Marrow Progenitor Cells Research Articles

Leukemogenesis of the EVI1/MEL1 Gene Family

Leukemia is a group of monoclonal diseases that arise from hematopoietic stem and progenitor cells in the bone marrow or other hematopoietic organs. Retroviral infections are one of the major events leading to leukemogenesis in mice, because retroviruses can induce hematopoietic disease via the insertional mutagenesis of oncogenes; therefore, the cloning of viral-integration sites in murine leukemia has provided valuable molecular tags for oncogene discovery. Transcription of the murine gene ecotropic viral-integration site 1 (Evi1) is activated by nearby viral integration. In humans, the Evi1 homologue EVI1 is activated by chromosomal translocations. This review discusses the roles of the overexpression of EVI1/MEL1 gene family members in leukemogenesis, the relationships of various translocations in EVI1 overexpression, and the importance of PR domains in tumor suppression and oncogenesis. The functions of EVI1/MEL1 members as transcription factors and the concept of EVI1-positive leukemia as a stem cell disease are also reviewed.

Transcription Profiles of Failing and Non-Failing Hearts after Two-Cycle RNA Amplification from Biopsy-Sized Samples

Introduction: Recent findings report the potential for bone marrow(BM) or cardiac-derived stem and progenitor cell mobilization and differentiation to repair the infarcted myocardium. Following myocardial infarction (MI), there is an influx of inflammatory cells and secretion of cytokines, leading to cardiomyocyte death and ventricular remodeling. Mobilized c-Kitþ progenitor cells, either from the BM or cardiac niches, play a key role in maintenance of the number of myocytes in aging or postMI. We focused on determining the role of the c-Kit receptor in long term remodeling post-MI. Methods and Results: Functional repair in c-kit mutant (W/W) and littermate wild type (WT) mice post-MI was compared by evaluating in vivo left ventricular (LV) pressure-volume performance and pathology at 5 and 24 weeks post-MI. Evaluation of LV end-systolic and diastolic volume (ESV and EDV) showed a significant worsening of heart function and greater left ventricular dilatation at 5 and 24 weeks post-MI in W/W mice compared to WT (Figure 1). Similarly, the W/W sham and MI operated groups developed greater LV dilatation at 24 weeks when compared to W/W sham and MI mice at 5 weeks. Interestingly, WT sham and MI mice did not demonstrate any significant worsening of heart function at 24 weeks when compared to 5 weeks post-MI. Conclusion: This demonstrates the importance of c-Kit receptors in maintaining heart function post-MI and in aging. These findings provide further evidence indicating the presence of BMor cardiac residentderived stem progenitor cells in maintenance of myocardial health with age and post-injury. 133

SDF1-3′A Gene Polymorphism Is Associated with Chronic Myeloproliferative Disease and Thrombotic Events

Stromal cell-derived factor-1 (SDF-1), a member of the CXC chemokine family, is constitutively produced by bone marrow stromal cells, and it induces chemotaxis on a variety of cell types that contain the G-protein–linked receptor CXCR4. SDF-1 plays a key role in hematopoiesis, and its involvement in migration, homing, and survival of hematopoietic progenitors has been well established. It is the first chemoattractant agent reported for human CD34+ progenitor cells (1), and it promotes CD34+ cell survival by counteracting apoptosis (2). SDF-1 may also have a role in neoangiogenesis. It increases production of vascular endothelial growth factor (VEGF), the main angiogenic factor, which in turn enhances the expression of CXCR4 receptor in endothelial cells, in an autocrine loop (3). SDF-1 may participate in atherothrombosis. It is a potent chemoattractant of monocytes and lymphocytes (4) and has a direct effect on platelet activation (5). SDF-1 is also involved in the neovascularization and angiogenesis response that occurs during formation of unstable atherosclerotic plaques (6), in which increased expression of this chemokine has been found (5). The 2 isoforms, SDF-1α and SDF-1β, arise from a single gene by alternative splicing (7). Sequence analysis reveals a common polymorphism in the 3′-untranslated region (3′-UTR), implicated in mRNA turnover regulation, of the SDF -1β gene transcript, which contains a G→A transition at position 801, designated SDF1-3′UTR-801G-A, abbreviated as SDF1-3′A (8). It has been reported that SDF1-3′A genotype action involves up-regulation of the quantity of SDF-1 protein available to bind CXCR4 (7). In addition, the SDF1-3′A polymorphism has been associated with high mobilization of CD34+ progenitor cells into peripheral blood (9). Philadelphia-negative chronic myeloproliferative disorders (CMDs) are characterized by clonal proliferation of hematopoietic progenitor cells in the bone marrow and by extramedullary hematopoiesis associated with increased CD34+ circulating cells (10)(11)(12 …

Protein Phosphatase2A Regulates Stromal Cell- Derived Factor-1 Mediated Responses of CD34+ Cord Blood Cells.

The chemokine stromal cell- derived factor-1(SDF-1) and its receptor, CXCR4, play a major role in migration, retention, and development of hematopoietic progenitors in the bone marrow. During steady-state hematopoiesis, CXCR4/SDF-1 interaction restricts hematopoietic stem progenitor cells in marrow and disruption of the SDF-1/CXCR4 axis leads to their egress into circulation. However, biological responses to SDF-1 are rapidly attenuated by overlapping mechanisms that include peptide degradation by cell surface proteases, receptor uncoupling from heterotrimeric G-proteins and receptor endocytosis. It is known that phosphorylation of G-protein coupled receptors (GPCRs) by several protein kinases including serine-threonine kinase, protein kinase C (PKC), can result in receptor desensitization. This prompted us to investigate whether the role of serine- threonine phosphatase in the regulation of SDF-1 induced responses of CD34+ cord blood (CB) cells. To investigate the role of serine- threonine phosphatase we evaluated the effect of okadaic acid, a specific serine-threonine phosphatase inhibitor, on SDF-1 induced chemotaxis and adhesion of CD34+ cells from cord blood. Pre-incubation of CD34+ CB cells with okadaic acid (100–1000nM) significantly reduced SDF-1 directed chemotaxis and adhesion of CD34+ CB cells. This correlated with an increase in PKC phosphorylation. Although primary SDF-1 induced calcium flux in CD34+CB cells was unaffected by okadaic acid, if the cells were pretreated with SDF-1 and then stimulated with SDF-1, calcium flux was significantly less in CD34+CB cells pretreated with okadaic acid compared to untreated cells. Further, confocal microscopy was used to demonstrate the co-localization of serine threonine phosphatase and PKC. Using antibodies specific to PP2A catalytic subunit (PP2Ac) and various classes of PKC super family, it was observed in unstimulated CD34+ CB cells PP2Ac was mainly localized in nucleus and some phosphor-PKC-delta was detected in the cytoplasm. However, upon SDF-1 stimulation, co-localization of PP2Ac and phospho-PKC-delta was observed on the membrane of CD34+CB cells. These results indicate that serine-threonine phosphatase regulate SDF-1 mediated responses in CD34+CB cells and may play an important role in the ability of hematopoietic stem progenitor cells to continually respond to SDF-1.

Role of Parenteral Nutrition in Cancer Patients Undergoing High-Dose Chemotherapy Followed by Autologous Peripheral Blood Progenitor Cell Transplantation

High-dose chemotherapy followed by autologous bone marrow or peripheral blood progenitor cell transplantation represents a recognized option in the treatment of solid tumors and hematologic diseases. Patients receiving high-dose chemotherapy are traditionally supported with parenteral nutrition with the aim to prevent malnutrition secondary to gastrointestinal toxicity and metabolic alterations induced by the conditioning regimens. Nevertheless, well-defined guidelines for its use in this clinical setting are lacking and there are several areas of controversy.

Uncaria tomentosa extract increases the number of myeloid progenitor cells in the bone marrow of mice infected with Listeria monocytogenes

In this study, we demonstrated that Uncaria tomentosa extract (UTE) protects mice from a lethal dose of Listeria monocytogenes when administered prophylactically at 50, 100, 150 and 200 mg/kg for 7 days, with survival rates up to 35%. These doses also prevented the myelosuppression and the splenomegaly caused by a sublethal infection with L. monocytogenes, due to increased numbers of granulocyte-macrophage progenitors (CFU-GM) in the bone marrow. Non-infected mice treated with 100 mg/kg UTE also presented higher numbers of CFU-GM in the bone marrow than the controls. Investigation of the production of colony-stimulating factors revealed increased colony-stimulating activity (CSA) in the serum of normal and infected mice pre-treated with UTE. Moreover, stimulation of myelopoiesis and CSA occurred in a dose-dependent manner, a plateaux being reached with the dose of 100 mg/kg. Further studies to investigate the levels of factors such as IL-1 and IL-6 were undertaken. We observed increases in the levels of IL-1 and IL-6 in mice infected with L. monocytogenes and treated with 100 mg/kg of UTE. White blood cells (WBC) and differential counting were also performed, and our results demonstrated no significant changes in these data, when infected mice were pre-treated with 100 mg/kg of UTE. All together, our results suggest that UTE indirectly modulates immune activity and probably disengages Listeria-induced supression of these responses by inducing a higher reserve of myeloid progenitors in the bone marrow in consequence of biologically active cytokine release (CSFs, IL-1 and IL-6).

673. Injection of Progenitor Cells from the Bone Marrow or Esophagus Prevents Irradiation-Induced Esophagitis

To demonstrate whether progenitor or stem cells exist in esophagus, which can prevent irradiation-induced esophagitis, esophageal stem cells were isolated from C57BL/6J mice using a side population (SP) technique used in the isolation of hematopoietic stem cells. To determine whether hematopoietic or esophageal stem cells migrated to areas of esophageal damage, C57BL/6J mice were irradiated to 29 Gy to the esophagus and injected with whole bone marrow as well as esophageal SP cells from GFP+ transgenic mice. Mice were sacrificed 14 days later, the esophagus removed, and green foci were detected in the esophagus, indicating that the donor cells migrated and proliferated in the esophagus. To determine whether injections of whole bone marrow cells protected against irradiation-induced damage to the esophagus, groups of C57BL/6J mice were irradiated to 29 Gy and then injected intravenously (IV) with whole bone marrow isolated from GFP+ mice (1×106, 5×105, 1×105, or 5×104 bone marrow cells per mouse), and followed for development of esophagitis. The results demonstrated a significant increase in survival in mice that were injected with either 1×106 or 5×105 whole bone marrow cells. GFP+ esophageal squamous cells were identified in the transplanted groups demonstrating that cells from whole bone marrow replace damaged squamous cells preventing the development of esophagitis. To demonstrate that esophageal SP cells repopulated the esophagus of irradiation damaged mice, C57BL/6J mice were irradiated to 29 Gy to the esophagus and injected IV with 1000 GFP+ esophageal SP cells and followed for development of esophagitis. Mice injected with GFP+ esophageal SP cells survived and did not develop esophagitis. In contrast, non-injected mice died of esophagitis. The results demonstrate that esophageal SP cells can prevent lethal irradiation-induced esophageal damage. It is also suggested that bone marrow stem or progenitor cells may provide a resource for treating esophageal irradiation injury.

The effect of immunosuppression on bone marrow-derived recipient cells in murine cardiac allografts

Cardiac TVD is the major cause of graft failure in patients surviving more than 1 year post-transplantation. TVD is believed to manifest from an interplay between immunological and non-immunological factors, including dyslipidemia and immunosuppressive drugs. Together, these factors trigger events resulting in vascular injury represented by the formation of concentric, lipid-rich plaques. Endothelial cell (EC) injury is an initiating step of the disease process and mounting evidence suggests ECs, as well as smooth muscle cells (SMC), in lesions of allograft vessels, as well as the organ intersitium, derive in part from recipient bone marrow (BM) progenitor cells. We hypothesize that recipient BM-derived cells contribute to the allogeneic and reparative response in the arteries and myocardium of cardiac allografts. This project is designed to characterize the abundance, localization and fate of recipient BM-derived cells in cardiac allografts, as well as the effect of immunosuppression on these recipient cells. 129J murine hearts were transplanted heterotopically into C57BL/6 mice expressing green fluorescent protein (GFP) in either BM cells. A subset of transplanted mice were immunosuppressed with FK506 (6 mg/kg/day). Mice were sacrificed on days 14 and 30. Hearts were examined using confocal and multi-photon microscopy. Recipient BM-derived cells localized to vessel walls and myocardium of transplanted hearts and demonstrated significantly increased seeding to the transplanted heart as compared to native heart controls in immunosuppressed and non-immunosuppressed allograft mice (p 0.05). Non-immunosuppressed and immunosuppressed mice showed 54% and 27% BM-derived recipient cell seeding to the transplanted heart, respectively, as determined on a denominator of total cardiac nuclei. Preliminary data suggests a subset of these cells also stain for vessel wall markers in allograft hearts. Thus, our data suggests recipient BM-derived cells migrate to the transplanted heart early after transplantation. Their functional roles and fate remains to be determined.

Anti-CD45–mediated cytoreduction to facilitate allogeneic stem cell transplantation

The CD45 antigen is present on all cells of the hematopoietic lineage. Using a murine model, we have determined whether a lytic CD45 monoclonal antibody can produce persistent aplasia and whether it could facilitate syngeneic or allogeneic stem cell engraftment. After its systemic administration, we found saturating quantities of the antibody on all cells expressing the CD45 antigen, both in marrow and in lymphoid organs. All leukocyte subsets in peripheral blood were markedly diminished during or soon after anti-CD45 treatment, but only the effect on the lymphoid compartment was sustained. In contrast to the prolonged depletion of T and B lymphocytes from the thymus and spleen, peripheral blood neutrophils began to recover within 24 hours after the first anti-CD45 injection and marrow progenitor cells were spared from destruction, despite being coated with saturating quantities of anti-CD45. Given the transient effects of the monoclonal antibody on myelopoiesis and the more persistent effects on lymphopoiesis, we asked whether this agent could contribute to donor hematopoietic engraftment following nonmyeloablative transplantation. Treatment with anti-CD45 alone did not enhance syngeneic engraftment, consistent with its inability to destroy progenitor cells and permit competitive repopulation with syngeneic donor stem cells. By contrast, the combination of anti-CD45 and an otherwise inactive dose of total-body irradiation allowed engraftment of H2 fully allogeneic donor stem cells. We attribute this result to the recipient immunosuppression produced by depletion of CD45(+) lymphocytes. Monoclonal antibodies of this type may therefore have an adjunctive role in nonmyeloablative conditioning regimens for allogeneic stem cell transplantation.

Effect of leukotactin-1 on the protection in vivo of myeloid progenitor cells against cytotoxic chemotherapeutics.

Leukotactin-1 (Lkn-1) is a human CC chemokine that induces chemotaxis of neutrophils, monocytes, eosinophils, and lymphocytes and suppresses colony formation of myeloid progenitor cells in vitro. Present studies evaluated the myeloprotective capabilities of Lkn-1 in vivo against Ara-C and 5-fluorouracil (5-FU). The effect of Lkn-1 on myelopoiesis was first assessed in vivo by injecting recombinant Lkn-1 in C3H/HeJ mice. Lkn-1 rapidly decreased cycling rates and absolute numbers of myeloid progenitor cells in marrow. Lkn-1 administration prior to and during the chemotherapeutics treatment resulted in increased progenitors for colony-forming units-granulocyte/macrophage (CFU-GM), colony-forming units-granulocyte/erythroid/megakaryocyte/macrophage (CFU-GEMM), and burst-forming units-erythroid (BFU-E) compared with a saline-treated group. The protective effects lasted until day 3 after the termination of Ara-C administration and until day 7 after the termination of 5-FU administration. The results indicate that Lkn-1 protects bone marrow myeloid progenitor cells when cytotoxic chemotherapeutics are used in a preclinical setting. These results may be of use in clinical treatment for myeloprotection.

176Time-temperature study of cell survival in Bone Marrow (BM) and Blood hematopoietic Progenitor Cell (HPC) grafts

176Time-temperature study of cell survival in Bone Marrow (BM) and Blood hematopoietic Progenitor Cell (HPC) grafts

HEMOPOIESIS-STIMULATING EFFECTS AND ENHANCED SURVIVAL OF IRRADIATED MICE AFTER PERORAL OR INTRAPERITONEAL ADMINISTRATION OF ULTRAFILTERED PIG LEUKOCYTE EXTRACT (UPLE, IMUNOR®)

ABSTRACTUltrafiltered pig leukocyte extract (UPLE, IMUNOR®, ImunomedicA, Ústí nad Labem, Czech Republic) administered perorally (p.o.) or intraperitoneally (i.p.) enhanced recovery of the pool of granulocyte-macrophage hemopoietic progenitor cells (GM-CFC) in the bone marrow of normal or sublethally irradiated mice and increased survival of mice exposed to a lethal radiation dose. In experiments in vitro, sera of mice treated with UPLE p.o. or i.p. induced GM-CFC colony formation in cultures of normal mouse bone marrow cells, i.e., produced colony-stimulating activity (CSA). UPLE alone did not induce GM-CFC colony growth, i.e., had no CSA. When UPLE alone or sera of mice administered UPLE p.o. or i.p. were added to bone marrow cultures containing suboptimal concentration of recombinant mouse interleukin-3 (rmIL-3), both UPLE and the sera increased the counts of GM-CFC colonies in comparison with cultures containing only rmIL-3, i.e., produced co-stimulating activity (CoSA). Based on the findings obtained in vitro, it can be hypothesized that the described CSA and CoSA of UPLE may play a role also under in vivo conditions; enhancement of the recovery of hemopoiesis suppressed by ionizing radiation may be due to co-operation of the stimulatory effects of UPLE with the action of cytokines endogenously produced in irradiated tissues.

Multiple-pool cell lifespan model of hematologic effects of anticancer agents.

The leukopenic effects of anticancer agents are described using a semi-physiologic multiple-pool cell lifespan model. The time course of myelosuppression in relation to the drug concentration vs. time profile was characterized using a three pool indirect model. The proliferation and maturation stages of myeloid cells in the bone marrow and cell removal from the circulation were quantitated with a cell life-span concept. Drug effects were assumed to take place in the bone marrow based on irreversible linear or capacity-limited cytotoxicity. Mathematical derivations and computer simulations (Adapt II) were used to examine the properties of the model. Data from the literature were also analyzed. Cell response profiles after therapy typically exhibit a lag period, reduction to a nadir, and return to baseline. The predicted values of the time periods of granulopoiesis were 10-14 days for proliferation, and 1-6 days for maturation of progenitor cells in the bone marrow. The proposed irreversible mechanism of cell killing by anticancer drugs explains previously observed relationships between leukocyte nadir counts and exposure to the drug and/or duration of drug concentrations above some threshold level. The model was applied to literature data for paclitaxel and etoposide effects on leukocyte counts. The predicted value of KC50 for paclitaxel ranged from 0.004 to 0.2 microgram/mL and for etoposide 2 micrograms/mL. The present model accounts for drug-induced leukopenia using a physiologic cell production and loss model and irreversible cytotoxicity in a precursor pool.

Effect of Continuous γ-Irradiation at Low Doses on Clonogenic Hemopoietic (CFU-S) and Stromal (CFU-F) Cells of Bone Marrow

We studied the effects of low doses of continuous γ-irradiation (Co60, 10 days, mean daily dose power 1.5-2.0 mGy, total dose 15 mGy) on hemopoietic and stromal progenitor cells of murine bone marrow. The content of hemopoietic clonogenic cells representing a “younger” (CFU-S-11) and more “mature” (CFU-S-7) categories in the compartment of stem cells was determined in the bone marrow. The state of bone marrow stroma was estimated by the method of in vitro cloning according to the number of progenitor cells that form colonies of fibroblasts (CFU-F) and by the method of ectopic transplantation according to the capacity of stroma of organizing and building new hemopoietic territories. Continuous γ-irradiation at low doses, that were by one order of magnitude lower than those inducing hermesis, exerted a stimulating effect on both hemopoietic (CFU-S) and stromal (CFU-F) progenitor cells. The number of CFU-S in the compartment of stem cells of the bone marrow markedly increased and they formed larger hemopoietic territories but these cells appeared to create a qualitatively different microenvironment, which stimulated the proliferation of CFU-S.

MODULATION BY ACANTHOSPERMUM AUSTRALE EXTRACTS OF THE TUMOR INDUCED HEMATOPOIETIC CHANGES IN MICE

ABSTRACTPrevious studies on the Ehrlich ascites tumor (EAT) model indicate that tumor progression is associated with reduced myelopoiesis and increased extramedullar hematopoiesis. In order to investigate the in vivo antitumor activity of Acanthospermum australe, its hydroalcoholic extract was partitioned with different solvents and the resulting extracts were monitored by their effects on bone marrow and spleen hematopoietic progenitor cell proliferation and differentiation in EAT-bearing mice. Oral treatment of tumor-bearing mice with 3 doses of 100, 500 and 1000 mg/kg of the crude hydroalcoholic extract and its chloroformic, butanolic and aqueous fractions significantly stimulated myelepoiesis and brought extramedullar hematopoiesis back to near control values. In normal mice, stimulation of myelopoiesis was only observed with the crude and the butanolic extracts. All the extracts at 500 mg/kg significantly increased survival of tumor-bearing mice, however a clear survival advantage in the group treated with the butanolic extract was observed. These results suggest that A. australe may exert effects on myelopoiesis that may be implicated in antitumor immune responses.

Alpha-2,6-sialylation regulation in CD34+ progenitor cells in the human bone marrow and granulocyte colony-stimulating factor mobilization.

The use of peripheral blood (PB) progenitor cells to reconstitute hematopoiesis after high-dose chemotherapy is now common practice in the treatment of malignancies. Growth factors such as granulocyte colony-stimulating factor (G-CSF) induce high proliferation and self-renewal within the stem cell compartment of the bone marrow. This is followed by the release of a very high number of progenitor cells into the peripheral blood, the mechanism of which remains unclear. Previously, we have showed that high cell-surface sialylation reduces matured myeloid attachment to the bone marrow stroma, which is likely to help their release into the peripheral blood. Consequently, we decided to investigate the possibility that sialylation may also contribute to the CD34(+) cell release into the peripheral blood during G-CSF treatment. We found that G-CSF-mobilized PB CD34(+) cells exhibit an increase in the sialyltransferase ST6Gal-I mRNA level. The analysis of the heterogeneity of ST6Gal-I transcripts showed a predominance of the Y form in both bone marrow (BM) and G-CSF PB CD34(+) cells. Higher levels in ST6Gal-I mRNA resulted in a higher expression of alpha-2,6-sialylation on the surface of G-CSF PB CD34(+) cells when compared to noninduced PB CD34(+) cells but not higher to BM CD34(+) cells. Our present results, together with recent observations, suggest that ST6Gal-I and alpha-2,6-sialylation expression in myeloid cells is tightly dependent on the presence of G-CSF in their environment. Up-regulation of ST6Gal-I together with alpha-2,6-sialylation of cell surface glycoproteins is likely to modulate cellular adherence and survival of progenitor cells.

Congenital amegakaryocytic thrombocytopenia (CAMT) - a defect of the thrombopoietin receptor c-Mpl

Congenital amegakaryocytic thrombocytopenia (CAMT) is a very rare bone marrow failure syndrome presenting with isolated hypomegakaryocytic thrombocytopenia at birth developing into a pancytopenia during the first years of life. Bone marrow transplantation is the only curative therapy for this disease so far. Thrombopoietin (TPO) is the most important hematopoietic growth factor for the regulation of megakaryopoiesis and thrombopoiesis. We investigated TPO production and reactivity in CAMT patients. TPO plasma levels were high like in other forms of thrombocytopenia due to ineffective megakaryopoiesis. However, we found a defective reactivity to TPO: Neither hematopoietic progenitor cells in the bone marrow nor platelets from the peripheral blood did respond to TPO. Flow cytometric investigations demonstrated a lack of expression of the TPO receptor c-Mpl on the surface of platelets. Accordingly, we found mutations in the c-mpl gene, which are predicted to lead to a complete or at least partial loss of function of the TPO receptor. TPO is not only involved in the regulation of megakaryocytopoiesis but also in early hematopoiesis. This seems to be the reason for the general defect in hematopoiesis in CAMT leading to the development of pancytopenia.

Bone marrow stromal cells of bcl-2 transgenic mice express widespread Bcl-2 protein and reduce apoptosis in a serum-free medium

P225 Stem cell transplantation is a vibrant area of research with the goal of treatment of uncurable diseases. Bone marrow stromal cells (MSCs, including marrow stem and progenitor cells) have been employed to improve neurological functional recovery after stoke and injury. Grafted cell death is a common feature of intracerebral transplantation. Bcl-2 has been shown to protect a variety of cell types from apoptoic cell death. We tested the hypothesis that MSCs harvested from bcl-2 transgenic mice promote survival and decrease apoptosis of cells in vitro. MSCs were harvested from adult male C57BL/6 transgenic mice (n=4) overexpressing bcl-2, and from normal control C57BL/6 mice (n=4). Cells were cultured in regular medium, Iscove s Modified Dulbecco s medium (IMDM) supplemented with 10% fetal bovine serum (FBS); and then isolated by their adherence to the plastic dishes from non-adherent hematopoietic cells at 72 h of incubation. To examine the effect of bcl-2 on cell death induced by serum deprivation. MSCs were washed and incubated with the serum-free medium. Immunostaining and Western blot were used to identify Bcl-2 protein expression, and TUNEL staining was used to identify in situ DNA fragmentation of apoptotic cells. Immunocytochemical staining indicated that 100% of MSCs harvested from bcl-2 transgenic mice express Bcl-2 protein. Western blot showed that Bcl-2 protein was 8 times more intense in the MSCs harvested from transgenic mice than that from normal control mice at 12 days cultured in regular medium. After 48 h cultured in the serum-free medium, 5.8% of MSCs from bcl-2 transgenic mice were presented apoptoic morphology, compared with 39% of MSCs from normal controls. Our finding demonstrates that the overexpression of bcl-2 gene in the MSCs reduces apoptotic cell death.

Influence of the joint treatment with granulocyte colony-stimulating factor and drugs elevating extracellular adenosine on erythropoietic recovery following 5-fluorouracil-induced haematotoxicity in mice.

The presented data address the problem of pleiotropic effects of granulocyte colony-stimulating factor (G-CSF) and suggest the ability of drugs increasing the level of extracellular adenosine to activate erythropoiesis when given jointly with G-CSF. To demonstrate these interactions, the effects of the drugs on the recovery from erythropoietic damage induced in mice by a single dose of 5-fluorouracil (5-FU) were investigated. Elevation of extracellular adenosine and thus activation of adenosine receptors was induced by joint administration of dipyridamole (DP), a drug inhibiting the cellular uptake of adenosine, and adenosine monophosphate (AMP), an adenosine prodrug. The drugs were injected in a 4-d treatment regimen starting 2 h after 5-FU injection. Both DP+AMP and G-CSF alone induced only weak effects. However, the combination of the three drugs produced significant elevation of erythrocytes in the peripheral blood which pertained in the posttreatment period. Stimulation of proliferation of erythroid progenitor cells (BFU-E) in femoral bone marrow and increased levels of reticulocytes in the peripheral blood were observed in the course of the 4-d treatment regimen. In addition, significantly decreased mean cell haemoglobin accompanying the elevated numbers of erythrocytes in the combination-treated mice was found. This effect could be interpreted as the result of a sublethal 5-FU-induced damage to erythroid progenitor and precursor cells forced to proliferate intensively by the combination therapy. The observed additivity and synergism of G-CSF with elevated extracellular adenosine in terms of erythropoiesis is an interesting finding with potential implications in clinical practice.

Cell cycle status and apoptosis of hematopoietic progenitor cells released into the peripheral blood after taxanes and granulocyte colony-stimulating factor in breast cancer patients.

Paclitaxel and its analogue docetaxel show a significant antitumor activity, particularly evident in breast cancer. Paclitaxel has also been proved to be effective as a peripheral blood progenitor cell (CPC) mobilizing agent. To optimize the use of active, disease-specific drugs as CPC priming, we have evaluated the effects of either paclitaxel or docetaxel both at standard dosages and followed by granulocyte colony-stimulating factor (G-CSF), on circulating CPC release and function in 18 patients with advanced breast cancer who had failed previous anthracycline-based regimens. The reported differences in biological behaviour between bone marrow and blood-derived hematopoietic progenitor cells and the ability of both paclitaxel and docetaxel to induce apoptosis, prompted us to simultaneously evaluate the cell cycle perturbations induced on CD34+ cells. Median CD34+ peaks were 24 microl (range: 10-58) in the paclitaxel-treated patients and 39 microl (range: 17-91), respectively, in the patients who received docetaxel. After paclitaxel, the percentage of CD34+ cells in S-phase was low (bromodeoxyuridine, BrdU, labelling index = 3.4+/-2%) with a concomitant presence of early apoptotic cells (8.1+/-3%). On the contrary, after docetaxel, the percentage of CD34+ cells in S-phase was higher (BrdU labelling index = 14.5+/-4%, p<0.05 vs. paclitaxel), while early apoptotic cells were detected at a similar rate (8. 6+/-3%, p = n.s. vs. paclitaxel). In conclusion, when used at standard dosages, with respect to paclitaxel + G-CSF, docetaxel + G-CSF is a more satisfactory tool to mobilize CPC and to induce them into the cell cycle. These data should be taken into account when combinations of docetaxel with other agents are explored as CPC mobilizing regimens for autografting.