Apoptosis in myelodysplastic syndromes : effects of hemopoietic growth factors

Abstract

Increased apoptosis of hernatopoietic progenitors is a hallmark of myelodysplastic syndromes (MDS) and results in ineffective hematopoiesis. Erythroid apoptosis is thought to be the main mechanism underlying the severe anemia observed in the low-risk subgroups, refractory anemia (RA) arid RA with ringed sideroblasts (RARS). Clinically, treatment with Epo alone or in combination with G-CSF may significantly improve anemia and reduce bone marrow apoptosis. The synergistic effect between Epo and G-CSF is more prominent in RARS.

The present study was designed to assess and characterize mechanisms underlying augmented apoptosis and ineffective erythropoiesis in MDS and to investigate the effects of hernatopoietic growth factors. Bone marrow cells from 55 patients were used in the experiments. RA and RARS cells showed significantly higher spontaneous initiator (caspase-8 and -9) and effector (caspase-3-like) activities than normal bone marrow (NBM). G-CSF inhibited Fasinduced caspase activity, restored proliferation, and improved erythroid colony growth in RARS. There was no significant effect of G-CSF on Fas-induced changes in RA mononuclear cells. Caspase-9 activation was shown to be an essential mediator of apoptosis in MDS, but not in NBM. Selective inhibition of caspase-9 reduced both spontaneous and Fas-induced caspase-3-like activity, indicating a role for mitochondria-dependent apoptosis in MDS. We then developed an assay in which CD34+ cells were cultured to mature erythroblasts for studies of mitochondrial function and signaling, as well as iron distribution during erythroid maturation.

While absent in NBM, significant release of cytochrome c was seen at all stages of erythroid differentiation i n RA and RARS, however, more prominent in RARS. This release was blocked by G-CSF during the whole culture period, with a peak effect between day 4 and 7, and by Epo during the latter phase of culture. We demonstrated that both freshly isolated glycophorin A+ BM cells and erythroblasts during the second week of cultivation retained the G-CSF receptor. The synergistic effect of Epo and G-CSF was assessed, and the in vitro and in vivo effects of these growth factors were compared. Cytochrome c release and caspase activation were significantly less pronounced in MDS progenitors obtained from successfully treated non-anemic patients, and showed no further response to G-CSF in vitro, indicating that data obtained in the erythroblast model reflect relevant biological events in vivo. Epo significantly promoted growth of cytogenetically normal cells from patients with 5q- syndrome, but had no effect on bone marrow from patients with other aberrations. RARS progenitors showed an aberrant accumulation of mitochondrial ferritin (MtF) already at day 4, and a continuous increase during the culture period. RA erythroblasts showed only few positive cells, and NBM was negative for MtF.

Hypothesizing a defect mitochondrial respiratory chain function in terms of ATP production in MDS, we developed a method for assessment of BM cells. However, no significant difference (all complexes or cytochrome cdependent complex IV) was found between MDS and NBM. We demonstrated a significant over-expression (mRNA) of the pro-apoptotic genes for cytochrome c, Bid and Bax, as well as up-regulation of genes involved in erythroid differentiation (beta-globin, GATA-1 and DLK-1) in MDS CD34+ cells. Beta-globin was adequately expressed during erythroid maturation, while GATA-1 expression was significantly decreased, indicating a block of erythroid maturation at the transcriptional level. G-CSF showed no effect on mitochondrial ATP production, cytochrome c mRNA, or MtF. In fact, MtF expression increased in some RARS cultures. This indicates that G-CSF allows survival of MDS erythroblasts by up-regulating compensatory mechanisms for cell survival, rather than by a direct effect on mitochondrial function or iron distribution.

In conclusion, we have shown strong evidence for mitochondria-dependent apoptosis in MDS and propose a model in which the increased sensitivity to external apoptotic stimuli is dependent on constitutive mitochondrial signaling mediated via cytochrome c release. Moreover, erythroid apoptosis, as well as defective erythroid maturation is initiated at the MDS stem cell level. Our data also provide a mechanistic explanation for the beneficial clinical effects of growth factor administration in MDS patients. We conclude that pro-erythroid growth factors can act both via inhibition of apoptosis, and via selection of cytogenetically normal progenitors.