Validation of a human cell based high-throughput genotoxicity assay 'Anthem's Genotoxicity screen' using ECVAM recommended lists of genotoxic and non-genotoxic chemicals.

A novel high throughput-enabled human cell based screen, Anthem's Genotoxicity screen, was developed to achieve higher specificity for predicting in vivo genotoxins by an in vitro method. The assay employs engineered human colon carcinoma cell line; HCT116 cells that are stably engineered with three promoter-reporter cassettes such that an increased reporter activity reflects the activation of associated signaling events in a human cell. The current study focuses on the evaluation of sensitivity and specificity of Anthem's Genotoxicity screen using 62 compounds recommended by the European Centre for the Validation of Alternative Methods (ECVAM). The concordance of Anthem's Genotoxicity screen with in vivo tests was 95.5% with sensitivity of 95.2% and specificity of 95.7%. Thus Anthem's Genotoxicity screen, a high-throughput mechanism based genotox indicator test can be employed by a variety of industries for rapid screening and early detection of potential genotoxins.

Effect of different UCOE-promoter combinations in creation of engineered cell lines for the production of Factor VIII.

BACKGROUND: The most common approach used in generating cell lines for the production of therapetic proteins relies on gene amplification induced by a drug resistance gene e. g., DHFR and glutamine synthetase. Practically, this results in screening large number of clones for the one that expresses high levels of the biologic in a stable manner. The inefficiency of mammalian vector systems to express proteins in a stable manner typically involves silencing of the exogenous gene resulting from modifications such as methylation of CpG DNA sequences, histone deacetylation and chromatin condensation. The use of un-methylated CpG island fragments from housekeeping genes referred to as UCOE (ubiquitous chromatin opening elements) in plasmid vectors is now well established for increased stability of transgene expression. However, few UCOE-promoter combinations have been studied to date and in this report we have tested 14 different combinations. FINDINGS: In this report we describe studies with two different UCOEs (the 1.5 Kb human RNP fragment and the 3.2 Kb mouse RPS3 fragment) in combination with various promoters to express a large protein (B domain deleted factor VIII; BDD-FVIII) in a production cell line, BHK21. We show here that there are differences in expression of BDD-FVIII by the different UCOE-promoter combinations in both attached and serum free suspension adapted cells. In all cases, the 1.5 Kb human RNP UCOE performed better in expressing BDD-FVIII than their corresponding 3.2 Kb mouse RPS3 UCOE. Surprisingly, in certain scenarios described here, expression from a number of promoters was equivalent or higher than the commonly used and industry standard human CMV promoter. CONCLUSION: This study indicates that certain UCOE-promoter combinations are better than others in expressing the BDD-FVIII protein in a stable manner in BHK21 cells. An empirical study such as this is required to determine the best combination of UCOE-promoter in a vector for a particular production cell line.

Inhibition of p53 by lentiviral mediated shRNA abrogates G1 arrest and apoptosis in retinal pigmented epithelial cell line.

We silenced p53 gene expression in ARPE-19, a human retinal pigmented epithelial cell line using RNA interference. The effect of silencing the p53 gene in proliferating ARPE-19 cells was studied. Four short hairpin RNAs (shRNAs) targeting different regions of human p53 mRNA were delivered individually into ARPE-19 cells using lentiviral vector to produce stable cell lines. p53 mRNA and protein levels were reduced to varying extents in the four shRNA-transduced ARPE-19 cell lines. The cell line that showed greatest reduction (85-90%) of p53 expression showed decreased p21 promoter activation after DNA damage with camptothecin, etoposide and MMS. Whereas treatment of wild type ARPE-19 cells with camptothecin resulted in apoptosis, silencing p53 expression increased their survival. Cell cycle analyses indicated that irradiation resulted in a G(1) arrest in ARPE-19 cells, and that the arrest was significantly reduced in p53-silenced cells. Thus, p53 plays a central role in the response of ARPE-19 cells to DNA damaging agents that act via different mechanisms. Additionally, ARPE-19 cells with reduced p53 expression behave similar to tumor cell lines with mutated or non-functional p53. The present data demonstrate the utility of lentiviral vectors to create stable isogenic cell lines with reduced expression of a specific gene, thereby permitting the study of the function of a gene, the pathways controlled by it, and the effect of therapeutics on a cell with altered genetic makeup in a pair-wise fashion.