Modulation of dihydrofolate reductase gene expression in methotrexate-resistant human leukemia CCRF-CEM/E cells by antisense oligonucleotides

An increase in the cellular levels of dihydrofolate reductase (DHFR) is one of the most common mechanisms of tumor resistance to methotrexate (MTX), a n antimetabolite that is widely used in the treatment of a variety of human malignancies. The MTX-resistant phenotype generally occurs as a consequenc e of DHFR gene amplification which in turn is responsible for DHFR gene ove rexpression. We have designed antisense oligodeoxynucleotides (aODNs) again st the DHFR mRNA and tested their in vitro effect on human leukemia CCRF-CE M/E cells, overexpressing the DHFR gene about 20-fold in comparison with th e CCRF-CEM/S parental cell line. An aODN complementary to a region encompas sing the AUG translation start (DHFR1) of DHFR mRNA and a mixture of two aO DNs complementary to the 5' untranslated region (DHFR2+DHFR3) have been use d. A DHFR1 scrambled-sequence ODN and a fully degenerated ODN were the cont rols. All ODNs had a phosphodiester backbone. DHFR1 and the relevant scramb led ODN were also capped with two phosphorothioate derivatives at both the 5' and 3' ends in order to increase ODN stability against serum nucleases. ODNs were vehiculated with a cationic lipid, N-[1-(dioleoyloxy)propyl]-N,N, N-trimethylammonium methyl sulfate (DOTAP), known to enhance ODN cell uptak e and biological activity. The effects of ODNs on DHFR gene expression were studied after a 4 day treatment by measuring both DHFR mRNA levels, using a semi-quantitative reverse transcription polymerase chain reaction method, and DHFR protein levels by flow cytometry. A marked reduction in DHFR mRNA levels (79.7 and 74.2%, respectively) was observed with both DHFR1 and DHF R2+DHFR3 aODNs, associated with a lower decrease in DHFR enzyme (44.8 and 6 1%, respectively). aODN effects on MTX cytotoxicity in CCRF-CEM/E cells wer e also assessed. No marked enhancement of in vitro MTX cytotoxicity was obs erved following co-exposure of cells with aODNs and the tested concentratio ns of the antifol (0.05 and 0.5 mu M), indicating that no substantial rever sal of the MTX-resistant phonotype was induced by the study aODNs. [(C) 200 0 Lippincott Williams & Wilkins.].