A real-time RT-PCR assay for the quantitiative determination of adenoviral gene expression in tumor cells

Abstract

Oncolytic adenoviruses are exploited as possible anticancer agents in clinical trails. To monitor adenoviral gene expression, a real-time RT-PCR method with a LightCycler™ was developed that allows the rapid and easy quantification of a number of early and late adenoviral genes in infected tumor cells. Primers were designed that can amplify the spliced forms of the genes encoding E1A13S, DNA polymerase (Pol), pre-terminal protein (pTP), adenoviral death protein (ADP), Hexon (Hex) and Penton (Pent) genes. Standard curves were generated using two-fold serial dilutions of cDNAs derived from non-small cell lung cancer (NSCLC) H460 cells infected for 24 h with wild-type adenovirus serotype 5. For all genes correlation coefficients of the standard curves of 0.984 or higher were obtained. The dynamic range of the assay was sufficient to allow the quantitative determination of adenoviral gene expression during a lytic cycle. This RT-PCR assay could be used as a research tool to study the effect of host-cell factors or exogenous treatments on adenoviral gene expression. As example, it is shown that the procedure is suitable to detect changes in adenoviral gene expression in infected H460 cells treated with paclitaxel that is known to enhance the antitumor effect of oncolytic adenoviruses.