REVERSAL OF MULTIPLE-DRUG RESISTANCE IN-VITRO BY PHOSPHORATHIOATE OLIGONUCLEOTIDES AND RIBOZYMES

In the present study we investigated the effectiveness of 14, 15 and 18 nucleotide antisense phosphorothioate oligonucleotides (S-ODNs) directed to four different regions of the published mdr-1 gene sequence to reduce the level of mdr-1 gene product (p170, P-glycoprotein) and its function in the over-expressing cell lines LoVoDx(R), S180Dx(R) and KBCh(R)8-5. The highest efficiency in reduction of multiple drug resistance was obtained at a concentration of 2 mu M. In proliferation assays a growth reduction of 50% was observed after exposure of doxorubicin-resistant cells to S-ODN3. p170 protein expression of the resistant cell line LoVoDx(R) was reduced to the level of the sensitive cell line LoVo as shown by Western blot analysis. S-ODN3 reduced the ID50 of the two human cell lines up to 60% (LoVoDx(R)) and 21% (KBCh(R)8-5), respectively, but showed no effect in the murine cell line S180Dx(R). In contrast, S-ODN1 was most effective in the murine system (67% reduction of the ID50), less effective in LoVoDx(R) (34%) and exhibited no effect in cell line KBCh(R)8-5. Based on the results with the antisense oligonucleotides, a ribozyme directed against the mRNA target region of S-ODN3 was designed. This ribozyme was able to reduce the mdr-1 mRNA in total RNA preparations from cell line LoVoDx(R) up to 80% after an incubation time of 6 h in the presence of 10 mM MgCl2 at pH 7.5. A modified ribozyme was investigated in cell culture and reduced chemo-resistance of the resistant cell line LoVoDx(R) and ex vivo cultured blasts of acute myeloid leukemia patients up to 50%.