Murine erythropoietic impairment induced by paclitaxel: interactions of GATA-1 and erythroid Krüppel-like transcription factors, apoptotic related proteins and erythropoietin receptor.

Paclitaxel, an antitumoral drug, was used in a single dose (29 mg/kg i.p.) as an injury agent for inducing transient suppression of hematopoiesis in a murine experimental model during 10days. The aim of this study focuses on erythropoietin (EPO) receptor, GATA binding protein 1 (globin transcription factor 1) (GATA-1) and erythroid Krüppel-like factor (EKLF) expressions related to the apoptotic events triggered by paclitaxel in bone marrow and the subsequent in vivo erythropoietic recovery. Results showed a massive impairment of erythropoiesis early post paclitaxel administration (1-2 days), which involved induction of high Bax/Bcl-x(L) ratio, caspase-3 activation, disruptions of the medullar niche and cell death by both apoptosis and necrosis. EPO receptor over-expression was noticed from day 3 onwards. It prompted the subsequent up-regulations of GATA-1 and EKLF transcription factors as well as of the anti apoptotic protein Bcl-x(L), crucial proteins in driving erythropoiesis. This study suggests that EPO receptor recovery is necessary for the subsequent bone marrow ability to accomplish the erythroid program through the modulation of apoptotic and survival events after a single paclitaxel insult. These findings contribute to new insights into the molecular mechanisms involved during in vivo erythropoiesis post paclitaxel administration. Therefore, the detailed knowledge of the injury elicited by this drug on red blood cell production may have clinical relevance to explore new therapeutic approaches.

Morphological and biochemical changes during formocresol induced cell death in murine peritoneal macrophages: apoptotic and necrotic features.

The present study was conducted to investigate the role of Formocresol (FC)-induced apoptosis and necrotic cell death in murine peritoneal macrophages (pMø). Macrophages were cultured with 1:100 FC for 2 to 24 h. The viability (trypan blue assay), cell morphology (scanning electronic microscope), and apoptotic and necrotic indexes (light and fluorescent microscopy) were determined at different scheduled times. Simultaneously, the expressions of proteins related to stress, survival, and cell death were measured by western blotting. FC-exposed macrophages exhibited maximal apoptosis from 2 to 6 h, coincident with Bax overexpression (P < 0.001). Additionally, Bcl-x(L) showed maximal expression between 12 and 24 h suggesting its survival effect in pMø. The lowest pMø viability and the increment of the necrotic rate from 4 to 12 h were observed in accordance to Fas and Hsp60 overexpressions. In summary, all the experimental data suggest that two different pathways emerge in pMø exposed to FC, one leading Bax-dependent apoptosis (2-6 h) and the other one favoring necrosis (4-18 h), related to Fas-receptor and Hsp60 stress signal.

Erythroid expansion and survival in response to acute anemia stress: the role of EPO receptor, GATA-1, Bcl-xL and caspase-3.

Erythropoietic stress occurs under conditions of tissular hypoxia, such as anemia. Functional relationships between erythroid bone marrow (BM) proliferation, differentiation, the expression of survival and apoptotic related proteins, as well as the features of the BM microenvironment upon acute anemic stress, are not fully elucidated. To achieve this aim, CF-1 Swiss mice were injected with a single dose of 5-fluorouracil (5-FU, 150 mg/kg ip) and a multiparametric analysis was conducted for 20 days. Apoptosis (TUNEL assay), BM architecture organization (scanning electronic microscopy), proliferation (DNA assay), differentiation (clonogenic cultures), expression of survival erythroid related proteins (EPO-R, GATA-1, Bcl-xL) as well as the expression of apoptotic- related proteins (Bax, activated Caspase-3) by Western blotting, were evaluated. Experimental data showed that apoptosis, arrest of cell proliferation and disruptions of BM architecture were maximal within the first period of acute stress (1-3 days). Bax and caspase-3 overexpressions were also coincident during this acute period. Moreover, from day 5 upon drug challenge BM responds to acute stress through the EPO-EPO-R system, prompting expressions of GATA-1 and Bcl-xL. Erythroid proliferation rates and red-cell-committed progenitors enhanced in a coordinated way to restore the size and function of the red cell compartment. A second overexpression wave of active caspase-3 was noticed during stress recovery. Together, these results indicate that in response to acute stress a dramatic increase in CFU-E (erythroid colony forming units) population is concomitant with upregulation of EPO-R, GATA-1 and Bcl-xL in the BM erythroid compartment, and that these concurrent processes are crucial for acquiring proper erythroid cell functionality without delayed response to tissular hypoxia.

EPO receptor, Bax and Bcl-x(L) expressions in murine erythropoiesis after cyclophosphamide treatment.

The effect of a single dose of cyclophosphamide (CY, 150 mg/kg i.p.) on the erythropoiesis using an "in vivo" murine model in a time course protocol (0-10 days) was studied through several experimental approaches. Total and differential bone marrow cellularities, apoptosis (TUNEL assays), bone marrow hematopoietic architecture (scanning electronic microscopy), proliferation (DNA assay), BM erythroid progenitors growth (semisolid clonogenic assays) and protein expressions for erythroid commitment and survival: erythropoietin receptor (EPO-R), Bcl-x(L), Bax (immunoblottings) were performed on the scheduled days. Most of the experiences were conducted comparing spontaneous with human recombinant (hr EPO) "ex vivo" stimulated bone marrow (BM) cells. Erythropoiesis was extremely affected by CY. Maximum apoptosis, minimal cellularities and severe disturbances of BM niche were noticed on the second day. During spontaneous recovery post-CY; EPO-R was expressed between 4 and 5 days. Following BM cells "ex vivo" hr EPO stimulation (2U/ml) EPO-R was expressed throughout the study except the period between the first and fourth day. Bax was noticeable all along the experience with and without hr EPO stimulation. Bcl-x(L) was barely detectable without hr EPO, but its expression showed a gradual enhancement from the fifth day onwards in hr EPO stimulated cells. This fact might be related to the end of the erythroid inhibitory stage and to the recovery of BM EPO-dependent proliferation between the fourth and fifth day, and the further recuperation of BFU-E and CFU-E colonies on days 6 and 7 post-CY, respectively. These findings suggest that the proliferation and differentiation of erythroid progenitor cells after the acute early injury inflicted by CY, is associated with changes in EPO-R expression during spontaneous recovery in this particular experimental system.

Characteristics of in vivo murine erythropoietic response to sodium orthovanadate.

Current knowledge about the effects of vanadium compounds on erythropoiesis is still reduced and even contradictory. The aim of this work was to evaluate the in vivo effects of a single dose of sodium orthovanadate (OV, 33 mg/kg i.p.) on CF-1 mice in a time course study (0-8 days). Murine erythropoiesis was assessed through a combinatory of experimental approaches. Classical peripheral and bone marrow (BM) hematological parameters were determined. Erythroid maturation in blood stream and hemopoietic tissues (59Fe uptake assays), BM erythroid progenitor frequency (clonogenic assays) and erythroid crucial protein expressions for commitment and survival: GATA-1, erythropoietin receptor (Epo-R) and Bcl-xL (immunoblottings) were evaluated. Neither BM cellularities nor BM viabilities changed noticeably during the study. Peripheral reticulocytes showed a biphasic increment on days 2 and 8 post-OV. hematocrits enhanced transiently between days 2 and 4. 59Fe uptake percentages enhanced in peripheral blood nearly two-fold over control values between 4 and 8 days (p<0.01) without changes in BM and spleen. Additionally, mature erythroid BM compartments: polychromatophilic erythroblasts and orthochromatic normoblasts increased by the eighth day. BFU-E colonies remained near basal values during the whole experience, whilst CFU-E colonies raised 60% over control at 8 days post-OV (p<0.05). GATA-1 and Epo-R were significantly over-expressed from the third until the end of the experimental protocol (p<0.01). Surprisingly, Bcl-xL showed a constitutive expression pattern without changes during the experience. Experimental data let us suggest that OV does not to cause bone marrow cytotoxicity and that it accelerates maturation of BM committed erythroid precursors. Moreover, there are significant correlations among erythroid-related protein expressions: GATA-1 and Epo-R and the frequency of CFU-E. In addition, Bcl-xL expression invariance during the time course study would indicate that the stimulatory effect of OV treatment on erythropoiesis was mainly exerted on the maturation of red cell precursors rather than on the antiapoptosis of erythroid terminal progenitors.