ANTICANCER RESEARCH 28 : 3785-3792 (2008)

Effects of 5-Fluorouracil on Human Mitogen-activated Peripheral Blood Lymphocytes from Healthy Individuals AVI EISENTHAL 1, KARIN EYTAN 2, ELI BRAZOWSKI 1, BEN-ZION KATZ 3, IDIT SHIRAZI 4 and YEHUDA SKORNICK 2

Departments of 1Pathology, 2Surgery “A” and 4Dermatology, and 3Hematology Institute, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Abstract. Background: The effect of 5-fluorouracil (5-FU) on eventually inhibiting DNA synthesis and cell replication (6). activated lymphocytes was explored. Materials and Methods: The role of TS as a target for 5-FU has been further The in vitro effects of 5-FU on DNA synthesis in mitogen- demonstrated in tumor cells overexpressing TS which renders activated lymphocytes from healthy volunteers were compared them more resistant to 5-FU (7, 8). Other antimetabolites to those of the antimetabolites doxorubicin, cyclophosphamide frequently employed in the chemotherapy of cancer patients and 6-mercaptopurine. These effects were assessed by act by inhibiting DNA synthesis through the activation of alterations in the phenotypic profile and the percentage of cells different mechanisms. Doxorubicin blocks DNA synthesis in various phases of the cell cycle, as well as by the secretion and transcription via the stabilization of topoisomerase of T helper (Th)1 and Th2 cytokines (ELISA). Results: Unlike II/DNA cleavable complexes (9). Cyclophosphamide inhibits 5-FU, the other antimetabolites failed to augment DNA DNA synthesis by crosslinking of DNA strands (10), and 6- synthesis in activated lymphocytes. The effect of 5-FU mercaptopurine reduces DNA synthesis by inhibiting purine correlated with an increase in the percentage of cells in the S- nucleotide synthesis and metabolism. Since 5-FU is widely phase and caused an increased in CD4 + cells, a decrease in used in cancer chemotherapy, its effects on the immune CD56 + cells and a shift of the cytokine secretion pattern from system, which mediates a variety of antitumor activities both Th2 to Th1. Conclusion: 5-FU exhibited a unique effect on in culture and in cancer patients (11, 12), is of substantial DNA synthesis in activated lymphocytes which was interest. In addition to its effect on tumor cells, 5-FU has accompanied by selective effects on various lymphocyte exhibited inhibitory activities on various immune responses, subpopulations. including NK cell-mediated cytotoxicity (13, 14) and mitogen-induced activation of lymphocytes (15). In previous Studies on cancer patients undergoing chemotherapy have experiments on human peripheral blood lymphocytes from shown the inhibitory effects of anticancer drugs on various healthy donors (16), we demonstrated by 3H-thymidine immune activities, including mitogen responses and cytokine incorporation that concentrations of 250-2500 μM of 5-FU secretion in the serum, as well as natural killer (NK) and substantially (up to 98%) reduced DNA synthesis in culture activated NK (ANK) cell activities (1, 2). One of the following lymphocyte activation. At a lower concentration antimetabolites frequently used in the treatment of colorectal (2.5 μM), however, 5-FU markedly increased DNA synthesis carcinoma and other malignancies of the digestive tract is the by 195%, 58% and 222% following stimulation with pyrimidine 5-fluorouracil (5-FU) (3-5). The target enzyme interleukin-2 (IL-2), phytohemagglutinin A (PHA) and of 5-FU is thymidylate synthase (TS) and the binding of pokeweed mitogen (PWM), respectively. While low these molecules forms a stabile tertiary complex which concentrations of 5-FU had no effect on the generation of inhibits the TS conversion of deoxyuridine monophosphate activated NK cells, concentrations of 250-2500 μM 5-FU (dUMP) to deoxythymidine monophosphate (dTMP), thus caused a marked decrease in the generation of IL-2-induced NK antitumor cytotoxic activity (16). Various cytokines, such as IL-10, prostaglandins and TGF-β have also been shown to modulate both proliferation and cytokine secretion by Correspondence to: Avi Eisenthal, Ph.D., Department of Pathology, lymphocytes (17-20). Tel Aviv Sourasky Medical Center, 6 Weizan Street, Tel Aviv 64239, Israel. Tel : +972 36974645, Fax: +972 36974648, e-mail: In the present study, the effects of 5-FU on lymphocyte [email protected] activation in culture was explored further by analyzing the kinetics of cell activation and percent age of cells at various Key Words: Lymphocytes, activation, 5-fluorouracil. cell cycle phases, by measuring the effect of other

0250-7005/2008 $2.00+.40 3785 ANTICANCER RESEARCH 28 : 3785-3792 (2008) antimetabolites on DNA synthesis and by analyzing the Phenotype analysis. The PBMCs were incubated for 120 h with 1000 effect of the 2.5 μM stimulatory concentration of 5-FU on U/ml IL-2 in the presence or absence of 2.5 μM of 5-FU. The cells were both the phenotypic profile and the T helper (Th)1 and Th2 then harvested, washed and incubated for 60 min with the following fluorescent mouse anti-human monoclonal antibodies (Becton cytokine secretion patterns in activated lymphocytes. Dickinson, San Jose, CA, USA): anti-CD3 (T lymphocytes), anti-CD4 (helper T lymphocytes), anti-CD8 (cytotoxic T lymphocytes), and anti- Materials and Methods CD56 (NK lymphocytes). The cells were then analyzed on a FACS Caliber (Becton Dickinson). Preparation of peripheral blood mononuclear cells (PBMCs). Twenty milliliters of heparinized blood obtained from healthy volunteers were Cell cycle measurements. PBMCs incubated in 1000 U/ml IL-2 diluted 1:1 in Hanks balanced salt solution (HBSS), pH 7.2. The alone or in the presence of 2.5 or 250 μM 5-FU were collected after blood was then layered on a 10-15 ml density-gradient solution 72 h and 96 h, washed twice in HBSS and 10 6 cells were incubated 3 (1.077 g/cm , Ficoll; Pharmacia, Uppsala, Sweden) and spun at 550 with 50 μl 1% Triton ® X-100/PBS (USB Corp, Cleveland, OH, xg for 15 min. The cells that accumulated at the interphase between USA) followed by 50 μl of 1 mg/ml propidium iodide (Sigma- the plasma and Ficoll layers were collected, washed twice in HBSS, Aldrich), and analyzed using a FACSort flow cytometer (Becton counted in 0.2% trypan blue solution to establish the percentage of Dickinson). viable cells and resuspended at the desired density in enriched medium (CM) as described elsewhere (21). Statistical analysis . Statistical analysis was performed by a two- tailed Student’s t-test. Antimetabolite drugs. 5-Flurouracil (50 mg/ml) was purchased from ABIC, Netanya, Israel, cyclophosphamide (20 mg/ml) was purchased from Endoxan-asta, Frankfurt Am Main, Germany, Results doxorubicin (2 mg/ml) was purchased from Pharmacia, Milan, Italy, Kinetics of 5-FU on mitogen-induced DNA synthesis. As and 6-mercaptopurine was purchased from Sigma-Aldrich, St. shown in Figure 1A, which summarizes six different Louis, MO, USA (Cat No. 852678-1G-A). All the drugs were experiments, incubation of the PBMCs for 72 h with 2.5 μM diluted in CM at the desired concentrations prior to use. 5-FU increased the response to IL-2 by 87% (from Mitogens. Human recombinant IL-2 (Cetus, Emeryville, CA, USA) 1130±243 to 2115±363 cpm). had a specific activity of 3×10 6 IU/mg protein and endotoxin levels To analyze the kinetics of the increase in DNA synthesis below 0.5 ng/mg. PHA (Israel Industries, Beit-Haemek, Israel) was induced by 2.5 μM 5-FU in the mitogen-activated cells, employed at 1:200 final dilution in CM, and PWM (Israel PBMCs were cultured with PHA for 24-96 h in the presence Industries) at 1:100 final dilution in CM. or absence of 2.5 μM 5-FU. As shown in Figure 1B, incubation of PBMC s with PHA in the presence of 5-FU Proliferation assay. The proliferation assay was carried out as detailed caused an increase in DNA synthesis from 2,051 (PHA alone) previously (21). PBMCs at 2×10 5/0.2 ml CM/well were incubated for 24-96 h in a 96-well round-bottom microplate with either 10 3 units to 4,464 cpm and 7,277 (PHA alone) to 13,119 cpm after 48 (U)/ml IL-2 or 1:200 PHA or 1:100 PWM in the presence or absence h and 72 h in culture, respectively, while 5-FU caused a of different concentrations of 5-FU, doxorubicin, cyclophosphamide decrease from 11,062 cpm without the drug to 6,294 cpm in or 6-mercaptopurine, after which 2 μCi/20 μl/well [ 3H]thymidine the presence of 5-FU at 96 h. The mitogens I L-2 and PHA (Sigma-Aldrich; cat No. T 5063) w ere added to each well for the last were chosen since similar results had been obtained with PHA 6 h of culture. The cells were then harvested and the radioactivity was and PWM (16). determined by a liquid scintillation counter (LKB, Mt Waverley, Australia). Proiferation index was calculated as following: Effects of doxorubicin, cyclophosphamide and 6-mercaptopurine Mean cpm of lymphocytes cultured with the mitogen on mitogen-induced DNA synthesis . To reveal the effect on Mean cpm of lymphocytes cultured in medium alone activated lymphocytes of other antimetabolites PBMCs were incubated for 72 h with IL-2, PHA or PWM in the presence or Cytokine measurement. The PBMCs were cultured at 10 6 cells/ml absence of either 10 –4 to 10 –1 μM doxorubicin, 10 –1 to 1,000 with PHA in the presence or absence of 2.5 μM 5-FU. Control μM cyclophosphamide or 10 –2 to 1,000 μM 6-mercaptopurine. cultures were incubated without PHA in the presence or absence of As shown in Figure 2A, doxorubicin at 10 –4 μM had no effect 5-FU. On days 1, 2, 3 and 4, 0.5 ml of supernatant from each culture was harvested and tested for both interferon-gamma (IFN-γ) and on DNA synthesis when any of the mitogens were used, while IL-10 secretion using the ELISA Ready-SET-Go kits (eBioscience, higher concentrations of the drug showed a gradual increase in San Diego, CA, USA) following the manufacturer’s instructions. the inhibitory effect on DNA synthesis. Similarly, Briefly, after coating 96-well plates with anti-IL-10 or IFN-γ cyclophosphamide (Figure 2B) substantially inhibited DNA antibody, 2- to 4 fold dilutions of the tested supernatant samples were synthesis at the highest concentrations used (1,000 μM), while applied, biotin-conjugated anti-IL-10 (clone JES3-12GB) or IFN-γ lower concentrations had no effect when any of the mitogens (clone 4S.B3) was added, the samples were incubated with avidin- were employed. Similarly, 6-mercaptopurine (Figure 2C) horse radish peroxidase (HRP) and the substrate solution tetramethylbenzidine was added at the end. The results were obtained inhibited IL-2-induced DNA synthesis at 10-1,000 μM, but there as pg or ng/10 6 PBMC /ml. was no effect when lower concentrations of the drug were tried.

3786 Eisenthal et al : 5-FU Effects on Activated Lymphocytes

Figure 1. DNA synthesis in activated lymphocytes. PBMCs were cultured for either (A) 72 h in the presence of 1000 U/ml IL-2 with or without 2.5 μM 5-FU or (B) 24-96 h in the presence of enriched medium (CM) with or without 1:200 diluted PHA or PHA plus 2.5 μM 5-FU. DNA synthesis was determined by the 3H-thymidine incorporation assay. Results are presented as the mean cpm of 3-4 wells. Figure A P value <0.005. Figure B PHA+5-FU compared to PHA: 48 h, p=0.001; 72 h, p=0.007; 96 h, p=0.011.

None of the antimetabolites showed any increase in DNA synthesis at any of the concentrations employed.

Effect of 5-FU on cell cycle. As shown in Figure 3A, the Figure 2. Effect of doxorubicin, cyclophosphamide and 6-mercaptopurine presence of 2.5 μM 5-FU increased the percentage of IL-2- on lymphocyte activation by various mitogens. PBMCs were incubated for activated lymphocytes in the S-phase from 5.96 to 7.48% (an 72 h with either IL-2 (1,000 U/ml) or PHA (1:200 dilution) or PWM (1:100 dilution) in the presence or absence of various concentrations of increase of 26%) following 72 h in culture, whereas 250 μM doxorubicin (A), cyclophosphamide (B) or 6-mercaptopurine (6-MP) 5-FU caused a marked decrease in cells in the S-phase (from (C). 6-Mercaptopurine was used with or without IL-2 only. Results are 5.96% to 0.62%, a reduction of 90%). After 96 h of presented as the proliferation index (See Materials and Methods). incubation (Figure 3B), 2.5 μM 5-FU increased the Doxorubicin: a, p=0.007; b, p=0.005; c, p=0.032; d, p=0.008; e, p= 0.001; percentage of cells in the S-phase from 5.94% to 7.82% (an f, p= 0.038; g, p=0.021; h, p=0.003. Cyclophosphamide: a, p=0.012; b, p=0.007. 6-Mercaptopurine: a, p=0.016; b, p=0.020. increase of 32%) while 250 μM 5-FU reduced the cells in S- phase from 5.94% to 0.64% (a reduction of 89%). In addition, the number of cells entering the G 2+M-phase increased between 72 h to 96 h from 2.3% to 3.14% (an than with 2.5 μM 5-FU (an increase of 32%) when compared increase of 37%) following their incubation with 2.5 μM 5- to cells incubated with IL-2 alone. FU, whereas 250 μM 5 FU led to a decrease from 2.58% to 1.54% (a reduction of 40%) in cells entering the G 2+M- Effects of 5-FU on the phenotypic profile of lymphocytes phase. The number of cells undergoing apoptosis increased undergoing 5 days’ activation with IL-2. As shown in a for both IL-2, IL-2 plus 2.5 μM 5-FU and IL-2 plus 250 μM representative experiment in Figure 4, on PBMCs IL-2 in the 5-FU between 72 and 96 h of incubation and this effect was presence of 5-FU had limited effects compared to IL-2 more prominent with 250 μM 5-FU (an increase of 80%) alone. 5-FU reduced the percentage of CD8 + cells from 16%

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Figure 3. Cell cycle distribution of PBMCs incubated with IL-2 and 5-FU. PBMCs were cultured in the presence of 2.5 or 250 μM 5-FU and 1,000 U/ml IL-2 for 72 h (A) or 96 h (B). Results are presented as the percent age of cells in each phase of the cell cycle as well as the percent age of apoptotic cells.

to 14% and increased the percentage of CD4 + cells from Discussion 49% to 53%, while increasing the percentage of CD3 + cells from 71% to 73% and decreasing the percentage of CD56 + The substantial increase in DNA synthesis in mitogen- lymphocytes from 8% to 3% (Figure 4). activated lymphocytes in the presence of a low (2.5 μM ) concentration of 5-FU was prominent between 48-72 h of Effects of 5-FU on the secretion of IFN-γ and IL-10 in incubation (Figure 1B), whereas there was a decrease in lymphocytes undergoing activation. As illustrated in a DNA synthesis at 96 h, which implied that 5-FU caused a representative experiment (Figure 5A), 5-FU increased shift in the kinetics of DNA synthesis in activated PHA-induced IL-10 secretion from 5,592 pg/10 6 cells to lymphocytes. The stimulatory effect on DNA synthesis of low 7,832 pg/10 6 cells (+40%) after 48 h of incubation, whereas concentration 5-FU was unique to this drug. High it reduced IL-10 secretion from 2,636 to 1,989 pg/10 6 cells concentrations of 5-FU as well as doxorubicin (10 –1-10 –3 (–25%), 10,281 to 7,548 pg/10 6 cells ( –27%) and 8,754 to μM), cyclophosphamide (1,000 μM) and 6-mercaptopurine 5,624 pg/10 6 cells ( –36%) at 24, 72 and 96 hours, (10-1,000 μM), substantially inhibited DNA synthesis in respectively. 5-FU increased IFN-γ secretion from 17,065 to activated lymphocytes (Figure 2). The increase in DNA 22,269 ng/10 6 (+30%) cells after 72 h of incubation and synthesis induced in activated lym phocytes by 2.5 μM 5-FU from 27,397 to 33,393 ng/10 6 cells (+22%) after 96 h of correlated with the percentage of cells in the S-phase of the incubation (Figure 5B). cell cycle (Figure 3A-B) which increased from 5.96%

3788 Eisenthal et al : 5-FU Effects on Activated Lymphocytes

Figure 4. Phenotypic profile of lymphocytes incubated with IL-2 + 2.5 μM 5-FU. PBMCs were cultured for 120 h with 1,000 U/ml of IL-2 in the presence (right) or absence (left) of 2.5 μM 5-FU. The cells were then harvested, incubated with anti-CD3 and anti-CD56 ( upper panel), or anti- CD4 and anti-CD8 ( lower panel) fluorescent-labeled monoclonal antibodies and analyzed by a flow cytometer. CD3: IL-2: 71%; IL-2+5-FU:73%. CD56: IL-2: 8%; IL-2+5-FU: 3%. CD4: IL-2: 49%; IL-2+5-FU:53%. CD8: IL-2: 16%; IL-2+5-FU: 14%.

(absence of 5-FU) to 7.48% (+26%) after 72 h in culture; 5-FU. The increased inhibition of cells in the S and G 2/M- 250 μM 5-FU caused a decrease in cells in the S-phase phases following the use of IL-2 plus 250 μM 5-FU (from 5.96% to 0.62%, –90%). Similar results were correlated with an increase of cells undergoing apoptosis, obtained at 96 h. Othe rs have shown an S-phase arrest after which was most prominent at 96 h (8.28%) when compared exposure of MDA435 breast cancer cells (8) and esophageal to the cells incubated with the lower concentration (2.5 μM) squamous cell carcinoma cells (22) to up to 1,540 μΜ of 5-FU (6.08%). 5-FU. When Yoshikawa et al. exposed SW480 and The mechanism underlying the increase in DNA synthesis COLO320DM colon carcinoma cells to 1,000 ng/ml 5-FU, induced by 2.5 μM 5-FU is still unknown. One possibility is arrest in the G1/S phase was accom panied by an increase in that a low concentration of 5-FU inactivates the expression the percentage of apoptotic cells throughout the experimental of p53 which was shown to abrogate cell cycle arrest and period (23), similar to the current findings (Figure 3A-B). apoptosis induced by 5-FU in breast tumor cells (7, 24). The present results also suggested that the increase of cells in Another mechanism might be a selective direction of DNA the S-phase induced by the low concentration of 2.5 μM synthesis via the salvage pathway (22) induced by low 5-FU 5-FU was not a mere accumulation of cells during this cell concentrations. Alternatively, 5-FU may increase both the cycle phase, since there had been an increase of 37% in the activity and synthesis of TS, thus leading to an increase in percentage of cells ( i.e. from 2.3 to 3.14%) entering the thymidine and DNA synthesis. Experiments for testing this G2/M-phase between 72 96 h of incubation, whereas a possibility are currently underway. decrease of 40% was observed during this period after The mitogens which were employed in the present study treatment of cells with IL-2 in the presence of 250 μM activate various lymphocyte subpopulations, including T and

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Th1 cells were more sensitive to apoptosis than Th2 cells (30, 31). Alternatively, since 2.5 μM 5-FU affected DNA synthesis only in activated lymphocytes (Eisenthal, unpublished observation), the observed effect of 5-FU might have been due to a different activation pattern that had been induced in Th1 and Th2 cells by the mitogens used in the current study. Finally, the ability of 5-FU at a 2.5 μM to increase DNA synthesis and modulate the Th1/Th2 cytokine secretion pattern raises the question of the possible effects of this drug on the regulation of the activities of various lymphocytes by other biomodulators, including TGF-β, prostaglandins and free oxygen radicals. Studies in search of such effects are currently being conducted in our laboratory.

Acknowledg ements

Esther Eshkol is thanked for her editorial assistance.

References

1 Eisenthal A, Barbarsteyn E, Gitstein G and Lifschitz-Mercer B: Inhibition of lymphokine-activated killer cells generation in vitro by soluble factors released from mixed human tumor and peripheral blood mononuclear (PBMC) adherent cells culture. Cancer Invest 24 : 28-34, 2006. 2 Baxevanis CN, Tsavaris NB, Papadhimitriou SI, Zarkadis IK, Papadopoulos NG, Bastounis EA and Papamichail M: Granulocyte-macrophage colony-stimulating factor improves Figure 5. Effect of 5-FU on cytokine secretion. IL-10 (A) and IFN-γ (B) immunological parameters in patients with refractory solid secretion by PBMCs cultured for various periods with 1:200 diluted tumors receiving second-line chemotherapy: correlation with PHA in the presence or absence of 2.5 μM of 5-FU. Results are clinical responses. Eur J Cancer 33 : 1202-1208, 1997. presented as the mean of 3 different samples. 3 Aballea S, Chancellor JV, Raikou M, Drummond MF, Weinstein MC, Jourdan S and Bridgewater J: Cost-effectiveness analysis of oxiliplatin compared with 5-fluorouracil/leucovorin in B lymphocytes as well as NK cells (25, 26). In this study, adjuvant treatment of stage III colon cancer in the US. Cancer 2.5 μM 5-FU reduced the percentage of CD56 + cells (from 109 : 1082-1089, 2007. 4 Sohn KJ, Croxford R, Yates Z, Lucock M and Kim YI: Effect of 8% to 3%) and increased that of CD4 + cells (from 49% to + the methylenetetrahydrofolate reductase C677T polymorphism 53%). The decrease in CD56 cells was in agreement with on chemosensitivity of colon and breast cancer cells to our previous study in which 2.5 μM 5-FU reduced the 5-fluorouracil and methotrexate. J Natl Cancer Inst 96 : 134-144, generation of NK cell cytotoxic activity by 12% following 2004. stimulation with IL-2 (16). The increase in CD4 + 5 Tong D, Poot M, Hu D and Oda D: 5-Fluorouracil-induced lymphocytes by 2.5 μM 5-FU could apparently be attributed apoptosis in cultured oral cancer cells. Oral Oncol 36 : 236-241, to an increase in Th1 cells as revealed by the increase in IFN- 2000. 6 Engelbrecht C, Ljungquist I, Lewan L, Yngner T: Modulation of γ secretion after 72-96 h of incubation (Figure 5B) . 5-fluorouracil metabolism by thymidine. In vivo and in vitro Similar to other modulators, including muramyl dipeptide studies on RNA-directed effects in rat liver and hepatoma. derivatives, cyclophosphamide and ribavirin (27-29), which Biochem Pharmacol 33 : 745-750, 1984. affect either the Th1 or Th2 cells, 2.5 μM 5-FU caused a 7 Longley DB, Boyer J, Allen WL, Latif T, Ferguson PR, Maxwell substantial shift from Th2 towards Th1 cells following 72- PJ, McDermott U, Lynch M, Harkin DP and Johnston PG: The 96 h incubation in the presence of the mitogen as revealed role of thymidylate synthase induction in modulating p53- by the cytokine secretion. The mechanism involved in this regulated gene expression in response to 5-fluorouracil and antifolates. Cancer Res 62 : 2644-2649, 2002. shift from Th2 to Th1 is still unknown. One possibility is 8 Longley DB, Ferguson PR, Boyer J, Latif T, Lynch M, Maxwell that 5-FU might exert different apoptotic signals to Th2 and P, Harkin DP and Johnston PG: Characterization of a Th1 cells, thus changing the Th2/Th1 cell balance. This thymidylate synthase (TS)-inducible cell line: a model system explanation seems less likely, however, considering the for studying sensitivity to TS- and non-TS-targeted results obtained by others who demonstrated that activated chemotherapies. Clin Cancer Res 7: 3533-3539, 2001.

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9 Lothstein L, Suttle DP, Roaten JB, Koseki Y, Israel M and 21 Eisenthal A, Barbarsteyn E, Gitstein G and Lifschitz-Mercer B: Sweatman TW: Catalytic inhibition of DNA topoisomerase II by Inhibition of lymphokine-activated killer cells generation in vitro N-benzyladriamycin (AD 288). Biochem Pharmacol 60 : 1621- by soluble factors released from mixed human tumor and 1628, 2000. peripheral blood mononuclear (PBMC) adherent cell culture. 10 Colvin OM: An overview of cyclophosphamide development and Cancer Invest 24 : 28-34, 2006. clinical applications. Curr Pharm Des 5: 555-560, 1999. 22 Dittmann H, Dohmen BM, Kehlbach R, Bartusek G, Pritzkow 11 Whiteside TL, Heo DS, Takagi S and Herberman RB: M, Sarbia M and Bares R: Early changes in [ 18 F]FLT uptake Characterization of novel antitumor effector cells in long-term after chemotherapy: an experimental study. Eur J Nucl Med Mol cultures of human tumor-infiltrating lymphocytes. Transplant Imaging 29 : 1462-1469, 2002. Proc 20 : 347-350, 1988. 23 Yoshikawa R, Kusunoki M, Yanagi H, Noda M, Furuyama JI, 12 Hilders CG, Ras L, van-Eendenburg JD, Nooyen Y and Fleuren Yamamura T and Hashimoto-Tamaoki T: Dual antitumor effects GJ: Isolation and characterization of tumor-infiltrating lymphocytes of 5-fluorouracil on the cell cycle in colorectal carcinoma cells: from cervical carcinoma. Int J Cancer 57 : 805-813, 1994. a novel target mechanism concept for pharmacokinetic 13 Okamoto M, Kasetan H, Kaji R, Goda H, Ohe G, Yoshida H and modulating chemotherapy. Cancer Res 61 : 1029-1037, 2001. Sato M: Cis -diaminedichloroplatinum and 5-fluorouracil are 24 Tokunaga E, Oda S, Fukushima M, Maehara Y and Sugimachi K: potent inducers of the cytokines and natural killer cell activity Differential growth inhibition by 5-fluorouracil in human colorectal in vivo and in vitro . Cancer Immunol Immunother 47 : 233-241, carcinoma cell lines. Eur J Cancer 36 : 1998-2006, 2000. 1998. 25 Sheldon PJ and Holborow EJ: Human erythrocyte rosette 14 Reiter Z, Ozes ON, Blatt LM and Taylor MW: A dual antitumor formation with mitogen-stimulated human lymphocytes – effect of a combination of interferon-alpha or interleukin-2 and A marker for the demonstration of activated T-cells. J Immunol 5-fluorouracil on natural killer (NK) cell-mediated cytotoxicity. Methods 7: 379-386, 1975. Clin Immunol Immunopathol 62 : 103-111, 1992. 26 Sasaki M, Sekizawa T, Takahashi H, Abo T and Kumagai K: 15 Meurette O, Fontaine A, Rebillard A, Le-Moigne G, LamyT, Heterogeneity of human T lymphocytes to bind sheep erythrocytes Lagadic-Gossmann D and Dimanche-Boitrel MT: Cytotoxicity and mitogenic responses of their subpopulations. J Immunol 115 : of TRAIL/anticancer drug combinations in human normal cells. 1509-1514, 1975. Ann NY Acad Sci 1090 : 209-216, 2006. 27 Gobec S, Sollner-Dolenc M, Urleb U, Wraber B, Simcic S and 16 Brazowski E, Eytan K and Eisenthal A: In vitro modulation of Filipic M: Modulation of cytokine production by some interleukin-2-mediated human peripheral mononuclear cell phthalimido-desmuramyl dipeptides and their cytotoxicity. proliferation and antitumor cytotoxicity by 5-fluorouracil. Farmaco 59 : 345-352, 2004. Anticancer Res 27 : 4135-4142, 2007. 28 Weiner HL: Immunosuppressive treatment in multiple sclerosis. 17 Zhu Z, Fu Q, Nightingale CH, Zhao YP, Liao Q and Li DK: J Neurol Sci 223 : 1-11, 2004. Penetration kinetics of 5-fluorouracil into pancreatic fluid in post 29 Kast RE: Ribavirin in cancer immunotherapies: controlling nitric pancreatoduodenectomy patients. Anticancer Drugs 9: 685-688, oxide augments cytotoxic lymphocyte function. Neoplasia 5: 3-8, 1998. 2003. 18 Gołb J, Zagozdzon R, Kaminski R, Kozar K, Gryska K, Izycki 30 Dzialo Hatton R, Milbrandt J, Hockett RD Jr and Weaver CT: D, Mackiewicz A, Stoklosa T, Giermasz A, Lasek W and Differential expression of Fas ligand in Th1 and Th2 cells is Jacobisiak M: Potentiated antitumor effectiveness of combined regulated by early growth response gene and NF-AT family chemo-immunotherapy with interleukin-12 and 5-fluorouracil of members. J Immunol 166 : 4534-4542, 2001. L1210 leukemia in vivo . Leukemia 15 : 613-620, 2001. 31 Roozendaal R, Vellenga E, de-Jong MA, Traanberg KF, Postma 19 Onocera H, Somers SS and Guillou PJ: Paradoxical effects of DS, de-Monchy JG and Kauffman HF: Resistance of activated 5-FU/folinic acid on lymphokine-activated killer (LAK) cell human Th2 cells to NO-induced apoptosis is mediated by induction in patients with colorectal cancer. Br J Cancer 62 : gamma-glutamyltranspeptidase. Int Immunol 13 : 519-528, 2001. 1042-1046, 1990. 20 Ehrnrooth E, Sorensen B, Poulsen JH, Sorensen BS and von der Maase H: Changes in thymidylate synthase mRNA in blood Received June 10, 2008 leukocytes from patients with colorectal cancer after bolus Revised August 12, 2008 administration of 5-fluorouracil. Acta Oncol 39 : 53-57, 2000. Accepted October 1, 2008

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