Identification of Putative New Tumor Suppressor Genes in Highly Purified CD34+ Bone Marrow Cells from Patients with Myelodysplastic Syndromes.

Activation of transcription of DNA by demethylation and hyperacetylation is known to cause hematologic improvement in patients with myelodysplastic syndromes (MDS). In this study we discriminated genes not expressed in CD34+ cells from untreated patients with MDS but activated by in vitro demethylation (2-aza-5-deoxycytidine, Decitabine) and hyperacetylation (suberoylanilide hydroxamic acid, SAHA). Highly purified CD34+ cells from normal individuals (n=3) and patients with low (n=3) and high (n=3) risk MDS were cultured with SCF (50 ng/ml), IL-3 (10 ng/ml) and GM-CSF (10 ng/ml). The cells were treated with 5 μmol Decitabine on day 1 and supplemented with 2.5 μmol SAHA on day 4 of culture. On day 5, global gene expression in these cells was compared to untreated cells (HG-U133A, Affymetrix, Santa Clara, CA). We identified 50 genes which are not expressed in untreated MDS CD34+ cells but 3-fold induced in all MDS samples by Decitabine and SAHA. Thirty-one of these genes were found to be expressed in normal CD34+ cells underlining the importance of such genes for normal hematopoiesis. This set of genes includes two genes for growth arrest and DNA damage control, the inducible protein beta (GADD45B), a regulator of growth and apoptosis and neural cell adhesion molecule 1 (NCAM1) that plays an important role in cell migration. Furthermore, hematological and neurological expressed 1 (HN1) which was not expressed in MDS CD34+ cells is known to have an anti-proliferative effect on tumor cell lines. N-myc downstream regulated 3 (NDRG3) is up-regulated during normal cell differentiation and suppressed in several tumor cells. In normal CD34+ cells, after in vitro treatment with Decitabine and SAHA we have discriminated 52 genes to be 3-fold up-regulated compared to untreated cells. Thirty-eight of these genes (73 %) were not inducible by demethylation and hyperacetylation in MDS CD34+ cells. These genes include chemokine receptor 3 (CCR3), a receptor for a C-C type chemokine involved in signal transduction, integrin beta-7 (ITGB7) that plays a role in adhesive interactions of leukocytes, preferentially expressed antigen in melanoma (PRAME) which is frequently expressed in human solid cancers and acute leukemia and tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) that recruits apoptotic suppressors and mediates most of the metabolic effects of TNF-alpha. The silencing of these genes is independent of methylation and acetylation state and might be due to other mechanisms. This study shows that in CD34+ cells from MDS patients several genes are suppressed by methylation and hypoacetylation but can be activated by treatment with Decitabine and SAHA. Some of these genes are present in normal untreated CD34+ cells which leads to the assumption that they might function as tumor suppressor genes. Low or absent expression of these genes may contribute to the clonal expansion of MDS CD34+ which can be overcome by treatment with Decitabine or SAHA. Furthermore, the knowledge about these target genes may enable a more specific evaluation of the mechanisms of action of demethylating/hyperacetylating agents.