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Functional analysis of altered reduced folate carrier sequence changes identified in osteosarcomas
Wayne F Flintoff1, Heather Sadlish, Richard Gorlick
1Department of Microbiology and Immunology, University of Western Ontario, Dental Sciences Bldg., Dock 15, London, Canada N6A 5C1. flintoff@uwo.ca
Biochimica Et Biophysica Acta
|October 8, 2004
Summary
Some osteosarcoma patients resist methotrexate due to altered reduced folate carrier (RFC) proteins. Specific RFC mutations impair methotrexate transport, contributing to treatment failure in these bone cancers.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Osteosarcomas are malignant bone tumors often resistant to methotrexate (Mtx).
- Reduced folate carrier (RFC) is the membrane transporter for Mtx.
- Sequence changes in RFC have been identified in osteosarcoma patient mRNA.
Purpose of the Study:
- To assess the functionality of altered RFC proteins in osteosarcoma.
- To determine if identified RFC alterations contribute to Mtx resistance.
Main Methods:
- Constructing fusion proteins of altered RFC with green fluorescent protein (GFP).
- Transfecting constructs into a hamster cell line null for RFC.
- Assessing protein localization via confocal microscopy and cell surface biotinylation.
- Evaluating Mtx transport complementation and binding.
Main Results:
- All tested altered RFC proteins localized to the cell membrane.
- The Leu291Pro alteration prevented Mtx transport, conferring resistance.
- Ser46Asn, Ser4Pro, and Gly259Trp alterations reduced Mtx transport rate (Vmax).
- Glu21Lys, Ala7Val, and combined Thr222Ile/Met254Thr alterations did not confer Mtx resistance.
Conclusions:
- Not all identified RFC alterations contribute to Mtx resistance in osteosarcomas.
- Specific RFC mutations, like Leu291Pro, directly cause Mtx resistance by blocking transport.
- Other RFC alterations can decrease Mtx transport efficiency, partially explaining resistance.