Transcriptional profiling of the effects of 25-hydroxycholesterol on human hepatocyte metabolism and the antiviral state it conveys against the hepatitis C virus.

BACKGROUND: Hepatitis C virus (HCV) infection is a global health problem. A number of studies have implicated a direct role of cellular lipid metabolism in the HCV life cycle and inhibitors of the mevalonate pathway have been demonstrated to result in an antiviral state within the host cell. Transcriptome profiling was conducted on Huh-7 human hepatoma cells bearing subgenomic HCV replicons with and without treatment with 25-hydroxycholesterol (25-HC), an inhibitor of the mevalonate pathway that alters lipid metabolism, to assess metabolic determinants of pro- and antiviral states within the host cell. These data were compared with gene expression profiles from HCV-infected chimpanzees. RESULTS: Transcriptome profiling of Huh-7 cells treated with 25-HC gave 47 downregulated genes, 16 of which are clearly related to the mevalonate pathway. Fewer genes were observed to be upregulated (22) in the presence of 25-HC and 5 genes were uniquely upregulated in the HCV replicon bearing cells. Comparison of these gene expression profiles with data collected during the initial rise in viremia in 4 previously characterized HCV-infected chimpanzees yielded 54 overlapping genes, 4 of which showed interesting differential regulation at the mRNA level in both systems. These genes are PROX1, INSIG-1, NK4, and UBD. The expression of these genes was perturbed with siRNAs and with overexpression vectors in HCV replicon cells, and the effect on HCV replication and translation was assessed. Both PROX1 and NK4 regulated HCV replication in conjunction with an antiviral state induced by 25-hydroxycholesterol. CONCLUSION: Treatment of Huh-7 cells bearing HCV replicons with 25-HC leads to the downregulation of many key genes involved in the mevalonate pathway leading to an antiviral state within the host cell. Furthermore, dysregulation of a larger subset of genes not directly related to the mevalonate pathway occurs both in 25-HC-treated HCV replicon harbouring cells as well as during the initial rise in viremia in infected chimpanzees. Functional studies of 3 of these genes demonstrates that they do not directly act as antiviral gene products but that they indirectly contribute to the antiviral state in the host cell. These genes may also represent novel biomarkers for HCV infection, since they demonstrate an outcome-specific expression profile.

Evaluation of chemical labeling strategies for monitoring HCV RNA using vibrational microscopy.

Raman and coherent anti-Stokes Raman scattering (CARS) microscopies have the potential to aid in detailed longitudinal studies of RNA localization. Here, we evaluate the use of carbon-deuterium and benzonitrile functional group labels as contrast agents for vibrational imaging of hepatitis C virus (HCV) replicon RNA. Dynamic light scattering and atomic force microscopy were used to evaluate the structural consequences of altering HCV subgenomic replicon RNA. Modification with benzonitrile labels caused the replicon RNA tertiary structure to partially unfold. Conversely, deuterium-modified replicon RNA was structurally similar to unmodified replicon RNA. Furthermore, the deuterated replicon RNA provided promising vibrational contrast in Raman imaging experiments. The functional effect of modifying subgenomic HCV replicon RNA was evaluated using the luciferase gene as a genetic reporter of translation. Benzonitrile labeling of the replicon RNA prevented translation in cell-based luciferase assays, while the deuterated replicon RNA retained both translation and replication competency. Thus, while the scattering cross-section for benzonitrile labels was higher, only carbon-deuterium labels proved to be non-perturbative to the function of HCV replicon RNA.

Synthesis and characterization of CN-modified protein analogues as potential vibrational contrast agents.

A recombinant VH single-domain antibody recognizing staphylococcal protein A was functionalized on reactive lysine residues with N-hydroxysuccimidyl-activated 4-cyanobenzoate. Surface plasmon resonance analysis of antibody-antigen binding revealed that modified and unmodified antibodies bound protein A with similar affinities. Raman imaging of the modified antibodies indicated that the benzonitrile group provides vibrational contrast enhancement in a region of the electromagnetic spectrum that is transparent to cellular materials. Thus, the modified single-domain antibody may be amenable to detecting protein A from samples of the human pathogen Staphylococcus aureus using vibronic detection schemes such as Raman and coherent anti-Stokes Raman scattering. The generality of this labeling strategy should make it applicable to modifying an array of proteins with varied structure and function.

Picoliter wells from selective growth of HEK293 cells on chemically modified PDMS surfaces.

In this study, a method for the rapid generation of a variety of bifunctional surfaces that can serve to quickly determine the selective adhesion of HEK293 cells towards different chemical functionalities has been established. Using the information about selective adhesion of HEK293 cells to bifunctional surfaces, we demonstrate the ability to construct stable, high density, and multi-welled surfaces where the mammalian cells form the walls of picoliter volume wells.

Simultaneous quantitative measurement of luciferase reporter activity and cell number in two- and three-dimensional cultures of hepatitis C virus replicons.

Hepatitis C virus (HCV) is a global health problem and an important human pathogen. The development of cell culture models for HCV infection has been difficult to accomplish, primarily because HCV is very sensitive to the host cell state. Future models will require the use of three-dimensional (3D) cultures that model the host cell state and environment more accurately. Higher information content screens for anti-HCV therapeutics will also involve 3D cell cultures. Here we report a method for screening cell models for HCV replication that involves normalizing luciferase reporter activity based on cell number in two-dimensional (2D) and 3D HCV replicon cultures. Human hepatoma cells stably replicating luciferase-containing HCV replicons were cultured in 2D monolayer culture and 3D spheroid culture. Optimization of cell lysis was performed so that cell lysates could be used to quantify both luciferase activity and cellular DNA content. Cellular DNA content was quantified using Hoechst 33258 dye and was converted to cell number. The method is straightforward, reproducible, and sensitive down to 5000 cells. This method enables low-throughput but high-information content screening of HCV replicons, with the potential for high-throughput screening in a variety of 3D cultures and cocultures.

Cyclic AMP in ovarian cancer cells both inhibits proliferation and increases c-KIT expression.

C-KIT encodes a tyrosine kinase receptor (KIT) that, when activated by its ligand (KL), stimulates proliferation, differentiation, migration, and survival. Greater than 70% of epithelial ovarian cancers coexpress c-KIT and KL. C-KIT and KL expression levels have been shown to be up-regulated by cAMP in some cell types. Additionally, cAMP is well-recognized for its anti-proliferative effects in cancer cells. The goal of these experiments was to investigate these seemingly contradictory consequences of cAMP treatment by: (1) confirming the growth inhibitory actions of cAMP on ovarian cancer cells; (2) investigating the ability of cAMP to affect c-KIT and KL expression in these cells; and (3) examining the possible role of endogenous and/or cAMP-regulated c-KIT and KL expression in ovarian cancer cell proliferation. HEY cells, an ovarian cancer cell line which expresses c-KIT and KL, were treated with dibutyryl cyclic AMP (dbcAMP), 8-bromo-cAMP, and cholera toxin over a range of concentrations. With all treatments, stimulation of cAMP signaling caused a dose-dependent inhibition of HEY cell proliferation by up to 40, 62, and 38%, respectively. This inhibition of proliferation correlated with a dose-dependent increase in c-KIT mRNA expression, yielding 4- to 7-fold elevations in transcript abundance; there were no changes in steady-state levels of KL transcripts. In order to determine whether KIT expression/activity was responsible for the observed decrease in proliferation, dbcAMP-treated HEY cells were exposed either to anti-KIT neutralizing antibodies or to the KIT inhibitor STI571. These experiments demonstrated that KIT inhibition did not alter the growth rate of cells or reverse the dbcAMP-induced inhibition of proliferation. These results suggest that cAMP signaling pathways regulate both cell proliferation and c-KIT expression in ovarian cancer cells; however, KIT is not assuming its well-established role as a growth factor.