The Role of Endogenous Stromal Cell-Derived Factor-1 in the Survival and Growth of Acute Myeloid Leukemia Cells

Abstract 1502

Stromal cell-derived factor-1 (SDF-1) is constitutively expressed and produced by stromal cells in the bone marrow (BM). The protein exerts its functions by binding to its receptor CXCR4, thus guiding the homing of hematopoietic stem/progenitor cells and leukemia cells to the BM. CD34+ cells purified from normal adult peripheral blood have been shown to express and produce SDF-1, which exerted an antiapoptotic effect on CD34+ cell through an autocrine/paracrine loop. Acute myeloid leukemia (AML) cells have also been shown to express and produce SDF-1, but the role of SDF-1 in this context is largely unknown. Thus, the aim of the present study was to determine the function of SDF-1 produced by AML cells. Using siRNA technology, SDF-1 was knocked-down in AML cells and subsequent biological alterations occurring in the cells were evaluated in vitro. All AML cell lines used in this study (U937, K562, KG1a, HL-60 and MO7e) as well as primary CD34+ cells obtained from the BM of 5 patients with AML expressed SDF-1 mRNA at various levels, as revealed by semi-quantitative and real time RT-PCR analysis. SDF-1 was detected by Western blotting analysis and ELISA allowed for the detection of SDF-1 in the supernatants of cultures of AML cells grown for 3 days in a serum-free medium, indicating that AML cells, similar to normal CD34+ cells, are able to produce and secrete SDF-1. Exogenous SDF-1 (added at a concentration of up to 200 ng/mL) alone had no discernible effects on the survival or proliferation of either the cell lines or the primary CD34+ AML cells maintained in a serum-free medium, but was able to induce the internalization of CXCR4 and CXCR7. Transfection of five AML cell lines with SDF-1 siRNA diminished the SDF-1 mRNA levels by up to 90%. The knock-down cells were stable for at least 3 days. SDF-1 knock-down did not affect the serum deprivation-induced apoptosis of the cells. Cell surface expression of CXCR4 was not changed, whereas cytoplasmic CXCR4 was significantly upregulated along with the upregulation of SDF-1 mRNA. Neither cell surface nor cytoplasmic expression of CXCR7 was affected by the knock-down of SDF-1. Conversely, the knock-down of CXCR7, but not CXCR4, upregulated the SDF-1 mRNA expression in the transfected cells. No changes in the transmembrane and trnasendothelial migration of the knock-down cells were observed. In contrast, the knock-down of SDF-1 inhibited the spontaneous proliferation of the U937, K562, and KG1a cells by 20–30% during a 3-day incubation in a serum-free medium. These results indicate that endogenous SDF-1 of AML cells functions as an autocrine growth factor.