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

Wayne F. Flintoff, Heather Sadlish, Richard Gorlick, Rui Yang, Frederick M.R. Williams

Research output: Contribution to journalArticle

20 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)110-117
Number of pages8
JournalBiochimica et Biophysica Acta - Molecular Basis of Disease
Volume1690
Issue number2
DOIs
StatePublished - Oct 14 2004

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Keywords

  • (E) GFP
  • (enhanced) green fluorescent protein
  • Biotin succinimide, sulfo-NHS-SS biotin
  • Biotinylation
  • Fusion protein
  • HRP
  • Methotrexate
  • Osteosarcomas
  • Reduced folate carrier
  • sulfosuccinimidyl-2- (biotinamido) ethyl-1,3-dithiopropionate

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology

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