Search

The self-renewal and differentiation of hematopoietic progenitors are regulated by the interaction between Notch receptors and Notch ligands. Since AML originates from dysregulated hematopoietic progenitors, some abnormalities in the Notch system may be involved in the abnormal proliferation of AML cells. However, the significance of the Notch system in AML is not known. We examined the functional roles of Notch activation on the in vitro growth of seven human AML cell lines using three kinds of recombinant Notch ligand proteins, Jagged-1, Delta-1 and Delta-4. The ligands significantly affected the growth of two cell lines. In TMD7 cells, Delta proteins promoted the short-term growth, however, suppressing the self-renewal capacity and long-term growth. In OCI/AML-6 cells, Delta proteins suppressed the growth and self-renewal capacity while inducing differentiation into macrophage-like cells. We additionally found that Notch ligands needed to be immobilized on culture wells to affect the cells. These findings were in contrast to our hypothesis that Notch activation in AML cells leads to excessive self-renewal capacity and proliferation. If the Notch system in AML cells is precisely understood, the control of Notch activation will become a novel therapeutic approach for AML.

We investigated the mechanisms of factor-independent growth of four human myeloid leukemia cell lines. The autocrine mechanisms were ruled out by RT-PCR method examining growth factor mRNA. The immunoblotting method showed that many proteins were tyrosine phosphorylated irrespective of the stimulation with growth factors (G-CSF and GM-CSF) in factor-independent cell lines while the phosphorylation was induced stimulation dependently in a factor-dependent cell line. MAP kinase was constitutively phosphorylated in factor-independent cell lines. JAK2 protein was not tyrosine phosphorylated before the stimulation. It was significantly phosphorylated after the stimulation in three factor-independent cell lines although the stimulation did not affect their growth. JAK1 protein was not phosphorylated either before or after the stimulation. In conclusion, constitutive phosphorylation of signaling proteins seemed to be related to factor-independent growth. MAP kinase was involved in the phosphorylation, while JAK1 and JAK2 were not.

In terms of growth, differentiation, and signaling pathways of hematopoietic factors, the effects of protein kinase C (PKC) activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and protein kinase A(PKA) activator, dibutyryl cyclic adenosine monophosphate (dbcAMP) were examined in vitro using three factor-responsive myeloid leukemia cell lines. TPA suppressed the growth of OCI/AML-1 and OCI/AML-5 cells but stimulated the proliferation of OCI/AML-4 cells. TPA differentiated OCI/AML-4 and OCI/AML-5 cells to macrophage-like cells. dbcAMP suppressed the growth of the three without differentiation. The stimulation of TPA induced tyrosine phosphorylation of some proteins in OCI/AML-4 and OCI/AML-5 cells. Their molecular weights were the same as those phosphorylated by hematopoietic factors. The patterns of phosphorylated proteins were different between the two. TPA induced the phosphorylation of MAP kinase, but not that of JAK2 protein in three cell lines. The stimulation of dbcAMP did not induce tyrosine phosphorylation in three cell lines. Overnight exposure of TPA inhibited the tyrosine phosphorylation induced by hematopoietic factors, although that of dbcAMP did not. We suggest a close relation of PKC to signaling pathways of hematopoietic factors, however, the ways of relation were diverse among the cell lines and the clear mechanism explaining its effects on growth and differentiation was not elucidated.

To examine the role of stroma cells on the proliferation and c-kit expression of acute myeloblastic leukemia (AML) cells, blast cells from patients were cultured with growth factors or on human marrow stroma cells. c-KIT protein detected by flow cytometry and the plating efficiencies of the cells cultured with growth factors decreased markedly, but those of the cells cultured on stroma cells were mostly preserved. Conditioned medium from the stroma cells and fibroblasts from fetal Sl/Sl mouse showed, to some extent, the same effects. The growth factors including kit ligand seemed to favor the terminal division more than the self renewal and stroma cells had the opposite tendency. Stoma cells may produce some factors other than kit ligand to maintain c-kit expression and high clonogenicity.