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Mizrahi, Lina. Hadassah Hebrew University Medical Center; IsraelFil: Pappo, Orit. Hadassah Hebrew University Medical Center; IsraelFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Galun, Eithan. Hadassah Hebrew University Medical Center; IsraelFil: Goldenberg, Daniel S.. Hadassah Hebrew University Medical Center; Israe

The H19 Non-Coding RNA Is Essential for Human Tumor Growth

Background. Mutations and epigenetic aberrant signaling of growth factors pathways contribute to carcinogenesis. Recent studies reveal that non-coding RNAs are controllers of gene expression. H19 is an imprinted gene that demonstrates maternal monoallelic expression without a protein product; although its expression is shut off in most tissues postnatally, it is reactivated during adult tissue regeneration and tumorigenesis. Moreover, H19 is highly expressed in liver metastasis derived from a range of carcinomas. The objective of this study is to explore the role of H19 in carcinogenesis, and to determine its identification as an anti-tumor target. Methodology / Principle Findings. By controlling oxygen pressure during tumor cell growth and H19 expression levels, we investigated the role of H19 expression in vitro and in vivo in hepatocellular (HCC) and bladder carcinoma. Hypoxia upregulates the level of H19 RNA. Ablations of tumorigenicity of HCC and bladder carcinomas in vivo are seen by H19 knockdown which also significantly abrogates anchorage-independent growth after hypoxia recovery, while ectopic H19 expression enhances tumorigenic potential of carcinoma cells in vivo. Knocking-down H19 message in hypoxic stress severely diminishes p57 kip2 induction. We identified a number of potential downstream targets of H19 RNA, including angiogenin and FGF18. Conclusions. H19 RNA harbors pro-tumorigenic properties, thus the H19 gene behaves as an oncogene and may serve as a potential new target for anti-tumor therapy

HCV Tumor Promoting Effect Is Dependent on Host Genetic Background

BACKGROUND: The hepatitis C virus (HCV) is one of the major risk factors for the development of hepatocellular carcinoma (HCC). Nevertheless, transgenic mice which express the whole HCV polyprotein (HCV-Tg) do not develop HCC. Whereas chronic HCV infection causes inflammation in patients, in HCV-Tg mice, the host immune reaction against viral proteins is lacking. We aimed to test the role of HCV proteins in HCC development on the background of chronic inflammation in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We crossed HCV-Tg mice that do not develop HCC with the Mdr2-knockout (Mdr2-KO) mice which develop inflammation-associated HCC, to generate Mdr2-KO/HCV-Tg mice. We studied the effect of the HCV transgene on tumor incidence, hepatocyte mitosis and apoptosis, and investigated the potential contributing factors for the generated phenotype by gene expression and protein analyses. The Mdr2-KO/HCV-Tg females from the N2 generation of this breeding (having 75% of the FVB/N genome and 25% of the C57BL/6 genome) produced significantly larger tumors in comparison with Mdr2-KO mice. In parallel, the Mdr2-KO/HCV-Tg females had an enhanced inflammatory gene expression signature. However, in the N7 generation (having 99.2% of the FVB/N genome and 0.8% of the C57BL/6 genome) there was no difference in tumor development between Mdr2-KO/HCV-Tg and Mdr2-KO animals of both sexes. The HCV transgene was similarly expressed in the livers of Mdr2-KO/HCV-Tg females of both generations, as revealed by detection of the HCV transcript and the core protein. CONCLUSION: These findings suggest that the HCV transgene accelerated inflammation-associated hepatocarcinogenesis in a host genetic background-dependent manner

Lack of galectin-1 exacerbates chronic hepatitis, liver fibrosis, and carcinogenesis in murine hepatocellular carcinoma model

Chronic liver inflammation (CLI) is a risk factor for development of hepatocellular carcinoma (HCC). Galectin-1 (Gal1) is involved in the regulation of inflammation, angiogenesis, and tumorigenesis, exhibiting multiple anti-inflammatory and protumorigenic activities. We aimed to explore its regulatory role in CLI and HCC progression using an established model of CLI-mediated HCC development, Abcb4 [multidrug-resistance 2 (Mdr2)]-knockout (KO) mice, which express high levels of Gal1 in the liver. We generated double-KO (dKO) Gal1- KO/Mdr2-KO mice on C57BL/6 and FVB/N genetic backgrounds and compared HCC development in the generated strains with their parentalMdr2-KO strains. Loss of Gal1 increased liver injury, inflammation, fibrosis, and ductular reaction in dKO mice of both strains starting from an early age. Aged dKO mutants displayed earlier hepatocarcinogenesis and increased tumor size compared with control Mdr2-KO mice. We found that osteopontin, a well-knownmodulator of HCC development, and oncogenic proteins Ntrk2 (TrkB) and S100A4 were overexpressed in dKO compared with Mdr2-KO livers. Our results demonstrate that in Mdr2-KO mice, a model of CLI-mediated HCC, Gal1-mediated protection from hepatitis, liver fibrosis, and HCC initiation dominates over its known procarcinogenic activities at later stages of HCC development. These findings suggest that anti-Gal1 treatmentsmay not be applicable at all stages of CLI-mediated HCC.—Potikha, T., Pappo, O., Mizrahi, L., Olam, D., Maller, S. M., Rabinovich, G. A., Galun, E., Goldenberg, D. S. Lack of galectin-1 exacerbates chronic hepatitis, liver fibrosis, and carcinogenesis in murine hepatocellular carcinoma model.Fil: Potikha, Tamara. Universidad Academica de Hadassa Jerusalem; IsraelFil: Pappo, Orit. Universidad Academica de Hadassa Jerusalem; IsraelFil: Mizrahi, Lina. Universidad Academica de Hadassa Jerusalem; IsraelFil: Olam, Devorah. Universidad Academica de Hadassa Jerusalem; IsraelFil: Maller, Sebastián M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Rabinovich, Gabriel Adrián. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Galun, Eithan. Universidad Academica de Hadassa Jerusalem; IsraelFil: Goldenberg, Daniel S.. Universidad Academica de Hadassa Jerusalem; Israe

Multiple adaptive mechanisms to chronic liver disease revealed at early stages of liver carcinogenesis in the Mdr2-knockout mice Cancer Res 66

Access the most recent version of this article at: doi: 10.1158/0008-5472.CAN-05-2937 Access the most recent supplemental material at

Systemic siRNA Nanoparticle-Based Drugs Combined with Radiofrequency Ablation for Cancer Therapy

Purpose Radiofrequency thermal ablation (RFA) of hepatic and renal tumors can be accompanied by non-desired tumorigenesis in residual, untreated tumor. Here, we studied the use of micelle-encapsulated siRNA to suppress IL-6-mediated local and systemic secondary effects of RFA. Methods: We compared standardized hepatic or renal RFA (laparotomy, 1 cm active tip at 70±2°C for 5 min) and sham procedures without and with administration of 150nm micelle-like nanoparticle (MNP) anti-IL6 siRNA (DOPE-PEI conjugates, single IP dose 15 min post-RFA, C57Bl mouse:3.5 ug/100ml, Fisher 344 rat: 20ug/200ul), RFA/scrambled siRNA, and RFA/empty MNPs. Outcome measures included: local periablational cellular infiltration (α-SMA+ stellate cells), regional hepatocyte proliferation, serum/tissue IL-6 and VEGF levels at 6-72hr, and distant tumor growth, tumor proliferation (Ki-67) and microvascular density (MVD, CD34) in subcutaneous R3230 and MATBIII breast adenocarcinoma models at 7 days. Results: For liver RFA, adjuvant MNP anti-IL6 siRNA reduced RFA-induced increases in tissue IL-6 levels, α-SMA+ stellate cell infiltration, and regional hepatocyte proliferation to baseline (p<0.04, all comparisons). Moreover, adjuvant MNP anti-IL6- siRNA suppressed increased distant tumor growth and Ki-67 observed in R3230 and MATBIII tumors post hepatic RFA (p<0.01). Anti-IL6 siRNA also reduced RFA-induced elevation in VEGF and tumor MVD (p<0.01). Likewise, renal RFA-induced increases in serum IL-6 levels and distant R3230 tumor growth was suppressed with anti-IL6 siRNA (p<0.01). Conclusions: Adjuvant nanoparticle-encapsulated siRNA against IL-6 can be used to modulate local and regional effects of hepatic RFA to block potential unwanted pro-oncogenic effects of hepatic or renal RFA on distant tumor

A Plant-Derived Recombinant Human Glucocerebrosidase Enzyme—A Preclinical and Phase I Investigation

Gaucher disease is a progressive lysosomal storage disorder caused by the deficiency of glucocerebrosidase leading to the dysfunction in multiple organ systems. Intravenous enzyme replacement is the accepted standard of treatment. In the current report, we evaluate the safety and pharmacokinetics of a novel human recombinant glucocerebrosidase enzyme expressed in transformed plant cells (prGCD), administered to primates and human subjects. Short term (28 days) and long term (9 months) repeated injections with a standard dose of 60 Units/kg and a high dose of 300 Units/kg were administered to monkeys (n = 4/sex/dose). Neither clinical drug-related adverse effects nor neutralizing antibodies were detected in the animals. In a phase I clinical trial, six healthy volunteers were treated by intravenous infusions with escalating single doses of prGCD. Doses of up to 60 Units/kg were administered at weekly intervals. prGCD infusions were very well tolerated. Anti-prGCD antibodies were not detected. The pharmacokinetic profile of the prGCD revealed a prolonged half-life compared to imiglucerase, the commercial enzyme that is manufactured in a costly mammalian cell system. These studies demonstrate the safety and lack of immunogenicity of prGCD. Following these encouraging results, a pivotal phase III clinical trial for prGCD was FDA approved and is currently ongoing.ClinicalTrials.gov NCT00258778