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Involvement of MDR1 P-glycoprotein in multifactorial resistance to methotrexate
M D Norris1, D De Graaf, M Haber
1Children's Leukaemia and Cancer Research Centre, Prince of Wales Children's Hospital, Sydney, Australia.
Abstract:
Cellular resistance to methotrexate (MTX) is believed to be unaffected by expression of MDR1 P-glycoprotein (Pgp), a pleiotropic efflux pump acting on different hydrophobic compounds that enter cells by passive diffusion. A series of human leukemic CCRF-CEM sublines, isolated by multi-step selection for very high resistance to MTX, exhibit multiple mechanisms of MTX resistance, including decreased carrier-mediated uptake of MTX and DHFR gene amplification. These sublines show cross-resistance to drugs of the multi-drug resistance (MDR) family, which is correlated with relative resistance to MTX. The MTX-selected sublines show increased expression and function of the MDR1 gene, based on the measurement of MDR1 mRNA, Pgp and rhodamine 123 accumulation. Sequence analysis of the MDR1 cDNA from MTX-selected CCRF-CEM cells revealed no mutations in the protein coding region. MTX resistance in these cell lines is partially reversible by a Pgp-specific monoclonal antibody (MAb) UIC2 and a monovalent FaB fragment of UIC2. Our results indicate that Pgp can contribute to multifactorial resistance to MTX.
Insights
P-glycoprotein (Pgp) can contribute to cellular resistance to methotrexate (MTX), contrary to previous beliefs. This study shows Pgp expression in MTX-resistant leukemia cells, suggesting a role in multifactorial drug resistance.
Area of Science:
- Molecular Biology
- Cancer Research
- Pharmacology
Background:
- Cellular resistance to methotrexate (MTX) is a complex phenomenon.
- P-glycoprotein (Pgp), encoded by the MDR1 gene, is a known efflux pump for hydrophobic drugs.
- The role of Pgp in MTX resistance has been previously underestimated.
Purpose of the Study:
- To investigate the potential contribution of P-glycoprotein (Pgp) to cellular resistance to methotrexate (MTX).
- To explore the mechanisms of MTX resistance in human leukemic CCRF-CEM sublines.
- To determine if Pgp expression and function are altered in MTX-resistant cells.
Main Methods:
- Development of multi-step MTX-selected CCRF-CEM sublines.
- Analysis of MDR1 gene expression (mRNA), Pgp protein levels, and Pgp function (rhodamine 123 accumulation).
- Assessment of MTX resistance reversibility using Pgp-specific monoclonal antibodies (UIC2) and Fab fragments.
Main Results:
- MTX-selected sublines exhibited cross-resistance to MDR drugs, correlated with MTX resistance.
- Increased MDR1 mRNA and Pgp expression/function were observed in MTX-resistant sublines.
- MTX resistance was partially reversed by Pgp-specific antibodies, indicating Pgp involvement.
Conclusions:
- P-glycoprotein (Pgp) can contribute to multifactorial resistance to methotrexate (MTX).
- This finding challenges the previous understanding of Pgp's role in MTX cellular resistance.
- Targeting Pgp may offer strategies to overcome MTX resistance in certain cancers.