Synergistic Interaction between Cisplatin and Gemcitabine in Vitro1 · ADDP, 12,000 cells/well;...

Vol. 2, 521-530, March 1996 Clinical Cancer Research 521

Synergistic Interaction between Cisplatin and Gemcitabine in Vitro1

Andries M. Bergman,

Veronique W. T. Ruiz van Haperen,

Gijsbert Veerman, Catharina M. Kuiper,

and Godefridus J. Peters2

Department of Oncology, Free University Hospital, P.O. Box 7057,

1007 MB Amsterdam, the Netherlands

ABSTRACT

2’,2’-Difluorodeoxycytidine (dFdC; gemcitabine) is a

new antineoplastic agent that is active against ovarian car-

cinoma, non-small-cell lung carcinoma, and head and neck

squamous cell carcinoma. cis-diamminedichioropiatinum

(CDDP; cisplatin) is used commonly for the treatment ofthese tumors. Because the two drugs have mechanisms of

action that might be complementary, we investigated a pos-

sible synergism between dFdC and CDDP on growth inhi-

bition. The combination was tested in the human ovarian

carcinoma cell line A2780, its CDDP-resistant variant ADDP

and its dFdC-resistant variant AG6000, the human head

and neck squamous cell carcinoma cell line UMSCC-22B,and the murine colon carcinoma cell line C26-10. The cellswere exposed to dFdC and CDDP as single agents and to

combinations in a molar ratio of 1:500 for 1, 4, 24, and 72 h

with a total culture time of 72 h. Synergy was evaluated

using the multiple drug effect analysis. In A2780 and ADDP

cells, simultaneous exposure to the drugs for 24 and 72 h

resulted in synergism, but shorter exposure times were an-

tagonistic. No synergism was found in the UMSCC-22B andC26-10 cell lines at prolonged simultaneous exposure. How-

ever, a preincubation with CDDP for 4 h followed by a dFdC

incubation for 1, 4, 24, and 72 h was synergistic in all cell

lines except C26-10 cells. A 4-h preincubation with dFdC

followed by an incubation with the combination for 20 and68 h was synergistic in all cell lines. Initial studies of themechanism of interaction concentrated on the effect of

CDDP on dFdCTP accumulation and DNA strand breakformation. In all cell lines, CDDP failed to increase dFdCTP

accumulation at 4- or 24-h exposure to dFdC; in two cell

lines, CDDP even tended to decrease dFdCTP accumulation.

Neither dFdC nor CDDP caused more than 25% double

strand break formation, whereas in the combination, CDDP

even tended to decrease this type of DNA damage. The

synergistic interaction between the two drugs is possibly the

result of dFdC incorporation into DNA and/or CDDP-DNA

adduct formation, which may be affected by each other.

INTRODUCTION

dFdC3 is a deoxycytidine analogue with two fluorine atoms

substituted for the two hydrogens at the 2’ position of the ribose

ring. The compound has established activity against solid tu-

mors, as described for several solid tumor models, including

ovarian and head and neck cancer (1-3). In the clinical setting,

dFdC has proven to be effective against ovarian cancer and

non-small cell lung cancer and moderately effective against

head and neck cancers (4-6). After entering the cell, the drug

has to be phosphorylated by dCK to dFdCMP and subsequently

to dFdCTP (7), which can be incorporated into both DNA and

RNA (8, 9). After incorporation of dFdCTP, one more de-

oxynucleotide can be incorporated, after which DNA polymer-

ization stops (9). Unlike ara-C, exonuclease activity is unable to

excise dFdCMP from the DNA (9). dFdC can be inactivated by

the action of deoxycytidine deaminase to 2’,2’-difluorode-

oxyuridine (7).

For the treatment of the above-mentioned tumors, CDDP, a

square planar coordination compound, is an important agent

(10). The antitumor activity of CDDP is the result of the

formation of adducts within the DNA (1 1-17). The binding of

the CDDP moiety with two adjacent guanines on the same DNA

strand is the most abundant lesion and is held responsible for the

antitumor activity (12, 13). This lesion is thought to introduce a

distortion in the DNA that is large enough to stop the division of

the cells without being recognized rapidly and, thus, removed

efficiently by repair enzymes (18). There is a possible relation

between CDDP-DNA adduct levels and cell growth inhibition in

cultured cells (14, 15).

Synergistic interactions of CDDP with ara-C and DAC,

two other deoxycytidine analogues, have been described previ-

ously. Studies on the mechanism of the synergism suggested

differences in the interactions between ara-C and CDDP and

DAC and CDDP (19, 20). CDDP-DNA adduct formation was

enhanced by DAC incorporation, but ara-C did not affect the

binding of CDDP to DNA. However, ara-C delayed the recov-

ery of DNA synthesis inhibited by CDDP markedly. Because

dFdC is related structurally to ara-C and DAC, it seemed ap-

propriate to investigate the combination of CDDP with this

compound too. One possible mechanism for synergy could be

the inhibitory effect of CDDP on ribonucleotide reductase (21),

possibly causing a depletion of dCTP pools, a potent feedback

inhibitor of dCK. Subsequently, this could lead to increased

dFdC phosphorylation. Alternatively, DNA might become more

accessible to DNA adduct formation by distortions caused by

Received 6/26/95; revised 1 1/15/95; accepted 12/5/95.I This work was supported by grants from the Dutch Cancer Society by

Grants IKA-VU 90-19 and VU 94-753, and by a grant from Eli Lilly,

Inc. (Nieuwegein, the Netherlands).2To whom requests for reprints should be addressed. Phone: + 31-20-

4442633; Fax: +31-20-4443844.

3 The abbreviations used are: dFdC, 2’,2’-difluorodeoxycytidine (gem-

citabine); dCK, deoxycytidine kinase; ara-C, i-�3-D-arabino furanosylcytosine; CDDP, cis-diamminedichloroplatinum (cisplatin); DAC, 2’-de-oxy-5-azacytidine; 22B, UMSCC-22B; TCA, trichloroacetic acid; SRB,

sulphorhodamine B; IC50, 50% inhibitory concentration; CI, combination

index; FA, fraction affected; Dm, dose required to produce a 50% growthinhibition; FADU, fluorometric analysis of DNA unwinding.

Research. on September 10, 2021. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

522 Synergistic Interaction between Cisplatin and Gemcitabine

dFdCTP incorporation. Furthermore, because both drugs have

different, nonoveriapping side effects, synergistic or even addi-

tive antitumor effects of the combination may be clinically

worthwhile. In addition, when using combinations, it is less

likely that resistance will develop.

Here, we describe the in vitro synergism between dFdC and

CDDP. Three human ovarian carcinoma cell lines, the dFdC-

and CDDP-sensitive A2780 cell line and its CDDP- and dFdC-

resistant variants ADDP and AG6000, respectively, the human

head and neck squamous cell carcinoma cell line 22B, and the

dFdC-insensitive murine colon tumor cell line C26-lO were

exposed to both drugs in different concentrations and schedules.

To investigate possible mechanisms of synergism, several pa-

rameters known to be important in the action of dFdC were

studied, and the effect of CDDP on the dFdCTP accumulation

and the number of DNA strand breaks induced by dFdC were

measured to clarify the mechanism of the synergistic interaction.

MATERIALS AND METHODS

Chemicals and Reagents. DMEM and RPMI 1640 were

purchased from Flow Laboratories (Irvine, United Kingdom);

FCS was from GIBCO (Grand Island NY), TCA, glutamine, and

gentamicin were from Merck (Darmstadt, Germany), trypsin,

and SRB was from Sigma Chemical Co. (St. Louis, MO). dFdC

was kindly supplied by Eli Lilly Research Labs (Indianapolis,

USA). CDDP was purchased from Bristol-Myers Squibb

(Weesp, the Netherlands). All other chemicals were of analyti-

cal grade and commercially available.

Cell Culture. The in vitro experiments were performed

with five different cell lines: A2780, a human ovarian cancer

cell line (22, 23), and two variants, ADDP, with a 50-fold

induced resistance to CDDP (kindly provided by Dr. K. J.

Scanlon City of Hope National Medical Center, Duarte, CA;

Ref. 22), and AG6000, with a 150,000-fold induced resistance

to dFdC (23); 22B, a human head and neck squamous cell

carcinoma cell line (24); and the murine colon carcinoma cell

line C26-lO (25). Doubling times of the cell lines were 21, 32,

37, 40, and 14 h, respectively. Cells were grown in monolayers

in DMEM supplemented with 5% heat-inactivated FCS, 1 mM

L-glutamine, and 250 ng/ml gentamicin, except for ADDP,

which was cultured in RPMI 1640 medium supplemented with

10% heat-inactivated FCS, 1 mM L-giutamine, and 250 ng/ml

gentamicin at 37#{176}Cat 5% CO2.

Chemosensitivity Testing. The determination of the

IC50 was performed using the SRB assay (26, 27). The assay

was performed using the National Cancer Institute protocol with

some small modifications (27). Culture conditions were opti-

mized for each cell line. The five cell lines were exposed to

dFdC and CDDP as separate agents and to a combination of

both. At day 1 , the cells were plated in 96-well plates in

different densities, depending on their doubling times. The

optimal plating number was the highest number of cells possible

to enable log linear growth for 72 h (A2780, 5,000 cells/well;

ADDP, 12,000 cells/well; AG6000, 18,000 cells/well; C26-lO,

1,500 cells/well; and 22B, 15,000 cells/well) in a volume of 100

�i.l/well. Of the 96-well plates, the upper and lower rows (rows

A and H) and the last two columns at the right side (columns 1 1

and I 2) were not used for cells, because we observed evapora-

tion in these wells earlier. Thus, the first column contained only

medium, the second column contained cells not exposed to

drugs, and eight columns contained cells exposed to increasing

concentrations of drugs. For each drug or drug combination,

three wells were used.

On day 2, dFdC and CDDP were added in a volume of 100

pal, resulting in series of final concentrations of 2.10 ‘ I_1.106

M dFdC and l.10_8_5,10_4 M of CDDP. The concentrations in

the combination had the same range as the separate agents, with

a dFdC:CDDP ratio of 1:500, which was based on the separate

IC50 values in the various cell lines. Similar to the National

Cancer Institute protocol, we chose the same culture time for all

cell lines. The cells were exposed for 1 , 4, 24, and 72 h. After

the exposure, the medium in the control and the drug-containing

wells was removed and replaced by fresh, drug-free medium

after a washing step with 50 �il PBS (pH 7.4), and the cells were

cultured until 72 h after the initial drug addition. Care was taken

to ensure that cell loss was minimal in the procedure; further-

more, control wells were also washed. Final A values in washed

and nonwashed wells were hardly different. At the end of the

culture, the cells were precipitated with 50 pi ice-cold 50%

(w/v) TCA and fixed for 60 mm, after which the SRB assay was

performed as described (27). Only proteins of intact cells are

stained, because proteins from killed cells would be degraded to

amino acids by endogenous proteases after lysis (27). Growth

inhibition curves were made relative to control As of every SRB

assay. Two control values were always included, that of the A of

cells at the day of drug administration (day 0, set at 0%) and that

of cells after 3 days (set at 100%). This enabled us to calculate

IC50 values (relative A at 50%), total growth inhibition (A of

drug-treated cells similar to the initial value, 0%) and cell killing

(A of drug treated cells lower than the initial value 0%). The

points were connected by straight lines, and the IC50 values

were determined from the interpolated graph (28). Also, the

method of Chou and Talalay (29) was used; for these cell lines

similar values (Dm) were achieved. The cells were not only

exposed to the combination simultaneously, but two sequential

schedules were also used: (a) 4-h preincubation with CDDP,

after which the cells were exposed to dFdC for 1, 4, 24, or 72 h;

and (b) 4-h preincubation with dFdC, followed by an incubation

with both dFdC and CDDP for 20 or 68 h (Fig. 1).

Dose-response interactions (antagonism, additivity, and

synergism) between dFdC and CDDP were expressed as a

nonexclusive case CI for every FA, using the method of Chou

and Talalay (29), processed by a computer program developed

by Chou and Chou (Biosoft, Cambridge, United Kingdom). This

program was chosen because it provided one of the few objec-

tive computerized evaluation procedures (30). The data are

supported by classic isobolograms made by the same computer

program, which were based on the original method of Steel and

Peckham (3 1 ). Other observations in favor for the choice of this

program were the sigmoidal forms of the growth curves, ideal

for median effect analysis. Thus, IC50 values (by interpolation)

and Dm values (by the computer program) were usually similar.

Measurable IC50 values for each drug separately are not re-

quired for computerized evaluation; Dm values would be calcu-

lated then by the program by extrapolation. For the separate

drugs, we had to introduce their respective growth inhibition

parameters, expressed as FA (e.g. , a FA of 0.25 is a growth



aim.

dFdC+

CDDP

coop

->dFdc

dFdC.>coop

-4 14 24 72

Clinical Cancer Research 523

I I

I I

‘Sf1 I

I I

�-

time (hr)

Fig. 1 Schematic representation of the drug exposure schedules of thegrowth inhibition experiments. sim. dFdC+CDDP, simultaneous expo-

sure to the combination of dFdC and CDDP; CDDP-*dFdC, preincu-

bation with CDDP followed by dFdC exposure; dFdC-*CDDP, prein-cubation with dFdC alone followed by exposure to the combination of

dFdC and CDDP. D, CDDP; �, dFdC; R, dFdC and CDDP; D. drug

free.

inhibition of 25%). The program was unable to evaluate values

of FA > 1, indicating cell killing or FA = 0, indicating no

growth inhibition. Growth inhibition of 0% of each drug sepa-

rately had to be introduced as a FA of 0.01, whereas total growth

inhibition of 100% had a FA of 0.99. For the final evaluation,

we excluded values of the combination for almost complete

growth inhibition (FA > 0.90) and hardly any effect (FA <

0. 10). The CI was calculated by the formula:

CI = [(D)l/(D5)lJ + [(D)2/(D5)2] + [a(D)l,2/(Dx)i,2]

Where a = 1 for mutually nonexclusive drugs; (D)l, (D)2, and

(D)l,2 are the doses of the separate drugs and their combination

in a fixed ratio of 1:500; and (D�)l, (D�)2 and (D�)l,2 are the

doses resulting in a growth inhibition of x%. These doses are

calculated by the formula: D = D, m X [(FA/l - FA)]/m,

where D� is the dose required to produce a 50% growth inhi-

bition, FA is the fraction affected, and m is the slope of the

median effect plot (a measure of sigmoidicity). A CI < I mdi-

cates synergism, > I indicates antagonism, and a CI of 1 mdi-

cates additivity. Results were also analyzed by isobologram

analysis using the same program as well by simple calculations

as comparisons of expected and observed growth inhibition or

cell killing. All three methods led to comparable conclusions,

underlining the significance of the data obtained with the mul-

tidrug effect analysis.

dFdCTP Accumulation. At day 1 , the cells were plated

in six-well plates in different densities, depending on their

doubling times (A2780, I .5 X 10� cells/well; ADDP, 1 .5 X I 0�

cells/well; AG6000, 2.5 X l0� cells/well; C26-l0, I X l0�

cells/well; and 22B, 1.0 X 106 cells/well) in a volume of 2

mi/well. At day 2, the cells were exposed to either 0. 1 p.M dFdC

or I �J.M dFdC as single agents or to combinations of 0. 1 pM

75’- I

dFdC with 20 �LM CDDP or to 1 J.LM dFdC with 100 p.M CDDP

and incubated at 37#{176}C.After 4 and 24 h of incubation, the cells

were harvested by trypsinization, washed, and counted while

kept on ice. Thereafter, the nucleotides were extracted using

TCA precipitation and alamine and freon neutralization (32).

Finally, the nucleotides were analyzed on high-performance

liquid chromatography using a Partisphere SAX (Whatman,

Clifton, NJ) column with a linear gradient between S m�i

NH4H2PO4 (pH 2.8; buffer A) and 0.5 M NH4H2PO4/0.25 M

KCL (pH 3.0; buffer B; 35-100% B over 30 mm) at a flow rate

of 1 .5 mi/mm. Samples were detected at 254 and 280 nm (32).

FADU Assay. To measure the dFdC-induced DNA dam-

age the FADU assay was used (33, 34). DNA damage was

assessed by exposing lysed cells to an alkaline environment,

allowing the DNA to unwind. The extent of strand breaks in the

DNA determines the extent of DNA unwinding at the end of

incubation. For this purpose, 3-5 X 106 A2780 or 22B cells

were incubated for 24 h at 37#{176}Cwith either I .5 nM dFdC as a

single agent or the combination of 1 .5 nM dFdC and 0.75 fLM

CDDP. For C26-lO cells, a higher drug concentration was

chosen, because for the concentrations used for the A2780 and

22B cell lines, no DNA damage could be detected. Three to S X

106 C26-l0 cells were incubated for 24 h with either 20 nM

dFdC as a single agent or the combination of 1 .5 nM dFdC and

0.75 �iM CDDP. Cells exposed to 50 p.M VP-16 (Etoposide) for

1 h were used as a positive control; cells not exposed to drugs

were used as a negative control.

After harvesting, using trypsmnization, cells were counted

and suspended in 0.25 M meso-inositol, 10 mr�i sodium phos-

phate, and I M MgCl, (pH 7.2) buffer (solution B; 1 .5-2.5 X 106

cells/mi). Every sample was distributed to three tubes (in dupli-

date, 0.2 mi/tube), in which the cells were lysed by 0.2 ml

solution of 9 M urea, 10 mrvi NaOH, 2.5 rn�i cyclohexanediami-

netetraacetate, and 0. 1% SDS (solution C). The cells were

exposed to an alkaline environment by adding 0. 1 ml solution D

(45% solution C and 55% 0.2 M NaOH) and 0.1 ml solution E

(40% solution C and 60% 0.2 M NaOH) gently to the tubes

without mixing and then kept on ice for 30 mm to achieve a

slowly increasing pH (final pH, 12.8). One of the three tubes

was protected against the alkaline environment by adding 0.4 ml

solution of I M glucose and 14 msi �3-mercaptoethanol (solution

F) before adding solutions D and E. In this tube, the DNA did

not unwind (100% dsDNA; tube T). One of the two other tubes

exposed to the alkaline environment was vortexed until no

dsDNA was left (0% dsDNA; tube B), whereas the third tube

represented the actual measurement (tube P). The cells were

incubated at 15#{176}C,allowing the DNA to unwind. For every cell

line, an optimal unwinding time had to be determined; for the

A2780 and 22B cells, an unwinding time of 60 mm was chosen,

and for the C26-lO cell line, the unwinding time was 30 mm.

The incubation was terminated by adding 0.4 ml solution F to

tubes P and B, which were mixed subsequently and chilled on

ice. The dsDNA was stained by adding I .5 ml solution of 6.7

p.g/ml ethidium bromide and 13.3 mtvi NaOH. Fluorescence was

measured on a SPEX F]uoromax fluorescence spectrometer

(Perkin Elmer/Cetus, Norwalk, CT; excitation, 520 nm; ana-

lyzer, 590 nm). The percentage of dsDNA was calculated by:

(P - B)/(T - B) X 100%.



Table I Summary of the sensitivity of the tested cells todFdC and CDDP

The cells were exposed continuously to the drugs for 1, 4, 24, and72 h, followed by culture in drug-free medium until 72 h after initialdrug addition. Values represent the mean IC50 ± SEM of three to eightexperiments


Cell line Exposure (h) dFdC (nM) CDDP (�i.M)

A2780 1

424

72

52.5 ± 8.4

12.4 ± 2.7

4.34 ± 2.16

2.16± 1.01

10.6 ± 2.84

2.86 ± 0.59

1.20 ± 0.48

1.96 ±0.77

ADDP 14

24

72

730 ± 213

239±117

193±43

625±154

460 ± 220

197±82

63±15

52±13

AG6000 14

24

72

>106

>106

>106

50.5 ± 20.2

54.2 ± 13.817.1 ± 4.5

4.70±0.88

4.51 ± 1.01

22B I

4

24

72

62.3 ± 12.5

14.0 ± 3.5

4.50 ± 1.21

0.86 ± 0.32

56.3 ± 15.6

14.7 ± 5.8

5.83 ± 0.52

4.10 ± 0.10

C26-l0 14

24

72

655 ± 141

254 ± 10924.7 ± 9.1

22.6 ± I 1 .6

23.8 ± 3.8

6.00 ± 1.082.95 ± 0.67

3.28 ± 0.74

RESULTS

Growth Inhibition Tests. The sensitivities of the fivecell lines to dFdC and CDDP, expressed as IC50 values, are

listed in Table 1 . Drug effects were easily detectable after 72 h

of drug exposure. The fast growing human ovarian cancer cell

line A2780 and the slow growing human head and neck squa-

mous cell carcinoma cell line 22B are the cell lines most

sensitive to dFdC. However, 22B cells were less sensitive to

CDDP than A2780 cells. As expected, ADDP was the least

sensitive cell line to CDDP, and AG6000 was the least sensitive

to dFdC. Furthermore, it seemed that AG6000 and ADDP cells

were also cross-resistant to both drugs, AG6000 about 4-fold to

CDDP and ADDP about 40-fold to dFdC (24-h exposure). In

most cell lines studied, the IC50 for 72-h exposure was less than

for shorter incubation times, consistent with the known cell

cycle specificity of dFdC. However, the IC50 value of ADDP

cells for dFdC is higher at 72 h than at 24 h of incubation. The

IC50 value of AG6000 cells for dFdC for the exposure times of

1 , 4, and 24 h was greater than the highest concentration used,

io� M. The murine colon cancer cell line C26-10 seemed to

have an intrinsic resistance to both drugs compared with A2780.

Fig. 2 shows representative relative growth inhibition

curves of ADDP and AG6000 cells when exposed to dFdC and

CDDP alone and to a combination of 1 :500 for 24 h. The curve

for dFdC in ADDP cells showed a rather flat pattern, in contrast

to CDDP; the combination curve, however, was shaped simi-

larly as that for CDDP, but with a shift to the lower concentra-

tion range. The AG6000 cells were exposed to the combination

of dFdC and CDDP simultaneously. dFdC did not affect the

growth of AG6000 cells. The growth inhibition curve of

AG6000 cells exposed to CDDP was similar to that of dFdC and

CDDP together, suggesting that the growth inhibition of the

combination is only due to CDDP exposure. At concentrations

of >30 ELM, CDDP induced cell killing. In a multiple drug effect

analysis, this effect is represented by a CI of about 1 . Fig. 3

shows representative CI/FA plots of A2780 and ADDP cells

exposed to dFdC and CDDP. In A2780 cells, the combination of

dFdC and CDDP was antagonistic (CI > 1) at exposure times of

1 and 4 h. The CIs varied over a range of fractions affected

studied; at a FA of 0.25, a high CI (CI = 5-10) was found, but

lower values (CI 2-4) were observed at a FA of 0.75 (Fig. 2).

Synergistic interaction (CI < 1) between dFdC and CDDP was

found in A2780 cells at an exposure time of 24 h. In the ADDP

cell line, CI/FA curves similar to those in A2780 cells were

found. However, in ADDP cells, synergism was observed at 4,

24, and 72 h. Also, analysis of the combination of dFdC and

CDDP with isobolograms was performed. Fig. 4 shows a rep-

resentative isobologram of ADDP cells exposed to dFdC and

CDDP for 24 h. The observed position of the combination point

on the left of the connecting line between the IC50 values of

dFdC and CDDP indicates synergism. For every experiment, the

isobologram analysis revealed the same dose-response interac-

tion as the method of Chou and Talalay (29).

Table 2 summarizes the CIs of the five cell lines exposed

to the simultaneous or sequential combination of dFdC and

CDDP. Only values at a FA of 0.5 are represented. When the

cells were exposed simultaneously to both drugs, very different

patterns were observed between the cell lines, with antagonism

in A2780 and ADDP cells but synergism for 22B cells at short

exposure times. At exposure durations of 4, 24, and 72 h,

however, the combination of dFdC and CDDP was not syner-

gistic in the 22B cell line, with increasing CIs at longer exposure

periods. In the C26-l0 cell line, interaction between the drugs

was modest, with only additive to slightly antagonistic effects.

In all three ovarian cancer cell lines, preincubation with

CDDP for 4 h followed by a dFdC incubation for 1, 4, 24, and

72 h resulted in synergism or additivity for all combinations.

This is in contrast to the simultaneous exposure schedule. Even

in the AG6000 cell line, synergism was observed. In the 22B

cell line, the pattern of the CIs per exposure time has changed

compared with the simultaneous exposure. For this schedule, the

1 - and 4-h exposure times were antagonistic, and the 24- and

72-h exposure times were synergistic. In C26-lO cells, all sched-

ules of sequential CDDP followed by dFdC were antagonistic.

In all cell lines for all exposure times, dFdC preincubation

for 4 h followed by exposure to the combination of dFdC and

CDDP was synergistic. Using this schedule, the lowest CIs were

found in the ADDP cell line. Again, in the dFdC-resistant

AG6000 cells, synergism was observed. Also, for the colon

cancer cell line C26-lO, for which no synergism was observed in

the other schedules, synergism was observed in the schedule of

preincubation with dFdC followed by schedule of the combination

of dFdC and CDDP. Surprisingly, in all cell lines, the 24-h expo-

sure time revealed a lower CI than the 72-h exposure time.

Effect of CDDP on dFdCTP Accumulation. To deter-

mine whether CDDP might influence the metabolism of dFdC,

we measured the accumulation of the main metabolite of dFdC,

dFdCTP. For this purpose, cells were exposed to 0. 1 p.M and

1 �LM dFdC as a single agent or in combination with 20 p.M and



A [dFdC](M)

I 20

I 00

10.6 10-s 10-a 10-s

BICDDPI(M)

[dFdCJ (M)

120

100

60

0

DI

a)

a)

� 0

-20

-40I 0�

100 �iM CDDP, respectively, for either 4 or 24 h. Results in four

cell lines are summarized in Fig. 5. In AG6000 cells, no

dFdCTP could be detected, consistent with its known dCK

deficiency (23); therefore, this cell line was not included in these

10-i 10.6 10� 10-

ICDDPJ (M)

studies. Despite the lower sensitivity of ADDP cells compared

with A2780 cells, this cell line showed at the 4-h exposure time

similar or greater accumulation of dFdCTP, both at 0.1 �.LM and

1 p.M dFdC. dFdCTP accumulation was similar in A2780 and


Fig. 2 Representative growthinhibition curves of the cell lines

ADDP (A) and AG6000 (B).Cells were exposed to dFdC (+),CDDP (A), and a combination ofboth simultaneously (#{149})for 24 h.After drug exposure, both cell

lines were cultured in fresh me-

dium. Total culture time was 72h. The lines had different sigmoi-dicity expressed as the m value,which was 1.315, 1.813, and1.646 for the ADDP cells, respec-tively, and 0, 1.084, and 1.080 forthe AG6000 cell line, respec-tively.

80

-

0

DI

4) 40

a)

80



A

10

I+ .+ + + + ..+ ..+. ‘-I- +#{149}#{149}+ #{149}+ #{149}+#{149}+. .+ +. .+ .+ +� .+

-V

-,--,�-.‘I--’.--v

��0

,� 1 I I

BFA

(U)

I 000

I 00

�3 10

1

0.1

V

+ -

. + + . + .+. .

...--.-.-.-.-_.--.-.-.-.--.--.--.-.-e-.--.-.

FA

1�00


0�1

0.00 0.25

0.00 0.25

0.50 0.75

050 0.75

Fig. 3 Synergy analysis of the

I 00 interaction between CDDP anddFdC in the cell lines A2780 (A)

and ADDP (B). Cells were ex-posed to dFdC and CDDP simul-taneously for 1 h (A), 4 h (#{149}),24

h (V), and 72 hr ( + ). The valuesof the CIs are: CI > 1, antago-nism; CI = I, additivity; and CI

< 1, synergism. Values are rep-

resentative of three to five inde-

pendent experiments.

22B cells, but in the murine colon carcinoma cell line C26-lO,

dFdCTP accumulation at 0. 1 ji.M dFdC was less. Also, exposure

to I �.LM dFdC resulted in similar accumulation of dFdCTP in

A2780 and 22B cells. In contrast, at the 24-h incubation time,

the A2780 cell line showed the highest accumulation of dFdCTP

both at 0.1 and 1 p.M dFdC, whereas in ADDP cells, the lowest

accumulation was found. At 0. 1 pM dFdC, the accumulation of

dFdCTP was undetectable in C26-lO cells.



15

10

C-)

U-0

5

00 100 200 300 400 500

CDDP (pM)


Fig. 4 Isobologram analysis of exposure of ADDP cells to the com-bination of dFdC and CDDP for 24 h. 1C5() values of the drugs as single

agents are plotted on the axes; when connected by a solid line, this

represents an additive effect in case the interaction would be mutuallyexclusive; when connected by a broken line, this represents an additiveeffect when the interaction would be mutually nonexclusive. The point

representing the concentrations of dFdC and CDDP resulting in a 50%growth inhibition of the combination is plotted on the left of both

connecting lines, indicating synergism.

At an exposure time of 4 h, CDDP did not increase the

dFdCTP accumulation in any of the five cell lines, neither at 0.1

�LM dFdC in combination with 20 pM CDDP nor at 1 p.M dFdC

in combination with 100 1JM CDDP. However, C26-10 cells

accumulated less dFdCTP in the presence of CDDP at 1 �LM

dFdC in combination with 100 (LM CDDP compared with dFdC

alone. Also, at an exposure time of 24 h, CDDP did not increase

accumulation of dFdCTP in C26-l0 cells. Under these condi-

tions, not only in C26-lO cells but also in A2780 cells, CDDP

decreased dFICTP accumulation. In A2780 and 22B cells,

dFdCTP concentrations could not be measured at the 24-h

incubation time due to the toxicity of the combination causing

cell death. In the more resistant ADDP and C26-lO cells,

dFdCTP accumulation could be measured at the higher concen-

trations also, but in ADDP cells, no effect was observed,

whereas in C26-lO cells, lower dFdCTP accumulation was

found.

DNA Damage. Another interaction between CDDP and

dFdC could be due to drug-induced DNA damage. This possi-

bility was evaluated with a DNA unwinding assay. Table 3

shows the relative amounts of dsDNA in A2780, 22B, and

C26-l0 cells after a 24-h exposure to 1.5 flM dFdC, 0.75 pM

CDDP, and a combination of both. Under these conditions,

dFdC alone did not cause DNA strand breaks in the A2780 and

C26-lO cells, but 20% DNA strand breaks were found in 22B

cells. After CDDP exposure, 15-25% DNA strand break forma-

tion was found in all three cell lines. However, the combination

of dFdC and CDDP did not result in an increase in DNA strand

break formation, because the effect of the combination was less

than could be expected based on an additive interaction.

Table 2 Summary of the CIs of the combination of dFdC and CDDP

Values represent CIs (non-mutually exclusive case) calculated at

the fraction affected of 0.5 of the combination of dFdC and CDDP andare means of three to five experiments; SEs were less than 30%. Cellswere exposed to the combination either simultaneously or preincubated

with CDDP for 4 h followed by a 1-, 4-, 24-, or 72-h dFdC incubation

or incubated with dFdC for 4 h followed by a combination of dFdC andCDDP for 20 or 68 h. The ratio dFdC:CDDP was 1 :500. CI > I

indicates antagonism; CI - 1 is additive; and CI < 1 is synergism.

Drug Incubationadministration (h) A2780 ADDP AG6000 22B C26-lO

CDDP and dFdC I 5.04 1 18.93 0.96 0.41 2.40

simultaneously 4 6.37 0.27 0.98 1 .33 2.5224 0.21 0.69 1.45 3.03 0.9972 1.15 0.57 1.81 13.35 1.92

CDDP 4 h I 0.06 0.59 0.86 153.24 6.50before dFdC 4 0.30 0.01 0.39 1.04 5.18

24 0.23 0.53 0.98 0.58 2.75

72 0.02 0.26 0.43 0.48 1.50

dFdC 4 h 24 0.28 0.04 0.06 0.36 0.17before dFdC 72 0.68 0.05 0.41 0.46 0.47

and CDDP

DISCUSSION

In this study, we show a definite synergism between dFdC

and CDDP on growth inhibition in a panel of five solid tumor

cell lines. The synergy is dependent on exposure duration and

sequence ofthe drugs. Exposure to the combination ofdFdC and

CDDP simultaneously was synergistic in A2780 and ADDP

cells. Preincubation with CDDP followed by dFdC was syner-

gistic in all cell lines except C26- 10 cells. Incubation with dFdC

followed 4 h later by the combination was synergistic in all cell

lines.

Mechanistic studies concentrated on dFdC metabolism,

with emphasis on accumulation of the active metabolite

dFdCTP. No consistent pattern of the effect of CDDP on

dFdCTP accumulation was observed. It is remarkable that in the

dFdC-insensitive ADDP cells, dFdCTP accumulation was corn-

parable to that of the parent cell line A2780. In contrast to other

cell lines, ADDP cells accumulated less dFdCTP with 24-h

exposure to the drug than with 4-h exposure. This early satura-

tion may explain the lower IC50 value at 4 and 24 h than at 72

h in ADDP cells. CDDP at the 4-h exposure time decreased

dFdCTP accumulation slightly in the ADDP cell line, whereas

the combination of dFdC and CDDP on growth inhibition was

synergistic. However, in C26-lO cells, CDDP decreased

dFdCTP accumulation considerably at both the 4- and 24-h

exposure times. The explanations for these phenomena are un-

clear, but possibly, CDDP inhibits dFdC uptake into cells and,

eventually, subsequent phosphorylation. Also, it was found pre-

viously that in a combination of DAC and CDDP, CDDP

decreased the cellular uptake of DAC ( 19, 35). We plan to

explore this hypothesis further in future studies.

To investigate the mechanism of interaction further, we

studied drug-induced DNA damage. In previous studies by

others, CDDP did not affect the levels of incorporated DAC into

DNA (35). Although CDDP did not inhibit the incorporation of

ara-C into normally replicating DNA, CDDP enhanced the



A2780 ADDP 22B C26-10

B


500

a,

� 4000

(0

Lu�. 300I-C-).5

IL

.5 2000E0�

100

0

1000

8000,

a)0

*0

� 600a-I-.

I 400200

0

A

A2780 ADDP 22B C26-10Fig. 5 Effect of CDDP on the accumulation of dFdCTP in A2780,ADDP, 22B, and C26-lO cells after 4-h (A) and 24-h (B) exposures to

dFdC with or without CDDP. Values are means ± SEM of three to fiveexperiments. I, 0. 1 �M dFdC; PA, 0. 1 p.M dFdC and 20 �i.M CDDP; �,I �LM dFdC; 0, 1 Ii.M dFdC and 100 �LM CDDP.

incorporation of ara-C into DNA undergoing repair synthesis

(36). In our study, dFdC did not cause DNA damage in A2780

and C26-l0 cells, whereas CDDP induced some damage, and

only in the 22B cell line did the combination dFdC and CDDP

cause considerable DNA damage after exposure to either drug

alone. The lack of an effect of dFdC in causing DNA damage

might be related to the observation that dFdCTP incorporation

Table 3 Effect of dFdC and CDDP on cellular dsDNA

The effect of 1-h exposure to 50 p.M VP-l6 was always included asa positive control for the assay, because VP- 16 is known to produce ds

breaks.DNA damage was evaluated as percentage of dsDNA. Values are

means ± SE of percentages of dsDNA in treated compared with that inuntreated cells, which was set at 100% in each experiment. The actual

amounts of dsDNA of cells not exposed to drugs were: A2780, 86.0 ±

5.2; 22B, 88.1 ± 7.0; and C26-lO, 73.0 ± 3.3% of fluoresence of

samples not exposed to an alkaline environment.Exposure was 24 h to dFdC (I .5 nM) and CDDP (0.75 pM) as single

agents and a combination of both agents (1 .5 n�i + 0.75 p.M). Theunwinding time in the FADU assay was 60 mm for A2780 and 22B, 30

mm for C26-I0.

Drug A2780 22B C26-lO”

dFdC 102 ± 6.7 79.2 ± 4.6 97.5 ± 6.6CDDP 89.3 ± 10.4 76.2 ± 6.7 85.8 ± 3.8

dFdC+CDDP 103 ± 8.2 83.5 ± 1 1.2 87.4 ± 6.5

VP-16 25.7 ± 15.4 32.3 ± 5.7 52.1 ± 27.3

a dFdC 20 flM and CDDP 2 �J.Mas single agents and in combination.

into DNA may inhibit exonuclease activity, thereby preventing

excision of misincorporated dFdCMP (9). Interestingly, in all

cell lines, the two drugs did not cause any obvious additive

DNA damage. Actually, in the combination, DNA damage was

always less than that caused by CDDP alone, and in A2780 and

22B cells, damage was also less than that caused by dFdC alone.

Not only might this be related to inhibition of dFdC-induced

DNA damage, but dFdC incorporated into DNA also might

result in inhibition of the repair of CDDP-DNA adducts, causing

apparent stabilization of DNA. Thus, the synergistic interaction

between dFdC and CDDP might be related to reduced DNA

repair. Moreover, dFdCTP inhibits the excision repair of intra-

strand and possibly interstrand cross-links of CDDP, which may

result in synergy (37). These mechanisms might clarify why no

increased DNA damage as a result of the combination of dFdC

and CDDP was found in the FADU assay, although these events

may enhance cytotoxicity. Further evidence that DNA repair

might play a role in the interaction of cytidine analogues with

CDDP was found in studies concentrating on the role of caffeine

(38-40). This methylated xanthine is capable of sensitizing

tumor cells to cytotoxic agents, probably by inhibiting DNA

repair. In a nude mouse model with human pancreatic adeno-

carcinoma, the combination of ara-C and CDDP did not enhance

the therapeutic effect. The addition of caffeine, however, to the

combination of ara-C and CDDP resulted in an early tumor

response followed by complete tumor regression. It is of interest

that this action of caffeine was seen only in the triple combina-

tion ara-C, CDDP, and caffeine, but not when caffeine was

combined only with either ara-C or CDDP. Probably, the addi-

tion of methylated xanthines can convert the ara-C-CDDP in-

teraction to synergism, due to inhibition of DNA repair by

caffeine. In contrast to dFdC, ara-C incorporation into DNA

does not lead to inhibition of DNA repair (41). Therefore, it is

likely that the observed interactions between dFdC and CDDP

are due to reduced DNA repair, in a manner similar to the

combination of ara-C, CDDP, and caffeine. The observation that

the sequential exposure of dFdC followed by CDDP was syn-

ergistic in all cell lines supports this hypothesis. Pretreatment of




cells with dFdC will allow sufficient dFdCTP accumulation and

incorporation into DNA, resulting in decreased repair when

CDDP-DNA adducts are formed.

Other studies of the mechanism of synergistic interaction in

the combination of cytidine analogues, including DAC, and

ara-C, and CDDP, suggest that the synergy is due to the incor-

poration of DAC into the DNA. DNA hypomethylated by DAC

bound greater amounts of CDDP than normally methylated

DNA (19). Hypomethylation could produce conformational

changes in DNA, which make potential sites of CDDP adduct

formation more accessible. Like dFdCTP, ara-CTP does not

hypomethylate DNA. It was found that ara-C had no effect on

the binding of CDDP to DNA, the induction of CDDP-induced

interstrand DNA cross-links, the excision of CDDP-induced

interstrand DNA cross-links, or the excision of total platinum

from DNA. However, ara-C delayed the recovery of DNA

synthesis inhibited by CDDP markedly (20). Similar mecha-

nisms may operate in the combination of dFdC and CDDP. It

seems likely that one site of interaction between dFdC and

CDDP is at the DNA level, because in AG6000 cells, which did

not accumulate dFdCTP, no synergism is observed with simul-

taneous exposure. The sequential effects of the combination, not

only in this cell line, but also in the other cell lines, suggest an

additional type of interaction between dFdC and CDDP not

necessarily requiring dFdC phosphorylation. The synergistic

effect of both sequences suggests not only an effect of dFdC (or

its metabolites) on CDDP uptake and intracellular distribution,

but also an effect of CDDP on dFdC metabolism. One such

interaction may be at the level of regulation of dFdC metabo-

lism. CDDP is known to inhibit ribonucleotide reductase (21),

which may lead to a decrease of dCTP and an imbalance in other

deoxyribonucleotides. This cannot only influence dFdC metab-

olism; it also affects DNA repair. Furthermore, differences in

enzyme parameters (Vmax and Km for ribonucleotide reductase

and DNA repair enzymes) in different cell lines might clarify

the difference in the interaction between dFdC and CDDP in the

cell lines (42). It remains to be investigated whether dFdCTP

affects CDDP, including adduct formation, or whether CDDP

affects dFdC. Both types of interaction may occur.

The combination of dFdC and CDDP is synergistic in vitro.

This synergism is time and schedule dependent. The most ef-

fective sequence is 4-h dFdC preincubation followed by the

combination of dFdC and CDDP, which was synergistic in all

cell lines. This indicates an enhanced effect of dFdC on CDDP

due to this preincubation. This sequential synergistic interaction,

in addition to nonoveriapping toxic side effects, is of interest for

future clinical studies, especially in tumor types such as non-

small cell lung cancer, ovarian cancer, and head and neck

cancer, in which both drugs have shown single agent activity.

ACKNOWLEDGMENTS

We thank Dr. S. Ackland (University of Newcastle, Australia) for

critical reading of the manuscript.

REFERENCES

1. Hertel, L. W., Boder, 0. B., Kroin, J. S., Rinzel, S. M., Poore, G. A.,Todd, G. C., and Grindey, G. B. Evaluation of the antitumor activity ofgemcitabine (2’,2’-difluoro-2’-deoxycytidine). Cancer Res., 50.- 4417-

4422, 1990.

2. Braakhuis, B. J. M., van Dongen, G. A. M. S., Vermorken, J. B., andSnow, G. B. Preclinical in vivo activity of 2’,2’-difluorodeoxycytidine

(gemcitabine) against head and neck cancer. Cancer Res., 51: 21 1-2 14,

1991.

3. Boven, E., Schipper, H., Erkelens, C. A. M., Hatty, S. A., andPinedo, H. M. The influence of the schedule and the dose of gemcitabineon the anti-tumour efficacy in experimental human cancer. Br. J. Can-cer, 68: 52-56, 1993.

4. Kaye, S. B. Gemcitabine: current status of Phase I and II trials. J.Clin. Oncol., 12: 1527-1531, 1994.

5. Abratt, R. P., Rezwoda, W., Faikson, G., Goedhals, L., and Hacking,D. Efficacy and safety profile of gemcitabine in non-small cell lungcancer. Phase II study. J. Clin. Oncol., 12: 1535-1540, 1994.

6. Lund, B., Hansen, 0. P., Theilade, K., Hansen, M., and Neijt, J. P.Phase II study of gemcitabine (2’,2’-difluorodeoxycytidine) in previ-ously treated ovarian cancer patients. J. Nail Cancer Inst.. 86: 1530-

1533, 1994.

7. Heinemann, V., Hertel, L. W., Grindey, G. B., and Plunkett, W.Comparison of the cellular pharmacokinetics and toxicity of 2’ .2’-

difluorodeoxycytidine and 1 -�3-D-arabinofuranosylcytosine. Cancer

Res., 48: 4024-4031, 1988.

8. Ruiz van Haperen, V. W. T., Veerman, G.. Vermorken, J. B., andPeters, 0. J. 2’,2’-difluoro-deoxycytidine (gemcitabine) incorporationinto RNA and DNA from tumour cell lines. Biochem. Pharmacol., 46:

762-766, 1993.

9. Huang, P., Chubb, S., Hertel, L. W., Grindey, G. B., and Plunkett, W.Action of 2’ ,2’-difluorodeoxycytidine on DNA synthesis. Cancer Res.,51: 6110-6117, 1991.

10. Muggia, F. Introduction: cisplatin update. Semin. Oncol., 18 (Suppl.3): 1-4, 1991.

I I . Roberts, J. J., Thomson, A. J. The mechanism of action of antitu-mour platinum compounds. Prog. Nucleic Acid Res. Mol. Biol., 22:

71-133, 1979.

12. Fichtinger-Schepman, A. M. J.. Van der Veer, J. L.. Den Hartog,J. H. J., Lohman, P. H. M., and Reedijk J. Adducts of the antitumor drugcis-diamminedichloroplatinum(II) with DNA: formation, identificationand quantitation. Biochemistry, 24: 707-713, 1985.

13. Fichtinger-Schepman, A. M. J. Variability in DNA-adduct levelsafter in vivo treatment with the antitumor drug cisplatin. J. Inorg.

Biochem., 39: 154-58, 1989.

14. Terheggen. P. M. A. B., Emondt, J. Y., Hoot, B. G. J., Dijkman. R.,Schrier, P. I., Den Engelse, L., and Begg A. C. Correlation between cellkilling by cis-diamminedichloroplatinum(II) in six mammalian cell lines

and binding of a cis-diamminedichloroplatinum(II)-DNA antiserum.

Cancer Res., 50: 3556-3561, 1990.

15. Terheggen, P. M. A. B., Begg, A. C., Emondt, J. Y., Dubbelman, R.,

Floot, B. G. J., and den Engelse L. Formation of interaction products ofcarboplatin with DNA in vitro and in cancer patients. Br. J. Cancer, 63:

195-200, 1991.

16. Parker, R. J., Gill, I., Tarone, R., Vionnet, J., Grunberg, S., Muggia,F., and Reed E. Platinum DNA-damage in leucocyte DNA of patients

receiving cisplatin and carboplatin chemotherapy, measured by atomic

absorption spectrometry. Carcinogenesis (Lond.), 12: 1253-1258, 1991.

17. Reed, E., Ozois, R. F., Tarone, R., Yuspa, S. H., and Poirier, M. C.Platinum DNA-damage in leucocyte DNA correlate with disease re-sponse in ovarian cancer patients receiving platinum-based chemother-

apy. Proc. NatI. Acad. Sci. USA, 84: 5024-5028, 1987.

18. Brabec, V., Kleinwachter, V., Butour, J. L., and Johnson, N. P.Biophysical studies of the modification of DNA by antitumour platinum

coordination complexes. Biophys. Chem., 35: 129-141, 1990.

19. Abbruzzese, J. L., and Frost, P. Studies on the mechanism of thesynergistic interaction between 2’-deoxy-5-azacytidine and CDDP.

Cancer Chemother. Pharmacol., 30: 31-36, 1982.

20. Fram, R. J., Robichaud, N., Bishov, S. D., and Wilson, J. M.Interactions of cis-diamminedichloroplatinum(II) with I 43-D-arabino-

furanosylcytosine in LoVo colon carcinoma cells. Cancer Res., 47:

3360-3365, 1987.




21. Smith, S. L., and Douglas, K. T. Stereoselective, strong inhibitionof ribonucleotide reductase from E. Coli by cisplatin. Biochem. Bio-

phys. Res. Commun., 162: 715-723, 1989.

22. Lu, Y., Han, J., and Scanlon, K. J. Biochemical and molecular

properties of cisplatin-resistant A2780 cells grown in folinic acid. J.Biol. Chem., 263: 4891-4894, 1988.

23. Ruiz van Haperen. V. W. T., Veerman, 0., Eriksson, S., Boven, E.,Stegmann, A. P. A., Hermsen, M., Vermorken, J. B., Pinedo, H. M., andPeters, G. J. Development and characterization of a 2’,2’-difluorode-oxycytidine-resistant variant of the human ovarian cancer cell lineA2780. Cancer Res., 54: 4138-4143, 1994.

24. Carey, T. E. Establishment of epidermoid carcinoma cell lines. In:

R. E. Wittes (ed.) Head and Neck Cancer, pp. 287-314. New York: JohnWiley & Sons, 1985.

25. Peters, G. J., Kraal, I., and Pinedo, H. M. In vitro and in vivo studies

on the combination of brequinar sodium (DUP 785, NSC 368390) with5-fluorouracil, effects of uridine. Br. J. Cancer, 65: 229-233, 1992.

26. Skehan, P., Storeng. R., Scudiero, D., Monks, A., McMahon, J.,

Vistica, D., Warren, J. T., Bokesch, H., Kenney, S., and Boyd, M. R.

New colorimetric cytotoxicity assay for anticancer drug screening. J.NatI. Cancer Inst., 82: 1 107-1 1 12, 1990.

27. Keepers, Y. P., Pizao, P. E., Peters, G. J., Van Ark-Otte, J.,

Winograd, B., and Pinedo, H. M. Comparison of the sulforhodamine B

protein and tetrazolium (MTT) assays for in vitro chemosensitivity

testing. Eur. J. Cancer, 27: 897-900, 1991.

28. Peters, 0. J., Wets, M., Keepers, Y. P. A. M., Oskam, R., VanArk-Otte, J.. Noordhuis, P., Smid, K., and Pinedo, H. M. Transformationof mouse fibroblasts with the oncogenes H-ras or trk is associated withpronounced changes in drug sensitivity and metabolism. Int. J. Cancer,

54: 450-455, 1993

29. Chou, T. C., Talalay P. Quantitative analysis of dose-effect rela-tionship: the combined effects of multiple drugs on enzyme inhibitors.

In: G. Weber (ed.) Advances in Enzyme Regulation, pp. 27-55. NewYork: Pergamon Press, 1983.

30. Greco, W. R., Bravo, G., and Parsons, J. C. The search for synergy:a critical review from a response surface perspective. Pharmacol. Rev.,

47: 331-385, 1995.

31. Steel, G. G., and Peckham, M. J. Exploitable mechanisms in com-

bined radiotherapy-chemotherapy: the concept of additivity. Int. J. Ra-

diat. Oncol. Biol. Phys., 5: 85-91, 1979.

32. Ruiz van Haperen, V. W. T., Veerman, 0., Boven, E., Noordhuis,P., Vermorken, J. B., and Peters 0. J. Schedule dependence of sensi-

tivity to 2’,2’-difluorodeoxycytidine (gemcitabine) in relation to accu-

mulation and retention of its triphosphate in solid tumor cell lines andsolid tumors. Biochem. Pharmacol., 48: 1327-1339, 1994.

33. Birnboim, H. C., and Jevcak, J. J. Fluorometric method for rapid

detection of DNA strand breaks in human white blood cells produced by

low doses of radiation. Cancer Res., 41: 1889-1892, 1981.

34. Meijer, C., Mulder, N. H., Timmer-Bosscha, H., Zijlstra, J. G., andDc Vries, E. G. E. Role of free radicals in an adriamycin-resistant

human small cell lung cancer cell line. Cancer Res., 47: 4613-4617,

1987.

35. Ellerhorst, J. A., Frost, P., Abbruzzese, J. L., Newman, R. A., andChernajovsky, Y. 2’-deoxy-5-azacytidine increases binding of cisplatin

to DNA by a mechanism independent of DNA hypomethylation. Br. J.

Cancer, 67: 209-215, 1993.

36. Greco, W. R., Hyoung, S. P., and Rustum, Y. M. Application to anew approach for the quantitation of drug synergism to the combination

of cis-diamminedichloroplatinum and 1 -�3-D-arabinofuranosylcytosine.Cancer Res., 50: 5318-5327, 1990.

37. Yang, L. Y., Li, L., Liu, X. M., Keating, M. J., and Plunkett, W.Gemcitabine suppresses the repair of cisplatin adducts in plasmid DNAby extracts of cisplatin-resistant human colon carcinoma cells. Proc.Am. Assoc. Cancer Res., 36: 357, 1995.

38. Bergerat, J. P., Drewinko, B., Cony, P., Barlogie, B., and Ho, D. H.Synergistic lethal effect of cis-diamminedichioroplatinum and l-�3-D-

arabino furanosyl cytosine. Cancer Res., 41: 25-30, 1981.

39. Waldren, C. A., and Rasko, I. Caffeine enhancement of X-raykilling in cultured human and rodent cells. Radiat Res., 73: 95-1 10,1987.

40. Byfield, J. E. Mice, men, mustards and methylated xanthines: thepotential role of caffeine and related drugs in the sensitization of human

tumours to alkylating agents. Br. J. Cancer., 43: 667-683, 1981.

41. Ross, D. D., and Cuddy, D. P. Molecular effect of 2’,2’-difluoro-

deoxycytidine (Gemcitabine) on DNA replication in intact HL-60 cells.Biochem. Pharmacol., 48: 1619-1630, 1994.

42. Jackson, R. C. Amphibolic drug combinations: the design of selec-tive antimetabolite protocols based upon the kinetic properties of mul-

tienzyme systems. Cancer Res., 53: 3998-4003, 1993.



1996;2:521-530. Clin Cancer Res A M Bergman, V W Ruiz van Haperen, G Veerman, et al. vitro.Synergistic interaction between cisplatin and gemcitabine in

Updated version

http://clincancerres.aacrjournals.org/content/2/3/521

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/2/3/521To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



Synergistic Interaction between Cisplatin and Gemcitabine in Vitro1 · ADDP, 12,000 cells/well;...

Documents

Transcript of Synergistic Interaction between Cisplatin and Gemcitabine in Vitro1 · ADDP, 12,000 cells/well;...