Reduced folate carrier gene (RFC1) expression and anti-folate resistance in transfected and non-selected cell lines
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J A Moscow1, T Connolly, T G Myers
1Medicine Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD 20892, USA. moscowj@pbmac.nci.nih.gov
Abstract:
Methotrexate transport deficiency due to decreased reduced folate carrier (RFC) activity has been observed in several cell lines selected for resistance to methotrexate (MTX). Since MTX resistance is multifactorial, however, it is difficult to quantify the relative importance of changes in RFC activity in selected cell lines and even more so to determine the relative contribution of naturally occurring RFC activity in the MTX sensitivity of non-selected cell lines. We examined the role of RFC in MTX resistance by studying a transport-deficient cell line transfected with the gene for human RFC, RFC1, and by correlating relative RFC1 expression with MTX and trimetrexate (TMTX) growth inhibition (GI50) in a panel of cell lines used in the NCI Anticancer Drug Screen. Clones of transport-deficient, MTX-resistant ZR-75-1 human breast cancer cells (MTX(R) ZR-75-1) transfected with RFC1 were 250-fold more sensitive to MTX and 300-fold more resistant to TMTX than control cell clones, showing that restoration of RFC activity has a significant impact on MTX and TMTX cytotoxicity. We also surveyed 40 of the 60 cell lines in the NCI drug screen panel for RFCI RNA levels by a quantitative RT-PCR assay. RFCI RNA levels varied over a range of 15-fold, with only 1 cell line found to be null in expression. Using data from the 6-day drug exposure assay, RFC1 correlated positively with MTX and negatively with TMTX cytotoxicity. As predicted by transfection studies, the calculated difference between MTX and TMTX potency was even more strongly correlated with RFC1 RNA levels of the cell lines. In addition, compounds in the NCI Anticancer Drug Screen database with cytotoxicity profiles which correlated with RFC1 RNA levels or with the calculated difference in MTX-TMTX potency were examined for MTX uptake inhibition and cytotoxicity in the RFC1-transfected MTX(R) ZR-75-1 cell line. Overall, our data demonstrate the importance of RFC1 in MTX resistance both as a transgene and as a constitutively expressed gene in non-selected cell lines.
Insights
Reduced folate carrier (RFC) activity significantly impacts methotrexate (MTX) resistance. Restoring RFC1 expression in cancer cells altered sensitivity to MTX and trimetrexate (TMTX), highlighting RFC1
Area of Science:
- Pharmacology
- Molecular Biology
- Cancer Research
Background:
- Methotrexate (MTX) resistance in cancer cells can stem from reduced activity of the reduced folate carrier (RFC).
- Quantifying RFC's role in MTX resistance is challenging due to multifactorial resistance mechanisms and natural variations in RFC activity.
- Understanding RFC's contribution to drug sensitivity is crucial for optimizing cancer chemotherapy.
Purpose of the Study:
- To investigate the functional significance of the reduced folate carrier 1 (RFC1) in mediating cellular sensitivity and resistance to methotrexate (MTX) and trimetrexate (TMTX).
- To correlate RFC1 expression levels with MTX and TMTX cytotoxicity in a panel of cancer cell lines.
- To elucidate the role of RFC1 as both a transgene and a naturally occurring gene in MTX resistance.
Main Methods:
- Transfection of MTX-resistant ZR-75-1 cells with the human RFC1 gene to restore RFC activity.
- Quantitative RT-PCR assay to measure RFC1 RNA levels in 40 NCI Anticancer Drug Screen cell lines.
- Assessing growth inhibition (GI50) and cytotoxicity of MTX and TMTX in transfected cells and the NCI panel.
- Analyzing correlations between RFC1 expression and drug potency.
Main Results:
- Restoration of RFC1 in transport-deficient cells significantly increased sensitivity to MTX (250-fold) and resistance to TMTX (300-fold).
- RFC1 RNA levels varied 15-fold across NCI panel cell lines, with most expressing detectable levels.
- RFC1 expression positively correlated with MTX cytotoxicity and negatively with TMTX cytotoxicity.
Conclusions:
- RFC1 plays a critical role in determining cellular response to MTX and TMTX.
- Natural variations in RFC1 expression contribute to differential MTX and TMTX sensitivity in cancer cell lines.
- The findings underscore the importance of RFC1 in MTX resistance, both when introduced as a transgene and when endogenously expressed.
