Search

INTRODUCTION AND OBJECTIVE:As a major component of ECM, Hyaluronic acid (HA) fragments different molecular sizes exhibit different biological actions and biological responses. While high molecular ...

The LIM and SH3 protein-1 (LASP-1) is a multi-domain protein that is involved in several malignant cancers. The role of LASP-1 in malignant phenotypes including high invasive properties and unrestricted cell proliferation, remain to be elucidated. The present study reported the association of LASP-1 expression with bladder cancer malignancy and its role in cancer cell invasion and proliferation. The immunohistochemical analysis of the expression status of LASP-1 in radical cystectomy specimens from invasive bladder cancer patients revealed that the LASP-1-positive patients demonstrated a decreased survival rate compared with the LASP-1-negative patients. The expression level of LASP-1 was increased in invasive bladder cancer cell lines compared with the non-invasive bladder cancer cell lines. Invasive cancer cells form invadopodia, the filamentous actin-based membrane protrusions that are essential in cancer cell invasion. Knockdown of LASP-1 reduced the ability to form invadopodia, resulting in decreased invasive capacity of the LASP-1 knockdown cells. In addition, knockdown of LASP-1 reduced cell proliferation. These results suggest that LASP-1 is important in invadopodia formation and cell proliferation of bladder cancer cells, promoting the malignant properties and resulting in poor-prognosis.

To investigate the molecular mechanisms of cancer metastasis, we have isolated a high-metastatic bladder cancer cell subpopulation from a low-metastatic cell line by using an in vivo selection system. Cells in the subpopulation showed a high ability to form invadopodia, the filamentous actin (F-actin)-based membrane protrusions that play an essential role in cancer cell invasion. Analysis of the gene expression profile revealed that the expression of an intermediate filament (IF) protein, vimentin and a cytoskeletal linker protein, plectin was up-regulated in the high-metastatic subpopulation compared with the low metastatic cell line. Here we report a novel role of vimentin IF and plectin in metastasis. In invasive bladder cancer cells, the vimentin IF-plectin-invadopodia F-actin link was formed. Disruption of this link severely impaired invadopodia formation, reducing the capacities of extracellular matrix degradation, transendothelial migration and metastasis. In addition, the vimentin assembly into the filaments was required for invadopodia formation. Our results suggest that plectin anchoring invadopodia to vimentin IF scaffolds and stabilizes invadopodia, which is a critical molecular process for cancer cell invasion and extravasation for metastasis.

INTRODUCTION AND OBJECTIVES: The two-step transcriptional amplification (TSTA) system was previously reported to enhance the tissue-specific gene expression driven by weak promoters, but the enhancement of the gene expression is limited to use in in vitro and in vivo experimental situations. METHODS: To achieve robust tissue-specific gene expression using the TSTA system, we developed an advanced TSTA system which includes polyglutamines and rat glucocorticoid receptor (GR) sequences between the GAL4 and VP16 sequences in the region of the first step of transcription. We herein evaluated this advanced TSTA system as a method to enhance the human telomerase reverse transcriptase (hTERT) promoter-driving cancer-specific transcription in various cancer cells. RESULTS: We constructed a luciferase gene encoding plasmid vector, where hTERT promoter-driving transcription is enhanced by the advanced TSTA (advanced TSTA-hTERT-Luc). The advanced TSTA enhanced cancer-specific luciferase gene expression in all of the examined 18 cancer cell lines. We then constructed a green fluorescent protein (GFP) gene encoding plasmid vector with the hTERT promoter and advanced TSTA, to utilize the system for the detection of viable bladder cancer cells. The advanced TSTA-hTERTGFP plasmid successfully induced cancer-specific gene expression, showing robust GFP expression in human bladder cancer cell lines, but no visible GFP expression in normal bladder urothelial cells. The advanced TSTA-hTERT-GFP plasmid allowed selective visualization of viable human bladder cancer cells in mixed cell culture containing 10and 100-fold more normal bladder urothelial cells. These findings indicate that the advanced TSTA-hTERT expressional system is a valuable tool for detecting viable bladder cancer cells. To further demonstrate the utility of the advanced TSTA system for cancerspecific gene expression and imaging, we constructed a luciferase gene encoding adeno-associated virus (AAV) vector in which the hTERT promoter-mediated expression was driven by the advanced TSTA systems. In an orthotopic liver tumor model, mice were treated with the vector via tail vein injection. An optical imaging device was used to visualize the in vivo luciferase expression in the orthotopic tumor. The advanced TSTA system significantly enhanced the luciferase expression compared with the one-step and conventional TSTA systems. CONCLUSIONS: This system can be applied for not only in vitro detection of the bladder and other types of cancer cells but also for the tumor imaging in vivo. Source of Funding: none

Core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) forms an N-acetylglucosamine branch in the O-glycans (core2 O-glycans) of cell surface glycoproteins. We previously revealed that the expression of C2GnT is positively correlated with poor prognosis in prostate cancer patients. However, the detailed mechanisms underlying their poor prognosis remain unclear. In the current study, we report that the core2 O-glycans carried by the surface MUC1 glyco- proteins of prostate cancer cells play an important role in the evasion of NK cell immunity. In C2GnT-expressing prostate cancer cells, the MUC1 core2 O-glycans are modified with poly-N-acetyllactosamine. MUC1 glycoproteins carrying poly-N-acetyllactosamine attenuated the interaction of the cancer cells with NK cells, resulting in decreased secretion of granzyme B by the NK cells. Poly-N-acetyllactosamine also interfered with the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to access the cancer cell surface. These effects of poly-N-acetyllactosamine on NK cells render C2GnT-expressing prostate cancer cells resistant to NK cell cytotoxicity. By contrast, C2GnT-deficient prostate cancer cells carrying a lower amount of poly-N-acetyllac- tosamine than the C2GnT-expressing prostate cancer cells were significantly more susceptible to NK cell cytotoxicity. Our results strongly suggest that C2GnT-expressing prostate cancer cells evade NK cell immunity and survive longer in the host blood circulation, thereby resulting in the promotion of prostate cancer metastasis.

During the process of hematogenous tumor metastasis, tumor cells that dissociated from the primary site enter the blood vessels and are exposed to innate immune systems in host blood circulation. In the innate immune systems, natural killer (NK) cells play a major role in rejecting tumors and suppressing metastasis. To establish metastasis, tumor cells therefore need to defend themselves against tumor rejection by NK cells. It has been recently discovered that some tumor cells develop defense systems against NK cell attack using certain types of cell-surface carbohydrates. The types of carbohydrates attached to cell-surface glycoproteins through serine or threonine residues contain a branch consisting of β-1,6-linkage of N-acetylglucosamine and N-acetylgalactosamine and are designated as core2 O-glycans. Tumor cells expressing core2 O-glycans evade NK cell-mediated tumor rejection, thereby surviving longer in host circulation and acquiring high-metastatic phenotypes. This review explains two types of tumor defense systems against NK cell immunity using core2 O-glycans.

The O-glycan branching enzyme, core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT), forms O-glycans containing an N-acetylglucosamine branch connected to N-acetylgalactosamine (core2 O-glycans) on cell-surface glycoproteins. Here, we report that upregulation of C2GnT is closely correlated with progression of bladder tumours and that C2GnT-expressing bladder tumours use a novel strategy to increase their metastatic potential. Our results showed that C2GnT-expressing bladder tumour cells are highly metastatic due to their high ability to evade NK cell immunity and revealed the molecular mechanism of the immune evasion by C2GnT expression. Engagement of an NK-activating receptor, NKG2D, by its tumour-associated ligand, Major histocompatibility complex class I-related chain A (MICA), is critical to tumour rejection by NK cells. In C2GnT-expressing bladder tumour cells, poly-N-acetyllactosamine was present on core2 O-glycans on MICA, and galectin-3 bound the NKG2D-binding site of MICA through this poly-N-acetyllactosamine. Galectin-3 reduced the affinity of MICA for NKG2D, thereby severely impairing NK cell activation and silencing the NK cells. This new mode of NK cell silencing promotes immune evasion of C2GnT-expressing bladder tumour cells, resulting in tumour metastasis.

Invadopodia (protrusions of the plasma membrane formed by invasive tumor cells) have an essential role in bladder tumor invasion. To understand the process of bladder tumor invasion it is crucial to investigate the molecular mechanisms of invadopodia formation. We found that invasive bladder tumor cells express FBP17. In this study we examined the role of FBP17 in bladder tumor cell invadopodia formation and invasion.We used the 3 bladder tumor cell lines YTS-1, T24 and RT4 (ATCC®), and primary culture of bladder tumors from patients. Cells were stained with phalloidin for invadopodia formation. FBP17 knockdown cells were tested for invadopodia formation and subjected to invasion assay using a Transwell® cell culture chamber. We also examined the role of the extended FER-CIP4 homology and Src homology 3 domains of FBP17 in invadopodia formation in FBP17 mutant constructs.Invadopodia formation was observed in invasive bladder tumor cells and FBP17 was localized to invadopodia in invasive cells. FBP17 knockdown decreased invadopodia formation in invasive cells to 13% to 14% (p0.0005) and decreased their invasive capacity to 14% to 16% (p0.001). The extended FER-CIP4 homology and Src homology 3 domains of FBP17 were necessary for invadopodia formation and invasion.Invadopodia formation requires membrane deformation activity and recruitment of dynamin-2 mediated by FBP17. FBP17 has a critical role in the process of bladder tumor cell invasion by mediating invadopodia formation.

Chemotactic migration of macrophages is critical for the recruitment of leukocytes to inflamed tissues. Macrophages use a specialized adhesive structure called a podosome to migrate. Podosome formation requires the Wiskott-Aldrich syndrome protein (WASP), which is a product of the gene defective in an X-linked inherited immunodeficiency disorder, the Wiskott-Aldrich syndrome. Macrophages from WASP-deficient Wiskott-Aldrich syndrome patients lack podosomes, resulting in defective chemotactic migration. However, the molecular basis for podosome formation is not fully understood. I have shown that the WASP interacting protein (WIP), a binding partner of WASP, plays an important role in podosome formation in macrophages. I showed that WASP bound WIP to form a complex at podosomes and that the knockdown of WIP impairs podosome formation. When WASP binding to WIP was blocked, podosome formation was also impaired. When WASP expression was reduced by small interfering RNA transfection, the amount of the complex of WASP with WIP decreased, resulting in reduced podosome formation. Podosomes were restored by reconstitution of the WASP-WIP complex in WASP knockdown cells. These results indicate that the WASP-WIP complex is required for podosome formation in macrophages. When podosome formation was reduced by blocking WASP binding to WIP, transendothelial migration of macrophages, the most crucial process in macrophage trafficking, was impaired. These results suggest that a complex of WASP with WIP plays a critical role in podosome formation, thereby mediating efficient transendothelial migration of macrophages.

The Wiskott-Aldrich syndrome protein (WASP) is a product of the gene defective in an Xid disorder, Wiskott-Aldrich syndrome. WASP expression is limited to hemopoietic cells, and WASP regulates the actin cytoskeleton. It has been reported that monocytes/macrophages from WASP-deficient Wiskott-Aldrich syndrome patients are severely defective in chemotaxis, resulting in recurrent infection. However, the molecular basis of such chemotactic defects is not understood. Recently, the WASP N-terminal region was found to bind to the three mammalian verprolin homologs: WASP interacting protein (WIP); WIP and CR16 homologous protein (WICH)/WIP-related protein (WIRE); and CR16. Verprolin was originally found to play an important role in the regulation of actin cytoskeleton in yeast. We have shown that WASP, WIP, and WICH/WIRE are expressed predominantly in the human monocyte cell line THP-1 and that WIP and WICH/WIRE are involved in monocyte chemotaxis. When WASP binding to verprolins was blocked, chemotactic migration of monocytes was impaired in both THP-1 cells and primary human monocytes. Increased expression of WASP and WIP enhanced monocyte chemotaxis. Blocking WASP binding to verprolins impaired cell polarization but not actin polymerization. These results indicate that a complex of WASP with mammalian verprolins plays an important role in chemotaxis of monocytes. Our results suggest that WASP and mammalian verprolins function as a unit in monocyte chemotaxis and that the activity of this unit is critical to establish cell polarization. In addition, our results also indicate that the WASP-verprolin complex is involved in other functions such as podosome formation and phagocytosis.

The Wiskott–Aldrich syndrome (WAS) is an X-chromosome-linked immunodeficiency disorder. The most common symptom seen in WAS patients is bleeding. One of the main causes of bleeding is defective platelet aggregation. The causative gene of WAS encodes WAS protein (WASP). Here, we show that WASP binds to the calcium- and integrin-binding protein (CIB) in platelets. CIB was originally identified as a protein binding to the αIIb cytoplasmic tail of platelet integrin αIIbβ3, which has a primary role in platelet aggregation. We also show that the WASP–CIB complex is important in αIIbβ3-mediated cell adhesion, and that in patients mutant forms of WASP are expressed at reduced levels or show lower affinities for CIB than wild-type WASP. Our results indicate that impaired complex formation between mutant WASPs and CIB reduces αIIbβ3-mediated cell adhesion and causes defective platelet aggregation, resulting in bleeding.

A new perfluoropolyether lubricant (LUB-A) with a 3-phenylpropyl functional group at both ends of the main chain was designed and synthesized by the authors for use in hard disk media, and its tribological performance was evaluated. Time-of-flight secondary ion mass spectrometry showed that LUB-A film on the disk lost none of its functional groups even after a 672 h exposure at 23 °C and 55% RH, although AM3001 lost 94% of its (3,4-dioxomethylenephenyl)methyl functional groups after the same period of exposure. It was found that cyclotriphosphazene (X-1P) was more than four times more soluble in 2 nm thick LUB-A film than in AM3001 film. The degradation ratio of read-back signals in the seek test was less than 5% over a wide range of X-1P content in LUB-A film and no micro-phase separation was induced, in contrast to AM3001 film that could not keep the degradation ratio at less than 5% without inducing micro-phase separation. LUB-A also showed better migration properties at 80 °C and 3% RH than AM3001. LUB-A was proved to be more chemically stable and to show better tribological performance than the currently popular AM3001 when it was used as a thin film mixed with X-1P.

αIIbβ3, a platelet-specific integrin, plays a critical role in platelet aggregation. The affinity of αIIbβ3 for its ligands such as fibrinogen and von Willebrand factor is tightly regulated in an uncharacterized intracellular process termed inside-out signaling. Calcium integrin-binding protein (CIB) has been identified as a protein interacting with the cytoplasmic tail of the αIIb subunit of αIIbβ3, but its physiological role has not been defined. In the present study, I demonstrate that CIB activates αIIbβ3 bothin vitro and in vivo. CIB interacts directly with the αIIb cytoplasmic tail, thereby increasing the affinity of αIIbβ3 for fibrinogen in anin vitro fibrinogen-binding assay. The interaction of CIB with the αIIb cytoplasmic tail is enhanced in a Ca2+-dependent manner. A physiological agonist, ADP, stimulates platelets, activating αIIbβ3. When the interaction of CIB with the αIIb cytoplasmic tail is blocked in native platelets by a permeable competing peptide, αIIbβ3activation is not detected even in the presence of ADP. This result indicates that direct interaction of CIB with the αIIbcytoplasmic tail converts αIIbβ3 from a resting to an active conformation. This suggests that CIB plays an important role in one of the pathways that modulate the affinity of αIIbβ3 for its ligand.

We innately have an ability to reject tumors, thereby limiting cancer progression and metastasis. The major effector lymphocytes in tumor rejection are natural killer (NK) cells. NK cells kill target cancer cells by two different rejection mechanisms, NK receptor-dependent killing and tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated killing. In spite of these tumor rejection systems, cancer cells make survival in host, facilitating the metastatic spread to other organs. The metastatic spread is still the major cause of cancer deaths. It has been revealed that some cancer cells acquire an ability to evade tumor rejection responses by NK cells to survive longer in host, thereby increasing the chance to metastasize. Several immune evasion strategies have been well documented. Recently, the immune evasion mechanisms from NK immunity using cell-surface glycans have been identified. The cancer cells use the certain types of cell-surface glycans to evade NK immunity in the following three ways: reducing NK activating receptor-mediated signaling, enhancing NK inhibitory receptor-mediated signaling, and modulating TRAIL-mediated killing. In this chapter, we will illustrate those evasion mechanisms in which cell-surface glycans play a central role.

Anti-tumour immunity by cytotoxic T-lymphocytes (CTLs) is essential to suppress tumour progression. Cancer cells that evade CTL immunity proliferate in the host, promoting metastasis, but mechanisms underlying this capacity remain unknown. Here we report that bladder cancer cells metastasized to lymph nodes evade CTL immunity by a new mechanism via altered glycosylation. CTLs normally recognize and kill cancer cells presenting antigenic peptides on human leukocyte antigen (HLA) class I. We show bladder cancer cells expressing the O-glycan processing enzyme, core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) exhibit HLA class I O-glycan modified with poly-N-acetyllactosamine and are highly susceptible to CTL. In those cells, poly-N-acetyllactosamine on HLA class I O-glycan binds galectin-3 to form a cell-surface molecular lattice, enabling efficient cell-surface retention of HLA class I. In contrast, bladder cancer cells in which C2GnT is downregulated show decreased levels of poly-N-acetyllactosamine on HLA class I O-glycans, attenuating lattice formation and reducing the cell-surface half-life of HLA class I. These tumour cells present antigenic peptides less efficiently, thereby evading CTL lysis and facilitating metastasis.

The chemical characteristics of perfluoropolyether lubricant films, which have arylalkyl groups at both ends of the main chain, were studied on hard-disk media. It was found by time-of-flight secondary ion mass spectroscopy (TOF-SIMS) that 94% of the functional groups of AM3001, (3,4-dioxomethylenephenyl)methyl, were eliminated from the main chain on a disk surface after a 672 h exposure, even in a regular clean room of 23°C and 55% RH. Regarding the lubricants synthesized by the authors, LUB-A having a benzyl functional group lost 35% of its functional group after a 768 h exposure. The 3-phenylpropyl functional group of LUB-B was not eliminated even after a 672 h exposure. It was suggested that hydrolysis easily occurred at the ether linkage between the main chain and the functional group, and removed the functional group from the main chain because the benzyl cation was chemically stable. The new lubricant having a 3-phenylpropyl functional group, which is chemically stable and does not decompose on a disk surface, was developed and proposed for use with hard-disk media.

The micro-phase separation of the additive, cyclotriphosphazene (X-1P), in perfluoropolyether (PFPE) lubricant films on hard disk media was studied. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) indicated that the small spots appearing on the disk surface consisted predominantly of X-1P. Observation using an atomic force microscope (AFM) revealed the micro-phase separation process to be the sudden, continuous appearance of new spots some time after coating the film. Some spots grew over previous ones, while some spots dissolved. Finally, they stopped growing and the number of spots became saturated. The solubility of X-1P in the lubricant film increased in the order of ZDIAC, ZDOL2000, ZDOL4000 and Z03, and that in bulk lubricant increased in the order of Z03, ZDOL4000, ZDOL2000 and ZDIAC. The order of solubility of X-1P in the film did not correspond to that in the bulk.

Many functional glycoproteins are expressed on the lymphocyte cell surface. Some of them carry O-linked oligosaccharides (O-glycans), which are conjugated through serine or threonine residues. During various biological processes, including T-cell activation, a tetrasaccharide on the T-cell surface is dramatically converted to a branched hexasaccharide, called core2 O-glycan. The same structural change in O-glycans is also found on the lymphocytes from patients with immunodeficiency conditions such as Wiskott-Aldrich syndrome and AIDS. Several studies revealing the roles of core2 O-glycans in immune responses show that this is a biologically significant change. In particular, core2 O-glycans expressed on the cell surface reduce cell-cell interactions, thereby regulating immune responses. Furthermore, core2 O-glycan is a key backbone structure in forming selectin ligands. Thus, O-linked oligosaccharides, in particular those containing core2 branches, play vital roles in immune responses and may play dual roles in certain situations. This review will summarize the results obtained from various studies investigating the roles of O-glycans in immunological processes. BioEssays 23:46-53, 2001.

Galectin-1 induces apoptosis of immature thymocytes and activated T cells, suggesting that galectin-1 regulates cell death in the thymus during selection and in the periphery following an immune response. Although it is known that galectin-1 recognizes lactosamine (Gal-GlcNAc) as a minimal ligand, this disaccharide is ubiquitously expressed on a variety of cell surface glycoproteins. Thus, susceptibility to galectin-1 may be regulated by the presentation of lactosamine on specific oligosaccharide structures created by specific glycosyltransferase enzymes. The core 2 beta-1, 6-N-acetylglucosaminyltransferase (core 2 GnT) creates a branched structure on O-glycans that can be elongated to present multiple lactosamine sequences. In the thymus, the core 2 GnT is expressed in galectin-1-sensitive thymocyte subsets. In the periphery, an oligosaccharide epitope created by the core 2 GnT is expressed on galectin-1-sensitive activated T-cells. In this report, we demonstrate that expression of the core 2 GnT was necessary and sufficient for galectin-1-induced death of murine T cell lines. In addition, overexpression of the core 2 GnT in mice increased the susceptibility of double positive thymocytes to galectin-1. These data demonstrate that expression of a specific glycosyltransferase can control susceptibility to galectin-1, suggesting that developmentally regulated glycosyltransferase expression may be a mechanism to modulate cell death during T cell development and function.

Mucin-type O -glycans on leukocytes acquire functions once they contain core 2 branches, which can be synthesized by core 2 β1,6- N -acetylglucosaminyltransferase (C2GnT). Recently, understanding the roles of mucin-type O -glycans has been significantly advanced by generating transgenic mice overexpressing C2GnT or knockout mice defective in C2GnT. This review article summarizes previous results implicating the roles of mucin-type O -glycans and the most recent studies to test such a hypothesis. These results, taken together, demonstrate that mucin-type O -glycans either facilitate or attenuate cell adhesion depending on the structures of non-reducing termini.

L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to unique carbohydrate ligands, sulfated sialyl Lewis x , which are expressed on high endothelial venules (HEV) in secondary lymphoid organs. The nature of the sulfotransferase(s) that contribute to sulfation of such L-selectin counterreceptors has been uncertain. We herein describe a novel L-selectin ligand sulfotransferase, termed LSST, that directs the synthesis of the 6-sulfo sialyl Lewis x on L-selectin counterreceptors CD34, GlyCAM-1, and MAdCAM-1. LSST is predominantly expressed in HEV and exhibits striking catalytic preference for core 2–branched mucin-type O -glycans as found in natural L-selectin counterreceptors. LSST enhances L-selectin–mediated adhesion under shear compared to nonsulfated controls. LSST therefore corresponds to an HEV-specific sulfotransferase that contributes to the biosynthesis of L-selectin ligands required for lymphocyte homing.

Aberrant expression of cell surface carbohydrates such as sialyl Lewis X is associated with tumor formation and metastasis. In order to determine the roles of sialyl Lewis X in tumor metastasis, mouse melanoma B16-F1 cells were stably transfected with alpha1, 3-fucosyltransferase III to express sialyl Lewis X structures. The transfected B16-F1 cells, B16-FTIII, were separated by cell sorting into three different groups based on the expression levels of sialyl Lewis X. When these transfected cells were injected into tail veins of C57BL/6 mice, B16-FTIII.M cells expressing moderate amounts of sialyl Lewis X in poly-N-acetyllactosamines produced large numbers of lung tumor nodules. Surprisingly, B16-FTIII.H cells expressing the highest amount of sialyl Lewis X in shorter N-glycans died in lung blood vessels, producing as few lung nodules as B16-FTIII.N cells which lack sialyl Lewis X. In contrast, B16-FIII.H cells formed more tumors in beige mice and NK cell-depleted C57BL/6 mice than did B16-FTIII.M cells. B16-FTIII.H cells bound to E-selectin better than did B16-FTIII.M cells, but both cells grew at the same rate. These results indicate that excessive expression of sialyl Lewis X in tumor cells leads to rejection by NK cells rather than tumor formation facilitated by attachment to endothelial cells.

Mammalian serine/threonine-linked oligosaccharides ( O -glycans) are commonly synthesized with the Golgi enzyme core 2 β-1,6- N -acetylglucosaminyltransferase (C2 GlcNAcT). Core 2 O -glycans have been hypothesized to be essential for mucin production and selectin ligand biosynthesis. We report that mice lacking C2 GlcNAcT exhibit a restricted phenotype with neutrophilia and a partial deficiency of selectin ligands. Loss of core 2 oligosaccharides reduces neutrophil rolling on substrata bearing E-, L-, and P-selectins and neutrophil recruitment to sites of inflammation. However, the diminished presence of L-selectin ligands on lymph node high endothelial venules does not affect lymphocyte homing. These studies indicate that core 2 oligosaccharide biosynthesis segregates the physiologic roles of selectins and reveal a function for the C2 GlcNAcT in myeloid homeostasis and inflammation.

The aberrant expression of core 2 O-glycans on T cell surface glycoproteins has been associated with various immunodeficient syndromes such as Wiskott-Aldrich syndrome and AIDS. To determine the effect of this aberrant expression of core 2 O-glycans on immune responses, we previously generated transgenic mice overexpressing core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT) in T cells, and demonstrated that T cell primary immune responses mediated through interaction between T cells and antigen-presenting cells are impaired in the transgenic mice (Tsuboi, S., and Fukuda, M. (1997) EMBO J. 16, 6364-6373). In this study, we determined whether overexpression of core 2 oligosaccharides on T cells leads to impaired humoral immune responses by B cells using the same transgenic mice. When T cells were activated, both T and B cells from the transgenic and control mice expressed an equivalent amount of CD40L and CD40, which are, respectively, the receptor and counter-receptor for the interaction between T and B cells. However, activated T cells from the transgenic mice induced B cell proliferation less efficiently than those from control mice, regardless of whether B cells were isolated from control or the transgenic mice. This suggests that overexpression of core 2 O-glycans on T cell surface glycoproteins renders T cell-B cell interaction inefficient. Moreover, in the transgenic mice both immunoglobulin isotype switching and germinal center formation were also impaired. Taken together, these results indicate that aberrant expression of core 2 O-glycans on T cell surface glycoproteins results in impaired humoral immune responses due to an impaired interaction between T and B cells.

Core 2 beta-1,6-N-acetylglucosaminyltransferase, C2GnT, is a key enzyme in O-linked oligosaccharide (O-glycan) biosynthesis and the resultant core 2 branch serves as a backbone for additional glycosylation to form oligosaccharide ligands such as sialyl Le(x). Since the expression of C2GnT is highly regulated during T-cell development and increases in pathological conditions such as the Wiskott-Aldrich syndrome, we have generated transgenic mice overexpressing C2GnT in the T-cell lineage. Surprisingly, T lymphocytes in the transgenic mice develop normally, but they exhibit a reduced immune response when assayed by delayed-type hypersensitivity, proliferation upon stimulation and cytokine production. Moreover, T lymphocytes from the transgenic mice adhere much less efficiently to ICAM-1 and fibronectin than do T lymphocytes from non-transgenic mice. These results indicate that overexpression of the core 2 branched O-glycans in T lymphocytes results in reduced immune responses due to impaired cell-cell interaction. Such an impaired immune response may be one of the causes for immunodeficiency in the Wiskott-Aldrich syndrome.

Muscle invasive bladder cancer is an aggressive type of epithelial tumor with a high rate of metastasis. For bladder cancer cells to reach the muscle layer, cells must invade through an urothelial cell monolayer (transurothelial invasion) and basement membrane. However, the process by which transurothelial invasion occurs has not been fully characterized. In this study we developed a novel method to evaluate the transurothelial invasion capacity and investigated its cellular and molecular processes using primary culture cells from bladder cancer patients. The analysis revealed that compared with the prognosis for patients with non‑muscle invasive bladder cancer that of patients with muscle invasive bladder cancer was particularly poor due to metastatic recurrence. Cancer cells from patients with muscle invasive bladder cancer exhibited a higher invasive capacity through the urothelial cell monolayer compared with those from non‑invasive bladder cancer patients. Furthermore, muscle invasive bladder cancer cells demonstrated a greater ability to form invadopodia, the filamentous actin‑based membrane protrusions required for matrix degradation and invasion compared with non‑invasive cells. Bladder cancer cell lines were established with reduced invadopodia formation by silencing the expression of cortactin, an essential component of invadopodia. The cortactin knockdown bladder cancer cells with reduced invadopodia formation demonstrated a markedly reduced ability to invade through the urothelial cell monolayer, indicating that invadopodia are essential for transurothelial invasion. The results indicate that invadopodia formation is required for muscle invasion of aggressive bladder cancer cells.

The majority of deaths associated with solid tumors are caused by tumor metastasis. To prevent metastasis, it is vital to understand its detailed process. In hematogenous metastasis of bladder cancer, some cancer cells disseminating into blood circulation extravasate into the lung tissues to form metastases. To study the molecular basis of the lung metastasis of bladder cancer, we employed an in vivo selection system that mimics hematogenous metastasis of bladder cancer on a low-metastatic bladder cancer cell line (KK-47). We have successfully isolated a high-metastatic bladder cancer subline, KK-47HM4, from KK-47 cells. We characterized KK-47HM4 in in vitro experimental systems. No significant difference in growth rate and susceptibility to NK cell attack between KK-47 and KK-47HM4 cells was observed. However, KK-47HM4 exhibited the higher capacities of Matrigel Matrix invasion and transendothelial invasion than KK-47. These results suggest that the extravasation of KK-47HM4 cells was enhanced among the multiple steps of the lung metastasis of bladder cancer. Our cDNA microarray analysis identified 67 genes whose expression was up- or downregulated in KK-47HM4 cells compared with KK-47 cells. This analysis data implied that one possible cause for enhanced extravasation of KK-47HM4 is its higher adhesion to extracellular matrix proteins. KK-47HM4 is the first bladder cancer subline with enhanced extravasation potential using the in vivo selection system. The information provided by our cDNA microarray analysis using KK-47HM4 will be useful for further investigation into the molecular basis of extravasation of cancer cells.

Invasive cancer cells form the filamentous actin‑based membrane protrusions known as invadopodia. Invadopodia are thought to play a critical role in cancer cell invasion and metastasis due to their ability to degrade the extracellular matrix. The present study assessed whether invadopodia formation is essential in extravasation of circulating bladder cancer cells and lung metastasis. To analyze the importance of invadopodia, bladder cancer cell lines with reduced invadopodia formation were established by silencing the expression of cortactin, an essential component of invadopodia, using cortactin short hairpin RNA. Bladder cancer cells with cortactin knockdown demonstrated a markedly decreased ability to form invadopodia, secrete matrix metalloproteinases and invade the extracellular matrix. In addition, the knockdown cells exhibited a reduced transendothelial invasion capacity and decreased formation of metastatic foci in the lungs. The present study demonstrated that bladder cancer cells with cortactin knockdown have a reduced capacity to extravasate into the lung from the circulation, due to the decreased invasive character of invadopodia. This suggests that invadopodia formation is a critical process for cancer cell extravasation.

In order to determine if a sulfated oligosaccharide on the cell surface can function as an L-selectin ligand, a novel approach for in vitro transfer of oligosaccharides was utilized (Srivastava, G., Kaun, K. J., Hindsgaul, O., and Palcic, M. M. (1992) J. Biol. Chem. 267, 22356-22361). CHO cells were incubated with synthetic 6'-sulfo sialyl Lex, NeuNAcalpha2-->3(sulfate-6)Galbeta1-->4(Fucalpha1-->3) GlcNAc or 6-sulfo sialyl Lex, NeuNAcalpha2-->3Galbeta1-->4[(Fucalpha1-->3)sulfate--> 6GlcNAc] oligosaccharide linked to C-6 of a fucose residue in GDP-fucose and a milk fucosyltransferase. The resultant CHO cells expressing 6'-sulfo sialyl Lex or 6-sulfo sialyl Lex on their cell surface were tested for adhesion to E-selectin and L-selectin chimeric proteins coated on plates. The results indicate that 6'-sulfo sialyl Lex supports L-selectin-mediated adhesion much better than sialyl Lex similarly tagged on the cell surface. In contrast, 6-sulfo sialyl Lex containing a sulfate group on the N-acetylglucosamine residue did not support adhesion with either selectin. These combined results suggest that 6'-sulfo sialyl Lex is a much better ligand than sialyl Lex oligosaccharide for L-selectin.

The occurrence of non-specific lipid transfer proteins (nsLTPs) in developing castor bean fruits was examined by immunoblot quantitation and immunogold electron microscopy. The levels of nsLTPs increased with fruit development, being higher in the pericarp than the seed. The distribution of nsLTPs was localized throughout the development in the peripheral tissues, the outer layer of the endosperm in the seed and the epicarp in the pericarp. nsLTPs in the endosperm were localized only in the dense vesicle in the cytoplasm at 14-days after flowering (14-DAF). At 28-DAF the dense vesicles containing nsLTPs were assembled to the central vacuole to form an aggregate, which in turn disappeared at 35-DAF. On the other hand, nsLTPs were scattered in the cell walls of sclereid surrounding the seed and in the cell walls of trichome and sclerenchyma in the pericarp. These results suggest that gene-expressed nsLTPs are transported by two routes; one is to the vacuole by the dense vesicles in the endosperm and the other is to the cell wall and around there in the pericarp, probably by Golgi route.

INTRODUCTION AND OBJECTIVES: To explore the detailed molecular mechanisms underlying bladder cancer metastasis, we focused on the role of cell-surface carbohydrates. We immunohistochemically analyzed paraffin-embedded bladder cancer specimens for the expression of a glycosyltransferase, core2 -1,6-N-acetylglucosminyltransferase (C2GnT) which forms core2 branch on O-glycans (A). We discovered that C2GnT expression was closely related with highly metastatic phenotypes of bladder cancer and that C2GnT-expressing cancer cells possessed high ability to evade NK cell immunity. We then aimed to elucidate their evasion mechanisms. There are two major ways of NK attacking cancer cells. Cancer cell-expressing ligands bind NK-activating receptors, thereby activating NK cells to attack cancer cells. NK cell-expressing ligands such as TNF-related apoptosis inducing ligand (TRAIL) bind death receptors in tumor cells, thereby inducing apoptosis of tumor cells. An NK-activating receptor, natural killer group 2 member D (NKG2D) and TRAIL play a major role in tumor rejection (B). METHODS: A primary ligand for NKG2D in cancer cells is MHC class I-related chain A (MICA). We analyzed O-glycans of MICA and MUC1 (a cancer cell-surface major mucin) by Western blotting and lectin column chromatography. We then examined the roles of Oglycans in evasion from NK cell immunity by tumor formation assay and cytotoxicity assay. RESULTS: In C2GnT-expressing cancer cells, poly N-acetyllactosamine was present on core2 O-glycans on MICA and MUC1, and galectin-3 bound MICA and MUC1 through this poly N-acetyllactosamine. We discovered that C2GnT-expressing bladder cancer cells took two different strategies to evade NK cell attack. One is that modification of MICA with poly N-acetyllactosamine and galectin-3 reduces the affinity of MICA for NKG2D, thereby severely impairing NK cell activation (C) (Tsuboi, S. et al. 2011 EMBO J 30:3173-3185). Another one is that modification of MUC1 with poly N-acetyllactosamine and galectin-3 reduces the accessibility of NK cells to the cancer cell surface, thereby shielding cancer cells from NK cell attack by TRAIL (D). CONCLUSIONS: The immunoevasion mechanisms using core2 O-glycans (C and D) allow bladder cancer cells to survive longer in the circulation, resulting in metastasis. Source of Funding: None

Core 2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) forms an N-acetylglucosamine branch in O-glycans (core 2 O-glycans) of cell surface glycoproteins. C2GnT-expressing bladder tumors acquire highly metastatic phenotypes by surviving longer in host blood circulation. However, the detailed mechanisms underlying this increased survival remain unclear. In this study, we report that the expression of C2GnT in bladder tumors positively correlates with tumor progression and that bladder tumor cell-surface mucin 1 (MUC1) carrying core 2 O-glycans plays an important role in the evasion from natural killer (NK) cell attack. In C2GnT-expressing bladder tumor cells, heavily core 2 O-glycosylated MUC1 carries poly-N-acetyllactosamine in its O-glycans and galectin-3 binds to MUC1 through this poly-N-acetyllactosamine. The binding of galectin-3 to poly-N-acetyllactosamine in MUC1 core 2 O-glycans attenuates the interaction of the tumor cells with NK cells and interferes with the access of tumor necrosis factor-related apoptosis-inducing ligand to the tumor cell surface. These effects of MUC1 carrying core 2 O-glycans on NK cell attack facilitate C2GnT-expressing tumor cells to evade NK cell immunity and survive longer in host blood circulation. We reveal that MUC1 carrying core 2 O-glycans thus functions as a molecular shield against NK cell attack, thereby promoting bladder tumor metastasis.

Despite the high prevalence of metastatic cancers and the poor outcome for patients, the processes of tumor metastasis still remain poorly understood. It has been shown that cell-surface carbohydrates attached to proteins through the amino acids serine or threonine (O-glycans) are involved in tumor metastasis, with the roles of O-glycans varying depending on their structure. Core2 O-glycans allow tumor cells to evade natural killer (NK) cells of the immune system and survive longer in the circulatory system, thereby promoting tumor metastasis. Core3 O-glycans or O-mannosyl glycans suppress tumor formation and metastasis by modulating integrin-mediated signaling. Here, we highlight recent advances in our understanding of the detailed molecular mechanisms by which O-glycans promote or suppress tumor metastasis.

A major cause of death in patients with bladder tumors is recurrence with metastasis. Bladder tumor metastasis is largely dependent upon the invasive capacity of tumor cells. Tumor cell invasion is mainly mediated by actin-rich protrusive membrane structures called invadopodia. The formation of invadopodia was observed in various types of invasive tumors such as breast cancer and melanomas. However, invadopodia formation so far has not been described in bladder tumor cells. We here report that human bladder tumor cells form functionally active invadopodia. By using a confocal laser scanning microscope, we demonstrated that invasive bladder tumor cell lines, YTS-1 and T24, with high Matrigel degradation activity form invadopodia but that noninvasive bladder tumor cell lines, RT4 and KK-47, form no detectable invadopodia. Invadopodia formed by YTS-1 cells had the ability to secrete matrix metalloproteases and degrade extracellular matrix to invade surrounding areas. Moreover, we observed that primary tumor cells obtained from patients with invasive bladder tumors also form invadopodia, validating the results from bladder tumor cell lines. Our results provide evidence that invasive human bladder tumor cells form invadopodia for tumor invasion.

For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.

The subcellular localization and several biochemical activities of nonspecific lipid transfer protein (nsLTP) were investigated. A section of a castor bean cotyledon cell was labeled with anti-nsLTP serum followed by protein A-gold. Gold particles were more abundant in the glyoxysome matrix and the vessel cell wall than in other areas. Cell fractionation analysis of 6-day-old castor bean cotyledons by sucrose density gradient centrifugation demonstrated that 13% of nsLTP was distributed in the glyoxysomal fraction, identified on the basis of catalase as a marker, and 87% in the soluble fraction near the top of the gradient. The location of castor bean nsLTP in glyoxysomes was further confirmed by in vitro import experiments. The synthesized precursor of nsLTP (pro-nsLTP-C) was incorporated into intact castor bean glyoxysomes and processed to the mature form after import into the glyoxysomes, but it was not imported into canine pancreatic microsomes. Castor bean nsLTP-A was found to possess the ability to bind oleic acid and oleoyl-CoA by means of a method involving Lipidex 1000. The dissociation constants (Kd) for oleic acid and oleoyl-CoA binding to nsLTP-A were 4.8 and 5.0 microM, respectively. The saturated binding capacities (Bmax) for oleic acid and oleoyl-CoA per mol of nsLTP-A were 1.1 and 1.2 mol, respectively. When acyl-CoA oxidase activity was assayed in the glyoxysomal fraction, marked enhancement of the activity was observed in the presence of nsLTP. These results suggest the possibility that nsLTP regulates fatty acid beta-oxidation through the enhancement of acyl-CoA oxidase activity in glyoxysomes. The occurrence of castor bean nsLTP in the vessel wall was discussed.

Four kinds of nonspecific lipid transfer proteins (nsLTP) were purified from different organs of castor bean (Ricinus communis L.) seedlings. Amino acid compositions and amino-terminal sequences of the four nsLTPs were determined and compared with those of castor bean isoforms, nsLTP-A, -B, and -C, previously reported [Takishima et al. (1986) Biochim. Biophys. Acta 870, 248-255; Takishima et al. (1988) Eur. J. Biochem. 177, 241-249]. Two isoforms from the cotyledons were identified as nsLTP-A and -C, one isoform from the endosperms as nsLTP-B, and the other was a new isoform from the axes. This new isoform was named nsLTP-D and its amino acid sequence was determined. These results demonstrated organ-specific occurrence of the nsLTP isoforms in castor bean seedlings. The isoforms nsLTP-A, -B, -C, and -D showed similar transfer activity not only for phosphatidylcholine and phosphatidylethanolamine but also for monogalactosyldiacylglycerol, although the homology among their amino acid sequences ranged from 70 to 30%. Two cDNA clones (pnsLTP-C and pnsLTP-D) for nsLTPs of castor bean seedlings were isolated and sequenced. pnsLTP-C was the cDNA clone for nsLTP-C expressed in the cotyledons, and pnsLTP-D was that for nsLTP-D in the axis. A coupled in vitro transcription-translation analysis of both cDNA clones revealed that pnsLTP-C encodes the full-length of nsLTP-C precursor (pro-nsLTP-C), while pnsLTP-D encodes a part of nsLTP-D precursor. PronsLTP-C contained a 24-amino acid pre-sequence preceding the mature nsLTP-C (92 amino acids).(ABSTRACT TRUNCATED AT 250 WORDS)

Phagocytosis is a vital first-line host defense mechanism against infection involving the ingestion and digestion of foreign materials such as bacteria by specialized cells, phagocytes. For phagocytes to ingest the foreign materials, they form an actin-based membrane structure called phagocytic cup at the plasma membranes. Formation of the phagocytic cup is impaired in phagocytes from patients with a genetic immunodeficiency disorder, Wiskott-Aldrich syndrome (WAS). The gene defective in WAS encodes Wiskott-Aldrich syndrome protein (WASP). Mutation or deletion of WASP causes impaired formation of the phagocytic cup, suggesting that WASP plays an important role in the phagocytic cup formation. However, the molecular details of its formation remain unknown. We have shown that the WASP C-terminal activity is critical for the phagocytic cup formation in macrophages. We demonstrated that WASP is phosphorylated on tyrosine 291 in macrophages, and the WASP phosphorylation is important for the phagocytic cup formation. In addition, we showed that WASP and WASP-interacting protein (WIP) form a complex at the phagocytic cup and that the WASP·WIP complex plays a critical role in the phagocytic cup formation. Our results indicate that the phosphorylation of WASP and the complex formation of WASP with WIP are the essential molecular steps for the efficient formation of the phagocytic cup in macrophages, suggesting a possible disease mechanism underlying phagocytic defects and recurrent infections in WAS patients.

The Wiskott-Aldrich syndrome (WAS) is an X-chromosome-linked immunodeficiency disorder. The most common symptom seen in WAS patients is bleeding. One of the main causes of bleeding is defective platelet aggregation. The causative gene of WAS encodes WAS protein (WASP). Here, we show that WASP binds to the calcium- and integrin-binding protein (CIB) in platelets. CIB was originally identified as a protein binding to the alphaIIb cytoplasmic tail of platelet integrin alphaIIb beta3, which has a primary role in platelet aggregation. We also show that the WASP-CIB complex is important in alphaIIb beta3-mediated cell adhesion, and that in patients mutant forms of WASP are expressed at reduced levels or show lower affinities for CIB than wild-type WASP. Our results indicate that impaired complex formation between mutant WASPs and CIB reduces alphaIIb beta3-mediated cell adhesion and causes defective platelet aggregation, resulting in bleeding.