Lenka Weiterová

Drugs elevating extracellular adenosine promote regeneration of haematopoietic progenitor cells in severely myelosuppressed mice: their comparison and joint effects with the granulocyte colony-stimulating factor.

Abstract: We tested capabilities of drugs elevating extracellular adenosine and of granulocyte colony‐stimulating factor (G‐CSF) given alone or in combination to modulate regeneration from severe myelosuppression resulting from combined exposure of mice to ionizing radiation and carboplatin. Elevation of extracellular adenosine was induced by joint administration of dipyridamole (DP), a drug inhibiting the cellular uptake of adenosine, and adenosine monophosphate (AMP), serving as an adenosine prodrug. DP + AMP, G‐CSF or all these drugs in combination were administered in a 4‐d treatment regimen starting on day 3 after induction of myelosuppression. Comparable enhancements of haematopoietic regeneration due to elevation of extracellular adenosine or to action of G‐CSF were demonstrated as shown by elevated numbers of haematopoietic progenitor cells for granulocytes/macrophages (GM‐CFC) and erythrocytes (BFU‐E) in the bone marrow and spleen in early time intervals after termination of the drug treatment, i.e. on days 7 and 10 after induction of myelosuppression. Coadministration of all the drugs further potentiated the restoration of progenitor cell pools in the haematopoietic organs. The effects of the drug treatments on progenitor cells were reflected in the peripheral blood in later time intervals of days 15 and 20 after induction of myelosuppression, especially as significantly elevated numbers of granulocytes and less pronounced elevation of lymphocytes and erythrocytes. The results substantiate the potential of drugs elevating extracellular adenosine for clinical utilization in myelosuppressive states, e.g. those accompanying oncological radio‐ and chemotherapy.

Meloxicam, a Cyclooxygenase 2 Inhibitor, Supports Hematopoietic Recovery in Gamma-Irradiated Mice

Abstract Hofer, M., Pospíšil, M., Znojil, V., Holá, J., Vacek, A., Weiterová, L., Štreitová, D. and Kozub;aaik, A. Meloxicam, a Cyclooxygenase 2 Inhibitor, Supports Hematopoietic Recovery in Gamma-Irradiated Mice. Radiat. Res. 166, 556–560 (2006). Meloxicam, a selective inhibitor of cyclooxygenase 2, a nonsteroidal anti-inflammatory drug with an improved side-effects profile in terms of gastrointestinal toxicity, has been found to stimulate hematopoiesis in whole-body γ-irradiated mice. A distinct corroboration of this positive action of meloxicam is an enhancement of the recovery of hematopoietic progenitor cells committed to granulocyte-macrophage and erythroid development, which has been demonstrated in sublethally irradiated animals treated with meloxicam at a dose of 20 mg/kg administered intraperitoneally either singly 1 h before irradiation or repeatedly after radiation exposure. The results suggest that meloxicam can be added to the list of biological response modifiers that can be used in the treatment of hematopoietic damage induced by ionizing radiation.

Drugs elevating extracellular adenosine enhance cell cycling of hematopoietic progenitor cells as inferred from the cytotoxic effects of 5-fluorouracil

OBJECTIVE Our previous studies showed that the combined administration of drugs elevating extracellular adenosine, i.e., dipyridamole and adenosine monophosphate (AMP), enhanced hematopoiesis in normal mice and increased hematopoietic recovery in irradiated mice. In the present study, we have examined the possibility that these effects are due to the adenosine-induced cycling of the hematopoietic progenitor cells. MATERIALS AND METHODS Experiments were performed under in vivo conditions using B10CBAF1 mice. The cycling status of hematopoietic progenitor cells (CFU-S(day 10), CFC-GM, and BFU-E) was determined on the basis of their sensitivity to 5-fluorouracil (5-FU), a cycle-specific cytotoxic agent. RESULTS Pretreatment of mice with dipyridamole + AMP enhanced the cytotoxic effects of a single bolus of 5-FU at a dose of 3 mg per mouse. Sensitizing effects of drugs occurred after a delay of several hours and attained a maximum of about 40-60% reduction of the progenitor cells surviving after 5-FU alone. The period of maximum sensitization of CFU-S by the combination of dipyridamole + AMP was shifted to later time intervals as compared with the effects on CFC-GM and BFU-E. Pretreatment of mice with the drugs also aggravated the 5-FU-induced lethality. Reduction of survival was found in mice exposed to two cycles of 3 mg of 5-FU following the pretreatment with dipyridamole + AMP at a time period characterized by the highest fraction of CFU-S in the S phase. CONCLUSIONS The results suggest that adenosine receptor signaling, induced by the administration of drugs elevating extracellular adenosine, enhances cycling of the hematopoietic progenitor cells. These effects might have pharmacological implications in the therapy of blood disorders.

Radioprotective action of extracellular adenosine on bone marrow cells in mice exposed to gamma rays as assayed by the micronucleus test

Abstract Hofer, M., Mazur, L., Pospíšil, M., Weiterová, L. and Znojil, V. Radioprotective Action of Extracellular Adenosine on Bone Marrow Cells in Mice Exposed to Gamma Rays as Assayed by the Micronucleus Test. The frequency of micronucleated polychromatic erythrocytes (PCEs) in mouse bone marrow was assessed after administration of dipyridamole and/or adenosine monophosphate (AMP) to nonirradiated mice or to mice irradiated 15 min later with a sublethal dose of 6.5 Gy γ rays. In nonirradiated mice, the administration of the drugs increased the frequency of micronucleated PCEs significantly (by 108%). In contrast, in irradiated mice, the number of radiation-induced micronucleated PCEs was significantly decreased if the mice had been pretreated with dipyridamole or AMP alone (by 24% after administration of each of the compounds) and in particular after administration of the drugs in combination (by 36%).

The role of adenosine receptor agonists in regulation of hematopoiesis.

The review summarizes data evaluating the role of adenosine receptor signaling in murine hematopoietic functions. The studies carried out utilized either non-selective activation of adenosine receptors induced by elevation of extracellular adenosine or by administration of synthetic adenosine analogs having various proportions of selectivity for a particular receptor. Numerous studies have described stimulatory effects of non-selective activation of adenosine receptors, manifested as enhancement of proliferation of cells at various levels of the hematopoietic hierarchy. Subsequent experimental approaches, considering the hematopoiesis-modulating action of adenosine receptor agonists with a high level of selectivity to individual adenosine receptor subtypes, have revealed differential effects of various adenosine analogs. Whereas selective activation of A1 receptors has resulted in suppression of proliferation of hematopoietic progenitor and precursor cells, that of A3 receptors has led to stimulated cell proliferation in these cell compartments. Thus, A1 and A3 receptors have been found to play a homeostatic role in suppressed and regenerating hematopoiesis. Selective activation of adenosine A3 receptors has been found to act curatively under conditions of drug- and radiation-induced myelosuppression. The findings in these and further research areas will be summarized and mechanisms of hematopoiesis-modulating action of adenosine receptor agonists will be discussed.

Two New Faces of Amifostine: Protector from DNA Damage in Normal Cells and Inhibitor of DNA Repair in Cancer Cells

Amifostine protects normal cells from DNA damage induction by ionizing radiation or chemotherapeutics, whereas cancer cells typically remain uninfluenced. While confirming this phenomenon, we have revealed by comet assay and currently the most sensitive method of DNA double strand break (DSB) quantification (based on γH2AX/53BP1 high-resolution immunofluorescence microscopy) that amifostine treatment supports DSB repair in γ-irradiated normal NHDF fibroblasts but alters it in MCF7 carcinoma cells. These effects follow from the significantly lower activity of alkaline phosphatase measured in MCF7 cells and their supernatants as compared with NHDF fibroblasts. Liquid chromatography-mass spectrometry confirmed that the amifostine conversion to WR-1065 was significantly more intensive in normal NHDF cells than in tumor MCF cells. In conclusion, due to common differences between normal and cancer cells in their abilities to convert amifostine to its active metabolite WR-1065, amifostine may not only protect in multiple ways normal cells from radiation-induced DNA damage but also make cancer cells suffer from DSB repair alteration.

The BRCA1 alternative splicing variant Δ14-15 with an in-frame deletion of part of the regulatory serine-containing domain (SCD) impairs the DNA repair capacity in MCF-7 cells

The BRCA1 gene codes for a protein involved in the DNA double strand break (DDSB) repair. Alongside the dominant full-length splicing form of BRCA1, numerous endogenously expressed alternative splicing variants of unknown significance have been described in various tissues. Some of them retain the original BRCA1 reading frame but lack several critical BRCA1 structural domains, suggesting an altered function of the resulting protein in the BRCA1-regulated processes. To characterize the effect of the BRCA1Δ14-15 splicing variant (with an in-frame deletion affecting the regulatory serine-containing domain) on the DDSB repair, we constructed the MCF-7 clones stably expressing the analyzed variant with/without a shRNA-mediated downregulation of the endogenous full-length wild-type BRCA1 expression. Our results show that the expression of the BRCA1Δ14-15 variant delays the γ-radiation-induced DDSB repair, alters the kinetics of irradiation-induced foci formation/decomposition and reduces the non-homologous end-joining capacity in MCF-7 cells. Therefore, the BRCA1Δ14-15 is not able to functionally replace the full-length wt BRCA1 in the DDSB repair. Our findings indicate that the endogenously expressed BRCA1 alternative splicing variants may negatively influence genome stability and support the growing evidence of the pathological potential of the sequence variants generated by an altered or misregulated alternative splicing in the process of mammary malignant transformation.

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.

Inhibition of cyclooxygenase-2 promotes the stimulatory action of adenosine A3 receptor agonist on hematopoiesis in sublethally γ-irradiated mice

Mouse hematopoiesis, suppressed by a sublethal dose of ionizing radiation, was the target for combined therapy with a cyclooxygenase-2 (COX-2) inhibitor meloxicam and an adenosine A₃ receptor agonist IB-MECA. The drugs were administered in an early postirradiation treatment regimen: meloxicam was given in a single dose 1hour after irradiation, IB-MECA in two doses 24 and 48hours after irradiation. Treatment-induced changes in several compartments of hematopoietic progenitor and precursor cells of the bone marrow were evaluated on day 3 after irradiation. Values of hematopoietic progenitor cells for granulocytes/macrophages and erythrocytes (GM-CFC and BFU-E, respectively), as well as those of proliferative granulocytic cells were found to be significantly higher in the mice treated with the drug combination in comparison to irradiated controls and attained the highest increase factors of 1.6, 1.6, and 2.6, respectively. The study emphasizes the significance of the combined treatment of suppressed hematopoiesis with more agents. Mechanisms of the action of the individual compounds of the studied drug combination and of their joint operation are discussed.

Stimulatory Action of Cyclooxygenase Inhibitors on Hematopoiesis: A Review

The presented review summarizes experimental data obtained with a mouse model when investigating the relationship between inhibition of prostaglandin production and hematopoiesis. While prostaglandin E2 acts in a negative feedback control of myelopoiesis, inhibition of cyclooxygenases, responsible for its production, shifts the feedback to positive control. Based on these relationships, agents inhibiting cyclo-oxygenases, known as non-steroidal anti-inflammatory drugs (NSAIDs), can activate hematopoiesis and be protective or curative under myelosuppressive states. The effectiveness of therapeutic use of NSAIDs in these situations is expressive especially under the selective inhibition of cyclooxygenase-2 (COX-2), when undesirable side effects of cyclooxygenase-1 inhibition, like gastrointestinal damage, are absent. The effects of the clinically approved selective COX-2 inhibitor, meloxicam, were investigated and demonstrated significant hematopoiesis-stimulating and survival-enhancing actions of this drug in sublethally or lethally γ-irradiated mice. These effects were connected with the ability of meloxicam to increase serum levels of the granulocyte colony-stimulating factor. It can be inferred from these findings that selective COX-2 inhibitors might find their use in the treatment of myelosuppressions of various etiologies.