RESUMEN
Recombinant human granulocyte colony-stimulating factor, rhG-CSF, is widely applied to ameliorate neutropenia following chemotherapy. However, rhG-CSF can exert negative effects on megakaryocytopoiesis that might cause a delay of megakaryocyte recovery. Therefore, the present study was designed to test different rhG-CSF administration protocols with regard to their megakaryocytic inhibitory potential in a 5-fluorouracil (5-FU)-induced experimental model system. Splenectomized B6D2F1 mice received a single injection of 5-FU (150 mg/kg) on day 0 followed by 50 micrograms/kg/day rhG-CSF given daily for either zero, four, or eight days. Five days after 5-FU, bone marrow and blood hematopoiesis were reduced significantly when compared with controls, independent of whether or not animals received rhG-CSF. However, nine days after 5-FU, granulopoietic recovery from 5-FU-induced toxicity was faster for rhG-CSF-treated versus untreated mice as demonstrated by higher values for colony forming unit-granulocyte macrophage (CFU-GM) and granulocytes (CFU-GM: 7.2 +/- 0.4 versus 5 +/- 0.6 x 10(4)/femur, granulocytes: 4.3 +/- 2 versus 1.4 +/- 0.4 x 10(5)/ml, respectively). Furthermore, significant mobilization of CFU-megakaryocyte (CFU-Meg) and CFU-GM into the peripheral blood was induced by the eight-day administration of rhG-CSF following 5-FU (day 9: 911 +/- 102 CFU-Meg/ml, 2330 +/- 152 CFU-GM/ml). However, megakaryocytic cells in these same mice were considerably lower when compared with those of animals receiving no rhG-CSF (CFU-Meg: 2.7 +/- 0.2 x 10(3) versus 4.2 +/- 0.2 x 10(3)/femur; small acetylcholinesterase positive (SAChE+) cells: 4.9 +/- 0.3 x 10(3) versus 7.3 +/- 0.9 x 10(3)/femur; megakaryocytes: 2.5 +/- 0.2 x 10(3) versus 4.1 +/- 0.7 x 10(3)/femur; platelets: 2.67 +/- 0.5 x 10(9) versus 3.1 +/- 0.5 x 10(9)/ml, respectively). On the other hand, the shortening of the rhG-CSF treatment from eight to four days caused a rapid granulopoietic recovery comparable to animals receiving eight days of G-CSF with no significant delay in megakaryocytic recovery when compared with mice treated with 5-FU alone; however, with four days of rhG-CSF, the mobilization of CFU into the peripheral blood was significantly less effective. Taken together, the results showed that a shortening of rhG-CSF treatment after chemotherapy is capable of ameliorating neutropenia without negatively affecting megakaryocytopoietic recovery. If, however, maximum recruitment of CFU into the peripheral blood circulation by rhG-CSF for subsequent harvest and transplantation is needed, any shortening of rhG-CSF administration is not advisable.
Asunto(s)
Fluorouracilo/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Inmunosupresores/administración & dosificación , Megacariocitos/efectos de los fármacos , Animales , Diferenciación Celular , Femenino , Granulocitos , Hematopoyesis , Humanos , Masculino , Megacariocitos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Proteínas Recombinantes , EsplenectomíaRESUMEN
The testing of new human leukemia-specific drugs for activity against primary acute myeloid leukemia (AML) blasts is severely limited by the low and variable clonogenic potential of primary human leukemias in culture. To circumvent this problem, we have modified a previously described flow cytometric approach to permit the simultaneous determination of live/dead cells, and the quantitation of the surviving cell fraction as a ratio of viable cells in treatment and control groups. The method utilizes the combination of calceinAM as a probe for intracellular esterase activity (green fluorescence,) which has the advantage over carboxyFDA of pH insensitivity and superior signal-to-noise ratio, and propidium iodide (red fluorescence) as an indicator of plasma membrane integrity. Suspension cultures of AML blood and marrow samples from patients were treated with known active agents as well as several new agents arising from a clonogenic disk assay screen. Quantitative dose-response values obtained from surviving cell fractions assayed by flow cytometry at 24 h following drug exposure demonstrated the utility of this assay for quantitating drug-induced cytotoxic effects on primary human AML cells in short-term culture.
Asunto(s)
Antineoplásicos/uso terapéutico , Leucemia Mielomonocítica Aguda/tratamiento farmacológico , Animales , Células de la Médula Ósea/fisiología , Recuento de Células , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citometría de Flujo , Fluoresceínas , Colorantes Fluorescentes , Humanos , Leucemia L1210/tratamiento farmacológico , Análisis Multivariante , Células Tumorales CultivadasRESUMEN
Increasing evidence especially stemming from peripheral blood progenitor transplantation studies points to a possible biologic difference between mobilized blood and bone marrow progenitor cells. The objective of this study was to compare the in situ radiation sensitivity of recombinant human granulocyte colony-stimulating factor (rhG-CSF)-recruited circulating granulopoietic (blood colony-forming unit-granulocyte-macrophage [CFU-GM(blood)]) and megakaryocytopoietic (blood CFU-megakaryocyte [CFU-Meg(blood)]) progenitors, with the nonmobilized fraction remaining in the bone marrow (CFU-GM(femur) and CFU-Meg(femur)). Splenectomized male B6D2F1 mice received 50 micrograms/kg/d rhG-CSF daily for 8 days to induce high levels of circulating progenitors, followed by either total body X-irradiation (TBI) or X-irradiation of the chest (CI) with 62.5, 125, 250, or 500 cGy. Progenitor cells were assayed 24 hours after irradiation. Circulating CFU-GM and CFU-Meg in the blood were decreased in a dose-dependent fashion by both TBI and CI, with TBI causing greater damage than CI. Average D0 values for TBI were 53 cGy for CFU-GM(blood) and 40 cGy for CFU-Meg(blood) D0 values for CI were 90 cGy for CFU-GM(blood) and 140 cGy for CFU-Meg(blood). As seen for blood progenitor cells, TBI caused a dose-dependent decrease of both CFU-GM(femur) (D0, 136 cGy) and CFU-Meg(femur) (D0, 148 cGy). However, radiation-induced bone marrow progenitor cell kill was significantly lower when compared with blood progenitors. Despite the fact that circulating blood elements only received a fraction of the total dose administered as Cl, the extent of blood progenitor kill caused by Cl was higher than the effects of identical TBI doses on bone marrow CFU. The results of this study showed that rhG-CSF-recruited CFU-Meg(blood) and CFU-GM(blood) were considerably more radiosensitive than femoral progenitors, thereby providing novel evidence for a biologic difference between rhG-CSF-recruited peripheral blood progenitors and the nonrecruited bone marrow CFU.
Asunto(s)
Células Sanguíneas/efectos de la radiación , Células de la Médula Ósea , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células Madre Hematopoyéticas/efectos de la radiación , Animales , Médula Ósea/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Relación Dosis-Respuesta en la Radiación , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Especificidad de Órganos , Tolerancia a Radiación , Radiografía Torácica , Proteínas Recombinantes/farmacología , Esplenectomía , Irradiación Corporal TotalRESUMEN
The aim of this study was to systematically characterize possible rhG-CSF effects on the murine megakaryocyte-platelet system (untreated and recovering from chemotherapy or extramedullary irradiation). In untreated, splenectomized male B6D2F1 mice, rhG-CSF treatment (50 micrograms/kg/d for up to 8 d) markedly decreased femoral megakaryocytopoiesis. CFU-Meg, small acetylcholinesterase-positive (SAChE) cells, and megakaryocytes were significantly reduced to 35-70%; platelets, however, were not affected. Peripheral CFU-Meg and CFU-GM increased up to 200-fold. Following a single injection of 5-FU (150 mg/kg) on day 0, rhG-CSF (50 micrograms/kg/d) on days 1-8 suppressed the megakaryocytopoietic recovery as indicated by significantly lower platelet numbers on day 9. Granulopoietic recovery was accelerated by rhG-CSF. When rhG-CSF treatment was started on day 5, no beneficial effect on granulopoietic recovery was observed, but again platelet levels were significantly lower on day 9, indicating that within the first 4 d of rhG-CSF application, recruitment or lineage competition was not a critical event. To test for the effects of extramedullary irradiation on circulating progenitors, mice pretreated with 50 micrograms/kg/d of rhG-CSF for 8 d received irradiation to the chest with 500 cGy resulting in a substantial kill of circulating CFU-Meg and CFU-GM of up to 99%. However, this striking decrease of blood progenitors did not significantly affect their total body contents. This study indicates that rhG-CSF treatment can impair bone marrow megakaryocytopoiesis, which might be an important consideration for those clinical situations that carry a high potential for treatment-induced thrombocytopenia.
Asunto(s)
Fluorouracilo/farmacología , Factor Estimulante de Colonias de Granulocitos/farmacología , Hematopoyesis/efectos de los fármacos , Megacariocitos/efectos de los fármacos , Animales , Plaquetas/efectos de los fármacos , Plaquetas/efectos de la radiación , Células de la Médula Ósea , Granulocitos/efectos de los fármacos , Granulocitos/efectos de la radiación , Hematopoyesis/efectos de la radiación , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de la radiación , Masculino , Megacariocitos/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Proteínas Recombinantes/farmacologíaRESUMEN
The immediate hematopoietic response to 3'-azido-3'-deoxythymidine (AZT) was characterized in an unperturbed and regenerating mouse marrow model to identify the in vivo hematopoietic targets of AZT and test whether AZT toxicity is dependent on the proliferative activity of the hematopoietic targets. B6D2F1 mice received intravenous (IV) bolus injections of 30, 60, 120, and 240 mg/kg AZT. None of the doses induced consistent changes in the number of hematopoietic stem and progenitor cells. However, identical cumulative doses administered as an intravenous 24-hour infusion led to marked changes. Spleen colony-forming units (CFU-S) per femur were diminished to about 60%. Burst-forming units-erythroid (BFU-E) and colony-forming units-erythroid (CFU-E) were substantially reduced to about 15 to 35% at the two highest doses, whereas the femoral content of colony-forming units-granulocyte/macrophage (CFU-GM) and colony-forming units-megakaryocyte (CFU-Meg) was unchanged. When administered to mice whose marrow was regenerating from total-body irradiation (TBI) and subsequent bone marrow transplantation (high proliferative fraction), 240 mg/kg AZT caused considerable reductions of all hematopoietic cell stages even when given as a single IV injection. The results indicate that (1) the mode of application is critical for AZT hematotoxicity; (2) erythropoietic progenitors are the most sensitive to AZT toxicity; and (3) hematotoxicity increases with increasing proliferative activity.