Inhibitory Effect of Nitric Oxide on Chemically Induced Differentiation of Human Leukemic K562 Cells

Abstract

International audienceThe effect of nitric oxide (NO) was investigated in the human K562 cell line during chemically induced erythroid differentiation. Butyric acid (BA) and the anthracycline antitumour drugs aclarubicin (ACLA) and doxorubicin (DOX) were used as differentiating agents. In all cases, cell hemoglobinization was dose dependently inhibited by NO donors such as sodium nitroprusside (SNP). A 50% inhibition of cell differentiation was obtained with 25 M SNP, which generated less than 2 M nitrite in 3-day culture media. Increasing SNP concentrations led to higher nitrite accumulation (up to 12 M with 1 mM SNP) and total inhibition of cell hemoglobinization, but did not have a significant effect on cell proliferation. As shown by Northern blotting, high concentrations of SNP (1 mM) reduced the expression of-globin and porphobilinogen deaminase, but did not change GATA-1 and NF-E2 mRNA levels in ACLA-and BA-treated cells. In contrast, hemin-induced erythroid differentiation was not affected by the presence of NO donors. Altogether, these results show that NO is able to inhibit cell differentiation induced by some (ACLA, DOX, BA), but not all (hemin), agents. The inhibitory effect of NO seems to take place downstream of the regulation of erythroid gene expression. BIOCHEM PHARMACOL 58;5:773-778, 1999. © 1999 Elsevier Science Inc. KEY WORDS. anthracyclines; butyric acid; erythroid differentiation; hemin; nitric oxide Erythroid differentiation of the human K562 leukemic cell line can be achieved by exposure to several pharmacological agents including hemin [1], BA † [2], and anthra-cyclines such as ACLA and DOX [3]. The molecular mechanisms involved in the differentiation process are quite different depending on the inducer. The transcrip-tional activation of the-globin gene has been demonstrated in BA-and ACLA-induced K562 cells [4, 5]. This has been related to the enhancement of DNA-binding activity and the expression of GATA-1 and NF-E2 transcription factors [6-9]. In addition, the heme synthesis pathway enzymes PBGD [5, 8], erythroid-aminolevulinate dehydratase [10, 11], and eALAS [12] were also shown to be up-regulated at the transcriptional level in response to ACLA and BA. In contrast, DOX-and hemin-induced differentiation did not involve the overexpression of GATA-1 and NF-E2 transcription factors [5, 7]. Moreover, the transcriptional activity of erythroid promoters, which is enhanced by BA and ACLA, is not affected by DOX and hemin [13]. Nitric oxide is synthesized by NO synthases in endothe-lial and inflammatory-activated cells [14]. NO has been demonstrated to modulate the RNA binding of IRP-1 and IRP-2 iron responsive element (IRE) binding proteins [15, 16], which are responsible for the posttranscriptional control of IRE-containing mRNAs related to cellular iron homeostasis (e.g. ferritin, transferrin receptor, and eALAS [16]). Therefore, the study of NO effects on erythroid-specific genes has been mainly restricted to iron homeo-stasis-related genes [17-19]. In addition, NO may disturb cell growth and differentiation by regulating the activity of cellular targets involved in signal transduction, ATP, and oxygen production. Indeed, NO activates the soluble guanylate cyclase and inhibits iron-cluster enzymes including aconitase and mitochondrial complex I and II enzymes [20]. In this work, the effect of NO on chemically induced erythroid differentiation was investigated in K562 cells using NO donors such as SNP, SIN-1, and SNAP. The release of NO from NO donors was shown to inhibit the differentiation of anthracycline-and BA-treated K562 cells with an IC 50 of 25 M for SNP and less than 100 M for SIN-1 and SNAP. Higher doses of SNP were needed to decrease the expression of erythroid mRNAs. In contrast, hemin-induced differentiation was not affected by NO