Upregulation of hGC-1 Expression by Hypomethylation, Retinoic Acid and Interferons in Human Myeloid Leukemia

hGC-1 (Granulocyte colony stimulating factor induced gene-1, also called GW112 and olfactomedin 4) was first identified in human myeloid precursor cells induced by granulocyte colony stimulating factor (G-CSF). It is a myeloid lineage and differentiation stage specific gene. Its expression, regulation and biological function, specifically in myeloid cells, are still poorly understood. In this study, we analyzed the hGC-1 gene expression in leukemia patients and further investigated the mechanism of hGC-1 gene regulation in leukemia cells. We found that hGC-1 was overexpressed in myeloid leukemia patients compared with normal individuals in peripheral blood leukocytes (p<0.01) and its expression in accelerated phase of chronic myeloid leukemia (CML) patients was significantly higher than that in chronic phase (p<0.01) using a dot blot and quantitative RT-PCR analysis. Hypomethylation of CpG sites in the promoter of hGC-1 gene were observed in CML patients by pyrosequence and 5-aza-2′-deoxycytidine induced hGC-1 expression in myeloid leukemia cells, suggesting that promoter CpG methylation status affects the expression of hGC-1 gene. All-trans-retinoic acid (ATRA) and interferons (IFNs) are active anti-leukemia agents. ATRA and IFNs have shown synergistic interactions in various experimental conditions and represent a potentially useful therapeutic combination in the treatment of various types of leukemia. However, the target genes and molecular basis of these interactions still needs to be further elucidated. Here, we identified that hGC-1 was a target gene of RA in myeloblastic leukemia cells. Treatment with ATRA induced hGC-1 expression in HL-60 cells and enhanced hGC-1 expression in AML-193 and GDM-1 cells. Deletion analysis led to the identification of a positive retinoic acid response element (DR5) and a negative response element (DR1) within hGC-1 promoter. Furthermore, electrophoretic mobility-shift assays demonstrated that RARa/RXRa binds to the DR5 site. Transfection study in COS-7 cells revealed RARa/RXRa mediated the RA induced transactivation of hGC-1 promoter. We also found that hGC-1 was an early responsive gene of IFN a and b in myeloid leukemia cells (HL-60, AML-193 and GDM-1). An effective interferon-stimulated response element (ISRE) was identified in the promoter of hGC-1 gene by examining the deletion mutants in luciferase reporter gene assay. Combined application of ATRA and IFNa and IFNb enhanced hGC-1 expression synergistically. Taken together, hGC-1 is identified as a novel target gene of methylation modification, RA and IFNs in myeloid leukemia cells. Our results suggest that hGC-1 is a potential marker for CML stage and may play a role in retinoic acid and interferon induced biological effects in leukemia cells.