Abstract

Refractory Anemia with Ringed Sideroblasts (RARS) is characterized by severe ineffective erythropoesis, cytochrome c release, and mitochondrial iron overload. (Tehranchi, 2003). Granulocyte-CSF inhibit erythroid apoptosis in vitro as well as in vivo (Tehranchi 2005) The molecular mechanisms underlying the erythroid apoptosis in RARS and the effects of G-CSF were studied by gene expression profiling of erythroblasts from 8 healthy controls and 6 RARS patients. CD34+ selected marrow cells were cultured for 7 days in Iscove’s medium with 15% BIT9500. At day 7 an aliquot of RARS cells were treated with G-CSF (100ng/ml) for 4 hours. The gene expression profiles were determined using Affymetrix, U133 Plus2.0 chips (Pellagatti, 2006). Statistical analysis showed that 1426 probe-sets were significantly differentially expressed (P<0.01) between untreated and G-CSF treated RARS samples, and healthy controls. Hierarchical clustering separated these groups into distinct clusters. 22 genes were significantly up-regulated by ≥2-fold in all RARS samples and included CCND2, DLK1, PPM1A and TBC1D8. 35 genes were significantly down-regulated by ≥2-fold, including LRIG1, ABCB7 and MLL3. RARS erythroblasts showed dysregulation of several genes involved in proliferation, apoptosis and iron transport. For instance CCND2 and TBC1D8, positive regulators of proliferation, were up-regulated in RARS, whereas LRIG1, a negative regulator of proliferation, was down-regulated. PPM1A, whose function leads to G2/M cell cycle arrest and apoptosis via p53 activation, was also up-regulated. ABCB7, involved in the transfer of iron from mitochondria to cytosol and in maturation of cytosolic Fe/S enzymes, and mutated in X-linked sideroblastic anemia with ataxia, was significantly down-regulated in RARS. Several dysregulated genes in RARS were restored to the range of normal expression after G-CSF treatment. Of these, MFN2, which was down-regulated in RARS, maintains mitochondrial membrane stability, and restores mitochondrial membrane potential and cell respiration. A normalization of MFN2 is thus consistent with the observed inhibitory effect on cytochrome c release.

Several dysregulated genes including BAX, FLIP and BAG1 were not altered by G-CSF treatment, indicating that G-CSF does not exert an unspecific anti-apoptotic effect through the Bcl2 family proteins. Only one, BCLAF1, a transcriptional repressor and pro-apoptotic member of BCL2 family was upregulated in RARS erythroblasts and down-regulated by G-CSF. G-CSF treatment down-regulated also the expression of interferon induced genes including IFIT1, IFIH1, IFI44, IFIT2, IFIT3, IRF7, IFRG28 and IFI78, several of which were previously shown to be upregulated in RARS CD34+ cells (Pellagatti, 2006). Since G-CSF specifically supports erythroblast survival in RARS through inhibition of mitochondria-mediated apoptosis, our findings may lead to further understanding of the molecular mechanisms in RARS and to the identification of candidate genes in this disease.

Author notes

Disclosure: No relevant conflicts of interest to declare.