P2X receptors are ATP-gated ion channels in the plasma membrane, but activation of the P2X 7 receptor also leads to rapid cytoskeletal re-arrangements such as membrane blebbing. We identi®ed 11 proteins in human embryonic kidney cells that interact with the rat P2X 7 receptor, by af®nity puri®cation followed by mass spectroscopy and immunoblotting [laminin a3, integrin b2, b-actin, a-actinin, supervillin, MAGuK, three heat shock proteins, phosphatidylinositol 4-kinase and receptor protein tyrosine phosphatase-b (RPTPb)]. Activation of the P2X 7 receptor resulted in its dephosphorylation. Whole-cell recordings from cells expressing P2X 7 receptors showed that this markedly reduced subsequent ionic currents and it also slowed membrane bleb formation. By mutagenesis, we identi®ed Tyr 343 in the putative second transmembrane domain as the site of phosphorylation. Thus, we have identi®ed a P2X 7 receptor signalling complex, some members of which may initiate cytoskeletal rearrangements following receptor activation. Others, such as RPTPb, might exert feedback control of the channel itself through its dephosphorylation. Keywords: ion channel/P2X receptors/receptor protein tyrosine phosphatase-b/signalling complex/tyrosine phosphorylation Introduction P2X receptors form a family of ATP-gated ion channels extensively distributed throughout the cells of vertebrates. They are ligand-gated ion channels, each with distinct pharmacological and/or physiological properties. They form as homomers and/or heteromers, and current biochemical evidence suggests that the channel has three or perhaps six subunits (Nicke et al., 1998; North and Surprenant, 2000). The P2X 7 receptor subunit has several features that set it apart from other members of the family (Surprenant et al., 1996). Co-immunoprecipitation experiments indicate that it is the only subunit that does not heteropolymerize with other P2X subunits (Torres et al., 1999). It is primarily localized to epithelia and immune cells, particularly antigen-presenting cells Mutini et al., 1999). Receptor activation requires concentrations of ATP that are 10±100 times higher than those required to activate other P2X receptors, but the agonist af®nity and maximum response can also be modulated 5-to 100-fold by alterations in external monovalent and divalent cations (Surprenant et al., 1996;Rassendren et al., 1997;Michel et al., 1999;Gudipaty et al., 2001). The ionic currents through P2X 7 receptors show considerable plasticity, in the sense that repeated applications of the agonist result in prominent changes in the amplitude and time course of the current elicited by subsequent applications (Surprenant et al., 1996;Rassendren et al., 1997;Hibell et al., 2000).Activation of native ATP receptors in macrophages and macrophage-like cell lines, in which P2X 7 subunits are predominately expressed, is fundamentally different from that observed for other ion channels because it initiates several cellular consequences further downstream. These include alterations in the cell morphology (Cohn...

A number of recent reports have demonstrated that only CD133-positive cancer cells of glioblastoma multiforme (GBM) have tumor-initiating potential. These findings raise an attractive hypothesis that GBMs can be cured by eradicating CD133-positive cancer stem cells (CSCs), which are a small portion of GBM cells. However, as GBMs are known to possess various genetic alterations, GBMs might harbor heterogeneous CSCs with different genetic alterations. Here, we compared the clinical characteristics of two GBM patient groups divided according to CD133-positive cell ratios. The CD133-low GBMs showed more invasive growth and gene expression profiles characteristic of mesenchymal or proliferative subtypes, whereas the CD133-high GBMs showed features of cortical and well-demarcated tumors and gene expressions typical of proneuronal subtype. Both CD133-positive and CD133-negative cells purified from four out of six GBM patients produced typical GBM tumor masses in NOD-SCID brains, whereas brain mass from CD133-negative cells showed more proliferative and angiogenic features compared to that from CD133-positive cells. Our results suggest, in contrast to previous reports that only CD133-positive cells of GBMs can initiate tumor formation in vivo CD133-negative cells also possess tumor-initiating potential, which is indicative of complexity in the identification of cancer cells for therapeutic targeting. A recent concept in brain tumor biology is that brain tumors arise from cancer stem cells (CSCs) that are CD133 positive (CD133 ( þ ) ). It has been reported that a small number of CD133 ( þ ) glioblastoma multiforme (GBM) cells are able to recapitulate the original tumor in vivo, whereas millions of CD133-negative (CD133 (À) ) cells could not produce brain tumor masses. 1-6 However, accumulating evidence suggests that CD133 (À) GBM cells can also regenerate heterogenous tumors in vivo, 7,8 and generation of the huge and rapidly growing tumors by only CD133 ( þ ) CSCs would be difficult because more than 50% of GBM patients have few CD133 ( þ ) cells. 9 As a majority of neurogenic astrocytes in the adult brain are not recognized by a CD133 antibody, 8 it is likely that CD133 might be newly expressed in GBM CSCs that are derived from CD133 (À) adult neural stem cells (NSCs) or terminally differentiated brain cells, such as astrocytes, neurons, and oligodendrocytes. Given that the gene expression profile is changed when GBM recurs after treatments, 10 it is plausible that new CD133 expression may occur if the characteristics of CSCs are changed or if some CSCs are selected by treatment. Furthermore, the wide-range variation in CD133 ( þ ) cell ratio (0.1-50% in GBM patients) 1-6 also suggests the existence of other GBM CSCs that do not express CD133.Therefore, we hypothesize that there are several kinds of CSCs in the tumor mass of GMB, and these diverse CSCs

The Wnt/Frizzled (FZD) signaling cascade is important for cell fate determination during embryonic development as well as maintaining tissue homeostasis in the adult. In addition to these physiologic roles, studies have shown that deregulation of Wnt/FZD signaling occurs during carcinogenesis. As an example, over 90% of the colorectal cancers have mutations in adenomatous polyposis coli (APC) or beta-catenin genes. In addition, hepatocellular carcinoma (HCC) is another tumor with frequent aberrant activation of beta-catenin signaling. Nuclear and/or cellular beta-catenin accumulation, a hallmark of the activated canonical Wnt/FZD signaling, has been observed in 33-67% of tumors. However, mutations of APC and/or beta-catenin genes are found only in about 20-30% of HCCs, suggesting that the predominant mechanism(s) activating Wnt/FZD signaling pathway may be different from that found in colorectal cancers. There is accumulating evidence to suggest that regulatory mechanisms other than mutations involving beta-catenin or proteins in its destruction complex, many of which involve upstream components of the Wnt/FZD cascade, are important in HCC. Furthermore, information on the target genes of Wnt/FZD signaling and their roles in hepatocarcinogenesis is limited despite the recent discovery of several candidate genes. This review focuses on the alterations of Wnt/FZD signaling pathways and their relationship to the pathogenesis of HCC. A better understanding of the precise mechanisms of altered Wnt/FZD signaling may provide new molecular targets for therapy of HCC.

Circulating tumor cells (CTCs) have great potential to provide minimally invasive ways for the early detection of cancer metastasis and for the response monitoring of various cancer treatments. Despite the clinical importance and progress of CTC-based cancer diagnostics, most of the current methods of enriching CTCs are difficult to implement in general hospital settings due to complex and time-consuming protocols. Among existing technologies, size-based isolation methods provide antibody-independent, relatively simple, and high throughput protocols. However, the clogging issues and lower than desired recovery rates and purity are the key challenges. In this work, inspired by antifouling membranes with liquid-filled pores in nature, clog-free, highly sensitive (95.9 ± 3.1% recovery rate), selective (>2.5 log depletion of white blood cells), rapid (>3 mL/min), and label-free isolation of viable CTCs from whole blood without prior sample treatment is achieved using a stand-alone lab-on-a-disc system equipped with fluid-assisted separation technology (FAST). Numerical simulation and experiments show that this method provides uniform, clog-free, ultrafast cell enrichment with pressure drops much less than in conventional size-based filtration, at 1 kPa. We demonstrate the clinical utility of the point-of-care detection of CTCs with samples taken from 142 patients suffering from breast, stomach, or lung cancer.