Apoptosis in the myelodysplastic syndromes : protective effect of G-CSF

Abstract

Increased apoptosis of hematopoietic progenitor cells is a hallmark for the myelodysplastic syndromes (MDS), and one of the main reasons for the ineffective hematopoiesis and subsequent cytopenia characteristic for the disease. In particular in low risk MDS, such as refractory anemia (RA) and refractory anemia with ringed sideroblasts (RARS), severe transfusion-dependent anemia leading to constant fatigue and weakness and therefore reduced quality of life often constitutes the main clinical problem. The development of specific treatment strategies for MDS is hampered by the lack of deep pathogenetic understanding of the disease. We have previously shown that treatment with granulocyte colony stimulation factor (G-CSF) and erythropoietin (EPO) reduces the number of apoptotic erythroid bone marrow precursors, and may abolish transfusion need in patients with low-risk MDS. Although our data suggest that G-CSF blockage of erythroid apoptosis occurs at the mitochondrial level, additional studies are required to understand the entire mechanism of G-CSF action. Thus, the present studies were initiated to explore mechanisms for apoptosis and the effects of G-CSF in cell lines and primary MDS progenitors.

Bone marrow cells from RARS cultured in vitro showed increased spontaneous apoptosis and a higher sensitivity to ligation of the Fas death receptor compared to progenitor cells from healthy individuals. Both erythroid and myeloid colony growth was reduced, indicating a defect at the stem cell level. Moreover, RARS cells displayed elevated caspase-3 activity, which could be reduced by the caspase inhibitor DEVD-fmk. Fas-antagonistic antibody fragments did not inhibit apoptosis in RARS cultures. Co-culture with G-CSF reduced caspase activity and generation of reactive oxygen species, while restoring mitochondrial potential and erythroid colony growth. No such effects were observed in normal bone marrow.

In order to further explore the anti-apoptotic effects of G-CSF, the myeloid P39 cell line, originally derived from an MDS patient, was treated with all-trans retinoic acid (ATRA) in the presence of G-CSF. ATRA induced differentiation and subsequent apoptosis. It affected mitochondrial functioning long before any signs of apoptosis appeared. ATRA-induced mitochondrial alterations were characterized by diminished mitochondrial oxygen consumption and decreased calcium uptake by mitochondria, leading to a lower mitochondrial matrix calcium concentration. Interestingly, G-CSF prevented mitochondrial deterioration. It partially restored respiration as well as the capacity of mitochondria to accumulate calcium. Mitochondrial alterations occurred at an early time point in ATRA-treated cells and were later followed by apoptosis, as detected by apoptotic nuclear morphology. G-CSF prevented both the early mitochondrial changes and the later apoptotic manifestations suggesting that mitochondrial dysfunction is an important step for ATRA-induced apoptosis.

Thus, the ability of ATRA and G-CSF to modulate mitochondrial respiration and intracellular calcium control are novel findings which help to give a better insight in their precise molecular mode of action. The restoration of mitochondrial functioning by G-CSF offers a new explanation for its anti-apoptotic function in the treatment of MDS.