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Modulation of dihydrofolate reductase gene expression in methotrexate-resistant human leukemia CCRF-CEM/E cells by
M Morganti1, M Coronnello, B Caciagli
1Dipartimento di Farmacologia Preclinica e Clinica, Università degli Studi di Firenze, Italy.
Abstract:
An increase in the cellular levels of dihydrofolate reductase (DHFR) is one of the most common mechanisms of tumor resistance to methotrexate (MTX), an antimetabolite that is widely used in the treatment of a variety of human malignancies. The MTX-resistant phenotype generally occurs as a consequence of DHFR gene amplification which in turn is responsible for DHFR gene overexpression. We have designed antisense oligodeoxynucleotides (aODNs) against the DHFR mRNA and tested their in vitro effect on human leukemia CCRF-CEM/E cells, overexpressing the DHFR gene about 20-fold in comparison with the CCRF-CEM/S parental cell line. An aODN complementary to a region encompassing the AUG translation start (DHFR1) of DHFR mRNA and a mixture of two aODNs complementary to the 5' untranslated region (DHFR2+DHFR3) have been used. A DHFR1 scrambled-sequence ODN and a fully degenerated ODN were the controls. All ODNs had a phosphodiester backbone. DHFR1 and the relevant scrambled 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 uptake 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 DHFR2+DHFR3 aODNs, associated with a lower decrease in DHFR enzyme (44.8 and 61%, respectively). aODN effects on MTX cytotoxicity in CCRF-CEM/E cells were also assessed. No marked enhancement of in vitro MTX cytotoxicity was observed following co-exposure of cells with aODNs and the tested concentrations of the antifol (0.05 and 0.5 microM), indicating that no substantial reversal of the MTX-resistant phenotype was induced by the study aODNs.
Insights
Antisense oligodeoxynucleotides (aODNs) targeting dihydrofolate reductase (DHFR) mRNA reduced DHFR levels in resistant leukemia cells. However, these DHFR-targeting aODNs did not significantly reverse methotrexate resistance in vitro.
Area of Science:
- Molecular Biology
- Cancer Research
- Pharmacology
Background:
- Increased cellular dihydrofolate reductase (DHFR) levels are a common mechanism of tumor resistance to methotrexate (MTX).
- DHFR gene amplification leads to DHFR overexpression, contributing to the MTX-resistant phenotype in malignancies.
- Methotrexate is a widely used antimetabolite in treating various human cancers.
Purpose of the Study:
- To design and test antisense oligodeoxynucleotides (aODNs) targeting DHFR mRNA in MTX-resistant human leukemia cells.
- To evaluate the in vitro efficacy of aODNs in reducing DHFR gene expression and reversing MTX resistance.
Main Methods:
- Designed aODNs targeting DHFR mRNA, including DHFR1 (translation start) and DHFR2+DHFR3 (5' untranslated region).
- Tested aODNs in human leukemia CCRF-CEM/E cells overexpressing DHFR, using scrambled ODNs as controls.
- Assessed DHFR mRNA and protein levels via RT-PCR and flow cytometry, respectively, after 4-day treatment.
- Evaluated the effect of aODNs on MTX cytotoxicity in co-exposure experiments.
Main Results:
- DHFR1 and DHFR2+DHFR3 aODNs significantly reduced DHFR mRNA levels (79.7% and 74.2%, respectively).
- A moderate decrease in DHFR enzyme levels was observed (44.8% and 61%, respectively).
- Co-exposure with aODNs and MTX did not result in a marked enhancement of MTX cytotoxicity.
- The study aODNs did not substantially reverse the MTX-resistant phenotype in the tested leukemia cells.
Conclusions:
- Antisense oligodeoxynucleotides targeting DHFR mRNA can effectively reduce DHFR expression in MTX-resistant leukemia cells.
- Despite reducing DHFR levels, the tested aODNs did not restore sensitivity to methotrexate in vitro.
- Further strategies may be needed to overcome DHFR-mediated MTX resistance in cancer therapy.