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.

Figure
View largeDownload slide

Figure

Topics:
aftercare, erythroblasts, gene expression profiling, granulocyte colony-stimulating factor, recombinant granulocyte colony stimulating factor, refractory anemia with sideroblasts, bcl-2 protein, cytochrome c, anemia, hereditary sideroblastic, ataxia

Author notes

Disclosure: No relevant conflicts of interest to declare.

2007, The American Society of Hematology
2007
Sign in via your Institution