Dusan Bartsch

Retinoic acid-mediated repression of human papillomavirus 18 transcription and different ligand regulation of the retinoic acid receptor beta gene in non-tumorigenic and tumorigenic HeLa hybrid cells.

Human papillomavirus type 18 (HPV18) belongs to the group of genital papillomaviruses involved in the development of cervical carcinomas. Since retinoic acid (RA) is a key regulator of epithelial cell differentiation and a growth inhibitor in vitro of HPV18‐positive HeLa cervical carcinoma cells, we have used HeLa and HeLa hybrid cells in order to analyse the effects of RA on expression of the HPV18 E6 and E7 oncogenes and of the cellular RA receptor genes RAR‐beta and ‐gamma. We show here that RA down‐regulates HPV18 mRNA levels apparently due to transcriptional repression. Transient cotransfection assays indicated that RARs negatively regulate the HPV18 upstream regulatory region and that the central enhancer can confer RA‐dependent repression on a heterologous promoter. RA treatment resulted in induction of RAR‐beta mRNA levels in non‐tumorigenic HeLa hybrid cells, but not in tumorigenic hybrid segregants nor in HeLa cells. No alterations of the RAR‐beta gene or of the HeLa RAR‐beta promoter could be revealed by Southern and DNA sequence analysis, respectively. As determined by transient transfection assays, however, the RAR‐beta control region was activated by RA more strongly in non‐tumorigenic hybrid cells than in HeLa cells, thus indicating differences in trans‐acting regulatory factors. Our data suggest that the RARs are potential negative regulators of HPV18 E6 and E7 gene expression, and that dysregulation of the RAR‐beta gene either causatively contributes to or is an indicator of tumorigenicity in HeLa and HeLa hybrid cells.

Quantitative analysis of conditional gene inactivation using rationally designed, tetracycline-controlled miRNAs

The combination of RNA interference (RNAi) with the tetracycline-controlled transcription activation (tet) system promises to become a powerful method for conditional gene inactivation in cultured cells and in whole organisms. Here, we tested critical sequence elements that originated from miRNA mR-30 for optimal efficiency of RNAi-based gene knockdown in mammalian cells. Rationally designed miRNAs, expressed conditionally via the tet system, led to an efficient knockdown of the expression of both reporter genes and the endogenous mitotic spindle protein TPX2 in HeLa cells. Quantitative studies of the tet-controlled gene inactivation revealed that the residual expression of the target gene is an intrinsic attribute of all cells that cannot be eliminated either by increasing the miRNA to target mRNA ratio or by simultaneous expression of miRNAs targeting different sequences within the transcript. The kinetic analysis of the reversibility of the miRNA mediated knockdown suggests that the recovery of target gene expression is primarily driven by cell division. Our miRNA design provides a useful tool for conditional gene inactivation in combination with the RNA-polymerase II based tet system. The identified characteristics of the conditional RNAi-mediated knockdown need to be considered for its application in cell culture or in vivo.