Concerning Antisense Inhibition of the Multiple Drug Resistance Gene

Abstract

Recently, Vasanthakumar and Ahmed reported (Vasanthakumar, G.; Ahmed, N.K., Cancer Communications 1:225-232; 1989) a complete inhibition of the multiple drug resistance gene (MDR1) in the K562/III erythroleukemia cells, using a 15 bases-long methylphosphonate oligodeoxynucleotide analog. The sequence used, however, contained three mismatches relative to the corresponding fragment of the human MDR1 gene and, hence, the results reported cannot at present be regarded as a classical antisense effect. We have made attempts to inhibit the expression of the MDR1 gene in MCF-7 human breast cancer cells selected for resistance to Adriamycin using phosphorothioate analogs of oligodeoxynucleotides. Studies with model 35S-labeled-phosphorothioates indicated poor uptake of the compounds into the cells; the radioactivity was located mainly in the soluble fraction (cytoplasm), but membranes and the nuclear fraction were also labeled. Unmodified oligodeoxynucleotides were toxic to the cells, whereas the phosphorothioates were not. The MDR1 inhibition with phosphorothioates was studied by measuring their effects on adriamycin toxicity and by immunocytochemical titration of P170. Elevation of adriamycin cytotoxicity consistent with a decreased drug resistance was observed with one antisense sequence, but the immunocytochemical assay indicated only slight inhibition of the synthesis of P170. In the wild type (drug sensitive) MCF-7 cells phosphorothioates decreased adriamycin toxicity in a sequence-independent manner. The results indicate that the effects of antisense oligodeoxynucleotides on cells are complex. Computer simulation of the secondary structure of MDR1 mRNA indicated not only extensive folding but, also, the presence of many regions not involved in intramolecular hybridization, which are of potential interest as targets for antisense oligodeoxynucleotides.