Effects of folate and folylpolyglutamyl synthase modulation on chemosensitivity of breast cancer cells

Effects of folate and folylpolyglutamyl synthasemodulation on chemosensitivity ofbreast cancer cells

Robert C. Cho,2 Peter D. Cole,6 Kyoung-Jin Sohn,1

Gregory Gaisano,1 Ruth Croxford,3

Barton A. Kamen,5 and Young-In Kim1,2,4

1Department of Medicine and 2Nutritional Sciences, Universityof Toronto, 3Clinical Epidemiology Unit, Sunnybrook HealthSciences Center, and 4Division of Gastroenterology, St. Michael’sHospital, Toronto, Ontario, Canada; 5Robert Wood JohnsonMedical School, University of Medicine and Dentistry of NewJersey, New Brunswick, New Jersey; and 6Department ofPediatrics, Albert Einstein College of Medicine, Bronx, New York

AbstractFolylpolyglutamyl synthase (FPGS) converts intracellularfolates and antifolates to polyglutamates. Polyglutamy-lated folates and antifolates are retained in cells longer andare better substrates than their monoglutamate counter-parts for enzymes involved in one-carbon transfer. FPGSmodulation affects the chemosensitivity of cancer cells toantifolates, such as methotrexate, and 5-fluorouracil (5FU)by altering polyglutamylation of antifolates and specifictarget intracellular folate cofactors. However, this effectmay be counterbalanced by FPGS modulation-inducedchanges in polyglutamylation of other intracellular folatecofactors and total intracellular folate pools. We generatedan in vitro model of FPGS overexpression and inhibition inbreast cancer cells by stably transfecting human MDA-MB-435 breast cancer cells with the sense FPGS cDNAor FPGS-targeted small interfering RNA, respectively, andinvestigated the effects of FPGS modulation on chemo-sensitivity to 5FU and methotrexate. FPGS modulation-

induced changes in polyglutamylation of both antifolatesand folate cofactors and in intracellular folate pools af-fected chemosensitivity of breast cancer cells to peme-trexed and trimetrexate whose cytotoxic effects do or donot depend on polyglutamylation, respectively, in a pre-dictable manner. However, the effects of FPGS modula-tion on the chemosensitivity of breast cancer cells to 5FUand methotrexate seem to be highly complex and dependnot only on polyglutamylation of a specific targetintracellular folate cofactor or methotrexate, respectively,but also on total intracellular folate pools and polygluta-mylation of other intracellular folate cofactors. Whether ornot FPGS modulation may be an important clinical determi-nant of chemosensitivity of breast cancer cells to 5FUand methotrexate-based chemotherapy needs furtherexploration. [Mol Cancer Ther 2007;6(11):2909–20]

IntroductionFolate mediates the transfer of one-carbon units necessaryfor thymidylate and purine biosynthesis and, hence, is anessential factor for DNA synthesis (1). In neoplastic cells,folate depletion and disrupted folate metabolism causeineffective DNA synthesis, resulting in inhibition of tumorgrowth (2). This has been the basis for cancer chemotherapyusing antifolates and 5-fluorouracil (5FU; ref. 2). Althoughmonoglutamates are the only circulating forms of folate inblood and the only form of folate that is transported acrossthe cell membrane, once taken up into cells, intracellularfolate exists primarily as polyglutamates (1). Intracellularfolate is converted to polyglutamates by folylpolygluta-myl synthase (FPGS), whereas g-glutamyl hydrolase (GGH)removes the terminal glutamates (1). Polyglutamylatedfolates are better retained in cells and are better substratesthan monoglutamates for intracellular folate-dependent en-zymes (3).Similarly, antifolates (e.g., methotrexate) are retained in

cells by FPGS-induced polyglutamylation and are exportedfrom cells after hydrolysis to monoglutamates by GGH(2, 3). As with folate, polyglutamylated antifolates are re-tained in cells longer, thereby increasing their cytotoxicityby extending the length of exposure (2, 3). Polyglutamy-lated antifolates generally have a higher affinity for and,hence, inhibit their target folate-dependent enzymes in thy-midylate and purine biosynthesis to a greater extent thanthe monoglutamate forms (2, 3). FPGS down-regulationseems to be a mechanism of resistance to methotrexate andother antifolates (4–10). Transfection of FPGS cDNA hasbeen shown to increase sensitivity to methotrexate andother antifolates in some mammalian cell lines (11, 12).FPGS-induced polyglutamylation may also affect the

sensitivity of tumor cells to other chemotherapeutic agents,

Received 7/18/07; accepted 9/14/07.

Grant support: Operating grant (14126) from the Canadian Institutes ofHealth Research (to Y.I. Kim). R.C. Cho is a recipient of a scholarship fromthe Natural Sciences and Engineering Research Council of Canada. P.D.Cole is a Damon Runyon-Lilly Clinical Investigator, supported in part by theDamon Runyon Cancer Research Foundation (CI-16-03). B.A. Kamen is anAmerican Cancer Society Professor.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

Note: Presented in part at the 2006 American Association for CancerResearch meeting, April 1 – 5, 2006, Washington, DC, and published inabstract form in the Proceedings of the American Association for CancerResearch 2006; 47: Abstract 2149.

Requests for reprints: Young-In Kim, Room 7258, Medical SciencesBuilding, University of Toronto, 1 King’s College Circle, Toronto, Ontario,Canada, M5S 1A8. Phone: 416-978-1183; Fax: 416-978-8765.E-mail: [email protected]

Copyright C 2007 American Association for Cancer Research.

doi:10.1158/1535-7163.MCT-07-0449

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Mol Cancer Ther 2007;6(11). November 2007

such as 5FU, not typically considered as antifolates. Onecytotoxic mechanism of 5FU is the formation of a ternarycomplex involving a metabolite of 5FU [5-fluoro-2-deoxy-uridine-5-monophosphate (FdUMP)], thymidylate synthase(TS) and 5,10-methylenetetrahydrofolate (5,10-methyle-neTHF), thereby inhibiting TS activity with consequentsuppression of DNA synthesis (13). Leucovorin (LV; 5-formylTHF), a precursor for 5,10-methyleneTHF, potenti-ates the cytotoxic effect of 5FU by stabilizing this inhibitoryternary complex (13). 5,10-MethyleneTHF with longerchain length polyglutamates is better retained intracellu-larly and is more efficient in the formation and stabilizationof this inhibitory ternary complex compared with shorterchain polyglutamates (14). Decreased FPGS activity haspreviously been shown to confer resistance to 5FU in somehuman cancer cell lines (15–17). We have previouslyreported that FPGS overexpression significantly increaseschemosensitivity of human HCT116 colon cancer cells to5FU (18).FPGS seems to play an important role in the sensitivity of

cancer cells to antifolates and 5FU, and thus, FPGS modu-lation might be a potential therapeutic target for increasingsensitivity of cancer cells to these chemotherapeutic agents.However, the effect of FPGS modulation on the chemo-sensitivity of breast cancer cells to antifolates and 5FU iscurrently unknown. Furthermore, FPGS modulation affectspolyglutamylation of not only antifolates and specific targetintracellular folate cofactors (e.g., 5,10-methyleneTHF for5FU), but also of all other intracellular folate cofactors.FPGS modulation also affects total intracellular folate con-centration, which is an important determinant of the cyto-toxic effects of antifolates and 5FU (2, 19, 20). Therefore,FPGS modulation-induced changes in intracellular folateconcentrations and folylpolyglutamylation may counter-balance the effects of FPGS modulation-induced changes inpolyglutamylation of antifolates and specific 5,10-methyl-eneTHF. In the present study, we generated an in vitromodel of FPGS modulation in breast cancer cells anddetermined the effects of FPGS modulation on chemo-sensitivity to methotrexate and 5FU. We also determinedwhether exogenous folate concentrations further influencethe effects of FPGS modulation on the chemosensitivity tomethotrexate and 5FU.

Materials andMethodsCell Line and CultureHuman breast adenocarcinoma MDA-MB-435 cells were

purchased from the American Type Culture Collection.Cells were grown either in standard RPMI 1640 (Invitro-gen) containing 2.3 Amol/L folic acid or in RPMI free offolic acid supplemented with either 20 or 100 nmol/L folicacid (Sigma). Growth medium was supplemented with10% dialyzed fetal bovine serum (FBS), which containsf0.6 nmol/L folic acid, 2 mmol/L L-glutamine, penicillinat 100 units/mL, and streptomycin at 100 mg/mL. Cultureswere maintained at 37jC in 5% CO2. The final concen-trations of folic acid in medium are therefore 2.3 Amol/L,100.6 nmol/L, and 20.6 nmol/L folate concentrations.

Construction and Transfection of FPGS ExpressionVectorsThe full-length human FPGS cDNA (21) was provided by

Dr. A. Bognar (University of Toronto, Canada). The full-length human FPGS cDNA was subcloned into the EcoRIsite of the eukaryotic expression vector pIRESneo (Clone-tech) in the sense orientation to generate the sense FPGSexpression vector as described (18).FPGS-targeted siRNA was designed according to the

manufacturer’s protocol and ligated into the vector be-tween the BamH1 and HindIII restriction sites of thepSilencer neo siRNA expression vector (Ambion). The oli-gonucleotides were designed encoding the desired siRNAstrand: GACGGGATTCTTAGCTCT (forward) and AGAG-CTAAAGAATCCCGTC (reverse), and AGAGCTAAAGA-ATCCCGTC (forward) and GACGGGATTCTTTAGCTCT(reverse).The pIRESneo vector containing sense cDNA and the

pSilencer vector containing FPGS-targeted siRNA werestably transfected into MDA-MB-435 cells using Lipofectin(Invitrogen) according to the manufacturer’s protocol. Inseparate transfections, MDA-MB-435 cells were stablytransfected with empty pIRESneo and pSilencer vectors ascorresponding controls expressing endogenous FPGS.Transfected cells were incubated with 500 Ag/mL of neo-mycin (Invitrogen) to select for cells that expressed thevarious constructs. After a population of cells was selected,individual clonal cell lines were isolated and expanded.Cells were maintained in complete medium supplementedwith 500 Ag/mL neomycin. Several (>10) clones expressingthe sense FPGS cDNA and FPGS-targeted siRNA andempty vectors were screened at random, and two inde-pendent clones of each construct were selected for furtheranalysis. Data from three experiments using two indepen-dent clones of each construct were similar, and thus, thedata from one experiment are presented. All analyses weredone in triplicate, and at least three replicate experimentswere done.

Western Blot AnalysisFPGS protein expression was determined by standard

Western blot analysis as described (18) using a rabbitpolyclonal antibody raised against a peptide sequencespanning amino acids 275 to 290 of human FPGS (ref. 22;Zymed) at a dilution of 1:1,000.

FPGS and GGHEnzymeActivityAssaysFPGS activity was determined by measuring the incor-

poration of [3H]-glutamate into the polyglutamate chain ofaminopterin as described (12). GGH activity was deter-mined by incubating protein in cell lysates with metho-trexate-glu4 as substrate and measuring methotrexate andits polyglutamates using high-performance liquid chroma-tography (HPLC) as described (23, 24).

Intracellular Folate Concentrations and GlutamateChain LengthsIntracellular folate concentrations for conjugase-treated

and untreated samples were determined by a standardmicrobiological assay to determine the extent of polyglu-tamylation as described (18, 25).

Folate, FPGS, and Breast Cancer Chemosensitivity2910


[3H]-Methotrexate and [3H]-5-MethylTHF Accu-mulationCells were plated at a density of 1 � 105 cells/mL in six-

well plates in 2 mL of RPMI free of folic acid supplementedwith 10% dialyzed FBS and 30 nmol/L of 5-methylTHF.After 3 to 4 days incubation, as the cells began to enterexponential growth, the cells were washed with HEPES-buffered saline solution, and the medium was replacedwith 1 mL of RPMI 1640, with no serum and no folate, pluseither [3H]-methotrexate (final concentration, 1 Amol/L)or [3H]-5-methylTHF (final concentration, 20 nmol/L).Thirty-five microliters of 10�4 mol/L unlabeled 5-meth-ylTHF was added to three wells for each sample to saturatenonspecific binding.After 24 h, the cells were washed with PBS, released by

incubation with trypsin, collected, and boiled for 5 min.Intracellular content of [3H]-methotrexate or 5-methylTHFwas determined by scintillation counting. Intracellularmethotrexate and folate polyglutamates were separatedon a reverse-phase C18 column as described (26).

DoublingTime CalculationEight thousand cells per well were plated in a 96-well

flat-bottom plate and grown in RPMI 1640 growth mediumwith 10% FBS for 72 h. The cell population was determinedusing the sulforhodamine B (SRB) absorbance measure-ment assay. The growth rate constant k was derived usingthe equation N/N0 = e

kt , where N0 is the A of cells at timezero, and N is the A of cells at 7 h. The same equation wasused to calculate doubling time t by setting N/N0 = 2.

In vitro ChemosensitivityAssayIn vitro chemosensitivity was determined using a

modification of the SRB protein assay as described (18,27, 28). Briefly, 8,000 cells per 100 AL RPMI 1640 per wellwere seeded in triplicate in 96-well flat-bottom plates. After24 h, an additional 100 AL of RPMI 1640 containing 5FU(InvivoGen) alone or in combination with LV (Sigma),methotrexate (Sigma), pemetrexed (Eli Lily), or trimetrex-ate (USB Pharma B.V.) were added, and cells were culturedfor an additional 72 h. The concentration of 5FU rangedfrom 1.5 � 10�6 to 25 � 10�6 mol/L, whereas the con-centration of LV was held constant at 5 � 10�6 mol/L. Theconcentration range was from 3 � 10�9 to 24 � 10�9 mol/Lfor methotrexate, from 0.5 � 10�6 to 24 � 10�6 mol/L forpemetrexed, and from 1 � 10�9 to 35 � 10�9 mol/L fortrimetrexate. After 72 h, cells were fixed with trichloro-acetic acid and stained with SRB protein dye. The dye wassolubilized, and the A of the solution was measured at595 nm. The results were expressed as the percentage of cellsurvival on the basis of the difference between A at the startand end of drug exposure according to the formula (29):

Survival ¼ ½ðAdrug=Astart drug exposureÞ � 1�=½ðAno drug=Astart drug exposureÞ � 1� � 100%:

IC50 values (i.e., the drug concentration that corre-sponded to a reduction in cell survival of 50% comparedwith survival of untreated control cells) were calculated

from plots of drug concentration versus proportion of cellsthat survived.

Statistical AnalysisFor continuous variables, comparisons between cells ex-

pressing the sense FPGS (Sense) and controls (Control-S)and between cells transfected with the FPGS-targetedsiRNA (siRNA) and controls (Control-si) were determinedusing Student’s t test. m2 analyses were used to examinedifferences in the distribution of intracellular methotre-xate- and 5-methylTHF-polyglutamates between the Senseand Control-S cells and between the siRNA and Control-sicells. For the in vitro chemosensitivity analyses, plots ofpercentage of survival versus dose showed S-shapedcurves, and therefore, the logit transformation [logit(p) =ln(p/[1 � p])] was used. Ordinary least-squares regressionwas used to model the effect of log(dose) of chemotherapyand cell type on the logit-transformed proportion of cellsthat survived at each dose. The interaction between celltype and log (dose) was included in the model to test thehypothesis that the cell types were differentially sensitiveto chemotherapy. IC50 doses and their 95% confidenceintervals were calculated on the log scale from the regres-sion results as described (30) and then back-transformed tothe original scale for reporting. Relationships betweenmedium folic acid levels and intracellular folate concen-trations were examined using Pearson correlations. For allanalyses, results were considered statistically significant iftwo-tailed P values were <0.05. Analyses were done usingSAS, version 8 (SAS Institute).

ResultsMDA-MB-435 cells expressing the sense FPGS and FPGS-targeted siRNA had significantly higher and lower steady-state levels of the FPGS protein, respectively, comparedwith corresponding controls expressing endogenous FPGS(P < 0.05; Figs. 1A and 2A). Cells expressing the sense FPGShad a 4.2-fold higher (P < 0.0001) and those transfectedwith the FPGS-targeted siRNA had a 1.3-fold lower (P =0.04) FPGS activity compared with corresponding controls(Figs. 1A and 2A).Following conjugase treatment (which allows the mea-

surement of total intracellular folate concentration, includ-ing short- and long-chain folylpolyglutamates), intracellularfolate concentration of cells expressing the sense FPGSwas 1.8- to 2.1-fold higher (P < 0.0001), whereas that of cellsexpressing the FPGS-targeted siRNA was 1.7- to 2.3-foldlower (P < 0.001), than that of controls at all three folic acidconcentrations (Figs. 1B and 2B). Intracellular folateconcentration of samples not treated with conjugase (whichallows the determination of short-chain folylpolygluta-mates) was significantly higher in cells expressing the senseFPGS than that of controls only at 20 nmol/L folic acid(P = 0.01; Fig. 1B). Intracellular concentration of short-chainfolylpolyglutamates was significantly lower in cells express-ing the FPGS-targeted siRNA than that of controls at100 nmol/L and 2.3 Amol/L (P = 0.023 and P = 0.0042,respectively), but not at 20 nmol/L folic acid (Fig. 2B). Total

Molecular Cancer Therapeutics 2911


intracellular folate concentrations correlated significantlywith medium folic acid concentrations in all four FPGSclones (r = 0.80–0.96; P < 0.01; Figs. 1B and 2B). In contrast,intracellular concentrations of short-chain folylpolygluta-mates correlated with medium folic acid concentrations incells expressing the sense FPGS (r = 0.92; P = 0.0004) andcorresponding controls (r = 0.62; P = 0.07), but not in cellsexpressing the FPGS-targeted siRNA and correspondingcontrols (Figs. 1B and 2B).Differences between mean folate concentration of con-

jugase-treated and untreated samples (which allows thedetermination of long-chain folylpolyglutamates) clearlyshow that cells expressing the sense FPGS had significantlyhigher concentrations of long-chain folylpolyglutamatesthan controls at all folic acid concentrations (at 2.3 Amol/L:Sense, 11.11 F 0.51 versus Control-S, 5.09 F 0.69; at100 nmol/L: Sense, 5.68 F 0.29 versus Control-S, 0.25 F0.62; at 20 nmol/L: Sense, 4.76 F 0.40 versus Control-S, 1.54F 0.58; P < 0.001; Fig. 1B). In contrast, cells expressing theFPGS-targeted siRNA had significantly lower concentra-tions of long-chain folylpolyglutamates than controls at100 nmol/L and 2.3 Amol/L (at 2.3 Amol/L: siRNA, 4.28 F0.30 versus Control-si, 10.57 F 1.05; at 100 nmol/L: siRNA,2.23F 0.17 versus Control-si, 3.07F 0.10; P < 0.002), but notat 20 nmol/L (siRNA, 1.02 F 0.54 versus Control-si, 3.22 F1.64) folic acid (Fig. 2B). Intracellular concentrations oflong-chain folylpolyglutamates correlated significantlywith medium folic acid concentrations in all four FPGSclones (r = 0.69–0.97; P < 0.05; Figs. 1B and 2B).

After 24-h exposure to 1 Amol/L [3H]-methotrexate, themajority of intracellular methotrexate was found as long-chain methotrexate-polyglutamates (methotrexate-glu3–6)in cells expressing the sense FPGS compared with con-trols in which the majority of intracellular methotrexatewas methotrexate-glu1 and methotrexate-glu2 (P < 0.01;Table 1). In both cells expressing the sense FPGS andcontrols, all of the intracellular 5-methylTHF was found aslong-chain polyglutamates (5-methylTHF-glu4 – 8) after24-h exposure to 20 nmol/L [3H]-5-methyTHF. FPGSoverexpression was associated with a shift toward the verylong-chain 5-methylTHF-polyglutamates (5-methylTHF-glu7–8; P < 0.01; Table 1). In contrast, after 24-h exposureto 1 Amol/L [3H]-methotrexate, the majority of intracellularmethotrexate was found as methotrexate-glu1 and metho-trexate-glu2 in cells transfected with the FPGS-targetedsiRNA compared with controls in which the majority ofintracellular methotrexate was found as long-chain meth-otrexate-polyglutamates (methotrexate-glu3–6; P < 0.01;Table 1). In both cells transfected with the FPGS-targetedsiRNA and controls, all of the intracellular 5-methylTHFwas found as long-chain polyglutamates (5-methylTHF-glu4–8) after 24-h exposure to 20 nmol/L [

3H]-5-methyTHF.FPGS inhibition was associated with a shift away from thevery long-chain 5-methylTHF-polyglutamates (5-meth-ylTHF-glu7–8; P < 0.01; Table 1).Cells expressing the sense FPGS had significantly lower

GGH activity compared with controls at 2.3 Amol/L and20 nmol/L (P = 0.01 and P = 0.03, respectively), but not at

Figure 1. MDA-MB-435 breast cancer cells transfected with the sense FPGS (Sense ) had significantly higher steady-state levels of the FPGS protein(A) and higher FPGS activity (A) compared with cells transfected with the vector alone (Control-S ; endogenous FPGS; *, P < 0.0001). Total intracellularfolate concentrations (i.e., folate levels after conjugase treatment) were significantly higher in the Sense cells than in the Control-S cells at all three mediumfolic acid concentrations (B; *, P < 0.0001). Intracellular concentrations of short-chain folylpolyglutamates (i.e., folate levels not treated with conjugase)were significantly higher in the Sense cells than in the Control-S cells only at the lowest medium folic acid concentration (20 nmol/L), but not at 100 nmol/Land 2.3 Amol/L (B; *, P = 0.01). Intracellular concentrations of long-chain folylpolyglutamates (i.e., differences between folate concentration ofconjugase-treated and untreated samples) were significantly higher in the Sense cells than in the Control-S cells at all three medium folic acidconcentrations (B; P < 0.01). c, P < 0.0001 compared with non – conjugase-treated cells of the same construct grown in the same medium folicacid concentration. Intracellular concentrations of total, long-chain, and short-chain folylpolyglutamates correlated significantly with medium folic acidconcentrations in both the Sense (**, r = 0.92 – 0.96, P < 0.001) and Control-S cells (**, r = 0.62 – 0.94, P < 0.05; B). GGH activity was significantlylower in the Sense cells than in the Control-S cells at 20 nmol/L and 2.3 Amol/L medium folic acid concentrations (C). *, P < 0.05. Columns, means;bars, SD.



100 nmol/L folic acid (Fig. 1C). In contrast, FPGS inhibitionhad no significant effect on GGH activity at all three folicacid concentrations (Fig. 2C).Cells expressing the sense FPGS exhibited a significantly

decreased doubling time compared with controls (43.4 F0.4 h versus 44.6 F 0.2 h; P < 0.0001), indicating a fastergrowth rate. Unexpectedly, cells transfected with the FPGS-targeted siRNA also had a significantly decreased doublingtime compared with controls (43.4 F 0.2 h versus 45.0 F0.5 h; P < 0.0001).As proof of principle, the effect of FPGS overexpression

and inhibition on chemosensitivity of breast cancer cells topemetrexed (positive control) and trimetrexate (negativecontrol) was determined at 2.3 Amol/L folic acid. Peme-trexed is a novel antimetabolite that inhibits multipleenzymes involved in thymidylate and purine biosynthesis(31). Pemetrexed is polyglutamylated to the active penta-glutamate by FPGS for its cytotoxic effects (31). Trimetrex-ate is a nonclassic antifolate that directly inhibitsdihydrofolate reductase (DHFR; ref. 32). Trimetrexate isnot polyglutamylated and, hence, does not depend onpolyglutamylation for its cytotoxic effects (32). Cells thatoverexpressed FPGS were more sensitive to pemetrexedcompared with controls (P < 0.05; Fig. 3A). This suggeststhat increased pemetrexed polyglutamylation by FPGSoverexpression significantly enhanced chemosensitivity topemetrexed. However, FPGS inhibition had no significanteffect on chemosensitivity to pemetrexed (Fig. 3A). Inter-estingly, cells that overexpressed FPGS were less sensitive

to trimetrexate, whereas cells in which FPGS was inhibitedwere more sensitive compared with controls (P < 0.05;Fig. 3B). Given the fact that cytotoxic effects of trimetrexatedo not depend on polyglutamylation, these observationssuggest that differential chemosensitivity to trimetrexatelikely resulted from different concentrations and polyglu-tamylation of intracellular folates mediated by FPGSmodulation.Chemosensitivity of cells expressing the sense FPGS to

5FU + LV and 5FU was significantly decreased com-pared with controls at 2.3 Amol/L folic acid (P < 0.05;Fig. 4A and D). However, this effect was not observed at100 nmol/L folic acid (Fig. 4B and E). At 20 nmol/L folicacid, chemosensitivity of cells expressing the sense FPGS to5FU + LV, but not to 5FU, was significantly increasedcompared with controls (P < 0.05; Fig. 4C and F). IC50values indicate significantly reduced chemosensitivity ofcells expressing the sense FPGS to 5FU + LV and 5FU at2.3 Amol/L folic acid, but significantly enhanced chemo-sensitivity to 5FU at 20 nmol/L folic acid compared withcontrols (Table 2).Chemosensitivity of cells transfected with the FPGS-

targeted siRNA to 5FU + LV and 5FU was significantlydecreased compared with controls at 2.3 Amol/L folic acid(P < 0.05; Fig. 5A and D). However, this effect was notobserved at 100 nmol/L folic acid (Fig. 5B and E). At20 nmol/L folic acid, cells transfected with the FPGS-targeted siRNA were significantly less sensitive to 5FU, butnot to 5FU + LV, compared with controls (Fig. 5C and F).

Figure 2. MDA-MB-435 breast cancer cells transfected with the FPGS-targeted siRNA (siRNA ) had significantly lower steady-state levels of the FPGSprotein (A) and lower FPGS activity (A; *P = 0.04) compared with cells transfected with the vector alone (Control-si ; endogenous FPGS). Totalintracellular folate concentrations (i.e., folate levels after conjugase treatment) were significantly lower in the siRNA cells than in the Control-si cells at allthree medium folic acid concentrations (B; *, P < 0.001). Intracellular concentrations of short-chain folylpolyglutamates (i.e., folate levels not treated withconjugase) were significantly lower in the siRNA cells than in the Control-si cells at 2.3 Amol/L and 100 nmol/L medium folic acid concentrations (*, P <0.05) but not at 20 nmol/L (B). Intracellular concentrations of long-chain folylpolyglutamates (i.e., differences between folate concentration of conjugase-treated and untreated samples) were significantly lower in the siRNA cells than in the Control-si cells at 2.3 Amol/L and 100 nmol/L medium folic acidconcentrations (P < 0.02), but not at 20 nmol/L (B). c, P < 0.0001 compared with non – conjugase-treated cells of the same construct grown in the samemedium folic acid concentration. Intracellular concentrations of total and long-chain folylpolyglutamates correlated significantly with medium folic acidconcentrations in both the siRNA (cc, r = 0.92 – 0.97, P < 0.005) and Control-si cells (cc, r = 0.80 – 0.83, P < 0.01; B). Intracellular concentrations ofshort-chain folylpolyglutamates did not correlate significantly with medium folic acid concentrations in either the siRNA or Control-si cells (B). FPGSinhibition did not significantly affect GGH activity in either the siRNA or Control-si cells (C). Columns, means; bars, SD.



IC50 values indicate significantly reduced chemosensitivityof cells transfected with the FPGS-targeted siRNA to5FU + LV and 5FU at 2.3 Amol/L folic acid and to 5FU at20 nmol/L folic acid compared with controls (Table 2).Chemosensitivity to methotrexate was significantly in-

creased in both cells expressing the sense FPGS and cellstransfected with the FPGS-targeted siRNA compared withcorresponding controls at all three folic acid concentra-tions (P < 0.05; Figs. 4G–I and 5G–I). IC50 values formethotrexate indicate significantly enhanced chemosensi-tivity of both cells expressing the sense FPGS and cellstransfected with the FPGS-targeted siRNA compared withcorresponding controls at all three folic acid concentrations(Table 2).Using the IC50 values obtained from corresponding

controls of cells transfected with the sense FPGS or FPGS-targeted siRNA treated with 5FU or methotrexate at eachfolic acid concentration (Table 2), the effects of FPGS modu-lation on chemosensitivity to a combination treatment of5FU and methotrexate was determined. Cells expressingthe sense FPGS were more sensitive to 5FU + methotrexatecompared with controls at all three folic acid concentra-tions (23%, 8%, and 11% lower survival at 2.3 Amol/L,100 nmol/L, and 20 nmol/L, respectively; P < 0.0001). Incontrast, cells transfected with the FPGS-targeted siRNA

were less sensitive to 5FU + methotrexate compared withcontrols only at 100 nmol/L folic acid (15% higher survival,P < 0.0001).

DiscussionWe developed an in vitro model of FPGS overexpressionand inhibition in MDA-MB-435 breast cancer cells withpredictable functional consequences. Compared with con-trols expressing endogenous FPGS, cells expressing thesense FPGS had significantly higher FPGS expression andactivity, shorter doubling time, higher concentrations oftotal intracellular folate, higher content of long-chainfolyl- and 5-methylTHF-polyglutamates, and a shift towardlong-chain methotrexate-polyglutamates. In contrast, cellstransfected with the FPGS-targeted siRNA had significantlylower FPGS expression and activity, lower concentrationsof total intracellular folate, lower content of long-chainfolyl- and 5-methylTHF-polyglutamates, and a shift awayfrom long-chain methotrexate-polyglutamates comparedwith controls expressing endogenous FPGS. Unexpectedly,however, cells transfected with the FPGS-targeted siRNAalso had a shorter doubling time compared with con-trols. The accelerated growth rate associated with FPGSoverexpression is consistent with the observed increased

Table 1. Distribution of 5-methylTHF- and methotrexate polyglutamates as a fraction of total intracellular 5-CH3-[3H]-THF and [3H]-methotrexate polyglutamates

(A) Effects of FPGS overexpression on 5-methylTHF- and methotrexate polyglutamate distribution

5CH3-THF-polyglutamate distribution (% of total 5CH3-THF)

Glu1 Glu2 Glu3 Glu4 Glu5 Glu6 Glu7 Glu8

Sense 0 0 0 0 14 40 34 12Control-S 0 0 0 3 26 53 17 0

Methotrexate-polyglutamate distribution (% of total methotrexate)

Glu1 Glu2 Glu3 Glu4 Glu5 Glu6

Sense 5 2 11 25 55 2Control-S 51 15 21 9 5 0

(B) Effects of FPGS inhibition on 5-methylTHF- and methotrexate polyglutamate distribution

5CH3-THF-polyglutamate distribution (% of total 5CH3-THF)

Glu1 Glu2 Glu3 Glu4 Glu5 Glu6 Glu7 Glu8

Control-si 0 0 0 0 3 53 39 6siRNA 0 0 0 1 52 44 3 0

Methotrexate-polyglutamate distribution (% of total methotrexate)

Glu1 Glu2 Glu3 Glu4 Glu5 Glu6

Control-si 18 17 28 14 22 0siRNA 60 20 16 4 0 0



intracellular folate concentrations and higher content oflong-chain folylpolyglutamates, which are better substratesthan short-chain folylpolyglutamates for folate-dependentenzymes involved in thymidylate and purine biosynthesis(3). This is also consistent with our prior observationsmade in human HCT116 colon cancer cells expressing thesense FPGS (18). However, the accelerated growth rateassociated with siRNA-based FPGS inhibition is not read-ily explained by the observed decrease in intracellularfolate concentrations and lower content of long-chainfolylpolyglutamates. In fact, this observation is in directcontrast to a slower growth rate and TS suppression ob-served with antisense-based FPGS down-regulation inHCT116 (18) and human CCRF-CEM leukemia (33) cells.Generally, most of the observed functional consequencesof FPGS overexpression and inhibition are consistent withthe known biological function of FPGS and provided anappropriate in vitro model to test the effect of FPGS modu-lation on chemosensitivity of breast cancer cells to 5FU andmethotrexate.We hypothesized that FPGS overexpression would up-

regulate GGH, whereas FPGS inhibition would down-regulate GGH to maintain intracellular homeostatic balanceof folate polyglutamylation. In contrast, cells expressing the

sense FPGS had significantly lower GGH activity comparedwith corresponding controls, whereas cells transfected withthe FPGS-targeted siRNA had no significant effect on GGHactivity compared with corresponding controls. We havepreviously reported that HCT116 cells expressing the senseFPGS had significantly higher GGH mRNA expression,whereas those expressing the antisense FPGS had signifi-cantly lower GGH mRNA expression compared with con-trols (18). These observations suggest a cell-specific effectof FPGS modulation on GGH unique to MDA-MB-435 cellsor post-transcriptional modifications.We first showed that the magnitude of FPGS overexpres-

sion induced in this model was sufficient to significantlyenhance chemosensitivity to pemetrexed, which is one ofthe best substrates for FPGS and whose cytotoxic effectsdepend on polyglutamylation by FPGS (31). However, thedegree of FPGS inhibition achieved in this model wasnot sufficient to significantly modulate chemosensitivityto pemetrexed. We also showed that different concentra-tions and polyglutamylation of intracellular folate med-iated by FPGS overexpression and inhibition significantlyreduced and enhanced chemosensitivity to trimetrexate,respectively, which is not polyglutamylated and, hence,does not depend on polyglutamylation for its cytotoxic

Figure 3. A, In vitro chemosensitivity to pemetrexed. Pemetrexed is a novel antimetabolite whose cytotoxic effects depend on polyglutamylation byFPGS. MDA-MB-435 breast cancer cells transfected with the sense FPGS (Sense ) were significantly more sensitive to pemetrexed compared with cellstransfected with the vector alone (Control-S; endogenous FPGS; left, *, P < 0.05). However, chemosensitivity of MDA-MB-435 breast cancer cellstransfected with the FPGS-targeted siRNA (siRNA ) to pemetrexed was not significantly different from that cells transfected with the vector alone (Control-si ; endogenous FPGS; right ). B, in vitro chemosensitivity to trimetrexate. Trimetrexate is a nonclassic antifolate that does not require polyglutamylationfor its cytotoxic effects; in fact, it cannot be polyglutamylated because there is no glutamate moiety. MDA-MB-435 breast cancer cells transfected with thesense FPGS (Sense) were significantly less sensitive to trimetrexate compared with cells transfected with the vector alone (Control-S ; endogenous FPGS;left ). In contrast, MDA-MB-435 breast cancer cells transfected with the FPGS-targeted siRNA (siRNA ) was more sensitive to trimetrexate compared withcells transfected with the vector alone (Control-si; endogenous FPGS; right ; *, P < 0.050). Points, means; bars, SD.



effects (32). These data collectively suggest that both poly-glutamylation of antifolates and folates as well as intra-cellular folate concentrations modulate chemosensitivity.FPGS overexpression was associated with decreased

chemosensitivity of breast cancer cells to 5FU + LV and5FU at 2.3 Amol/L folic acid concentration. However, thisdecreased cytotoxic effect of 5FU + LV and 5FU was notobserved at 100 nmol/L folic acid concentration, whereas at20 nmol/L folic acid concentration, FPGS overexpressionwas associated with enhanced chemosensitivity to 5FU +LV but not to 5FU. Our a priori hypothesis was that FPGSoverexpression would enhance the cytotoxic effect of 5FUby increasing relative intracellular concentration of long-chain 5,10-methyleneTHF-polyglutamates, resulting inmore efficient formation and stabilization of the inhibi-tory 5,10-methyleneTHF-TS-FdUMP ternary complex. At2.3 Amol/L folic acid concentration, it is possible that FPGSoverexpression greatly increased intracellular folate con-centrations and contents of long-chain polyglutamates ofother intracellular folate cofactors to a greater extent than

long-chain 5,10-methyleneTHF-polyglutamates, resultingin increased thymidylate and purine biosynthesis. Thismight have overridden the TS-inhibiting effect of increasedlong-chain 5,10-methyleneTHF-polyglutamates, resultingin decreased chemosensitivity to 5FU + LV and 5FU. At20 nmol/L folic acid concentration, the magnitude ofincreased contents of long-chain 5,10-methyleneTHF-polyglutamates resulting from FPGS overexpression wassufficient to overcome the effect of increased intracellularfolate concentrations and contents of long-chain polygluta-mates of other intracellular folate derivatives, resulting inTS inhibition and increased chemosensitivity to 5FU. How-ever, this enhanced cytotoxic effect was observed onlywhen a precursor for 5,10-methyleneTHF (LV) was sup-plied exogenously. At 100 nmol/L folic acid concentration,the competing effects of increased DNA synthesis associ-ated with increased intracellular folate concentrations andcontent of long-chain folylpolyglutamates and TS inhibi-tion associated with increased long-chain 5,10-methyle-neTHF-polyglutamates seem to have canceled each other out.

Figure 4. In vitro chemosensitivity of MDA-MB-435 breast cancer cells transfected with the sense FPGS (Sense) to 5FU plus LV (A–C), 5FU alone(D–F), or methotrexate (G– I) in comparison to cells transfected with the vector alone (Control-S; endogenous FPGS). Cells were grown in mediumcontaining 2.3 Amol/L (A, D, and G), 100 nmol/L (B, E, and H), or 20 nmol/L (C, F, and I) folic acid. Points, means; bars, SD. *, P < 0.05, statisticallysignificant compared with corresponding control cells.



FPGS inhibition was associated with decreased chemo-sensitivity of breast cancer cells to 5FU + LV and 5FU at2.3 Amol/L folic acid concentration. However, this de-creased cytotoxic effect of 5FU + LV and 5FU was not ob-served at 100 nmol/L folic acid concentration, whereasat 20 nmol/L folic acid concentration, FPGS inhibition wasalso associated with decreased chemosensitivity to 5FU +LV but not to 5FU. Our a priori hypothesis was that FPGSinhibition would decrease the cytotoxic effect of 5FU bydecreasing relative intracellular concentrations of long-chain 5,10-methyleneTHF-polyglutamates, resulting in lessefficient formation and stabilization of the 5,10-methyle-neTHF-TS-FdUMP ternary complex. It seems that de-creased long-chain 5,10-methyleneTHF-polyglutamatesmight have been the predominant effect associated withFPGS inhibition, overriding the counterbalancing effectassociated with decreased total intracellular folate concen-trations and contents of long-chain polyglutamates of otherintracellular folate derivatives and leading to decreasedchemosensitivity to 5FU.Our 5FU chemosensitivity data from the FPGS inhibition

studies are generally consistent with our hypothesis,whereas those from the FPGS overexpression studies areconsistent only at 20 nmol/L folic acid concentration andmostly opposite to our hypothesis. A previous study hasshown that the cytotoxic effect of 5FU is directly correlatedwith FPGS activity in 14 human cancer cell lines (17).Another study has shown that human HCT8 colon cancercells become rapidly resistant to 5FU over time owing to aprogressive decrease in FPGS mRNA expression andactivity (16). We have previously reported that FPGSoverexpression significantly enhances chemosensitivity ofhuman HCT116 colon cancer cells to 5FU, whereas anti-sense FPGS inhibition decreases chemosensitivity of human

HCT116 colon cancer cells to 5FU (18). However, thesein vitro studies were conducted only in media containingz2.3 Amol/L folic acid concentrations. In another study, theeffect of LV, which is converted to 5,10-methyleneTHF andenters the folate pathway (13), on the chemosensitivityof human leukemia CCRF-CEM (the parent cell line withproficient polyglutamylation) and CCRF-CEM/P (a cellline with impaired ability to form polyglutamates) to 5FUwas compared (15). Both CCRF-CEM and CCRF-CEM/Pcells accumulated 5,10-methyleneTHF in the presence ofLV in a dose-dependent manner (15). However, at a doseof 5FU that produced only a slight decrease in cell growth,the addition of LV further inhibited the cell growth inCCRF-CEM cells, but not in CCRF-CEM/P cells (15), sug-gesting that the impaired polyglutamylation of 5,10-methyleneTHF was likely responsible for the lack ofpotentiation of the cytotoxic effect of 5FU by LV inCCRF-CEM/P cells. In contrast, three human studies haveshown conflicting results concerning the role of FPGS inpredicting treatment response to 5FU + LV and survival inpatients with colon cancer (34–36). Our data suggest thatthe effect of FPGS modulation on the chemosensitivity ofbreast cancer cells to 5FU is highly complex and dependson intracellular folate concentrations and polyglutamyla-tion of not only 5,10-methyleneTHF but also other folatecofactors.FPGS overexpression resulted in increased chemosensi-

tivity of breast cancer cells to methotrexate at all three folicacid concentrations, consistent with our a priori hypothesisthat predicted that increased methotrexate polyglutamyla-tion would enhance the cytotoxic effect of methotrexate.IC50 values indicate that an increased concentration ofmethotrexate was required to achieve 50% inhibitionamong cells grown in 2.3 Amol/L compared with lower

Table 2. IC50 values of 5FU and methotrexate in MDA-MB-435 breast cancer cells transfected with the sense FPGS and with theFPGS-targeted siRNA in comparison with corresponding control cells expressing endogenous FPGS

Folate concentration IC50 (95% CI)

2.3 Amol/L 100 nmol/L 20 nmol/L

5FU + LV (Amol/L)Sense 14.14* (12.66, 15.99) 12.67 (11.36, 14.30) 10.48* (9.29, 11.94)Control-S 6.61 (5.75, 7.54) 14.52 (12.59, 17.14) 18.94 (16.37, 22.53)Control-si 11.77 (10.51, 13.35) 4.84 (4.01, 5.69) 7.50 (6.84, 8.22)siRNA 22.85* (19.99, 26.70) 5.49 (4.71, 6.31) 7.07 (6.56, 7.61)5FU (Amol/L)Sense 25.73* (22.92, 29.35) 7.43 (6.69, 8.25) 15.33 (13.65, 17.51)Control-S 12.50 (11.20, 14.12) 6.05 (5.26, 6.89) 14.30 (12.66, 16.43)Control-si 12.61 (10.78, 15.14) 7.08 (6.26, 7.99) 5.63 (4.94, 6.35)siRNA 34.66* (27.78, 45.92) 8.78 (7.95, 9.72) 9.98* (9.00, 11.14)Methotrexate (nmol/L)Sense 21.52* (19.39, 23.97) 11.87* (11.22, 12,57) 14.83* (13.91, 15.88)Control-S 28.07 (25.92, 30.63) 15.18 (14.47, 15.97) 17.60 (16.35, 19.09)Control-si 41.01 (35.22, 49.45) 10.75 (10.03, 11.51) 16.09 (15.16, 17.13)siRNA 26.63* (24.15, 29.85) 7.28* (6.39, 8.14) 12.81* (11.98, 13.71)

*P < 0.05, compared with corresponding controls.



concentrations of folic acid. This suggests that increasedtotal intracellular folate concentrations and higher contentsof folylpolyglutamates associated with FPGS overexpres-sion, which would provide an increased amount ofsubstrates for nucleotide biosynthesis, had a competingeffect on increased methotrexate polyglutamylation, whichwould enhance chemosensitivity to methotrexate. Further-more, methotrexate and folate share cell entry mechanisms,and folate is a higher affinity substrate for DHFR thanmethotrexate (2, 3). Therefore, at high medium folate con-centrations, more folate will enter cells and bind prefe-rentially to DHFR. As a result, higher concentrations ofmethotrexate are required for its cytotoxic effects in highfolate environments.In contrast to our a priori hypothesis that predicted that

FPGS inhibition would decrease chemosensitivity of breastcancer cells to methotrexate, FPGS inhibition enhancedthe cytotoxic effect of methotrexate at 20 and 100 nmol/Lfolic acid concentrations. This suggests that decreased intra-cellular folate concentrations and contents of long-chain

folylpolyglutamates induced by FPGS inhibition over-whelmed the effect of decreased methotrexate polygluta-mylation and increased chemosensitivity to methotrexate.However, this effect was not observed at 2.3 Amol/L folicacid concentration, suggesting that the magnitude ofintracellular folate depletion associated with FPGS inhibi-tion at this folic acid concentration was not sufficient tomodulate chemosensitivity to methotrexate.Decreased antifolate polyglutamylation due to quantita-

tive (4–7) or qualitative (8–10) alterations in FPGS activityhas been shown to be a mechanism of resistance tomethotrexate and other antifolates in human and murineleukemia cell lines. Transfection of FPGS cDNA has beenshown to increase sensitivity to methotrexate in varianthamster cells that lack endogenous FPGS activity (11).FPGS gene transfer into rat and human glioma, gliosar-coma, and glioblastoma cell lines already expressing FPGSsignificantly enhanced cytotoxic sensitivity to methotrexateand other antifolates in vitro and in vivo (12). We havepreviously reported that sense FPGS overexpression and

Figure 5. In vitro chemosensitivity of MDA-MB-435 breast cancer cells transfected with the FPGS-targeted siRNA (siRNA ) to 5FU plus LV (A–C), 5FUalone (D–F), or methotrexate (G– I) in comparison with cells transfected with the vector alone (Control-si ; endogenous FPGS). Cells were grown inmedium containing 2.3 Amol/L (A, D, and G), 100 nmol/L (B, E, and H), or 20 nmol/L (C, F, and I) folic acid. Points, means; bars, SD. *, P < 0.05,statistically significant compared with corresponding control cells.



antisense FPGS inhibition do not significantly affect che-mosensitivity of human HCT116 colon cancer cells tomethotrexate at the same supraphysiologic folic acidconcentration (18). Our present data suggest that the effectof FPGS modulation on the chemosensitivity of breastcancer cells to methotrexate depends not only on metho-trexate polyglutamylation but also on folylpolyglutamyla-tion and intracellular folate pools. FPGS modulation affectsboth polyglutamylation of antifolates and folate cofactors(37). Consequently, alterations in polyglutamylation ofintracellular folate cofactors as well as intracellular folatepools may diminish or even abolish the effects associatedwith alterations in polyglutamylation of antifolates, inparticular for antifolates that are highly dependent onpolyglutamylation for their cytotoxic effects (37–42).5FU and methotrexate are combined with cyclophospha-

mide in the CMF (cyclophosphamide; methotrexate; 5-fluorouracil) regimen for breast cancer treatment (43). Ourdata suggest that FPGS overexpression increases chemo-sensitivity of breast cancer cells to 5FU + methotrexate at allthree folic acid concentrations, whereas FPGS inhibitiondecreases chemosensitivity to 5FU + methotrexate only at100 nmol/L folic acid concentration. The effect of FPGSmodulation on the chemosensitivity of breast cancer cells to5FU + methotrexate likely depends on a complex interplayof intracellular folate concentrations, polyglutamylation of5,10-methyleneTHF and other folate cofactors, and metho-trexate polyglutamylation.In conclusion, as proof of principle, we provide func-

tional evidence that FPGS modulation affects the chemo-sensitivity of breast cancer cells to 5FU and methotrexate.Our data suggest that the effect of FPGS modulation on thechemosensitivity of breast cancer cells to 5FU and metho-trexate is highly complex and depends on intracellularfolate concentrations and polyglutamylation of intracellularfolate cofactors and methotrexate. Furthermore, our datasuggest that the effect of FPGS modulation on the chemo-sensitivity to antifolates, whose cytotoxic effects depend onpolyglutamylation, cannot be predicted solely based on thepolyglutamylation of antifolates. Whether or not FPGSmodulation may be an important clinical determinant ofchemosensitivity of breast cancer cells to 5FU- and metho-trexate-based chemotherapy needs further exploration.

References

1. Shane B. Folate chemistry and metabolism. In: Bailey LB, editor. Folatein health and disease. New York: Marcel Dekker; 1995. p. 1 – 22.

2. Kamen B. Folate and antifolate pharmacology. Semin Oncol 1997;24:S18 – 30-S18 – 39.

3. Moran RG. Roles of folylpoly-g-glutamate synthetase in therapeuticswith tetrahydrofolate antimetabolites: an overview. Semin Oncol 1999;26:24 – 32.

4. Pizzorno G, Mini E, Coronnello M, et al. Impaired polyglutamylation ofmethotrexate as a cause of resistance in CCRF-CEM cells after short-term,high-dose treatment with this drug. Cancer Res 1988;48:2149 – 55.

5. McCloskey DE, McGuire JJ, Russell CA, et al. Decreased folylpolyglu-tamate synthetase activity as a mechanism of methotrexate resistance inCCRF-CEM human leukemia sublines. J Biol Chem 1991;266:6181 – 7.

6. Mauritz R, Peters GJ, Priest DG, et al. Multiple mechanisms ofresistance to methotrexate and novel antifolates in human CCRF-CEM

leukemia cells and their implications for folate homeostasis. BiochemPharmacol 2002;63:105 – 15.

7. Liani E, Rothem L, Bunni MA, Smith CA, Jansen G, Assaraf YG. Loss offolylpoly-g-glutamate synthetase activity is a dominant mechanism ofresistance to polyglutamylation-dependent novel antifolates in multiplehuman leukemia sublines. Int J Cancer 2003;103:587 – 99.

8. Sanghani SP, Sanghani PC, Moran RG. Identification of three key activesite residues in the C-terminal domain of human recombinant folylpoly-g-glutamate synthetase by site-directed mutagenesis. J Biol Chem 1999;274:27018 – 27.

9. Zhao R, Titus S, Gao F, Moran RG, Goldman ID. Molecular analysis ofmurine leukemia cell lines resistant to 5,10-dideazatetrahydrofolateidentifies several amino acids critical to the function of folylpolyglutamatesynthetase. J Biol Chem 2000;275:26599 – 606.

10. Roy K, Egan MG, Sirlin S, Sirotnak FM. Posttranscriptionally mediateddecreases in folylpolyglutamate synthetase gene expression in some folateanalogue-resistant variants of the L1210 cell. Evidence for an alteredcognate mRNA in the variants affecting the rate of de novo synthesis ofthe enzyme. J Biol Chem 1997;272:6903 – 8.

11. Kim JS, Lowe KE, Shane B. Regulation of folate and one-carbonmetabolism in mammalian cells. IV. Role of folylpoly-g-glutamate synthe-tase in methotrexate metabolism and cytotoxicity. J Biol Chem 1993;268:21680 – 5.

12. Aghi M, Kramm CM, Breakefield XO. Folylpolyglutamyl synthetasegene transfer and glioma antifolate sensitivity in culture and in vivo . J NatlCancer Inst 1999;91:1233 – 41.

13. Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms ofaction and clinical strategies. Nat Rev Cancer 2003;3:330 – 8.

14. Radparvar S, Houghton PJ, Houghton JA. Effect of polyglutamylationof 5,10-methylenetetrahydrofolate on the binding of 5-fluoro-2¶-deoxyur-idylate to thymidylate synthase purified from a human colon adenocarci-noma xenograft. Biochem Pharmacol 1989;38:335 – 42.

15. Romanini A, Lin JT, Niedzwiecki D, Bunni M, Priest DG, Bertino JR.Role of folylpolyglutamates in biochemical modulation of fluoropyrimidinesby leucovorin. Cancer Res 1991;51:789 – 93.

16. Wang FS, Aschele C, Sobrero A, Chang YM, Bertino JR. Decreasedfolylpolyglutamate synthetase expression: a novel mechanism of fluoro-uracil resistance. Cancer Res 1993;53:3677 – 80.

17. Cheradame S, Etienne MC, Chazal M, et al. Relevance of tumoralfolylpolyglutamate synthetase and reduced folates for optimal 5-fluoro-uracil efficacy: experimental data. Eur J Cancer 1997;33:950 – 9.

18. Sohn KJ, Smirnakis F, Moskovitz DN, et al. Effects of folylpolyglu-tamate synthetase modulation on chemosensitivity of colon cancer cells to5-fluorouracil and methotrexate. Gut 2004;53:1825 – 31.

19. Backus HH, Pinedo HM, Wouters D, et al. Folate depletion increasessensitivity of solid tumor cell lines to 5-fluorouracil and antifolates. Int JCancer 2000;87:771 – 8.

20. Zhao R, Gao F, Goldman ID. Marked suppression of the activity ofsome, but not all, antifolate compounds by augmentation of folatecofactor pools within tumor cells. Biochem Pharmacol 2001;61:857 – 65.

21. Garrow TA, Admon A, Shane B. Expression cloning of a human cDNAencoding folylpoly(g-glutamate) synthetase and determination of itsprimary structure. Proc Natl Acad Sci U S A 1992;89:9151 – 5.

22. McGuire JJ, Russell CA. Folylpolyglutamate synthetase expression inantifolate-sensitive and -resistant human cell lines. Oncol Res 1998;10:193 – 200.

23. Cole PD, Kamen BA, Gorlick R, et al. Effects of overexpression of g-glutamyl hydrolase on methotrexate metabolism and resistance. CancerRes 2001;61:4599 – 604.

24. Panetta JC, Wall A, Pui CH, Relling MV, Evans WE. Methotrexateintracellular disposition in acute lymphoblastic leukemia: a mathematicalmodel of g-glutamyl hydrolase activity. Clin Cancer Res 2002;8:2423 – 9.

25. Tamura T. Microbiologicl assay of folate. In: Picciano MF, StokstadELR, Gregory III JF, editors. Folic acid metabolism in health and disease.New York: Wiley-Liss; 1990. p. 121 – 37.

26. Kamen BA, Wang MT, Streckfuss AJ, Peryea X, Anderson RG.Delivery of folates to the cytoplasm of MA104 cells is mediated by a sur-face membrane receptor that recycles. J Biol Chem 1988;263:13602 – 9.

27. Skehan P, Storeng R, Scudiero D, et al. New colorimetric cytotoxicityassay for anticancer-drug screening. J Natl Cancer Inst 1990;82:1107– 12.

28. Sohn KJ, Croxford R, Yates Z, Lucock M, Kim YI. Effect of the



methylenetetrahydrofolate reductase C677T polymorphism on chemo-sensitivity of colon and breast cancer cells to 5-fluorouracil andmethotrexate. J Natl Cancer Inst 2004;96:134 – 44.

29. Peters GJ, Wets M, Keepers YP, et al. Transformation of mousefibroblasts with the oncogenes H-ras OR trk is associated withpronounced changes in drug sensitivity and metabolism. Int J Cancer1993;54:450 – 5.

30. Draper NR, Smith H. Applied regression analysis. 2nd ed. New York:John Wiley & Sons; 1981.

31. Chattopadhyay S, Moran RG, Goldman ID. Pemetrexed: biochemicaland cellular pharmacology, mechanisms, and clinical applications. MolCancer Ther 2007;6:404 – 17.

32. Lin JT, Bertino JR. Update on trimetrexate, a folate antagonist with anti-neoplastic and antiprotozoal properties. Cancer Invest 1991;9:159–72.

33. Liu Y, Raghunathan K, Hill C, et al. Effects of antisense-basedfolypoly-g-glutamate synthetase down-regulation on reduced folates andcellular proliferation in CCRF-CEM cells. Biochem Pharmacol 1998;55:2031 – 7.

34. Chazal M, Cheradame S, Formento JL, et al. Decreased folylpolyglu-tamate synthetase activity in tumors resistant to fluorouracil-folinic acidtreatment: clinical data. Clin Cancer Res 1997;3:553 – 7.

35. Mini E, Biondi C, Morganti M, et al. Marked variation of thymidylatesynthase and folylpolyglutamate synthetase gene expression in humancolorectal tumors. Oncol Res 1999;11:437 – 45.

36. Odin E, Wettergren Y, Nilsson S, et al. Altered gene expression of

folate enzymes in adjacent mucosa is associated with outcome ofcolorectal cancer patients. Clin Cancer Res 2003;9:6012 – 9.

37. Jansen G, Peters GJ, Pinedo HM, Priest DG, Assaraf YG. Re:Folylpolyglutamyl synthetase gene transfer and glioma antifolate sensitiv-ity in culture and in vivo. J Natl Cancer Inst 1999;91:2047 – 50.

38. Jansen G, Barr H, Kathmann I, et al. Multiple mechanisms ofresistance to polyglutamatable and lipophilic antifolates in mammaliancells: role of increased folylpolyglutamylation, expanded folate pools, andintralysosomal drug sequestration. Mol Pharmacol 1999;55:761 – 9.

39. Jansen G, Mauritz R, Drori S, et al. A structurally altered humanreduced folate carrier with increased folic acid transport mediates a novelmechanism of antifolate resistance. J Biol Chem 1998;273:30189 – 98.

40. Peters GJ, Smitskamp-Wilms E, Smid K, Pinedo HM, Jansen G.Determinants of activity of the antifolate thymidylate synthase inhibitorsTomudex (ZD1694) and GW1843U89 against mono- and multilayeredcolon cancer cell lines under folate-restricted conditions. Cancer Res1999;59:5529 – 35.

41. Tse A, Moran RG. Cellular folates prevent polyglutamation of 5, 10-dideazatetrahydrofolate. A novel mechanism of resistance to folateantimetabolites. J Biol Chem 1998;273:25944 – 52.

42. O’Dwyer PJ, Nelson K, Thornton DE. Overview of phase II trials ofMTA in solid tumors. Semin Oncol 1999;26:99 – 104.

43. Colozza M, de Azambuja E, Cardoso F, Bernard C, Piccart MJ. Breastcancer: achievements in adjuvant systemic therapies in the pre-genomicera. The Oncologist 2006;11:111 – 25.



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Effects of folate and folylpolyglutamyl synthase modulation on chemosensitivity of breast cancer cells