Bcl-XL is required for heme synthesis during the chemical induction of erythroid differentiation of murine erythroleukemia cells independently of its antiapoptotic function

Abstract

Bcl-XL is essential for the survival and normal maturation of erythroid cells, especially at the late stage of erythroid differentiation. It remains unclear whether Bcl-XL serves only as a survival factor for erythroid cells or if it can induce a signal for differentiation. We have previously shown that dimethyl sulfoxide (DMSO) induction of erythroid differentiation in murine erythroleukemia (MEL) cells correlates with delay of apoptosis and specific induction of Bcl-XL. In this study, we investigate the contribution of Bcl-2 and Bcl-XL to survival and erythroid differentiation by generating stable MEL transfectants expressing these antiapoptotic regulators. Overexpression of Bcl-2 completely prevented apoptosis of MEL cells before and after DMSO induction, whereas overexpression of Bcl-XL only delayed it. Overexpression of Bcl-2 or Bcl-XL neither induced spontaneous erythroid differentiation nor accelerated DMSO-induced differentiation. Inhibition of Bcl-XL by antisense transcripts accelerated apoptosis in DMSO-treated MEL cells and blocked the synthesis of hemoglobin without altering the growth arrest associated with terminal erythroid differentiation. An antisense oligonucleotide to Bcl-XL did not induce apoptosis in MEL cells overexpressing Bcl-2 but greatly decreased their hemoglobin synthesis when treated with DMSO, suggesting that Bcl-XL is necessary for erythroid differentiation independently of its antiapoptotic function. Importantly, Bcl-XL antisense transcripts prevented heme synthesis but not globin mRNA induction in DMSO-treated MEL cells. Furthermore, inhibition of hemoglobin synthesis by Bcl-XLantisense was reversed by addition of exogenous hemin. Finally, Bcl-XL localized to mitochondria during MEL erythroid differentiation, suggesting that it may mediate a critical mitochondrial transport function related to heme biosynthesis.

Bcl-xL prevents apoptosis of late-stage erythroblasts but does not mediate the antiapoptotic effect of erythropoietin

“…In murine erythroleukemia (MEL) cells induced to differentiate by dimethyl sulfoxide, cell loss appeared to be at an earlier stage than reticulocytes, specifically the mature erythroblast, because inhibition of Bcl-x L resulted in loss of both benzidine-positive cells and heme biosynthesis. 15 Loss at this mature erythroblast stage is consistent with the death of mature cells in erythroid colonies derived from ES cells, 13 the evidence of cell debris in the spleens of conditional bcl-x Ϫ/Ϫ mice, 14 and our previous studies, showing that Bcl-x L is up-regulated at the terminal stages of erythroblast differentiation, 8 when hemoglobin synthesis is maximal. 16 We have previously used a system of explanted erythroid cells from spleens of mice infected with the anemia-inducing strain of Friend leukemia virus (FVA cells) to study the EPO-dependent and subsequent stages of erythropoiesis in vitro.…”

“…15 We quantified heme synthesis by analyzing the incorporation of 59 Fe into heme in erythroblasts cultured with EPO ( Figure 5). The amount of 59 Fe incorporated by cultured cells over 2-hour periods was measured at various times and expressed as counts per minute per 10 6 viable cells.…”

Erythropoietin treatment of human ovarian cancer cells results in enhanced signaling and a paclitaxel‐resistant phenotype

“…8). Among the 4 apoptosis-related proteins surveyed, the expression of Bax and Bcl-xL (known to mediate Epo's direct antiapoptotic action [28][29][30][31][32][33][34][35] ) were virtually identical in the paclitaxel-sensitive A2780 and the relatively paclitaxel resistant A2780 2m35U cells. However, expression of both Bcl-2 and Bcl-10 was substantially lower in the A2780 2m35U cells, and paclitaxel treatment may have reduced these levels further.…”

Terminalia catappa Extract Enhances Erythropoiesis in Adult Balb C Mice

“…Terminalia catappa improves PCV levels in adult Balb C mice Terminalia catappa extract was administered at a dosage of 80mg/kg. mice were sacrificed by humane decapitation, and their blood collected in EDTA and PCV was determined by benzidine spectrophotometric assay method as previously described by Khalid et al (2003) P< 0.05 for both folic acid and T. catappa …”

“…Haemoglobin was assayed as previously described Khalid et al, (2003) with slight modifications. Briefly; cells were washed twice with phosphate-buffered saline (PBS), resuspended in 0.5 mL distilled water, and lysed under hypotonic conditions for 10 minutes.…”