Targeting MDR1 gene: synthesis and cellular study of modified daunomycin-triplex-forming oligonucleotide conjugates able to inhibit gene expression in resistant cell lines.

Reversal of the multidrug-resistant (MDR) phenotype is very important for chemotherapy success. In fact, the expression of the MDR1 gene-encoded P-glycoprotein (P-gp) actively expels antitumor agents such as daunomycin (DNM) out of the cells, resulting in drug resistance. We show that upon conjugation to triplex-forming oligonucleotides, it is possible to address DNM in resistant cells (MCF7-R and NIH-MDR-G185).

Modulation of the efficiency of a siRNA directed against MDR1 expression in MCF7-R cells when combined with a second siRNA.

Effective silencing of MDR1, one of the genes involved in the multidrug resistance phenotype, can be achieved by the use of an efficient siRNA transfected into the doxorubicin-selected MCF7-R human cell line, alone or combined with a moderately efficient siRNA. On the contrary, there is no MDR1 silencing when it is co-transfected with a control siRNA that does not target the human genome. This results from the limited amount of RISC (RNA-Induced Silencing Complex) in human cells, leading to competition between siRNAs.

Solution structure of a purine rich hexaloop hairpin belonging to PGY/MDR1 mRNA and targeted by antisense oligonucleotides.

A preferential target of antisense oligonucleotides directed against human PGY/MDR1 mRNA is a hairpin containing a stem with a G*U wobble pair, capped by the purine-rich 5'r(GGGAUG)3' hexaloop. This hairpin is studied by multidimensional NMR and restrained molecular dynamics, with special emphasis on the conformation of south sugars and non-standard phosphate linkages evidenced in both the stem and the loop. The hairpin is found to be highly structured.

Modulation of MDR1 gene expression in multidrug resistant MCF7 cells by low concentrations of small interfering RNAs.

MDR1 overexpression is one form of the multidrug resistance (MDR) phenotype, which can be acquired by patients initially responsive to chemotherapy. Because of the high toxicity of the inhibitors of P-glycoprotein (P-gp), the protein encoded by MDR1, attention has been focused on selective modulation of the MDR1 gene. Small interfering RNAs (siRNAs) were shown to be powerful tools for such a purpose, even when used at low concentrations (< or =20 nM) in order to avoid sequence nonspecific effects.

The reduction of P-glycoprotein expression by small interfering RNAs is improved in exponentially growing cells.

Small interfering RNAs (siRNAs) are powerful tools in specifically silencing gene expression. Nevertheless, their efficiency can be limited when targeting proteins with an unusually long half-life, such as P-glycoprotein (P-gp), which is involved in the multidrug resistance phenomenon. P-gp is characterized by a long half-life, which may vary depending on the cell line and, for some of them, on serum deprivation or high cell density.