Cancer Research TCM Europe 2010 Workshops
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by van der Wilt, C. L.
Right arrow Articles by Peters, G. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van der Wilt, C. L.
Right arrow Articles by Peters, G. J.
[Cancer Research 61, 3675-3681, May 1, 2001]
© 2001 American Association for Cancer Research

Experimental Therapeutics

Modulation of Both Endogenous Folates and Thymidine Enhance the Therapeutic Efficacy of Thymidylate Synthase Inhibitors1

Clasina L. van der Wilt, Harold H. J. Backus, Kees Smid, Lizzy Comijn, Gijsbert Veerman, Dorine Wouters, Daphne A. Voorn, David G. Priest, Marlene A. Bunni, Fraser Mitchell, Ann L. Jackman, Gerrit Jansen and Godefridus J. Peters2

Department of Medical Oncology, University Hospital Vrije Universiteit, 1007 MB Amsterdam, the Netherlands [C. L. v. d. W., H. H. J. B., K. S., L. C., G. V., D. W., D. A. V., G. J., G. J. P.]; Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425 [D. G. P., M. A. B.]; and CRC Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey, SM2 5N9 England [F. M., A. L. J.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasma levels of folates and thymidine in mice are about 10-fold higher than in humans and may influence the therapeutic efficacy of thymidylate synthase (TS) inhibitors, such as 5-fluorouracil (5FU) and the antifolates pemetrexed (MTA) and raltitrexed (RTX). Therefore, we tested their therapeutic efficacy in various murine tumor models, grown in mice on a normal and a folate-depleted diet, with high and low thymidine kinase (TK) levels. MTA and RTX were inactive against Colon-26-10 [doubling times gained by treatment; growth delay factor (GDF), 0.5 and 0.3, respectively], whereas 5FU was very active (GDF, >10; complete cures). Colon-26-10/F, grown in mice on a folate-depleted diet, was more sensitive to RTX and MTA (GDF, 2.1 and 1.3, respectively) but not to 5FU (GDF, 1.2); however, leucovorin reversed the effect leading to cures. Folate depletion did not reverse resistance of Colon-26A and Colon-26G (low TK) to MTA and RTX, whereas leucovorin only enhanced the 5FU effect in Colon-26A and Colon-26A/F. Folic acid at 15 mg/kg did not improve the therapeutic efficacy of MTA in folate-deficient mice. The folate-depleted diet decreased the reduced folates in Colon-26A/F and Colon-26-G/F tumors less (4–5-fold; P < 0.01) than in Colon-26-10/F tumors (8-fold; P < 0.001). Folate depletion increased TS levels 2–3-fold in all of the models and TK levels 6-fold (P < 0.01) in Colon-26G/F, explaining the lack of activity of MTA and RTX in Colon-26G/F. In contrast, TK-deficient FM3A/TK tumors were much more sensitive to RTX, MTA, and 5FU than parent FM3A tumors, which have comparable TS levels. The rate of thymidine phosphorylysis varied considerably in all of the tumors without a clear relation to antitumor activity.

In conclusion, tumor folates may potentiate (5FU) or protect (antifolates). Murine tumor models should combine low folates and low thymidine rescue to optimize preclinical testing of antifolates.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In vivo tumor models are an essential step in the preclinical evaluation of anticancer drugs to determine the pharmacokinetic and toxicity parameters (1) . However, these models have some major disadvantages that can influence the sensitivity of drugs such as TS3 (2, 3, 4, 5) . TS is an essential enzyme in the DNA synthesis. It catalyzes the reductive methylation of dUMP in the final step in the only de novo pathway for synthesis of dTMP. 5,10-CH2-H4-folate serves as a methyl donor and is converted into dihydrofolate (6) .

Murine models have 10-fold higher plasma levels of folates, predominantly 5-CH3-H4-folate, than human plasma (7 , 8) . This may abrogate the activity of folate-based TS inhibitors such as RTX (Tomudex, ZD1694) and MTA (LY231514; Ref. 9 ). More frequent and higher doses will be required to compete with natural folates at different levels, including the uptake into the cell via the reduced folate carrier and the linkage of polyglutamate side chains by the enzyme folylpolyglutamate synthetase, which would enhance antifolate retention in the cell and, finally, the binding to the target enzyme TS (10) .

High folate levels in mice can also mask the effect of LV (5-CHO-H4-folate) on 5FU treatment (4) . 5FU is a precursor of a nucleotide-based inhibitor of TS. The 5FU metabolite, FdUMP, binds to TS, and together with 5,10-CH2-H4-folate, a ternary complex is formed (11) . 5,10-CH2-H4-folate stabilizes this ternary complex, and when folate pools are limiting, they can be elevated by administration of LV (4) . Subsequently, the inhibition of TS by the 5FU will be enhanced, and the antitumor activity will be increased. Depending on the tumor model (12) , such an effect might be not observed in mice because of their high plasma folate levels. In other in vivo studies (13 , 14) , it has been shown that LV and TdR can abrogate both the antitumor effect and the toxicity of RTX. The effects of MTA in vitro can be reversed by TdR in combination with hypoxanthine (15) . The latter is related to the other targets of MTA, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase.

The 10-fold higher level of TdR in murine plasma compared with human plasma (3 , 14 , 16 , 17) is another problem of in vivo models. TdR is converted into dTMP in a reaction catalyzed by TK. This can salvage dTMP depletion caused by inhibition of TS and may reduce the antitumor activity of 5FU and folate-based TS inhibitors (5 , 10 , 14) .

Several approaches have been used to solve these two problems of murine models. The folate levels in mice can be reduced by feeding the mice a folate-deficient diet. The effects of a folate-deficient diet have been studied extensively in another rodent, the rat (18) . Mice can also be kept on such a diet, efficiently reducing folate levels (7) . Studies with C3H mice on the activity of 5FU and LV (4) and another antifolate drug, lometrexol (7 , 19) , showed that the intrinsic level of folates in mice on normal diet play an important role. The toxicity of lometrexol increased by three orders of magnitude in folate-deficient mice, mimicking some unexpected toxicity seen in humans.

A combination of folate-depleted diet and coadministration of FA has been tested to manipulate the therapeutic window of some antifolates. This approach allowed a precise control of the folate status of murine plasma and tissues. Studies with the antifolate TS inhibitor GW1843U89 (17) have shown that this concept works very well in a preclinical setting. Administration of FA allowed an increase of the antifolate dose; the antitumor activity improved, and the FA protected normal tissues against toxic side effects, whereas no protection of antitumor activity was observed up to a dose of 366 mg/kg FA (oral administration). A similar effect was observed with lometrexol and FA (19) . Lometrexol had a limited therapeutic activity in folate-deficient mice with C3H mammary tumors compared with that of mice on a standard diet. When FA was coadministered p.o. to mice that were mildly folate-deficient, antitumor activity was observed, and toxicity was reduced. The range of lometrexol doses that allowed safe therapeutic use of this drug increased at higher folate intake, but excessively high folate intake reversed the antitumor activity of lometrexol.

The effect of high plasma TdR levels can be circumvented in two ways, either by using a tumor that is TK deficient (14 , 20, 21, 22) or by feeding the mice a conjugate of polyethylene glycol-thymidine phosphorylase (5 , 17) . The TK- will prevent any interference of TdR with antitumor activity of a TS inhibitor, whereas systemic toxic side effects of the drug might be slightly reduced. The polyethylene glycol-thymidine phosphorylase will catabolize all of the TdR in the plasma into thymine and deoxyribose-1-phosphate, and thus plasma TdR levels will be decreased.

In this study, we used three variants of the murine colon tumor model Colon 26 to evaluate the influence of folates on the antitumor effect of 5FU, 5FU/LV, and the antifolates MTA and RTX. Balb/C mice were put on a folate-deficient diet, and we developed low folate variants of the tumors. One variant with a very low TK activity and a TK-deficient variant of the murine mammary tumor FM3A were used to evaluate the role of TdR levels. The antitumor activity of drugs was related to the effect of folate depletion on several relevant biochemical properties, such as TS activity, TK activity, and TP activity.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
5FU was obtained from PCH Pharmachemie (Haarlem, the Netherlands). d,l-LV and FA were provided by the Pharmacy Department of the University Hospital Vrije Universiteit (Amsterdam, the Netherlands). These drugs were diluted to appropriate concentrations in 0.9% NaCl. RTX was from AstraZeneca Pharmaceuticals (Macclesfield, United Kingdom). MTA was generously supplied by Lilly Research Laboratories (Indianapolis, IN). These drugs were diluted in a 50 mM NaHCO3 buffer.

[6-3H]FdUMP (specific activity, 20 Ci/mmol) and [2-14C]TdR (specific activity, 59.2 Ci/mmol) were obtained from Moravek Biochemicals, Inc. (Brea, CA). [5-3H]dUMP (specific activity, 10.9 Ci/mmol) was from Amersham International (Buckinghamshire, United Kingdom). TS (3.2 units/mg protein) was purified from the Escherichia coli strain that overproduces Lactobacillus casei TS (23) . The E. coli strain was a gift from D. V. Santi (University of California, San Francisco, CA). 5,10-CH2-H4-folate reductase (0.52 units/mg protein) and 10-formyltetrahydrofolate dehydrogenase (0.2 units/mg protein) were purified from pig (24 , 25) . Methylenetetrahydrofolate synthetase (0.1 units/mg protein) was purified from rabbit liver. All of the other chemicals were from analytical grade and commercially available.

FA-deficient chow (0 mg of FA/100 kg) to which no FA was added and standard rodent chow (3.65 mg of FA/100 kg) were obtained from Hope Farms (Woerden, the Netherlands).

Animals.
Female BALB/c and C3H/He mice (Harlan, Zeist, the Netherlands) 6–10 weeks of age (18–20 g) were used for dose-finding studies and antitumor activity experiments. The mice were housed six to eight/cage with normal sawdust bedding in a room with a controlled light-dark cycle (8 h light, 16 h dark). Water, normal chow, and folate-deficient chow were provided ad libitum. To establish a mouse model with low folate levels, mice were fed the folate-deficient chow. During 2 weeks, we monitored food intake, weight, hematocrit, reticulocytes, plasma folate, TdR levels, and general behavior. Blood (100–200 µl) was collected from the mice by a cut in the tip of the tail vein.

Dose-finding studies were performed with mice that did not bear tumors. The MTD and schedule for 5FU and LV in BALB/c was known from previous experiments (100 mg/kg 5FU; 2 x 50 mg/kg LV; q7d x 2; Refs. 12 , 26 ), and the same 5FU dose/schedule was tested in C3H/He mice. RTX and MTA were given in a 5 times daily schedule (q1d x 5) at start doses of 10 mg/kg and 80 mg/kg, respectively. The RTX and MTA dose were escalated further by 1.3-fold steps. MTD studies in low folate mice were initiated with the standard 5FU dose and 10-fold reduced doses of MTA and RTX as compared with the MTD for normal mice. MTD studies on MTA with FA supplementation in mice on a low folate diet were initiated at 50 and 15 mg/kg, respectively. The FA dose was kept constant, whereas we increased the MTA dose by 1.3-fold until the MTD was reached. The MTD was defined as a weight loss of maximally 10% in mice without tumors. The doses are indicated further by subscripts in the text, so 5FU100, is 100 mg/kg.

For the antitumor activity experiments, tumors were excised from a previous passage in mice and cut into 1-mm3 pieces. The tumor pieces were implanted s.c. in the left and right flank with a 16-gauge trochar. Colon 26 variants were transplanted into female BALB/c mice. The following Colon 26 variants were used: Colon 26A was regarded as the parental tumor; Colon 26G was derived from Colon 26A by in vivo selection with gemcitabine and has a very low total TK activity (12% of Colon 26A; Ref. 27 ); and Colon 26-10 was derived from a cell line C26-10 (12) . FM3A and FM3A/TK- mammary carcinoma cells were a gift from Dr. J. Balzarini at the Rega Institute (Leuven, Belgium). The original FM3A/TK- cells were developed by Ayusawa et al. (28) . Tumors were established by s.c. injection of 106 mammary carcinoma cells into female C3H/He mice. The Colon 26 variants were also transplanted into low folate BALB/c mice. All of the variants (indicated as Colon 26../F) grew well in these mice and were subsequently maintained in low folate mice. At a tumor size of approximately 100 mm3, treatment was started according to MTD of the drugs. Mice were randomized in treatment groups and a control group of six to eight animals, depending on the number of tumors. Weight and tumor volume were measured twice weekly. Antitumor activity was evaluated by the GDF, which indicated the gain of doubling time by treatment (12) . Toxic side effects were evaluated by weight loss. Statistical analysis was performed with the Student t test for unpaired data. All of the animal experiments were approved by the Scientific and Animal Ethical Committees of the Vrije Universiteit.

Tissue Preparation.
Blood was collected in hematocrit capillaries, smears for reticulocytes were made, and the remaining part was centrifuged immediately. Hematocrit was determined, and the plasma was collected and diluted with an equal volume of cold 50 mM Tris/HCl buffer (pH 7.4) containing 50 mM sodium ascorbate and 1 mM EDTA. This was stored at -70°C until analysis. Tumor and liver tissue were excised from the mice, washed with cold PBS, frozen in liquid nitrogen, and stored at -70°C. The frozen tissues were pulverized by means of a microdismembrator (29) .

Folate Analysis.
The ternary complex assay used to measure reduced folates is based upon enzymatic cycling of reduced folates to 5,10-CH2-H4-folate followed by entrapment into a stable ternary complex with excess L. casei TS and [3H]FdUMP. Estimation of 5,10-CH2-H4-folate + H4-folate, 5-CH3- H4-folate, were described previously (7) using this approach. The frozen pulverized tissue was suspended in cold 50 mM Tris-HCl buffer (pH 7.4) containing 50 mM sodium ascorbate and centrifuged at 10,000 x g for 3 min at 4°C to remove cell debris. An aliquot of the supernatant was used for protein determination (30) . The remainder of the supernatant was immediately placed in a boiling water bath for 3 min and centrifuged to remove precipitated proteins. The resultant supernatants were used for folate estimation. Because 5,10-CH2-H4-folate can potentially be dissociated to H4-folate and formaldehyde under these conditions, the sum of these folates is reported.

Thymidine Analysis.
Plasma TdR levels were determined by HPLC. Briefly, 75-µl samples of mouse plasma were diluted with 1.0 ml of water, and the plasma proteins were precipitated by the addition of 50 µl of ice-cold percholic acid. After centrifugation, the obtained supernatant was treated with potassium carbonate to remove the perchlorate as the insoluble potassium salt. The sample was then purified by solid-phase extraction using columns packed with 100 mg of a high capacity reverse-phase polymeric sorbent. The column-bound TdR was eluted with methanol, and the eluates were evaporated to dryness in a vacuum centrifuge. The dried residues were reconstituted in 150 µl of water and stored frozen until analyzed by HPLC. The analysis was carried out using 250-mm Supelcosil C18 column using a mobile phase of 2% acetonitrile in 0.05% trifluoroacetic acid at a flow rate of 1.25 ml/min. The retention time of the TdR peak was approximately 8.5 min, and it was measured at 267 nm using a 5-point standard curve ranging from 0.2 to 4 µM and corrected for sample processing losses. This correction factor was obtained by spiking samples with radiolabeled TdR and measuring the difference in activity after sample preparation.

Enzyme Assays.
The enzyme activities of TS, TK, and TP in tumor tissues were determined according to previously published methods. For each assay, the frozen pulverized tissue was suspended in the appropriate assay buffer, and an aliquot was taken for protein measurement.

TS activity was determined as the conversion rate of [5-3H]dUMP into dTMP, releasing tritiated water (31) . The pulverized tissue was suspended in 0.2 M Tris/HCl (pH 7.4) buffer containing 20 mM mercaptoethanol, 100 mM NaF, and 15 mM CMP. The reaction mixture consisted of 25 µl of 10,000 g supernatant, 5 µl 6.5 mM of 5,10-CH2-H4-folate, 10 µl of Tris/HCl buffer, and 10 µl of [5-3H]dUMP (final concentration, 10 µM; specific activity, 50 mCi/mol) and was incubated for 30 min at 37°C. The reaction was stopped by addition of 50 µl of ice-cold 35% trichloroacetic acid and 250 µl of 10% neutral activated charcoal. After centrifugation, 150 µl of the resultant supernatant was used for liquid scintillation counting of radioactivity.

Total TK activity consisting of cytosolic TK1 and mitochondrial TK2 was measured as described by Ruiz van Haperen et al. (32) . A 50 mM Tris/1 mM EDTA (pH 7.4) buffer was used, and the assay mixture consisted of 25 µl of 10,000 g supernatant and 25 µl of substrate solution [21.9 µM TdR, final specific activity, 1.8 Ci/mmol; 20 mM ATP; 10 mM MgCl2; 10 mM Tris/200 µM EDTA (pH 7.4)]. When only TK1 was determined, 10 mM dCTP was added to the substrate solution, as a specific inhibitor of TK2 (33) . The mixture was incubated at 37°C for 30 min and stopped by heating the mixture at 95°C for 3 min, followed by the addition of 10 µl of 5 mM TdR/5 mM thymine. Substrate (TdR) and product (dTMP) were separated by TLC on polyethylene imine cellulose layers, with distilled water as eluent. The spots could be visualized under UV, marked, and cut out. Radioactivity was estimated by liquid scintillation counting.

TP activity was measured with TdR as a substrate by means of a nonradioactive assay (34) . TdR phosphorylysis in murine tissue may also be catalyzed by uridine phosphorylase. TP activity as measured here also represents the contribution of uridine phosphorylase. The assay buffer was similar to that of the TK activity assay. Fifty µl of 10,000 g supernatant was mixed with 10 µl of 0.8M K2HPO4, 10 µl of 1 mM TdR, and 130 µl of Tris/EDTA (pH 7.4) buffer and incubated for 30 min at 37°C. The reaction was stopped by the addition of 50 µl of 40% trichloroacetic acid. The samples were left on ice for 20 min and then centrifuged for 5 min at 10,000 x g at 4°C and neutralized with 400 µl of trioctyl amine/1,1,2,-trichlorotrifluoro ethane (1:4). The upper fraction was stored at -20°C until HPLC analysis, which was performed with a µBondapak C18 column. TdR and thymine were separated using isocratic elution with PicB7 reagens (25 ml/liter; pH 3.1) at a flow of 1 ml/min. The injection volume was 20–50 µl. Concentrations of thymine and TdR were calculated from the peak heights at 254 nm using external standards.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dose-Finding Studies.
Development of the low folate mouse model allowed us to study the impact of dietary folate on the antitumor activity of TS inhibitors. There was a large difference in MTD for the folate-based TS inhibitors (Table 1)Citation , which is not observed for 5FU. Furthermore, a mouse strain-dependent difference in MTD was observed. The dose of MTA in low folate mice could be increased to nearly the level of normal diet mice, when 15 mg/kg FA was administered as a rescue agent. The weight loss observed at the MTD in nontumor-bearing mice was always below 10% (data not shown), but the weight loss indicated in Table 1Citation accounted for the weight loss in tumor-bearing mice during therapy. This weight loss was a good indication for toxicity of the treatment. During the development of the low folate mouse model, we observed that the food consumption of folate-deficient chow was stable (±3 gram/mouse/day). Weight of the mice was stable (±20 g), but they did not grow anymore, whereas mice of the same age on a normal diet did grow. The formation of RBCs in the bone marrow is highly dependent on folates, but we saw no significant changes in hematocrit or reticulocytes during 5 weeks on folate-deficient diet.


View this table:
[in this window]
[in a new window]

  		Table 1 MTD of TS inhibitors in C3H/He mice and Balb/c mice on normal and low folate diet

 
Folate Pools.
In mice kept on a folate-deficient diet, a marked reduction in plasma folate levels (8–28-fold; Table 2Citation ) and in the colon tumor tissue (4–10-fold) was noted. Interestingly, the 5,10-CH2-H4-folate and H4-folate pools in parent Colon 26-10 tumors were 2.6-fold (P < 0.001) higher than the pools of Colon 26A and 26G (Table 2)Citation . Among the low folate variants, Colon 26-10/F had the highest 5,10-CH2-H4-folate and H4-folate pools. There appeared to be a shift in the folate pools, because the 5-CH3-H4-folate pools were significantly lower in Colon 26-10 tumors compared with the other two colon tumors. The decrease in 5,10-CH2-H4-folate and H4-folate levels of Colon 26-10/F was 8-fold compared with the parent Colon 26-10. The other colon tumors showed smaller changes, 5-fold and 4-fold for Colon 26A and 26G, respectively. The mammary tumor FM3A/TK- had 1.6-fold higher 5,10-CH2-H4-folate and H4-folate pools (P < 0.01) than FM3A tumors.


View this table:
[in this window]
[in a new window]

  		Table 2 Folate levels in murine plasma and tissue

 
TdR Levels.
The TdR levels in plasma of Balb/c mice on a normal diet and that of Balb/c mice on a folate-depleted diet were comparable. TdR in the plasma sample of two Balb/c mice on a normal diet were 0.57 and 0.56 µM; in a Balb/C mouse on low folate diet, 0.64 µM TdR was measured in plasma. The other mouse strain C3H/He also had comparable TdR levels. Measurements on two plasma samples from different C3H/He mice showed 0.29 and 0.73 µM TdR.

Antitumor Effects.
The doubling time of the tumors Colon 26A and 26G did not change under dietary folate conditions, but Colon 26-10/F grew two times slower than Colon 26-10 (P < 0.001; Table 3Citation ). The antitumor activities of the various drugs in mice on a normal and folate-free diet are summarized in Table 3Citation . Colon 26-10 tumors were much more sensitive to 5FU100 and 5FU100 + LV100 than Colon 26A or Colon 26G. In the folate-depleted mice, the low folate variant Colon 26-10/F was more resistant to 5FU100, but the sensitivity to 5FU100 + LV100 was unchanged. The Colon 26-10 tumor was rather resistant to RTX10 and MTA100 compared with Colon 26A and Colon 26G. The tumor-doubling time of Colon 26-10 tumors treated with these drugs did not differ from control as expressed by the low GDF.


View this table:
[in this window]
[in a new window]

  		Table 3 Antitumor activity in mice on normal and low folate diet

 
The decrease of folate levels in Colon 26A/F, Colon 26G/F, and Colon 26-10/F tumors as compared with their high folate variants resulted only in Colon 26-10/F tumors in a change to higher sensitivity to RTX0.2 and MTA1 (GDF, 2.1 and 1.3, respectively). For the other two variants, the antitumor activity was less when GDF was calculated. However, the therapeutic efficacy of RTX0.2 and MTA1 in Colon 26-10/F-bearing mice was not superior to 5FU100 or LV100 and 5FU100, because weight loss was worse (see Table 1Citation ).

The effect of MTA1 might be improved by the use of folate depletion and supplementation of FA. In mice bearing the Colon 26A/F and Colon 26G/F tumors, the antitumor activity did not increase (Table 3)Citation , despite the higher MTA80 dose. However, the toxicity of MTA80 treatment was diminished, for less dramatic weight loss effects were observed (see Table 1Citation ).

The FM3A tumor was not very sensitive to 5FU100, RTX25, or MTA300 treatment (Table 3)Citation . In contrast, the TK- variant treated with the same doses was very sensitive to 5FU100, RTX25, and MTA300 treatment, resulting in remissions of the FM3A/TK- tumors. In the case of 5FU100, regrowth of the tumors occurred after 4 to 8 weeks, but for RTX25 and MTA300, the remissions remained complete cures.

Enzyme Assays.
The activities of enzymes involved in thymidine metabolism of the different murine colon tumors showed remarkable differences, whereas folate depletion was also associated with several changes. Total TK activity and especially TK2 of Colon 26G was low compared with the other variants, but this appeared to be an unstable phenotype (Table 4)Citation . Every other passage of the tumor in Balb/c mice had to be treated with gemcitabine to keep a constant phenotype. When the mice were fed a folate-depleted diet and no gemcitabine injections were given, the low TK activity was reversed, because Colon 26G/F had a total TK activity comparable with Colon 26A and Colon 26-10 tumors. In contrast, the other two variants, Colon 26A/F and 26–10/F, showed a 3-fold lower total TK activity than Colon 26A and 26-10 tumors, respectively. The total TK activity of the FM3A/TK- tumor was still measurable but much lower than in the parental FM3A tumor. The FM3A/TK- cell line, which was used for the initiation of the solid tumors, had no TK1 activity and relatively high TK2 activity, as compared with the parental FM3A cells (data not shown). The remaining activity of TK1 in FM3A/TK- tumors was 4-fold lower than that of the TK1 activity of FM3A tumors. This change was similar to the 4-fold difference in total TK activity of the two tumors. A possible explanation for the remaining TK activity is infiltration of murine cells with normal TK activity into the tumor tissue.


View this table:
[in this window]
[in a new window]

  		Table 4 TK activity in murine tumorsa

 
TS activity of the Colon 26 variants under normal diet conditions was comparable (Table 5)Citation . A 2–3-fold increase was observed for the Colon 26... /F variants. The mammary tumor FM3A had a slightly higher TS activity than the standard Colon 26 variants, whereas the TS activity of the FM3A/TK- was comparable with FM3A tumors.


View this table:
[in this window]
[in a new window]

  		Table 5 TS activity in murine tumors measured at a substrate concentration of 10 µM dUMPa

 
TP activity of Colon 26 tumors appeared to be very different in the three variants (Table 6)Citation . TP activity was lowest in Colon 26-10 tumors and did not differ significantly from its low folate variant. Colon 26A/F had a 5-fold reduced TP activity as compared with Colon 26A and reached a level of activity similar to Colon 26-10/F. Colon 26G/F had the highest TP activity also compared with its parental tumor Colon 26G. The activity in Colon 26G and Colon 26G/F was significantly higher (P = 0.00002) than in the other colon tumors. The FM3A tumors had a 6-fold higher TP activity than the FM3A/TK- (P = 0.02).


View this table:
[in this window]
[in a new window]

  		Table 6 TP activity in murine tumorsa

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The influence of endogenous plasma and tumoral levels of both folates and thymidine metabolism on the antitumor activity of TS inhibitors in vivo was substantial. The adaptation of the mice to low folate diet and initial differences in folate status were associated with sensitivity to 5FU, MTA, or RTX in the colon tumors. The role of TdR was evident from the increased antitumor activity of 5FU, MTA, and RTX in TK-deficient FM3A mammary tumors compared with parental FM3A tumors.

The MTD for RTX in Balb/c mice (10 mg/kg) was in the same range as the dose that was used by Clarke et al. (14) . However, for the various strains and for both RTX and MTA, large differences in MTD were observed (14 , 35) . MTA required a larger dose reduction for mice on a low folate diet than RTX required. In contrast to experimental head and neck tumors (5) , the therapeutic index of RTX was not superior to that of 5FU and LV in nearly all of the subtypes of Colon 26. This is consistent with the results of clinical Phase II (breast) and Phase III (colon) trials (36) .

One of the biochemical effects of a low folate diet was a 2–3-fold increase in TS activity. A similar increase in TS mRNA expression and FdUMP-binding sites in murine breast tumors was already reported by Priest et al. (37) . The fundamental process underlying these changes could well be at the level of DNA methylation. Folate depletion caused diminished DNA methylation at least at one site within the TS promoter (37) .

Folate depletion was associated with a decrease of the total TK activity in Colon 26A/F and Colon 26-10/F. In Colon 26G/F, total TK activity increased, which may be attributable to the omission of the selection pressure by gemcitabine, which is a substrate for TK2 (33) . These changes suggest that TK is differently regulated in this tumor when compared with the other Colon 26 subtypes.

Also, the total rate of TdR phosphorylysis did not show a consistent pattern of changes under low folate conditions. TP is also known as a platelet-derived endothelial cell growth factor that plays a role in tumor vascularization and proliferation (38) . Although there was a large variation in TP activity, the proliferation rate of the tumors, except Colon 26-10/F, was similar. Ackland and Peters (38) suggested that folate-based TS inhibitors may be more effective in tumors with a high TP because of increased degradation of TdR. We observed that Colon 26G with a high TP activity in this panel of the tumors was not sensitive to MTA or RTX, indicating that other factors are more dominant for achievement of the antitumor effect.

The role of TdR in antifolate resistance has already been recognized by Pinedo et al. (2) . The TdR levels in plasma of Balb/c mice on normal diet and folate-depleted diet and of C3H/He mice were comparable, but large differences were observed in the activity of the enzymes TK, TP, and TS. Their role is rather complex, especially when a drug like 5FU is involved because the metabolism to the active nucleotide FdUMP may also proceed via TP and TK. However, the present data provide additional evidence that metabolism of 5FU is not mediated by this pathway (11) because the TK- tumor showed an increased sensitivity to 5FU. Apparently in the parent FM3A tumor, TK protected the tumor against 5FU, possibly by providing dTTP via the salvage pathway.

The pattern of enzyme activities could not completely explain the difference in antitumor activity of the Colon 26 subtypes such as resistance to 5FU in Colon 26A and Colon 26G and complete remissions of Colon 26-10, all of which have comparable TS activity. The pool of 5,10-CH2-H4-folate and H4-folate in the tumors appeared to be important for the antitumor activity, especially of 5FU. The other differences that have been described for Colon 26-10 and Colon 26A were the longer duration of TS inhibition (39) and retention of [18F]5FU and its metabolites (40) in Colon 26-10. The activity of enzyme dihydropyrimidine dehydrogenase responsible for 5FU degradation and also related to 5FU sensitivity was comparable in Colon 26A and Colon 26-10 (40) . The activity of the 5FU-activating enzyme orotate phosphoribosyl-transferase is high in the parental Colon 26A tumors (41) , whereas synthesis of FdUMP catalyzed by uridine phosphorylase and uridine kinase was >10 higher than that of FdUMP catalyzed by TP and TK. Therefore, it is unlikely that TP is a major 5FU-activating enzyme in this tumor. The low total TK activity of Colon 26G compared with the other colon tumors did not increase the sensitivity to 5FU, RTX, or MTA, but the relatively low folate pools might account for the 5FU resistance. However, in Colon 26-10/F, the decreased TK activity together with reduced 5,10-CH2-H4-folate and H4-folate pools were associated with increased sensitivity to RTX and MTA. The importance of TK activity in folate depletion was illustrated by Colon 26G/F, which has an increased TK activity and increased resistance to RTX and MTA as compared with Colon 26G. Also, the low TK activity of FM3A/TK- was associated with an increased antitumor activity for all of the compounds. This was in agreement with a study showing that treatment with MTA (300 mg/kg) cured 4 of 10 mice bearing GC3/TK- colon tumors, whereas only a growth inhibition was seen in mice bearing the parental GC3 tumors (19) . In the same GC3/TK- tumors, a repeated dose of 5FU (75 mg/kg) and LV (800 mg/kg) induced a significant tumor reduction but no cures (42) . The 5FU activity against these TK- tumors was not superior to that observed in parental tumors, but the activity of 5FU and LV was (42) . This suggests a typical TS inhibition induced antitumor activity. RTX was curative in mice with L5178Y TK-/- lymphoma (10) . In our study, FM3A/TK- tumors still had TK activity, which, together with the relatively high 5,10-CH2-H4-folate and H4-folate levels compared with FM3A, might explain why the effects were not always optimal with all of the drugs. These relatively high folate levels may support 5FU activity. MTA activity is believed to be additionally mediated by inhibition of dihydrofolate reductase and glycinamide ribonucleotide formyltransferase (15 , 21) , which may contribute to its antitumor effect.

The role of a low folate diet in the antitumor activity of 5FU has been studied previously (4) , showing that LV potentiated 5FU antitumor activity when mice were put on a low folate diet. This is in agreement with our data, which showed a decreased antitumor effect of 5FU on Colon 26-10/F, but in combination with LV all of the tumors regressed. This is due to the TS-dependent 5FU antitumor effect, because in folate-depleted tumors 5,10-CH2-H4-folate pools are too low to stabilize the FdUMP-TS-5,10-CH2-H4-folate complex. The limiting 5,10-CH2-H4-folate pools in the tumor will be restored by treatment with LV.

Adaptation of mice to a low folate diet necessitated a reduction in the doses of both RTX and MTA, which were probably too low to achieve sufficient antifolate levels in the tumor to induce TS inhibition. FA supplementation enabled the administration of a higher dose of MTA, but this may have rescued the antitumor effect. Also, other studies (35) showed that supplementation with 15 mg/kg FA at a low folate diet allowed increase of the MTA dose from 1 mg/kg to 30–1000 mg/kg i.p. A combination of RTX (10 mg/kg) and FA (300 mg/kg) in DBA mice on a normal diet was less successful because reduction of antitumor activity was observed (43) , but reduction of the FA dose and adaptation of the diet might improve the efficacy. The successful combination of GW1843U89 with FA to prevent gut toxicity was based on the properties of GW1843U89 (17) , which is a noncompetitive inhibitor of TS and only competes with natural folates for transport and polyglutamylation. MTA is a competitive inhibitor of TS (15) , and RTX is a mixed noncompetitive inhibitor of TS with a tendency toward competitive (13) . Moreover, RTX uptake and polyglutamylation were reduced by LV (13) , and the antiproliferative activity of MTA was prevented by LV (15) . In vivo FA is metabolized into reduced folates, such as 5-CH3-H4-folate, which is also rendered by LV. This implies that combining these folate-based TS inhibitors with FA can reduce the antitumor activity as seen for RTX (43) and our experiments with MTA. It is clear that regulation of folate homeostasis by diet and FA administration can affect the therapeutic efficacy of antifolates (44) . The present data underline a drug- and tumor-dependent effect of folate homeostasis on the antitumor effect of antifolates. When total folate levels in the tumors are high, only small amounts of MTA and RTX polyglutamates will be formed, because of competition by the natural folates, as has been shown for cell lines (44 , 45) in which a 7-fold increase in the total folate pool resulted in a 510-fold increase of resistance to RTX. The major advantage of FA supplementation seems to be protection of toxicity. It may enlarge the therapeutic window of drug dosing, enabling a control in toxicity without affecting or even improving the antitumor activity. In animals, coadministration of FA provides a means for further optimization and reproducibility of antifolate testing and therapy, whereas in patients, shifts in folate pools because of diet and vitamin intake may be prevented.

In conclusion, these studies show that reduction of folate levels and TK activity in mice have a large impact on the antitumor activity of TS inhibitors. A murine tumor model combining both low folate levels equivalent to those found in humans (8) and TK deficiency would be a more accurate and predictable system for the preclinical testing of TS inhibitors with therapeutic potential against human tumors.


   ACKNOWLEDGMENTS
 
This study was performed at the KDL (clinical animal laboratory) of the Vrije Universiteit.


   FOOTNOTES
 
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 Supported by the Dutch Cancer Society (Grant IKA92-88, VU96-1240) and the BIOMED II program of the European Community (Grant BMH4-CT96-0479). Back

2 To whom requests for reprints should be addressed, at Department of Medical Oncology, University Hospital Vrije Universiteit, P. O. Box 7057, 1007 MB Amsterdam, the Netherlands. Phone: 31-20-4442633; Fax: 31-20-4443844; E-mail: gj.peters@azvu.nl. Back

3 The abbreviations used are: TS, thymidylate synthase; 5,10-CH2-H4-folate, 5,10-methylenetetrahydrofolate; 5-CH3-H4-folate, 5-methyl-tetrahydrofolate; RTX, raltitrexed; MTA, pemetrexed; LV, leucovorin; FdUMP, 5-fluoro-dUMP; TdR, thymidine; TK, thymidine kinase; TP, thymidine phosphorylase; lometrexol, (6-R)5,10-dideazatetrahydrofolate; FA, folic acid; MTD, maximum tolerated dose; GDF, growth delay factor; HPLC, high-performance liquid chromatography. Back

Received 8/23/00. Accepted 2/28/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Newell D. R., Burtles S. S., Fox B. W., Jodrell D. I., Connors T. A. Evaluation of rodent-only toxicology for early clinical trials with novel cancer therapeutics. Br. J. Cancer., 81: 760-766, 1999.[Medline]
  2. Pinedo H. M., Zaharko D. S., Bull J. M., Chabner B. A. The reversal of methotrexate cytotoxicity to mouse bone marrow cells by leucovorin and nucleosides. Cancer Res., 36: 4418-4424, 1976.[Abstract/Free Full Text]
  3. Jackman A. L., Taylor G. A., Calvert A. H., Harrap K. R. Modulation of anti-metabolite effects. Effects of the thymidine on the efficacy of the quinazoline-based thymidylate synthetase inhibitors, CB3717. Biochem. Pharmacol., 33: 3269-3275, 1984.[Medline]
  4. Raghunathan K., Schmitz J. C., Priest D. G. Impact of schedule on leucovorin potentiation of fluorouracil antitumor activity in dietary folic acid deplete mice. Biochem. Pharmacol., 53: 1197-1202, 1997.[Medline]
  5. Cao S., McGuire J. J., Rustum Y. M. Antitumor activity of ZD1694 (Tomudex) against human head and neck cancer in nude mouse models: role of dosing and schedule and plasma thymidine. Clin. Cancer Res., 5: 1925-1934, 1999.[Abstract/Free Full Text]
  6. Carreras C. W., Santi D. V. The catalytic mechanism and structure of thymidylate synthase. Annu. Rev. Biochem., 64: 721-762, 1995.[Medline]
  7. 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. Biochem. Pharmacol., 48: 319-325, 1994.[Medline]
  8. Priest D. G., Schmitz J. C., Bunni M. A., Stuart R. K. Pharmacokinetics of leucovorin metabolites in human plasma as a function of dose administered orally and intravenously. J. Natl. Cancer Inst. (Bethesda), 83: 1806-1812, 1991.[Abstract/Free Full Text]
  9. Jackman A. L., Calvert A. H. Folate-based thymidylate synthase inhibitors as anticancer drugs. Ann. Oncol., 6: 871-881, 1995.[Abstract/Free Full Text]
  10. Jackman A. L., Farrugia D. C., Gibson W., Kimbell R., Harrap K. R., Stephens T. C., Azab M., Boyle F. T. ZD1694 (Tomudex) a new thymidylate synthase inhibitor with activity in colorectal cancer. Eur. J. Cancer, 31: 1227-1282, 1995.
  11. Peters G. J., Köhne C. H. Fluoropyrimidines as antifolate drugs Jackman A. L. eds. . Antifolate Drugs in Cancer Therapy, : 101-145, Humana Press Totowa, NJ 1999.
  12. Van Laar J. A. M., Rustum Y. M., Van der Wilt C. L., Smid K., Pinedo H. M., Peters G. J. Tumor size and origin determine antitumor activity of cisplatin or 5-fluorouracil and its modulation by leucovorin in murine colon carcinomas. Cancer Chemother. Pharmacol., 39: 79-89, 1996.[Medline]
  13. Jackman A. L., Taylor G. A., Gibson W., Kimbell R., Brown M., Calvert A. H., Judson I. R., Hughes L. R. ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that is a potent inhibitor of L1210 tumor cell growth in vitro and in vivo: a new agent for clinical study. Cancer Res., 51: 5579-5586, 1991.[Abstract/Free Full Text]
  14. Clarke S. J., Farrugia D. C., Aherne G. W., Pritchard D. M., Benstead J., Jackman A. L. Balb/c mice as a preclinical model for raltitrexed-induced gastrointestinal toxicity. Clin. Cancer Res., 6: 285-296, 2000.[Abstract/Free Full Text]
  15. 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., Andis S. L., Bewley J. R., Rayl E. A., Moroson B. A., Beardsley G. P., Kohler W., Ratnam M., Schultz R. M. LY231514, a Pyrrolo[2,3-d]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes. Cancer Res., 57: 1116-1123, 1997.[Abstract/Free Full Text]
  16. Taylor G. A., Jackman A. L., Calvert A. H., Harrap K. R. Plasma nucleoside and base levels following treatment with the new thymidylate synthase inhibitor CB 3717. Adv. Exp. Med. Biol., 165: 379-382, 1984.
  17. Smith G. K., Amys H., Boytos C. M., Duch D. S., Ferone R., Winson H. Enhanced antitumor activity for thymidylate synthase inhibitor 1843U89 through decreased host toxicity with oral folic acid. Cancer Res., 55: 6117-6125, 1995.[Abstract/Free Full Text]
  18. Walzem R. L., Clifford A. J. Folate deficiency in rats fed diets containing free amino acids or intact proteins. J. Nutr., 118: 1089-1096, 1988.
  19. Alati T., Worzalla J. F., Shih C., Bewley J. R., Lewis S., Moran R. G., Grindey G. B. Augmentation of therapeutic activity of Lometrexol [(6-R)5,10-dideazahydrofolate] by oral folic acid. Cancer Res., 56: 2331-2335, 1996.[Abstract/Free Full Text]
  20. Houghton P. J., Houghton J. A., Germain G., Torrance P. M. Development and characterization of a human colon adenocarcinoma xenograft deficient in thymidine salvage. Cancer Res., 47: 2117-2122, 1987.[Abstract/Free Full Text]
  21. Schultz R. M., Patel V. F., Worzalla J. F., Shih C. Role of thymidylate synthase in the antitumor activity of the multitargeted antifolate, LY231514. Anticancer Res., 19: 437-444, 1999.[Medline]
  22. Jackman A. L., Kimbell R., Aherne G. W., Brunton L., Jansen G., Stephens T. C., Smith M. N., Wardleworth J. M., Boyle F. T. Cellular pharmacology and in vivo activity of a new anticancer agent, ZD9331: a water-soluble, nonpolyglutamatable, quinazoline-based inhibitor of thymidylate synthase. Clin. Cancer Res., 3: 911-921, 1997.[Abstract]
  23. Pinter K., Davisson V. J., Santi D. V. Cloning, sequencing, and expression of the Lactobacillus casei thymidylate synthase gene. DNA (NY), 7: 235-241, 1988.[Medline]
  24. Mathews R. G. Methylenetetrahydrofolate reductase from pig liver. Methods Enzymol., 122: 372-381, 1986.[Medline]
  25. Rios-Orlandi E. M., Zarkadas C. G., Mackenzie R. E. Formyltetrahydrofolate dehydrogenase-hydrolase from pig liver: simultaneous assay of the activities. Biochem. Biophys. Acta, 871: 24-35, 1986.[Medline]
  26. Peters G. J., Van Dijk J., Nadal J. C., Van Groeningen C. J., Lankelma J., Pinedo H. M. Diurnal variation in the therapeutic efficacy of 5-fluororuacil against murine colon cancer. In Vivo (Athens), 1: 113-118, 1987.
  27. Ruiz van Haperen V. W. T., Veerman G., Van Moorsel C. J. A., Peters G. J. Induction of in vivo resistance against gemcitabine (dFdC, 2',2'-difluoro-deoxycytidine). Adv. Exp. Med. Biol., 431: 637-640, 1998.[Medline]
  28. Ayusawa D., Koyama H., Iwata K., Seno T. Resistance to methotrexate in thymidylate synthetase-deficient mutants of cultured mouse mammary tumor FMA3A cells. Cancer Res., 41: 1497-1501, 1981.[Abstract/Free Full Text]
  29. Peters G. J., Laurensse E., Leyva A., Pinedo H. M. Tissue homogenization using a microdismembrator for the measurement of enzyme activities. Clin. Chim. Acta, 158: 193-198, 1986.[Medline]
  30. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254, 1986.
  31. Van der Wilt C. L., Pinedo H. M., Smid K., Peters G. J. Elevation of thymidylate synthase levels following 5-fluorouracil treatment is prevented by the addition of leucovorin in murine colon tumors. Cancer Res., 52: 4922-4928, 1992.[Abstract/Free Full Text]
  32. Ruiz van Haperen V. W. T., Veerman G., Eriksson S., Boven E., Stegmann A. P. A., Hermsen M., Vermorken J. B., Pinedo H. M., Peters G. J. Development and molecular characterization of a 2',2'-difluorodeoxycytidine-resistant variant of the human ovarian carcinoma cell line A2780. Cancer Res., 54: 4138-4143, 1994.[Abstract/Free Full Text]
  33. Eriksson S., Arnér E., Spasokoukotskaja T., Wang L., Karlsson A., Brosjö O., Gunvén P., Julisson G., Liliemark J. Properties and levels of deoxynucleoside kinases in normal and tumor cells; implications for chemotherapy. Adv. Enzyme Regul., 34: 13-25, 1994.[Medline]
  34. Laurensse E. J., Pinedo H. M., Peters G. J. A sensitive non-radioactive assay for pyrimidine nucleoside phosphorylase using reversed-phase high performance liquid chromatography. Clin. Chim. Acta, 178: 71-78, 1988.[Medline]
  35. Shih C., Thornton D. E. Preclinical pharmacology studies and the clinical development of a novel multitargeted antifolate, MTA (LY231514) Jackman A. L. eds. . Anticancer Drug Development, : 183-201, Humana Press, Inc. Totowa, NJ 1999.
  36. Beale P., Clark S. Tomudex. Clinical development Jackman A. L. eds. . Anticancer Drug Development, : 167-181, Humana Press, Inc. Totowa, NJ 1999.
  37. Priest D. G., Raghunathan K., Liu Y., Bunni M. A. Modulation of thymidylate synthase expression by dietary folic acid induced DNA hypomethylation in a mouse model tumor Pfleiderer W. Rokos H. eds. . Chemistry and Biology of Pteridines and Folates, : 387-392, Blackwell Scientific Publications Berlin 1997.
  38. Ackland S. P., Peters G. J. Thymidine phosphorylase: its role in sensitivity and resistance to anticancer drugs. Drug Resistance Updates., 2: 205-214, 1999.[Medline]
  39. Codacci-Pisanelli G., Pinedo H. M., Van der Wilt C. L., Smid K., Franchi F., Peters G. J. Improved therapeutic efficacy of high-dose 5-fluorouracil (5FU) with uridinediphosphoglucose (UDPG) rescue in mice: the role of prolonged inhibition of thymidylate synthase (TS). Proc. Am. Assoc. Cancer Res., 37: 407 1996.
  40. Visser G. W. M., Van der Wilt C. L., Wedzinga R., Peters G. J., Herscheid J. D. M. 18F-radiopharmacokinetics of [18F]-5-fluorouracil in a mouse bearing two colon tumors with a different 5-fluorouracil sensitivity. A study for a correlation with oncological results. Eur. J. Nucl. Med., 23: 333-342, 1996.
  41. Peters G. J., Braakhuis B. J. M., De Bruijn E. A., Laurensse E. J., Van Walsum M., Pinedo H. M. Enhanced therapeutic efficacy of 5'deoxy-5-fluorouridine in 5-fluorouracil resistant head and neck tumours in relation to 5-fluorouracil metabolizing enzymes. Br. J. Cancer, 59: 327-334, 1989.[Medline]
  42. Houghton J. A., Williams L. G., Loftin S. K., Cheshire P. J., Morton C. L., Houghton P. J., Dayan A., Jolivet J. Factors that influence the therapeutic activity of 5-fluorouracil [6RS]leucovorin combinations in colon adenocarcinoma xenografts. Cancer Chemother. Pharmacol., 30: 423-432, 1992.[Medline]
  43. Rees C. R., Kimbell R., Valenti M., Brunton L., Farrugia D., Jackman A. L. Effects of leucovorin and folic acid on the cytotoxicity of the TS inhibitors Tomudex (ZD1694) and ZD9331. Proc. Am. Assoc. Cancer Res., 38: 476 1997.
  44. Jansen G., Mauritz R. M., Drori S., Sprecher H., Kathmann I., Bunni M., Priest D. G., Noordhuis P., Schornagel J. H., Pinedo H. M., Peters G. J., Assaraf Y. G. A structurally altered human reduced folate carrier with increased folic acid transport mediates a novel mechanism of antifolate resistance. J. Biol. Chem., 273: 30189-30198, 1998.[Abstract/Free Full Text]
  45. Jansen G., Barr H., Kathmann I., Bunni M. A., Priest D. G., Noordhuis P., Peters G. J., Assaraf Y. G. Multiple mechanisms of resistance to polyglutamatable and lipophilic antifolates in mammalian cells: role of increased folylpolyglutamylation, expanded folate pools, and intralysosomal drug sequestration. Mol. Pharmacol., 55: 761-769, 1999.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
R. G. Pillai, M. Forster, M. Perumal, F. Mitchell, J. Leyton, F. I. Aibgirhio, O. Golovko, A. L. Jackman, and E. O. Aboagye
Imaging Pharmacodynamics of the {alpha}-Folate Receptor-Targeted Thymidylate Synthase Inhibitor BGC 945
Cancer Res., May 15, 2008; 68(10): 3827 - 3834.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
R. Sharma, J. M. Hoskins, L. P. Rivory, M. Zucknick, R. London, C. Liddle, and S. J. Clarke
Thymidylate Synthase and Methylenetetrahydrofolate Reductase Gene Polymorphisms and Toxicity to Capecitabine in Advanced Colorectal Cancer Patients
Clin. Cancer Res., February 1, 2008; 14(3): 817 - 825.
[Abstract] [Full Text] [PDF]

Home page
 The OncologistHome page
S. Chattopadhyay, R. Tamari, S. H. Min, R. Zhao, E. Tsai, and I. D. Goldman
Commentary: A Case for Minimizing Folate Supplementation in Clinical Regimens with Pemetrexed Based on the Marked Sensitivity of the Drug to Folate Availability
Oncologist, July 1, 2007; 12(7): 808 - 815.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by van der Wilt, C. L.
Right arrow Articles by Peters, G. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van der Wilt, C. L.
Right arrow Articles by Peters, G. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online