Abstract. The RCC1 gene, a regulator for the onset of chromosome condensation was found to encode a protein with a molecular mass of 45 kD, determined using the antibody against the synthetic peptides prepared according to the amino acid sequence of the putative RCC1 protein. The p45 located in the nuclei was released from the isolated nuclei, either by DNase I digestion or by treatment with 0.3 M NaCI. Consistently, p45 bound to the DNA-cellulose column was eluted with 0.3 M NaCI. After sequential treatment with DNase I and 2 M NaCI, almost all of the RCC1 protein were released from the nuclei. Thus, RCC1 protein locates on the chromatin and is not a component of the nuclear matrix. In mitotic cells, 1M5 is dispersed into the cytoplasm. Presumably, RCC1 protein plays a role in regulating the onset of chromosome condensation, at the level of transcription or of mRNA maturation.
Recent studies (1-3) provide increasing evidence of roles for lysosphingolipids as mediators to elicit a variety of physiological and pathophysiological responses. Thus, the lysosphingolipids SP 1 and SPC have been shown to evoke diverse cellular responses in various cell types, including mitogenesis (1, 2), inhibition of migration (4, 5), cell shape change (6), and microfilament reorganization (6, 7). Stimulation of cells with the lysosphingolipids triggers the activation of multiple intracellular signaling molecules, including phospholipase C (2, 5, 8, 9), phospholipase D (8), PKC (10), MAPK (5, 11), and K ϩ channel (muscarinic K ϩ current) (12). Many of the lysosphingolipidinduced responses are demonstrated to be inhibited by PTX pretreatment (5, 8 -13). In addition, either an increase or a decrease in cellular cAMP content in response to SP has been reported, depending on cell types used (5, 13). These observations suggest the existence of multiple G protein-coupled cell surface receptors for SP and SPC.Recently, the orphan G protein-coupled receptor EDG2 was identified as a functional receptor for LPA (14). Moreover, EDG4 was very recently identified to be the second LPA receptor (15). EDG2 and EDG4 are members of the EDG family of receptors comprising EDG1 (16), EDG3 (17), and AGR16 (18)/ H218 (19), which have 36 -58% homology in amino acid sequences with each other. SP is related in its structure to LPA, and in some cell types, LPA and SP have been suggested to share a cell surface receptor (20, 21). These observations prompted us to examine the possibility that members of the EDG family receptors could function as a receptor for the lysosphingolipids. Many of cell lines usually used for expression of exogenous genes, including COS, NIH3T3 and HEK293 cells, respond to SP (13), which hampered expression cloning of SP receptor gene and functional analysis of cloned SP receptor gene. In the present study, by using carefully selected mammalian cell expression systems, we found that EDG1 is a functional receptor with a high specificity and affinity for SP. We demonstrate that EDG1 is coupled via a G i/o protein to multiple effector pathways, including phospholipase C, adenylate cyclase, and Ras/MAPK. MATERIALS AND METHODS Cells-CHO-K1(CHO) and HEL cells, obtained from RIKEN CellBank and the Japanese Cancer Research Resources Bank (Tokyo, Japan), respectively, were grown in Ham's F-12 (CHO) and RPMI (HEL) media supplemented with 10% fetal calf serum (Equitech-Bio, Ingram, TX), 100 units/ml penicillin, and 100 g/ml streptomycin (Wako Pure Chemicals, Osaka, Japan). Before each experiment, cells were switched to the respective medium supplemented with 1% fetal calf serum.
Activated blood platelets and macrophages metabolize prostaglandin H2 into thromboxane A2 and 12(S)-hydroxyheptadeca-5Z, 8E, 10E–trienoic acid (12-HHT) in an equimolar ratio through the action of thromboxane synthase. Although it has been shown that 12-HHT is abundant in tissues and bodily fluids, this compound has long been viewed as a by-product lacking any specific function. We show that 12-HHT is a natural ligand for leukotriene B4 (LTB4) receptor-2 (BLT2), a G protein–coupled receptor that was originally identified as a low-affinity receptor for LTB4. BLT2 agonistic activity in lipid fractions from rat small intestine was identified as 12-HHT using high-performance liquid chromatography and mass spectrometry. Exogenously expressed BLT2 in mammalian cells was activated by synthetic 12-HHT, as assessed by guanosine 5′-O-(3-thio) triphosphate binding, the activation of intracellular signaling pathways, and chemotaxis assay. Displacement analysis using [3H]LTB4 showed that 12-HHT binds to BLT2 with a higher affinity than LTB4. Lipid extracts from cyclooxygenase 1–deficient mice failed to activate BLT2. Bone marrow–derived mast cells (BMMCs) isolated from wild-type mice migrated toward a low concentration of 12-HHT, whereas BMMCs from BLT2-deficient mice did not. We conclude that 12-HHT is a natural lipid agonist of BLT2 in vivo and induces chemotaxis of mast cells.
In the present study, we determined the agonist specificity and the signalling mechanisms of a putative sphingosine 1-phosphate (S1P) receptor, AGR16. In CHO cells transiently transfected with an AGR16 expression vector, but not in cells transfected with an empty vector, the addition of a low concentration of S1P (1 nM) caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i) by mobilization of Ca2+ from both intra- and extra-cellular pools. To determine the spectrum of agonists for AGR16, we employed K562 cells, which in the naive state do not respond at all to either S1P or structurally related lipids with an increase in [Ca2+]i. In K562 cells stably expressing AGR16, S1P and sphingosylphosphorylcholine (SPC) dose-dependently increased [Ca2+]i with half-maximal values of 3 nM and 100 nM respectively. In CHO cells stably expressing AGR16 (CHO-AGR16), but not in parental CHO cells, we observed specific binding of [32P]S1P, which was displaced by unlabelled S1P and SPC. In CHO-AGR16 cells, but not in parental CHO cells, S1P stimulated the production of inositol phosphates and Ca2+ mobilization which was only 30% inhibited by pertussis toxin (PTX), different from the case of the recently identified S1P receptor EDG1. Also in CHO-AGR16 cells, but not in CHO cells, S1P at higher concentrations activated mitogen-activated protein kinase (MAPK) in a PTX-sensitive and Ras-dependent manner. S1P also induced the activation of two stress-activated MAPKs, c-Jun N-terminal kinase and p38, in a manner that was totally insensitive to PTX. In CHO-AGR16 cells, S1P induced stress-fibre formation, with an increase in myosin light chain phosphorylation, in a PTX-insensitive and Rho-dependent manner. S1P also induced an increase in the cellular cAMP content in CHO-AGR16 cells, which contrasts sharply with the case of EDG1. These results establish that the S1P receptor AGR16 is coupled via both PTX-sensitive and -insensitive G-proteins to multiple effector pathways.
BACKGROUND.To propose ‘never‐smoking nonsmall cell lung cancer (NSCLC)’ as a separate entity, the clinicopathologic differences of operable NSCLC between never‐smoking patients and patients with a history of smoking were investigated.METHODS.The medical records of 1405 patients with primary NSCLC who underwent a complete resection at the study institution from 1974 through 2004 were reviewed for clinicopathologic variables and postoperative survival.RESULTS.The proportion of never‐smoking patients with NSCLC has been significantly increasing over 30 years, from 15.9% in the 1970s to 32.8% in the 2000s. A significantly greater proportion of female patients or adenocarcinoma patients was found in the ‘never‐smoking NSCLC’ group in comparison to the ‘smoking NSCLC’ group (85.8% vs 11.2% and 87.8% vs 49.1%, respectively). The proportion of pathologic stage IA disease for the ‘never‐smoking NSCLC’ group was significantly higher than that for the ‘smoking NSCLC’ group (40.1% vs 25.4%; P < .0001). With regard to both overall and cancer‐specific survival, the ‘never‐smoking NSCLC’ patient group was significantly superior to the ‘smoking NSCLC’ group. In addition to smoking status, the factors found to be significantly associated with the postoperative survival rate were sex, histologic type, T classification, and N classification by univariate analyses. A multivariate analysis revealed never‐smoking status to be an independent prognostic factor in addition to pathologic T and N classification.CONCLUSIONS.The differences in the clinicopathologic factors and survivals between the ‘never‐smoking NSCLC’ patient group and the ‘smoking NSCLC’ group suggest that NSCLC in never‐smokers should be considered a separate disease entity. Cancer 2008. © 2008 American Cancer Society.
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