Flt3 ligand stimulates proliferation and inhibits apoptosis of acute myeloid leukemia cells: regulation of Bcl-2 and Bax

Abstract

Flt3/flk-2 ligand (flt3-L) is a potent costimulator of normal bone marrow (BM) myeloid progenitors. Flt3-L is produced by BM stromal cells and its receptor is expressed in the majority of acute myeloid leukemia (AML) cases. Therefore, flt3-L may play a role in the paracrine and/or autocrine loops sustaining leukemic cell growth. We evaluated the effects of recombinant human flt3-L on proliferation, apoptosis, and Bcl-2 and Bax expression in primary AML cells and compared them with those of stem cell factor (SCF). Mononuclear BM cells from patients with newly diagnosed AML were cultured in serum-free conditions with flt3-L, SCF, granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage-colony-stimulating factor (GM-CSF) alone and in combination. In 9 of 10 samples, flt3-L significantly increased [3H]thymidine uptake (geometric mean stimulation index, 7.5; range, 2.4 to 41.5). Flt3-L also increased the number of AML blast colonies by 126% (range, 61% to 181%). In these 9 samples, flt3-L significantly enhanced the proliferative response triggered by G-CSF or GM-CSF. Flt3- L prevented apoptosis in AML blasts. It reduced the number of apoptotic cells by 36% +/- 3.9% compared with control cultures. Combining flt3-L with G-CSF or GM-CSF doubled the antiapoptotic effect. Cellular Bcl-2 and Bax levels were determined separately for apoptotic and nonapoptotic cells by flow cytometry. Cells undergoing spontaneous apoptosis had low Bcl-2 and high Bax levels, whereas nonapoptotic cells had high Bcl-2 and low Bax levels. Flt3-L alone or in combination with G-CSF or GM-CSF did not upregulate Bcl-2. However, Bax expression decreased in viable cells in the presence of these cytokines and the lowest level was achieved when a combination of flt3 and GM-CSF was used. Proliferative and viability effects of flt3-L were similar to those of SCF. Our results demonstrate that flt3-L acts as a stimulatory factor for primary AML cells. The antiapoptotic effects of flt3-L or its combinations with G-CSF or GM-CSF correlate with their ability to prevent upregulation of Bax.