The amount ofCD7transcripts is relatively constant in the lin-CD34+cells from all four normal bone marrow samples, whereas theCD7transcript levels in the corresponding subset of CML cells show marked variation among samples. promoter did not correlate with its expression pattern in lin-CD34+cells from chronic phase CML patient samples even though there was clear differential DNA methylation of this locus inELA2-expressing and non-expressing cell lines. In contrast, we found a strong relation between CD7 expression and transcription-permissive chromatin modifications, both at the level of DNA methylation and histone acetylation with evidence of hypomethylation of theCD7promoter region in the lin-CD34+cells from CML patients with high CD7 expression. == Conclusion == These findings indicate a link between ZLN024 epigenetic modifications ZLN024 and CD7 expression in primitive CML cells. == Background == A feature of chronic myeloid leukaemia (CML) is the Philadelphia chromosome (Ph). This abnormal chromosome results from a reciprocal translocation between chromosome 9 and 22 that gives rise to theBCR-ABLfusion gene that is now the accepted defining hallmark of the disease [1-3]. CML is typically diagnosed in an initial chronic phase (CP) which is characterized by the formation of a multi-lineage clone of Ph+/BCR-ABL+leukemic cells that typically dominates the entire hematopoietic system by the time of diagnosis. This clone includes a selectively enlarged compartment of myeloid progenitors that produce an elevated number of normally differentiated granulocytes [4]. Targeted therapy of CML with imatinib mesylate (IM) or other inhibitors of the BCR-ABL oncoprotein is the current therapy of choice for patients with CP disease, although IM does not always produce durable remissions [5,6]. The most common cause of a poor response outcome is the appearance of IM-resistant cells. This resistance might be BCR-ABL-dependent, such as mutations in the kinase domain of BCR-ABLand genomic amplification of theBCR-ABLlocus or BCR-ABL-independent mechanisms such as ZLN024 constitutive activation of downstream pathways [7-9]. Other genetic causes of disease heterogeneity as well as the inherent resistance of the leukemic progenitor/stem cells to IM may be additional contributing factors [2,10,11]. Identification and characterization of epigenetic changes that control the properties of CML cells, especially those of CML stem/progenitor (lin-CD34+) cells may be useful ZLN024 for the design of suitable therapies for IM-refractory CML [12]. As with other cancers, various markers divide CP CML into different subclasses that are associated with different patient survival probabilities.CD7and elastase 2 (ELA2)are two such genes that show differential expression patterns in CML cells [13]. Various studies indicate expression ofCD7to be upregulated in CML cells (and various other leukaemias and lymphomas) where it has been associated with poor survival [14,15].ELA2is clustered with two other serine protease gene family members, azurocidin 1 and proteinase 3 (PRTN3, also known as Myeloblastin) genes, at chromosome 19pter [16]. These three genes are expressed co-ordinately and their products packaged together Rabbit polyclonal to AMPK gamma1 into azurophil granules during neutrophil differentiation. High expression of these three genes in CML is associated with a favourable prognosis [13]. CD7is located on chromosome 17 and encodes a cell surface glycoprotein member of the immunoglobulin superfamily. The protein is found on thymocytes and mature NK and T-cells [17,18]. It plays an essential role in T-cell interactions and also in T-cell/B-cell interactions during early lymphopoeisis. ELA2is one of six structurally similar human elastase genes,ELA1, 2, 2A, 2B, 3Aand3B. Elastases form a subfamily of serine proteases that hydrolyze many proteins in addition to elastin. ELA2 hydrolyzes proteins within specialized neutrophil lysosomes, called azurophil granules, as well as proteins of the extracellular matrix following the protein’s release from activated neutrophils. In addition, ELA2 may play a role in degenerative and inflammatory diseases by its proteolysis of collagen-IV and elastin of the extracellular matrix. This protease degrades the outer membrane protein A (OmpA) of E. coli as well as the virulence factors of such bacteria as Shigella, Salmonella and Yersinia [19]. Mutations in theELA2gene are associated with cyclic neutropenia and severe congenital neutropenia (SCN) [20]. The role of epigenetic mechanisms in the transcriptional control ofCD7andELA2is unknown. In this study we have examined the transcription pattern of these two genes in normal adult ZLN024 human bone marrow and primary CML cells. We specifically compared the degree of 5′ region DNA methylation withCD7andELA2transcript levels and assessed levels of histone acetylation at the promoter regions ofCD7andELA2in expressing and non-expressing cell lines. == Materials and methods == == Cells == The human cell line THP-1 (monocytic leukemia) was cultured in RPMI plus 10% fetal calf serum (FCS), 10 mM Hepes, 2 mM glutamine and penicillin/streptomycin (pen/strep). The human cell lines ALL-SIL (T-cell leukemia) and RPM1 (T-ALL) were cultured in RPMI plus 10% FCS, 1% sodium pyruvate, 1% glutamine and pen/strep. Normal adult human bone marrow cells and CP CML patient samples were obtained with informed consent according to protocols approved by the Research Ethics.