Cancer Letters, 66 (1992) 29-34 Elsevier Scientific Publishers Ireland
29 Ltd.
Increased survival of L1210 leukemic mice by prevention of the utilization of extracellular polyamines. Studies using a polyamineuptake mutant, antibiotics and a polyamine-deficient diet Anders Ask”, Lo Perssonb Departments
of “Oncology
Deoelopmentaf (Received (Accepted
Biology,
and Olle Heby”
and bPhysiology, University of Lund, S-22362 University of Ume& S-90187 Ume: (Sweden)
Lund;
and ‘Deportment
of Cellular and
20 May 1992) 22 June 1992)
Summary When l.1210 leukemia cells are inhibited in their polyamine synthesis by treatment with adijluoromethylornithine (DFMO), their growth in culture is strongly suppressed. In striking contrast, the suruiual of LIZ10 leukemic mice is only marginally prolonged by DFMO treatment. This inconsistency is due to the fact, that in the mouse the tumor ceils can utilize extracellular polyamines to compensate for the decrease in putrescine and spermidine synthesis caused by DFMO treatment. In the present study, we demonstrate that a reduction in the transport ojpolyamines into the tumor cells is a more effective means of increasing the therapeutic ejject of DFMO than is a reduction in the supply of extracellular polyamines. DFMO treatment cured 30 - 75 % of leukemic mice bearing mutant LUIO-MGBG’ cells dejicient in polyamine uptake, but only slightly increased the suruiual time of leukemic mice bearing the parental IA210 cells despite the fact that the supply of extracellular polyamines was reduced (by feeding the mice a polyamine-deficient diet containing antibiCorrespondence Developmental
to: Olle Heby, Department Biology, University of Umeb,
of Cellular and S-90187 Ume:,
Sweden.
0304-3835/92/$05.00 Printed and Published
0 1992 Elsevier Scientific Publishers in Ireland
otics). The effectiveness by which DFMO cured leukemic mice bearing L1210-MGBG’ cells appeared to be sex dependent. Thus, 58% of the female mice, as compared to 30% of the male mice, were cured by DFMO treatment.
Keywords:
uptake; polyamine difluoromethylornithine; polyamine-cieficiezi diet; antibiotics; antileukemic effect
Introduction Synthesis of the polyamines putrescine, spermidine and spermine is essential for proliferating cells [4,9]. Therefore, inhibitors of the rate-limiting enzymes in the polyamine biosynthetic pathway, ornithine decarboxylase (ODC) and S- a d enosylmethionine decarboxylase are potential cancer chemotherapeutic agents [9]. We have previously shown that depletion of putrescine and spermidine by treatment with a-difluoromethylornithine (DFMO) causes a strong inhibitory effect on the growth of L1210 leukemia cells in culture, but has little effect on the survival of L1210 leukemic mice [2,10]. This discrepancy is not due to degradation or inability of DFMO to reach the leukemia cells Ireland Ltd
30
in the mouse. Instead, the finding can be explained by the fact that in the mouse the leukemia cells can use extracellular polyamines in overcoming the block in pofyamine production caused by DFMO treatment. In culture, however, the amount of polyamines present in the medium is insufficient to rescue the cells from polyamine deprivation [lo]. The importance of extracellular polyamines in protecting tumor cells against the antiproliferative effect of DFMO is also indicated by the results of a series of studies carried out by Seiler and coworkers [ll, 121. They demonstrated that the therapeutic effect of DFMO could be improved if tumor-bearing mice were provided with a polyamine-deficient diet, supplemented with antibiotics in order to at least partly decontaminate the gastrointestinal tract from polyamine-producing bacteria. In the present study, we have used a mutant L1210 leukemia cell line (L1210-MGBG’), deficient in polyamine transport [lo], to further investigate the importance of extraceUular polyamines when using DFMO as a therapeutic agent against tumors. Materjal and Wetbods
Experimental animals Female and male B6D2F1 (C57BL/6 x DBA/Z F,) mice, weighing 18 - 20 g, were purchased from Gl. Bomholtg&d, Ry, Denmark. The mice were initially given standard laboratory chow and tapwater ad libitum. Cell culture
L1210 leukemia cells and mutant L1210MGBG’ cells, deficient in polyamine uptake, were grown in suspension cultures using RPM1 1640 medium supplemented with 10% fetal calf serum, 50 units/ml of penicillin, 50 pg/ml of streptomycin and 50 PM 2-mercaptoethanol. The cells were routinely subcultured twice weekly by dilution in fresh medium to 1.0 x lo5 cells per ml. The LlBlO-MGBG’ cells were isolated by selecting for cells with resistance to the cytotoxicity of methylglyoxal bis(guanylhydrazone) (MGBG) [lo], which is
taken up by the same transport system as the polyamines [8]. Polyamine uptake The polyamine transport capacities of the cells were determined by measuring the uptake of radioactive polyamines. Exponentially growing L1210 cells and LlZlO-MGBG’ cells were supplemented with either [ 14C]putrestine (107 mCi/mmol), [3H]spermidine (250 mCi/mmoI) or [ 14C]spermine (110 mCi/ mmol) at a concentration of 1 PM. After a 20min incubation at 37OC, the uptake was stopped by rapidly chilling the cells on ice followed by centrifugation at 1000 x g for 5 min at 4OC. The cells were washed twice with icecold phosphate-buffered saline containing 10 mM unlabeled putrescine, spermidine or spermine. The amounts of radioactivity in the cells were determined after solubilizing the cells in 500 ~1 of Soluene 350 (Packard Instruments). The small amount of radiolabel bound to the cell surface was adjusted for by using parallel cultures incubated on ice. Tumor models and drug regimen B6D2F1 female and male mice were inoculated i.p. with 1.0 x lo6 parental L1210 cells or mutant L1210-MGBG’ cells suspended in 0.2 ml of 0.9% NaCl solution. The mice were given a polyamine-deficient diet (Table I), supplemented with neomycin (2 g/kg) and metronidazole (34 mg/kg), starting 2 - 3 weeks before tumor inoculation. DFMO was included in the drinking water (3%)) beginning 1 day after tumor inoculation. The average daily intake of DFMO was 4.2 to 4.4 g/kg [lo]. From day 40 on, the surviving mice Table 1. Composition
of polyamine-deficient References
Main constituents (per kg of diet) Wheat starch Casein Arachis oil Salt mixture Vitamin mixture
diet.
630 g 190 g 100 g 40 3 20 g
Hubbel et al. [6] Kahlson et al. [7]
31
received standard laboratory chow and tap water ad libitum. Surviving mice were inspected for a total of 60 days. B4SSUltS The median survival time of female B6DZFr mice inoculated with 1.0 x lo6 parental L1210 cells was 11 days when they were fed
a standard laboratory chow and 12 days when instead provided with a polyamine-deficient diet containing antibiotics (Fig. 1A). If the mice were also subjected to DFMO treatment, by supplementing the drinking water with the drug, there was a further prolongation of the survival time to 17 days (Fig. 1B). DFMO by itself had a slight effect on the survival time, which increased to 13 days (Fig. 1B). In
I -
0
10
20
30
40
50
60
40
so
60
DFMO,MSFUO
Days 100
0 0
10
20
30 Days
0
10
20
30
40
50
60
Days
Fia. 1. Effects of limitation of extracellular polyamines on the survival of female B6D2FI mice inoculated with 1 .O x lo6 parental L1210 cells (A,B) or MGBG-resistant (L1210-MGBG’) L1210 cells (C,D). One hundred percent corresponds to 19 - 20 mice. (A) Mice inoculated with parental L1210 leukemia cells and fed a standard laboratory chow (solid line) or a polyamine-deficient diet containing antibiotics (described in Materials and Methods) (dashed line). (B) Mice inoculated with parental L1210 leukemia cells, given DFMO in their drinking water (3%) from day 1 on and fed a standard laboratory chow (solid line) or a polyamine-deficient diet containing antibiotics (dashed line). (C) Mice inoculated with Ll210-MGBG’ leukemia cells and fed a standard laboratory chow (solid line) or a polyamine-deficient diet containing antibiotics (dashed line). (D) Mice inoculated with L1210-MGBG’ leukemia cells, given DFMO in their drinking water (3%) from day 1 on and fed a standard laboratory chow (solid line) or a polyamine-deficient diet containing antibiotics (dashed line). MST, median survival time (days). Since less than 50% of the mice died in the experiments shown in D no MST values could be determined. Each figure is based on two separate experiments.
32
Table II.
Uptake of polyamines into MGBG-resistant (LlZlO-MGBG’) and parental L1210 cells. Polyamine uptake (pmol/106 cells
x
20 min)
Putrescine
Spermidine
Spermine
L1210
123.8 f 33.2 (100%)
129.9 l 18.7 (100%)
71.4 zt 7.8 (100%)
L1210-MGBG’
5.1 f 0.7 (4.1%)
2.2 l 0.4 (1.7%)
0.5 f 0.1 (0.7%)
The cells were grown as suspension cultures and exposed to radiolabeled polyamines for a 20-min period while in logarithmic growth (1 day after seeding). The rates of uptake of the various polyamines were compared and are presented as percent of the parental L1210 cells. Data shown are means ( •t S.E.M.) of 3 experiments.
addition to slightly prolonging the survival time of the whole population of leukemic mice, the polyamine-deficient diet containing antibiotics resulted in long-term survival or cure of 1 - 3 mice in each experimental group of 10 mice. An L1210 cell line (LlZlO-MGBG’), deficient in the transport of spermidine, has been isolated by selecting for the resistance to the cytotoxic effect of MGBG [lo]. MGBG and spermidine bear a structural resemblance and are taken up by the same transport system (81. However, as shown in Table II, the uptakedeficiency of the LlZlO-MGBG’ cells was not limited to spermidine. Also the capacities to transport putrescine and spermine were markedly reduced in the mutant cell line. The median survival time for female B6D2Fr mice inoculated with 1.0 x lo6 LlZlO-MGBG’ leukemia cells was 12.5 days when they were fed a standard laboratory chow (Fig. 1C). The provision of a polyaminedeficient diet containing antibiotics to the mice gave a slight prolongation of the survival time (Fig. 1C). Contrary to the minor increase in survival caused by the special diet, there was a dramatic therapeutic effect by supplementing the drinking water with DFMO (Fig. lC,D). Thus, 58% of the leukemic mice bearing LlZlO-MGBG’ cells were cured of their disease (Fig. 1D). Providing the mice with a polyamine-deficient diet, containing antibiotics, increased the cure rate obtained by DFMO treatment to 75% (two series; 60% and 90%) respectively) (Fig. 1D). When tak-
ing together all the results obtained with mice bearing L1210-MGBG’ cells, it became apparent that provision of the special diet gave rise to a significant increase in survival time. The effectiveness by which DFMO cured leukemic mice bearing LlZlO-MGBG’ cells appeared to be dependent on the sex of the mice. Thus, the therapeutic effect of DFMO was much better for female than for male mice; 58% of the female mice and 30% of the male mice were cured (Figs. 1D and 2).
-
100T-7.‘:
coamllbdsnm
01 0
10
20
30
40
50
60
Days Fig. 2.
Survival of male B6D2FI mice inoculated with 1.0 x lo6 MGBG-resistant (L1210-MGBG’) leukemia cells and given drinking water without supplements (solid line) or containing 3% DFMO (dashed line). DFMO was provided from day 1 on. One hundred percent corresponds to 10 mice. MST, median survival time (days).
33
Depletion of putrescine and spermidine by treatment with DFMO has been found to reduce the growth rate of all cell lines studied, that is, when the cells are grown in culture. In vivo, however, most cell lines respond poorly to the same treatment. This is not due to DFMO degradation or inability of the drug to reach and enter the target cells in the host. Instead, the poor effect is due to the fact that DFMO treatment and the resulting putrescine and spermidine deficiency elicits a compensatory uptake of extracellular polyamines [l] . This was clearly demonstrated in a previous study, in which we used a polyamine uptake mutant selected from mutagenized L1210 leukemia cells [lo]. Thus, leukemic mice bearing L1210 cells severely deficient in polyamine uptake exhibited a cure rate of 33% when blocked in their polyamine synthesis by DFMO. This should be compared with the 2day increase in survival time resulting from DFMO treatment of leukemic mice bearing the parental L1210 cells with their efficient uptake of extracellular polyamines [lo]. Thus, tumor cells seem to have a very efficient polyamine uptake system, due to which the therapeutic effectiveness of inhibitors of endogenous polyamine synthesis may be severely compromised. In an attempt to limit the availability of extracellular polyamines, Seiler et al. [12] provided tumor-bearing mice with a polyaminedeficient diet containing antibiotics and inhibitors of ODC and polyamine oxidase. The inhibitor of polyamine oxidase was included to prevent polyamine-interconversion reactions [12] and the antibiotics to decontaminate the gastrointestinal from tract polyamineproducing bacteria [5]. The gastrointestinal tract is considered to be the most important exogenous source of polyamines. The approach used by Seiler et al. [12] resulted in a markedly reduced growth rate of intramuscularly inoculated Lewis lung carcinomas and prevented the formation of lung metastases. In the case of L1210 leukemic mice, the antiproliferative effect obtained by
simultaneously limiting the supply of extracellular polyamines and inhibiting polyamine synthesis with DFMO, was not as marked and there were no survivors [ 121. A more impressive therapeutic effect was obtained in the present study, where about 60% of the L1210 leukemic mice survived as a result of prevention of polyamine uptake. Thus, instead of reducing the external pool of polyamines a more effective approach would be to reduce the transport of the polyamines into the tumor cells. However, so far no specific inhibitors of the polyamine transport system have been described. Notably, there was a slight prolongation of the survival time when the mice inoculated with LlZlO-MGBG’ cells were provided with the polyamine-deficient diet containing antibiotics. Since the LlZlO-MGBG’ cells are deficient in their polyamine uptake their utilization of external polyamines should be low. However, although effectively reduced, the uptake of polyamines was still measurable in the mutant cells, which may explain why extracellular polyamine depletion caused an increase in host survival. Another explanation would be that the therapeutic effect of this diet is caused, at least partly, by a mechanism unrelated to the uptake of extracellular polyamines. Neomycin, for example, has been shown to interfere with the generation of second messengers, which may affect cell proliferation [3]. On the basis of our present knowledge it seems that polyamine synthesis inhibitors have strong therapeutic potential provided that the compensatory uptake of extracellular polyamines can be prevented or reduced. Therefore, it is important to characterize the polyamine-uptake system, such that new means may be devised for specific interference with the uptake mechanism. Acknowledgements
This study was supported by the Swedish Natural Science Research Council, the Swedish Cancer Society, the Medical Faculty (University of Lund) and the John and
34
Augusta Persson, the Carl Tesdorpf and the BeAa Kamprad Research Foundatidns. DFMO was a generous gift from the Marion Merrell Dow Research Institute, Cincinnati, Ohio.
L.,
Seppiinen,
P.
and
Janne,
induces increased uptake of the natural polyamines methylglyoxal-bis(guanylhydrazone).
Biochem.
J.,
Persson,
L.,
Oredsson,
Synergistic anttleukemic
synthesis inhibitors. Carney,
D.H.,
(1985)
inhibits
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and
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Scott,
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kinetic
D.L.,
Gordon,
Phosphoinositides
R.B.,
E.A.
Mendel,
Int.
J.
Cancer,
L.B. and Wakeman,
and Persson, L. (1990)
turnover
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43,
A.J.
(1937)
diets. J Nutr.,
Molecular
Hessels, J., Kingma, A.W.,
Physiol.,
103.
143, 91-
Mandel, J.-L. and Flintoff, W.F.
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Pegg, A.E.
Keij, J. and Muskiet, F.A.J.
(1989)
Microbial flora
ln-
rat. J.
(1978) Isolation of mutant transport.
J. Cell.
(1988) Polyamine metabolism and its impor-
in
neoplastic
12
growth
and
Cancer Res., 48,
L.,
effect of DL-2-difluoromethylornithine
bearing
mutant
Ask,
L1210
uptake.
S.,
I.,
a
Persson,
Sarhan,
Holm,
as
target
for
759 - 774.
Curative
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and Heby,
leukemia
cells
0.
(1988) on mice
deficient
in
Cancer Res., 48, 4807 - 4811.
Knodgen,
gastrointestinal
genetics of
H., van den Berg,
in the pregnant
cells altered in polyamine
tract
B. as
and
Seiler,
polyamine
growth. Anticancer
Res., 9, 215-224.
Seiler, N., Sarhan,
S., Grauffel,
B. and Moulinoux, Ferwerda,
E. and Westling, H. (1958)
of histamine
polyamine
Cell, 42, 479 -488.
synthesis in eukaryotic cells. Trends Biochem.
G., Rosengren,
creased formation
chemotherapy.
and LaBelle
phosphoinositide
Kahlson.
tance
in mitogenesis: neomycin
Sci., 15, 153-158. G.A.,
vivo.
Physiol., 97, 335 - 344.
37, 465 -470.
thrombin-stimulated 0.
Hubbel,
mammalian
effect of two polyamine
and initiation of cell proliferation. polyamine
SM.
Host survival and cell-cycle
analysis. Int. J. Cancer,
Heby,
8
9
A.,
in
1164.
A new salt mixture for use in experimental
and
491-501.
E.F.
6
J.
Intracellular putrescine and spermidine deprivation
(1986)
1155-
7
Alhonen-Hongtsto,
Ask,
difluoromethylomithine
14, 273 - 285.
References (1980)
in the gastrointestinal tract abolishes cytostatic effects of a-
J.-P.
N.
(1989)
source
for
C., Jones, R., Kniidgen,
(1990)
Endogenous
ogenous polyamines in support of tumor growth. Res., 50, 5077 - 5083.
The tumor
and exCancer
29 Ltd.
Increased survival of L1210 leukemic mice by prevention of the utilization of extracellular polyamines. Studies using a polyamineuptake mutant, antibiotics and a polyamine-deficient diet Anders Ask”, Lo Perssonb Departments
of “Oncology
Deoelopmentaf (Received (Accepted
Biology,
and Olle Heby”
and bPhysiology, University of Lund, S-22362 University of Ume& S-90187 Ume: (Sweden)
Lund;
and ‘Deportment
of Cellular and
20 May 1992) 22 June 1992)
Summary When l.1210 leukemia cells are inhibited in their polyamine synthesis by treatment with adijluoromethylornithine (DFMO), their growth in culture is strongly suppressed. In striking contrast, the suruiual of LIZ10 leukemic mice is only marginally prolonged by DFMO treatment. This inconsistency is due to the fact, that in the mouse the tumor ceils can utilize extracellular polyamines to compensate for the decrease in putrescine and spermidine synthesis caused by DFMO treatment. In the present study, we demonstrate that a reduction in the transport ojpolyamines into the tumor cells is a more effective means of increasing the therapeutic ejject of DFMO than is a reduction in the supply of extracellular polyamines. DFMO treatment cured 30 - 75 % of leukemic mice bearing mutant LUIO-MGBG’ cells dejicient in polyamine uptake, but only slightly increased the suruiual time of leukemic mice bearing the parental IA210 cells despite the fact that the supply of extracellular polyamines was reduced (by feeding the mice a polyamine-deficient diet containing antibiCorrespondence Developmental
to: Olle Heby, Department Biology, University of Umeb,
of Cellular and S-90187 Ume:,
Sweden.
0304-3835/92/$05.00 Printed and Published
0 1992 Elsevier Scientific Publishers in Ireland
otics). The effectiveness by which DFMO cured leukemic mice bearing L1210-MGBG’ cells appeared to be sex dependent. Thus, 58% of the female mice, as compared to 30% of the male mice, were cured by DFMO treatment.
Keywords:
uptake; polyamine difluoromethylornithine; polyamine-cieficiezi diet; antibiotics; antileukemic effect
Introduction Synthesis of the polyamines putrescine, spermidine and spermine is essential for proliferating cells [4,9]. Therefore, inhibitors of the rate-limiting enzymes in the polyamine biosynthetic pathway, ornithine decarboxylase (ODC) and S- a d enosylmethionine decarboxylase are potential cancer chemotherapeutic agents [9]. We have previously shown that depletion of putrescine and spermidine by treatment with a-difluoromethylornithine (DFMO) causes a strong inhibitory effect on the growth of L1210 leukemia cells in culture, but has little effect on the survival of L1210 leukemic mice [2,10]. This discrepancy is not due to degradation or inability of DFMO to reach the leukemia cells Ireland Ltd
30
in the mouse. Instead, the finding can be explained by the fact that in the mouse the leukemia cells can use extracellular polyamines in overcoming the block in pofyamine production caused by DFMO treatment. In culture, however, the amount of polyamines present in the medium is insufficient to rescue the cells from polyamine deprivation [lo]. The importance of extracellular polyamines in protecting tumor cells against the antiproliferative effect of DFMO is also indicated by the results of a series of studies carried out by Seiler and coworkers [ll, 121. They demonstrated that the therapeutic effect of DFMO could be improved if tumor-bearing mice were provided with a polyamine-deficient diet, supplemented with antibiotics in order to at least partly decontaminate the gastrointestinal tract from polyamine-producing bacteria. In the present study, we have used a mutant L1210 leukemia cell line (L1210-MGBG’), deficient in polyamine transport [lo], to further investigate the importance of extraceUular polyamines when using DFMO as a therapeutic agent against tumors. Materjal and Wetbods
Experimental animals Female and male B6D2F1 (C57BL/6 x DBA/Z F,) mice, weighing 18 - 20 g, were purchased from Gl. Bomholtg&d, Ry, Denmark. The mice were initially given standard laboratory chow and tapwater ad libitum. Cell culture
L1210 leukemia cells and mutant L1210MGBG’ cells, deficient in polyamine uptake, were grown in suspension cultures using RPM1 1640 medium supplemented with 10% fetal calf serum, 50 units/ml of penicillin, 50 pg/ml of streptomycin and 50 PM 2-mercaptoethanol. The cells were routinely subcultured twice weekly by dilution in fresh medium to 1.0 x lo5 cells per ml. The LlBlO-MGBG’ cells were isolated by selecting for cells with resistance to the cytotoxicity of methylglyoxal bis(guanylhydrazone) (MGBG) [lo], which is
taken up by the same transport system as the polyamines [8]. Polyamine uptake The polyamine transport capacities of the cells were determined by measuring the uptake of radioactive polyamines. Exponentially growing L1210 cells and LlZlO-MGBG’ cells were supplemented with either [ 14C]putrestine (107 mCi/mmol), [3H]spermidine (250 mCi/mmoI) or [ 14C]spermine (110 mCi/ mmol) at a concentration of 1 PM. After a 20min incubation at 37OC, the uptake was stopped by rapidly chilling the cells on ice followed by centrifugation at 1000 x g for 5 min at 4OC. The cells were washed twice with icecold phosphate-buffered saline containing 10 mM unlabeled putrescine, spermidine or spermine. The amounts of radioactivity in the cells were determined after solubilizing the cells in 500 ~1 of Soluene 350 (Packard Instruments). The small amount of radiolabel bound to the cell surface was adjusted for by using parallel cultures incubated on ice. Tumor models and drug regimen B6D2F1 female and male mice were inoculated i.p. with 1.0 x lo6 parental L1210 cells or mutant L1210-MGBG’ cells suspended in 0.2 ml of 0.9% NaCl solution. The mice were given a polyamine-deficient diet (Table I), supplemented with neomycin (2 g/kg) and metronidazole (34 mg/kg), starting 2 - 3 weeks before tumor inoculation. DFMO was included in the drinking water (3%)) beginning 1 day after tumor inoculation. The average daily intake of DFMO was 4.2 to 4.4 g/kg [lo]. From day 40 on, the surviving mice Table 1. Composition
of polyamine-deficient References
Main constituents (per kg of diet) Wheat starch Casein Arachis oil Salt mixture Vitamin mixture
diet.
630 g 190 g 100 g 40 3 20 g
Hubbel et al. [6] Kahlson et al. [7]
31
received standard laboratory chow and tap water ad libitum. Surviving mice were inspected for a total of 60 days. B4SSUltS The median survival time of female B6DZFr mice inoculated with 1.0 x lo6 parental L1210 cells was 11 days when they were fed
a standard laboratory chow and 12 days when instead provided with a polyamine-deficient diet containing antibiotics (Fig. 1A). If the mice were also subjected to DFMO treatment, by supplementing the drinking water with the drug, there was a further prolongation of the survival time to 17 days (Fig. 1B). DFMO by itself had a slight effect on the survival time, which increased to 13 days (Fig. 1B). In
I -
0
10
20
30
40
50
60
40
so
60
DFMO,MSFUO
Days 100
0 0
10
20
30 Days
0
10
20
30
40
50
60
Days
Fia. 1. Effects of limitation of extracellular polyamines on the survival of female B6D2FI mice inoculated with 1 .O x lo6 parental L1210 cells (A,B) or MGBG-resistant (L1210-MGBG’) L1210 cells (C,D). One hundred percent corresponds to 19 - 20 mice. (A) Mice inoculated with parental L1210 leukemia cells and fed a standard laboratory chow (solid line) or a polyamine-deficient diet containing antibiotics (described in Materials and Methods) (dashed line). (B) Mice inoculated with parental L1210 leukemia cells, given DFMO in their drinking water (3%) from day 1 on and fed a standard laboratory chow (solid line) or a polyamine-deficient diet containing antibiotics (dashed line). (C) Mice inoculated with Ll210-MGBG’ leukemia cells and fed a standard laboratory chow (solid line) or a polyamine-deficient diet containing antibiotics (dashed line). (D) Mice inoculated with L1210-MGBG’ leukemia cells, given DFMO in their drinking water (3%) from day 1 on and fed a standard laboratory chow (solid line) or a polyamine-deficient diet containing antibiotics (dashed line). MST, median survival time (days). Since less than 50% of the mice died in the experiments shown in D no MST values could be determined. Each figure is based on two separate experiments.
32
Table II.
Uptake of polyamines into MGBG-resistant (LlZlO-MGBG’) and parental L1210 cells. Polyamine uptake (pmol/106 cells
x
20 min)
Putrescine
Spermidine
Spermine
L1210
123.8 f 33.2 (100%)
129.9 l 18.7 (100%)
71.4 zt 7.8 (100%)
L1210-MGBG’
5.1 f 0.7 (4.1%)
2.2 l 0.4 (1.7%)
0.5 f 0.1 (0.7%)
The cells were grown as suspension cultures and exposed to radiolabeled polyamines for a 20-min period while in logarithmic growth (1 day after seeding). The rates of uptake of the various polyamines were compared and are presented as percent of the parental L1210 cells. Data shown are means ( •t S.E.M.) of 3 experiments.
addition to slightly prolonging the survival time of the whole population of leukemic mice, the polyamine-deficient diet containing antibiotics resulted in long-term survival or cure of 1 - 3 mice in each experimental group of 10 mice. An L1210 cell line (LlZlO-MGBG’), deficient in the transport of spermidine, has been isolated by selecting for the resistance to the cytotoxic effect of MGBG [lo]. MGBG and spermidine bear a structural resemblance and are taken up by the same transport system (81. However, as shown in Table II, the uptakedeficiency of the LlZlO-MGBG’ cells was not limited to spermidine. Also the capacities to transport putrescine and spermine were markedly reduced in the mutant cell line. The median survival time for female B6D2Fr mice inoculated with 1.0 x lo6 LlZlO-MGBG’ leukemia cells was 12.5 days when they were fed a standard laboratory chow (Fig. 1C). The provision of a polyaminedeficient diet containing antibiotics to the mice gave a slight prolongation of the survival time (Fig. 1C). Contrary to the minor increase in survival caused by the special diet, there was a dramatic therapeutic effect by supplementing the drinking water with DFMO (Fig. lC,D). Thus, 58% of the leukemic mice bearing LlZlO-MGBG’ cells were cured of their disease (Fig. 1D). Providing the mice with a polyamine-deficient diet, containing antibiotics, increased the cure rate obtained by DFMO treatment to 75% (two series; 60% and 90%) respectively) (Fig. 1D). When tak-
ing together all the results obtained with mice bearing L1210-MGBG’ cells, it became apparent that provision of the special diet gave rise to a significant increase in survival time. The effectiveness by which DFMO cured leukemic mice bearing LlZlO-MGBG’ cells appeared to be dependent on the sex of the mice. Thus, the therapeutic effect of DFMO was much better for female than for male mice; 58% of the female mice and 30% of the male mice were cured (Figs. 1D and 2).
-
100T-7.‘:
coamllbdsnm
01 0
10
20
30
40
50
60
Days Fig. 2.
Survival of male B6D2FI mice inoculated with 1.0 x lo6 MGBG-resistant (L1210-MGBG’) leukemia cells and given drinking water without supplements (solid line) or containing 3% DFMO (dashed line). DFMO was provided from day 1 on. One hundred percent corresponds to 10 mice. MST, median survival time (days).
33
Depletion of putrescine and spermidine by treatment with DFMO has been found to reduce the growth rate of all cell lines studied, that is, when the cells are grown in culture. In vivo, however, most cell lines respond poorly to the same treatment. This is not due to DFMO degradation or inability of the drug to reach and enter the target cells in the host. Instead, the poor effect is due to the fact that DFMO treatment and the resulting putrescine and spermidine deficiency elicits a compensatory uptake of extracellular polyamines [l] . This was clearly demonstrated in a previous study, in which we used a polyamine uptake mutant selected from mutagenized L1210 leukemia cells [lo]. Thus, leukemic mice bearing L1210 cells severely deficient in polyamine uptake exhibited a cure rate of 33% when blocked in their polyamine synthesis by DFMO. This should be compared with the 2day increase in survival time resulting from DFMO treatment of leukemic mice bearing the parental L1210 cells with their efficient uptake of extracellular polyamines [lo]. Thus, tumor cells seem to have a very efficient polyamine uptake system, due to which the therapeutic effectiveness of inhibitors of endogenous polyamine synthesis may be severely compromised. In an attempt to limit the availability of extracellular polyamines, Seiler et al. [12] provided tumor-bearing mice with a polyaminedeficient diet containing antibiotics and inhibitors of ODC and polyamine oxidase. The inhibitor of polyamine oxidase was included to prevent polyamine-interconversion reactions [12] and the antibiotics to decontaminate the gastrointestinal from tract polyamineproducing bacteria [5]. The gastrointestinal tract is considered to be the most important exogenous source of polyamines. The approach used by Seiler et al. [12] resulted in a markedly reduced growth rate of intramuscularly inoculated Lewis lung carcinomas and prevented the formation of lung metastases. In the case of L1210 leukemic mice, the antiproliferative effect obtained by
simultaneously limiting the supply of extracellular polyamines and inhibiting polyamine synthesis with DFMO, was not as marked and there were no survivors [ 121. A more impressive therapeutic effect was obtained in the present study, where about 60% of the L1210 leukemic mice survived as a result of prevention of polyamine uptake. Thus, instead of reducing the external pool of polyamines a more effective approach would be to reduce the transport of the polyamines into the tumor cells. However, so far no specific inhibitors of the polyamine transport system have been described. Notably, there was a slight prolongation of the survival time when the mice inoculated with LlZlO-MGBG’ cells were provided with the polyamine-deficient diet containing antibiotics. Since the LlZlO-MGBG’ cells are deficient in their polyamine uptake their utilization of external polyamines should be low. However, although effectively reduced, the uptake of polyamines was still measurable in the mutant cells, which may explain why extracellular polyamine depletion caused an increase in host survival. Another explanation would be that the therapeutic effect of this diet is caused, at least partly, by a mechanism unrelated to the uptake of extracellular polyamines. Neomycin, for example, has been shown to interfere with the generation of second messengers, which may affect cell proliferation [3]. On the basis of our present knowledge it seems that polyamine synthesis inhibitors have strong therapeutic potential provided that the compensatory uptake of extracellular polyamines can be prevented or reduced. Therefore, it is important to characterize the polyamine-uptake system, such that new means may be devised for specific interference with the uptake mechanism. Acknowledgements
This study was supported by the Swedish Natural Science Research Council, the Swedish Cancer Society, the Medical Faculty (University of Lund) and the John and
34
Augusta Persson, the Carl Tesdorpf and the BeAa Kamprad Research Foundatidns. DFMO was a generous gift from the Marion Merrell Dow Research Institute, Cincinnati, Ohio.
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