Reversal of multiple drug resistance in vitro by phosphorothioate 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 Lo-VoDxR, S180DxR and KBChR8-5. The highest efficiency in reduction of multiple drug resistance was obtained at a concentration of 2 microM. 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 LoVoDxR 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% (LoVoDxR) and 21% (KBChR8-5), respectively, but showed no effect in the murine cell line S180DxR. In contrast, S-ODN1 was most effective in the murine system (67% reduction of the ID50), less effective in LoVoDxR (34%) and exhibited no effect in cell line KBChR8-5. 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 LoVoDxR 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 LoVoDxR and ex vivo cultured blasts of acute myeloid leukemia patients up to 50%.
Bertram
J
J
Department of Hematology/Oncology, University Clinics, Göttingen, Germany.
Palfner
K
K
Killian
M
M
Brysch
W
W
Schlingensiepen
K H
KH
Hiddemann
W
W
Kneba
M
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000235:genetics; D052138:Genes, Neoplasm / Q000187:drug effects*; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D016337:RNA, Catalytic / Q000494:pharmacology*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors; D012334:RNA, Neoplasm / Q000037:antagonists & inhibitors; D013629:Tamoxifen / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D009363:Neoplasm Proteins; D016376:Oligonucleotides, Antisense; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D013873:Thionucleotides; D013629:Tamoxifen; D004317:Doxorubicin; D014700:Verapamil