Bcl-XL induction during terminal differentiation of Friend erythroleukaemia cells correlates with delay of apoptosis and loss of proliferative capacity but not with haemoglobinization

Abstract

Friend murine erythroleukaemia (F-MEL) cells are a useful model for studying the processes that regulate erythroid differentiation since exposure of these cells to chemical inducers (DMSO or HMBA) results in commitment to terminal cell division and synthesis of haemoglobin. This study examined the relationship between differentiation and apoptosis in DMSO sensitive and resistant F-MEL cells. Clear apoptosis was not observed in DMSO-treated sensitive F-MEL (strain 745A) cells during the induction of differentiation. In contrast, DMSO-induced 745A cells exhibited delayed apoptosis compared to uninduced cells. Since the Bcl-2 family members play a major role in the control of apoptosis and/or differentiation, we determined their expression before and after DMSO or HMBA treatment. Neither untreated nor chemically-induced 745A cells expressed the Bcl-2 protein. The levels of Bax and Bad proteins remained relatively constant during DMSO-induced differentiation. DMSO or HMBA treatment of 745A cells induced a marked increase of Bcl-XL expression during the late phase of differentiation which persisted even when the cells began to die. This upregulation of Bcl-XL was independent of cell density but was correlated with cell arrest in G0/G1. DMSO treatment induced a similar delay of apoptosis and enhancement of Bcl-XL expression in F-MEL (strain TFP10) cells which fail to synthesize haemoglobin in the presence of DMSO. Dexamethasone, which blocks DMSO-induced differentiation of F-MEL cells, prevented the induction of Bcl-XL. Inhibitors such as imidazole or succinylacetone, which inhibit haemoglobin synthesis but not commitment to terminal cell division, did not suppress Bcl-XL induction in DMSO-induced cells. Taken together, these results indicate that DMSO treatment of F-MEL cells induces a marked increase in Bcl-XL expression suggesting a role for this anti-apoptotic protein in the process of erythroid differentiation in F-MEL cells. Moreover, induction of Bcl-XL during this process seems to be associated with loss of proliferative capacity rather than with haemoglobin synthesis.

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

“…We have previously shown an up-regulation of Bcl-X L in MEL cells induced by DMSO or hexamethylene bisacetamide (HMBA). 14 We show here that inhibition of Bcl-X L expression by antisense transcripts or antisense oligonucleotide results in an acceleration of apoptosis in differentiating MEL cells associated with an inhibition of hemoglobin synthesis. This indicates that Bcl-X L is essential to delay apoptosis during MEL erythroid differentiation.…”

“…14 Incubation of MEL cells with DMSO inducer leads to the stimulation of 2 programs: one responsible for hemoglobin synthesis and the other for inhibition of cell growth. Several studies have indicated that the coordination of both programs is not obligatory.…”

“…Moreover, DMSO-induced erythroid differentiation of MEL cells is associated with an enhancement of Bcl-X L expression correlated with a delay of apoptosis. 14 To investigate the potential role of these 2 antiapoptotic proteins in regulating erythroid differentiation, we introduced Bcl-2 or Bcl-X L sense expression vectors into 745A MEL cells. Cells were transfected by electroporation with linearized plasmid DNA from one of the expression vectors.…”

“…12,13 We have previously reported that DMSO induction of terminal erythroid differentiation in MEL cells correlates with a delay of apoptosis and a specific induction of the Bcl-X L protein. 14 Here, we have created MEL cell lines which overexpressed Bcl-2 or Bcl-X L and a variant cell line transfected with a Bcl-X L antisense construct. Our results demonstrate that Bcl-2 prevents apoptosis in differentiated MEL cells, whereas Bcl-X L only delays it.…”

CD44 ligation induces apoptosis via caspase- and serine protease-dependent pathways in acute promyelocytic leukemia cells

“…Protein lysates were separated by 8 or 12% SDSpolyacrylamide gel electrophoresis (SDS-PAGE), blotted onto PolyScreen membranes (NEN, Paris, France) and processed for Western blot analysis as described previously. 19 After incubation of membranes with primary antibodies and secondary antimouse or anti-rabbit antibody conjugated with horseradish peroxidase, the protein bands were detected as described in the ECL protocol (NEN) using Reflection autoradiography film.…”