Regulation of the acute myeloid leukemia cell line OCI/AML-2 by endothelial nitric oxide synthase under the control of a vascular endothelial growth factor signaling system

It is generally accepted that the vascular endothelial growth factor (VEGF) signal system has no role in the maintenance of normal blood cell formatio n, although it obviously regulates the development of primitive hematopoies is during an early stage of embryogenesis. The VEGF signaling pathway, howe ver, might have some role in malignant hematopoiesis, since malignant hemat opoietic cells, including acute myeloid leukemia (AML) cells, have been sho wn to express VEGF and its receptors. In endothelial cells, the VEGF/Flk-1/ KDR signal system is a very important generator of nitric oxide (NO) throug h the activation of its downstream effectors phosphatidylinositol-3-OH-kina se (P13-K), Akt kinase and endothelial NO synthase (eNOS). It is known that NO regulates hematopoiesis and modulates AML cell growth. The role of the VEGF signaling pathway in the control of AML cell growth through eNOS, howe ver, has not been studied. By using the OCI/AML-2 cell line, which expresse s VEGF receptor-2, ie Flk-1/KDR, eNOS and VEGF, as analyzed by flow cytomet ry, and produces VEGF into growth medium, as analyzed by ELISA, we showed t hat the Akt kinase and NOS activities in these cells were decreased by the inhibitors of VEGF, Flk-1/KDR and P13-K, and NOS activity also by the direc t inhibitor of NOS. The decreased NOS activity led to inhibition of clonoge nic cell growth and, to some extent, induction of apoptosis. We also found that blast cells of bone marrow samples randomly taken from 14 AML patients uniformly expressed FIk-1/KDR and to varying degrees eNOS and VEGF, as ana lyzed by immunohistochemistry. We conclude that autocrine VEGF through FIk- 1/KDR, by activating eNOS to produce NO through P13-K/Akt kinase, maintains clonogenic cell growth in the OCI/AML-2 cell line. Since the patient samp es did not express VEGF in all cases, it is possible that in vivo the regul atory connection between these two signal systems is also mediated via endo crine VEGF in addition to autocrine or paracrine VEGF.