cytotoxicity and dna fragmentation associated with ... · 2812 cytotoxicity with sequential...

Vol. 4, 2811-2818, November 1998 Clinical Cancer Research 2811

Cytotoxicity and DNA Fragmentation Associated with Sequential

Gemcitabine and 5-Fluoro-2’-deoxyuridine in HT-29 Colon

Cancer Cells

Qianfang Ren, Vivian Kao, and Jean L. Grem’

Developmental Therapeutics Department, Medicine Branch, Division

of Clinical Sciences, National Cancer Institute, National NavalMedical Center, Bethesda, Maryland 20889-5105

ABSTRACT

The combined cytotoxic effects of the antimetabolites

gemcitabine (dFdCyd) and 5-fluoro-2’-deoxyuridine

(FdUrd) were studied. Cytotoxicity, biochemical pertur-

bations, and DNA damage seen with dFdCyd and FdUrd

alone and in combination were evaluated in HT-29 human

colon cancer cells. A 4-h exposure to dFdCyd followed by

FdUrd for 24 h produced more than additive cytotoxicity

and marked S-phase accumulation. Cells progressedthrough the cell cycle, however, after a 22-h drug-freeinterval. [3H]dFdCyd was rapidly metabolized to the 5’-

triphosphate and incorporated into DNA. [3H]FdUrd was

anabolized exclusively to FdUrd monophosphate, andpreexposure to dFdCyd did not affect FdUrd monophos-

phate formation. Thymidylate synthase catalytic activity

was inhibited by 48% after a 4-h exposure to 10 flM FdUrd

and by 80% after exposure to the combination. Sequential

4-h exposures to 15 n�i dFdCyd and 10 n�i FdUrd led togreater depletion of dTTP pools (29% of control) than

with either drug alone. Greater effects on nascent DNA

integrity were seen with sequential dFdCyd followed by

FdUrd. Although parental DNA damage was not evidentimmediately after exposure to 15 nM dFdCyd for 4 h

followed by 10 nM FdUrd for 24 h, high molecular massDNA fragmentation was evident 72-96 h after drug re-

moval. Sequential dFdCydlFdUrd was associated with

prominent disturbance of the cell cycle, dTTP pool deple.

tion, dATP/dTTP imbalance, and nascent DNA damage.

Induction of double-strand parental DNA damage and

cell death was delayed, consistent with postmitotic

apoptosis.

Received 1/30/98; revised 8/4/98; accepted 8/5/98.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertise,nent in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

I To whom requests for reprints should be addressed. at NCI-NavyMedicine Branch, NNMC, Building 8, Room 5101, 8901 Wisconsin

Avenue, Bethesda, MD 20889-5105. Phone: (301 ) 496-0901 : Fax:

(301) 496-0047; E-mail: [email protected].

INTRODUCTION

dFdCyd2 (gemcitabine) is a pyrimidine analogue of deoxy-

cytidine in which the deoxyribose moiety contains two fluorine

atoms in place of the hydrogens at the 2’ position. The drug

enters cells by the facilitated nucleoside transport mechanism

and undergoes phosphorylation to the 5’-monophosphate form

(dFdCMP) by deoxycytidine kinase (1-3). The drug is subse-

quently phosphorylated to the 5’-diphosphate (dFdCDP) and

5’-triphosphate derivatives (dFdCTP), respectively. Inhibition

of ribonucleotide reductase by dFdCDP decreases the physio-

logical deoxyribonucleotide triphosphate pools (4, 5). Incorpo-

ration of dFdCTP into DNA appears to be a critical event for

induction of programmed cell death (6-9). dFdCyd is converted

to the inactive metabolite 2’,2’-difluorodeoxyuridine by cytidine

deaminase. in vitro primer extension studies indicate that

dFdCTP competes with dCTP for incorporation into the C sites

of the growing DNA strand by purified DNA polymerases a and

C (involved in DNA replication and repair; Ref. 6). After incor-

poration of the dFdCMP residue, the primer extends by one

deoxynucleotide before a major pause in the polymerization

process occurs. Incorporation of a dFdCyd molecule into a DNA

template affects both the kinetics and fidelity of base insertion

by the Khenow fragment of bacterial DNA polymerase I (10).

Inhibition of DNA synthesis may therefore result from both

perturbations of deoxynucleotide pools and interference by the

incorporated dFdCyd residues with DNA chain elongation. Of

particular interest is the clinical activity of dFdCyd in various

human solid tumors (1 1-1 4).

FUra, a fluorinated analogue of uracil, is used in a variety

of chemotherapeutic combinations in the treatment of solid

tumors (15). Incorporation of FUTP into RNA affects normal

RNA processing and function. Another active metabolite,

FdUMP, competes with the normal substrate, dUMP, for bind-

ing to the nucleotide site of TS, thus resulting in enzyme

inhibition and depletion of d’lTP pools. Incorporation of the

fraudulent nucheotides dUTP and FdUTP into DNA stimulates

excision repair by uracil-DNA glycosylase. In the setting of

decreased dTTP and increased dUTP, a futile cycle may ensue,

accompanied by interference with nascent DNA synthesis and

integrity.

The combination of dFdCyd and FUra represents a reason-

able therapeutic strategy for the treatment of malignancies in

which both drugs are active because their mechanisms of action

and spectra of clinical toxicity are different. We have demon-

strated previously a striking sequence-dependent interaction be-

2 The abbreviations used are: dFdCyd, 2’.2’-difluorodeoxycytidine:

FUra. 5-fluorouracil: Ara-C, 1 -3-D-arabinofuranosylcytosine: FdUrd.

5-fiuoro-2’-deoxyuridine: TS. thymidylate synthase.

Research. on February 10, 2020. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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2812 Cytotoxicity with Sequential Gemcitabine and FdUrd

tween Ara-C and FUra in human colon cancer cells ( 17). Ex-

posure to FUra for 24 h followed by Ara-C was markedly

antagonistic. FUra-mediated inhibition of DNA synthesis prior

to Ara-C exposure significantly decreased the amount of Ara-C

incorporated into DNA, thus accounting for the antagonism. In

contrast, initial exposure to Ara-C followed by FUra was ac-

companied by greater inhibition of DNA synthesis compared

with either Ara-C or FUra alone and led to more than additive

cytotoxicity. Because incorporation of both Ara-C and dFdCyd

into DNA are essential prerequisites for induction of pro-

grammed cell death, it seemed reasonable to select sequential

exposure to dFdCyd followed by FdUrd for the present studies

to avoid potential interference with incorporation of dFdCyd

into DNA. FdUrd was used in place of FUra because it produces

more selective inhibition of TS and DNA-directed cytotoxic

effects.

MATERIALS AND METHODS

Materials. dFdCyd and [3H]dFdCyd (18.6 Ci/mmol)

were kindly provided by Lilly Research Laboratories (Indian-

apohis, IN). Other isotopes were obtained from Moravek Bio-

chemicals (Brea, CA). Calcium- and magnesium-free PBS (pH

7.4) was purchased from Biofluids, Inc. (Rockvihle, MD). Un-

less otherwise stated, chemicals were obtained from Sigma

Chemical Co. (St. Louis, MO).

Cytotoxicity. HT-29 cells were grown in RPMI 1640

supplemented with 10% dialyzed fetal bovine serum (both ob-

tamed from Life Technologies, Inc., Grand Island, NY) and 2

mM glutamine. For cell growth experiments, 20,000 cells were

plated in replicate in 6-well plates. To assess survivorship, 500

cells were plated in replicate in 6-well plates. After 48 h, when

the cells had entered exponential growth, either PBS (controls)

or dFdCyd was added at concentrations ranging from 3 to 40 nM.

After 4 h, the medium was gently aspirated, the cells were

washed with 3 ml of cold PBS, and fresh medium was added.

PBS or FdUrd at various concentrations ranging from 3 to 1 8 ns�

was then added. After an additional 24 h, the medium was

aspirated, and the cells were washed with 3 ml of cold PBS and

incubated in fresh, drug-free medium. The cell number was

determined at 72 h in a Coulter Multisizer II (Miami, FL), at

which time the control cell number was 319,330 ± 20,290. On

day 10, colonies of 50 or more cells were stained and enumer-

ated; the average control colony number was 100 ± 7. To assess

any interaction between the two drugs, various combinations of

sequential dFdCyd and FdUrd at fixed concentration ratios were

evaluated. The fraction of affected cells for each drug alone and

the combination were calculated, and the data were analyzed

using CalcuSyn version 1 . 1 software (Biosoft, Ferguson, MO),

assuming a mutually nonexclusive model. The combination

index was used to signify antagonism (>1), additivity (1), or

synergism (<1).

dFdCyd and FdUrd Metabolism and Incorporation intoDNA. Exponentially growing cells were exposed to [3H]-

dFdCyd ( 15 nM; specific activity, 1 �i.Ci/225 pmoh) for 4 h, after

which the medium was aspirated and the cells were washed with

cold PBS. The cells were then extracted with 0.5 N perchloric

acid immediately or after an additional 4-h incubation with

either drug-free medium or 10 nrvi FdUrd. For FdUrd metabo-

lism, HT-29 cells were preincubated with either drug-free me-

dium or 15 nM dFdCyd for 4 h; after the cells were washed,

FdUrd ( 10 nM; specific activity, 1 p.Ci/150 pmol) was added for

4 h, and the cells were then extracted with 0.5 N perchloric acid.

The acid-soluble fraction was isolated, neutralized, and lyoph-

ilized. The radioactivity in an ahiquot of the reconstituted sample

was determined, and the distribution of [3H]dFdCyd and [3H]-

FdUrd metabohites was measured by anion exchange and re-

verse-phase high-performance liquid chromatography meth-

ods, respectively, with in-line scintillation detection (Packard

Instruments, Mount Prospect, IL; Refs. 16-18).

To assess incorporation of dFdCyd into DNA, HT-29 cells

were exposed to [3H]dFdCyd (15 nM; 1 �i.Ci/300 pmoh) for 4 h,

following which the cells were either harvested immediately or

washed and incubated in fresh medium with either 10 nM FdUrd

or no drug for 24 h. Alternatively, cells were exposed to [3H]-

FdUrd (10 nM; I p.Ci/200 pmol) for 24 h with or without a 4-h

preexposure to 15 ntvi dFdCyd. At the desired times, the DNA

was extracted using the DNA Stat-30 kit (Tel-Test “B”, Inc.,

Friendswood, TX) as recommended by the manufacturer, and

trichloroacetic acid-precipitable counts retained on 0.45 p.m HA

filters were determined.

Measurement of Deoxynucleotide Pools. Exponentially

growing cells were exposed to 15 nM dFdCyd (4 h) and 10 nM

FdUrd (24 h) individually or sequentially. The cells were ex-

tracted with 0.5 N perchhoric acid, neutralized, and lyophihized,

and the samples were stored at -70#{176}Cuntil the time of analysis.

Deoxyribonucleotide triphosphate pools were determined by a

DNA polymerase (Escherichia co/i, Klenow fragment) assay

using synthetic ohigonucleotides as template and primers (19).

TS Assays. TS catalytic activity in cellular hysates was

determined by tritium release from [5-3H]dUMP as described

previously (20). The proportion of the total cellular lysate used

in the assay was 16 ± 2%. Protein was quantified by the

Bio-Rad protein assay kit. For analysis of total TS content, equal

amounts of protein were resolved by PAGE using TS 106 mono-

chonal antibody as the primary antibody (21 , 22). The protein

bands were developed with an enhanced chemiluminescence kit

(ECL kit; Amersham, Buckinghamshire, UK). The relative

quantities of TS in the bound and unbound state were deter-

mined by densitometry using Sigmagel software (Jandel Scien-

tific, San Rafael, CA). The position of the TS protein band (-35

kDa) was identified both by protein standard markers and by

inclusion of hysate from NCI-H63OIR1O cells, a line that highly

overexpresses TS protein (2 1).

Cell Cycle Analysis. After the desired drug exposure,

cell nuclei were isolated, incubated in propidium iodide and

DNase-free RNase for 60 mm, and then gently filtered through

35-pm strainer caps into 12 X 75 mm polystyrene tubes. DNA

histogram data were collected using a FACScan (Becton Dick-

inson, San Jose, CA). The list mode files were analyzed with

“ModFit LT for Win32” version 2.0 software (Verity Software

House, Inc., Topsham, ME).

Detection of Single-Strand and Double-Strand Breaks

in DNA. Induction of single-strand breaks in DNA was deter-

mined by alkaline ehution at a fixed pH of 12.1 as described

previously (22, 23). The cells were either labeled with [‘4CJthy-

midine (0.05 �xCi/ml) for the final 4 h of drug exposure (to

assess nascent DNA) or prelabeled for 24 h prior to drug



0 Colony number (day 10)U Cell number (72 hr)

0 Colony number (day 10). Cell number (72 hr)

N.JON

Fig. 1 Cytotoxicity of dFdCyd 100 #{149}� 100and FdUrd in HT-29 cells. Ex-ponentially growing cells were

exposed to either dFdCyd or 80 80FdUrd at the indicated concen-trations for 4 or 24 h, respec-tively, after which the cells 60 60were washed and incubated in

drug-free medium. Cell numberand colony formation were de- �termined at 72 h and 10 days,

respectively. The data are themeans; bars, SE. The cell 20 20growth data are from five toseven separate experiments

done in duplicate, and colony ____________________________________

fourseparateexperirnentsdone #{176} � 10 20 30 � 40 ‘ 0 5 10 15 20in duplicate. Gemcltabine (nM) hr 0 - 4 FdUrd (nM) hr 4-28

1.25 � dFdCyd:FdUrd (1:1) 1.25 dFdCyd:FdUrd (1.67:1)

�!:.� �o.5o

C�

0.25 � 0.25

0.00 � � I I I 0.00 � � I I I

0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0

Fraction Affected Fraction Affected(Cell Growth at 72 hr) (Colony Formation at 10 days)

Fig. 2 Median effect analysis of dFdCyd followed by FdUrd. Exponentially growing cells were exposed to no drugs, dFdCyd from h 0 to 4, FdUrdfrom h 4 to 28, or the combination at five different concentrations with a fixed ratio of either 1 : 1 (growth) or 1: I .67 (colony formation) in threeseparate experiments, each done in duplicate. The results for each combination for all three experiments were averaged and subjected to median effectanalysis using a mutually nonexclusive model; the results are presented as the combination index versus fraction affected. #{149},observed data points

for the five drug combinations; , the combination index plot predicted by the software. A combination index < 1 .0 indicates synergism.

1.00 -

. . ..

Clinical Cancer Research 2813

0

C

0C.)

C.)5<0

0

C.)

exposure (to assess parental DNA). The total radioactivity was

defined as the sum of the dpm in each elution fraction plus that

retained on the filter, minus the background. Double-strand

breaks were determined by a filter binding assay at a nondena-

turing pH (23). The total radioactivity was determined by com-

bining the counts from the eluting fractions (loading, wash,

lysis, and EDTA wash) plus the dpm retained on the filter. DNA

fragmentation was calculated by dividing the dpm in the eluting

fractions by the total dpm.

Detection of High Molecular Weight DNA Fragmenta-

tion. After the desired drug exposure, intact cells were em-

bedded in 0.5% low melting point agarose plugs (100,000

cells/plug), digested for 48 h with five volumes of a buffer

containing 0.5 M EDTA (pH 9.0), 1% sodium lauroyl sarcosine,

and 0.5 mg/ml proteinase K at 50#{176}C,and then washed and stored

at 4#{176}Cas recommended by the manufacturer (Bio-Rad Labora-

tories; Refs. 18 and 23). Individual plugs were placed on the

teeth of a gel comb, and a 1 .0% chromosomal grade agarose gel

0

C

0C.)

C.)5<0

0

C-)

(160 ml) was cast. A previously described Chef Mapper (Bio-

Rad Laboratories) program was run for 17.5 h, during which

Tris-borate-EDTA buffer (0.5 X , 4 liters) was recirculated at

14#{176}C(23). The gels were subsequently stained with ethidium

bromide.

RESULTSdFdCyd- and FdUrd-mediated Growth Inhibition and

Lethality. In the clinical setting, dFdCyd is most commonly

administered as a short infusion, and extending the duration

of infusion leads to a marked reduction in the maximum

tolerated dose (24, 25). In preliminary experiments, we com-

pared growth inhibition following either 4- or 24-h exposure

to dFdCyd. The IC50 for a 4-h dFdCyd exposure was 15-20

nM. Extending the duration of exposure to 24 h led to a

marked increase in cytotoxicity (IC50 < 1 nM). Therefore, a

4-h exposure to dFdCyd was selected for subsequent exper-



Table 2 dFdCyd and FdUrd DNA incorporation

Exponentially growing cells were exposed to the conditions indicated in the heft column. The DNA was extracted as outlined in “Materials and

Methods.” The data, shown as the mean ± SE, are from four to seven separate experiments done in duplicate.

Condition

15 nM [3H]dFdCyd X 4 h

15 nM [3H]dFdCyd X 4 h to no drug X 24 h15 nM [3H]dFdCyd X 4 h to 10 nM FdUrd X 24 h

No drug to 10 nM [3H]FdUrd X 24 h15 nM dFdCyd X 4 h to 10 n�i [3H}FdUrd X 24 h


Table 1 dFdCyd and FdUrd metabolism

Exponentially growing cells were exposed to dFdCyd and FdUrd as indicated, and the distribution of radiohabehed metabohites was determined

by high-performance liquid chromatography analysis with in-line scintillation detection. The data are presented as the mean ± SE and are from six([3H]dFdCyd) and eight ([3H]FdUrd) separate experiments, each done in duplicate. FdUMP was the only tritiated nucleotide metabolite of FdUrdformed under the experimental conditions.

Concentration (pmol/l06 cells)

Condition dFdCDP’1 dFdCTP FdUMP

15 nM [3H]dFdCyd x 4 h 0.36 ± 0.10 8.08 ± 1.32 NA5

15 nM [3H]dFdCyd X 4 h to 4 h no drug 0.18 ± 0.05 4.07 ± 0.99 NA15 nM [3H]dFdCyd X 4 h to 10 nM FdUrd X 4 h 0.14 ± 0.36 3.70 ± 0.77 NA

10 nM [3H]FdUrd X 4 h NA NA 1.56 ± 0.02

15 nM dFdCyd X 4 h to 10 nM [3H]FdUrd X 4 h NA NA 1.81 ± 0.03

a dFdCDP, deoxycytidine diphosphate; dFdCTP, deoxycytidine triphosphate; FdUMP, 5-fluoro-2’-deoxyuridine monophosphate.S NA. not applicable.

dFdCyd-DNA(fmol/p.g DNA)

1.12 ± 0.44

2.52 ± 1.06

2.52 ± 1.32

NA

NA

FdUrd-DNA

(fmol/p.g DNA)

NA”

NA

NA

1.60 ± 0.58

1.16 ± 0.46

I, NA, not applicable.

iments. The cytotoxicity of FdUrd is also strongly influenced

by duration of exposure; we therefore tested a 24-h exposure

to FdUrd. Following single-agent exposure to either dFdCyd

(4 h) or FdUrd (24 h), the concentration-response curves

were similar as measured by both growth inhibition and

interference with colony formation (Fig. 1).

We next evaluated the effects of sequential exposure to

dFdCyd (4 h) followed by FdUrd (24 h). As shown in Fig. 2,

sequential dFdCyd/FdUrd led to more than additive cytotoxic

effects in both cell growth and colony formation assays. Pro-

tection from 5-fluoropyrimidine-mediated toxicity by thymidine

is generally taken as evidence of rescue from DNA-directed

cytotoxic effects. We found that although 10 �iM thymidine

(4-28 h) had no effect on growth inhibition associated with an

initial 4-h exposure to 15 nrvi dFdCyd, it provided partial pro-

tection against growth inhibition from both 10 flM FdUrd alone

(h 4-28; 86 ± 17% versus 61 ± 14%, mean ± SD, n = 3) and

dFdCyd-sFdUrd (84 ± 1 1% versus 45 ± 10%). These results

suggest that DNA-directed effects of FdUrd are important for

the cytotoxicity seen with the combination.

dFdCyd and FdUrd Metabolism and Incorporation into

DNA. The 5’-triphosphate derivative dFdCTP, the predomi-

nant [3H]dFdCyd metabohite, was -22-fold higher than the

diphosphate pool size (dFdCDP; Table 1). After drug removal,

the dFdCTP pool size decreased by one-half at 4 h, and subse-

quent exposure to FdUrd did not appreciably alter this. With

[3H]FdUrd, FdUMP was the only phosphorylated metabolite

identified. [3H]dFdCyd incorporation into DNA after a 4-h

exposure to 15 nM was 1. 1 fmol/p.g (Table 2). Interestingly,

[3H]dFdCyd content in DNA was -2.2-fold higher 24 h after

drug removal, with or without subsequent exposure to FdUrd,

suggesting continued incorporation of dFdCTP into DNA.

[3H]FdUrd incorporation into DNA reached 1.6 fmoh/pg after a

24-h exposure to 10 nM. Although the amount was somewhat

lower when cells where preexposed to dFdCyd (72% of FdUrd

alone), the differences were not significant (P = 0.397).

Biochemical and Cell Cycle Effects of dFdCyd and

FdUrd. Immediately after a 4-h exposure to 15 nM dFdCyd,

an 82% reduction in dATP pools (to 18% of control) was

evident (Table 3). dGTP pools were also decreased by 32% (to

68% of control), whereas dCTP and dTFP pools were un-

changed. With a 4-h exposure to 10 nM FdUrd, dTTP and dGTP

pools were diminished, whereas dATP and dCTP pools were

somewhat higher than control. Sequential exposure to dFdCyd

and FdUrd led to greater dTl’P depletion than seen with FdUrd

alone, whereas dGTP pools were similar to those seen with

either drug alone. In contrast, dATP and dCTP pools were close

to control values, suggesting that sequential drug exposure led to

offsetting effects on these two deoxynucleotide pools. An in-

crease in the dATP:dTI’P ratio pools provides an index of

deoxynucleotide imbalance (26-28). In cells treated with FdUrd

alone or with preexposure to dFdCyd, the dATP:dTTP ratio was

2.9- and 3.4-fold higher than control, respectively.

TS catalytic activity in control cells averaged 40 pmoll

mm/mg (Fig. 3). TS activity was 1.6-fold higher after a 4-h

exposure to dFdCyd, but this difference was not significant (P =

0. 17, t test). Apparent TS activity was reduced by 48% after a

4-h exposure to 10 ntvt FdUrd, whereas sequential dFdCyd

followed by a 4-h exposure to FdUrd was associated with 80%

TS inhibition. When the duration of FdUrd exposure was ex-

tended to 24 h, TS catalytic activity was reduced by 67 and 74%

without and with dFdCyd preexposure, respectively. TS activity



control

dFdC hrO.4

� FdUrd hr 4.8

� dFdChrO.4 ..�. FdUrdhr4-28

dFdC -e FdUrd hr 4.28


Table 3 Deoxyribonucleotide triphosphate pools

Exponentially growing cells were exposed to no drug. dFdCyd, FdUrd, or sequential dFdCyd plus FdUrd as indicated. Deoxyribonucleotide

levels were determined by an enzymatic DNA pohymerase assay. The assay mixture was incubated for 15 mm at 37#{176}C,and the calibration curve foreach dRTP was linear between 1 and 40 pmol of substrate. The data are presented as the mean ± SE and are from at least four separate experiments,

each done in duplicate.

Concentration (pmolIlO6 cells)

Condition dATP dCTP dGTP dTl’P dATP:dTTP ratio

Control 44.7 ± 3.0 48.8 ± 6.0 27.2 ± 4.2 82.4 ± 10.2 0.54

dFdCyd 15 flM h 0-4 7.9 ± 2.0 46.7 ± 6.6 18.4 � 3.0 80.1 ± 5.8 0.10

(0.18)” (0.96) (0.68) (0.97) (0.19)FdUrd 10 flM X h 4-8 59.6 ± 14.1 70.8 ± 12.0 15.3 ± 3.2 37.6 ± 11.8 1.59

(1.33) (1.45) (0.56) (0.46) (2.94)

dFdCyd -ti FdUrd 43.7 ± 14.5 43.9 ± 8.7 18.6 ± 3.6 24.1 ± 7.8 1.81

(0.98) (0.90) (0.68) (0.29) (3.35)

a Pooh size as a fraction of control.

80

�l 60

>

�40

Cl)

I- 20

0

Fig. 3 FdUrd-mediated inhibition of TS. TS catalytic activity in ccl-lular hysates was determined by tritium release from [5-3H]dUMP. The

concentrations of dFdCyd (dFdC) and FdUrd were 15 and 10 nM,

respectively, and the exposure times are shown. The data, presented as

the means, are from five or more separate experiments; bars, SE. TScatalytic activity was significantly different from control for both Se-

quential exposures of dFdCyd followed by FdUrd h 4-8 (P = 0.036)and h 4-28 (P = 0.034, t test).

was significantly lower than control with sequential dFdCyd and

FdUrd (4- and 24-h exposures; P < 0.04, t test). Because

enzyme activity in the catalytic assay is assessed by the release

of tritium from [5-3H]dUMP, potential expansion of endoge-

nous dUMP pools due to TS inhibition might lead to overesti-

mation of the extent of TS inhibition. Therefore, Western im-

munoblot analysis was also used to assess TS ternary complex

formation. A single band migrating at 35 kDa was evident in

control cells and cells treated with dFdCyd alone, reflecting free

TS. In contrast, a slightly higher molecular weight band that

represented bound TS was seen in addition to the 35-kDa band

in cells treated with FdUrd (data not shown). After a 4-h

exposure to FdUrd alone or with preexposure to dFdCyd, den-

sitometric analysis showed that the percentage of bound TS was

54 ± 6% and 75 ± 2% (mean ± range/2, n 2 experiments).

After a 24-h exposure to FdUrd, bound TS accounted for 67 ±

1% with FdUrd alone (mean ± SD, n = 3) and 79 ± 2% with

dFdCyd followed by FdUrd. These results are consistent with

the catalytic assay.

The effects of drug exposure on cell cycle distribution over

time are shown in Table 4. For these experiments, cells were

exposed to either diluent or to 15 n�i dFdCyd for the initial 4 h.

After drug removal, FdUrd or dihuent was added for either an

additional 4 h or 24 h. The cells were then harvested at the

indicated times. No major effects were evident at 8 h; however,

S-phase accumulation was evident at 28 h with dFdCyd and

FdUrd given individually. The most striking effects were ob-

served with the two drugs given sequentially. The S-phase

accumulation had largely dissipated at h 50 (46 h and 22 h after

removal of dFdCyd and FdUrd, respectively), indicating that the

cells had progressed through the cell cycle.

DNA Damage. The effect of drug exposure on the fra-

gility of newly synthesized DNA was determined by fixed pH

alkaline elution. Increasing single-strand breaks are reflected by

a decreasing proportion of the total labeled DNA species re-

tamed on the filter relative to control. When the cells were

labeled and harvested immediately after a 4-h exposure to

dFdCyd at concentrations ranging from 10 to 40 nsi, the per-

centage retained on the filter decreased with increasing drug

concentration from 79% of control to 39% of control, respec-

lively (,2 0.91 ; data not shown). In addition, the extent of

nascent DNA damage was inversely related to colony formation

(r2 0.78; data not shown). With a 4-h exposure to 15 nrvi

dFdCyd followed by a 24-h drug-free period, however, the

extent of single-strand breaks in nascent DNA was not as great

(88% of nascent DNA retained versus control; Fig. 4). This

observation suggests that interference with nascent DNA syn-

thesis at this concentration of dFdCyd is greatest during drug

exposure, consistent with prior reports (8). After a 24-h expo-

sure to FdUrd, with the cells labeled for the final 4 h, an inverse

correlation was also noted between nascent DNA retained and

FdUrd concentration (r� = 0.83; data not shown). After expo-

sure to 10 nM FdUrd for 24 h, only 78% of the nascent DNA was

retained compared with control, whereas nascent DNA damage

was greatest after sequential 10 rtM dFdCyd at h 0-4 to 10 flM

FdUrd at h 4-28. The alkaline-labile DNA strand breaks in

newly synthesized DNA are presumably due to both deoxyri-

bonucleotide triphosphate imbalance, which may interfere with



100

� 90

� 80

asXC.)

� 70aCasC.)� 60z

50I

Oligonucleosomal DNA laddering was not detected using con-

ventional agarose electrophoresis for up to 72 h after drug

removal (data not shown). Because traditional DNA purification

techniques result in extensive shearing of DNA, induction of

high molecular mass DNA fragments might be overlooked.

DNA was therefore purified from intact cells, embedded in

agarose plugs and analyzed by pulsed-field gel electrophoresis.

Delayed appearance of parental DNA fragmentation of < 1-Mb

fragments was observed in cells treated with sequential

dFdCyd-sFdUrd 72 and 96 h after drug exposure (Fig. 5).

DISCUSSION

We found that sequential exposure to dFdCyd for 4 h

followed by FdUrd for 24 h led to more than additive growth

inhibition and interference with colony formation. To simplify

the analyses, the IC50 concentrations were used to study possible

mechanism(s) of interaction between the drugs. Our results

indicate that giving dFdCyd first allowed its metabolism and

incorporation of dFdCTP into DNA to proceed without inter-

ference. Furthermore, preexposure to dFdCyd did not perturb

FdUrd metabolism. The opposite sequence was not studied

because we established previously that fluoropyrimidine admin-

istration prior to Ara-C decreased Ara-C DNA incorporation

and was associated with marked antagonism in cytotoxicity

assays (16). Because incorporation of both Ara-C and dFdCyd

are essential prerequisites for induction of programmed cell

death, it seemed logical to avoid an antagonistic sequence in the

present studies. The present results are consistent with our prior

study in that sequential administration of a deoxycytidine ana-

logue followed by a 24-h exposure to a 5-fluoropyrimidine

resulted in more than additive cytotoxicity. Exposure to FdUrd

after dFdCyd avoided any potential interference with dFdCyd

metabolism and DNA incorporation. Whereas preexposure to

dFdCyd did not affect free FdUMP formation, the extent of TS

inhibition early during FdUrd exposure was greater than that

observed with FdUrd alone. The basis for this observation is not

clear, but the dTFP pool data corroborated this phenomenon.

Acute biochemical and metabolic derangements seen with

the combination included pronounced effects on the cell cycle

with S-phase accumulation, depletion of dTl’P, an increase in

the dATP:dTFP ratio, and induction of single-strand breaks in

0 dFdC hr 0-4

FdUrd FdUrd hr 4-28

Fig. 4 Induction of single-strand breaks in nascent DNA. Exponen-

tially growing cells were exposed to either no drug or to 15 nivi dFdCyd(dFdC) and/or 10 nM FdUrd as indicated. The cells were incubated with

[‘4Clthymidine (0.05 p.Ci/ml) from h 24 to 28 and then were harvestedand subjected to alkaline elution (pH 12.1) for 15 h. For each condition,the counts retained on the filter were determined relative to the totalamount of counts on the filter and in the eluting fractions. The fractionretained on the filter for drug-treated cells was then expressed as a

percentage of control for each experiment. The data are presented as the

means and are from five or more separate experiments done in duplicate:

bars, SE. In control cells, the fraction of [t4C]thymidine�labeled DNAretained on the filter was 78.9 ± 4.0%.

both DNA synthesis and repair, and incorporation of fraudulent

nucleotides into DNA either directly (as with dFdCyd) or mdi-

recthy (as with FdUrd by stimulating excision/repair).

There was no evidence of parental DNA damage as meas-

ured by either single-strand or double-strand breaks immedi-

ately after exposure to dFdCyd alone, FdUrd alone, or the

combination. Induction of double-strand breaks in parental

DNA is a hallmark of programmed cell death. We therefore

monitored the cells for delayed induction of double-strand pa-

rental DNA damage at 24-h intervals after exposure to sequen-

tial 15 flM dFdCyd at h 0-4 to 10 nM FdUrd at h 4-28.


Table 4 Changes in cell cycle distribution

The cell cycle distribution was determined at the indicated times following exposure to either no drug, 15 flM dFdCyd for the initial 4 h. and 10nM FdUrd for either h 4-8 or h 4-28. The data for the 8-h and 50-h time points are from a single experiment. The results for the 24-h time point,shown as the mean ± range/2, are from two separate experiments. The data for controls are presented as the mean and SD and are pooled results from

these four separate experiments. Information on 10,000-20,000 cells was collected for each sample.

First exposure h 0-4 Second exposure Time of analysis % in G1 % in G,/M % in S

No drug No drug 8. 28, and 50 h 54 ± 8 13 ± 2 33 ± 9dFdCyd No drug h 4-8 8 h 60 6 34

No drug FdUrd h 4-8 8 h 48 6 45

dFdCyd FdUrd h 4-8 8 h 58 2 40

dFdCyd No drug h 4-28 28 h 9 ± 7 27 ± 1 63 ± 7

No drug FdUrd h 4-28 28 h 8 ± 8 33 t 6 59 ± 2

dFdCyd FdUrd h 4-28 28 h 6 ± 4 13 ± 2 81 ± 3

dFdCyd No drug h 4-50 50 h 38 27 25No drug FdUrd h 4-28 50 h 49 10 40

dFdCyd FdUrd h 4-28 50 h 38 35 27

dFdC0



A B C D E F C


Fig. 5 Delayed induction of high molecular mass DNA damage. Cellswere exposed to either no drug or sequentially to 15 nM dFdCyd during

h 0-4. followed by 10 nsi FdUrd at h 4-28. The cells were harvestedeither immediately after drug exposure or after incubation in drug-free

medium at 24-h intervals thereafter. Intact cells were embedded in

agarose plugs (200.000 cells/plug) and digested in situ at 37#{176}Cfor 48 h

to remove protein and RNA. Lane A. Saccharornvces cerevisiae mark-

ers; Lane B, DNA from control cells: Lanes C-G, DNA from cellstreated with dFdCyd-eFdUrd and harvested 0 h (Lane C). 24 h (Lane

D), 48 h (Lane E), 72 h (Lane F). or 96 h (Lane G) after drug removal.

Similar results were seen in separate experiments.

nascent DNA, leading to growth imbalance. The observation

that dFdCyd-mediated interference with nascent DNA integrity

was greater during drug exposure than after a drug-free interval

is consistent with previous reports that over time the growing

DNA strand can extend beyond the incorporated dFdCMP res-

idue in intact cells (8). Thus. dFdCyd appears to function as a

relative, rather than as an absolute, chain terminator in intact

cells. Induction of programmed cell death after drug exposure in

these cells appeared to be a delayed event. The time course and

pattern of parental DNA fragmentation produced by dFdCyd

and FdUrd in HT-29 cells thus differs markedly from that

reported for human leukemic cells (7, 9, 29). In HL-60 myeloid

heukemic cells, for example, a 6-h exposure to 50 nM dFdCyd

was sufficient to induce classic signs of apoptosis (29). HL-60

cells are representative of the many cell types, particularly of

hematopoietic origin, that undergo apoptosis rapidly (within a

few hours) after exposure to relatively high concentrations of

cytotoxic agents (30). Previous investigators have reported that

exposure of epithelial cancer cell lines to various DNA-damag-

ing agents, including FdUrd, produced high molecular mass

DNA fragmentation in the 50- to 300-kb range in the absence of

nucheosomal haddering (23. 31-33). The absence of ohigonucheo-

somal laddering in the previously reported epithehial malignan-

cies and in the HT-29 cells in the present report may signify that

the cells either do not contain or do not activate that specific

calcium- and magnesium-dependent endonuclease under the

experimental conditions used. Furthermore, the delayed appear-

ance of such double-strand DNA fragmentation has generally

occurred when certain epithelial cancer cells are exposed to

relatively low concentrations of a cytotoxic agent and then

allowed to grow in drug-free medium (30, 32, 34, 35). In this

scenario, sometimes referred to as “postmitotic apoptosis,” pa-

rental DNA fragmentation occurs in the cell cycle(s) subsequent

to the one in which the cells were initially exposed to the

- 3.13 mb damaging agent (30). The observation that the HT-29 cellsprogressed through the cell cycle by h 50 after recovering from

- 2.35 the initial drug exposure is consistent with this mechanism.

HT-29 cells have a mutant p53 genotype which may in part

account for the relative inability of moderate genotoxic stress to

trigger rapid induction of programmed cell death. Although the

precise basis for delayed apoptosis is not clear, one possible

- I .05 explanation is that originally sublethal damage to genes that are

essential for cell survival may ultimately lead to cell death with

subsequent rounds of DNA replication (30).

In summary, these data indicate that sequential administra-

tion of dFdCyd followed by FdUrd did not interfere with the

metabolic activation of either nucleoside analogue. and the

combination produced acute biochemical derangements. nascent

DNA damage, and delayed induction of high molecular mass

DNA fragmentation. These results provide a rationale for chin-

ical evaluation of this drug combination. Whether any potential

sequence-dependent interactions will be observed in the clinical

setting with 5-fluorouracil given either by bolus injection or by

infusion either immediately before or after a conventional 30-

mm dFdCyd infusion remain to be determined.

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1998;4:2811-2818. Clin Cancer Res Q Ren, V Kao and J L Grem cells.gemcitabine and 5-fluoro-2'-deoxyuridine in HT-29 colon cancer Cytotoxicity and DNA fragmentation associated with sequential

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