RhoBTB proteins are atypical members of the Rho family of small GTPases. Two of the three RhoBTB proteins, RhoBTB1 and RhoBTB2, have been proposed as tumor suppressors and might function as adaptors of Cul3-dependent ubiquitin ligase complexes. Using yeast two-hybrid analysis and co-immunoprecipitation we show that all three RhoBTB proteins interact with Cul3. The interaction requires the N-terminal region of Cul3 and the first BTB domain of RhoBTB. RhoBTB3, the only RhoBTB with a prenylation motif, associates with vesicles that are frequently found in the vicinity of microtubules, suggesting a participation in some aspects of vesicle trafficking. We also show that RhoBTB2 and RhoBTB3 are capable of homo- and heterodimerizing through the BTB domain region. The GTPase domain, which does not bind GTP, is able to interact with the BTB domain region, thus preventing proteasomal degradation of RhoBTB. This fits into a model in which an intramolecular interaction maintains RhoBTB in an inactive state, preventing the formation or the functionality of Cul3-dependent complexes. We also report a significantly decreased expression of RHOBTB and CUL3 genes in kidney and breast tumor samples and a very good correlation in the expression changes between RHOBTB and CUL3 that suggests that these genes are subject to a common inactivation mechanism in tumors.

Summary. We examined the sphingolipid metabolism of peripheral blood cells, i.e. platelets, erythrocytes, neutrophils and mononuclear cells. A distinguishing characteristic of sphingolipid metabolism in these highly differentiated cells was their high sphingosine (Sph) kinase activity. The occurrence of [ 3 H]sphingosine 1-phosphate (Sph-1-P) from [ 3 H]Sph (actively incorporated from the outside) in the blood cells was strong, long-lasting, and independent of cell activation. Hence, the possibility of Sph-1-P playing a second messenger role is remote in these cells. About 40% of platelet Sph-1-P could be released extracellularly by 12-Otetradecanoylphorbol 13-acetate, possibly through mediation by protein kinase C. On the other hand, in erythrocytes, neutrophils and mononuclear cells a signi®cant percentage of Sph-1-P formed inside the cell was discharged without stimulation, whereas the stimulation-dependent release was marginal. In contrast to active [ ]sphingomyelin was barely detectable in the blood cells; this was especially true for anucleate platelets and erythrocytes. The Sph ! Sph-1-P pathway may become predominant over the Sph ! Cer ! sphingomyelin pathway during late-stage differentiation into platelets or erythrocytes. Sph and its methylated derivative, N,N-dimethylsphingosine, induced apoptosis not only in neutrophils but also in mononuclear cells, whereas Sph-1-P elicited Ca 2 mobilization in platelets. Our results suggest that all blood cells may remove plasma Sph, which is harmful or suppressive to cellular functions, and change it into Sph-1-P, acting as the source of plasma Sph-1-P, which may play a variety of important roles in blood vessels.

To analyze the possibility that immunological alteration in asbestos-related diseases (ARDs) such as asbestosis (ASB) and malignant mesothelioma (MM) may affect the progression of cancers, a human adult T cell leukemia virus-immortalized T cell line (MT-2Org) was continuously exposed to 10 µg/ml of chrysotile-B (CB), an asbestos. After at least 8 months of exposure, the rate of apoptosis in the cells became very low and the resultant subline was designated MT-2Rst. The MT-2Rst cells were characterized by (i) enhanced expression of bcl-2, with regain of apoptosis-sensitivity by reduction of bcl-2 by siRNA, (ii) excess IL-10 secretion and expression, and (iii) activation of STAT3 that was inhibited by PP2, a specific inhibitor of Src family kinases. These results suggested that the contact

Because patients with silicosis who are chronically exposed to silica particles develop not only pulmonary fibrosis, but also complications involving autoimmune diseases such as rheumatoid arthritis and systemic sclerosis, exposure to asbestos may affect the human immune system. This immunologic effect may impair antitumor immune function because cancer complications such as lung cancer and malignant mesothelioma are found in patients exposed to asbestos. To elucidate the antitumor immune status caused by CD4(+) T cells exposed to asbestos, an in vitro T-cell model of long-term and low-level exposure to chrysotile asbestos was established from a human adult T-cell leukemia virus-1-immortalized human polyclonal T cell line, MT-2, and the resulting six sublines showed resistance to asbestos-induced apoptosis after more than 8 months of continuous exposure. The results of DNA microarray analysis showed that the expression of 139 genes was altered by long-term and low-level exposure to asbestos, and the profile was almost similar among the six sublines when compared with the original MT-2 cells that had never been exposed to asbestos. Pathway and network analysis indicated a down-regulation of IFN-γ signaling and expression of CXC chemokine receptor 3 (CXCR3) in the sublines, whereas ELISA and flow cytometry analysis demonstrated a reduction in Th1-related IFN-γ production and cell-surface CXCR3 expression. These findings suggest that chronic exposure to asbestos may reduce antitumor immune status in CD4(+) T cells, and that an in vitro T-cell model may be useful in identifying molecules related to the impairment of antitumor immune function.