Combination effects of alkylphosphocholines and gemcitabine inmalignant and normal hematopoietic cells

Milka C. Georgievaa,b, Spiro M. Konstantinova,c,Margarita Topashka-Anchevad, Martin R. Bergera,*

aUnit of Toxicology and Chemotherapy, German Cancer Research Center, 280 Im Neuenheimer Feld, 69120 Heidelberg, GermanybDepartment of Oncogenesis, National Oncology Center, 6 Plovdivsko pole Street, 1156 Sofia, Bulgaria

cLaboratory for Experimental Chemotherapy, Department of Pharmacology, Faculty of Pharmacy, Medical University of Sofia,

2 Dunav Street, 1000 Sofia, BulgariadInstitute of Zoology, Bulgarian Academy of Sciences, 1 Tsar Osvoboditel Boulevard, 1000 Sofia, Bulgaria

Received 17 August 2001; received in revised form 5 February 2002; accepted 7 February 2002

Abstract

Cytosine arabinoside (ara-C) and 2 0,2 0-difluorodeoxycytidine (Gem) were compared in leukemia cells, with Gem being more

potent than ara-C. Gem was combined with hexadecylphosphocholine (HPC) or erucylphospho-N,N,N-trimethylpropanolamine

(ErPC3) in resistant CML cells. Supra-additive effects were seen in K-562 cells after concomitant and sequential exposure of

Gem followed by HPC. The reverse sequence resulted in antagonism. Both effects were more significant when HPC was

exchanged for ErPC3. Gem or HPC failed to induce DNA laddering in K-562 cells, but apoptotic signals were transferred by the

Gem-exposed SKW-3 cytosolic fraction to K-562 nuclei. HPC did not increase the clastogenicity of Gem and counteracted its

mitotic inhibition in murine bone marrow. Thus, the combination of Gem and an alkylphosphocholine is advantageous in terms

of their complementary mode of action, resulting in increased cytotoxicity and lowered myelotoxicity. q 2002 Elsevier

Science Ireland Ltd. All rights reserved.

Keywords: Gemcitabine; Alkylphosphocholine; Combination effect; Cell-free apoptosis system; CML cell

1. Introduction

Alkylphosphocholines are membrane seeking anti-

neoplastic drugs [1]. They are bone marrow-stimulat-

ing agents with pronounced anti-leukemic activity and

exert their influence by modulating signal transduction

pathways [2,3]. Structural variations have shown that

many of their properties are correlated with the length

of their alkyl chain and presence of a double bond. For

example, the hemolytic and micellar activities are

strongly decreased in compounds with an unsaturated

alkyl chain longer than 20 carbon atoms. The proto-

types of this i.v. injectable subgroup are erucylpho-

sphocholine [4] and its congener erucylphospho-

N,N,N-trimethylpropanolamine (ErPC3). The only

alkylphosphocholine which has been registered for

clinical purposes is hexadecylphosphocholine (milte-

fosine, HPC), which is used to control the growth of

breast cancer skin metastases following topical admin-

istration and recently has been found to eradicate leish-

maniasis following oral application [5–7]. Although

alkylphosphocholines exert high anti-leukemic activ-

ity in various types of leukemia they are distinctly less

effective against BCR-ABL-positive leukemia cell

Cancer Letters 182 (2002) 163–174

0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0304-3835(02)00088-5

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* Corresponding author. Tel.: 149-6221-423310; fax: 149-6221-

423313.

E-mail address: m.berger@dkfz-heidelberg.de (M.R. Berger).

lines [3,8–10]. However, a suitable combination with

other cytostatics could help to overcome this draw-

back. Combined with cytosine arabinoside (ara-C), a

synergistic enhancement of antileukemic activity has

been described in HL-60 and Raji cells [11]. ara-C

belongs to the nucleoside anti-metabolites that are

active only after uptake into the cell and phosphoryla-

tion to nucleotide forms (di- and triphosphates) [12].

Within the subgroup of clinically active pyrimidine

analogues, 2 0,2 0-difluorodeoxycytidine (gemcitabine,

Gem) differs from ara-C by the presence of two fluorine

atoms at the 2 0 position of the sugar ring and the

pentose type, i.e. deoxyribose instead of arabinose.

Metabolic activation of Gem proceeds through the

same pathway as for ara-C. In addition, Gem possesses

a unique self-potentiating mechanism of action: its

diphosphate inhibits ribonucleotide reductase, while

incorporation of the triphosphate into DNA leads to

masked termination of DNA chain elongation since

the altered base sequence cannot be efficiently repaired

[13–18]. Various cytostatic combinations with Gem

have been investigated for synergistic anti-tumor

activity [19,20] but since their dose-limiting toxicity

is myelosuppression and thrombocytopenia [12,21],

this side effect is expected to occur more frequently

following the use of Gem with classical cytostatics

such as cisplatin, doxorubicin, or taxanes. Our hypoth-

esis was that the combination of Gem with an alkylpho-

sphocholine would diminish the myelosuppression

caused by Gem and show at least an additive antineo-

plastic effect. We used a panel of cell lines that

included both sensitive (HL-60, SKW-3) and resistant

BCR-ABL-expressing (LAMA-84, AR-230, K-562)

leukemia cell lines for assessing the anti-leukemic

effects. Gem was combined with HPC and the newly

developed ErPC3. In order to determine the resulting

hematological toxicity in vivo, sequential treatment

schedules of Gem and HPC were investigated for

induction of chromosomal aberrations in normal

mouse bone marrow cells.

2. Materials and methods

2.1. Drugs

Gem was provided by Eli Lilly (Bad Homburg,

Germany) at a quality for clinical application. Stock

solutions were prepared immediately before use. Only

freshly prepared stock solutions were used. ara-C,

provided by Mack Co. (Illertissen, Germany), was

of the same quality and working dilutions were

prepared before use. HPC and ErPC3 were synthesized

by Professor H. Eibl (MPI for Biophysical Chemistry,

Gottingen, Germany), dissolved in ethanol/phos-

phate-buffered saline (PBS) (1:1, v/v) and stored at

4 8C.

2.2. Cell lines and culture conditions

The following cell lines of the DKFZ tumor bank

were used: acute myeloid leukemia HL-60 cells, T-

cell CLL-derived SKW-3 cells and the BCR-ABL

expressing CML-derived lines LAMA-84, AR-230

and K-562. All lines were passaged and kept in log

phase as previously described [9]. Briefly, cells were

grown as suspension cultures (RPMI-1640 medium

supplemented with 10% fetal bovine serum) at 37

8C in an incubator with humidified atmosphere and

5% CO2. Cells were passaged two or three times a

week.

2.3. MTT assay for cell survival

Cells were seeded in 96-well or 24-well plates (flat

bottom, Becton Dickinson, Heidelberg, Germany) or

tissue culture flasks (25 cm2, Integra Biosciences,

Fernwald, Germany) at a density of 1 £ 105 cells/ml

(100 ml/well for 96-well plates; 1.6 ml/well for 24-

well plates; 5.4 ml for flasks) and exposed to various

concentrations of the compounds for time periods as

indicated below. The cell survival fraction was deter-

mined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide) dye-reduction assay as

described by Mosmann [22], with some modifications

[9]. In brief, after the incubation period with the test

compounds, MTT-solution (10 mg/ml in PBS) was

added (10 ml/well). Plates were further incubated for

4 h at 37 8C and the formazan crystals formed were

dissolved by adding 110 ml/well acidified 2-propanol

(0.04 N HCl). Absorption was measured by an auto-

mated microtiter plate spectrophotometer (Anthos,

2001) at 540 nm, reference filter 690 nm. For each

concentration at least eight wells were used. Complete

medium (100 ml), MTT stock (10 ml) and 0.04 N HCl

in 2-propanol (110 ml) was used as blank solution.

M.C. Georgieva et al. / Cancer Letters 182 (2002) 163–174164

2.4. DNA extraction and gel electrophoresis

DNA was extracted from the cytosolic fraction as

described previously [10]. About 5 £ 106 treated or

untreated cells were washed in PBS and lysed in

buffer (0.3% Triton X-100, 20 mM Tris–HCl, 1 mM

EDTA, pH 7.4). Lysates were centrifuged at 13 000 £

g for 20 min. Supernatants were kept and processed by

adding 0.187 ml 6 M NaCl solution and 0.937 ml 2-

propanol. Probes were incubated at 220 8C overnight,

thereafter centrifuged at 13 000 £ g for 20 min and the

DNA pellets were washed with 70% ethanol, air dried,

re-dissolved in distilled water and analyzed by elec-

trophoresis in a 0.8% agarose gel.

2.5. Nuclear staining with Hoechst 33342

After treatment, cells were washed in PBS, stained

with 5 mM Hoechst 33342 solution and spun on slides

as previously described [23]. Until analysis with a

fluorescence microscope, slides were kept in the

dark at 4 8C.

2.6. Flow cytometry

HL-60 cells were exposed to 5 mM Gem for 24 h

and then analyzed by FACS in comparison with

untreated cells according to a previously described

protocol [3]. Briefly, cells were washed in PBS,

centrifuged and re-suspended in 1 ml solution of 50

mg/ml propidium iodide in 0.1% Triton X-100 and

0.1% sodium citrate. Probes were incubated at 4 8C

for 1 h just prior to flow cytometric analysis with a

Coulter Counter flow cytometer. Red fluorescence

staining was evaluated using a 4-decade linear scale

for fluorescence detection. Ten thousand cells from

each sample were analyzed, and arithmetic histogram

plots were performed.

2.7. Cell-free system for apoptosis

The system is based on the visualization of the

ongoing DNA fragmentation in nuclei from untreated

cells that are incubated with the cytosolic fraction

from induced cells. The experimental procedure has

been described previously [10]. Briefly, treated and

untreated cells were lysed in buffer (1 mM KH2PO4,

150 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 0.1 mM

4-(aminoethyl)-benzenesulphonyl fluoride hydro-

chloride (AEBSF), 0.15 units/ml aprotinin, 1 mM

Na3VO4, 5 mM HEPES, 10% glycerol, 0.3% Triton

X-100, pH 7.4). Lysates were spun for 10 min at

10 000 rev./min. Supernatants were used as cytosolic

fraction. Nuclear pellets were additionally washed

with the same buffer, but without Triton X-100. Incu-

bation with the untreated nuclei was started immedi-

ately under continuous shaking at 37 8C. Following

intervals of 20 min, aliquots from the nuclear pellets

were taken, centrifuged to sediment the nuclei and

processed for DNA isolation using spin columns

(QIAmp DNA Blood mini, Qiagen, Hilden,

Germany). DNA from the cytosolic and nuclear frac-

tion was analyzed by gel electrophoresis as described

above.

2.8. Cytogenetic investigation on bone marrow cells

in vivo

The dosages of Gem and ara-C were chosen from

clinically used dose levels which translated to doses

below the maximum tolerated dose in rodents. The

dosage of HPC was chosen as described in a previous

study [3] which demonstrated very low to no clasto-

genic effects. The cytogenetic investigation was

performed as previously described [3]. In brief, male

and female C57BL6 mice were treated as indicated

below. After ending the treatment period, animals

were injected with colchicine (100 mg/kg body

weight). One hour later animals were killed by cervical

dislocation and bone marrow cells were isolated from

their femurs. After hypotonization and fixation, slides

were prepared and stained with Giemsa solution. At

least 50 metaphase plates from each animal were

analyzed for the incidence of chromosomal aberra-

tions.

2.9. Evaluation of combination effects and statistics

Predicted theoretical values were calculated

according to the equation:

c ¼ a £ b=100

where a and b are cell survival values with single

agents, presented as percent of untreated control. For

each concentration applied theoretical values were

calculated and compared with the real value of the

combination: For cmeasured ¼ ccalculated the combination

effect is additive; for cmeasured , ccalculated the combina-

M.C. Georgieva et al. / Cancer Letters 182 (2002) 163–174 165

tion effect is synergistic; and for cmeasured . ccalculated

the combination effect is antagonistic. Cell survival

values significantly lower (,70%) than the value

calculated indicated the presence of synergistic

potentiation [20,24,25]. Cytotoxicity and cytogenetic

data were calculated as mean values with corre-

sponding confidence limits or standard deviation.

Significance was evaluated by Student’s t-test

(P , 0:05).

3. Results

3.1. DNA fragmentation and morphological changes

In contrast to the clearly visible ladder formation

after 24 h in DNA from HL-60 cells exposed to ara-C,

Gem or ErPC3 (Fig. 1a), a less pronounced fragmenta-

tion was observed in bcr-abl expressing LAMA-84

cells after exposure to different concentrations of

Gem for a longer period of time (48 h; Fig. 1c).

DNA staining with Hoechst 33342 showed similar

nuclear fragmentation in both cell lines after treatment

with Gem (Fig. 1b,d).

3.2. Cell-free system

Previous experiments indicated that Gem and HPC

are potent inductors of apoptosis in the CLL-derived

SKW-3 cell line but fail to cause DNA laddering in K-

562 cells (data not shown). As shown in Fig. 2 (lanes

1–3) oligonucleosomal DNA fragmentation was

induced by incubation with 5 mM Gem for 24 h in

SKW-3 cells, which were used to prepare a cytosolic

fraction. After 20 min incubation of K-562 nuclei with

the cytosolic fraction of apoptotic SKW-3 cells, visi-

ble fragmentation of the nuclear DNA occurred and

was detected for periods up to 1 h, as well (Fig. 2,

lanes 5–7).

3.3. Flow cytometry

Untreated and treated (5 mM Gem) promyelocytic

HL-60 cells were processed for flow cytometry analy-

sis of DNA content (data not shown). Gem treated

cells reacted with the nearly complete disappearance

of their S-phase fraction (1.7%) as compared with

untreated cells (48% of cells in S phase). These data

demonstrate that Gem is almost exclusively effective

on cells in the S phase of the cell cycle.

3.4. Cytotoxic activity of ara-C, Gem and HPC or

ErPC3

The cytotoxic activity of rising concentrations of

ara-C and Gem was measured by the MTT dye reduc-

tion assay. Interpolated from experimental concentra-

tion–effect curves, IC50 and IC80 values are presented

in Table 1. On a molar basis, Gem showed higher

cytotoxic efficacy than ara-C in any cell line exam-

ined. The most resistant cell line was K-562, as

evidenced by the fact that neither ara-C nor Gem

effected enough cytotoxicity to obtain IC50 values.

This cell line was chosen for determining the combi-

nation effects of HPC and ErPC3 with Gem. HPC

effected only negligible cytotoxic activity after 72 h

up to a 25 mM concentration (Fig. 3). The anti-meta-

bolite Gem showed more pronounced activity in the

concentration range investigated, but reached a

plateau in its cell kill at 6.25 mM. Interestingly, the

simultaneous application of the two drugs led to a

cytotoxic effect which was greater than expected: At

the highest concentration tested (25 mM), the combi-

nation exerted more efficacy than Gem alone and this

effect was somewhat greater than the theoretical value

calculated (Fig. 3). The efficacy of a consecutive treat-

ment of K-562 cells with HPC and Gem is shown in

Fig. 4. Exposure of K-562 cells for 24 h to HPC at 25

and 50 mM concentrations resulted in 14 and 18%

growth inhibition (Fig. 4a); equal concentrations of

Gem caused 28 and 33% growth inhibition (Fig.

4b). Consecutive exposure to HPC and Gem at 25

and 50 mM resulted in 50 and 54% growth inhibition,

respectively, which was below the expected combina-

tion effect at the 25 mM level (Fig. 4a), whereas an

additive effect was observed at the 50 mM level. The

reversed sequence was associated with an additive

effect at 25 mM, but with a synergistic potentiation

at 50 mM, resulting in 74% growth inhibition (Fig.

4b). For investigating this potentiation in greater

detail, two other CML cell lines (LAMA-84 and

AR-230) and a more effective alkylphosphocholine

(ErPC3) were used as well (Table 2 and Fig. 5).

Sequential exposure for 48 h to HPC (first drug) and

Gem (second drug) resulted in antagonism at low

HPC concentrations. Higher concentrations of HPC

M.C. Georgieva et al. / Cancer Letters 182 (2002) 163–174166

were associated with additive effects of anti-leukemic

activity. The reverse sequence, however, was more

effective and resulted in clear synergism at concentra-

tions of 0.18 and 0.6 mM Gem followed by 50 mM

HPC, respectively (Table 2). Exchanging of HPC for

ErPC3 resulted in a more pronounced synergy when

Gem preceded the alkylphosphocholine (Fig. 5,

bottom), but in increased antagonism for the reverse

sequence (Fig. 5, top).

The combination effects of HPC and Gem in

LAMA-84 and AR-230 cells are given in Table 2.

Interestingly, no significant deviation from additivity

M.C. Georgieva et al. / Cancer Letters 182 (2002) 163–174 167

Fig. 1. DNA laddering and nuclear fragmentation in HL-60 and LAMA-84 cells: (a) Oligonucleosomal DNA fragmentation in HL-60 cells

treated for 24 h with ara-C, 20 and 40 mM (lanes 1 and 2); alkylphosphocholine, 20 and 40 mM (lanes 3 and 4); Gem, 0.5, 2.5, 5 and 10 mM

(lanes 5–8); untreated control (lane 9); size marker (lane M). (b) Nuclear fragmentation in HL-60 cells after 24 h treatment with 5 mM Gem

(Hochst 33342 staining: fluorescence microscopy, £500Þ; fragmented nuclei are indicated by arrows. (c) DNA fragmentation in LAMA-84 cells

treated for 48 h with Gem, 0.5, 5, 10 and 20 mM (lanes 1–4, respectively) or ara-C, 0.5 and 20 mM (lanes 5 and 6); size marker (lane M);

untreated control (lane 7). (d) Nuclear fragmentation in LAMA-84 cells after treatment with 5 mM Gem for 48 h (Hochst 33342 staining,

fluorescence microscopy, £500Þ; fragmented nuclei are indicated by arrows.

was observed in LAMA-84 cells. On the other hand,

in AR-230 cells no antagonism but synergy was found

following exposure to HPC followed by Gem.

3.5. Clastogenic effect on normal murine bone

marrow cells

The clastogenic activity of Gem and ara-C was

compared at clinically relevant dosages in C57BL6

mice. As shown in Table 3, there was a comparably

slight increase in aberrant metaphases for both agents

(4 and 6.4%, respectively) and an insignificant reduc-

tion of the mitotic index (10.2 and 11.3 ‰, respec-

tively). In order to determine the optimally tolerated

treatment schedule, HPC and Gem were alternated in

sequence of their combined administration, as indi-

cated in Table 3. Both combination schedules did

not lead to increased induction of chromosomal aber-

rations as compared to Gem alone. Interestingly, the

M.C. Georgieva et al. / Cancer Letters 182 (2002) 163–174168

Fig. 3. Concentration–effect curves of Gem, HPC and their combi-

nation in K-562 cells as measured by MTT assay after drug expo-

sure for 72 h. The calculated additive combination effect (dotted

line) is shown in comparison with the observed combination effect

that was found following concomitant exposure. Bars denote stan-

dard deviation.

Fig. 2. DNA fragmentation in the cell-free system consisting of the

cytosolic fraction of Gem pre-treated SKW-3 cells and K-562

nuclei. Lanes 1–3, DNA isolated from aliquots of the cytosolic

fraction taken at 0, 40 and 60 min following start of co-incubation;

lanes 4–7, DNA isolated from aliquots of the nuclear fraction taken

at 0, 20, 40 and 60 min following start of co-incubation.

Table 1

Inhibition of leukemia cell growth by cytosine arabinoside (ara-C) and gemcitabine (Gem) following incubation for 48 h

Cell line Drug IC50 (mM) IC80 (mM)

HL-60 ara-C 1.54 (1.28–1.92)a 2.56 (2.31–3.08)a

Gem 0.59 (0.51–0.69) 2.54 (2.31–2.95)

LAMA-84 ara-C 2.05 (1.92–2.56) .50

Gem 2.00 (1.79–2.57) 4.36 (3.08–5.90)

K-562 ara-C .100 .100

Gem .25 .25

SKW-3 Gem 2.78 (2.68–2.99) 4.94 (4.89–5.05)

a 95% confidence limits.

treatment regimen of HPC followed by Gem led to a

certain inhibition of the mitotic activity (mitotic

index ¼ 8.9‰ versus 13.5‰ in controls). By contrast,

the combined regimen of gem followed by HPC

recorded the highest mitotic activity (15.3 ‰).

4. Discussion

Alkylphosphocholines are potent anti-leukemic

agents which at least in part act through induction of

apoptosis [3,8–10,23]. Due to their bone marrow-

stimulating properties they have been used experimen-

tally for alleviating myelosuppression induced by

cytostatic therapy [26]. In this respect, the combination

of an alkylphosphocholine (e.g. HPC or ErPC3) with an

anti-metabolite appeared promising because both

groups of agents have a different mechanism of action

and the myelosuppression induced by the anti-metabo-

lite could be ameliorated by the alkylphosphocholine.

Therefore, we hypothesized that the combination of an

anti-metabolite with an alkylphosphocholine may be

advantageous in terms of an increased therapeutic

ratio.

For selecting the most suitable anti-metabolite Gem

and ara-C were compared in a panel of sensitive and

resistant leukemia cell lines. The data show that Gem

induces programmed cell death more potently than

ara-C and, on a molar basis, shows greater anti-leuke-

mic efficacy in vitro (Fig. 1a and Table 1). However,

the presence of BCR-ABL is the probable reason for a

less pronounced and delayed DNA laddering as

shown for the response of LAMA-84 cells to Gem

(Fig. 1c) and that of K-562 cells to ErPC3 [10].

The mode how BCR-ABL delays an apoptotic

response is not clear, but this fact has been described

repeatedly [10,27–29]. Therefore we used a cell-free

system to determine the sensitivity of nuclei from the

most resistant BCR-ABL expressing cell line (K-562)

towards the stimuli of pre-induced cytosol of sensitive

cells (SKW-3). Our results show a time-dependent

induction of oligonucleosomal DNA fragmentation

starting after co-incubation for 20 min. It can be

deduced that the causal factor(s) for resistance to

apoptosis in response to treatment with Gem are loca-

lized in the cytoplasm. This holds true for BCR-ABL

and is in line with the finding that BclXL overexpres-

sion may result from the presence of BCR-ABL

[30,31] and that specific inhibition of BCR-ABL

expression leads to enhanced chemosensitivity ([32];

Konstantinov et al., submitted). In addition to the

expression of BCR-ABL, the sensitivity of K-562

cells to Gem is influenced by the activity of nucleo-

side transporters that are required for Gem influx [33]

and by induction of resistance leading to karyotypic

abnormalities [34]. When comparing the sensitivity of

K-562 cells reported by others ([33,34]; IC50 ¼ 1:5

and 57 nM) with that found in our experiments

(IC50 . 25 mM), it appears that the K-562 variant

used in this study was distinctly more resistant. In

part, this difference could be accounted for by

variances in the technique used (exposure time 72 h

vs. 48 h in this study, no baseline subtraction of cell

growth at the time of drug addition performed in this

study), but the major part probably can be attributed to

an increased resistance of our variant.

Concomitant exposure of these highly resistant

M.C. Georgieva et al. / Cancer Letters 182 (2002) 163–174 169

Fig. 4. Cell survival of K-562 cells treated with sequential schedules

in percent of untreated control: For schedule A (HPC followed by

Gem, 24 h exposure, respectively) the optimal concentration is 25

mM and for schedule B (Gem followed by HPC, 24 h exposure,

respectively) the optimal concentration is 50 mM. Calculated addi-

tive effects are presented as columns without bars. A significant

difference to the calculated additive effect is marked by an asterisk

(P # 0:05). Bars denote standard deviation.

M.C. Georgieva et al. / Cancer Letters 182 (2002) 163–174170

Table 2

Combination of alkylphosphocholines and gemcitabine in CML cell lines

Cell line Concentration (mM) of

1st drug (name)

Concentration (mM) of

2nd drug (name)

Duration of experiment

(h)

T/C%a (observed) T/C% (expected)

K-562 12.5 (HPC) – 48 99 ^ 2.7 –

25 (HPC) – 48 101 ^ 1.2 –

50 (HPC) – 48 96 ^ 3.5 –

12.5 (HPC) – 96 106 ^ 3.9 –

25 (HPC) – 96 108 ^ 3.5 –

50 (HPC) – 96 112 ^ 7.0 –

0.06 (Gem) – 48 98 ^ 1.6 –

0.18 (Gem) – 48 86 ^ 0.9 –

0.6 (Gem) – 48 81 ^ 2.4 –

0.06 (Gem) – 96 124 ^ 17.7 –

0.18 (Gem) – 96 61 ^ 1.3 –

0.6 (Gem) – 96 64 ^ 1.9 –

12.5 (HPC) 0.06 (Gem) 96 113 ^ 5.9 104

12.5 (HPC) 0.18 (Gem) 96 110 ^ 3.2 91

12.5 (HPC) 0.6 (Gem) 96 108 ^ 6.8 86

25 (HPC) 0.06 (Gem) 96 85 ^ 7.0 107

25 (HPC) 0.18 (Gem) 96 84 ^ 2.5 93

25 (HPC) 0.6 (Gem) 96 84 ^ 5.3 88

50 (HPC) 0.06 (Gem) 96 78 ^ 6.8 110

50 (HPC) 0.18 (Gem) 96 87 ^ 2.0 96

50 (HPC) 0.6 (Gem) 96 92 ^ 5.0 91

0.06 (Gem) 12.5 (HPC) 96 109 ^ 3.5 124

0.06 (Gem) 25 (HPC) 96 109 ^ 2.8 126

0.06 (Gem) 50 (HPC) 96 112 ^ 8.3 119

0.18 (Gem) 12.5 (HPC) 96 60 ^ 1.9 61

0.18 (Gem) 25 (HPC) 96 62 ^ 1.0 62

0.18 (Gem) 50 (HPC) 96 41 ^ 2.5b 59

0.6 (Gem) 12.5 (HPC) 96 62 ^ 2.6 64

0.6 (Gem) 25 (HPC) 96 68 ^ 1.4 65

0.6 (Gem) 50 (HPC) 96 43 ^ 4.1b 62

K-562 12.5 (ErPC3) – 48 99 ^ 4.0 –

25 (ErPC3) – 48 93 ^ 5.5 –

50 (ErPC3) – 48 74 ^ 4.5 –

12.5 (ErPC3) – 96 114 ^ 5.3 –

25 (ErPC3) – 96 100 ^ 3.7 –

50 (ErPC3) – 96 97 ^ 4.1 –

0.06 (Gem) – 48 86 ^ 2.0 –

0.18 (Gem) – 48 86 ^ 6.1 –

0.6 (Gem) – 48 84 ^ 1.2 –

0.06 (Gem) – 96 57 ^ 1.7 –

0.18 (Gem) – 96 58 ^ 2.1 –

0.6 (Gem) – 96 55 ^ 3.8 –

LAMA-84 12.5 (HPC) – 48 102 ^ 2.0 –

25 (HPC) – 48 103 ^ 4.1 –

50 (HPC) – 48 90 ^ 3.0 –

12.5 (HPC) – 96 88 ^ 2.7 –

25 (HPC) – 96 90 ^ 1.4 –

50 (HPC) – 96 59 ^ 1.7 –

0.06 (Gem) – 48 88 ^ 3.0 –

0.18 (Gem) – 48 88 ^ 2.6 –

0.6 (Gem) – 48 87 ^ 1.9 –

0.06 (Gem) – 96 46 ^ 1.1 –

0.18 (Gem) – 96 25 ^ 2.0 –

0.6 (Gem) – 96 32 ^ 2.8 –

12.5 (HPC) 0.06 (Gem) 96 71 ^ 3.0 78

12.5 (HPC) 0.18 (Gem) 96 70 ^ 2.3 78

12.5 (HPC) 0.6 (Gem) 96 67 ^ 2.7 76

cells to Gem and HPC resulted in a supra-additive

effect. To explore this synergy in greater detail we

performed sequential exposure experiments to

describe the optimum concentration, sequence and

incubation time of the drugs. The Gem (first drug)

HPC (second drug) sequence showed significant

synergy at 50 mM concentrations and 24 h incubation

periods (Fig. 4). This result in K-562 cells was corro-

borated when using a longer incubation period (48 h,

respectively) or exchanging HPC for ErPC3. For this

latter agent, the synergistic and antagonistic effects

became distinctly more apparent, in line with its

greater potency. When transferring the exposure sche-

dule to other cell lines, LAMA-84 cells showed only

M.C. Georgieva et al. / Cancer Letters 182 (2002) 163–174 171

Table 2 (continued)

Cell line Concentration (mM) of

1st drug (name)

Concentration (mM) of

2nd drug (name)

Duration of experiment

(h)

T/C%a (observed) T/C% (expected)

25 (HPC) 0.06 (Gem) 96 70 ^ 5.3 80

25 (HPC) 0.18 (Gem) 96 67 ^ 5.3 80

25 (HPC) 0.6 (Gem) 96 64 ^ 5.0 79

50 (HPC) 0.06 (Gem) 96 53 ^ 3.3 52

50 (HPC) 0.18 (Gem) 96 54 ^ 3.1 52

50 (HPC) 0.6 (Gem) 96 54 ^ 3.0 51

0.06 (Gem) 12.5 (HPC) 96 42 ^ 2.3 47

0.06 (Gem) 25 (HPC) 96 40 ^ 2.1 47

0.06 (Gem) 50 (HPC) 96 37 ^ 2.1 41

0.18 (Gem) 12.5 (HPC) 96 33 ^ 0.6 25

0.18 (Gem) 25 (HPC) 96 32 ^ 1.7 26

0.18 (Gem) 50 (HPC) 96 29 ^ 1.9 22

0.6 (Gem) 12.5 (HPC) 96 27 ^ 1.2 32

0.6 (Gem) 25 (HPC) 96 28 ^ 1.1 32

0.6 (Gem) 50 (HPC) 96 29 ^ 1.5 28

AR-230 12.5 (HPC) – 48 98 ^ 2.3 –

25 (HPC) – 48 91 ^ 4.4 –

50 (HPC) – 48 93 ^ 8.0 –

12.5 (HPC) – 96 92 ^ 4.6 –

25 (HPC) – 96 78 ^ 4.0 –

50 (HPC) – 96 67 ^ 1.6 –

0.06 (Gem) - 48 96 ^ 1.8 –

0.18 (Gem) – 48 84 ^ 3.1 –

0.6 (Gem) – 48 55 ^ 3.3 –

0.06 (Gem) – 96 90 ^ 6.3 –

0.18 (Gem) – 96 72 ^ 3.1 –

0.6 (Gem) – 96 43 ^ 1.4 –

12.5 (HPC) 0.06 (Gem) 96 70 ^ 4.0 88

12.5 (HPC) 0.18 (Gem) 96 52 ^ 3.6b 77

12.5 (HPC) 0.6 (Gem) 96 31 ^ 2.4b 51

25 (HPC) 0.06 (Gem) 96 52 ^ 3.4 74

25 (HPC) 0.18 (Gem) 96 44 ^ 2.0b 65

25 (HPC) 0.6 (Gem) 96 35 ^ 1.8 43

50 (HPC) 0.06 (Gem) 96 51 ^ 1.6 64

50 (HPC) 0.18 (Gem) 96 45 ^ 1.6 56

50 (HPC) 0.6 (Gem) 96 36 ^ 2.0 37

0.06 (Gem) 12.5 (HPC) 96 84 ^ 4.8 88

0.06 (Gem) 25 (HPC) 96 83 ^ 3.1 81

0.06 (Gem) 50 (HPC) 96 74 ^ 3.3 83

0.18 (Gem) 12.5 (HPC) 96 59 ^ 2.1 71

0.18 (Gem) 25 (HPC) 96 60 ^ 3.1 66

0.18 (Gem) 50 (HPC) 96 50 ^ 2.4 67

0.6 (Gem) 12.5 (HPC) 96 41 ^ 2.0 42

0.6 (Gem) 25 (HPC) 96 38 ^ 1.3 39

0.6 (Gem) 50 (HPC) 96 36 ^ 2.5 40

a Percent of untreated control ^ SD.b Synergistic effect (observed value , 70% of predicted value).

additive interactions. The more slowly proliferating

cell line AR-230 interestingly showed synergistic

interaction following the reverse sequence (HPC

followed by Gem). We hypothesize that AR-230

cells were initially stimulated by HPC and this

might have enhanced the S-phase specific cytotoxicity

of Gem. In analogy to data published on the interac-

tion of HPC and ara-C, the synergistic effect might

also result from lipid composition disturbance caused

by Gem [11].

Our considerations on the mechanism of action of

sequentially administered Gem and HPC are

confirmed by the mitotic indices obtained in normal

murine bone marrow cells (Table 3): Gem followed

M.C. Georgieva et al. / Cancer Letters 182 (2002) 163–174172

Fig. 5. Cell survival in percent of untreated control. Exposure to optimal schedules for K562 cells (top: ErPC3 followed by Gem, 48 h exposure,

respectively; bottom: Gem followed by ErPC3, 48 h exposure, respectively). Calculated additive effects are presented as columns without bars.

A significant difference to the calculated additive effect is marked by an asterisk (P # 0:05). Bars denote standard deviation.

by HPC caused the highest mitotic index (15.3‰)

whereas the reverse schedule was associated with a

distinctly lower number of metaphase plates (8.9‰).

Pharmacokinetic data from a phase I study investi-

gating the systemic administration of HPC show that

steady-state concentrations were reached in patients

after 2 weeks of daily treatment with 50–200 mg. The

corresponding serum concentrations ranged from 100

to 200 mM (C. Unger, personal communication).

Thus, the concentrations of HPC used in this study

(up to 50 mM) are pharmacologically achievable in

leukemia patients and the same holds true for Gem

[35]. This could be beneficial not only to patients with

relapsed AML but also to CML blast crisis patients

not responding to ara-C standard therapy.

In conclusion, our data indicate that Gem is a more

potent anti-leukemic agent than cytosine arabinoside

and that the combination of Gem and an alkylpho-

sphocholine is advantageous in terms of their comple-

mentary mode of action, resulting in synergistic

cytotoxicity. In addition, the main dose-limiting side

effect of Gem (myelosuppression) can be lowered by

the combination with bone marrow-stimulating alkyl-

phosphocholines and may enable the administration

of higher, more effective Gem doses.

Acknowledgements

The authors thank Ms. Carola Seuffert of Lilly

Deutschland GmbH for kindly providing gemcitabine

(Gemzar). This study was supported in part by DKFZ

postdoctoral fellowship grants to M.G. and S.K.

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