Fluorodeoxyuridine-induced radiosensitization and inhibition of DNA double strand break repair in human colon cancer cells

Inr J Rodroiwn Onoh~yl’ Bid P/tn Vol. Printed in the IJ.S.A. All rights reserved.

19, pp.

141 l-1411 ~opynght

0360.3016190 $3.00 + .oO c 1990 Pergamon Press plc

??Original Contribution FLUORODEOXYURIDINE-INDUCED RADIOSENSITIZATION AND INHIBITION OF DNA DOUBLE STRAND BREAK REPAIR IN HUMAN COLON CANCER CELLS CHARLES

E. BRUSO,

AND THEODORE

M.D.,*

DONNA

S. LAWRENCE,

S. SHEWACH, M.D.,

PH.D.+

PH.D.*

University of Michigan Medical Center, 133 I E. Ann Street, Ann Arbor, MI 48 109-0582 The halogenated pyrimidine, fluorodeoxyuridine (FdUrd), has been used in combination with radiation for the treatment of human neoplasms. In an attempt to improve the clinical use of this combination, FdUrd-radiation interactions were studied in vitro using human HT29 colon cancer cells. It was found that FdUrd produced radiosensitization at clinically achievable (l-100 nM) concentrations. Sensitization depended critically on the timing of exposure. When cells were irradiated after a 12-hr exposure to 100 nM FdUrd, marked sensitization was produced (mean inactivation dose (MID) = 2.01 + 0.01, compared to control of 4.35 If:0.16, p < .Ol). No radiosensitization occurred when cells were irradiated 4 hr prior to incubation (MID = 3.95 f 0.05, p > 0.4). Radiosensitization appeared to result from an inhibition of thymidylate synthase since concentrations of FdUrd which produced radiosensitization depleted intracellular ‘ITP pools and blocked the incorporation of deoxyuridine into DNA. Furthermore, radiosensitization was completely inhibited by co-incubation with thymidine. FdUrd also decreased the repair, but not the formation, of radiation-induced DNA double strand breaks (DSB’s). These data are consistent with the hypothesis that FdUrd produces radiosensitization by depleting thymidine pools which leads to a decreased rate of DNA DSB repair. Furthermore, they suggest that in clinical trials FdUrd should be infused at least 8 hr before irradiation. Colorectal carcinoma, 5_fluorodeoxyuridine,

FdUrd, FUdR, Radiation, DNA damage, Thymidylate

tion by 5-FU occurs only when the cells are exposed to the drug after irradiation (9). This seemed unexpected based on the known mechanism of action of these agents. Both 5-FU and FdUrd are metabolized to the active intermediate, FdUMP, and inhibit thymidylate synthase ( 17. 20). Thus, a likely mechanism for the interaction of 5-FU or FdUrd and radiation would be through the depletion of thymidine pools. However, sensitization by this mechanism would appear to require drug treatment prior to irradiation. Therefore. it was elected to examine the dependence of radiosensitization on the timing of drug exposure with respect to irradiation. When it was discovered that greater sensitization resulted when cells were preincubated with FdUrd. the mechanism of sensitization was investigated.

INTRODUCTION The halogenated pyrimidine nucleoside, 5-fluorodeoxyuridine (FdUrd), has been used in Phase II and III trials for the treatment of colorectal cancer metastatic to liver (3, I 1, 19, 28). Although response rates have varied between 29% and 88% with FdUrd alone ( 19) few complete remissions are produced. In the search for more efficacious treatment of colorectal carcinoma metastatic to the liver, some investigators have combined FdUrd with radiation (4, 8, 23). This combined modality treatment shows promise. However, neither the optimal timing for administration of these two antineoplastic agents nor the mechanism of interaction of FdUrd and radiation is understood. Although little is known about FdUrd-radiation interactions, some data exist concerning the relationship between the time of administration of 5 fluorouracil(5-FU) with respect to radiation and the resulting radiosensitization. One previous report has suggested that sensitiza-

METHODS

authors wish to acknowledge

AND

MATERIALS

Cell cultl~rc~

Human colon adenocarcinoma from the American Type Culture

Presented in part at the 31st Annual American Society of Therapeutic Radiology and Oncology Meeting, San Francisco, California, l-6, October 1989. * Dept. of Radiation Oncology. + Dept. of Pharmacology. Reprint requests to: Theodore S. Lawrence, M.D., Ph.D. .4cknowledgements-The

synthase.

cells (HT29) obtained Collection (Rockville.

Lichter, M.D. for helpful discussions and reviewing the manuscript, Diane Dolinshek and Mary Davis, Ph.D.. for technical assistance, and Annise Johnson for manuscript preparation. This work was supported by an ASCO Young Investigator award, the Phoenix Memorial Fund, and by NIH Grants CA4276 I and CA441 73. Accepted for publication 2 1 June 1990.

Allen S. 1411

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1. J. Radiation Oncology 0 Biology 0 Physics

MD) were maintained in RPM1 medium with penicillin (100 U/ml), streptomycin (100 /*g/ml), and 10% calf serum which was heat-inactivated for 1 hr at 56°C. Cells were grown in a humidified atmosphere of 7% CO2 and 93% air at 37°C. Under these conditions, the plating efficiency was 60-90%. and the doubling time was 22 hr. Experiments were carried out using the same lot of calf serum containing 2 pm thymidine. Therefore, the final concentration of thymidine in the medium for all experiments was approximately 0.2 FM.

Drug treatment FdUrd* ( 10 mg/ml) and thymidine? ( 1 mM) stocks were made every 3 months, frozen at -2O”C, and diluted with medium to the appropriate concentration at the start of the incubation. Cell survival assay After treatment with drugs and/or radiation, cells were removed from the dishes with 0.03% trypsin and 0.27 mM EDTA. They were plated into culture dishes to yield between 20-200 colonies/dish. Each plating was done in triplicate. After 9-l 1 days of incubation the plates were fixed with methanol/acetic acid and stained with crystal violet. Colonies containing more than 50 cells were counted. All data from FdUrd treated irradiated plates were corrected for plating efficiency using an unirradiated control treated with the same FdUrd concentration. Cell survival curves were fitted using the linear quadratic equation, and the mean inactivation dose (MID) was calculated as outlined by Fertil et a/. (13).

Irradiation conditions Cells were irradiated at room temperature’ at a dose rate of l-1.1 Gy/min. An ionization chamber” with an electrometer system traceable to that of the National Bureau of Standards was used for calibration.

Flow cytometry HT29 cells were exposed to FdUrd as in the cell survival experiments. They were then trypsinized, washed, resuspended in Hank’s buffered saline (HBSS), fixed by dropwise addition of 2.5 volumes of cold 70% ethanol, and stored at 4°C until the day of analysis. The cells were washed with HBSS and then suspended in 1 ml of HBSS containing 16.7 pug/ml of propidium iodide and 40 pg/ ml of Ribonuclease A. The percentage of cells in each phase of the cell cycle was determined using an Epics C Coulter cytometer and PARA 1 Analysis.**

Estimation of thymidylate synthase activity Thymidylate synthase activity was estimated in a semiquantitative fashion by measurement of the thymidylate

* Roche Laboratories, Nutley, NJ. + Sigma Chemical Company, St. Louis, MO. * Using a Theratron 80 (60Co). § Baldwin-Farmer.

December 1990, Volume 19, Number 6

synthase dependent incorporation of [6-3H]-deoxyuridine++ into DNA. Cells were first exposed to medium containing 0.03 pCi/ml [2-‘4C]-thymidinett (59.3 Ci/mmol) for 48 hr to label the DNA and to allow correction for recovery. After a 12-hr chase with unlabeled medium, cells were exposed to FdUrd for 12 hr. During the last 2 hr of the FdUrd incubation, the cells were incubated with 0.3 pCi/ml of [6-3H]-deoxyuridine (26.7 Ci/mmol). They were then removed from the dishes with trypsin, centrifuged, and lysed using 0.4 ml of 10 mM Tris, 1 mM Na2EDTA, and 0.01% SDS at a pH of 8.0. The cell suspension was triturated with an 18 gauge needle and the protein digested with proteinase K.*$ DNA and RNA were extracted with chloroform and phenol and precipitated with ice cold 95% ethanol. The extracted DNA was assessed for 3H and 14C using standard liquid scintillation counting procedures.

Neutral elation assay HT29 cells which had been incubated for 48 hr with [2-‘4C]-thymidine (0.03 &i/ml) were chased for 8-24 hr in medium without label. Cultures were then treated with FdUrd under the same conditions as were used to determine cell survival. To assess the induction of DSB’s, cells were scraped from their plates, suspended in ice cold Ca+*, Mg+2 free PBS with 15 mM EDTA, and then irradiated. To measure the repair of DNA DSB’s, cells were irradiated and incubated at 37°C on the culture dishes for 1 or 2 hr prior to processing for elution. Neutral elution was performed according to the method of Bradley and Kohn (7) with minor modifications as described previously (23). The data from individual elutions are expressed as the fraction of 14C-thymidine retained on the filter. DNA damage is expressed as Gy equivalents (Gy Eq) determined from a calibration curve (6). The DSB enhancement ratio is defined as the ratio of DSB produced by the same dose of radiation under experimental conditions, divided by that produced under control conditions.

Statistical analysis Results of individual experiments are shown in the figures unless otherwise indicated. For the cell survival curves, each symbol is the average of triplicate platings. The standard error was less than 15% of the mean and is within the size of the symbol unless otherwise indicated. For the estimation of thymidylate synthase and for neutral elution, assays were performed in duplicate, which were within 10% of the mean. All averages in the text and tables are expressed as the mean + the standard error (number of experiments). All experiments were performed at least twice. Means were compared using Student’s t-test for paired samples (two-tailed).

** Coulter Cytometry, Hialeah, FL. ++New England Nuclear Research Products, Boston, MA. *$ Bethesda Research Laboratories, Life Technologies Inc.. Gaithersburg. MD.

FdUrd and radiation 0 C. E. BRUSO PIal

1o-4' 0

I

I

3

6

I 12

1 9

I

1O-5' 0

I

I

3

6

9

I 12

Dose (Gy)

Dose(Gy)

Fig. 1. FdUrd-mediated radiosensitization depends on the concentration and the duration of exposure. HT29 cells were incubated in medium alone (III) or with FdUrd for (A) 8 hr with IO nM (m) or 100 nM (0) FdUrd or (B) 24 hr with I nM (m) or 10 nM (0) FdUrd. After irradiation, cells were incubated for 4 hr and assessed for cell survival.

RESULTS Initial experiments were conducted to determine the time course and dose-response characteristics of FdUrd mediated radiosensitization of HT29 cells. For these studies, cells were incubated in medium containing FdUrd, irradiated, and incubated in FdUrd for 4 additional hr before being assessed for clonigenicity. FdUrd sensitization was a function of both time of FdUrd exposure and of FdUrd concentration (Figure 1, Table 1). No significant radiosensitization was produced after a 2hr exposure with FdUrd up to a concentration of 400 nM. By contrast, a preradiation FdUrd incubation period of 8 hr or 24 hr caused a significant degree of radiosensitization even with drug concentrations as low as 1 nM. Radiosensitization increased both with concentration and with duration of exposure. At subcytotoxic concentrations of FdUrd. increasing the pre-radiation FdUrd incubation time from 24 to 48 hr did not contribute further to radiosensitization (data not shown). The cytotoxicity produced by these conditions was also determined (Table 2). For FdUrd incubation times of 2-

Table 1. Mean inactivation

12 hr, there was no significant HT29 cell cytotoxicity at concentrations of less than 100 nM. Incubation times of 24 hr or greater were associated with increasing cytotoxicity as the concentration of FdUrd increased. Experiments were then performed to determine the influence of the timing of exposure on the resulting radiosensitization. For these experiments, the duration of FdUrd exposure was held constant at 12 hr, and the concentration was fixed at 100 nM. while the timing of the radiation was varied with respect to the drug exposure interval. These conditions were felt to be optimal for two reasons. First, the 12-hr exposures described above produced minimal cytotoxicity when FdUrd alone was administered. This lack of cytotoxicity allowed an unambiguous estimation of radiosensitization. Second, an FdUrd concentration of 100 nM was chosen because it was within the range of clinically achievable FdUrd concentrations ( 12). It was found that FdUrd-mediated radiosensitization was critically dependent on the timing of exposure with respect to irradiation (Fig. 2). Maximum sensitization was produced when the entire 12-hr exposure was followed by irradiation (MID = 2.0 I -t 0.0 I ; I, < 0.0 I ).

dose of HT29 cells exposed [FdUrd]

Time (hr) 3 8 24

4.35 f 0.16 (‘1) -

40

100

400

ND*

ND

ND

3.45 -t 0.40+ (4) 2.66 f 0.68+ (3)

3.18 k 0.36* (4) 2.54 f 0.48’ (7)

3.43 f 0.38 (3) 2.97 * 0.30$ (5) ND

4.09 + 0. I’ (3) 2.33 S 0.26’ (5) ND

Data are presented as the mean f the standard error (number of experiments). * Not determined. +p < 0.05 compared to control (paired 1 test). * p < 0.0 1 compared

to control

(nM)

10

1

Control

to FdUrd

(paired f test).

2.56 + 0.15$ (5) 1.54 + 0.04* (5)

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1. J. Radiation Oncology 0 Biology 0 Physics Table 2. Surviving

fraction

December 1990. Volume 19, Number 6

of HT29 cells after exposure

to FdUrd*

FdUrd (nM)

2 hr

8 hr

12 hr

24 hr

48 hr

I

ND*

ND

10

ND 0.92 + 0.10 (3) ND

ND

0.70 * 0.09 (4) 0.53 + 0.13+ (4) ND

0.58 f 0.05 (3) ND

40

0.87 * 0.03 (4) 0.79 f 0.04 (4) 0.70 f 0.04 (5) 0.76 f 0.08 (5) 0.47 f 0.06+ (5)

0.74 + 0.05 (3) ND

0.13 * 0.051’ (5) ND

0.05 Ck0.05+ (3) ND

100 400

0.64 + 0.07 (3)

ND

Data are presented as the mean of the uncorrected plating efficiency efficiency of untreated cells was 0.77 + 0.02 (IV = 3 1). * Not determined. +p < 0.05 compared to control (paired t test). *p -C 0.01 compared to control (paired t test).

When cells were exposed to the same concentration (100 nM) of FdUrd for 12 hr beginning 4 hr after irradiation, no sensitization occurred (MID = 3.95 k 0.05; p > 0.4 compared to control). In addition, neither alpha nor beta was significantly changed. For this series of experiments, under control conditions alpha was 0.032 ?Z0.006 Gy-’ (N = 3) and beta was 0.056 f 0.012 Gy-* (N = 3). These values were not significantly different from those obtained when incubation with FdUrd commenced 4 hr after irradiation (alpha = 0.043 + 0.006 Gy-’ (N = 3): beta = 0.030 k 0.030 Gyp2 (N = 3). Thus, under FdUrd exposure conditions which produce minimal drug cytotoxicity, preincubation was critical in producing radiosensitization. FdUrd is known to inhibit thymidylate synthase and thus interfere with the conversion of deoxyuridylate (dUMP) to thymidylate (TMP), which is needed for DNA synthesis. Therefore, the incorporation of deoxyuridylate

f the standard

g L

0.8

L ._

,I:

5

I

I

3

6

9

c 12

of experiments).

The plating

into DNA is a semiquantitative assay of thymidylate synthase activity. To confirm that FdUrd inhibited thymidylate synthase activity under the conditions used in these experiments, cells were assessed for [6-3H]-deoxyuridine incorporation into DNA after FdUrd exposure as described in Methods and Materials. Exposure of cells to FdUrd under the same conditions which led to radiosensitization produced a dose-dependent decrease in the incorporation of 3H into DNA (Fig. 3), suggesting decreased thymidylate synthase activity. Furthermore, preliminary results using a sensitive HPLC assay (31) suggest that FdUrd depletes thymidine triphosphate (TTP) pools under these conditions. When cells were exposed to 100 nM FdUrd for 14 hr, intracellular TTP levels decreased to 42 t- 3% (N = 3) of the values obtained under control conditions. If the radiosensitizing effect of FdUrd were mediated

b

1

error (number

ND

‘3 a

)’ t \ I

04. t 0.2 1

Dose (Gy) Fig. 2. FdUrd-mediated radiosensitization depends on the timing of exposure with respect to irradiation. HT29 cells were incubated in medium alone (0) or with LOOnM FdUrd for 12 hr. For cells that were exposed to FdUrd, treatment began 4 hr after irradiation (W), 8 hr before irradiation (0), or 12 hr before irradiation (0).

Fig. 3. FdUrd decreases thymidylate synthase activity. HT29 cells were exposed to varying concentrations of FdUrd and assessed for thymidylate synthase activity as described in Methods and Materials. Data are expressed as a fraction of the 3H incorporated in the absence of FdUrd.

1415

FdUrd and radiation 0 C. E. BRUSO et al. FdUrd + Thymidine

Fig. 4. Exogenous thymidine reverses FdUrd-induced radiosensitization. HT29 cells were incubated for 12 hr in medium alone (Cl). with 100 nM FdUrd (m). or with 100 nM FdUrd in the presence of IO FM thymidine (0).

by depletion of the TTP available for DNA repair, the addition of thymidine would be predicted to inhibit radiosensitization. To examine this possibility, radiosensitization was assessed in cells exposed to FdUrd either alone or in the presence of thymidine. The addition of thymidine completely eliminated radiosensitization (Fig. 4). Because changes in the induction and repair of DNA DSB’s have been found for other radiosensitizers, it was of interest to assess the effect of FdUrd on these parameters. When the cells were treated with FdUrd at concentrations of IO nM and 100 nM for 8 hr prior to irradiation. no enhancement of DNA DSB was found (1.04 + .05 and I .O1 k 0.07 respectively). To determine if the repair of DNA DSB’s was affected by FdUrd, the cells were allowed a 1-hr or 2-hr interval after irradiation before they were assessed. In contrast to its lack of influence on the formation of radiation-induced

8 iL

-

o.2o

This study demonstrates that FdUrd radiosensitizes HT29 human colon cancer cells at clinically achievable concentrations. FdUrd needed to be present for at least 8 hr prior to radiation to cause radiosensitization; neither post-incubation nor a short (2-hr) preincubation with postincubation produced sensitization. Two lines of evidence suggest that FdUrd-induced radiosensitization resulted from the depletion of intracellular thymidine pools. First, FdUrd inhibited thymidylate synthase and depleted TTP pools under the same conditions which produced sensitization. Second, elevation of exogenous thymidine completely reversed the effect of FdUrd. The inhibition of thymidylate synthase and the presumed resulting depletion of thymidine pools did not appear to affect the in-

100 nM FdUrd

A .

DISCUSSION

Control

FdUrd

-=

DNA DSBs, FdUrd decreased the rate of DSB repair (Fig. 5). This decrease in repair was dependent upon the concentration of FdUrd and occurred at concentrations in the same range as those which produced radiosensitization. Since FdUrd is known to block the progression of cells at the G l/S interface (5), it was possible that the radiosensitization described above could, in part, result from an increased proportion of cells being held in the more radiosensitive early S phase ( 16). Analysis of cells incubated with FdUrd at concentrations of 0. l- 100 nM for 8 hr indicated that cell cycle redistribution does occur (Table 3). With an 8-hr FdUrd incubation period there is a slight decrease in the fraction of cells in G2/M and GO/G 1 and a corresponding increase in the percentage of cells in S phase, although these differences were small. Longer incubations produced more profound changes of the same type.

100 nM FdUrd 2’

.

4’

*

6’

.

8’

Elution time (hr)

. 10’

’

0

30

60

90

120

Repair time (min)

Fig. 5. FdUrd inhibits the repair of DNA DSBs. (A) HT29 cells were incubated for 8 hr with medium alone (0) or with 100 nM FdUrd (m) prior to receiving 100 Gy. Cells were allowed an hour to repair and were processed for neutral elution. The elution profiles for unirradiated cells that were prepared identically in medium alone (0) or with 100 nM FdUrd (0) are also shown and are overlapping. (B) Multiple experiments similar to those described in (A), were performed using cultures exposed to medium alone (0) 10 nM FdUrd (W). or 100 nM FdUrd (0) and assessed 1 or 2 hr after irradiation. Data are expressed in terms of the fraction of the total number of DSBs.

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I. J. Radiation Oncology 0 Biology 0 Physics

December 1990. Volume 19. Number 6

Table 3. Cell cycle phase of HT29 cells exposed

to FdUrd 24 hr incubation

8 h incubation FdUrd (nM) 0 0.1 1 10 100

GO/G 1 49.9 44.6 36.6 48.0 42.0

_+ 3.2 4 3.4 t 3.9 + 4.5 f 8.7

S 19.5 29.0 42.8 27.8 36.7

t + f + *

G2/M 2.2 7.7 1.9 6.4 9.8

Data are presented as the mean percent f the standard * p < 0.05 compared to control (paired t test).

_

30.6 26.4 20.7 24.3 21.4

GO/G 1

f 1.5 + 3.5 f 4.3 +- 2.5* + 0.8*

54.1 44.4 20.3 17.8

* f * +

7.2 6.7 3.9* 2.2*

S

18.8 17.4 53.9 60.0

i f -t &

G7/M

3.3 3.1 6.1* 0.9*

26.5 ? 38.2 * 25.8 i --.333+37 -

3.2 6.8 4.2 _

error of three experiments.

duction of DNA DSB by radiation, but it did slow the repair of these breaks. Although the authors are unaware of antecedent literature regarding FdUrd-radiation interactions, previous investigators have looked at the relationship between the administration of 5-FU and radiation. The results presented here superficially contrast with those reported by Byfield and colleagues (9), who found that 5-FU caused maximal radiosensitization when administered 24-48 hr after irradiation. However, there are several methodological differences between these studies that could account for the different findings. First, unlike the previous study, the conditions of drug exposure in the present study were selected to produce only slight cytotoxicity. This approach was chosen because it is difficult to determine the radiosensitizing properties of a drug when the drug itself produces significant toxicity (32). In addition, the previous study used only a 60-minute preincubation period, which, in the present investigation, was also insufficient to produce sensitization. Third, it is possible that FdUrd and 5FU act differently. Both FdUrd and 5-FU can be converted to FdUMP, which inhibits thymidylate synthase ( 17, 18). However, FdUrd can become incorporated into DNA (21), which does not occur with 5-FU. Similarly. 5-FU, but not FdUrd, can be incorporated into RNA ( 18, 25,29). Note that, as was found in the present study with FdUrd, preincubation with 5-FU has also been reported to produce radiosensitization in other systems (2,27,33). The actual mechanism of sensitization is not yet clear. Although these experiments suggest that the depletion of thymidine pools plays a role in radiosensitization, other factors may also influence radiosensitivity. For instance, FdUrd caused cell cycle redistribution (34) which would be anticipated to affect radiation sensitivity ( 16). However, sensitization was produced by conditions that only minimally perturbed the cell cycle (10 nM at 8 hr and 1 nM at 24 hr), suggesting that changes in the cell cycle alone might not completely account for increased radiation sensitivity. Studies using synchronized cells will be re-

quired to clarify the relative contribution of cell cycle redistribution to FdUrd radiosensitization. It was found that FdUrd slowed repair of radiationinduced DNA DSB. Although previous work with human cervix cancer cells exposed to 5-FU did not show inhibition of repair of DNA single strand breaks (SSB’s) ( IO), it is possible that DSB’s and SSB’s are repaired by different mechanisms. The finding reported here that radiation sensitivity is associated with altered repair. rather than induction of DNA DSB, parallels the results from studies of human head and neck squamous cell carcinomas (30). In that system, both resistant and sensitive cell lines showed the same degree of induction of radiation-induced DSB, but the sensitive lines were found to repair radiationinduced DSB more slowly than resistant lines. It would be of interest to determine if FdUrd or 5-FU could reverse radiation resistance by slowing the more rapid repair mechanism ofthe resistant cells. However, all studies using neutral elution to assess the repair of radiation-induced DNA DSB’s must use doses of radiation in excess of those used in cell survival experiments. These data suggest that a minimum of 8 hr of drug infusion is needed prior to the initiation of radiation therapy to obtain greatest tumor sensitization. The short plasma half life of FdUrd (< 10 min (12)) suggests a rationale for administration by continuous infusion. These concepts have been used in our ongoing clinical protocol using external beam irradiation and intraarterial FdUrd in the treatment of patients with malignancies of the liver and porta hepatis (23). However, it is not yet known if such timing will produce the greatest therapeutic index. Experiments using a nude mouse xenograft model have been initiated to determine the most effective method of using 5-FU and FdUrd as clinical radiosensitizers. In addition, the effects of other modulators of fluoropyrimidine action, such as leucovorin (1, 24, 26) and dipyridamole ( 14, 15) on FdUrd-mediated radiosensitization of human tumor cells are also under investigation.

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Oderman,

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