RESUMO
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.
Assuntos
Antineoplásicos Fitogênicos/toxicidade , Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eritropoese/efeitos dos fármacos , Genes myb/genética , Paclitaxel/toxicidade , Receptores da Eritropoetina/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Sobrevivência Celular , Ensaio de Unidades Formadoras de Colônias , Células Precursoras Eritroides/efeitos dos fármacos , Fatores de Ligação de DNA Eritroide Específicos , Feminino , Fator de Transcrição GATA1 , Expressão Gênica/efeitos dos fármacos , Genes bcl-1/genética , Processamento de Imagem Assistida por Computador , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Cinética , Camundongos , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2 , Receptores da Eritropoetina/biossínteseRESUMO
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.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Células Precursoras Eritroides/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Paclitaxel/farmacologia , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/sangue , Divisão Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Relação Dose-Resposta a Droga , Eritropoetina/farmacologia , Feminino , Radioisótopos de Ferro/metabolismo , Camundongos , Células Mieloides/efeitos dos fármacos , Paclitaxel/administração & dosagem , Paclitaxel/sangue , Proteínas Recombinantes , Baço/citologia , Baço/efeitos dos fármacosRESUMO
Hypoxia is the best physiological stress to disturb the erythropoietic steady state. The present study has been undertaken in the aim to analize the splenic erythropoietic proliferative response with different doses of recombinant human erythropoietin under hypoxic conditions along 18 days using the DNA synthesis assay. Normoxic splenic progenitors failed to show significative erythroid replication at 0 days. A clearly rh Epo response was noticed from 2 to 8 days of hypoxia. Splenic proliferation returned to basal pattern from 10 days to the end of the experience. The highest proliferative activity, 25 fold increase over control (p < 0.001), was found at 6 days from 62.5 to 250 mU/ml rh Epo. These results support suggestions that hypoxia induces a transiently erythroid splenic proliferative response changing its quantitative parameters in the Epo dose-response relationship during the physiological adaptation.
Assuntos
Eritropoese/fisiologia , Eritropoetina/administração & dosagem , Hipóxia/fisiopatologia , Baço/patologia , Animais , Técnicas de Cultura de Células , Divisão Celular , DNA/biossíntese , Células Precursoras Eritroides/fisiologia , Feminino , Hipóxia/patologia , Camundongos , Camundongos Endogâmicos , Fatores de TempoRESUMO
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
Assuntos
Feminino , Camundongos , Animais , Baço/citologia , Eritropoese/fisiologia , Eritropoetina/administração & dosagem , Hipóxia/fisiopatologia , Adaptação Fisiológica , Camundongos Endogâmicos , Técnicas de Cultura de Células , Células Precursoras Eritroides/fisiologia , DNA/biossíntese , Fatores de TempoRESUMO
Hypoxia is the best physiological stress to disturb the erythropoietic steady state. The present study has been undertaken in the aim to analize the splenic erythropoietic proliferative response with different doses of recombinant human erythropoietin under hypoxic conditions along 18 days using the DNA synthesis assay. Normoxic splenic progenitors failed to show significative erythroid replication at 0 days. A clearly rh Epo response was noticed from 2 to 8 days of hypoxia. Splenic proliferation returned to basal pattern from 10 days to the end of the experience. The highest proliferative activity, 25 fold increase over control (p < 0.001), was found at 6 days from 62.5 to 250 mU/ml rh Epo. These results support suggestions that hypoxia induces a transiently erythroid splenic proliferative response changing its quantitative parameters in the Epo dose-response relationship during the physiological adaptation.
RESUMO
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 (AU)
Assuntos
Feminino , Camundongos , Animais , Hipóxia/fisiopatologia , Eritropoese/fisiologia , Eritropoetina/administração & dosagem , Baço/citologia , Células Precursoras Eritroides/fisiologia , Adaptação Fisiológica , DNA/biossíntese , Técnicas de Cultura de Células , Fatores de Tempo , Camundongos EndogâmicosRESUMO
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.
Assuntos
Anemia/sangue , Sangue , Fatores Estimuladores de Colônias/sangue , Células Precursoras Eritroides/efeitos dos fármacos , Animais , Células da Medula Óssea , Bufonidae , Cromatografia em Gel , Feminino , Masculino , Camundongos , Aves DomésticasRESUMO
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.