The possible role of the peripheral cannabinoid receptor (CB2) in neutrophil migration was investigated by using human promyelocytic HL60 cells differentiated into neutrophil-like cells and human neutrophils isolated from whole blood. Cell surface expression of CB2 on HL60 cells, on neutrophil-like HL60 cells, and on human neutrophils was confirmed by flow cytometry. Upon stimulation with either of the CB2 ligands JWH015 and 2-arachidonoylglycerol (2-AG), neutrophil-like HL60 cells rapidly extended and retracted one or more pseudopods containing F-actin in different directions instead of developing front/rear polarity typically exhibited by migrating leukocytes. Activity of the Rho-GTPase RhoA decreased in response to CB2 stimulation, whereas Rac1, Rac2, and Cdc42 activity increased. Moreover, treatment of cells with RhoA-dependent protein kinase (p160-ROCK) inhibitor Y27632 yielded cytoskeletal organization similar to that of CB2-stimulated cells. In human neutrophils, neither JWH015 nor 2-AG induced motility or morphologic alterations. However, pretreatment of neutrophils with these ligands disrupted N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced front/rear polarization and migration and also substantially suppressed fMLP-induced RhoA activity. These results suggest that CB2 might play a role in regulating excessive inflammatory response by controlling RhoA activation, thereby suppressing neutrophil migration.The peripheral cannabinoid receptor (CB2) 2 was cloned in 1993 (1) after cloning of the central cannabinoid receptor (CB1) in 1990 (2). It has been suggested that the gene encoding CB2 is a protooncogene and that aberrant expression of CB2 in myeloid precursor cells results in the development of leukemia by blocking neutrophil differentiation (3, 4). CB2 is expressed predominantly in immune cells (5), and because of the diversity of immune cells, it is assumed that CB2 is involved in various activities in addition to inhibition of neutrophil differentiation (6 -9). Steffens et al. (10) recently reported that doses of ⌬ 9 -tetrahydrocannabinol (the most psychoactive component of marijuana) too low to have psychotropic effects inhibit the progression of atherosclerosis via immunomodulatory effects on lymphoid and myeloid cells. This report indicates that CB2 may be involved in a wide range of physiologic phenomena related to immunity and that some CB2 ligands may have application in the treatment of inflammatory disease. However, research into CB2 is still in its early stages. In particular, the involvement of only a few molecules, G␣ i /G␣ o protein, phosphatidylinositol 3-kinase (PI3K), and members of the mitogen-activated protein kinase and nuclear factor-B families, in the CB2 signaling pathways has been reported (6 -8, 11).Among the possible roles of CB2 in immunity is the induction of leukocyte migration to sites of infection and inflammation, an important step in the host defense against pathogenic microorganisms. CB2 is a seven-transmembrane, G␣ i /G␣ o protein-coupled receptor, as ar...

Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell disorders characterized by refractory cytopenias and susceptibility to leukemic transformation. On a subset of MDS patients with deletion of the long arm of chromosome5 (del(5q)), lenalidomide exerts hematological and cytogenetic effects, but the underlying pharmacological mechanisms are not fully understood. In this study, we have investigated the in vitro effects of lenalidomide on an MDS-derived cell line, MDS-L, which carries del(5q) and complex chromosome abnormalities. We found that the growth of MDS-L cells was specifically suppressed mainly by apoptosis, and in addition, multinucleated cells were frequently formed and finally died out in the presence of lenalidomide. Time-lapse microscopic observation and the DNA ploidy analysis revealed that lenalidomide does not affect DNA synthesis but inhibits cytokinesis of MDS-L cells. The gene expression profile showed decreased expression of M phase-related genes such as non-muscle myosin heavy-chain 10, polo-like kinase 1, aurora kinase B, citron kinase and kinesin family member 20A(KIF20A). Interestingly, KIF20A is located at 5q31. These data contribute to the understanding of action mechanisms of lenalidomide on MDS with del(5q) and complex abnormalities.

Summary The neuron cytoplasmic protein gene product 9·5 (PGP9·5)/ubiquitin‐C‐terminal hydrolase 1 (UCHL‐1) protein is a thiol protease that recognizes and hydrolyzes a peptide bond at the C‐terminal of ubiquitin, and is involved in the processing of ubiquitin precursors and ubiquinated proteins. Although this molecule is known as a specific tissue marker for the neuroendocrine system, many reports have indicated that PGP9·5 is a marker for certain tumour types, such as cancer of the lung, colon, and pancreas. The expression of PGP9·5 in myeloma cells was examined. PGP9·5 seemed to be expressed specifically in myeloma cells as compared with other haematological malignant cells. In addition, in myeloma cells subjected to growth‐factor starvation, the upregulation of PGP9·5 was observed in association with that of p27Kip1, a cyclin‐dependent‐kinase inhibitor, although the upregulation caused by irradiation was milder. In contrast, the hypoxic culture of myeloma cells induced down‐regulation of PGP9·5. These results suggested that PGP9·5 may be a good marker for myeloma among haematological malignancies. In addition, it may indicate certain cellular features of myeloma cells, such as sensitivity to proteasome inhibitors.

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Treatment outcomes for acute myeloid leukemia and myelodysplastic syndromes (MDS) remain unsatisfactory despite progress in various types of chemotherapy and hematopoietic stem cell transplantation. Therefore, there is a need for the development of new treatment options. We investigated the growth‐suppressive effects of withaferin A (WA), a natural plant steroidal lactone, on myelodysplasia and leukemia cell lines. WA exhibited growth‐suppressive effects on the cell lines, MDS‐L, HL‐60, THP‐1, Jurkat and Ramos, and induction of cell cycle arrest at G2/M phase at relatively low doses. Evaluation by annexin V/PI also confirmed the induction of partial apoptosis. Gene expression profiling and subsequent gene set enrichment analysis revealed increased expression of heme oxygenase‐1 (HMOX1). HMOX1 is known to induce autophagy during anticancer chemotherapy and is considered to be involved in the treatment resistance. Our study indicated increased HMOX1 protein levels and simultaneous increases in the autophagy‐related protein LC3A/B in MDS‐L cells treated with WA, suggesting increased autophagy. Combined use of WA with chloroquine, an autophagy inhibitor, enhanced early apoptosis and growth suppression. Together with the knowledge that WA had no apparent suppressive effect on the growth of human normal bone marrow CD34‐positive cells in the short‐term culture, this drug may have a potential for a novel therapeutic approach to the treatment of leukemia or MDS.