Cyclooxygenase (COX)-1 or -2 and prostaglandin (PG) synthases catalyze the formation of various PGs and thromboxane (TX) A 2. We have investigated the expression and activity of COX-1 and -2 during human megakaryocytopoiesis. We analyzed megakaryocytes from bone marrow biopsies and derived from thrombopoietin-treated CD34 ؉ hemopoietic progenitor cells in culture. Platelets were obtained from healthy donors and patients with high platelet regeneration because of immune thrombocytopenia or peripheral blood stem cell transplantation. By immunocytochemistry, COX-1 was observed in CD34 ؉ cells and in megakaryocytes at each stage of maturation, whereas COX-2 was induced after 6 days of culture, and remained detectable in mature megakaryocytes. CD34 ؉ cells synthesized more PGE 2 than TXB2 (214 ؎ 50 vs. 30 ؎ 10 pg͞10 6 cells), whereas the reverse was true in mature megakaryocytes (TXB 2 8,440 ؎ 2,500 vs. PGE 2 906 ؎ 161 pg͞10 6 cells). By immunostaining, COX-2 was observed in <10% of circulating platelets from healthy controls, whereas up to 60% of COX-2-positive platelets were found in patients. A selective COX-2 inhibitor reduced platelet production of both PGE 2 and TXB2 to a significantly greater extent in patients than in healthy subjects. Finally, we found that COX-2 and the inducible PGE-synthase were coexpressed in mature megakaryocytes and in platelets. We conclude that both COX-isoforms contribute to prostanoid formation during human megakaryocytopoiesis and that COX-2-derived PGE 2 and TXA2 may play an unrecognized role in inflammatory and hemostatic responses in clinical syndromes associated with high platelet turnover.

The role of the tumor necrosis factor (TNF) superfamily member receptor activator of nuclear factor kappa B ligand (RANKL) in promoting the differentiation of osteoclasts has been extensively characterized. In this study, we have investigated the effect of TNF-related apoptosisinducing ligand (TRAIL), a member of the TNF superfamily of cytokines, in osteoclastogenesis, by using human peripheral blood mononuclear cells and the RAW264.7 murine monocytic cell line. Both cell models differentiate into osteoclast-like cells in presence of RANKL plus macrophage-colony-stimulating factor (M-CSF), as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Unexpectedly, when added in culture in combination with RANKL plus M-CSF, TRAIL inhibited osteoclastic differentiation in both cell models. To investigate the molecular mechanism underlining such inhibitory activity, we analyzed the effect of TRAIL on the mitogen-activated protein kinases (MAPKs) pathways, which play a key role in osteoclastogenesis. Treatment with RANKL plus M-CSF activated both the ERK1/2 and p38/MAPK pathways, which are essential for proliferation and differentiation of preosteoclasts, respectively. Of note, the addition of TRAIL to RANKL plus M-CSF did not affect ERK1/2 but it profoundly inhibited p38/MAPK phosphorylation. Thus, our data demonstrate that TRAIL blocks osteoclastic differentiation and suggest that inhibition of the p38/ MAPK pathway by TRAIL likely plays an important role in this process. (Blood.

In order to investigate the biologic activity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on human erythropoiesis, glycophorin A (GPA) ؉ erythroid cells were generated in serum-free liquid phase from human cord blood (CB) CD34 ؉

BackgroundAlthough multimodality treatment can induce high rate of remission in many subtypes of non-Hodgkin's lymphoma (NHL), significant proportions of patients relapse with incurable disease. The effect of human bone marrow (BM) mesenchymal stem cells (MSC) on tumor cell growth is controversial, and no specific information is available on the effect of BM-MSC on NHL.Methodology/Principal FindingsThe effect of BM-MSC was analyzed in two in vivo models of disseminated non-Hodgkin's lymphomas with an indolent (EBV− Burkitt-type BJAB, median survival = 46 days) and an aggressive (EBV+ B lymphoblastoid SKW6.4, median survival = 27 days) behavior in nude-SCID mice. Intra-peritoneal (i.p.) injection of MSC (4 days after i.p. injection of lymphoma cells) significantly increased the overall survival at an optimal MSC∶lymphoma ratio of 1∶10 in both xenograft models (BJAB+MSC, median survival = 58.5 days; SKW6.4+MSC, median survival = 40 days). Upon MSC injection, i.p. tumor masses developed more slowly and, at the histopathological observation, exhibited a massive stromal infiltration coupled to extensive intra-tumor necrosis. In in vitro experiments, we found that: i) MSC/lymphoma co-cultures modestly affected lymphoma cell survival and were characterized by increased release of pro-angiogenic cytokines with respect to the MSC, or lymphoma, cultures; ii) MSC induce the migration of endothelial cells in transwell assays, but promoted endothelial cell apoptosis in direct MSC/endothelial cell co-cultures.Conclusions/SignificanceOur data demonstrate that BM-MSC exhibit anti-lymphoma activity in two distinct xenograft SCID mouse models of disseminated NHL.

Treatment of the human HL-60 cell line with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resulted in rapid (6-24 hours) cytotoxicity associated with progressive maturation of the surviving cells along the monocytic lineage. The occurrence of monocytic maturation was demonstrated by a significant increase of both CD14 and CD11b surface expression, the acquisition of morphologic features typical of mature monocytes, and phagocytic capacity in TRAIL-treated cultures. By using selective pharmacologic inhibitors, it was possible to demonstrate that activation of the caspase cascade played a crucial role in mediating TRAIL cytotoxicity and monocytic maturation of HL-60 cells. Moreover, experiments performed using agonistic polyclonal antibodies, which mimic the interactions between TRAIL and each TRAIL receptor, indicated that TRAIL-R1 was responsible for mediating the TRAILinduced maturation. Importantly, the maturational effects of TRAIL were observed also in primary normal CD34 ؉ cells, seeded in serum-free liquid cultures for 4 to 8 days in the presence of SCF ؉ GM؊CSF. After treatment with TRAIL for 3 additional days, a significant increase in CD14 and CD11b expression, coupled with an increased number of mature monocytes and macrophages, was noticed in the absence of cytotoxicity. These data disclose a novel role for TRAIL as a positive regulator of myeloid differentiation. Moreover, the dichotomous effect of TRAIL on malignant cells (early induction of apoptosis and monocytic maturation of the surviving cells) might have important therapeutic implications for the treatment of acute myeloid leukemia.

Osteoprotegerin (OPG) is a soluble tumor necrosis factor receptor family member, which potently inhibits RANKL-mediated osteoclastogenesis. Numerous constructs have been created for therapeutic purposes in which the heparin-binding and death homology domains of OPG were removed and the remaining peptide (amino acids 22-194) was fused to the Fc domain of human IgG1 (OPG-Fc). The administration of OPG-Fc efficiently counteracted bone loss in a variety of preclinical models of cancers. However, several in vitro studies have shown that native or recombinant full-length OPG not only neuralizes RANKL, but also the death-inducing ligand TRAIL, suggesting that OPG might potentially counteract the anti-tumor activity of TRAIL. Additional evidence suggests that full-length OPG possesses RANKL- and TRAIL-independent biological properties, mainly related to the promotion of endothelial cell survival and angiogenesis. Finally, breast tumor cells overexpressing OPG have shown increased bone metastatic potential in vivo. The relevance of these apparently conflicting findings in tumor cell biology is highlighted.

The small molecule inhibitor of the MDM2/ p53 interaction Nutlin-3 significantly upregulated the steady-state mRNA and protein levels of Notch1 in TP53 wild-type (OCI, SKW6.4) but not in TP53 deleted (HL-60) or TP53 mutated (BJAB) leukemic cell lines. A direct demonstration that NOTCH1 was a transcriptional target of p53 in leukemic cells was obtained in experiments carried out with siRNA for p53. Moreover, inhibition of Notch1 expression using Notch1-specific siRNA significantly increased cytotoxicity in TP53 wild-type leukemic cells. IntroductionThe activation of p53 is tightly regulated by the human homolog of murine double minute 2 (MDM2) gene, 1 which is an E3 ubiquitin ligase for p53 and itself and controls p53 half-life mainly via ubiquitindependent degradation. In response to a variety of stimuli, such as cellular stress, the p53-MDM2 interaction is disrupted and p53 rapidly accumulates within the cell. 1 Potent and selective small molecule inhibitors of the p53-MDM2 interaction, the Nutlins, have been recently reported. 2,3 These compounds bind MDM2 in the p53 binding pocket with high selectivity and can release p53 from negative control, leading to effective stabilization of p53 and activation of the p53 pathway. 2,3 It has been demonstrated that treatment with the active enantiomer Nutlin-3a results in rising levels of p53 protein and subsequent induction of cell cycle arrest and apoptosis in a variety of tumor cells. 2 In contrast to most solid tumors, TP53 is mutated in approximately 10% to 15% of both myeloid and lymphoid leukemias at diagnosis, 4 and several recent studies have demonstrated that Nutlin-3 induces ex vivo cytotoxic cell death of most TP53 wild-type primary hematologic malignancies, including acute myeloid leukemias, multiple myeloma, B-chronic lymphocytic leukemias (B-CLL), and B-cell lymphomas. [5][6][7][8][9][10][11][12][13][14][15] Although it is well established that p53 mediates a variety of cellular functions, such as cell cycle arrest, cellular senescence, and, only as ultimate choice, apoptosis, 16,17 some experimental evidence suggests that inhibition of the transcriptional activity of p53 might paradoxically result in an increase of the p53-mediated proapoptotic activity, 18 also in B-CLL cells. 19 Therefore, the aim of this study was to further investigate the relationship between the transcriptional and cytotoxic activities induced by Nutlin-3 and to elucidate whether Nutlin-3 promotes the transcription of antiapoptotic genes in leukemic cells, which might hamper and/or reduce its potential therapeutic efficacy. In this respect, it has recently emerged that a potential target gene of the p53 pathway is NOTCH1, 20 which belongs to an evolutionarily conserved pathway that profoundly impacts mammalian development. Of note, a recent study published while this article was under preparation has demonstrated that circulating B-CLL cells overexpress Notch1 and Notch2 family members with respect to circulating normal B lymphocytes. 21 The same study suggested that constitutively...

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