Adenosine A3 receptor agonist acts as a homeostatic regulator of bone marrow hematopoiesis

Biomedicine & Pharmacotherapy 61 (2007) 356e359 www.elsevier.com/locate/biopha

Original article

Adenosine A3 receptor agonist acts as a homeostatic regulator of bone marrow hematopoiesis Michal Hofer a,*, Milan Pospı´sˇil a, Vladimı´r Znojil b, Jirˇina Hola´ a, Antonı´n Vacek a, Denisa Sˇtreitova´ a a

Laboratory of Experimental Hematology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kra´lovopolska´ 135, CZ-61265 Brno, Czech Republic b Institute of Pathological Physiology, Medical Faculty, Masaryk University, Komenske´ho na´m. 2, CZ-66243 Brno, Czech Republic Received 1 February 2007; accepted 12 February 2007 Available online 8 March 2007

Abstract The present study was performed to define the optimum conditions of the stimulatory action of the adenosine A3 receptor agonist, N6-(3iodobenzyl)adenosine-50 -N-methyluronamide (IB-MECA), on bone marrow hematopoiesis in mice. Effects of 2-day treatment with IBMECA given at single doses of 200 nmol/kg twice daily were investigated in normal mice and in mice whose femoral bone marrow cells were either depleted or regenerating after pretreatment with the cytotoxic drug 5-fluorouracil. Morphological criteria were used to determine the proliferation state of the granulocytic and erythroid cell systems. Significant negative correlation between the control proliferation state and the increase of cell proliferation after IB-MECA treatment irrespective of the cell lineage investigated was found. The results suggest the homeostatic character of the induced stimulatory effects and the need to respect the functional state of the target tissue when investigating effects of adenosine receptor agonists under in vivo conditions. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Adenosine A3 receptor; Cell proliferation; Hematopoiesis

1. Introduction Adenosine is now accepted as an agent participating in the regulation of a wide range of physiological functions including cell proliferation and differentiation [1,2]. In the extracellular space it acts through the cell surface receptors differing by their physiological roles and classified as A1, A2A, A2B and A3 subtypes [3]. It has been previously shown that the pharmacologically mediated activation of adenosine receptors can influence hematopoietic cell populations even under in vivo conditions and in particular its stimulatory effects have deserved attention. Enhancement of hematopoiesis could be induced in normal and myelosuppressed mice either by the Abbreviations: IB-MECA, N6-(3-iodobenzyl)adenosine-50 -N-methyluronamide; 5-FU, 5-fluorouracil. * Corresponding author. Tel.: þ420 5 412 1293; fax: þ420 5 4121 1293. E-mail address: [email protected] (M. Hofer). 0753-3322/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2007.02.010

elevation of extracellular adenosine [4e7] or more recently by the administration of the selective adenosine A3 receptor agonist [8e11]. These studies have indicated that adenosine A3 receptors could be promising targets for treatment of hematological disorders. However, the goal of therapeutical exploration of adenosine receptor modulation can be achieved only after understanding the optimum conditions of the agonist’s action. Because the adenosine receptor signalling is in general homeostatic [12], it can be hypothesized that the potency of the agonist will depend on the functional state of the cell system, i.e. on its different requirements for activating stabilizing regulatory mechanisms. To address this problem, effects of adenosine A3 receptor agonist, N6-(3-iodobenzyl)adenosine-50 -N-methyluronamide (IB-MECA), on the proliferation potential of the bone marrow granulocytic and erythroid progenitor cells were investigated in normal mice and in mice whose hematopoiesis was either depressed or regenerating after pretreatment with the cytotoxic drug 5-fluorouracil

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(5-FU). The results of experiments indicate a negative correlation between the intensity of cell proliferation in the control groups and the proliferation enhancing effects of the IBMECA treatment for the both cells lineages investigated and thus support the above given working hypothesis.

comparisons. In addition, Spearman’s coefficient of rank correlation (rs) was used. The significance level was set at P < 0.05.

2. Materials and methods

Three variants of experiments were performed. These included the investigation of IB-MECA action on femoral bone marrow hematopoiesis in normal mice, whose hematopoiesis operated in a steady state, and in mice which were treated with 5-FU either 3 or 7 days before bone marrow sampling. In all experiments, IB-MECA (200 nmol/kg in a single injection) or the vehicle were administered twice daily for 2 days and the bone marrow sampling was performed 24 h after the last injection. As shown in Fig. 1, total counts of nucleated cells in femoral bone marrow decreased to about 27% of the norm on day 3 after 5-FU administration and exhibited tendency to regeneration at day 7 (about 41% of the norm). Treatment with IB-MECA did not influence these values. The same applies to the values of the granulocytic and erythroid cells, each contributing to the femoral cellularity by about 40 to 50% (data not given). This suggests that the balance between the influx of cells from the progenitor cell compartment and efflux of mature cells into the peripheral blood is not influenced by IB-MECA under the conditions used. To consider effects of the investigated treatments on the proliferation state of the bone marrow cells, proliferation indices calculated as the ratios of the counts of morphologically identifiable proliferative cells to total cell counts were used, separately for the granulocytic and erythroid lineages. The data are summarized in Table 1. In accordance with the expectation, the values of control proliferation indices deeply decreased at day 3 after 5-FU administration because this drug is preferentially toxic to rapidly proliferating cells. At day 7 after 5-FU administration the proliferation indices attained the norm in the case of erythroid cells and exhibited a distinct

B10CBAF1 male mice aged 3 months and weighing in average 30 g were used. The mice were kept under controlled conditions; standardized pelleted diet and HCl-treated tap water were available ad libitum. The use and treatment of the animals followed the European Community Guidelines as accepted principles for the use of experimental animals. The experiments were performed with the approval of the Institute’s Ethics Committee. 2.2. Drugs N6-(3-iodobenzyl)adenosine-50 -N-methyluronamide (IBMECA), the agonist of adenosine A3 receptors, was dissolved initially in dimethyl sulphoxide, then diluted with sterile saline and injected intraperitoneally (i.p.) at single doses of 200 nmol/kg in a volume of 0.2 ml. The final concentration of dimethyl sulphoxide was 2%. The choice of the dose of IB-MECA was based on our former experiments showing that this dose induced cycling of murine hematopoietic progenitor cells under in vivo conditions [10]. 5-Fluorouracil was diluted in saline and injected i.p. at a single dose of 100 mg/kg in a volume of 0.2 ml. The corresponding drug vehicles were used for control injections. All the drugs were obtained from Sigma (St. Louis, MO, USA). 2.3. Hematological methods Mice were sacrificed by cervical dislocation. The femurs were dissected and marrow cells flushed from the bone. Numbers of nucleated cells of the bone marrow were determined using a Coulter Counter (model ZF; Coulter Electronics, UK). Differential counts were performed on marrow smears stained with the May-Gru¨nwald-Giemsa method. For the granulocytic lineage myeloblasts through myelocytes were designated as proliferative cells, metamyelocytes through segmented stages as non-proliferative ones. In case of erythroid lineage proerythroblasts through basophilic erythroblasts were classified as proliferative cells, polychromatic and orthochromatic erythroblasts as non-proliferative ones. Total counts of granulocytic or erythroid cells per femur represent the sum of proliferative and non-proliferative cells. 2.4. Statistics

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NUCLEATED CELLS PER FEMUR X 106

2.1. Animals

3. Results

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15

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DAYS AFTER 5-FLUOROURACIL

The data are given as means  SEM. Experiments were repeated twice to four times and the data were pooled. Manne Whitney rank sum test was used for comparison of the effects and the Holm‘s method was applied to correct for multiple

Fig. 1. Mean (SEM) values of total counts of nucleated cells in femoral bone marrow of normal mice (0) and mice pretreated with 5-FU. Ten to 20 mice per group were used. Open columns relate to control mice, full columns to mice treated with IB-MECA.

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Table 1 Effects of IB-MECA on proliferation indices of bone marrow cells Experimental conditions

Control

IB-MECA

Factor of increase

Granulocytic cells A Normal steady state B Day 3 after 5-FU, cell depletion C Day 7 after 5-FU, cell regeneration

0.120  0.009 0.020  0.005 0.385  0.049

0.174  0.011* 0.183  0.053** 0.386  0.034

1.452  0.148 9.079  3.645 1.002  0.158

0.203  0.012

0.208  0.012

1.025  0.088

0.043  0.007 0.224  0.028

0.135  0.025* 0.289  0.028

3.083  0.770 1.292  0.197

Erythroid cells A Normal steady state B Day 3 after 5-FU, cell depletion C Day 7 after 5-FU, cell regeneration

Proliferation indices were calculated as the ratios of the counts of morphologically recognizable proliferating cells to total cell counts. Data are given as means SEM; 10 to 20 mice per group were used. Statistical significance: *P < 0.01, **P < 0.001 vs. vehicle-treated control. When comparing the control proliferation indices, significant differences (P < 0.001) were found in the case of granulocytic cells between groups AeB, AeC, and BeC, in the case of erythroid cells between groups AeB and BeC. In all calculations P values were corrected for multiple comparisons by the Holm’s procedure.

overshoot in the case of granulocytic cells, probably due to the higher demands to produce mature short lived granulocytes. Treatment with IB-MECA strongly increased proliferation indices in both cell lineages in the state of cell depletion on day 3 after 5-FU administration and moderately also in granulocytic cells of normal mice. Proliferation indices in the state of cell regeneration on day 7 after 5-FU administration were not influenced by IB-MECA treatment. Thus, the results indicate that the proliferation-enhancing effects of IB-MECA are dependent on the functional state of the cell system. In order to demonstrate clearly the homeostatic character of the stimulatory effects of IB-MECA, factors of increase were related to the control proliferation indices irrespective of the experimental conditions used and cell lineages investigated. As shown in Fig. 2, significant negative correlation between these two parameters was observed. 4. Discussion and conclusions

FACTOR OF INCREASE AFTER IB-MECA TREATMENT

Our interpretation of the presented findings is based on the presumption that the expression of adenosine receptors is variable and depends on the functional state of the cell system. Such a possibility has been proposed and verified by findings in the 10

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areas of cardiac physiology [13,14] and tumor pathology [15]. It is tempting to speculate that in the cell depletion phase of bone marrow hematopoiesis higher levels of extracellular adenosine generated by cellular damage and impairment of the balance of energy supply/demand trigger higher expression of adenosine A3 receptors by the surviving cells. Consequently, the external administration of the agonist can be more effective. Interestingly, the positive effects of IB-MECA on cell proliferation were found in both the granulocytic and erythroid cell lineages. This suggests the role of some lineage- non-specific regulatory mechanisms controlling cell proliferation. Some earlier experiments [8,9] have shown that treatment with IB-MECA enhances serum levels of the granulocyte colony-stimulating factor and indicate that this mechanism might be responsible for the granulopoiesis-stimulating effects of this adenosine A3 receptor agonist. However, the findings presented here, which suggest the cell lineage-independent action of IB-MECA, leave the question of its cell proliferation-enhancing mechanisms as a still open problem. It is worth noting that IB-MECA has been found to induce cytostatic effects in various tumor cell systems in vitro and in vivo [16]. The association of the stimulatory action of IBMECA on hematopoiesis with its inhibitory effects on tumors is interesting not only from practical point of view (myelosuppression belongs to undesirable complications of chemo- and radiotherapy of tumors) but raises also fundamental questions concerning the role of A3 receptor signalling in normal and malignant cell renewal systems. In conclusion, our data indicate that IB-MECA acts in the hematopoietic system as a homeostatic regulator whose cell proliferation-stimulating effects are dependent on the functional state of the cell system. The phenomenon of the homeostatic efficiency of the receptor activation is probably not specific for hematopoiesis and might be considered as generally applicable to other functions modulated by adenosine receptor signalling.

CONTROL PROLIFERATION INDEX Fig. 2. Relationship between the mean control proliferation indices and mean factors of increase after IB-MECA treatment (data from Table 1). Open circles relate to granulocytic cells, full circles to erythroid cells. Correlation coefficient rs ¼  0.943 (P < 0.05).

Acknowledgements The work was supported by the grants from the Grant Agency of the Czech Republic (grant no. 305/06/0015) and

M. Hofer et al. / Biomedicine & Pharmacotherapy 61 (2007) 356e359

from the Grant Agency of the Academy of Sciences of the Czech Republic (grant no. AV0Z50040507). The authors thank Ms. Kveˇta La´nı´kova´ for her excellent technical assistance. References [1] Abbracchio MP. P1 and P2 receptors in cell growth and differentiation. Drug Dev Res 1996;39:393. [2] Schulte G, Fredholm BB. Signalling from adenosine receptors to mitogen-activated protein kinases. Cell Signal 2003;15:813. [3] Fredholm BB, Arslan G, Halldner L, Kull B, Schulte G, Wasserman W. Structure and function of adenosine receptors and their genes. NaunynSchmiedeberg’s Arch Pharmacol 2000;362:364. [4] Pospı´sˇil M, Hofer M, Znojil V, Va´cha J, Netı´kova´ J, Hola´ J. Synergistic effect of granulocyte colony-stimulating factor and drugs elevating extracellular adenosine on neutrophil production in mice. Blood 1995;86:3692. [5] Hofer M, Pospı´sˇil M, Netı´kova´ J, Znojil V, Va´cha J. Granulocyte colonystimulating factor and drugs elevating extracellular adenosine act additively to enhance the hemopoietic spleen colony formation in irradiated mice. Physiol Res 1999;48:37. [6] Pospı´sˇil M, Hofer M, Vacek A, Netı´kova´ J, Hola´ J, Znojil V, et al. Drugs elevating extracellular adenosine enhance cell cycling of hematopoietic progenitor cells as inferred from the cytotoxic effects of 5-fluorouracil. Exp Hematol 2001;29:557. [7] Hofer M, Pospı´sˇil M, Znojil V, Vacek A, Weiterova´ L, Hola´ J, et al. Drugs elevating extracellular adenosine promote regeneration of haematopoietic progenitor cells in severely myelosuppressed mice: their

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