Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Nov 1;367(Pt 3):741–750. doi: 10.1042/BJ20020801

Resistance to multiple novel antifolates is mediated via defective drug transport resulting from clustered mutations in the reduced folate carrier gene in human leukaemia cell lines.

Lilah Rothem 1, Ilan Ifergan 1, Yotam Kaufman 1, David G Priest 1, Gerrit Jansen 1, Yehuda G Assaraf 1
PMCID: PMC1222927  PMID: 12139489

Abstract

We have studied the molecular basis of resistance of multiple human leukaemia CCRF-CEM sublines to the novel antifolates ZD9331, GW1843, AG2034, PT523 and edatrexate, which use the reduced folate carrier (RFC) as their main cellular uptake route and that target different folate-dependent enzymes. Antifolate-resistant sublines established by stepwise and single-step selections displayed up to 2135-fold resistance to the selection drug, and up to 2323-fold cross-resistance to various hydrophilic antifolates. In contrast, these sublines were up to 17- and 20-fold hypersensitive to the lipophilic antifolates AG377 and trimetrexate, respectively. The total reduced folate pool of these antifolate-resistant sublines shrunk by 87-96%, resulting in up to 42-fold increased folic acid growth requirement. These sublines lost 92-97% of parental [(3)H]methotrexate influx rates. Genomic PCR single-strand conformational polymorphism analysis and sequencing revealed that most of these drug-resistant sublines harboured RFC mutations that surprisingly clustered in two confined regions in exons 2 and 3. The majority of these mutations resulted in frame-shift and/or premature translation termination and lack of RFC protein expression. The remaining mutations involved single amino acid substitutions predominantly residing in the first transmembrane domain (TMD1). Some RFC-inactivating mutations emerged during the early stages of antifolate selection and were stably retained during further drug selection. Furthermore, some sublines displayed a markedly decreased or abolished RFC mRNA and/or protein expression. This constitutes the first demonstration of clustering of multiple human RFC mutations in TMD1, thereby suggesting that it plays a functional role in folate/antifolate binding and/or translocation. This is the first molecular characterization of human RFC-associated modalities of resistance to various novel antifolates in multiple leukaemia sublines.

Full Text

The Full Text of this article is available as a PDF (252.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Assaraf Y. G., Borgnia M. J. Probing the interaction of the multidrug-resistance phenotype with the polypeptide ionophore gramicidin D via functional channel formation. Eur J Biochem. 1994 Jun 15;222(3):813–824. doi: 10.1111/j.1432-1033.1994.tb18928.x. [DOI] [PubMed] [Google Scholar]
  2. Assaraf Y. G., Goldman I. D. Loss of folic acid exporter function with markedly augmented folate accumulation in lipophilic antifolate-resistant mammalian cells. J Biol Chem. 1997 Jul 11;272(28):17460–17466. doi: 10.1074/jbc.272.28.17460. [DOI] [PubMed] [Google Scholar]
  3. Borgnia M. J., Eytan G. D., Assaraf Y. G. Competition of hydrophobic peptides, cytotoxic drugs, and chemosensitizers on a common P-glycoprotein pharmacophore as revealed by its ATPase activity. J Biol Chem. 1996 Feb 9;271(6):3163–3171. doi: 10.1074/jbc.271.6.3163. [DOI] [PubMed] [Google Scholar]
  4. Boritzki T. J., Barlett C. A., Zhang C., Howland E. F. AG2034: a novel inhibitor of glycinamide ribonucleotide formyltransferase. Invest New Drugs. 1996;14(3):295–303. doi: 10.1007/BF00194533. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Brigle K. E., Spinella M. J., Sierra E. E., Goldman I. D. Characterization of a mutation in the reduced folate carrier in a transport defective L1210 murine leukemia cell line. J Biol Chem. 1995 Sep 29;270(39):22974–22979. doi: 10.1074/jbc.270.39.22974. [DOI] [PubMed] [Google Scholar]
  7. Diaz G. A., Banikazemi M., Oishi K., Desnick R. J., Gelb B. D. Mutations in a new gene encoding a thiamine transporter cause thiamine-responsive megaloblastic anaemia syndrome. Nat Genet. 1999 Jul;22(3):309–312. doi: 10.1038/10385. [DOI] [PubMed] [Google Scholar]
  8. Drori S., Jansen G., Mauritz R., Peters G. J., Assaraf Y. G. Clustering of mutations in the first transmembrane domain of the human reduced folate carrier in GW1843U89-resistant leukemia cells with impaired antifolate transport and augmented folate uptake. J Biol Chem. 2000 Oct 6;275(40):30855–30863. doi: 10.1074/jbc.M003988200. [DOI] [PubMed] [Google Scholar]
  9. Drori S., Sprecher H., Shemer G., Jansen G., Goldman I. D., Assaraf Y. G. Characterization of a human alternatively spliced truncated reduced folate carrier increasing folate accumulation in parental leukemia cells. Eur J Biochem. 2000 Feb;267(3):690–702. doi: 10.1046/j.1432-1327.2000.01049.x. [DOI] [PubMed] [Google Scholar]
  10. Dutta B., Huang W., Molero M., Kekuda R., Leibach F. H., Devoe L. D., Ganapathy V., Prasad P. D. Cloning of the human thiamine transporter, a member of the folate transporter family. J Biol Chem. 1999 Nov 5;274(45):31925–31929. doi: 10.1074/jbc.274.45.31925. [DOI] [PubMed] [Google Scholar]
  11. Eudy J. D., Spiegelstein O., Barber R. C., Wlodarczyk B. J., Talbot J., Finnell R. H. Identification and characterization of the human and mouse SLC19A3 gene: a novel member of the reduced folate family of micronutrient transporter genes. Mol Genet Metab. 2000 Dec;71(4):581–590. doi: 10.1006/mgme.2000.3112. [DOI] [PubMed] [Google Scholar]
  12. Ferguson P. L., Flintoff W. F. Topological and functional analysis of the human reduced folate carrier by hemagglutinin epitope insertion. J Biol Chem. 1999 Jun 4;274(23):16269–16278. doi: 10.1074/jbc.274.23.16269. [DOI] [PubMed] [Google Scholar]
  13. Fleming J. C., Tartaglini E., Steinkamp M. P., Schorderet D. F., Cohen N., Neufeld E. J. The gene mutated in thiamine-responsive anaemia with diabetes and deafness (TRMA) encodes a functional thiamine transporter. Nat Genet. 1999 Jul;22(3):305–308. doi: 10.1038/10379. [DOI] [PubMed] [Google Scholar]
  14. Freisheim J. H., Ratnam M., McAlinden T. P., Prasad K. M., Williams F. E., Westerhof G. R., Schornagel J. H., Jansen G. Molecular events in the membrane transport of methotrexate in human CCRF-CEM leukemia cell lines. Adv Enzyme Regul. 1992;32:17–31. doi: 10.1016/0065-2571(92)90006-l. [DOI] [PubMed] [Google Scholar]
  15. Fry D. W., Besserer J. A. Characterization of trimetrexate transport in human lymphoblastoid cells and development of impaired influx as a mechanism of resistance to lipophilic antifolates. Cancer Res. 1988 Dec 15;48(24 Pt 1):6986–6991. [PubMed] [Google Scholar]
  16. Goldman I. D. The characteristics of the membrane transport of amethopterin and the naturally occurring folates. Ann N Y Acad Sci. 1971 Nov 30;186:400–422. doi: 10.1111/j.1749-6632.1971.tb46996.x. [DOI] [PubMed] [Google Scholar]
  17. Gong M., Yess J., Connolly T., Ivy S. P., Ohnuma T., Cowan K. H., Moscow J. A. Molecular mechanism of antifolate transport-deficiency in a methotrexate-resistant MOLT-3 human leukemia cell line. Blood. 1997 Apr 1;89(7):2494–2499. [PubMed] [Google Scholar]
  18. Henderson G. B., Zevely E. M. Anion exchange mechanism for transport of methotrexate in L1210 cells. Biochem Biophys Res Commun. 1981 Mar 16;99(1):163–169. doi: 10.1016/0006-291x(81)91727-7. [DOI] [PubMed] [Google Scholar]
  19. Jackman A. L., Calvert A. H. Folate-based thymidylate synthase inhibitors as anticancer drugs. Ann Oncol. 1995 Nov;6(9):871–881. doi: 10.1093/oxfordjournals.annonc.a059353. [DOI] [PubMed] [Google Scholar]
  20. Jansen G., Kathmann I., Rademaker B. C., Braakhuis B. J., Westerhof G. R., Rijksen G., Schornagel J. H. Expression of a folate binding protein in L1210 cells grown in low folate medium. Cancer Res. 1989 Apr 15;49(8):1959–1963. [PubMed] [Google Scholar]
  21. Jansen G., Mauritz R., Drori S., Sprecher H., Kathmann I., Bunni M., Priest D. G., Noordhuis P., Schornagel J. H., Pinedo H. M. A structurally altered human reduced folate carrier with increased folic acid transport mediates a novel mechanism of antifolate resistance. J Biol Chem. 1998 Nov 13;273(46):30189–30198. doi: 10.1074/jbc.273.46.30189. [DOI] [PubMed] [Google Scholar]
  22. Jansen G., Pieters R. The role of impaired transport in (pre)clinical resistance to methotrexate: insights on new antifolates. Drug Resist Updat. 1998;1(3):211–218. doi: 10.1016/s1368-7646(98)80042-3. [DOI] [PubMed] [Google Scholar]
  23. Jansen G., Westerhof G. R., Jarmuszewski M. J., Kathmann I., Rijksen G., Schornagel J. H. Methotrexate transport in variant human CCRF-CEM leukemia cells with elevated levels of the reduced folate carrier. Selective effect on carrier-mediated transport of physiological concentrations of reduced folates. J Biol Chem. 1990 Oct 25;265(30):18272–18277. [PubMed] [Google Scholar]
  24. Labay V., Raz T., Baron D., Mandel H., Williams H., Barrett T., Szargel R., McDonald L., Shalata A., Nosaka K. Mutations in SLC19A2 cause thiamine-responsive megaloblastic anaemia associated with diabetes mellitus and deafness. Nat Genet. 1999 Jul;22(3):300–304. doi: 10.1038/10372. [DOI] [PubMed] [Google Scholar]
  25. Matherly L. H., Angeles S. M., Czajkowski C. A. Characterization of transport-mediated methotrexate resistance in human tumor cells with antibodies to the membrane carrier for methotrexate and tetrahydrofolate cofactors. J Biol Chem. 1992 Nov 15;267(32):23253–23260. [PubMed] [Google Scholar]
  26. Mendelsohn L. G., Shih C., Schultz R. M., Worzalla J. F. Biochemistry and pharmacology of glycinamide ribonucleotide formyltransferase inhibitors: LY309887 and lometrexol. Invest New Drugs. 1996;14(3):287–294. doi: 10.1007/BF00194532. [DOI] [PubMed] [Google Scholar]
  27. Pao S. S., Paulsen I. T., Saier M. H., Jr Major facilitator superfamily. Microbiol Mol Biol Rev. 1998 Mar;62(1):1–34. doi: 10.1128/mmbr.62.1.1-34.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rajgopal A., Edmondnson A., Goldman I. D., Zhao R. SLC19A3 encodes a second thiamine transporter ThTr2. Biochim Biophys Acta. 2001 Nov 29;1537(3):175–178. doi: 10.1016/s0925-4439(01)00073-4. [DOI] [PubMed] [Google Scholar]
  29. Roy K., Tolner B., Chiao J. H., Sirotnak F. M. A single amino acid difference within the folate transporter encoded by the murine RFC-1 gene selectively alters its interaction with folate analogues. Implications for intrinsic antifolate resistance and directional orientation of the transporter within the plasma membrane of tumor cells. J Biol Chem. 1998 Jan 30;273(5):2526–2531. doi: 10.1074/jbc.273.5.2526. [DOI] [PubMed] [Google Scholar]
  30. Santi D. V., McHenry C. S., Perriard E. R. A filter assay for thymidylate synthetase using 5-fluoro-2'-deoxyuridylate as an active site titrant. Biochemistry. 1974 Jan 29;13(3):467–470. doi: 10.1021/bi00700a011. [DOI] [PubMed] [Google Scholar]
  31. Schmitz J. C., Grindey G. B., Schultz R. M., Priest D. G. Impact of dietary folic acid on reduced folates in mouse plasma and tissues. Relationship to dideazatetrahydrofolate sensitivity. Biochem Pharmacol. 1994 Jul 19;48(2):319–325. doi: 10.1016/0006-2952(94)90103-1. [DOI] [PubMed] [Google Scholar]
  32. Shih C., Chen V. J., Gossett L. S., Gates S. B., MacKellar W. C., Habeck L. L., Shackelford K. A., Mendelsohn L. G., Soose D. J., Patel V. F. LY231514, a pyrrolo[2,3-d]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes. Cancer Res. 1997 Mar 15;57(6):1116–1123. [PubMed] [Google Scholar]
  33. Wong S. C., Zhang L., Witt T. L., Proefke S. A., Bhushan A., Matherly L. H. Impaired membrane transport in methotrexate-resistant CCRF-CEM cells involves early translation termination and increased turnover of a mutant reduced folate carrier. J Biol Chem. 1999 Apr 9;274(15):10388–10394. doi: 10.1074/jbc.274.15.10388. [DOI] [PubMed] [Google Scholar]
  34. Zhao R., Assaraf Y. G., Goldman I. D. A mutated murine reduced folate carrier (RFC1) with increased affinity for folic acid, decreased affinity for methotrexate, and an obligatory anion requirement for transport function. J Biol Chem. 1998 Jul 24;273(30):19065–19071. doi: 10.1074/jbc.273.30.19065. [DOI] [PubMed] [Google Scholar]
  35. Zhao R., Gao F., Wang Y., Diaz G. A., Gelb B. D., Goldman I. D. Impact of the reduced folate carrier on the accumulation of active thiamin metabolites in murine leukemia cells. J Biol Chem. 2001 Jan 12;276(2):1114–1118. doi: 10.1074/jbc.M007919200. [DOI] [PubMed] [Google Scholar]
  36. Zhao R., Russell R. G., Wang Y., Liu L., Gao F., Kneitz B., Edelmann W., Goldman I. D. Rescue of embryonic lethality in reduced folate carrier-deficient mice by maternal folic acid supplementation reveals early neonatal failure of hematopoietic organs. J Biol Chem. 2001 Mar 30;276(13):10224–10228. doi: 10.1074/jbc.c000905200. [DOI] [PubMed] [Google Scholar]
  37. Zhao R., Sharina I. G., Goldman I. D. Pattern of mutations that results in loss of reduced folate carrier function under antifolate selective pressure augmented by chemical mutagenesis. Mol Pharmacol. 1999 Jul;56(1):68–76. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES