Effects of Extracellular NM23 Protein on In Vitro Survival of Acute Myeloid Leukemia Cells and Normal Peripheral Blood Mononuclear Cells.

Overexpression of the nm23 gene is found in many hematological malignancies, and predicts poor treatment outcome. An elevated serum level of NM23-H1 protein is also a poor prognostic factor in patients with these diseases. To investigate the potential pathological link between the elevated serum level of this protein and poor prognosis, we examined the extracellular effects of recombinant NM23-H1 protein (rNM23-H1) on the in vitro survival of primary cultured AML cells and normal peripheral blood mononuclear cells (PBMNC) at concentrations equivalent to the levels found in AML patients. First, we examined the effects of extracellular rNM23 proteins on in vitro survival of primary cultured leukemic cells obtained from 14 patients with AML. The rNM23 proteins promoted survival of the cells from 11 of these 14 patients, and various cytokines were found in the conditioned medium of the leukemic cells. Furthermore, some cytokine antibodies inhibited the NM23-promoted survival of the leukemic cells. These findings suggest that by the stimulation of rNM23-H1 the leukemic cells released cytokines that may have promoted the survival of leukemic cells. rNM23-H1 activated p38 MAPK, Erk1/2, STAT1, and STAT3 in the leukemic cells. Inhibitors specific for these signaling pathways suppressed the survival-promoting activity of rNM23-H1. Next, normal PBMNC (1x10⁶ cells/ml) were cultured with or without rNM23-H1 at 10–1000 ng/ml. Although rNM23-H1 hardly affected the survival of PBMNC at day 7 assessed by MTT assay, the number of adherent cells significantly decreased. These adherent cells were shown to be CD68− and NSE-positive monocytes. These findings indicate that extracellular NM23 protein affects the in vitro survival of normal monocytes. It is known that the survival of PBMNC is regulated by various cytokines. Therefore, we examined cytokine expression patterns in PBMNC treated with or without rNM23-H1, using multiplex RT-PCR, cDNA microarray, cytokine antibody array, and ELISA. Concentrations of various cytokines (IL-1β, IL-6, TNFα. IL-8, GM-CSF, I-309, RANTES, IL-10) significantly increased and that of IP-10 profoundly reduced in conditioned medium of rNM23-H1-treated cells. These findings indicate that extracellular rNM23-H1 could modulate the induction of various cytokines including inflammatory cytokines in the normal PBMNC. These cytokines activated monocytes, and the activation resulted in apoptosis of the cells. These findings are similar to those shown in tumor-associated macrophages. Taken together, these observations suggest that extracellular NM23-H1 may play an important role in the malignant progression of leukemia through cytokine pathways of leukemic cells and normal monocytes. A reduction of extracellular NM23-H1 protein concentration or inhibitors of extracellular actions of these proteins should be evaluated for the therapeutic potential to combat these malignancies.