Functional analysis of altered reduced folate carrier sequence changes identified in osteosarcomas Journal Article

  


Authors: Flintoff, W. F.; Sadlish, H.; Gorlick, R.; Yang, R.; Williams, F. M. R.
Article Title: Functional analysis of altered reduced folate carrier sequence changes identified in osteosarcomas
Abstract: Osteosarcomas are common primary malignant bone tumors that do not respond to conventional low-dose treatments of methotrexate (Mtx), suggesting an intrinsic resistance to this drug. Previous work has shown that cDNAs generated from osteosarcoma mRNA from a fraction of patients contain sequence changes in the reduced folate carrier (RFC), the membrane protein transporter for Mtx. In this study, the functionality of the altered RFC proteins was assessed by fusing the green fluorescent protein (GFP) to the C-terminal, and examining the ability of the transfected constructs to complement a hamster cell line null for the carrier. Confocal microscopy and cell surface biotinylation indicated that all altered proteins were properly localized at the cell membrane. Only one of those examined, Leu291Pro, was unable to complement the null carrier line, but did bind Mtx at the cell surface. Thus, this alteration confers drug resistance since the carrier is unable to translocate the substrate across the cell membrane. Three alterations, Ser46Asn, Ser4Pro and Gly259Trp, while able to complement the carrier null line, conferred some degree of resistance to Mtx via a decreased rate of transport (Vmax). Another set of alterations, Glu21Lys, Ala7Val, and the combined changes Thr222Ile, Met254Thr, complemented the carrier null line and did not confer resistance to Mtx. Thus, some, but not all of these identified alterations in the RFC may contribute to the lack of responsiveness of osteosarcomas to Mtx treatment. © 2004 Elsevier B.V. All rights reserved.
Keywords: osteosarcoma; controlled study; nonhuman; methotrexate; binding affinity; protein function; protein localization; animal cell; phenotype; animals; amino acid substitution; confocal microscopy; microscopy, confocal; serine; carboxy terminal sequence; green fluorescent protein; cell line; drug resistance, neoplasm; cell line, tumor; transfection; blotting, western; dna; amino acid sequence; molecular sequence data; genetic transfection; kinetics; recombinant fusion proteins; rna, messenger; cell membrane; isoleucine; protein structure, tertiary; threonine; folic acid; alanine; leucine; glutamic acid; drug transport; methionine; lysine; glycine; asparagine; valine; tryptophan; proline; dna, complementary; biotinylation; biotin; cell surface; cricetinae; transport kinetics; epitopes; fusion protein; osteosarcomas; membrane transport proteins; reduced folate carrier; humans; priority journal; article; hrp; (e) gfp; (enhanced) green fluorescent protein; biotin succinimide, sulfo-nhs-ss biotin; sulfosuccinimidyl-2- (biotinamido) ethyl-1,3-dithiopropionate
Journal Title: Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease
Volume: 1690
Issue: 2
ISSN: 0925-4439
Publisher: Elsevier B.V.  
Date Published: 2004-10-14
Start Page: 110
End Page: 117
Language: English
DOI: 10.1016/j.bbadis.2004.05.008
PROVIDER: scopus
PUBMED: 15469899
DOI/URL:

http://www.scopus.com/inward/record.url?eid=2-s2.0-4644251592&partnerID=40&md5=eab72df8b8c017b986ac1801e400db8b
Notes: Biochim. Biophys. Acta Mol. Basis Dis. -- Cited By (since 1996):15 -- Export Date: 16 June 2014 -- CODEN: BBADE -- Source: Scopus
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MSK Authors
  1. Richard G Gorlick
    121 Gorlick
  2. Rui Yang
    23 Yang
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