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Platelets play central role in thrombosis and haemostasis. Platelets store adenine nucleotides in their dense granules, which are released upon agonist-stimulation. Level of these nucleotides in extracellular fluid is regulated by activities of ectonucleotidases such as ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5'-nucleotidase (CD73) expressed on platelet surface. Here we demonstrate that, expression of surface-bound ectonucleotidases rose significantly in platelets, concomitant with upregulation of their enzymatic activities, when cells were stimulated with thrombin. Interestingly, inhibition of CD73 in thrombin-treated platelets led to enhanced tyrosine phosphorylation of proteins and rise in intracellular free calcium, [Ca2+]i, thus signifying the inhibitory role of the ectonucleotidase on agonist-mediated platelet signaling.

Mesothelin (MSLN) has been reported to be overexpressed in ovarian cancer and may be an ideal target for immunotherapy. Recent studies have suggested that natural killer (NK) cells may be better chimeric antigen receptor (CAR) drivers because of their favorable innate characteristics, such as directly recognizing and killing tumor cells, resulting in a graft-versus-tumor effect but irresponsible for graft-versus-host disease (GVHD). The therapeutic effects of CAR-engineered NK cells targeting MSLN in ovarian cancer have not been evaluated. In this study, MSLN- and CD19-targeted CAR NK-92 (MSLN- and CD19-CAR NK) cells were constructed. Both MSLN- and CD19-CAR molecules were highly expressed on the surface of NK-92 cells following lentiviral gene transduction. MSLN-CAR NK cells specifically killed MSLN-positive ovarian cancer cells (OVCAR-3 and SK-OV-3), rather than MSLN-negative cells (SK-HEP-1), in vitro. Moreover, compared with parental NK-92 cells and CD19-CAR NK cells, stronger cytokine secretion was detected in MSLN-CAR NK cells cocultured with OVCAR-3 and SK-OV-3. Furthermore, MSLN-CAR NK cells effectively eliminated ovarian cancer cells in both subcutaneous and intraperitoneal tumor models; these cells also significantly prolonged the survival of intraperitoneally tumor-bearing mice. These results demonstrate that MSLN-CAR NK cells have robust specific antitumor activity, both in vitro and in vivo, suggesting that mesothelin could be a potential target for CAR NK cells and could be applied in the treatment of ovarian cancer.

Sleep deprivation (SD) has detrimental effects on the physiological function of the brain. However, the underlying mechanism remains elusive. In the present study, we investigated the expression of candidate plasticity-related gene 15 (cpg15), a neurotrophic gene, and its potential role in SD using a REM-SD mouse model. Immunofluorescent and Western blot analysis revealed that the expression of cpg15 protein decreased in the hippocampus, ventral group of the dorsal thalamus (VENT), and somatosensory area of cerebral cortex (SSP) after 24–72 h of REM-SD, and the oxidative stress in these brain regions was increased in parallel, as indicated by the ratio of glutathione (GSH) to its oxidative product (GSSG). Over-expression of cpg15 in thalamus, hippocampus, and cerebral cortex mediated by AAV reduced the oxidative stress in these regions, indicating that the decrease of cpg15 might be a cause that augments oxidative stress in the sleep deprived mouse brain. Collectively, the results imply that cpg15 may play a protective function in the SD-subjected mouse brain via an anti-oxidative function. To our knowledge, this is the first time to provide evidences in the role of cpg15 against SD-induced oxidative stress in the brain.

The immune system plays important roles in pancreatic cancer. MHC class I-chain-related proteins A and B (MICA/B) and UL16-binding proteins (ULBPs) are known natural killer group 2D (NKG2D) ligands. Soluble NKG2D ligands can inhibit the activation of Natural killer (NK) cells. In pancreatic cancer, soluble ULBPs are relatively unstudied in contrast to soluble MICA/B. We examined the significance of soluble ULBPs, especially ULBP2, in pancreatic cancer. Soluble ULBP2 but neither soluble ULBP1 nor soluble ULBP3, was etected in the supernatants of pancreatic cancer cells. Soluble ULBP2 derived from pancreatic cancer cells could reduce the cytotoxicity of NK cells. Multivariate analysis demonstrated that serum soluble ULBP2 was a significant independent factor associated with poor overall survival (OS) in all pancreatic cancer patients, specifically in stage IV patients. In conclusion, pancreatic cancer-derived soluble ULBP2 might affect the prognosis in pancreatic cancer.

Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors some proteins to the plasma membrane. This anchoring is catalyzed by a transamidase complex (TAC) composed of five subunits: PIG-K, GAA1, PIG-U, PIG-T, and PIG-S (Fig. 1A). PIG-K and GAA1 are predicted to catalyze the first and second steps during attachment of proproteins of GPI-anchored proteins (GPI-APs) to GPI. GPI may be delivered by PIG-U, and PIG-T is required for stability of all TAC subunits when overexpressed in cultured cells. However, protein stability of TAC has not been analyzed using loss-of-function mutants for each subunit. Herein, we analyzed the stability of TAC in knockout and/or knockdown mutants for each subunit. PIG-T and PIG-U, or PIG-T and GAA1, were mutually required for stability, and all three subunits were stable without PIG-S or PIG-K. However, these three subunits were essential for the stability of both PIG-S and PIG-K. By contrast, loss of PIG-S reduced the stability of PIG-K and left the other subunits unaffected. Reduction of PIG-K did not impact any of the other subunits. Thus, PIG-T, PIG-U, and GAA1 may form a core complex associated by PIG-S, and these four subunits may stabilize PIG-K, triggering GPI anchoring reactions. Instability of PIG-K in the absence of the other four subunits may ensure that GPI anchoring is catalyzed only by the completely assembled complex.

BST2 is an antiviral factor that inhibits the release of enveloped virus at the plasma membrane via an unusual topology in which its N-terminal is in the cytosol while its C-terminal is anchored by glycophosphatidylinositol (GPI). BST2-deficient cells showed substantially higher release of virions than wild type cells. Influenza-infected BST2-deficient cells showed greatly reduced cytopathic effect (CPE) than wild type cells despite their generally robust virus production. This finding prompted us to determine whether BST2 was involved in the apoptotic process of virus-infected host cells. Our results revealed that BST2 might be involved in IRE1α-mediated ER stress pathway by increasing spliced form XBP-1. Consequently, levels of cytochrome C, caspase-3, caspase-9, and PARP as representative molecules of apoptosis were significantly increased in wild type cells than those in BST2-deficient cells. These results suggest that BST2 might participate in innate host defense by augmenting ER-stress-induced apoptotic signaling to inhibit the replication and spread of virus.

Signal peptides (SPs) consist of short peptide sequences present at the N-terminal of newly synthesizing proteins and act as a zip code for the translocation of the proteins to the endoplasmic reticulum (ER). It was thought that the SPs are intracellularly degraded after translocation to the ER; however, recent studies showed cleaved SPs have diverse roles for controlling cell functions in auto- and/or intercellular manners. In addition, it still remains obscure how SP fragments translocate away from the site where they are produced. Extracellular vesicles (EV) are important for intercellular communication and can transport functional molecules to specific cells. In this study, we show that SPs are involved in EV from T-REx AspALP cells that were transfected with a human APP SP-inducible expression vector. There was no difference in the average particle size or particle concentration of EV collected from T-REx AspALP cells and T-REx Mock cells. When the SP content in the EV was examined by mass spectrometry, the C-terminal fragment of APP SP was identified in the exosomes (SEV) of T-REx AspALP cells. In our preparation of SEV fractions, no ER-specific proteins were detected; therefore, SPs may be included in SEV but not in the debris of degraded ER. This is the first indication that SPs are secreted from cells via EV.

The endoplasmic reticulum (ER) is an organelle with high protein density and therefore prone to be damaged by protein aggregates. One proposed preventive measure is a pre-emptive quality control pathway that attenuates ER import during protein folding stress. ER resident proteins are targeted into the ER via signal peptides cleaved rapidly upon ER insertion by the ER signal peptidase. Here we show that the ER insertion and cleavage of the ER-targeting peptide of the prostate carcinoma antigen prostate stem cell antigen (PSCA) is retarded and strongly reduced when the proteasome is inhibited or genetically silenced. Also overexpression of the C-terminally extended ubiquitin variant Ub2-UBB+1 or oxidative stress attenuated signal peptide processing. Proteasome inhibition likewise protracted ER signal processing of the ER targeted hormone leptin and the MHC class I molecule H-2Dd. These findings, which are consistent with a pre-emptive ER quality control pathway, may explain why an immunodominant MHC class I peptide ligand of PSCA spanning its ER signal peptidase cleavage site is efficiently generated in the cytoplasm from PSCA precursors that fail to reach the ER.

Modifying T cells to attack tumors using engineered chimeric receptors display powerful new therapeutic capabilities. Unfortunately, the effectiveness of therapeutic T cells is limited due to the inherent T cell responses: certain facets of endogenous response programs may be toxic, and the ability to overcome the immunosuppression in TME is deficient. Here we developed a Notch receptor based synNotch T cell platform that is able to response to target tumor cells and selectively lead to CXCL10 production. Further study showed that the administration of synNotch T cells significantly inhibited the tumor growth in a humanized murine model, accompanied by the increased infiltration of CD3+T cells and elevated level of CXCL10 and IFN-γ in the tumor site. A slightly increased level of CXCL10 and limited IFN-γ were found in the serum in mice received synNotch T cells, suggesting a high security of this treatment. Finally, we demonstrated that CXCL10 is sufficient and indispensable for the synNotch T cells induced anti-tumor effect. This study provided theoretical and experimental bases for the clinical implication of CXCL10 encoding synNotch T cells.

Exposure to chemicals and other environmental stressors can differentially impact the expression of Acetylcholinesterase (AChE) splice variants. Surprisingly, despite the widespread use of the rat model in toxicological studies and the wealth of literature on this important biomarker of neurotoxicity, AChE coding exons and splice variants are not yet fully annotated in this species. To address this knowledge gap, a short problematic region of the rat AChE genomic DNA present in GenBank was first re-sequenced. This revised genomic sequence was then aligned to rat AChE RefSeq mRNA and compared to orthologous mammalian sequences, in order to map the coding exon and intron boundaries of the rat AChE gene. Based on these bioinformatics analyses, a sequence was predicted for the yet-unannotated rat Acetylcholinesterase readthrough (AChE-R) splice variant. PCR primers designed to specifically amplify rat AChE-R were used to confirm its expression in rat PC12 cells. Compared to the canonical AChE-S splice variant, AChE-R was expressed at much lower levels but presented distinct regulation patterns in PC12 cells and rat primary cerebral granule cells (CGCs) following exposure to Chlorpyrifos (a well-known neurotoxic organophosphate pesticide). Taken together, these observations point to the evolutionary conservation of the AChE-R splicing event between rodents and human and to the distinct regulation of AChE splice variants in response to toxicological challenges.

The natural killer group 2D (NKG2D) receptor on natural killer (NK) cells play an important role in immunosurveillance to cancer cells, which could mediate the eradication of tumor cells through specific interactions with NKG2D ligands on tumor cells. Here we report one natural compound aurovertin B from basidiomycete Albatrellus confluens significantly stimulates the expression of NKG2D ligands on tumor cells, which greatly sensitizes its recognition and lysis by NK cell. It is completely a novel role for aurovertin B to target tumor cells to death mediated by NK cells and our findings indicate aurovertin B may deserve further development as sensitizing agent in NK cell mediated cancer immunotherapy.

CD177 is considered to represent neutrophils. We analyzed mRNA expression level of CD177 and clinical follow-up survey of PDAC to estimate overall survival (OS) from Gene Expression Omnibus (GEO) dataset (GSE21501, containing samples from 102 PDAC patients) by R2 platform (http://r2.amc.nl). We also analyzed correlated genes of CD177 by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to predict the potential relationship between neutrophils and prognosis of PDAC. We then performed hematoxylin and eosin (HE) staining and immunohistochemical staining of surgical specimens to verify infiltration of neutrophils in PDAC tissues. After analyzing mRNA expression data and clinical follow-up survey provided in the GEO dataset (GSE21501, containing samples from 102 PDAC patients) and clinicopathological data of 23 PDAC patients, we demonstrated that CD177 was correlated with poor prognosis. The univariate Kaplan-Meier survival analysis revealed that OS was inversely associated with increased expression of CD177 (P = 0.012). Expression of phosphodiesterase (PDE)4D was positively related to CD177 in gene correlation analysis (R = 0.413, P 0.001) by R2 platform. HE staining and immunohistochemistry of CD177 in 23 PDAC surgical samples showed accumulation of neutrophils in the stroma and blood vessels around the cancer cells. In addition, immunohistochemical staining showed that CD177 was highly expressed in the stroma and blood vessels around tumor tissues of PDAC, which was similar to HE staining. Expression of CD177 can be used to represent infiltration of neutrophils, which may have potential prognostic value in PDAC.

Although several etiological factors contribute to the complexity of the aging process, the ultimate component of macromolecular damage and consequent cell death involves the altered redox balance inclined towards increased ROS production and/or decreased antioxidant protection. Given that, the chronic dihydrotachysterol (DHT) intoxication in rats induce Hutchinson Gilford progeria like syndrome, the present study provides the evidence for altered redox balance as evidenced by alteration in parameters of oxidative stress in blood plasma and erythrocytes including MDA, GSH, FRAP AOPP PMRS, AGEs, AChE and osmotic fragility which substantiate the suitability of the model for aging studies.

Our previous analysis of gene expression profiles in the peripheral blood from patients with influenza A (H1N1) pdm09 pneumonia revealed elevated transcription levels of the vanin-1 (vascular non-inflammatory molecule 1, VNN1) gene, which encodes an epithelial ectoenzyme with pantetheinase activity involved in recycling coenzyme A. Here, to elucidate the role of VNN1 in influenza A virus (IAV) H1N1 infection, we investigated the change of VNN1 expression in the context of IAV infection and the effects of its related substances, i.e., its direct substrate pantetheine and its two metabolites pantothenic acid and cysteamine on the replication of IAV in the human alveolar epithelial carcinoma cell line A549. The messenger RNA expression of VNN1 in A549 cells was significantly increased (by 4.9-fold) after IAV infection under an elevated concentration of pantetheine. Moreover, VNN1 mRNA levels were elevated by > 100-fold in response to pro-inflammatory cytokines, especially TNF-α and IL-1β. Pantetheine significantly reduced the IAV replication and IAV Matrix 1 (M1) mRNA levels when it was administered prior to and during infection. In addition, cysteamine treatment during IAV infection significantly reduced the viral replication and IAV M1 mRNA levels, whereas pantothenic acid did not. These findings suggest that the metabolic pathway catalyzed by VNN1 pantetheinase plays a suppressive role in IAV infection in the respiratory tract, especially in severe conditions under hypercytokinemia.

Hepatitis B virus (HBV) surface antigen (HBsAg) loss is one of the treatment goals of chronic HBV infection. Bone marrow stromal cell antigen 2 (BST2) is one of the interferon (IFN)-stimulated genes (ISGs) and inhibits the release of various enveloped viruses. Here we examined the effects of antiviral treatment on HBsAg levels and its intracellular mechanism in HBsAg-producing hepatocytes. In PLC/PRF/5 and Huh1, IFNα-2a treatment decreased HBsAg levels in their conditioned media. Upregulation of interleukin 8 (IL8), toll-like receptor 2 (TLR2) and interferon gamma-induced protein 10 (IP10) mRNAs was associated with the reduction of HBsAg in both PLC/PRF/5 and Huh1. The HBsAg level was upregulated by knockdown of IL8, TLR2 or IP10. Exogenous addition of IL8 enhanced BST2 promoter activity and BST2 mRNA expression. Additionally, knockdown of IL8 could lead to the downregulation of BST2 mRNA. Transfection of poly(I-C) enhanced IL8 and BST2 mRNA expression and inhibited HBsAg secretion from PLC/PRF/5 cells. In conclusion, IL8 might play an important role in the enhancement of BST2 and be involved in HBsAg eradication.

Neuronal growth regulator 1 (NEGR1) is a newly identified raft-associated protein, which has recently been spotlighted as a new locus related to human obesity. Niemann-Pick disease Type C2 (NPC2) protein functions as a key player in the intracellular cholesterol trafficking, and its defect is linked to a fatal human neurodegenerative disease, NPC. In this study, we identified that NEGR1 interacts with NPC2 and increases its protein stability. Ectopically expressed NEGR1 proteins relieved an abnormal cholesterol accumulation in endosomal compartments. Importantly, NEGR1-defective mouse embryonic fibroblast cells exhibit increased cholesterol levels and triglyceride contents. These findings provide the first insight into the role of NEGR1 in intracellular cholesterol homeostasis, possibly explaining the missing link between NEGR1 with human obesity.

Glycosylphosphatidylinositol-anchored proteins were reported to be involved in many developmental progresses in Arabidopsis. Here I report that, a group of homologous glycosylphosphatidylinositol-anchored proteins from SKU5-Similar family regulate seedling root development of Arabidopsis through controlling cell polar expansion and cell wall synthesis. Due to the irregular expansion of root cells and the defective synthesis of cell walls, their knockout mutants generated shorter roots with irregularly shaped root cells, and thicker cell walls.

Exosomes are 50-100-nm-diameter membrane vesicles released from various types of cells. Exosomes retain proteins, mRNAs and miRNAs, which can be transported to surrounding cells. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, and is released from the cell surface to the culture medium in vitro. Recently, it was reported that secreted CD109 from the cell surface downregulates transforming growth factor-β signaling in human keratinocytes. In this study, we revealed that CD109 is a component of the exosome in conditioned medium. FLAG-tagged human CD109 (FLAG-CD109) in conditioned medium secreted from HEK293 cells expressing FLAG-CD109 (293/FLAG-CD109) was immunoprecipitated with anti-FLAG affinity gel, and the co-precipitated proteins were analyzed by mass spectrometry and western blotting. Exosomal proteins were associated with CD109. We revealed the presence of CD109 in exosome fractions from conditioned medium of 293/FLAG-CD109. Moreover, the localization of CD109 in the exosome was demonstrated using immuno-electron microscopy. When we used HEK293 cells expressing FLAG-tagged truncated CD109, which does not contain the C-terminal region, the association of truncated CD109 with exosomes was not detected in conditioned medium. These findings indicate that CD109 is an exosomal protein and that the C-terminal region of CD109 is required for its presence in the exosome.

Long noncoding RNA (OGFRP1) has been reported to be involved in the progression of non-small cell lung cancer (NSCLC). However, the expression pattern, functions and molecular mechanisms of OGFRP1 in NSCLC remains unclear. In the present study, we found that OGFRP1 expression was significantly up-regulated in both NSCLC tissues and cell lines, and the upregulation of OGFRP1 expression is a powerful predictor of advanced clinical stage, lymph nodes metastasis and poor prognosis for NSCLC patients. Loss-of-function assay indicated that knockdown of OGFRP1 inhibited proliferation, migration and invasion, and induced apoptosis in vitro. Mechanistically, OGFRP1 could directly bind to miR-124-3p and effectively act as a competing endogenous RNA (ceRNA) for miR-124-3p to promote the expression of the target gene LYPD3. Taken together, OGFRP1 contributed to progression of NSCLC at least partly through upregulating LYPD3 expression by sponging miR-124-3p, indicating that OGFRP1 may be a novel prognostic biomarker and therapeutic target in NSCLC.

Oxidation of low-density lipoproteins (ox-LDL) plays a critical role in endothelial dysfunction and the pathological progression of atherosclerosis by causing leukocyte attachment to endothelial surfaces. Omentin-1, an important adipokine primarily secreted by stromal vascular cells, has displayed various biological functions in diverse tissues. However, little information regarding the effects of omentin-1 on ox-LDL- induced endothelial dysfunction has been reported before. In the current study, we found that omentin-1 significantly reduced the attachment of the leukocyte THP-1 cells to human umbilical vein endothelial cells (HUVECs) in a dose dependent manner. Additionally, omentin-1 treatment prevented the expression of cell adhesion molecules such as VCAM-1 and E-selectin at both the mRNA level and the protein level. Notably, we found that omentin-1 significantly restored ox-LDL-induced reduction of KLF2, an important transcriptional factor and regulator of endothelial function. Also, omentin-1 promoted the expression of KLF2 target genes eNOS and PAI-1. Mechanistically, our results indicate that the effects of omentin-1 on KLF2 expression are mediated by p53. These results highlight the potential of omentin-1 in preventing endothelial dysfunction and atherosclerosis.

Scavenger receptor, class B type I (SR-BI) is a physiologically relevant regulator of high density lipoprotein (HDL) metabolism. Low HDL is a common feature of patients with non-alcoholic fatty liver disease (NAFLD). Here, hepatic SR-BI expression was analyzed in human and murine NAFLD. In primary human hepatocytes NAFLD relevant factors like inflammatory cytokines, lipopolysaccharide and TGF-β did not affect SR-BI protein. Similarly, oleate and palmitate had no effect. The adipokines chemerin, adiponectin, leptin and omentin did not regulate SR-BI expression. Accordingly, hepatic SR-BI was not changed in human and murine fatty liver and non-alcoholic steatohepatits. SR-BI was higher in type 2 diabetes patients but not in those with hypercholesterolemia. The current study indicates a minor if any role of SR-BI in human and murine NAFLD.

CD109 is a glycosylphosphatidylinositol-anchored cell surface protein that is frequently detected in squamous cell carcinomas. CD109 is a negative regulator of TGF-β1 signaling in human keratinocytes, and the N-terminal fragment of CD109 secreted from cells after cleavage by the furin protease is important for modulating TGF-β1 signaling. Previously, we found that CD109 is expressed in human glioblastoma cells; however, the role of CD109 in glioblastoma cells is not established. Here, we describe the effects of CD109 in human glioblastoma cell lines. Three glioblastoma cell lines, SK-MG-1, U251MG and MG178, were tested and CD109 overexpression attenuated TGF-β1 signaling and enhanced EGF signaling in SK-MG-1, but not in U251MG or MG178. The N-terminal CD109 fragment in SK-MG-1 was hyperglycosylated compared with that in MG178 or U251MG. The conditioned medium of CD109-overexpressing SK-MG-1, containing the secreted N-terminal CD109, had a negative effect on TGF-β1 signaling in wild-type SK-MG-1 and MG178, whereas it did not show any effect on EGF signaling. In addition, cell surface CD109 interacts with EGF receptor in SK-MG-1 overexpressing CD109, and exhibited enhanced cell migration and invasion. These findings suggest that CD109 attenuates TGF-β1 signaling and enhances EGF signaling in SK-MG-1 cells and that the membrane-anchored CD109 may play major roles in the EGF signaling pathway.

Malignant mesothelioma (MM) is a fatal disease mostly associated with asbestos exposure and difficult to detect by non-invasive methods. This study aimed to use recombinant fragments of the megakaryocyte potentiating factor (MPF) for the development of cost-effective MPF ELISAs. Three polypeptides spanning the MPF region (MPF1-148, MPF 34-288, MPF/MSLN254-400) were produced in E.coli as maltose-binding protein hybrids. After isolation, Factor Xa digest, and purification, the polypeptides were used for the generation of rabbit antibodies and development of ELISAs. Forty-one MM patients with known histological subtype before tumor-specific treatment and 70 asbestos-exposed individuals free of any cancer were matched according to age, gender, and smoking. Plasma of all subjects was tested with the three newly developed polyclonal antibody-based ELISAs and a commercial mesothelin assay (MESOMARK™). The latter differentiated patients (median concentration 1.95 nM) from controls (median 1.07 nM, p < 0.0001) and showed an area under curve (AUC) of 0.77 in receiver operating characteristics (ROC) analysis. Of the MPF variants, exclusively the ELISA based on antibodies against the MPF34-288 fragment displayed significantly (p = 0.0002) higher values in patients than in controls (median 1.61 nM versus 0.88 nM; AUC = 0.72). The combination of the MPF34-288 and mesothelin displayed an improved ROC performance (AUC = 0.80). The MPF34-288 ELISA could be a cost-effective and minimal-invasive contribution to support a diagnosis of mesothelioma, especially in regions with a limited medical care.

Colorectal carcinoma (CRC) is one of the most frequently diagnosed cancers and the leading cause of cancer-related death for both men and women. Recent studies have revealed that a small sub-population of cancer cells, termed cancer stem-like cells (CSCs)/cancer-initiating cells (CICs), are endowed with tumor-initiating ability, self-renewal ability and differentiation ability. CSCs/CICs are resistant to current therapies including chemotherapy and radiotherapy. Thus, CSCs/CICs are responsible for recurrence and metastasis, and eradication of CSCs/CICs is essential to cure cancer. In this study, we isolated CR-CSCs/CICs as sphere-cultured cells and found that a product derived from LY6/PLAUR domain containing 3 (LYPD3) is preferentially expressed in CSCs/CICs. Gene overexpression and gene knockdown experiments revealed that LYPD3 has a role in the maintenance of CR-CSCs/CICs. The findings provide a novel molecular insight into CR-CSCs/CICs.

Farnesoid X receptor (FXR) belongs to the ligand-activated nuclear receptor superfamily, and functions as a transcription factor regulating the transcription of numerous genes involved in bile acid homeostasis, lipoprotein and glucose metabolism. In the present study, we identified RECK, a membrane-anchored inhibitor of matrix metalloproteinases, as a novel target gene of FXR in mouse liver. We found that FXR agonist substantially augmented hepatic RECK mRNA and protein expression in vivo and in vitro. FXR regulated the transcription of RECK through directly binding to FXR response element located within intron 1 of the mouse RECK gene. Moreover, FXR agonist reversed the down-regulation of RECK in the livers from mice fed a methionine and choline deficient diet. In summary, our data suggest that RECK is a novel transcriptional target of FXR in mouse liver, and provide clues to better understanding the function of FXR in liver.

International audience; Human adipose-derived stem cell populations express cell surface markers such as CD105, CD73, CD146 and CD140a/PDFGRα. However, it was unclear whether these markers could discriminate subpopulations of undifferentiated cells and whether the expression of these markers is modulated during differentiation. To address this issue, we analyzed the immunophenotype of cultured human multipotent adipose derived stem (hMADS) cell populations at different adipocyte differentiation steps. We found that 100% of undifferentiated cells expressed CD73 and CD105. In contrast, CD146 and CD140a/PDFGRα marked two different subpopulations of cells. CD140a/PDGFRα subpopulation was regulated by FGF2, a critical factor of human adipose-derived stem cell self-renewal. During differentiation, CD73 was maintained and marked lipid-laden cells, whereas CD105 expression was inhibited in fully differentiated cells. The percentage of CD146- and CD140a/PDFGRα-positive cells declined as soon as cells had undergone differentiation. Altogether, these data support the notion that expanded adipose-derived stem cells are heterogeneous mixtures of cells and cell surface markers studied can discriminate subpopulations.

Macroautophagy (herein termed autophagy) works as a protective mechanism in tumorigenesis and development under metabolic stress condition. Multitudes of genes have been found involved in this process during past decades. In the present study, we report that Wilm's tumor suppressor1 (WT1) is involved in autophagy in osteosarcoma (OS) cells. WT1, a transcription factor with multitude of target genes, expresses in a majority of cancer types. Though wide-ranging effect of WT1 is now well documented, the function of WT1 in tumors remains poorly defined. In this chapter, it is found that high expression of WT1 positively correlates with active autophagy in human osteosarcoma cells. And further study on cell signaling pathway illustrates that Akt/JNK pathway acts as a positive regulator of autophagy induced by WT1. Here, we present evidence that WT1 modulates Akt/JNK signaling pathway mediated autophagy by controlling the expression of growth arrest-specific 1 (Gas1). We show that WT1 is required for Gas1 transcription in osteosarcoma cells. And Gas1 is upregulated followed WT1 overexpression in a time-dependent manner. Loss of Gas1 results in a reduction of WT1-induced autophagy.

Nogo-A and its receptor (NgR) were first described as myelin-associated inhibitors of neuronal regeneration in response to injury. In recent years, knowledge about the important role of the Nogo-A protein in several neuronal pathologies has grown considerably. Here, we employed a neonatal cortex freeze-lesion (NFL) model in neonatal rats and measured the expression of Nogo-A and NgR in the resulting cerebrocortical microdysgenesis 5-75 days after freezing injury. We observed marked upregulation of Nogo-A and NgR in protein levels. Furthermore, the migration of neural precursor cells (NPCs) derived from the subventricular zone (SVZ) toward the sits of injury was perturbed by treatment of NgR antagonist peptide NEP1-40. In vitro analysis showed that the knockdown of NgR by lentivirus-delivered siRNA promoted in axonal regeneration and SVZ-derived neural stem cell/progenitor cell (SVZ-NPCs) adhesion and migration, findings which were similar to the effects of NEP1-40. Taken together, our results indicate an important role for NgR in regulating the physiological processes of SVZ-NPCs. The observation of upregulated Nogo-A/NgR in lesion sites in the NFL model suggest that the effects of the perturbed Nogo-A are a key feature during the development and/or the progression of cortical malformation.

BST-2 (bone marrow stromal cell antigen 2) is an interferon-inducible protein that inhibits the release of a variety of enveloped viruses by tethering viral particles to the cell surface. Xenotropic murine leukemia virus-related virus (XMRV) is a gamma-retrovirus that was derived from the recombination of two endogenous murine leukemia viruses during the production of a prostate cell line in mice. In this study, we observed that XMRV was highly sensitive to the inhibition by human BST-2. We were able to determine the structural domains of BST-2 that are essential to restrict XMRV, including the transmembrane domain, the coiled-coil ectodomain, the C-terminal glycosylphosphatidylinositol (GPI) anchor, the two putative N-linked glycosylation sites, and the three extracellular cysteine residues. Protease treatment effectively released XMRV particles into the supernatant, supporting the notion that BST-2 tethered nascent particles to the cell surface. These data suggest that BST-2 poses a strong restriction toward XMRV production.

In the present study, we investigated whether omentin affected the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-α (TNF-α) induced human umbilical vein endothelial cells (HUVECs). Our data showed that omentin decreased TNF-α-induced expression of ICAM-1 and VCAM-1 in HUVECs. In addition, omentin inhibited TNF-α-induced adhesion of THP-1 cells to HUVECs. Further, we found that omentin inhibited TNF-α-activated signal pathway of nuclear factor-κB (NF-κB) by preventing NF-κB inhibitory protein (IκBα) degradation and NF-κB/DNA binding activity. Omentin pretreatment significantly inhibited TNF-α-induced ERK activity and ERK phosphorylation in HUVECs. Pretreatment with PD98059 suppressed TNF-α-induced NF-κB activity. Omentin, NF-kB inhibitor (BAY11-7082) and ERK inhibitor (PD98059) reduced the up-regulation of ICAM-1 and VCAM-1 induced by TNF-α. These results suggest that omentin may inhibit TNF-α-induced expression of adhesion molecules in endothelial cells via blocking ERK/NF-κB pathway.

The therapeutic applications of neural stem cells (NSCs) have potential to promote recovery in many obstinate diseases in central nervous system. Regulation of certain gene expressions using siRNA may have significant influence on the fate of NSC. To achieve the optimum gene silencing effect of siRNA, non-viral vector polyethylene glycol-polyethyleneimine (PEG-PEI) was investigated in the delivery of siRNA to NSCs. The characteristics of PEG-PEI/siRNA polyplexes were detected by scanning electron microscopy (SEM). The effects of nanoparticles on cell viability were measured via CCK-8 assay. In addition, the transfection efficiency was evaluated by fluorescence microscope and flow cytometry, and real-time PCR and Western Blot were employed to detect the gene inhibition effect of siRNA delivered by PEG-PEI. The SEM micrographs showed that PEG-PEI could condense siRNA to form diffuse and spherical nanoparticles. The cytotoxicity of PEG-PEI/siRNA nanocomplexes (N/P=15) was significantly lower when compared with that of Lipofectamine 2000/siRNA (P0.05). Moreover, the highest transfection efficiency of PEG-PEI/siRNA nanoparticles was obtained at an N/P ratio of 15, which was better than that achieved in the transfection using Lipofectamine 2000 (P0.05). Finally, the gene knockdown effect of PEG-PEI/siRNA nanoparticles was verified at the levels of mRNA and protein. These results suggest that PEG-PEI may potentially be used as a siRNA delivery vector for neural regeneration therapy.

The Acrodysplasia (Adp) mutation arises from the insertion of a transgene containing a mouse metallothionein-promoted bovine papilloma virus and human growth hormone-releasing factor gene. Although the transgene is not expressed, mice that are hemizygous for the transgene show skull and paw deformities when the progeny receive the transgene paternally. To elucidate the molecular mechanisms underlying the mutant phenotype and the modified transmission pattern of the Adp phenotype, a junctional fragment around the transgene integration site was cloned. The transgene was inserted into the intronic sequences between exon 3 and exon 4 of the Mdga2 gene and the degree of methylation of the transgene and the severity of the phenotype were reciprocally related in that the transgene was highly or under methylated in normal and deformed mice, respectively. Thus, methylation of the transgene appears to regulate phenotypic expression and imprinting of Adp.

The repulsive guidance molecule-a (RGMa) is a membrane-associated glycoprotein that has diverse functions in the developing and adult central nervous system. Here, we show that RGMa suppresses new blood vessel formation. Treatment of human umbilical artery endothelial cells (HUAEC) on Matrigel with recombinant RGMa inhibits vascular endothelial growth factor (VEGF)-induced and VEGF-independent tubular formation and migration. RGMa enhances adhesion presumably through dephosphorylation of focal adhesion kinase (FAK) at tyrosine-397. Neogenin, an RGMa receptor, in HUAEC is required for the effect of RGMa. In vivo Matrigel plug assay reveals that treatment with recombinant RGMa suppresses angiogenesis. Thus, we conclude that RGMa inhibits angiogenesis in vitro and in vivo suggesting that its manipulation would be an efficient therapeutic strategy for pro-angiogenic conditions.

The rapid turnover rate of hyaluronan (HA), the major unbranched glycosaminoglycan of the extracellular matrix, is dependent on hyaluronidases. One of them, hyaluronidase-2 (Hyal2), degrades HA into smaller fragments endowed with specific biological activities such as inflammation and angiogenesis. Yet the cellular environment of Hyal2, a purported glycosylphosphatidylinositol (GPI)-anchored protein, remains uncertain. We have examined the membrane association of Hyal2 in MDA-MB231 cancer cells where it is highly expressed and in COS-7 cells transfected with native or fluorescent Hyal2 constructs. In both cell types, Hyal2 was strongly associated with cell membrane fractions from which it could be extracted using a Triton X-114 treatment (hydrophobic phase) but not an osmotic shock or an alkaline carbonate solution. Treatment of membrane preparations with phosphatidylinositol-specific phospholipase C released immunoreactive Hyal2 into the aqueous phase, confirming the protein is attached to the membrane through a functional GPI anchor. Hyal2 transfected in COS-7 cells was associated with detergent-resistant, cholesterol-rich membranes known as lipid rafts. The cellular immunofluorescent pattern of Hyal2 was conditioned by the presence of a GPI anchor. In summary, the strong membrane association of Hyal2 through its GPI anchor demonstrated in this study using biochemical methods suggests that the main activity of this enzyme is located at the level of the plasma membrane in close contact with the pericellular HA-rich glycocalyx, the extracellular matrix, or possibly endocytic vesicles.

Omentin is a recently identified adipocytokine with insulin-sensitizing effect. While lack of omentin may be related to the pathogenesis of obesity-related cardiovascular diseases, its effect in vasculature is largely unknown. We examined effects of omentin on vascular endothelial inflammatory states. Western blotting was performed to analyze inflammatory signal transduction in cultured vascular endothelia cells. The cyclic guanosine monophosphate (cGMP) content was measured using enzyme immunoassay. Treatment of human umbilical vein endothelial cells with omentin (300 ng/ml, 20 min) induced phosphorylation of 5′-AMP-activated protein kinase (AMPK) (Thr 172) and endothelial nitric oxide (NO) synthase (eNOS) (Ser 1177). Consistently, omentin increased the cGMP level. Pretreatment with omentin (300 ng/ml, 30 min) significantly inhibited the phosphorylation of JNK as well as expression of cyclooxygenase (COX)-2 by TNF-α (5 ng/ml, 20 min–24 h). An inhibitor of JNK significantly inhibited the TNF-α-induced COX-2 expression. Inhibitory effect of omentin on TNF-α-induced COX-2 was reversed by a NOS inhibitor. The present results demonstrate for the first time that omentin plays an anti-inflammatory role by preventing the TNF-α-induced COX-2 expression in vascular endothelial cells. Omentin inhibits COX-2 induction via preventing the JNK activation presumably through activation of AMPK/eNOS/NO pathways.

Cripto-1 (CR-1) is a glycosylphosphatidylinositol-anchored glycoprotein which acts as an obligate co-receptor of a TGFβ family ligand, Nodal. Previous studies have demonstrated that CR-1 functions in a paracrine fashion by a cellular mechanism which has not been fully described. This paracrine activity was observed only when CR-1 was expressed as a membrane-bound form and was abolished when CR-1 was expressed in a soluble form. In the current study, we found that there were few biochemical differences in post-translational modifications between membrane-anchored and soluble forms of CR-1. Flow cytometric analysis revealed an intercellular transfer of the membrane-bound form of CR-1 between cells. CR-1-expressing cells formed unique membrane extensions, generated more membrane fragments than control cells, and exhibited enhanced cellular adhesion. Thus, expression of CR-1 may alter the physiochemical properties of the plasma membrane resulting in an enhancement of intercellular transfer of cellular signaling components which may account for the paracrine activity of CR-1.

Omentin is a recently identified adipose tissue-derived cytokine and is implicated in obesity-related cardiovascular disorders. In the present study, we tested the hypothesis that omentin could directly affect vascular reactivity of isolated blood vessels. In endothelium-intact rat isolated aorta, pretreatment with omentin (300 ng/ml, 30 min) inhibited noradrenaline (NA; 1 nM-1 microM)-induced concentration-dependent contraction. In NA (100 nM)-pre-contracted aorta, omentin (1-300 ng/ml) directly induced an endothelium-dependent relaxation. While a nitric oxide (NO) synthase (NOS) inhibitor, N(G)-nitro-l-arginine methyl ester (100 microM, 30 min) inhibited the relaxation, a PI3K/Akt inhibitor, LY294002 (10 microM, 30 min) or a tyrosine kinase inhibitor, genistein (30 microM, 30 min) was ineffective. Omentin (300 ng/ml, 5 min) induced a phosphorylation of endothelial NOS at serine 1177 but not a phosphorylation of Akt at serine 473. Omentin (1-300 ng/ml) also relaxed NA pre-contracted mesenteric artery. Present study for the first time demonstrated that omentin has a vasodilating effect on isolated blood vessels, which is mediated through endothelium-derived NO.

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease, which is usually diagnosed in an advanced stage. Animal PDA models which reflect the human condition are clearly necessary to develop early diagnostic tools and explore new therapeutic approaches. We have established transgenic rats carrying a mutated H- or K-ras gene (Hras250 and Kras327) controlled by Cre/loxP activation. These animals develop PDA which are histopathologically similar to that in humans. We utilized this model to identify biomarkers to detect early PDA. We report here that serum levels of Erc/Mesothelin are significantly higher in rats bearing PDA than in controls. Importantly, the levels are significantly elevated in rats before grossly visible carcinomas develop. Even in rats with very small microscopic ductal carcinoma lesions, elevated serum Erc/Mesothelin can be detected. We believe this is the first report of a pancreas tumor animal model in which pre-symptomatic lesions can be diagnosed.

ERC/mesothelin is expressed on the normal mesothelium and some cancers such as mesothelioma or ovarian carcinoma. A splicing isoform of ERC/mesothelin (known as SMRP), which has an 82-bp insertion and codes for a C-terminus with a hydrophilic, presumably soluble, tail instead of a GPI-anchoring signal, has been reported as a useful marker for the diagnosis of mesothelioma. However, the existence of SMRP has not yet been demonstrated in the serum of mesothelioma patients. To elucidate the existence of SMRP, we have established a new enzyme-linked immunosorbent assay (ELISA) system for SMRP. The ELISA study revealed that N- and C-ERC/mesothelin were detected in sera from mesothelioma patients, but not SMRP, even in these samples. This result showed that the SMRP detected with MESOMARK kit should be lack of soluble C-terminus and indistinguishable from C-ERC/mesothelin. Further study might be necessary to demonstrate the relationship between SMRP and mesothelin.

GPI-80 is a glycosylphosphatidylinositol (GPI)-anchored protein that is mainly expressed in human neutrophils. Previous studies using 3H9, a monoclonal antibody (mAb) against GPI-80, suggested that GPI-80 regulates leukocyte adherence and migration through Mac-1. GPI-80, which is anchored at the plasma membrane in resting neutrophils, moves into the pseudopodia and is released from activated human neutrophils. Here, we demonstrate that neutrophil activation affects GPI-80 dynamics using a new anti-GPI-80 mAb, designated 4D4, which is directed against the form of GPI-80 found on resting human neutrophils. Similar to 3H9, 4D4 influences Mac-1-dependent neutrophil adhesion. Treatment of purified GPI-80 with periodic acid and trypsin indicated that 3H9 and 4D4 recognize peptide and carbohydrate moieties, respectively. Stimulation with fMLP decreased the binding of 4D4 to GPI-80 on the neutrophil surface but increased the overall expression of GPI-80, as visualized by the 3H9 signal. Confocal laser microscopy revealed the 4D4 signal mainly on cell bodies and at a low level on pseudopodia during migration toward increasing concentrations of fMLP, whereas the 3H9 signal was observed in both areas. In addition, soluble GPI-80 released from activated neutrophils did not bind 4D4. These results suggest that there are two populations of GPI-80 that differ in the ability to bind 4D4. The 4D4-recognized form may regulate Mac-1-dependent neutrophil adhesion, and may subsequently be converted to a 4D4-unrecognized form during neutrophil activation.

Splenocytes of wild-type (Prnp(+/+)) and prion protein gene-deficient (Prnp(-/-)) mice were treated with various activation stimuli such as T cell mitogen concanavalin A (ConA), phorbol 12-myristate 13-acetate (PMA)+ionomycin (Io), or B cell mitogen lipopolysaccharide (LPS). Cellular prion protein (PrP(C)) expression was enhanced following ConA stimulation, but not PMA+Io or LPS in Prnp(+/+) splenocytes. Rikn Prnp(-/-) splenocytes elicited lower cell proliferations than Prnp(+/+) or Zrch I Prnp(-/-) splenocytes after LPS stimulation and showed sporadic nerve cells in the cerebral cortex and deeper structure. Around the degenerated nerve cells, mild vacuolation in the neuropil was observed. This neural alteration correlated well to the suppressed response of B cells in the spleen. The finding that discrete lesions within the central nervous systems induced marked modulation of immune function probably indicates the existence of a delicately balanced neural-endocrine network by PrP(C) and PrPLP/Doppel.

We identified RECK, a membrane-anchored glycoprotein negatively regulating the activities of MMPs, as a molecule interacting with Tgat oncoprotein consisting of RhoGEF domain and the unique C-terminal 15 amino acids. The Tgat increased the invasive potential of NIH3T3 cells expressing endogenous mouse RECK and this effect was partially inhibited by the co-expression of human RECK. On the contrary, the expression of exogenous human RECK in HT1080 cell line lacking the endogenous RECK expression reduced its invasive activity, which was recovered by the Tgat co-expression. Moreover, a Tgat mutant lacking the C-terminal region lost the potential to compete the function of RECK in HT1080 cells. These findings indicate that Tgat is the functional inhibitor of RECK, and the activation of MMPs induced by Tgat is likely to enhance invasive activities of cancer cells expressing Tgat.

Recently, we identified a cell-surface marker protein, TEX101, that is unique to male and female germ cells. On/off switching of TEX101 expression in germ cells is closely linked to the kinetics of gametogenesis. In the present study, we isolated testicular proteins by immunoprecipitation with anti-TEX101 antibody and identified the proteins using liquid chromatography/tandem mass spectrometry. Of three proteins identified (annexin 2, ly6k, and cellubrevin), a biochemical association between TEX101 and cellubrevin was confirmed by immunoprecipitation-Western blotting experiments. Immunohistochemistry using a cellubrevin-specific antibody indicated that the molecule is abundant on spermatocytes and early-stage spermatids, whereas negligible amounts are found in Sertoli cells, spermatogonia, spermatozoa, and late-stage spermatids. Most of the intracellular cellubrevin appeared to be juxtaposed with intracellular TEX101, and membrane-associated cellubrevin was docked near TEX101-positive plasma membranes on the cytoplasmic side. This close association was never observed on the outer surface of the plasma membrane. From these results we concluded that cellubrevin-dependent membrane trafficking is involved in TEX101-transport to the surface of male germ cells.

Serum levels of intestinal alkaline phosphatase (IAP), a protein implicated in transcellular transport of chylomicrons, vary among ABO blood groups. In rat enterocytes, IAP is associated with chylomicron secretion, but the rat expresses only blood group A. It is not known whether chylomicron secretion may be affected in humans who express multiple blood group types. Serum samples from 40 healthy subjects were obtained after overnight fast and 3h after a high-fat meal, and assayed for IAP and apolipoprotein B-48 (apoB-48), both proteins exclusive to intestine, although only apoB-48 is found in chylomicrons. The two proteins were greater in subjects without blood antigen A (B and O) than in those with this antigen (A and AB); 2.4- and 4.7-fold for IAP and 1.5- and 2.0-fold for apoB-48 before and after the meal, respectively. Moreover, IAP and apoB-48 levels were strongly correlated in the subjects with the secretor phenotype (r > 0.81). These results indicate that IAP is strongly involved in chylomicron formation and fatty acid metabolism might change among ABO blood type. In addition, ABO blood type classification in apoB-48 measurement would improve the diagnostic value in the evaluation of metabolic syndrome.

The Nogo-66 receptor (NgR) plays a pivotal role in the inhibition of neuroregeneration as the receptor for multiple neurite outgrowth inhibitors such as Nogo-A. We have previously shown that NgR undergoes zinc metalloproteinase-mediated ectodomain shedding in neuroblastoma cells. Here, we demonstrate that the NgR-related protein NgR homologue-1 is released from neuroblastoma cells as a full-length ectodomain (NgRH1-ecto) and an N-terminal fragment (NTF-NgRH1) containing the leucine-rich repeat region of the protein. Inhibitors of the major protease classes failed to block the release of NgRH1-ecto, suggesting that this occurs via a protease-independent mechanism, presumably by a phospholipase-like enzyme. The release of NTF-NgRH1 was blocked by a hydroxamate-based zinc metalloproteinase inhibitor and tissue inhibitor of metalloproteinases-2 and -3, but not -1, implicating the involvement of membrane-type matrix metalloproteinases in this process. Our findings thus highlight the parallels between the ectodomain shedding of NgRH1 and that previously described for NgR.

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an immunoglobulin superfamily member with a diversity of extracellular ligands that is implicated in the initiation and progression of a variety of malignancies. We sought to characterize the effects of CEACAM6 crosslinking on pancreatic adenocarcinoma cellular interaction with the extracellular matrix (ECM) components fibronectin and vitronectin. Antibody-mediated CEACAM6 crosslinking was performed and the ability of BxPC3 cells, which inherently overexpress CEACAM6, to adhere to fibronectin and vitronectin was quantified. The roles of the archetypal fibronectin (alpha5beta1 integrin) and vitronectin (alphavbeta3 integrin) receptors were determined. The effects of c-Src inhibition were investigated using the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and c-Src specific RNA interference. CEACAM6 crosslinking initiates c-Src-dependent cross-talk between CEACAM6 and alphavbeta3 integrin, leading to increased ECM component adhesion. CEACAM6-mediated signaling events may contribute to the invasive and metastatic potential of pancreatic adenocarcinoma cells by promoting their interaction with ECM components.

Immature secretory granules (ISG's) are sites of segregation of proteins destined for secretion by unregulated pathways from those stored in mature secretory granules in endocrine cells. To determine whether significant soluble protein sorting occurs in ISG's, the secretion of soluble versions of the pancreatic protein GP2 (GP2-GPI − ) and placental alkaline phosphatase (SEAP) was analyzed in NIT-1 cells. By immunofluorescence microscopy, neither protein localized to SG's in transfected cells. Their secretion was secretagogue-independent in pulse-chase radiolabeling experiments even at early times of chase, while a small increase in the secretion of amylase, which is known to enter ISG's, could be detected. Finally, in sucrose gradient fractionation experiments, SEAP was present in light density fractions. We conclude that while some proteins, such as amylase, have a limited intrinsic capacity to enter ISG's, the segregation of proteins secreted via the constitutive pathway from SG content proteins occurs primarily in the trans Golgi network.

In a search for novel genes expressed in human cancers, we identified one gene from an assembled expressed sequence tag database. Northern blot analysis revealed that the gene, termed alcan, was expressed in various types of human cancer cell lines and in the fetus, but not in normal tissues. The alcan gene is located on chromosome 6 and is encoded on a 246-amino-acid protein with weak homology to classical major histocompatibility complex class I. Its gene product, ALCAN, had hydrophobic amino acid clusters at both the N- and C-terminal regions and was predicted to be a glycosylphosphatidylinositol (GPI)-anchored membrane protein. Flow cytometric analysis revealed that ALCAN was detected on the surface of human cancer cells and on alcan-transfected CHO-K1 cells. ALCAN was also secreted from these cells, suggesting that some portion of the molecules was secreted by enzymatic cleavage by, for example, phospholipases. Mutational analysis of ALCAN suggested that the GPI-anchored position was the Ser(216) residue. These findings indicate that ALCAN may be a potential target for cancer diagnosis or therapy.