In vivo erythroid recovery following paclitaxel injury: correlation between GATA-1, c-MYB, NF-E2, Epo receptor expressions, and apoptosis.

Paclitaxel (Px) is a cancer chemotherapeutic agent that causes bone marrow (BM) cytotoxicity by microtubule stabilization and by modifications in the expression of several genes. Hematopoietic progenitors show severe alterations following Px injury. Erythropoietic recovery should be accompanied by changes in the expression of transcription factors such as c-MYB, GATA-1, NF-E2, Bcl-x(L), and erythropoietin receptor (Epo-R). The aim of this work was to study the in vivo recovery of erythropoiesis and to correlate transcription factors, Bcl-x(L), and Epo-R expressions to apoptosis and changes in proliferation of murine erythroid progenitors following a single dose of Px (29 mg/kg, i.p.). BM total and differential cellularities, apoptosis (TdT-mediated dUTP Nick-End Labeling [TUNEL] assay), clonogenic assays, and immunoblots for transcription factors, Epo-R, and Bcl-x(L) were performed each day for 5 days post-injury. Apoptosis (24 +/- 0.81%, P < 0.01), inhibition of colony growth (burst-forming units-erythroid [BFU-E] and granulocyte-erythroid-macrophage [GEM]), and decrease in BM cellularities (28 +/- 4.2% of control) were maximal at 24 h following Px. The highest apoptosis was concomitant with the lowest BM cellularities. Apoptosis returned to normal values (3.08 +/- 0.61%) by day 3 post-Px. Up-regulation of c-MYB, GATA-1, Epo-R, and Bcl-x(L) expressions were observed between 24 and 48 h following Px. Correlations among c-MYB, GATA-1, Bcl-x(L), and Epo-R were extremely significant. Maximal expression of NF-E2 was observed on day 3 concomitant with the rise (threefold) of early erythroid precursors (BFU-E). Thus, cells that survive injury seem to be stimulated to produce early (24-48 h) erythroid-related and antiapoptotic proteins. Therefore, the results suggest an in vivo interplay between specific transcription factors and Bcl-x(L) during progenitor cell survival and proliferation; mechanisms triggered to restore size and composition of the erythroid compartment.

Hematotoxicity induced by paclitaxel: in vitro and in vivo assays during normal murine hematopoietic recovery.

Paclitaxel is a drug widely used in several oncological trials. Like other antineoplastics, it causes severe neutropenia. However, its effects on erythropoiesis are not as well known. This study analyzes the recovery of normal murine hematopoiesis after single dose paclitaxel administration (29 mg/kg i.p.) over 20 days. Different assays were used to analyze the restorative kinetics from primitive early progenitors to mature functional erythroid cells. Proliferation of the erythroid compartment was assessed by DNA microculture assays in medullar and splenic cells stimulated with recombinant human erythropoietin (rh Epo 0-500 mU/ml). Enhancement of early hematopoietic progenitors was determined using clonogenic assays and erythroid terminal maturation by 59Fe incorporation. Peripheral hematologic parameters and cellularities in both tissues were also determined on each day of the experimental schedule. At 2 days post-paclitaxel treatment, medullar cellularity diminished drastically (> 90%) and 59Fe incorporation decreased in all compartments. DNA assay revealed maximum sensitivity to Epo (p < 0.05 with 15 mU/ml) while clonogenic cultures failed to show significant results. At 5 days both bone marrow and spleen semisolid cultures showed great expansion of early hematopoietic progenitors (about 5- and 83-fold. respectively). Hormonal sensitivity decreased progressively along the experiment. Splenic cultures showed a linear dose-response to rh Epo at day 5 post-paclitaxel administration (p < 0.05 with 125 mU/ml). Medullar and splenic total progenitor colony-forming units (CFU) scorings with and without rh Epo revealed a notable enhancement at 5 days post-paclitaxel treatment. Data from this study suggest that paclitaxel causes deep injury in the erythropoietic compartment, including temporary variations of Epo sensitivity in late bone marrow erythroid progenitors, early multilineage hematopoietic explosion and terminal erythroid precursors depletion as a result of a complex microenvironmental restitutive regulation.

Expression of scinderin in megakaryoblastic leukemia cells induces differentiation, maturation, and apoptosis with release of plateletlike particles and inhibits proliferation and tumorigenesis.

Rapid proliferation of atypical megakaryoblasts is a characteristic of megakaryoblastic leukemia. Cells from patients with this disorder and cell lines established from this type of leukemia showed the presence of gelsolin but the absence of scinderin expression, 2 filamentous actin-severing proteins present in normal megakaryocytes and platelets. Vector-mediated expression of scinderin in the megakaryoblastic cell line MEG-01 induced a decrease in both F-actin and gelsolin. This was accompanied by increased Rac2 expression and by activation of the PAK/MEKK.SEK/JNK/c-jun, c-fos transduction pathway. The Raf/MEK/ERK pathway was also activated in these cells. Transduction pathway activation was followed by cell differentiation, polyploidization, maturation, and apoptosis with release of platelet-like particles. Particles expressed surface CD41a antigen (glycoprotein IIb/IIIa or fibrinogen receptor), had dense bodies, high-affinity serotonin transport, and circular array of microtubules. Treatment of particles with thrombin induced serotonin release and aggregation that was blocked by CD41a antibodies. PAC-1 antibodies also blocked aggregation. Exposure of cells to PD98059, a blocker of MEK, inhibited antigen CD41a expression, increases in cell volume, and number of protoplasmic extensions. Cell proliferation and cell ability to form tumors in nude mice were also inhibited by the expression of scinderin. MEG-01 cells expressing scinderin had the same fate in vivo as in culture. Thus, when injected into nude mice, they entered apoptosis and released platelet-like particles. The lack of scinderin expression in megakaryoblastic leukemia cells seems to be responsible for their inability to enter into differentiation and maturation pathways characteristic of their normal counterparts.

Myristoylated alanine-rich C kinase substrate phosphorylation is involved in thrombin-induced serotonin release from platelets.

Stimulation of platelets by thrombin induces protein kinase C (PKC) activation, phosphorylation of pleckstrin, aggregation and serotonin release. Here, we demonstrate that, in human platelets, thrombin stimulation also induced phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) and serotonin release in intact and digitonin-permeabilized platelets. MARCKS is known to bind actin and cross-link actin filaments, and this is inhibited by PKC-evoked MARCKS phosphorylation. MARCKS phosphorylation and serotonin release in response to increasing concentrations of thrombin have a similar EC50 and time course and, in permeabilized platelets, peptide MPSD, with an amino acid sequence corresponding to the phosphorylation site domain of MARCKS, blocked both responses. However, pleckstrin and myosin light chain phosphorylations were not modified. Ala-MPSD, in which the four serine residues of MPSD were substituted by alanines was ineffective. The results suggest a role for MARCKS in platelet secretion. The fact that pleckstrin phosphorylation has a different time course and was not modified in the presence of MPSD when MARCKS phosphorylation and serotonin release were inhibited would suggest either that pleckstrin phosphorylation is unrelated to secretion or that it might only be involved upstream in the events leading to secretion.

[In vitro and in vivo studies of murine erythropoietic recovery after treatment with cyclophosphamide]. / Estudios in vitro e in vivo de la recuperación eritropoyética murina post-tratamiento con ciclofosfamida.

OBJECTIVES: The aim of this study is to analyse functional changes in murine erythropoietic tissues over 20 days post cyclophosphamide (CY) treatment. The project was focused on the capability of femoral and splenic erythropoietic responsive cells (ERC) to proliferate with human recombinant erythropoietin (rh Epo) stimulation after cytotoxic treatment. MATERIALS AND METHODS: CF-1 mice were injected i.p. with CY (200 mg/Kg). Individual lots were studied at 0, 2, 5, 7, 10, 14, 17 and 20 days post cytotoxic treatment. Haematologic parameters [packed red cells (PRC) reticulocytes, white blood cells] were scored. Erythropoietic differentiation was assessed by the 59Fe uptake assay and the proliferative profiles of erythropoietic progenitors were determined by 3H-thymidine incorporation assay with several doses of rh Epo (0-250 mU/mL). Total and differential cellularities were scored in bone marrow (BM) and spleen. RESULTS: A drastical decrease of total nucleated BM cells was noticed at 2 days post CY, although cellularity was restored by the 7th day. Spleen, however, failed in showing significant decrease. The maintenance of PRC was achieved through a deep erythropoietic reorganization. 59Fe uptake revealed changes in erythroid differentiation in both tissues. Spleen maturative contribution to whole erythropoiesis was always less than medullary supply, except on day 10 post CY when a transient compensatory red cell contribution was noticed. Proliferative assays revealed that erythropoietic recovery in BM post CY was delayed in comparison with myelopoietic restoration. Splenic erythroid proliferative pattern correlated with differentiation data. CONCLUSIONS: Myelopoietic and erythropoietic progenitors showed different recovery patterns post CY administration in BM and spleen. Medullary hemopoietic lineages restoration described a particular sequence: myelopoiesis restitution was previous to the erythroid one. Medullary erythropoiesis occurred without drastic changes in erythropoietin sensitivity while the spleen showed a transient dramatic increment on 10 days post CY red proliferation. Experimental data strongly suggest that erythropoietic recovery after CY insult mainly depends on microenvironmental regulations rather than on hormone titers.

Effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on murine bone marrow and spleen erythropoiesis.

100 micrograms/kg of recombinant human granulocyte colony-stimulating factor was injected twice daily into normal adult CF1 female mice for a period of 15 days. After that time we have observed a decrease 59Fe marrow incorporation with a parallel increase in the spleen. During the first 9 days the marrow plus spleen erythroid cells number decreased to 60% of control approximately, but recovered thereafter and were not significantly different from normal values at 12-15 days. In addition, our studies demonstrate that the spleen erythropoiesis is quantitatively more important at the final time than marrow erythropoiesis. For this reason, splenic compensatory erythropoiesis maintained the hematocrit values between normal ranges. Regarding the granulocytic compartment, 15 days of rhG-CSF treatment produce a marked increase in total count of splenic granulocytes (a 7.7 fold rise from control values). Marrow granulocytes shows a 2-fold increment, but considering the absolute counts, bone marrow still was predominant as a granulopoieitc organ. Our results indicate that the spleen is a more important erythropoietic organ than marrow after 15 days of rhG-CSF treatment.

Effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on murine bone marrow and spleen erythropoiesis

100 mug/kg of recombinant human granulocyte colony - stimulating factor was injected twice daily into normal adult CF1 female mice for a period of 15 days. After that time was have observed a decrease (59)Fe marrow incorporation with a parallel increase in the spleen. During the first 9 days the marrow plus spleen erythroid cells number decreased to 60 percent of control approximately, but recovered thereafter and were not significantly different from normal values at 12 - 15 days. In addition, our studies demonstrate that the spleen erythropoiesis is quantitatively more important at the final time tham marrow erythropoiesis. For this reason, splenic compensatory erythropoiesis maintained the hematocrit values between normal ranges. Regarding the granulocytic compartment, 15 days of rhG-CSF treatment produce a marked increase in total count os splenic granulocytes (a 7.7 fold rise from control values). Marrow granulocytes shows a 2 - fold increment, but considering the absolute counts, bone marrow still was predominant as a granulopoieitc organ. Our results indicate that the spleen is a more important erythropoietic organ than marrow after 15 days of rhG-CSF treatment.

Murine splenic proliferative response to human recombinant erythropoietin along hypoxia

La hipoxia constituye el mejor stress fisiológico para la pertubación del estado estacionario eritropoyético. El present estudio tiende a analizar la respuesta proliferativa eritropoyética esplénica con diferentes dosis de eritropoyentina humana recombinante bajo condiciones hipóxicas a lo largo 18 días mediante el ensayo de síntesis del DNA. Los progenitores esplénicos normóxicos no sufren proliferación eritroide significativa al día 0. Una clara respuesta proliferativa a rh Epo se verificó entre los 2 y 8 días de hipoxia. La proliferación de los progenitores eritroides esplénicos hipóxicos retornó a un patrón basal desde los 10 días hasta el final de la experiencia. La mayor creatividad proliferativa, 25 veces sobre el control (p<0.001), se produjo a los 6 días de condicionamiento desde 62.5 hasta 250mU/ml de rh Epo. estos resultados son concordantes con el concepto que durante la daptación fisiológica a la hipoxia, las células progenitoras eritroides esplénicas modifican transitoriamente su tasa proliferativa observable por variaciones en la relación dosis-respueta a Epo

Comparative studies of erythroid growth factors.

Representative specimens from two classes of Vertebrata Sub-Phyllum, Bufo paracnemis (amphibian) and Gallus domesticus (avian) were made anemic by phenylhydrazine treatment. Appearance of serum factors able to stimulate the proliferation of mammalian erythroid cells was tested. Normal and anemic sera from Gallus domesticus and Bufo paracnemis were fractioned by alcoholic treatment and assayed by the post-hypoxic mice method, showing null uptake of 59Fe. When assayed in semisolid cultures using bone marrow murine cells at different times of incubation (CFU-E and BFU-E colonies), anemia Gallus domesticus serum showed high stimulatory activity, while anemic Bufo paracnemis serum was unable to enhance erythroid proliferation. Gel filtration chromatography of partially purified avian samples on Sephadex G-150 showed three molecular entities responsible for biological activity in vitro, with an apparent molecular weight of 29, 14 and 10 KD respectively. They were submitted to several treatments and then tested for biological activity. All factors were heat stable, sensitive to neuraminidase treatment, while dithiothreitol caused loss of activity on low molecular weight proteins. These results suggest at least under these experimental conditions, the presence of analogous erythroid factors among homeotherms amniotas.

In vitro assay of erythropoietin in fetal mouse liver cultures. II. Effects of human transferrin bound iron and of serum on the stimulation of incorporation of 3H-thymidine into DNA.

Iron bound to human transferrin but not apotransferrin, increases the effect of erythropoietin in stimulating incorporation of 3H-thymidine into DNA in fetal mouse liver cells in vitro. The effect of erythropoietin, with or without transferrin-iron is blocked by pre-incubation of the erythropoietin with rabbit anti erythropoietin serum. Human sera contain factors in addition to erythropoietin and transferin-iron which may modify the stimulation of incorporation of 3H-thymidine into fetal mouse liver DNA induced by erythropoietin. Heat treatment of sera at 56 degrees C for 30 min does not necessarily destroy these factors. Acid heat treatment of sera (pH 5.5 and 100 degrees C for 5 min) may destroy inhibitory factors and can result in an apparent increase in serum erythropoietin activity assessed in this fetal mouse liver system.