Induction and characterization of taxol-resistance phenotypes with a transiently expressed artificial transcriptional activator library

Abstract

Phenotype-based functional genomic methods are useful for the identification of genes that are related to a particular biological function or disease. Essential to this approach is the ability to regulate the expression of selected genes. Artificial transcription factors (ATFs) are key molecular tools that selectively regulate gene expression in vivo. Here, we use an ATF library to identify genes that participate in rendering a cell resistant to the drug Taxol, a potent anti-cancer drug that binds to tubulin and inhibits cell division. The library, which encodes ATFs that activate (rather than inhibit) transcription, was introduced into a HeLa cell line, and Taxol-resistant cells were selected. After eight rounds of selection, we identified two ATFs that significantly increased the level of Taxol resistance (TR) in HeLa cells. Gene expression microarray experiments using these ATFs identified 37 co-regulated genes, including genes already known to participate in TR. This study demonstrates that ATF libraries can be used to induce phenotypic alterations in eukaryotic cells and then identify specific genes that are associated with the phenotype of choice.

Figure 1

Procedure for screening an ATF library for ATFs that induce TR in HeLa cells. (a) An ATF library was transiently transfected into HeLa cells, and the transfected cells were incubated in the presence of Taxol. Colonies of cells that survived the Taxol treatment were pooled, and genomic DNA was isolated and used to transform E.coli for plasmid amplification. A selected ATF library was prepared from a pool of E.coli transformants and used again for HeLa cell transfection. The whole procedure was repeated eight times, and two ATFs were finally isolated from individual E.coli transformants. (b) Conditions of Taxol treatment for each selection cycle. To maximize the effect of the screening, Taxol concentrations and exposure times were increased as the cycles proceeded.

Figure 2

Expression of two ATFs, TR17-p65 and TR216-p65, increased TR in HeLa cells. (a) HeLa cells grown in a 12-well plate were transfected with 500 ng of a plasmid encoding one of the ATFs and 200 ng of a lacZ reporter plasmid and were treated with Taxol at 100 nM for 48 h. Cells were stained for lacZ activity to differentiate transfected cells from untransfected ones. (b) The survival rate of HeLa cells transfected with each of the ATF-encoding plasmids was calculated as follows: Survival rate (%) = the number of blue cells in samples treated with Taxol/the number of blue cells in samples not treated with Taxol × 100.

Figure 3

The survival rates of HeLa cells transfected with TR17-p65 intact and mutated plasmids. The TR17-p65 sequence was mutated such that either the RDNQ-encoding sequences in the first two ZFDs were mutated to yield ADNQ (mTR17-p65) or the p65 functional domain was deleted (TR17-no FD). Two control plasmids, empty vector (Vector-no FD) and vector expressing only p65 without any ZFDs (Vector-p65), were also tested. The transfections and Taxol treatments were carried out as shown in Figure 2.