| Popis: |
A novel gene, named as the PHDX gene, had been previously identified while screening genes for their involvement in resistance to the chemotherapeutic drug etoposide and to hydrogen peroxide, using the methodology of retrovirus promoter trap mutagenesis. This study was undertaken for the purpose of testing whether the loss of PHDX gene is responsible for drug resistance in CHO-E-126 cell line which was created from Chinese hamster ovary cells having a retroviral receptor and selected for etoposide resistance. The PHDX gene resides on mouse chromosome 11 and has homology with the prolyl hydroxylase gene family. We hypothesized that the inactivation of the PHDX gene by promoter trap mutagenesis will confer resistance to etoposide and hydrogen peroxide in the E-126 cell line. In addition, the alteration of the cellular hydroxyproline levels by the loss of the gene might influence the drug response through the production of oxygen free radicals. To study the involvement of the PHDX gene in etoposide and hydrogen peroxide induced drug-resistance, we used two experimental approaches to modulate the function of the gene in cells. First, we silenced the expression of this gene by RNA interference (RNAi) through stable and transient knockdown experiments in the parental Chinese hamster ovary (CHO-K1)cells, and second, we overexpressed the gene in CHO-Cl-22 and CHO-E-126 cells. We assessed the effect of the knockdown by RT-PCR. The effect of etoposide and hydrogen peroxide was determined by the clonogenic crystal violet staining assay and the MTT assay. Through our siRNAi knockdown studies, we were able to demonstrate the involvement of the gene in drug resistance. We were unable to show that the overexpression of the gene was capable of reverting to the drug sensitive phenotype. |
