Arsenic trioxide (As2O3) is a potent inducer of apoptosis of leukemic cells in vitro and in vivo, but the precise mechanisms by which it mediates such effects are not well defined. We provide evidence that As2O3 induces activation of the mitogen- and stress-activated kinase 1 (MSK1) and downstream phosphorylation of its substrate, histone H3, in leukemia cell lines. Such activation requires upstream engagement of p38 MAPK, as demonstrated by experiments using pharmacological inhibitors of p38 or p38alpha knock-out cells. Arsenic-induced apoptosis was enhanced in cells in which MSK1 expression was decreased using small interfering RNA and in Msk1 knock-out mouse embryonic fibroblasts, suggesting that this kinase is activated in a negative feedback regulatory manner to regulate As2O3 responses. Consistent with this, pharmacological inhibition of MSK1 enhanced the suppressive effects of As2O3 on the growth of primary leukemic progenitors from chronic myelogenous leukemia patients. Altogether, these findings indicate an important role for MSK1 downstream of p38 in the regulation of As2O3 responses.

The p38 mitogen-activated protein kinase (MAPK) pathway is activated by IFNs and other cytokines to mediate signals for important cellular functions, including transcriptional regulation and apoptosis. We examined the role of the p38 pathway in the generation of the effects of myelosuppressive cytokines on human hematopoiesis. Pharmacologic inhibition of p38 using BIX-01208 resulted in reversal of IFN-, tumor necrosis factor-A (TNF-A)-, and transforming growth factor-B (TGF-B)-mediated suppression of human erythroid (blast-forming unit-erythroid) and myeloid (granulocyte-macrophage colony-forming unit) colony formation, consistent with a key role for p38 in the generation of myelosuppressive signals by different cytokines. Similarly, the myelosuppressive effects of TNF-A and TGF-B were reversed by small interfering RNAs targeting p38A expression, further establishing the requirement of this kinase in the induction of myelosuppressive responses. As TNF overproduction has been implicated in the pathophysiology of bone marrow failure states, we determined whether pharmacologic inhibition of p38 reverses the hematopoietic defects seen in bone marrows from patients with myelodysplastic syndromes (MDS) and the anemia of chronic disease. Addition of pharmacologic inhibitors of p38 on such bone marrows resulted in increased numbers of erythroid and myeloid progenitors. Similarly, inhibition of the activity of the downstream effectors of p38, MAPK activated protein kinase-2, and mitogen and stress activated kinase 1 partially restored the hematopoietic defect seen in these bone marrows. Taken altogether, our data implicate the p38 MAPK in the pathophysiology of myelodysplasias and suggest that p38 pharmacologic inhibitors may have therapeutic applications in the treatment of MDS. (Cancer Res 2005; 65(19): 9029-37)

Proteinase inhibitor 9 (PI-9) inhibits caspase-1 (interleukin (IL)-1␤-converting enzyme) and granzyme B, thereby regulating production of the pro-inflammatory cytokine IL-1␤ and susceptibility to granzyme B-induced apoptosis. We show that cellular PI-9 mRNA and protein are induced by IL-1␤, lipopolysaccharide, and 12-O-tetradecanoylphorbol-13-acetate. We identified functional imperfect nuclear factor-B (NF-B) sites at ؊135 and ؊88 and a consensus activator protein-1 (AP-1) site at ؊308 in the PI-9 promoter region. Using transient transfections in HepG2 cells to assay PI-9 promoter mutations, we find that mutational ablation of the AP-1 site or of either NF-B site reduces IL-1␤-induced expression of PI-9 by ϳ60%. Mutational ablation of the two NF-B sites and of the AP-1 site nearly abolishes both basal and IL-1␤-induced expression of PI-9. Nuclear extracts from IL-1␤-treated HepG2 cells exhibited strong, IL-1␤-inducible binding to the NF-B sites and to the AP-1 site. Electrophoretic mobility shift assays show that after IL-1␤ treatment c-Jun/c-Fos and JunD bind to the AP-1 site, whereas the p50/p65 heterodimer binds to the two NF-B sites. Estrogens induce PI-9, but induction of PI-9 by estrogens and IL-1␤ is not synergistic. In transiently transfected, estrogen receptor-positive HepG2ER7 cells, estrogens do not interfere with IL-1␤ induction, whereas IL-1␤ exhibits dose-dependent repression of estrogen-inducible PI-9 expression. Our surprising finding that the pro-inflammatory cytokine IL-1␤ strongly induces PI-9 suggests a novel mechanism for regulating inflammation and apoptosis through a negative feedback loop controlling expression of the anti-inflammatory and anti-apoptotic protein, PI-9.

The mechanisms by which retinoids regulate initiation of mRNA translation for proteins that mediate their biological effects are not known. We have previously shown that all-trans-retinoic acid (ATRA) induces mTOR-mediated activation of the p70 S6 kinase, suggesting the existence of a mechanism by which retinoids may regulate mRNA translation. We now demonstrate that treatment of acute promyelocytic leukemia (APL)-derived NB4 cells with ATRA results in dissociation of the translational repressor 4E-BP1 from the eukaryotic initiation factor eIF4E, and subsequent formation of eIF4G-eIF4E complexes. We also show that siRNA-mediated inhibition of 4E-BP1 expression enhances ATRA-dependent upregulation of p21 Waf1/Cip1 , a protein that plays a key role in the induction of retinoid-dependent responses. Our data also establish that ATRA-or cis-RA-dependent p21 Waf1/Cip1 protein expression is enhanced in mouse embryonic fibroblasts with targeted disruption of the 4e-bp1 gene, in the absence of any effects on the transcriptional regulation of the p21 Waf1/Cip1 gene. Moreover, generation of ATRA-or cis-retinoic acid (cis-RA)-antiproliferative responses is enhanced in 4E-BP1 knockout cells. Altogether, these findings strongly suggest a key regulatory role for the translational repressor 4E-BP1 in the generation of retinoid-dependent functional responses.