Mutation of the K-ras gene is an early event in the development of pancreatic adenocarcinoma and, therefore, RNA interference (RNAi) directed toward mutant K-ras could represent a novel therapy. In this study, we examine the phenotypic and molecular consequences of exposure of pancreatic tumor cells to mutant-specific K-ras small interfering RNA. Specific reduction of activated K-ras via RNAi in Panc-1 and MiaPaca-2 cells resulted in cellular changes consistent with a reduced capacity to form malignant tumors. These changes occur through distinct mechanisms but likely reflect an addiction of each cell line to oncogene stimulation. Both cell lines show reduced proliferation after K-ras RNAi, but only MiaPaca-2 cells showed increased apoptosis. Both cell lines showed reduced migration after K-ras knockdown, but changes in integrin levels were not consistent between the cell lines. Both cell lines showed alteration of the level of GLUT-1, a metabolism-associated gene that is downstream of c-myc, with Panc-1 cells demonstrating decreased GLUT-1 levels, whereas MiaPaca-2 cells showed increased levels of expression after K-ras knockdown. Furthermore, after K-ras RNAi, there was a reduction in angiogenic potential of both Panc-1 and MiaPaca-2 cells. Panc-1 cells increased the level of expression of thrombospondin-1, an endogenous inhibitor of angiogenesis, whereas MiaPaca-2 cells decreased the production of vascular endothelial growth factor, a primary stimulant of angiogenesis in pancreatic tumors. We have found that silencing mutant K-ras through RNAi results in alteration of tumor cell behavior in vitro and suggests that targeting mutant K-ras specifically might be effective against pancreatic cancer in vivo. (Mol Cancer Res 2005; 3(7):413-23)

Abstract-Elevated plasma homocysteine is an independent risk factor for atherosclerosis. We hypothesized that homocysteine enhances monocyte/human aortic endothelial cell (HAEC) interactions, a pivotal early event in atherogenesis, by upregulating endothelial adhesion molecules. After incubation of cultured HAECs with reduced DL-homocysteine for up to 24 hours, adhesion of human monocytes to homocysteine-stimulated HAECs was significantly upregulated in a time-and dose-dependent fashion. Pretreatment of HAECs with 100 mol/L homocysteine caused a 4.5-fold increase in the adhesion of normal human monocytes (PϽ0.001). Similarly, adhesion of monocytic U937 cells was maximally elevated by 3.5-fold at 100 mol/L homocysteine (PϽ0.001). In support of our hypothesis, vascular cell adhesion molecule (VCAM)-1 mRNA expression increased 5-fold in HAECs after 3 hours of treatment with 100 mol/L homocysteine, as assessed by quantitative reverse transcription-polymerase chain reaction. Neutralizing antibody studies confirmed the involvement of VCAM-1 in mediating monocyte adhesion to homocysteine-stimulated HAECs. Coincubation of HAECs with homocysteine and tumor necrosis factor-␣ synergistically elevated monocyte adhesion as well as VCAM-1 protein expression, with the latter evaluated by flow cytometry. Preincubation of HAECs with cyclooxygenase inhibitors completely abrogated homocysteine-induced monocyte adhesion, whereas scavenging reactive oxygen species and the elevation of NO caused partial inhibition only. These data support the notion that the proinflammatory effects of homocysteine may have important implications in atherogenesis. Key Words: human aortic endothelial cells Ⅲ homocysteine Ⅲ monocyte adhesion Ⅲ vascular cell adhesion molecule-1 Ⅲ cyclooxygenase H omocysteine (Hcy) is a sulfhydryl amino acid metabolite of dietary methionine. Elevated plasma Hcy is an independent cardiovascular risk factor that is associated with accelerated atherosclerosis and increased cerebrovascular/ischemic heart disease. 1 Without intervention, nearly 50% of the patients with congenital hyperhomocystinuria (ie, total plasma Hcy [tHcy] levels Ͼ200 mol/L versus 7 to 14 mol/L in normal individuals) will experience a major cardiovascular event by the age of 30 years. 2 However, more frequently seen in the general population are modestly elevated tHcy levels, which are nonetheless strong predictors of existent and future development of vascular pathologies. 3 Elevated Hcy appears to contribute to cardiovascular disease, in part, by inducing endothelial cell (EC) dysfunction. In vivo, moderately elevated tHcy causes EC damage, 4 exacerbates hypertension-related atherosclerosis, 4 and impairs flowmediated arterial dilation. 5 In vitro, Hcy-thiolactone is cytotoxic to ECs, 6 and the free reduced thiol (HcyH) alters the endothelial expression of bioactive molecules, such as NO, interleukin (IL)-8, and tissue factor. 7,8 Additionally, HcyH increases leukocyte adhesion to cultured human umbilical vein ECs, 9 and elevated tHcy induces adhesion m...

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The expression of genetic material governs brain development, differentiation, and function, and targeted manipulation of gene expression is required to understand contributions of gene function to health and disease states. Although recent improvements in CRISPR/dCas9 interference (CRISPRi) technology have enabled targeted transcriptional repression at selected genomic sites, integrating these techniques for use in non-dividing neuronal systems remains challenging. Previously, we optimized a dual lentivirus expression system to express CRISPR-based activation machinery in post-mitotic neurons. Here we used a similar strategy to adapt an improved dCas9-KRAB-MeCP2 repression system for robust transcriptional inhibition in neurons. We find that lentiviral delivery of a dCas9-KRAB-MeCP2 construct driven by the neuron-selective human synapsin promoter enabled transgene expression in primary rat neurons. Next, we demonstrate transcriptional repression using CRISPR sgRNAs targeting diverse gene promoters, and show superiority of this system in neurons compared to existing RNA interference methods for robust transcript specific manipulation at the complex Brain-derived neurotrophic factor (Bdnf) gene. Our findings advance this improved CRISPRi technology for use in neuronal systems for the first time, potentially enabling improved ability to manipulate gene expression states in the nervous system.

Using an orthotopic model of tumor growth and metastasis identifies distinct metastatic profiles associated with molecular alterations of the TGF-beta/Smad4 pathway and provides insight with regard to the biologic consequences of these changes.