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Cytotoxic effect of extracellular ATP on L1210 leukemic cells and normal hemopoietic stem cells
~
)Pergamon
Leukemia Research Vol. 18, No. 8, pp. 637-641, 1994
Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0145-2126/94 $7.00 + 0.00
0145-2126(94)E0039-C
CYTOTOXIC EFFECT OF EXTRACELLULAR ATP ON L1210 LEUKEMIC CELLS AND NORMAL HEMOPOIETIC STEM CELLS YOSHIHIRO HAT]?A,* SHIN AIZAWA,t TAKEYOSHIITOH,* MASUMI BABA* and TAKASHI HORIE* *First Department of Internal Medicine, School of Medicine, Nihon University, 30-1, Oyaguchi Kami-machi, Itabashi-ku, Tokyo, 173 Japan; and ?First Department of Internal Medicine, Tokyo Medical College, 6-7-1, Shinjuku, Shinjuku-ku, Tokyo, 160 Japan (Received 20 June 1993. Revision accepted 8 February 1994) Abstract--The cytotoxic effect of extracellular adenosine triphosphate (ATP) was examined on normal murine hemopoietic stem cells and a representative leukemic ceil line (L1210). After L1210 cells were incubated with 4 mM ATP for 3 h, 3H-thymidine incorporation was almost completely inhibited. The number of viable L1210 cells was also significantly decreased and L1210 colony formation was suppressed to approximately 30% of the control level after treatment. The CFU-GM survival rate was reduced to 70%, however, CFU-S and marrow nucleated cell numbers were not changed after the same treatment with ATP. All mice that were injected with the untreated mixture of normal marrow cells (3.3 × 104) and L1210 cells (3.3 × 10 3) died of leukemia within 18 days. On the contrary, 85% of the recipients given ATPtreated grafts survived more than 70 days. These findings indicate that ATP extra vivotreatment is useful for purging the residual leukemic cells in autologous bone marrow transplantation.
Key words: CFU-GM, CFU-S, cell membrane, permeability.
[5]. A similar cytotoxic effect of exogenous ATP on some tumor cell lines (9-L glioma cell [6], EL-4 tumor cell [7], P-815 mastocytoma cell [7], YAC- 1 lymphoma cell [5], and MBL-2 lymphoma cell [5]) has been also revealed. These reports suggest that exogenous ATP may be used as a cytotoxic agent to affect tumor cells. However, the effect of exogenous ATP should be examined on normal hemopoietic stem cells before its clinical application, especially in patients with hematological disorders. No reports have been found about the effect of exogenous ATP on normal hemopoietic stem cells. The effects of chemotherapy agents on hemopoietic stem cells [8, 9] and on leukemic colony-forming stem cells [10, 11] have been studied using semi-solid clonal cultures. L1210 is a murine lymphocytic leukemia cell line, that has been widely used to investigate the in vitro efficacy of anti-tumor agents [12-14] and has been used as a model of purging in autologous bone marrow transplantation [15] because this cell line easily forms colonies in semi-solid cultures [12]. In the present study, we examined the sensitivity to extracellular ATP of L1210 cells and hemopoietic stem cells (CFU-GM and CFU-S) using a clonogenic assay technique. The possibility of in vitro purging of leukemic cells by ATP was revealed.
Introduction ADENOSINE triphosphate (ATP) is one of the nucleotides which is contained mainly in muscle tissue. In ATP, one adenosine molecule binds to three molecules of phosphoric acid, two of which have hyperenergy acid bindings. ATP plays an important role in the intracellular metabolism as a source of high energy inorganic phosphoric acids. On the other hand, the role of extracellular ATP has not been established sufficiently. Recently, several biological activities of extracellular ATP have been found, including the stimulation of mitogenesis in immature murine thymocytes [1], the stimulation of lysozomal enzyme secretion of rabbit polymorphonuclear leukocytes and the promotion of histamine release from mast cells [2, 3], and the inhibition of phagocytosis in human polymorphonuclear leukocytes [4]. A cytotoxic effect of exogenous ATP on lymphocytes was also reported Abbreviations: CFU-GM, colony-forming unit granulocyte/macrophage; CFU-S, colony-forming unit in spleen. Correspondence to: Yoshihiro Hatta, First Department of Internal Medicine, School of Medicine, Nihon University, 30-1, Oyaguchi Kami-machi, Itabashi-ku, Tokyo, 173 Japan.
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638 Materials and M e t h o d s
Cells L1210 cells were maintained in RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum (FCS), 20 ~tU/ml penicillin (GIBCO, Grand Island, NY), and 20 ~tg/ml streptomycin (GIBCO, Grand Island, NY) at 37°C in a fully humidified atmosphere of 5% COz in air. Bone marrow mononuclear cells (BMMNC) were isolated from normal ll-week-old BDF1 male mice after sacrifice by cervical dislocation. The femurs and tibias were aseptically removed and marrow cells were gently flushed out by repeated injection of alpha-MEM (Flow Lab Inc., U.S.A.) through a syringe with a 23-gauge needle. The BMMNC were then isolated by Ficoll-Paque density centrifugation as described previously [16], and were used as a source of hemopoietic stem cells.
Treatment with A TP Adenosine triphosphate (ATP, Sigma Chemical Co., U.S.A.) was dissolved in RPMI 1640 or alpha-MEM, adjusted to pH 7.40 and stored at -40°C until use. Cells supplemented with 10% FCS in medium (1 x 106 cells/ml) were incubated with various concentrations of ATP at 37°C in a 5% CO2 humidified incubator. After 30 min, 3 h and 6 h of incubation, the number of viable cells was counted by trypan blue dye exclusion. Cells were then examined in a 3H-thymidine incorporation assay or in a clonogenic assay.
3H-thymidine incorporation To estimate the leukemic cell activities, 3H-thymidine incorporation was assayed and the result was expressed as the percentage of incorporated radioactivity into the ATPtreated cells compared with that of untreated cells. ATPtreated L1210 cells were washed twice with RPMI 1640 and
2 mM ATP~ 75
~o .~ 50
3mArP
resuspended in the medium with 10% FCS. A 0.2 ml aliquot of this cell suspension was plated into 96-well flat-bottomed culture trays (Coster, U.S.A.) and incubated for 6 h at 37°C under 5% CO2 in air with 0.5 ~tCi of 3H-thymidine (Amersham, U.K.). Then the cells were harvested and the incorporated radioactivity was counted using a liquid scintillation counter.
Clonogenic assay of L1210 cells ATP-treated L1210 cells were washed twice with RPMI 1640, and then incubated in a semi-solid culture medium comprising 10% FCS and 0.8% methylcellulose (Shin-etsu Kagaku, Japan) in RPMI 1640. After 11 days, colonies formed in the dishes were counted by inverted microscopy.
Clonogenic assay of CFU-GM and CFU-S The semi-solid culture technique was used for the CFUGM assay. BMMNC were incubated at 37°C under 5% CO2 in air with alpha-MEM containing 20% FCS, 10% L-cellconditioned medium as a source of colony stimulating factor, and 0.3% agar. After 7 days, the aggregates of 40 or more cells were enumerated on an inverted microscope as CFUGM. The CFU-S assay was performed according to the method of Till & McCullough [17]. Briefly, 11-week-old BDF1 male mice were irradiated with 9 Gy followed by injection of ATP-treated or untreated BMMNC via the tail vein. On day 13, the spleens were removed and fixed in Bouin's solution, and then macroscopic colonies were counted.
Bone marrow transplantation Bone marrow cells (3.3 x 104) from normal male BDF1 mice were mixed with L1210 leukemia cells (3.3 x 103), and were incubated with or without ATP (4 mM) for 3 h as described above. Then the cells were washed with RPMI 1640, injected into lethally irradiated (9 Gy) recipient mice of the same strain via the tail vein. They were not sacrificed and their survival was monitored. When the mice had expired, dissection was performed to determine the cause of death.
Competitive effect of ADP-fl-S on the biological activity of ATP ADP-fl-S is an analogue of ATP and is known to compete with ATP to increase intracellular calcium [18]. To determine whether or not ADP-fl-S inhibited the ATP-mediated cell death competitively, the effects on L1210 cells combining these drugs were examined. L1210 cells were incubated with 4 mM or 40 mM ADP-fl-S (Sigma Chemical Co., USA) in the presence of 4 mM ATP for 3 h. After treatment with ATP and ADP-fl-S, a L1210 clonogenic assay was performed as described previously.
Statistical analysis
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FIG. 1. Effect of ATP on 3H-thymidine incorporation of L1210 cells. The cells were incubated with 1 mM ( . . . . ), 2mM ( ), 3 m M ( ), 4 m M ( ) and 6 m M ( - - . - - ) ATP for 0.5, 3 and 6 h. The 3H-thymidine incorporation is expressed as a percentage of the control value (mean + S.D.).
All experiments were performed at least three times in triplicate or quadruplicate. Values are expressed as the mean +- S.D. The statistical significance of differences was calculated using Wilcoxon's test. Less than 1% difference was considered to be statistically significant. Results
Effect of A TP on 3H-thymidine incorporation T h e 3 H - t h y m i d i n e i n c o r p o r a t i o n o f L1210 cells
Cytotoxic effect of ATP on L1210
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FIG. 2. Effect of ATP on the viable cell number of L1210 cells. Cells were incubated with or without 4 mM ATP for 0.5, 3 and 6 h. The initial cell number was i x 106/ml. Data represent the mean +--S.D.
3 Exposure time (hr)
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FIG. 3. Effect of ATP on the viable cell number of BMMNC. Bone marrow mononuclear cells were incubated with or without 4 mM ATP for 0.5, 3 and 6 h. The initial cell number was 1 × 106/ml. Data represent the mean --- S.D.
1(10 after t r e a t m e n t with various concentrations of A T P is shown in Fig. 1. After incubation for 3 and 6 h with 4 and 6 m M A T P , 3H-thymidine incorporation was almost completely inhibited c o m p a r e d with the untreated control. After 6 h of treatment with A T P , 3H-thymidine incorporation decreased in a concent r a t i o n - d e p e n d e n t manner.
Effect of A TP on cell numbers As shown in Fig. 2, the n u m b e r of viable L1210 cells decreased to 8.40 -+ 1.07 x 105/ml, 8.01 -2-_2.07 x 105/ml, and 7.15 --- 2.26 x 105/ml after t r e a t m e n t with 4 m M A T P for 30 min, 3 and 6 h, respectively. H o w e v e r , a time-dependent decrease was not seen. In untreated cultures, cell numbers in creased to 1.21 --- 0.40 x 106/ml, 1.58 --- 0.19 x 106/ml, and 1.54 +- 0.17 x 106/ml after 30 min, 3 and 6 h of incubation, respectively. The n u m b e r of B M M N C was not altered by 4 m M A T P , even after incubation for 6 h (Fig. 3).
Cytotoxic effect of A TP on L1210 clonogenic cells Following exposure to 4 m M A T P for 3 or 6 h, the n u m b e r of L1210 colonies was reduced to 32.7 --- 9 . 5 % , and 30.4 -+ 2.1% of the untreated level, respectively (Fig. 4). H o w e v e r , the colony size was not smaller than that in the untreated control culture.
Survival of hemopoietic stem cells (CFU-GM and CFU-S) after A TP treatment Figure 5 shows the growth of C F U - G M and CFU-S after incubation of B M M N C at 37°C for up to 6 h in
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FIG. 4. Effect of ATP on L1210 colony growth. L1210 cells were incubated with 4 mM ATP for 3 and 6 h. The results are expressed as the mean +- S.D. of the percent colony growth compared with the control cultures.
the presence of 4 m M A T P . A T P did not alter the colony-forming capacity of CFU-S, but the n u m b e r of C F U - G M decreased with the incubation period. A f t e r incubation with 4 m M A T P for 3 and 6 h, C F U - G M d e v e l o p e d a t 8 1 . 9 +- 6.2% and58.5 +-- 8.7% o f t h e c o n trol level, respectively.
Survival of bone marrow transplanted mice All mice that were injected with the u n t r e a t e d mixture of normal m a r r o w cells (3.3 x 104) and L1210 leukemic cells (3.3 x 103) died within 18 days with
Y. HATTAet al.
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Time (days) FIG. 6. Survival curves of lethally irradiated BDF1 mice transplanted with a mixture of 3.3 × 1 0 4 normal marrow cells and 3.3 x 1 0 3 L1210 leukemic cells. - - - -: graft of a mixture of normal marrow and L1210 cells that had been treated without ATP for 3 h (n = 12). -: graft of a mixture of normal marrow and L1210 cells that had been treated with 4 mM ATP for 3 h (n = 13).
6 Exposure period (hr)
FIG. 5. Effect of 4 mM ATP on normal hemopoietic stem cells (CFU-S and CFU-GM). Bone marrow mononuclear cells were incubated with 4 mM ATP for 3 and 6 h. The results are expressed as the mean -+ S.D. of the percent colony growth compared with the control cultures.
obvious signs of leukemia, that is, enlargement of spleens. H o w e v e r , 85% of recipients given A T P treated mixture cells survived for m o r e than 70 days (Fig. 6).
Effect of ADP-fl-S on A TP-mediated cell death A single administration of ADP-fl-S also suppressed L1210 colony formation in a dose-dependent manner. But this suppression was milder than that of ATP. C o m b i n a t i o n of A T P and ADP-fl-S resulted in synergistic inhibition of L1210 clonogenic cells (Table 1). Discussion T h e cytotoxicity of A T P for a murine lymphocytic leukemia cell line (L1210) and for hemopoietic stem cells ( C F U - G M and CFU-S) was investigated in this study. 3H-thymidine incorporation of L1210 cells was distinctly inhibited in the presence of 4 and 6 m M
ATP. F u r t h e r m o r e , the viable leukemic cell n u m b e r after 4 m M A T P treatment for 3 h was reduced to about 80%, whereas that of untreated leukemic cells increased to about 150%. Since the doubling time of L1210 cells was reported by S h i m o y a m a & Kimura as 1 2 . 2 - 1.2h [12] and that of our cell line was 16.9---3.3 h, this increase of untreated cells was reasonable. Following A T P treatment, the decrease in the n u m b e r of L1210 colonies was greater than that of the total n u m b e r of viable cells. T h e reasons for this discrepancy may be explained by the assumptions that: (1) A T P killed mainly L1210 clonogenic cells; and (2) L1210 cells remained viable immediately after A T P treatment, but later b e c a m e d a m a g e d and died. In our experimental system, it is unclear which of these assumptions was the main factor. There was a smaller inhibitory effect of A T P against hemopoietic stem cells ( C F U - G M and CFU-S) than against L1210 cells. C F U - G M was significantly decreased in a time-dependent m a n n e r by incubation with 4 m M ATP. A dose-dependent reduction of C F U - G M by incubation with A T P had already been reported [16]. C F U - G M was m o r e vulnerable to A T P than CFU-S. The cytotoxic activity of A T P for some t u m o r cell lines has been reported [5-7]. In addition, a similar
TABLE 1. EFFECT OF ATP AND ADP-fl-S ON L1210 CLONOGENICCELLS Agents
Colonies (percent of control)
Control ATP 4 mM ADP-fl-S 4 m M ADP-fl-S 40 mM ATP 4 mM and ADP-fl-S 4 mM ATP 4 mM and ADP-fl-S 40 mM
100.0 32.7 - 9.5 54.3 - 1.6 0 0 0
Cytotoxic effect of ATP on L1210 cytotoxic effect of A T P against lymphoma cells has also been demonstrated to operate through the alteration of m e m b r a n e permeability [5]. It is also known that extracellular A T P induces cation influx, depolarization, and increased cell membrane permeability [19,20]. Furthermore, putative A T P 4 receptors have been reported to be related to ATPmediated cell death. These findings suggest that extracellular A T P alters the potential and permeability of the cell m e m b r a n e in a process, which is mediated by putative A T P 4 - receptors, and that target cells are damaged as a consequence. ADP-/3-S increases intracellular calcium on human blood platelets through P2T-purinoceptors [18]. This effect is competitively inhibited by A T P [18]. In our experiments, ADP-/3-S could not inhibit the suppression of L1210 colony formation induced by ATP. Moreover, A T P and ADP-/3-S act synergistically on L1210 leukemic cells. It is reported that the P2T-purinoceptor is different from the A T P 4 - receptor which is classified as a P2Z-purinoceptor [21]. From these aspects, the inhibitory effects of A T P on L1210 cells are presumed to be mediated by A T P 4 - receptors rather than P2T-purinoceptors. It is unclear why the sensitivity to A T P was different between the leukemic cell line and hemopoietic stem cells. Possible differences in the number or the binding affinity of A T P 4 - receptors on L1210 cells and hemopoietic stem cells should be studied. A T P at high concentrations induced irreversible damage o f leukemic cells without injuring normal hemopoietic stem cells, suggesting the potential clinical application of extracellular A T P for leukemia or other hematological disorders. Treatment with 4 m M A T P for 3 h was the optimum condition in the present study. Most mice injected with a mixture of normal marrow cells and L1210 leukemic cells (10 : 1) treated by this m e t h o d survived long-term. In contrast, all the mice administered untreated mixture cells died early. Although A T P was not entirely successful in purging residual tumor cells, these data favor the use of A T P in autologous bone marrow transplantation. References 1. Gregory S. M. & Kern M. (1978) Adenosine and adenine nucleotides are mitogenic for mouse thymocytes. Biochem. biophys. Res. Commun. 83, 1111. 2. Cockcroft S. & Comperts B. D. (1980) The ATP4receptor of rat mast cells. Biochem. J. 188, 789. 3. DahlquistR. &DiamamntB. (1974) InteractionofATP and calcium on the rat mast cell: effect on histamine release. Acta Pharmac. Toxic. 34, 368. 4. McCord J. M., Petrone W. F. & Jones H. P. (1985) Ecto-ATPase-mediated inhibition of human neutrophil function. Adv. Inflammation Res. 19, 21.
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5. Virgillio F. D., Bronte V., Collavono D. & Zanovello P. (1989) Responses of mouse lymphocytes to extracellular adenosine 5'-triphosphate (ATP). J. Immun. 143, 1955. 6. Miyagi A. (1987) Cell membrane mechanism for regulation of ion changes and uptake of antitumor metal complex in the malignant tumor cells. J. Nihon Univ. Med. Ass. 46, 231 (abstract in English) 7. Fillipini A., RolfE. T., Agui T. & Sitovsky M. V. (1990) Ecto-ATPase activity in cytolytic T-lymphocytes. J. biol. Chem. 265, 334. 8. Ogawa M., Bersagel D. E. & McCulloch E. A. (1973) Sensitivity of human and murine hemopoietic precursor cells to chemotherapeutic agents assessed in cell culture. Blood 42, 851. 9. Horikoshi A., Martin J. & Murphy M. J. Jr (1982) Comparative effects of chemotherapeutic drugs on human and murine hematopoietic progenitors in vitro. Chemotherapy 28,480. 10. Gustavsson A. & Olofsson T. (1984) Prediction of response to chemotherapy in acute leukemia by in vitro drug sensitivity testing on leukemic stem cells. Cancer Res. 44, 4648. 11. Marie J.-P., Zittoun R., Delmer A., Thevenin D. & Sulberville A.-M. (1987) Prognostic value of clonogenic assay for induction and duration of complete remission in acute myelogenous leukemia. Leukemia 1, 121. 12. ShimoyamaM. &KimuraK. (1972) Quantitative clonal growth of mammalian cells. Nippon Kagaku-ryoho Gakkai Zasshi (Chemotherapy) 20, 787 (abstract in English). 13. Takamizawa A., Matsumoto S., Iwata Y., Katagiri K., Tachino Y. & Yamaguchi K. (1973) Studies on cyclophosphamide metabolites and their related compounds I. Preparation of an active species of cyclophosphamide and some related compound. J. A m . Chem. Soc. 95,985. 14. Skipper H. E. (1981) Resultsandinterpretations oftrials in which animals bearing known tumor burdens and mixes of sensitive and drug-resistant leukemia cells were treated with single drug combinations: Publication No.22 (Southern Research Institute. Birmingham, AL). 15. Sieber F., Spivak J. L. & Sutcliffe A. M. (1984) Selective killing of leukemic cells by merocyanine 540-mediated photosensitization. Proc. natn. Acad. Sci. 81, 7584. 16. Hatta Y., Aizawa S., Horikoshi A., Baba M. & Horie T. (1993) Selective killing of murine leukemic cells by adenosine triphosphate (ATP): a study of the value of autologous bone marrow transplantation. Int. Med. 32, 768. 17. Till J. E. & McCulloch E. A. (1961) A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14, 213. 18. Hall D. A. & Hourani S. M. O. (1993) Effects of analogues of adenine nucleotides on increases in intracellular calcium mediated by P2T-purinoceptors on human blood platelets. Br. J. Pharmac. 108, 728. 19. Steinberg T. H. & Silverstein S. C. (1987) Extracellular ATP4- promotes cation fluxes in the J774 mouse macrophage cell line. J. biol. Chem. 262, 3118. 20. Willey J. S. &DuybakG. R. (1989) Extracellular adenosine triphosphate increases cation permeability of chronic lymphocytic leukemic lymphocytes. Blood 73, 1316. 21. Gordon J. L. (1986) Extracellular ATP: effect, sources and fate. Biochem. J. 233, 309.
)Pergamon
Leukemia Research Vol. 18, No. 8, pp. 637-641, 1994
Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0145-2126/94 $7.00 + 0.00
0145-2126(94)E0039-C
CYTOTOXIC EFFECT OF EXTRACELLULAR ATP ON L1210 LEUKEMIC CELLS AND NORMAL HEMOPOIETIC STEM CELLS YOSHIHIRO HAT]?A,* SHIN AIZAWA,t TAKEYOSHIITOH,* MASUMI BABA* and TAKASHI HORIE* *First Department of Internal Medicine, School of Medicine, Nihon University, 30-1, Oyaguchi Kami-machi, Itabashi-ku, Tokyo, 173 Japan; and ?First Department of Internal Medicine, Tokyo Medical College, 6-7-1, Shinjuku, Shinjuku-ku, Tokyo, 160 Japan (Received 20 June 1993. Revision accepted 8 February 1994) Abstract--The cytotoxic effect of extracellular adenosine triphosphate (ATP) was examined on normal murine hemopoietic stem cells and a representative leukemic ceil line (L1210). After L1210 cells were incubated with 4 mM ATP for 3 h, 3H-thymidine incorporation was almost completely inhibited. The number of viable L1210 cells was also significantly decreased and L1210 colony formation was suppressed to approximately 30% of the control level after treatment. The CFU-GM survival rate was reduced to 70%, however, CFU-S and marrow nucleated cell numbers were not changed after the same treatment with ATP. All mice that were injected with the untreated mixture of normal marrow cells (3.3 × 104) and L1210 cells (3.3 × 10 3) died of leukemia within 18 days. On the contrary, 85% of the recipients given ATPtreated grafts survived more than 70 days. These findings indicate that ATP extra vivotreatment is useful for purging the residual leukemic cells in autologous bone marrow transplantation.
Key words: CFU-GM, CFU-S, cell membrane, permeability.
[5]. A similar cytotoxic effect of exogenous ATP on some tumor cell lines (9-L glioma cell [6], EL-4 tumor cell [7], P-815 mastocytoma cell [7], YAC- 1 lymphoma cell [5], and MBL-2 lymphoma cell [5]) has been also revealed. These reports suggest that exogenous ATP may be used as a cytotoxic agent to affect tumor cells. However, the effect of exogenous ATP should be examined on normal hemopoietic stem cells before its clinical application, especially in patients with hematological disorders. No reports have been found about the effect of exogenous ATP on normal hemopoietic stem cells. The effects of chemotherapy agents on hemopoietic stem cells [8, 9] and on leukemic colony-forming stem cells [10, 11] have been studied using semi-solid clonal cultures. L1210 is a murine lymphocytic leukemia cell line, that has been widely used to investigate the in vitro efficacy of anti-tumor agents [12-14] and has been used as a model of purging in autologous bone marrow transplantation [15] because this cell line easily forms colonies in semi-solid cultures [12]. In the present study, we examined the sensitivity to extracellular ATP of L1210 cells and hemopoietic stem cells (CFU-GM and CFU-S) using a clonogenic assay technique. The possibility of in vitro purging of leukemic cells by ATP was revealed.
Introduction ADENOSINE triphosphate (ATP) is one of the nucleotides which is contained mainly in muscle tissue. In ATP, one adenosine molecule binds to three molecules of phosphoric acid, two of which have hyperenergy acid bindings. ATP plays an important role in the intracellular metabolism as a source of high energy inorganic phosphoric acids. On the other hand, the role of extracellular ATP has not been established sufficiently. Recently, several biological activities of extracellular ATP have been found, including the stimulation of mitogenesis in immature murine thymocytes [1], the stimulation of lysozomal enzyme secretion of rabbit polymorphonuclear leukocytes and the promotion of histamine release from mast cells [2, 3], and the inhibition of phagocytosis in human polymorphonuclear leukocytes [4]. A cytotoxic effect of exogenous ATP on lymphocytes was also reported Abbreviations: CFU-GM, colony-forming unit granulocyte/macrophage; CFU-S, colony-forming unit in spleen. Correspondence to: Yoshihiro Hatta, First Department of Internal Medicine, School of Medicine, Nihon University, 30-1, Oyaguchi Kami-machi, Itabashi-ku, Tokyo, 173 Japan.
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638 Materials and M e t h o d s
Cells L1210 cells were maintained in RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum (FCS), 20 ~tU/ml penicillin (GIBCO, Grand Island, NY), and 20 ~tg/ml streptomycin (GIBCO, Grand Island, NY) at 37°C in a fully humidified atmosphere of 5% COz in air. Bone marrow mononuclear cells (BMMNC) were isolated from normal ll-week-old BDF1 male mice after sacrifice by cervical dislocation. The femurs and tibias were aseptically removed and marrow cells were gently flushed out by repeated injection of alpha-MEM (Flow Lab Inc., U.S.A.) through a syringe with a 23-gauge needle. The BMMNC were then isolated by Ficoll-Paque density centrifugation as described previously [16], and were used as a source of hemopoietic stem cells.
Treatment with A TP Adenosine triphosphate (ATP, Sigma Chemical Co., U.S.A.) was dissolved in RPMI 1640 or alpha-MEM, adjusted to pH 7.40 and stored at -40°C until use. Cells supplemented with 10% FCS in medium (1 x 106 cells/ml) were incubated with various concentrations of ATP at 37°C in a 5% CO2 humidified incubator. After 30 min, 3 h and 6 h of incubation, the number of viable cells was counted by trypan blue dye exclusion. Cells were then examined in a 3H-thymidine incorporation assay or in a clonogenic assay.
3H-thymidine incorporation To estimate the leukemic cell activities, 3H-thymidine incorporation was assayed and the result was expressed as the percentage of incorporated radioactivity into the ATPtreated cells compared with that of untreated cells. ATPtreated L1210 cells were washed twice with RPMI 1640 and
2 mM ATP~ 75
~o .~ 50
3mArP
resuspended in the medium with 10% FCS. A 0.2 ml aliquot of this cell suspension was plated into 96-well flat-bottomed culture trays (Coster, U.S.A.) and incubated for 6 h at 37°C under 5% CO2 in air with 0.5 ~tCi of 3H-thymidine (Amersham, U.K.). Then the cells were harvested and the incorporated radioactivity was counted using a liquid scintillation counter.
Clonogenic assay of L1210 cells ATP-treated L1210 cells were washed twice with RPMI 1640, and then incubated in a semi-solid culture medium comprising 10% FCS and 0.8% methylcellulose (Shin-etsu Kagaku, Japan) in RPMI 1640. After 11 days, colonies formed in the dishes were counted by inverted microscopy.
Clonogenic assay of CFU-GM and CFU-S The semi-solid culture technique was used for the CFUGM assay. BMMNC were incubated at 37°C under 5% CO2 in air with alpha-MEM containing 20% FCS, 10% L-cellconditioned medium as a source of colony stimulating factor, and 0.3% agar. After 7 days, the aggregates of 40 or more cells were enumerated on an inverted microscope as CFUGM. The CFU-S assay was performed according to the method of Till & McCullough [17]. Briefly, 11-week-old BDF1 male mice were irradiated with 9 Gy followed by injection of ATP-treated or untreated BMMNC via the tail vein. On day 13, the spleens were removed and fixed in Bouin's solution, and then macroscopic colonies were counted.
Bone marrow transplantation Bone marrow cells (3.3 x 104) from normal male BDF1 mice were mixed with L1210 leukemia cells (3.3 x 103), and were incubated with or without ATP (4 mM) for 3 h as described above. Then the cells were washed with RPMI 1640, injected into lethally irradiated (9 Gy) recipient mice of the same strain via the tail vein. They were not sacrificed and their survival was monitored. When the mice had expired, dissection was performed to determine the cause of death.
Competitive effect of ADP-fl-S on the biological activity of ATP ADP-fl-S is an analogue of ATP and is known to compete with ATP to increase intracellular calcium [18]. To determine whether or not ADP-fl-S inhibited the ATP-mediated cell death competitively, the effects on L1210 cells combining these drugs were examined. L1210 cells were incubated with 4 mM or 40 mM ADP-fl-S (Sigma Chemical Co., USA) in the presence of 4 mM ATP for 3 h. After treatment with ATP and ADP-fl-S, a L1210 clonogenic assay was performed as described previously.
Statistical analysis
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FIG. 1. Effect of ATP on 3H-thymidine incorporation of L1210 cells. The cells were incubated with 1 mM ( . . . . ), 2mM ( ), 3 m M ( ), 4 m M ( ) and 6 m M ( - - . - - ) ATP for 0.5, 3 and 6 h. The 3H-thymidine incorporation is expressed as a percentage of the control value (mean + S.D.).
All experiments were performed at least three times in triplicate or quadruplicate. Values are expressed as the mean +- S.D. The statistical significance of differences was calculated using Wilcoxon's test. Less than 1% difference was considered to be statistically significant. Results
Effect of A TP on 3H-thymidine incorporation T h e 3 H - t h y m i d i n e i n c o r p o r a t i o n o f L1210 cells
Cytotoxic effect of ATP on L1210
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FIG. 2. Effect of ATP on the viable cell number of L1210 cells. Cells were incubated with or without 4 mM ATP for 0.5, 3 and 6 h. The initial cell number was i x 106/ml. Data represent the mean +--S.D.
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FIG. 3. Effect of ATP on the viable cell number of BMMNC. Bone marrow mononuclear cells were incubated with or without 4 mM ATP for 0.5, 3 and 6 h. The initial cell number was 1 × 106/ml. Data represent the mean --- S.D.
1(10 after t r e a t m e n t with various concentrations of A T P is shown in Fig. 1. After incubation for 3 and 6 h with 4 and 6 m M A T P , 3H-thymidine incorporation was almost completely inhibited c o m p a r e d with the untreated control. After 6 h of treatment with A T P , 3H-thymidine incorporation decreased in a concent r a t i o n - d e p e n d e n t manner.
Effect of A TP on cell numbers As shown in Fig. 2, the n u m b e r of viable L1210 cells decreased to 8.40 -+ 1.07 x 105/ml, 8.01 -2-_2.07 x 105/ml, and 7.15 --- 2.26 x 105/ml after t r e a t m e n t with 4 m M A T P for 30 min, 3 and 6 h, respectively. H o w e v e r , a time-dependent decrease was not seen. In untreated cultures, cell numbers in creased to 1.21 --- 0.40 x 106/ml, 1.58 --- 0.19 x 106/ml, and 1.54 +- 0.17 x 106/ml after 30 min, 3 and 6 h of incubation, respectively. The n u m b e r of B M M N C was not altered by 4 m M A T P , even after incubation for 6 h (Fig. 3).
Cytotoxic effect of A TP on L1210 clonogenic cells Following exposure to 4 m M A T P for 3 or 6 h, the n u m b e r of L1210 colonies was reduced to 32.7 --- 9 . 5 % , and 30.4 -+ 2.1% of the untreated level, respectively (Fig. 4). H o w e v e r , the colony size was not smaller than that in the untreated control culture.
Survival of hemopoietic stem cells (CFU-GM and CFU-S) after A TP treatment Figure 5 shows the growth of C F U - G M and CFU-S after incubation of B M M N C at 37°C for up to 6 h in
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FIG. 4. Effect of ATP on L1210 colony growth. L1210 cells were incubated with 4 mM ATP for 3 and 6 h. The results are expressed as the mean +- S.D. of the percent colony growth compared with the control cultures.
the presence of 4 m M A T P . A T P did not alter the colony-forming capacity of CFU-S, but the n u m b e r of C F U - G M decreased with the incubation period. A f t e r incubation with 4 m M A T P for 3 and 6 h, C F U - G M d e v e l o p e d a t 8 1 . 9 +- 6.2% and58.5 +-- 8.7% o f t h e c o n trol level, respectively.
Survival of bone marrow transplanted mice All mice that were injected with the u n t r e a t e d mixture of normal m a r r o w cells (3.3 x 104) and L1210 leukemic cells (3.3 x 103) died within 18 days with
Y. HATTAet al.
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Time (days) FIG. 6. Survival curves of lethally irradiated BDF1 mice transplanted with a mixture of 3.3 × 1 0 4 normal marrow cells and 3.3 x 1 0 3 L1210 leukemic cells. - - - -: graft of a mixture of normal marrow and L1210 cells that had been treated without ATP for 3 h (n = 12). -: graft of a mixture of normal marrow and L1210 cells that had been treated with 4 mM ATP for 3 h (n = 13).
6 Exposure period (hr)
FIG. 5. Effect of 4 mM ATP on normal hemopoietic stem cells (CFU-S and CFU-GM). Bone marrow mononuclear cells were incubated with 4 mM ATP for 3 and 6 h. The results are expressed as the mean -+ S.D. of the percent colony growth compared with the control cultures.
obvious signs of leukemia, that is, enlargement of spleens. H o w e v e r , 85% of recipients given A T P treated mixture cells survived for m o r e than 70 days (Fig. 6).
Effect of ADP-fl-S on A TP-mediated cell death A single administration of ADP-fl-S also suppressed L1210 colony formation in a dose-dependent manner. But this suppression was milder than that of ATP. C o m b i n a t i o n of A T P and ADP-fl-S resulted in synergistic inhibition of L1210 clonogenic cells (Table 1). Discussion T h e cytotoxicity of A T P for a murine lymphocytic leukemia cell line (L1210) and for hemopoietic stem cells ( C F U - G M and CFU-S) was investigated in this study. 3H-thymidine incorporation of L1210 cells was distinctly inhibited in the presence of 4 and 6 m M
ATP. F u r t h e r m o r e , the viable leukemic cell n u m b e r after 4 m M A T P treatment for 3 h was reduced to about 80%, whereas that of untreated leukemic cells increased to about 150%. Since the doubling time of L1210 cells was reported by S h i m o y a m a & Kimura as 1 2 . 2 - 1.2h [12] and that of our cell line was 16.9---3.3 h, this increase of untreated cells was reasonable. Following A T P treatment, the decrease in the n u m b e r of L1210 colonies was greater than that of the total n u m b e r of viable cells. T h e reasons for this discrepancy may be explained by the assumptions that: (1) A T P killed mainly L1210 clonogenic cells; and (2) L1210 cells remained viable immediately after A T P treatment, but later b e c a m e d a m a g e d and died. In our experimental system, it is unclear which of these assumptions was the main factor. There was a smaller inhibitory effect of A T P against hemopoietic stem cells ( C F U - G M and CFU-S) than against L1210 cells. C F U - G M was significantly decreased in a time-dependent m a n n e r by incubation with 4 m M ATP. A dose-dependent reduction of C F U - G M by incubation with A T P had already been reported [16]. C F U - G M was m o r e vulnerable to A T P than CFU-S. The cytotoxic activity of A T P for some t u m o r cell lines has been reported [5-7]. In addition, a similar
TABLE 1. EFFECT OF ATP AND ADP-fl-S ON L1210 CLONOGENICCELLS Agents
Colonies (percent of control)
Control ATP 4 mM ADP-fl-S 4 m M ADP-fl-S 40 mM ATP 4 mM and ADP-fl-S 4 mM ATP 4 mM and ADP-fl-S 40 mM
100.0 32.7 - 9.5 54.3 - 1.6 0 0 0
Cytotoxic effect of ATP on L1210 cytotoxic effect of A T P against lymphoma cells has also been demonstrated to operate through the alteration of m e m b r a n e permeability [5]. It is also known that extracellular A T P induces cation influx, depolarization, and increased cell membrane permeability [19,20]. Furthermore, putative A T P 4 receptors have been reported to be related to ATPmediated cell death. These findings suggest that extracellular A T P alters the potential and permeability of the cell m e m b r a n e in a process, which is mediated by putative A T P 4 - receptors, and that target cells are damaged as a consequence. ADP-/3-S increases intracellular calcium on human blood platelets through P2T-purinoceptors [18]. This effect is competitively inhibited by A T P [18]. In our experiments, ADP-/3-S could not inhibit the suppression of L1210 colony formation induced by ATP. Moreover, A T P and ADP-/3-S act synergistically on L1210 leukemic cells. It is reported that the P2T-purinoceptor is different from the A T P 4 - receptor which is classified as a P2Z-purinoceptor [21]. From these aspects, the inhibitory effects of A T P on L1210 cells are presumed to be mediated by A T P 4 - receptors rather than P2T-purinoceptors. It is unclear why the sensitivity to A T P was different between the leukemic cell line and hemopoietic stem cells. Possible differences in the number or the binding affinity of A T P 4 - receptors on L1210 cells and hemopoietic stem cells should be studied. A T P at high concentrations induced irreversible damage o f leukemic cells without injuring normal hemopoietic stem cells, suggesting the potential clinical application of extracellular A T P for leukemia or other hematological disorders. Treatment with 4 m M A T P for 3 h was the optimum condition in the present study. Most mice injected with a mixture of normal marrow cells and L1210 leukemic cells (10 : 1) treated by this m e t h o d survived long-term. In contrast, all the mice administered untreated mixture cells died early. Although A T P was not entirely successful in purging residual tumor cells, these data favor the use of A T P in autologous bone marrow transplantation. References 1. Gregory S. M. & Kern M. (1978) Adenosine and adenine nucleotides are mitogenic for mouse thymocytes. Biochem. biophys. Res. Commun. 83, 1111. 2. Cockcroft S. & Comperts B. D. (1980) The ATP4receptor of rat mast cells. Biochem. J. 188, 789. 3. DahlquistR. &DiamamntB. (1974) InteractionofATP and calcium on the rat mast cell: effect on histamine release. Acta Pharmac. Toxic. 34, 368. 4. McCord J. M., Petrone W. F. & Jones H. P. (1985) Ecto-ATPase-mediated inhibition of human neutrophil function. Adv. Inflammation Res. 19, 21.
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5. Virgillio F. D., Bronte V., Collavono D. & Zanovello P. (1989) Responses of mouse lymphocytes to extracellular adenosine 5'-triphosphate (ATP). J. Immun. 143, 1955. 6. Miyagi A. (1987) Cell membrane mechanism for regulation of ion changes and uptake of antitumor metal complex in the malignant tumor cells. J. Nihon Univ. Med. Ass. 46, 231 (abstract in English) 7. Fillipini A., RolfE. T., Agui T. & Sitovsky M. V. (1990) Ecto-ATPase activity in cytolytic T-lymphocytes. J. biol. Chem. 265, 334. 8. Ogawa M., Bersagel D. E. & McCulloch E. A. (1973) Sensitivity of human and murine hemopoietic precursor cells to chemotherapeutic agents assessed in cell culture. Blood 42, 851. 9. Horikoshi A., Martin J. & Murphy M. J. Jr (1982) Comparative effects of chemotherapeutic drugs on human and murine hematopoietic progenitors in vitro. Chemotherapy 28,480. 10. Gustavsson A. & Olofsson T. (1984) Prediction of response to chemotherapy in acute leukemia by in vitro drug sensitivity testing on leukemic stem cells. Cancer Res. 44, 4648. 11. Marie J.-P., Zittoun R., Delmer A., Thevenin D. & Sulberville A.-M. (1987) Prognostic value of clonogenic assay for induction and duration of complete remission in acute myelogenous leukemia. Leukemia 1, 121. 12. ShimoyamaM. &KimuraK. (1972) Quantitative clonal growth of mammalian cells. Nippon Kagaku-ryoho Gakkai Zasshi (Chemotherapy) 20, 787 (abstract in English). 13. Takamizawa A., Matsumoto S., Iwata Y., Katagiri K., Tachino Y. & Yamaguchi K. (1973) Studies on cyclophosphamide metabolites and their related compounds I. Preparation of an active species of cyclophosphamide and some related compound. J. A m . Chem. Soc. 95,985. 14. Skipper H. E. (1981) Resultsandinterpretations oftrials in which animals bearing known tumor burdens and mixes of sensitive and drug-resistant leukemia cells were treated with single drug combinations: Publication No.22 (Southern Research Institute. Birmingham, AL). 15. Sieber F., Spivak J. L. & Sutcliffe A. M. (1984) Selective killing of leukemic cells by merocyanine 540-mediated photosensitization. Proc. natn. Acad. Sci. 81, 7584. 16. Hatta Y., Aizawa S., Horikoshi A., Baba M. & Horie T. (1993) Selective killing of murine leukemic cells by adenosine triphosphate (ATP): a study of the value of autologous bone marrow transplantation. Int. Med. 32, 768. 17. Till J. E. & McCulloch E. A. (1961) A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14, 213. 18. Hall D. A. & Hourani S. M. O. (1993) Effects of analogues of adenine nucleotides on increases in intracellular calcium mediated by P2T-purinoceptors on human blood platelets. Br. J. Pharmac. 108, 728. 19. Steinberg T. H. & Silverstein S. C. (1987) Extracellular ATP4- promotes cation fluxes in the J774 mouse macrophage cell line. J. biol. Chem. 262, 3118. 20. Willey J. S. &DuybakG. R. (1989) Extracellular adenosine triphosphate increases cation permeability of chronic lymphocytic leukemic lymphocytes. Blood 73, 1316. 21. Gordon J. L. (1986) Extracellular ATP: effect, sources and fate. Biochem. J. 233, 309.