Extracellular NM23 protein promotes the growth and survival of primary cultured human acute myelogenous leukemia cells

Abstract

An elevated serum level of NM23-H1 protein is found in acute myelogenous leukemia (AML), and predicts a poor treatment outcome in AML patients. 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 on the in vitro growth and survival of primary cultured AML cells at concentrations equivalent to the levels found in the serum of AML patients. Extracellular NM23-H1 protein promoted the in vitro growth and survival of AML cells and this activity was associated with the cytokine production and activation of the MAPK and signal transducers and activators of transcription signaling pathways. Inhibitors specific to MAPK signaling pathways inhibited the growth- and survival-promoting activity of NM23-H1. These findings indicate the novel biological action of extracellular NM23-H1 and its association with poor prognosis, and suggest an important role for extracellular NM23-H1 in the malignant progression of leukemia and a potential therapeutic target for these malignancies.

Figure 1

Growth‐ and survival‐promoting activity of extracellular recombinant (r) NM23 proteins on primary cultured acute myelogenous leukemia (AML) cells. (a) We used 14 cases of AML. +, NM23‐responsive cases; –, unresponsive cases. (b) AML cells (1 × 10⁶ cells/mL, n = 14) were incubated with or without 100 ng/mL rNM23‐H1 protein. The effects on cell growth and survival were measured by MTT assay. Box plots: upper and lower lines indicate 10th and 90th percentiles, boxes indicate 25th and 75th percentiles. The line through each box indicates the median. Asterisk represents significant differences (P < 0.05) using Student's t‐test. (c–d) Representative growth‐ and survival‐promoting activity of rNM23‐H1 and rNM23‐H2 proteins in the presence or absence of polymixin B to eliminate any potential effect of LPS. (c) Case 9 on day 3; (d) case 4 on day 6. (e) Effect of LPS inhibitor, polymixin B (1.25 µg/mL), on growth‐ and survival‐promoting activity of extracellular rNM23‐H1 (100 ng/mL) or LPS (1 and 10 ng/mL) (case 2 on day 5). (f) Growth‐ and survival‐promoting activity of mutant NM23‐H1^His protein (100 ng/mL), which does not have nucleoside diphosphate kinase (NDPK) activity (case 2 on day 5). Error bars, SD. FAB, French–American–British classification; MDS, myelodysplastic syndrome.

Figure 2

Cytokine‐inducing activity of extracellular recombinant (r) NM23‐H1 protein on primary cultured acute myelogenous leukemia (AML) cells. (a) Growth‐ and survival‐promoting activity (dose response) of rNM23‐H1 protein of four representative cases on days 2–10 (left panels), and the cytokines detected in conditioned medium (CM) of AML cells treated with or without rNM23‐H1 (100 ng/mL) for 48 h, using human inflammation antibody array (right panels), refer to (b). 1, GM‐CSF; 2, I‐309; 3, IL‐1β; 4, IL‐6; 5, IL‐8; 6, IL‐10; 7, RANTES; 8, TNFα. (c) Various cytokine levels in CM (2 h for TNFα, IL‐6 and 48 h for IL‐1β, GM‐CSF, IL‐8, IP‐10) of AML cells treated with or without rNM23‐H1 (100 ng/mL), were measured by sandwich ELISA. Error bars, SD.

Figure 2

Cytokine‐inducing activity of extracellular recombinant (r) NM23‐H1 protein on primary cultured acute myelogenous leukemia (AML) cells. (a) Growth‐ and survival‐promoting activity (dose response) of rNM23‐H1 protein of four representative cases on days 2–10 (left panels), and the cytokines detected in conditioned medium (CM) of AML cells treated with or without rNM23‐H1 (100 ng/mL) for 48 h, using human inflammation antibody array (right panels), refer to (b). 1, GM‐CSF; 2, I‐309; 3, IL‐1β; 4, IL‐6; 5, IL‐8; 6, IL‐10; 7, RANTES; 8, TNFα. (c) Various cytokine levels in CM (2 h for TNFα, IL‐6 and 48 h for IL‐1β, GM‐CSF, IL‐8, IP‐10) of AML cells treated with or without rNM23‐H1 (100 ng/mL), were measured by sandwich ELISA. Error bars, SD.

Figure 3

Inhibitory effects of cytokine antibodies on the growth‐ and survival‐promoting activity of NM23‐H1. (a) Growth‐ and survival‐promoting activity of NM23‐H1 in the presence or absence of cytokine antibodies (TNFα antibody, IL‐6 antibody, IL‐1β antibody). IgG, control immunoglobulin. (b) Inhibitory effects induced by the combination of two cytokine antibodies (case 2 on day 6). (c) Anti‐GM‐CSF antibody inhibited the growth‐ and survival‐promoting activity of recombinant (r) NM23‐H1 and rGM‐CSF (case 1 on day 6). Error bars, SD.

Figure 4

Signal transduction pathways induced by extracellular recombinant (r) NM23‐H1 protein in primary acute myelogenous leukemia (AML) cells. (a) AML cells (case 2 and 9) were treated with extracellular rNM23‐H1 protein (100 ng/mL) for the indicated hours. Activation (phosphorylation) of MAPK (p38 and Erk1/2) was investigated by SDS‐PAGE and immunoblotting. (b) Activation (phosphorylation of Tyr and Ser) of signal transducers and activators of transcription (STAT) 1, STAT3, and STAT5 in AML cells (case 9) by treatment with extracellular rNM23‐H1 protein.

Figure 5

Effects of inhibitors of MAPK and signal transducers and activators of transcription (STAT) 3 on the recombinant (r) NM23‐H1‐promoted growth and survival of acute myelogenous leukemia (AML) cells. AML cells (case 2) were cultured with or without rNM23‐H1 protein (100 ng/mL) and various inhibitors for 5 days. Viable cells were determined by MTT assay. The values are the means ± SD for four independent samples. SB202190 and SKF86002, p38 MAPK inhibitor; PD98059, MEK inhibitor; Curcumin, STAT3 inhibitor, but not specific to STAT3. Right panels have an expanded MTT scale to show the direct toxicity of these inhibitors on AML cells under conditions without rNM23‐H1.

Figure 6

Extracellular function of NM23‐H1 protein derived from tumor cells on primary cultured acute myelogenous leukemia (AML) cells. Overexpression of the NM23 gene found in many hematological malignancies predicts a poor treatment outcome, and the elevated serum level of NM23‐H1 protein is also a poor prognostic factor in patients with these diseases. Extracellular NM23‐H1 protein promotes the growth and survival of primary AML cells mediated by MAPK activation, signal transducers and activators of transcription (STAT) activation and cytokine release. Taken together, these observations suggest that extracellular NM23‐H1 may play an important role in the malignant progression of leukemia, and that inhibitors of these protein or inhibitors of the extracellular functions of these proteins should be evaluated for their treatment potential.