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J. Biol. Chem., Vol. 283, Issue 30, 20687-20695, July 25, 2008

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The Reduced Folate Carrier (RFC) Is Cytotoxic to Cells under Conditions of Severe Folate Deprivation

RFC AS A DOUBLE EDGED SWORD IN FOLATE HOMEOSTASIS^*

Ilan Ifergan, Gerrit Jansen, and Yehuda G. Assaraf¹

From the The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel and the Department of Rheumatology, Vrije Universiteit University Medical Center, 1081 Amsterdam, The Netherlands

The reduced folate carrier (RFC), a bidirectional anion transporter, is the major uptake route of reduced folates essential for a spectrum of biochemical reactions and thus cellular proliferation. However, here we show that ectopic overexpression of the RFC, but not of folate receptor , a high affinity unidirectional folate uptake route serving here as a negative control, resulted in an 15-fold decline in cellular viability in medium lacking folates but not in folate-containing medium. Moreover to explore possible mechanisms of adaptation to folate deficiency in various cell lines that express the endogenous RFC, we first determined the gene expression status of the following genes: (a) RFC, (b) ATP-driven folate exporters (i.e. MRP1, MRP5, and breast cancer resistance protein), and (c) folylpoly--glutamate synthetase and -glutamate hydrolase (GGH), enzymes catalyzing folate polyglutamylation and hydrolysis, respectively. Upon 3–7 days of folate deprivation, semiquantitative reverse transcription-PCR analysis revealed a specific 2.5-fold decrease in RFC mRNA levels in both breast cancer and T-cell leukemia cell lines that was accompanied by a consistent fall in methotrexate influx, serving here as an RFC transport activity assay. Likewise a 2.4-fold decrease in GGH mRNA levels and 19% decreased GGH activity was documented for folate-deprived breast cancer cells. These results along with those of a novel mathematical biomodeling devised here suggest that upon severe short term (i.e. up to 7 days) folate deprivation RFC transport activity becomes detrimental as RFC, but not ATP-driven folate exporters, efficiently extrudes folate monoglutamates out of cells. Hence down-regulation of RFC and GGH may serve as a novel adaptive response to severe folate deficiency.

Received for publication, April 14, 2008 , and in revised form, May 20, 2008.

^* This work was supported by Fred Wyszkowski Cancer Research Fund Grant 2007346 (to Y. G. A.) as well as by Dutch Arthritis Association Grant NRF-030-I-40 (to G. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. E-mail: assaraf{at}tx.technion.ac.il.

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