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.
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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
Research. on February 10, 2020. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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-
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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
2814 Cytotoxicity with Sequential Gemcitabine and FdUrd
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
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control
dFdC hrO.4
� FdUrd hr 4.8
� dFdChrO.4 ..�. FdUrdhr4-28
dFdC -e FdUrd hr 4.28
Clinical Cancer Research 2815
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
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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.
2816 Cytotoxicity with Sequential Gemcitabine and FdUrd
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
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A B C D E F C
Clinical Cancer Research 2817
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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