the effects of the fluorinated pyrimidines fur, fudr,...

Investigative Ophthalmology & Visual Science. Vol. 32, No. 9, August 1991Copyright © Association for Research in Vision and Ophthalmology

The Effects of the Fluorinated Pyrimidines FUR, FUdR,FUMP, and FdUMP on Human Tenon's Fibroblasts

David A. Lee, Saeedeh Shapourifar-Tehrani, Thomas R. Stephenson, and Shinichi Kitada

5-Fluorouracil (5-FU) has effectively inhibited fibroblast proliferation to prevent scar formation andbleb failure after glaucoma filtering surgery. To identify more potent but less toxic antiproliferativedrugs, the authors studied cell attachment and proliferation of 5-FU metabolites: 5-fluorouridine(FUR), 5-fluorodeoxyuridine (FUdR), 5-fluorouridine-5'-monophosphate (FUMP), and 5-fluorode-oxyuridinc-5'-monophosphatc (FdUMP) on human Tenon's fibroblasts in vitro. Invest Ophthalmol VisSci 32:2599-2609, 1991

Human Tenon's fibroblasts were treated withvarious drug concentrations in the presence of 5% fe-tal bovine serum (FBS). Coulter counting (ModelZM, Coulter Electronics Inc., Hialeah, FL), a colori-metric assay based on hexosaminidase enzyme, andradionucleotide-labeling assays using 3H-adenosineand 3H-thymidine were used to measure attachment(at day 0) and proliferation (on days 1, 2, 3, 6, and 9).

None of the tested drugs had an effect on attach-ment. The most potent pyrimidine analog was 5-fluorouridine (FUR), with a 50% inhibitory dose(ID50) of 0.0019 Mg/ml for the Coulter counter at 9incubation days, followed by 5-fluorouridine-5'-monophosphate (FUMP) > 5-fluorodeoxyuridine(FUdR) = 5-fluorodeoxyuridine-5'-monophosphate(FdUMP). Our results show that all four fluorinatedpyrimidines that were tested are more potent than5-fluorouracil (5-FU). with immediate effects on fibro-blast proliferation. The delayed antiproliferative ef-fects of 5-FU may be due to conversion to these morepotent metabolites of 5-FU. These metabolites maybe useful in clinical applications to control excessivewound healing.

To enhance bleb survival in glaucoma filtering sur-gery, it is desirable to inhibit excessive postoperativescarring caused by fibroblast proliferation and sub-

From the Jules Stein Eye Institute and the Department of Oph-thalmology, UCLA School of Medicine, Los Angeles, California.

Supported in part by grants EY07026-I3, EY07701, andEY003I, from the National Institute of Health, Bethcsda, Mary-land, from Research to Prevent Blindness. New York, New York,and from the Lucille Ellis Simon Glaucoma Research Fund, LosAngeles, CA.

Submitted for publication: November 19, 1990: accepted April 2.1991.

Reprint requests: David A. Lee. MD, University of California,Los Angeles, Jules Stein Eye Institute. 100 Stein Plaza. Los Angeles,CA 90024-7004.

conjunctival fibrosis.'-2The pyrimidine analog. 5-FU,improved the success of glaucoma filtering surgery inmonkeys and humans. The mechanism of action of5-FU is probably the inhibition of cell proliferationand subsequent decrease in scar tissue formation.1"5

In previous studies, we reported delayed inhibitoryeffects of 5-FU on human ocular fibroblasts by de-creasing 50% inhibitory dose (ID50) with increasingincubation time.6 This finding may be due to the in-tracellular conversion of 5-FU into more potentantimetabolites, such as 5-fluorouridine (FUR), 5-fluorouridine 5-monophosphate (FUMP), 5-fluoro-deoxyuridine (FUdR), and 5-fluorodeoxyuridine5-monophosphate (FdUMP). The fluorinated pyri-midines produce their inhibitory and cytotoxic effectsby two separate biochemical mechanisms (Table 1 .)7

This study evaluated the relative antiproliferativeeffects of these fluoropyrimidines on human ocularfibroblasts in tissue culture.

Materials and Methods

Tissue Culture

Human Tenon's tissue from patients who under-went various operative procedures including cataract,strabismus, or glaucoma surgery were processed andminced within 24 hr after surgery on 35-mm tissueculture dishes (Falcon tissue culture, Becton Dickin-son, Lincoln Park, NJ). Explants were grown in Ea-gle's Minimal Essential Medium (MEM) that con-tained 20% FBS (Flow Laboratories, McLean, VA),penicillin G (100 IU/ml), streptomycin (100 fig/m\),and amphotericin B (0.25 Mg/ml) (Sigma ChemicalCo., St. Louis, MO) and were incubated at 37°C in ahumidified atmosphere of 5% CO2. After growing toconfluence, fibroblasts were enzymatically detachedwith 0.05% trypsin (Sigma) at 37°C for 10 min, and

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2600 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Augusr 1991 Vol. 32

Table J. Schematic representation of the biochemical mechanism of action of fluoropyrimidine

FUR

FUMP FUTP

FUdR

10 RNA incorporation—^suppressed maturation

and altered function

Enzymes

1 -Uridine phosphorylase2-Uridine kinase3-Phosphoribosyl transferase4-Thymidine phosphorylase5-Thymidine kinase6-Thymidylate synthetase7-Pyrimidine monophosphate kinase8-Pyrimidine diphosphate kinase9-Ribonucleotide reductase10-RNA polymerase

_ dTTP

dTMP

Substrate/Products

DNAbreakage

cell death

FUR=5-fluorouridineFUMP=5-fluoro-5 monophosphateFUdR=5-fluoro-2 deoxyuridineFdUMP=5-fluoro-2 deoxy-5 monophsphateFUDP=5-fluorouridine-5 diphosphateFUTP=5-fluorouridine-5 triphosphateFdUDP=5-fluorodeoxyuridine-5 diphosphateFdUTP=5-fluorodeoxyuridine-5 triphosphatedUMP=Deoxyuridine-5 monophosphatedTMP=Deoxythymidine-5 monophosphatedTTP=deoxythymidine-5 triphosphate

trypsinization was stopped by the addition of 10%FBS that contained MEM. The suspended fibroblastswere centrifuged at 1200 rpm for 10 min. The pelletswere first subcultured in a 75-cm2 flask that containedculture media. After confluency, the cells were pas-saged into the larger, 150-cm2 flask. Fibroblasts werereplenished with media twice a week and were periodi-cally screened for mycoplasma contamination withfluorescent Hoechst 33258 stain (Flow Laboratories).

Cell Seeding

Fibroblasts were trypsinized and washed with phos-phate-buffered saline (PBS) (Dulbecco minus magne-sium and calcium, Flow Laboratories). The fibro-blasts were then transferred from 150-m2 flasks tocentrifuge tubes and were centrifuged at 1200 rpm for10 min. The cells were washed and centrifuged twice.Cell viability and number were determined after theywere stained with 0.008% trypan blue and werecounted with a hemocytometer. A 50-Ail aliquot of thestained cell suspension was then used to calibratethreshhold settings on a Coulter counter (Model ZM,Coulter). Then, one thousand viable cells/100 /A ofMEM that contained 10% FBS were plated into 96-

well tissue culture plates (Costar, Van Nuys, CA) for agiven drug experiment.

Drug Treatment

To test cell attachment, 100 n\ of MEM that con-tained various concentrations of FUdR, FdUMP,FUR (Sigma), and FUMP (Calbiochem, La Jolla, CA)(final concentration range: 1000-0.00001 Mg/ml)were added to the plates immediately after they wereseeded. To test cell proliferation, 100 ti\ of these drugsin the same final concentrations was added 22 hr aftercell attachment to the plate and incubated for 1, 2, 3,6, and 9 days in the incubator. The final concentra-tion of FBS was 5% in all wells. Each drug concentra-tion was tested in four wells on each experimentalincubation day.

Coulter Counter Assays

Before they were counted, plates were inverted,blotted, and washed with PBS to remove all unat-tached cells and residual media. Cells were then tryp-sinized for 10 min at 37°C. MEM that contained 10%FBS was added to stop trypsinization. Immediatelyafter trypsinization was stopped, cells were trans-


No. 9 THE EFFECTS OF FLUORINATED PYRIMIDINES ON FIBRODLASTS / Lee er ol 2601

FUR120 -i COULTER COUNTER 120 i HEXOSAMINIDASE

.00001 0001 .001 .01 .1 1Flg.1(a) Concentration

100 1000 .00001.0001 .001 .01 .1 1 10 100 1000

Flg.1(b) Concentration (ng/ml)

ADENOSINE UPTAKE

.00001.0001 .001 .01 .1 1 10 100 1000

Flg.1(c) Concentration (fig/ml)

Fig. 1. Inhibitory effect of various concentrations of FUR on human Tenon's fibroblast growth at different incubation times. Each pointrepresents mean (±SE) of three experiments, performed in quadruplicate, with 16 determinations at each experiment for Coulter counter (a), 6determinations at each experiment for hexosaminidase (b), and 4 determinations at each experiment for 3H-adenosinc (c).

ferred to Coulter counter vials that contained 9.950ml of filtered isotone (Fisher Scientific, Pittsburgh,PA) and were mixed together. Four measurements ofeach well were made, with four wells per drug concen-tration per day. The average of those numbers was thecell count for each drug concentration on a particularexperimental day.

Hexosaminidase Assay

Plates for the hexosaminidase assay were similarlyblotted and washed with PBS. Fifty microliters of7.5-mM p-nitrophenyl-N-acetyl-/3-D-glucosaminide(Sigma) with 0.25% Triton X-100 in 0.1 M citratebuffer (pH 5.0) was then added to each well and incu-bated at 37°C. After 2 hr, the reaction was stopped byadding 100 1̂ of 150-mM glycine/5-mM edetic acid

(EDTA) buffer (pH 10.4, Sigma) to each well. Theoptical density of each well was then read at 405 nmby an enzyme-linked immunosorbent assay reader(Titertek Multiskan, Flow Laboratories). Four wellswere read for each drug concentration for a given day,and the average of those measurements was the opti-cal density (OD) for each drug concentration on agiven experimental day.

3H-Adenosine Uptake Assays

Each 96-well plate received the same series of con-centrations of each of the four tested drugs and wasincubated for 1, 2, 3, 6, and 9 days. To examine theeffect of the four fluorinated pyrimidines on deoxyri-bonucleic acid (DNA) and ribonucleic acid (RNA)synthesis, fibroblasts in each 96-well plate were pulsed


2602 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / August 1991 Vol. 32

120 -i

FUMPCOULTER COUNTER

.00001.0001 .001 .01 .1 1 10FIG.2(a) Concentration (ng/ml)

100 1000

120 -i

100-

8 0 -

6 0 -

HEXOSAMINIDASE

4 0 -

20 -

.00001.0001 .001 .01 .1 1 10 100 1000Flg.2(b) Concentration (ng/ml)

120 -i

100

ADENOSINE UPTAKE

day 1

day 3

day 6

day 9

.00001.0001 .001Fig.2(c)

.01 .1 1Concentration

10 100(ng/ml)

1000

Tig. 2. Inhibitory cflect of various concentrations ofFUMP on human Tenon's libroblast growth at different incubation times. Each pointrepresents mean (±SE) of three experiments, performed in quadruplicate, with 16 determinations at each experiment for Coulter counter (a), 6determinations at each experiment for hexosaminidase (b), and 4 determinations at each experiment for 3H-adcnosinc (c).

for 22 hr with 1 nC\ of 3H-adenosine (position 6-la-beled, specific activity 50 Ci/mmol; New EnglandNuclear, DuPont Co., Wilmington, DE) before theincubation was terminated. At the end of each incu-bation, cells were washed with PBS, trypsinized, anddeposited onto a glass fiber paper of 3-mn pore-size(Whittaker Corp., Walkersville, MA) by an automaticcell harvester (Otto Hiller Co., Madison, WI). Thepaper was washed again with PBS three times, thenwashed at 4°C with 5% cold trichloroacetic acid(TCA, ICN Radiochemicals, Irvine, CA) and absoluteethanol. The glass fiber filter paper was then trans-ferred to scintillation vials (Packard Instrument Co.,Downers, IL) and was air-dried. One hundred micro-liters of 0.01 N NaOH was added to each vial, andafter 30 min, 3.5 ml of scintillation liquid (Ready Pro-tein+, Beckman Inc., Fullerton, CA) was added toeach vial. Scintillation vials were allowed to standovernight in the dark, after which the disintegrations

per minute (DPM) were measured by a 1900 CA Tri-carb liquid scintillation analyzer (Packard). Fourwells were read for each drug concentration for agiven day and the average of those measurements wasthe DPM count for each drug concentration on a par-ticular experimental day.

3H-Thymidinc Uptake Assays

The same procedure was conducted to measure in-corporation of 3H-thymidine (position 6-labeled, spe-cific activity 15.0 Ci/mmol) into fibroblast DNA. Fi-broblasts were incubated with drugs for 1, 2, 3. and 6days.

Statistical Analysis of ID50 Within and Between theExperiments

To compute ID50 for a given day and experiment,the empiric method described by Finney8 was used.


No. 9 THE EFFECTS OF FLUORINATED PYRIMIDINES ON FIDRODLASTS / Lee er ol 2603

120 -i

FUdRCOULTER COUNTER

40 -

20-

.00001.0001 .001 .01 .1 1 10 100 1000FIG.3(a) Concentration (\xglm\)

120-i HEXOSAMINIDASE

.00001.0001 .001 .01 .1 1 10

Flg.3(b) Concentration fag/ml)100 1000

ADENOSINE UPTAKE

.00001 .0001 .001 .01 .1Flg.3(c) Concentration

1 10(fig/ml)

100 1000

Fig. 3. Inhibitory effect of various concentrations of FUdR on human Tenon's fibroblast growth at different incubation times. Each pointrepresents ±SEM of three experiments, performed in quadruplicate, with 16 determinations at each experiment for Coulter counter (a), 6determinations at each experiment for hexosaminidase (b). and 4 determinations at each experiment for 3H-adenosinc (c).

To compute the ID50 based on cytostatic effect, thepoint between the maximum (control) and minimum(concentration that caused complete inhibition) wasdetermined on the semi-log scale. The average 1D5O ofthree experiments for each incubation time was com-puted as the antilog of the mean log ID50 value acrossexperiments. Confidence intervals were computed bytaking the antilog of the log 1D5O confidence interval.

The paired t-test was used to test for statistical signif-icance between the different drugs and different as-says. A probability of P < 0.05 was accepted as beingstatistically significant.

ResultsThe effect of FUR on human Tenon's fibroblast

proliferation on different days as measured by theCoulter counter, hexosaminidase, and 3H-adenosineuptake assays is shown in Figure 1. By the adenosineuptake assay, 0.1 /xg/ml of FUR inhibited fibroblast

growth to 46% of control value at day 1 and to 23% ofcontrol value by day 6 and longer (Fig. 1C). At thehigher concentrations (1-1000 /ig/ml), cell growthwas completely inhibited.

The effect of FUMP on the growth of fibroblastswith three assays is shown in Figure 2. FUMP exerteddose-response curves that were similar to thoseof FUR.

The effects of FUdR on fibroblast proliferation areshown in Figure 3. As indicated, 10 Mg/ml of FUdRinhibited fibroblast DNA and RNA synthesis to 78%of control values at day 1, to 38% of control values atday 3, and to 25% of control values at day 6 and later(Fig. 3C).

The effect of FdUMP on the fibroblast growth mea-sured by three assays is shown in Figure 4. FUdR andFdUMP had similar dose-response curves. Both drugsinhibited fibroblast growth completely at concentra-tions of 100 jug/ml or greater.



120

FdUMPCOULTER COUNTER

.00001.0001 .001 .01 .1 1 10 100Flg.4(a) concentration (fig/ml)

1000

120 -i HEXOSAMINIDASE

.00001.0001 .001 .01 .1 1 10 100Flg.4(b) Concentration (\ig/m\)

1000

120 i ADENOSINE UPTAKE

.00001.0001 .001 .01 .1 1 10Flg.4(c) Concentration (fxg/ml)

100 1000

Fig. 4. Inhibitory effect of various concentrations of FdUMP on human Tenon's fibroblast growth at different incubation times. Each pointrepresents ± SEM of three experiments, performed in quadruplicate, with 16 determinations at each experiment for Coulter counter (a). 6determinations at each experiment for hexosaminidase (b). 4 determinations at each experiment for 3H-adcnosinc (c), or 4 determinations for3H-thymidine uptake (d).

A comparison of the effects of these four fluori-nated pyrimidines on human Tenon's fibroblast pro-liferation is shown in Figure 5. The data were takenafter 9 days of incubation by the Coulter counter, hex-osaminidase, and 3H-adenosine uptake assays andafter 3 days of incubation by the 3H-thymidine uptakeassay. FUR and FUMP showed similar inhibitory ac-tivity, and they were 100-1000 times more potentthan FUdR and FdUMP. Addition of 1 n\ of 6.6X 10"5 M 3H-thymidine to FUdR or FdUMP in-creased the uptake of labeled thymidine by 190-500%of control values, with the peak at 0.01 -0.001 Mg/ml ofthese drugs up to day 3 (Fig. 5D). This effect was re-versed after 6 days of incubation with both drugs.

The average ID50 with 95% confidence interval foreach drug from at least three different cell lines overvarious incubation times by all four assays is shown in

Table 2. None of the fluorinated pyrimidines testedhad any effect on attachment of fibroblasts (incuba-tion day 0).

FUR and FUMP had similar ID50 values. The 50%inhibition of DNA synthesis and cell growth was ob-tained at 0.04 and 0.01 //g/ml of FUR and FUMP,respectively, as early as day 1, decreased about ten-fold at day 2, and remained almost constant withlonger incubation time when measured with adeno-sine uptake assay. The ID50 measured with thymidineuptake assay (0.0017 and 0.002 /ig/ml for FUR andFUMP, respectively) was also obtained as early as day1. When measured by Coulter counter and hexosa-minidase assay, the earliest detectable ID50 for bothdrugs was at day 2.

FUdR and FdUMP also showed similar ID50 valuesand were 100-1000 times less potent than FUR and


No. 9 THE EFFECTS OF FLUORINATED PYRIMIDINES ON FIBRODLASTS / Lee er ol 2605

120-1 COULTER COUNTER

.00001 .0001 .001 .01 .1 1 10Flg.S(a) Concentration (fig/ml)

ADENOSINE UPTAKE

100 1000

0.00001 1000

120 -i

100-3

O 8 0 -

HEXOSAMINIDASE

6 0 -Q

2°5

4 0 -

2 0 -

.00001 .0001 .001 .01 .1 1 10

Flg.5(b) Concentration

100 1000

FURFUMP

FUdRFdUMP

• jOoc

8Ul

UJ

a

FURFUMPFUdRFdUMP

THYMIDINEUPTAKE

000001-.00001 .0001 .001 .01 .1 1 10Flg.5(d) Concentration (fig/ml)

100

Fig. 5. Comparative effects of fluorinated pyrimidines (FUR. FUMP, FUdR, and FdUMP) on human Tenon's fibroblasts from a 4-yr-oldwoman who had undergone strabismus surgery. The inhibitory eftcct was measured by Coulter counter (a), hexosaminidase (b), 3H-adcnosincuptake (c) at day 9, and 3H-thymidinc (d) at day 3.

FUMP. The ID50 at day 1 was between 32.7 and 80.59Mg/ml for FUdR and between 42.30 and 32 forFdUMP with adenosine and thymidine uptake as-says, respectively. When the ID50 was measured withCoulter counter and hexosaminidase assays, earliestdetectable ID50 was 0.55-3.1 Mg/ml for FUdR and1.66-14.4 Mg/ml for FdUMP at day 2. The ID50 ofFUdR and of its monophosphate FdUMP decreasedas a function of incubation time as measured with allfour assays.

Toxic effects of all fluorinated pyrimidine wereshown by trypan blue uptake. The earliest detectablecytotoxicity with morphologic change in the appear-ance of the cells occurred at day 1 with 1000 /xg/ml ofFUR or FUMP. After longer incubation, all cells weredead at 1 /xg/ml of FUR or FUMP by day 9, whereasFUdR or FdUMP manifested toxicity after day 2 with1000 Mg/ml. All cells were dead at the concentrationof 100 Mg/ml by day 9. Figures 6A and 6B show mor-phologic differences between the spheric dead fibro-

blasts that were exposed to 1000 Mg/ml of all fluori-nated pyrimidines and normal spindle-shaped fibro-blasts with no drug exposure.

Comparative ID50 values of all four fluorinated pyr-imidine drugs on fibroblasts at day 6 are shown inFigure 7. FUR or FUMP had significantly lower ID50

values than FUdR or FdUMP (P < 0.001). For FURand FUMP, the adenosine uptake assay showed bettercorrelation with the Coulter counter, whereas the thy-midine uptake assay showed lower ID50 values com-pared with the hexosaminidase assay (P < 0.01).

Discussion

Among the fluorinated pyrimidines. 5-FU was stud-ied for the treatment of experimental intraocular pro-liferative disorders910 and was used as an adjuncttreatment after glaucoma filtering surgery was per-formed with poor prognosis.3 One of the majorgrowth-inhibitory effects of fluorinated pyrimidines,



Table 2. Average ID50 from at least three different cell lines measured by all four assays

Drug

FUR

FUMP

FUdR

FdUMP

IncubationDays

012369012369012369012369

Coulter CounterID50 (fig/ml)

Mean

NDND

0.00810.0070.00230.0019

NDND

0.00850.0390.0030.003

NDND

0.5520.1620.0550.043

NDND

1.660.1440.0510.045

(CD*

(.005-.01)(.004-.07)(.002-.007)(.0005-.0039)

(.001-.019)(.008-. 112)(.OO3O-.O58)(.OO2-.OO5)

(.19-1.59)(.05-.467)(.01-.160)(.01-.126)

(.21-1.69)(.05-.416)(.01-. 148)(.01-. 131)

Hexosaminidase1D5O (ng/ml)

Mean

NDND0.1070.030.0690.021NDND0.7650.1100.0480.039NDND3.11.980.8060.479NDND

14.46.74.070.721

(CD*

(.01-.290)(.01-.57)(.04-.21)(.004-.037)

(.012-1.52)(.027-.202)(.020-.29)(.008-. 11)

(2.1-4.3)(.215-13.8)(.027-10.03)(.04-2.04)

(11.9-27.6)(1.57-21.3)(1.57-11.13)(.4-2.02)

AdenosineID50 fag/ml)

Mean

_0.0400.00430.00190.00100.003

—0.0110.0030.0050.0040.004

—32.7

2.890.700.800.50

—42.30

4.551.630.420.56

(CD*

(.0037-.08)(.0006-.008)(.0004-.007)(.0001-.002)(.0006-.015)

(.001-.08)(.OO2-.OO5)(.OO3-.OO8)(.OO3-.OO5)(.001-.011)

(7.9-135)(1.32-6.31)(.04-1.2)(.05-1.4)(.25-.99)

(19.8-90.3)(.9-18.5)(.48-5.55)(.009-18.52)(.004-.72)

Mean

_0.00170.000250.000140.00004

——

0.0020.00050.00450.0005

——

80.595.461.980.122

——

32.0112.23.90.207

—

Thymidine1D5O (ng/ml)

(CD*

(.001-.0022)(.00004-.00038)(.00004-.00017)(.000014-.00009)

(.OO13-.OO31)(.00002-.001I)(.001-.009)(.00001-.0006)

(9.81-661.7)(2.80-10.6)(.11-35.7)(.076-. 195)

(26.7-38.3)(4.48-23.72)(1.5-5.3)(.021-1.99)

* CI, 95% confidence interval.ND. not detectable.

—, not done.

which binds covalently to thymidylate synthetase andthereby blocks DNA synthesis, was associated withFdUMP. l l12 To elicit its growth-inhibitory effect, 5-FU must first be converted to an active nucleotidederivative713 (Table 1). In vivo, 5-FU is rapidly con-verted to FUR or FUdR by uridine or thymidinephosphorylase, respectively, enzymes that are foundin most normal and neoplastic tissues and inplasma.14 Our laboratory reported a delayed antipro-liferative effect of 5-FU on human Tenon's fibroblastsin tissue culture.6 Other active metabolites of 5-FUsuch as FUR, FUMP, FUdR, and FdUMP, may bemore potent. We measured the relative inhibitory ef-fects of these four fluorinated pyrimidines on cells intissue culture by four different assays and at six differ-ent incubation times.

Heidelbergcr and associates15 showed that, at anequivalent dose, FUR is more effective than FUdR or5-FU in prolonging the lives of mice that are afflictedwith Ehrlich ascites tumor, but is less effective inblocking the methylation reaction. Reports fromother investigators showed that human colon carci-noma cells (HT-29) and Hela cells are 100 times moresensitive to the lethal effects of FUR than FUdR or5-FU.16'17 Complete growth-inhibition of human cer-vical carcinoma cells (H.Ep.# 1) was obtained by 0.01/ug/ml of FUR.18 These data are consistent with ourID50 values. The addition of equivalent concentra-

tions of thymidine (0.01 mg/ml or 100 times higher)did not reverse the growth-inhibitory effect caused byFUR.18 Similar results were obtained in our experi-ments where the addition of 1 /zl of 1 X 10"5 M of3H-thymidine to the fibroblasts that were exposed todifferent concentrations of the FUR or FUMP did notreverse the inhibitory effect of this drug, suggesting anessential metabolic block in addition to the methyl-ation block.18 This additional mechanism probablyinvolves the incorporation of FUR into RNA, leadingto suppression of RNA maturation. Other investiga-tors19 suggest that the primary action of FUR is RNA-directed toxicity and the difference in effectivenessbetween FUR and FUdR is probably due to thegreater transport and incorporation of FUR intoRNA. Wilkinson and associates20 also showed thatthe addition of 1 X 10"4 M thymidine did not preventthe growth inhibition that was caused by a 1-hr expo-sure to 1 X 10"5 M FUR in Novikoff hepatoma cells.In contrast, they also showed that the addition of thesame concentration of thymidine prevented thegrowth-inhibitory effects of 1 X 10"7 M FUR, suggest-ing that thymidine can compete with the low concen-tration of FUR in preventing inhibition of thymidy-late synthetase. FUR and its monophosphate FUMPare much more potent inhibitors of fibroblasts, retinalpigment epithelial cell proliferation, collagen synthe-sis, and contraction.21-22 These two drugs can be incor-


No. 9 THE EFFECTS OF FLUORINATED PYRIMIDINE5 ON FIDRODLA5T5 / Lee er ol 2607

Fig. 6. Phase-contrast micrograph (X32) of human Tenon's ftbro-blasts with 1000 jig/ml of all fluorinated pyrimidines (a) and withno drug (control) (b).

porated into mRNA, rRNA, and tRNA and can alsointerfere with rRNA maturation.1923"25 Houghtonand associates26 also showed that the order of gastroin-testinal toxicity in the mouse was FUR > 5-FU> FUdR. Reports by Blumenkranz and associates27

showed that the ID50 value of FUR was 0.0047 Mg/mlon human dermal fibroblasts, which is consistent withthe ID50 value (0.001 /ig/ml) in our assays. Based onthe ID50 determination of FUR in our experiments(0.001 Mg/ml), this drug is 10-100 times more effec-tive for inhibiting fibroblasts and approximately 100-1000 times more cytotoxic than 5-FU, FUdR, andFdUMP. This finding agrees with the findings of Blu-menkranz and others. l41617'21 Our experimentsshowed that the ID50 of FUR or FUMP did not de-crease as a function of incubation time after day 2(measured with adenosine uptake), and our findingsagreed with those of Currie and colleagues, which in-dicated that the toxicity of FUR, unlike that of FUdRand 5-FU, is not time-dependent.28

FU^u_\ another pyrimidine analog, may be phos-phorylated to FUDP and FUTP, which is incorpo-

rated into mRNA and causes miscoding29 or interfer-ence with ribosomal RNA (rRNA) processing andmaturation.25 In addition, FUDP may undergo a con-version to FdUDP and subsequently, to FdUMP,which can inhibit thymidylate synthetase.30'31 May-baum and associates indicated that the RNA-directedcomponent, such as FUMP, causes growth inhibitionand cell killing after one doubling time in asynchro-nous cultures.32 The observations of Skuta and asso-ciates33 suggested that a subconjunctival administra-tion of liposomal FUMP can reduce fibrous prolifera-tion and can delay wound healing. Although theirprocedure encountered systemic toxicity, such asbone marrow aplasia and corneal complications, theysuggest that lower doses of FUMP may be more suit-able and may reduce systemic toxicity. In our in vitrostudy, FUMP showed cytotoxic and cytostatic effectson human Tenon's fibroblasts that were similar toFUR. Our data suggest that FUR may be a particu-larly effective agent among the fluorinated pyrimi-dines for the success of glaucoma filtering surgery.

FUdR acts as a substrate for thymidine kinase withresultant conversion to FdUMP. FUdR has been usedin the treatment of certain types of cancer, such asbreast and gastrointestinal tract,34"36 and improvedtherapeutic response from regional chemotherapy byhepatic artery infusion has been reported.37"39 Richand associates18 reported complete inhibition ofH.Ep. #1 cell growth at 0.01 ^g/ml of FUdR. Theyalso showed that the growth inhibition caused byFUdR was completely reversed by administration of0.5 Mg/ml of thymidine. Prolonged survival time ofmice who had Ehrlich ascites tumors after treatmentwith FUdR was also reported by Bosch and asso-ciates.40 In our experiments, FUdR showed the ID50

of 0.043 fig/ml measured with the Coulter counterand 0.47-0.5 fig/m\ measured with hexosaminidaseand adenosine uptake assay for day 9, respectively.These values agree with the data of Rich and asso-ciates. The addition of 1 /ul of 6.6 X 10~5 M of tritiatedthymidine to fibroblasts that were exposed to concen-trations lower than 1 /ig/ml of FUdR stimulated theuptake of 3H-thymidine, with the peak at a FUdR orFdUMP concentration of 0.01 Mg/ml by day 3. Inhibi-tion of the methylation reaction is the principal causeof growth inhibition and cytostatic effect of FUdR onEhrlich tumor in mice and H.Ep. #1 cells in tissueculture.151840 Maybaum and associates32 reportedthat 10 MM of FUdR inhibited the growth of S-49 cellswithout lysis within 24 hr of exposure, indicating thatFUdR manifested DNA-directed toxicity in this cellline. Trypan blue uptake assays, which stain the deadcells, and morphologic observation showed that onlythe highest concentration of FUdR or FdUMP (> 100/xg/ml) killed the fibroblasts at day 6. The concentra-



1000-3

100

10

.00001

• Coulter Counter• HexosaminidaseCD Adenosine Uptake{£] Thymidine Uptake

FUMP FUdR MUMP

Fig. 7. Comparative ID50 of fluorinated pyrimidincs (FUR, FUMP, FUdR, and FdUMP) on human Tenon's fibroblasts on day 6. ID^, is theaverage of three experiments.

tion below 100 ng/m\ was only inhibitory to cell prolif-eration. Our data are consistent with the report byPaul and associates,41 who indicated that FUdR ini-tially inhibits DNA synthesis and is nonlethal, thenlater decreases the synthesis of nuclear RNA, nuclearprotein, cytoplasmic RNA, and cytoplasmic protein,and eventually causes cell lysis.

Myers found30 that the nucleotide FdUMP is anextremely potent inhibitor of the enzyme thymidylatesynthetase. In our experiments, FdUMP, like FUdR,showed both cytostatic and cytotoxic effects. Addi-tion of 1 M! of 6.6 X 10"5 M of 3H-thymidine reversedthe inhibitory effect of FdUMP at concentrationslower than 1 /ug/ml by day 3.

Among the four assays that were used, the 3H-thy-midine uptake assay showed the lowest ID50 value inthe case of FUR or FUMP, probably because this as-say only measured those cells that actively synthesizeDNA before division and was the most sensitive indetecting the effect of antimetabolites. The other twoassays measured total cell numbers directly (Coultercounter) or indirectly by colorimetric assay (hexosa-minidase), including living and dead cells. The ID50

values that were determined by these two assays areconsistently higher than the values determined by the3H-thymidine uptake assay. Because addition of 3H-thymidine reversed the inhibitory effects of FUdR orFdUMP on fibroblasts by day 3, we used 3H-adeno-sine uptake to measure the antiproliferative effect atthe level of both DNA and RNA. The uptake of 3H-adenosine decreased as early as day 1 for all fourfluorinated pyrimidines. In conclusion, we showedthat FUR and FUMP are more potent than 5-FU,FUdR, and FdUMP, and have immediate inhibitoryeffects, probably because they do not require conver-sion to a more potent form. Because FUR showed thehighest potency and is less expensive than FUMP,

FUR may be a promising antimetabolite among thefluorinated pyrimidines to be used in controlling ocu-lar wound healing after surgery.42

Key words: 5-fluorouridine (FUR), 5-fluorouridinc-5'-monophosphate (FUMP), 5-fluorodeoxyuridine (FUdR),5-fluorodeoxyuridine-5'-monophosphate (FdUMP), hu-man Tenon's fibroblasts

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