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.