Impaired post-irradiation survival of cyclooxygenase-2-deficient mice.

We investigated and evaluated post-irradiation survival in cyclooxygenase-2-deficient (COX-2 KO) mice. Thirty-day survival following exposure of COX-2 KO mice to a lethal dose of 8.5 Gy of gamma-rays was observed to be statistically significantly lower in both males and females, as well as when the sexes were merged, in comparisons with their wild-type counterparts. These findings were related to the previous observations concerning the detrimental influence of the COX-2 genetic disruption on hematopoiesis in sublethally irradiated mice. Deteriorated post-irradiation survival of COX-2 KO mice confirmed the previously anticipated conclusion regarding negative influence of the antiinflammatory action of COX-2 deficiency under the conditions of exposure of the animals to ionizing radiation.

Hematological findings in non-treated and gamma-irradiated mice deficient for MIC-1/GDF15.

Several members of the TGF-beta family are known to effectively regulate the fate of hematopoietic progenitor cells in a complex and context-dependent manner. Growth differentiation factor-15 (GDF15) is a divergent member of the TGF-beta family. This stress-induced cytokine has been proposed to possess immunomodulatory functions and its high expression is often associated with progression of a variety of pathological conditions. GDF15 is also induced by chemotherapy and irradiation. Very few fundamental studies have been published regarding the effect of GDF15 in hematopoiesis. In this study, we analyzed the hematological status of untreated and gamma-irradiated mice deficient for GDF15 as a result of genetic knock-out (KO), in order to clarify the regulatory role of GDF15 in hematopoiesis. Significant differences between GDF15 KO mice and their pertinent WT controls were found in the parameters of blood monocyte numbers, blood platelet size, and distribution width, as well as in the values of bone marrow granulocyte/macrophage progenitor cells. Different tendencies of some hematological parameters in the GDF15 KO mice in normal conditions and those under exposure of the mice to ionizing radiation were registered. These findings are discussed in the context of the GDF15 gene function and its lack under conditions of radiation-induced damage.

Hematological profile of untreated or ionizing radiation-exposed cyclooxygenase-2-deficient mice.

We investigated hematopoiesis in untreated and ionizing radiation-exposed cyclooxygenase-2-deficient (COX-2 KO) mice. We performed a complex hematological analysis of 16 parameters in untreated COX-2 KO male mice or COX-2 KO male mice irradiated with the dose of 4 Gy of gamma-rays and their wildtype littermates. At baseline, hematopoiesis was increased in COX-2-deficient mice, but attenuated by irradation in COX-2-deficient mice compared to wildtype. To conclude, the anti-inflammatory action of the COX-2 genetic disruption plays a positive role in hematopoiesis under basal conditions but is detrimental following radiation exposure.

Hematopoiesis in 5-fluorouracil-treated adenosine A(3) receptor knock-out mice.

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.

Erythropoiesis- and thrombopoiesis-characterizing parameters in adenosine A3 receptor knock-out mice.

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.

IB-MECA, an adenosine A(3) receptor agonist, does not influence survival of lethally γ-irradiated mice.

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.

Expression of mRNA for adenosine A(1), A(2a), A(2b), and A(3) receptors in HL-60 cells: dependence on cell cycle phases.

The present studies investigated changes in expression of mRNA for adenosine A(1), A(2a), A(2b), and A(3) 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 A(1), A(2a), and A(3) receptors is dependent on the cell cycle phase. G(0)/G(1) and G(2)/M phases were characterized by a higher mRNA expression of adenosine A(1) receptors and a lower one of adenosine A(2a) and A(3) receptors whereas the opposite was true for the S phase. Interestingly, expression of mRNA of the adenosine A(2b) 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.

A single dose of an inhibitor of cyclooxygenase 2, meloxicam, administered shortly after irradiation increases survival of lethally irradiated mice.

This study extends earlier findings of the authors demonstrating that meloxicam, a selective inhibitor of cyclooxygenase 2, supports hematopoietic recovery in sublethally irradiated mice and is radioprotective when given before irradiation. We report here that when meloxicam was administered in a single dose 1 h after a lethal 9-Gy whole-body dose, an increased 30-day survival was achieved. Additional studies showed that administration of meloxicam 24 h after lethal irradiation is ineffective and its repeated administration deleterious. Possible mechanisms of the therapeutic effects of meloxicam administered early after irradiation are discussed.

Effects of adenosine on the growth of murine G:5:113 fibrosarcoma cells in vitro.

It has been observed that adenosine suppresses the growth of G:5:113 murine fibrosarcoma cells in vitro with EC50 of 178 mM. Changes in the cell cycle including decreased percentage of cells in S-phase, increased portion of cells in G0/G1-phase, as well as prolonged generation time were found to be responsible for the growth suppression. Dipyridamole, a drug inhibiting the cellular uptake of adenosine, enhanced the growth suppression induced with adenosine in concentrations of 100 and 200 microM. It follows from these results that the action of adenosine on the G:5:113 cells is extracellular, mediated by adenosine receptors. Elevation of extracellular adenosine might serve potentially as an anticancer therapeutic agent.

Administration of liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE) and diclofenac in the combination attenuates their anti-tumor activities.

The anti-tumor effects of i.p. administered cyclooxygenase inhibitor - diclofenac and i.v. administered liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE) were investigated using a s.c. growing murine fibrosarcoma tumor. Tumor growth was assessed by measuring tumor volumes and survival of the mice. Both of the drugs were administered either alone or in combination. Repeated application of diclofenac in two schedules (150 microg/mouse/day for 14 consecutive days or 2 x 150 microg/mouse/week for 4 weeks) or application of liposomal MTP-PE (2 x 20 microg/mouse/week for 4 weeks) starting on day 5 after tumor cell transplantation significantly suppressed the tumor growth and increased the percentage of surviving mice. However, the volume of tumors and the survival time in tumor bearing mice treated with the two agents were similar to untreated counterparts. Thus, these data suggest the anti-tumor activity of either of the two drugs is lost when they are used in combination. Hematological examinations confirmed previously observed hematopoiesis-stimulating activities of the drugs when given alone. However, mutually potentiating effects after combined administration of liposomal MTP-PE and diclofenac were observed only exceptionally. Our findings corroborate the recommendation that the interactions of drugs used for the treatment of tumors must be carefully checked, if the drugs are applied in combination.

Lipoxygenase inhibitors suppress proliferation of G5:113 fibrosarcoma cells in vitro but they have no anticancer activity in vivo.

Nordihydroguaiaretic acid (NDGA) and esculetin, both nonspecific inhibitors of lipoxygenases (LOX), were found to suppress expressively the in vitro proliferation of fibrosarcoma cells G5:113 in concentrations ranging from 10 to 50 microM. Subsequent flow-cytometric analysis of the cell cycle showed that both these drugs significantly decreased the percentage proportion of cells in the G0/G1-phase and simultaneously increased significantly this proportion in the S-phase. No apoptosis was detected in the whole range of concentrations studied, from 2.5 to 50 mM. On the contrary, in experiments in vivo, neither NDGA nor esculetin had any curative effect if they were repeatedly injected intraperitoneally (i.p.) into mice bearing tumors growing from subcutaneously (s.c.) transplanted G5:113 cells. Pretreatment of the fibrosarcoma cells with NDGA or esculetin in vitro preceding their s.c. transplantation into mice did not result in suppression of the tumor growth, either. Finally, if G5:113 cells were injected intravenously and the mice were subsequently treated repeatedly with i.p. injections of NDGA, decreased survival and increased number of surface lung metastases were observed in the NDGA-treated group. Thus the suppressive action of inhibitors of LOX on the growth of fibrosarcoma cells in vitro was not reflected in their anti-tumor effects in vivo.

Lipoxygenase inhibitors induce arrest of tumor cells in S-phase of the cell cycle.

Inhibitors of the lipoxygenase pathway of arachidonic acid metabolism represent a potential anti-tumor drugs. These compounds have been found to inhibit the growth and induce the apoptosis of various tumor cells both in vitro and in vivo. In this study, the effects of the lipoxygenase inhibitors esculetin and nordihydroguaiaretic acid (NDGA) on the progression of the cell cycle were investigated in eight mammalian cell lines of different origin. Flow cytometric analyses of cell cycle distribution after staining of DNA with propidium iodide or 7-aminoactinomycin D and DNA synthesis using incorporation of 5-bromo-2'-deoxy-uridine showed that both esculetin and NDGA suppress cell growth by interrupting the progression of cells through S-phase that results in their accumulation in this phase of the cell cycle. The possible mechanisms of these effects and the significance of the findings for the improvement of anticancer therapy targeted on cell cycle is discussed.

Hematopoiesis-stimulating and anti-tumor effects of repeated administration of diclofenac in mice with transplanted fibrosarcoma cells.

Positive effects of repeated administration of diclofenac, an inhibitor of prostaglandin synthesis, in terms of prevention of tumor development and stimulation of hematopoiesis have been observed in C3H mice transplanted subcutaneously with G:5:113 fibrosarcoma cells. Fourteen-day treatment with diclofenac (3.75 microg/kg/day) started from day 5 after tumor cell transplantation. Measurements of tumors and hematological examinations were performed on day 30. The results strongly suggest the possibility that inhibitors of prostaglandin synthesis (non-steroidal anti-inflammatory drugs) may be used in oncological practice where the observed effects are highly desirable.

Hemopoiesis-stimulating action of adamantylamide dipeptide: kinetics of increase of GM-CFC in femur and co-stimulating activity of serum, role of bone marrow stromal cells.

Chief part of hemopoietic stromal cells in mediating hemopoiesis-stimulating effects of adamantylamide dipeptide (AdDP), a synthetic immunomodulatory compound, has been determined in a series of combined in vivo/in vitro studies. Indirect stimulatory effect of AdDP on proliferation of hemopoietic progenitor cells for granulocytes and macrophages (GM-CFC) was proved to be mediated by the cells of hemopoietic microenvironment growing as adherent stromal cell populations in vitro. These results supplement previously reported findings of a positive role which is played by AdDP at modulating the interplay among stimulatory cytokines and their cellular sources, and are in consent with the idea to introduce AdDP as a constituent of the hemopoiesis- and immunity-stimulating supportive medical care.

Influence of age and K, Mg aspartate (Cardilan) on murine haemopoiesis.

We have studied the effect of age as well as the effect of short-term and long-term intake of K and Mg salts of aspartic acid (Cardilan) on haemopoiesis in ICR mice strain. The cellularity of the bone marrow does not change with aging, but the number of granulocyte-macrophage colony-forming cells (GM-CFC) and also the number of spleen colony-forming units (CFU-S) and erythroid burst-forming units (BFU-E) in two-year-old mice increased in the bone marrow. In two-year-old mice the number of leukocytes decreased in the peripheral blood with aging, mainly as a result of a decrease in mononuclear cells. Short-term drinking (STD) of Cardilan caused increased numbers of CFU-S and BFU-E in bone marrow and increased numbers of reticulocytes in the peripheral blood of one-year old animals (STD/12 months old). In the oldest mice (STD/24) increased weight and cellularity of the spleen and rapid increase of leukocytes and reticulocytes in the peripheral blood was recorded. After long-term drinking (LTD) of Cardilan the number of spleen GM-CFC rose markedly in one-year-old mice (LTD/12) and in two-year-old mice (LTD/24) the number of reticulocytes in the peripheral blood rose. Our results indicate that K and Mg salts of aspartic acid influence erythropoietic activity most widely.