Adenosine A3 receptor knockout (A3AR KO) mice and their wild-type (WT) counterparts were compared from the point of view of their abilities to survive exposures to lethal doses of γ-radiation belonging to the range of radiation doses inducing the bone marrow acute radiation syndrome. Parameters of cumulative 30-day survival (experiment using a midlethal radiation dose) or cumulative 11-day survival (experiment using an absolutely lethal radiation dose), and of mean survival time were evaluated. The values of A3AR KO mice always reflected their higher survival in comparison with WT ones, the P values being above the limit for statistical significance after the midlethal radiation dose and standing for statistical significance after the absolutely lethal radiation dose. This finding was considered surprising, taking into account the previously obtained findings on defects in numbers and functional properties of peripheral blood cells in A3AR KO mice. Therefore, previous hematological analyses of A3AR KO mice were supplemented in the present studies with determination of serum levels of the granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin. Though distinct differences in these parameters were observed between A3AR KO and WT mice, none of them could explain the relatively high postirradiation survival of A3AR KO mice. Further studies on these mice comprising also those on other than hemopoietic tissues and organs can help to clarify their relative radioresistance.
The purpose of the study was to describe and compare normal and 5-fluorouracil (5-FU)-suppressed hematopoiesis in adenosine A(3) receptor knock-out (A(3)AR KO) mice and their wild-type (WT) counterparts. To meet the purpose, a complex hematological analysis comprising nineteen peripheral blood and bone marrow parameters was performed in the mice. Defects previously observed in the peripheral blood erythrocyte and thrombocyte parameters of the A(3)AR KO mice were confirmed. Compartments of the bone marrow progenitor cells for granulocytes/macrophages and erythrocytes were enhanced in the control, as well as in the 5-FU-administered A(3)AR KO mice. 5-FU-induced hematopoietic suppression, evaluated on day 2 after the administration of the cytotoxic drug, was found to be significantly deeper in the A(3)AR KO mice compared with their WT counterparts, as measured at the level of the bone marrow progenitor cells. The rate of regeneration, as assessed between days 2 and 7 after 5-FU administration, was observed in the population of the granulocyte/macrophage progenitor cells to be higher in the A(3)AR KO mice in comparison with the WT ones. The increased depth of 5-FU-induced suppression in the compartments of the hematopoietic progenitor cells in the A(3)AR KO mice represents probably a hitherto undescribed further consequence of the lack of adenosine A(3) receptors and indicates its synergism with the pharmacologically induced cytotoxic action of 5-FU.
- MeSH
- antimetabolity antitumorózní farmakologie MeSH
- biochemická analýza krve MeSH
- buňky kostní dřeně účinky léků MeSH
- fluorouracil farmakologie MeSH
- hematopoéza účinky léků MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši MeSH
- počet erytrocytů MeSH
- počet leukocytů MeSH
- receptor adenosinový A3 genetika MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The role of the adenosine A3 receptor in hematopoiesis was studied using adenosine A3 receptor knockout (A3AR KO) mice. Hematological parameters of peripheral blood and femoral bone marrow of irradiated and untreated A3AR KO mice and their wild-type (WT) counterparts were investigated. Irradiation of the mice served as a defined hematopoiesis-damaging means enabling us to evaluate contingent differences in the pattern of experimentally induced hematopoietic suppression between the A3AR KO mice and WT mice. Defects were observed in the counts and/or functional parameters of blood cells in the A3AR KO mice. These defects include statistically significantly lower values of blood neutrophil and monocyte counts, as well as those of mean erythrocyte volume, mean erythrocyte hemoglobin, blood platelet counts, mean platelet volume, and plateletcrit, and can be considered to bear evidence of the lack of a positive role played by the adenosine A3 receptor in the hematopoietic system. Statistically significantly increased values of the bone marrow parameters studied in A3AR KO mice (femoral bone marrow cellularity, granulocyte/macrophage progenitor cells, and erythrocyte progenitor cells) can probably be explained by compensatory mechanisms attempting to offset the disorders in the function of blood elements in these mice. The pattern of the radiation-induced hematopoietic suppression was very similar in A3AR KO mice and their WT counterparts.
- MeSH
- hematopoetické kmenové buňky metabolismus MeSH
- hematopoéza fyziologie MeSH
- leukocyty mononukleární metabolismus MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši MeSH
- receptor adenosinový A3 nedostatek MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
There exists a requirement for drugs which would be useful in therapy of an acute radiation damage of a mammalian organism. The aim of the study was to evaluate survival parameters in mice exposed to a lethal γ-ray dose of 8.5 Gy and treated with single doses of an adenosine A(3) receptor agonist, IB-MECA, or a cyclooxygenase-2 (COX-2) inhibitor, meloxicam, administered alone or in a combination early after irradiation, i.e., 0.5 and 1 h post-irradiation, respectively. The assessed parameters were the mean survival time (MST) and the cumulative percentage 30-day survival (CPS). Administrations of single intraperitoneal doses of either IB-MECA 0.5 h post-irradiation or meloxicam 1 h post-irradiation resulted in statistically significant increases of MST in comparison with the control irradiated mice. Combined administration of IB-MECA and meloxicam was found to be the only treatment statistically enhancing the parameter of CPS and to lead to the most expressive increase in MST of the experimental mice. The findings add new knowledge on the action of an adenosine A3 receptor agonist and a COX-2 inhibitor in an irradiated mammalian organism and suggest the potential of both the investigated drugs in the treatment of the acute radiation damage.
- MeSH
- adenosin analogy a deriváty farmakologie MeSH
- agonisté adenosinového receptoru A3 farmakologie MeSH
- časové faktory MeSH
- celotělové ozáření škodlivé účinky MeSH
- cyklooxygenasa 2 metabolismus MeSH
- inhibitory cyklooxygenasy 2 farmakologie MeSH
- lékové interakce MeSH
- míra přežití MeSH
- myši MeSH
- radioprotektivní látky farmakologie MeSH
- receptor adenosinový A3 metabolismus MeSH
- thiaziny farmakologie MeSH
- thiazoly farmakologie MeSH
- záření gama škodlivé účinky MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Influence of the regulatory system mediated by adenosine A(3) receptors on the functioning of erythropoiesis and thrombopoiesis was studied by means of evaluation of the numbers and attributes of peripheral blood erythrocytes and platelets, as well as of erythroid bone marrow progenitor cells in adenosine A(3) receptor knock-out (Adora3(tm1Jbsn)/Adora3(tm1Jbsn), A(3)AR((-/-))) mice and their wild-type C57BL/6 counterparts, both males and females. Minor but statistically significant disturbances in the properties of erythrocytes, namely in the parameters of mean erythrocyte volume and mean erythrocyte hemoglobin were observed in A(3)AR((-/-)) mice. In addition, adenosine A(3) receptor knock-out mice were found to exhibit an expressive, statistically significant decrease of their blood platelet count, amounting to 17 % and 21 % in males and females, respectively. This decrease in platelet levels was accompanied by a significant 17 % decline in the plateletcrit in both sexes. The obtained data can help to define therapeutic applications based on the principle of adenosine receptor signaling.
- MeSH
- erytrocyty cytologie fyziologie MeSH
- erytropoéza fyziologie MeSH
- mezenchymální kmenové buňky cytologie fyziologie MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši MeSH
- receptor adenosinový A3 genetika metabolismus MeSH
- signální transdukce fyziologie MeSH
- trombocyty cytologie fyziologie MeSH
- trombopoéza fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The question as to whether A3 adenosine receptor (A3AR) agonists, N (6)-(3-iodobenzyl)-adenosine-5'-N- methyluronamide (IB-MECA) and 2-chloro-N (6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), could exert cytotoxic effects at high concentrations with or without the involvement of A3AR has been a controversial issue for a long time. The initial findings suggesting that A3AR plays a crucial role in the induction of cell death upon treatment with micromolar concentrations of IB-MECA or Cl-IB-MECA were revised, however, the direct and unequivocal evidence is still missing. Therefore, the sensitivity of Chinese hamster ovary (CHO) cells transfected with human recombinant A3AR (A3-CHO) and their counter partner wild-type CHO cells, which do not express any of adenosine receptors, to micromolar concentrations of IB-MECA and Cl-IB-MECA was studied. We observed that IB-MECA and Cl-IB-MECA exhibited a strong inhibitory effect on cell proliferation due to the blockage of cell cycle progression at G1/S and G2/M transitions in both A3-CHO and CHO cells. Further analysis revealed that IB-MECA and Cl-IB-MECA attenuated the Erk1/2 signalling irrespectively to A3AR expression. In addition, Cl-IB-MECA induced massive cell death mainly with hallmarks of a necrosis in both cell lines. In contrast, IB-MECA affected cell viability only slightly independently of A3AR expression. IB-MECA induced cell death that exhibited apoptotic hallmarks. In general, the sensitivity of A3-CHO cells to micromolar concentrations of IB-MECA and Cl-IB-MECA was somewhat, but not significantly, higher than that observed in the CHO cells. These results strongly suggest that IB-MECA and Cl-IB-MECA exert cytotoxic effects at micromolar concentrations independently of A3AR expression.
- MeSH
- adenosin analogy a deriváty farmakologie MeSH
- agonisté adenosinového receptoru A3 farmakologie MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- cytotoxiny farmakologie MeSH
- kontrolní body buněčného cyklu účinky léků MeSH
- lidé MeSH
- mitogenem aktivovaná proteinkinasa 1 antagonisté a inhibitory genetika metabolismus MeSH
- mitogenem aktivovaná proteinkinasa 3 antagonisté a inhibitory genetika metabolismus MeSH
- proliferace buněk účinky léků MeSH
- protoonkogenní proteiny c-akt genetika metabolismus MeSH
- receptor adenosinový A3 genetika metabolismus MeSH
- regulace genové exprese MeSH
- signální transdukce účinky léků MeSH
- transfekce MeSH
- viabilita buněk účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This study continues our earlier findings on the hematopoiesis-modulating effects of adenosine A1 and A3 receptor agonists that were performed on committed hematopoietic progenitor and precursor cell populations. In the earlier experiments, N (6)-cyclopentyladenosine (CPA), an adenosine A1 receptor agonist, was found to inhibit proliferation in the above-mentioned hematopoietic cell systems, whereas N (6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), an adenosine A3 receptor agonist, was found to stimulate it. The topic of this study was to evaluate the possibility that the above-mentioned adenosine receptor agonists modulate the behavior of early hematopoietic progenitor cells and hematopoietic stem cells. Flow cytometric analysis of hematopoietic stem cells in mice was employed, as well as a functional test of hematopoietic stem and progenitor cells (HSPCs). These techniques enabled us to study the effect of the agonists on both short-term repopulating ability and long-term repopulating ability, representing multipotent progenitors and hematopoietic stem cells, respectively. In a series of studies, we did not find any significant effect of adenosine agonists on HSPCs in terms of their numbers, proliferation, or functional activity. Thus, it can be concluded that CPA and IB-MECA do not significantly influence the primitive hematopoietic stem and progenitor cell pool and that the hematopoiesis-modulating action of these adenosine receptor agonists is restricted to more mature compartments of hematopoietic progenitor and precursor cells.
- MeSH
- agonisté purinergního receptoru P1 farmakologie MeSH
- hematopoetické kmenové buňky účinky léků fyziologie MeSH
- hematopoéza účinky léků fyziologie MeSH
- multipotentní kmenové buňky účinky léků fyziologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- průtoková cytometrie MeSH
- receptor adenosinový A1 metabolismus MeSH
- receptor adenosinový A3 metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In our previous studies, IB-MECA, an adenosine A(3) receptor agonist, was found to stimulate proliferation of hematopoietic progenitor and precursor cells in mice. This property of IB-MECA was considered to be responsible for its ability to support regeneration of suppressed hematopoiesis after irradiation with sublethal doses of γ-rays when the drug was given in a post-irradiation treatment regimen. This study was aimed at assessing the ability of IB-MECA to influence a 30-day survival of lethally irradiated mice. In a series of experiments, IB-MECA was administered following various lethal radiation doses in various numbers of drug doses and various administration routes. Though in some of these experiments a moderate increase in 30-day survival was observed in IB-MECA-treated mice, the differences in comparison with the controls were not significantly different. It can be inferred from these results and those of previous studies assessing the effects of IB-MECA after sublethal radiation doses that IB-MECA can probably influence only a substantially preserved hematopoiesis like that remaining after sublethal irradiation. Future studies should be aimed at evaluation of the abilities of IB-MECA to influence post-irradiation survival when administered as a part of combined treatment regimens.
- MeSH
- adenosin aplikace a dávkování analogy a deriváty farmakologie MeSH
- agonisté adenosinového receptoru A3 aplikace a dávkování farmakologie MeSH
- experimentální radiační poranění metabolismus mortalita MeSH
- hematopoéza účinky léků účinky záření MeSH
- inbrední kmeny myší MeSH
- myši MeSH
- receptor adenosinový A3 metabolismus MeSH
- záření gama MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We studied effects of 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA) on apoptosis induction in the K562/Dox cell line, which overexpressed P-glycoprotein (P-gp, ABCB1, MDR1). We found that the K562/Dox cell line was significantly more resistant to Cl-IB-MECA than the maternal cell line K562, which did not express P-gp. Although both cell lines expressed the A3 adenosine receptor (A3AR), cytotoxic effects of Cl-IB-MECA were not prevented by its selective antagonist MRS1523 (3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2 phenyl-4-propyl-3-pyridine carboxylate). Analysis of cell extracts revealed that the intracellular level of Cl-IB-MECA was significantly lower in the K562/Dox cell line than in the maternal cell line K562. The downregulation of P-gp expression using shRNA targeting ABCB1 gene led to increased intracellular level of Cl-IB-MECA and restored cell sensitivity to this drug. Similarly, valspodar (PSC-833), a specific inhibitor of P-gp, restored sensitivity of the K562/Dox cell line to Cl-IB-MECA with concomitant increase of intracellular level of Cl-IB-MECA in the resistant cell line, while it affected cytotoxicity of Cl-IB-MECA in the sensitive cell line only marginally. An enzyme based assay provided evidence for interaction of P-gp with Cl-IB-MECA. We further observed that cytotoxic effects of Cl-IB-MECA could be augmented by activation of extrinsic cell death pathway by Apo-2L (TRAIL) but not FasL or TNF-α. Our results revealed that Cl-IB-MECA induced an increase in expression of TRAIL receptors in K562 cells, which could sensitize cells to apoptosis induction via an extrinsic cell death pathway. Importantly, these effects were inversely related to P-gp expression. In addition, MRS1523 did not affect Cl-IB-MECA induced expression of TRAIL receptors.
- MeSH
- adenosin analogy a deriváty farmakologie MeSH
- adenosintrifosfatasy metabolismus MeSH
- agonisté adenosinového receptoru A3 farmakologie MeSH
- buněčná membrána účinky léků metabolismus MeSH
- buněčná smrt MeSH
- chemorezistence fyziologie MeSH
- leukemie farmakoterapie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- P-glykoprotein genetika metabolismus MeSH
- receptor adenosinový A3 metabolismus MeSH
- regulace genové exprese u nádorů MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The present studies investigated changes in expression of mRNA for adenosine A1, A2a, A2b, and A3 receptors in samples of HL-60 promyelocytic cells differing in the actual presence of cells in various phases of the cell cycle induced by the double thymidine block method. Real-time PCR technique was used for obtaining data on mRNA expression. Statistical analysis of the data revealed that the mRNA expression of adenosine A1, A2a, and A3 receptors is dependent on the cell cycle phase. G0/G1 and G2/M phases were characterized by a higher mRNA expression of adenosine A1 receptors and a lower one of adenosine A2a and A3 receptors whereas the opposite was true for the S phase. Interestingly, expression of mRNA of the adenosine A2b receptors was independent on the cell cycle phase. The results indicate the plasticity of mRNA expression of adenosine receptors in the investigated promyelocytic cells and its interaction with physiological mechanisms of the cell cycle.
- MeSH
- buněčný cyklus genetika MeSH
- HL-60 buňky MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- messenger RNA metabolismus MeSH
- purinergní receptory P1 genetika metabolismus MeSH
- receptor adenosinový A1 genetika metabolismus MeSH
- receptor adenosinový A2A genetika metabolismus MeSH
- receptor adenosinový A2B genetika metabolismus MeSH
- receptor adenosinový A3 genetika metabolismus MeSH
- S fáze MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
