Exosomes are nano-vesicles secreted by a wide range of mammalian cell types. These vesicles are abundant in serum and other extracellular fluids and contain a large repertoire of proteins, mRNA and microRNA. Exosomes have been implicated in cell to cell communication, the transfer of infectious agents, and neurodegenerative diseases as well as tumor progression. However, the precise mechanisms by which they are internalized and/or secreted remain poorly understood. In order to follow their release and uptake in breast tumor cells in real time, cell-derived exosomes were tagged with green fluorescent protein (GFP)-CD63 while human serum exosomes were rhodamine isothiocynate-labeled. We show that detachment of adherent cells from various substrata induces a rapid and substantial secretion of exosomes, which then concentrate on the cell surfaces and mediate adhesion to various extracellular matrix proteins. We also demonstrate that disruption of lipid rafts with methyl-beta-cyclodextrin (MβCD) inhibits the internalization of exosomes and that annexins are essential for the exosomal uptake mechanisms. Taken together, these data suggest that cellular detachment is accompanied by significant release of exosomes while cellular adhesion and spreading are enhanced by rapid uptake and disposition of exosomes on the cell surface.

Minichromosome maintenance (MCM) complex replicative helicase complexes play essential roles in DNA replication in all eukaryotes. Using a tandem affinity purification-tagging approach in human cells, we discovered a form of the MCM complex that contains a previously unstudied protein, MCM binding protein (MCM-BP). MCM-BP is conserved in multicellular eukaryotes and shares limited homology with MCM proteins. MCM-BP formed a complex with MCM3 to MCM7, which excluded MCM2; and, conversely, hexameric complexes of MCM2 to MCM7 lacked MCM-BP, indicating that MCM-BP can replace MCM2 in the MCM complex. MCM-BP-containing complexes exhibited increased stability under experimental conditions relative to those containing MCM2. MCM-BP also formed a complex with the MCM4/6/7 core helicase in vitro, but, unlike MCM2, did not inhibit this helicase activity. A proportion of MCM-BP bound to cellular chromatin in a cell cycle-dependent manner typical of MCM proteins, and, like other MCM subunits, preferentially associated with a cellular origin in G 1 but not in S phase. In addition, down-regulation of MCM-BP decreased the association of MCM4 with chromatin, and the chromatin association of MCM-BP was at least partially dependent on MCM4 and cdc6. The results indicate that multicellular eukaryotes contain two types of hexameric MCM complexes with unique properties and functions.The initiation of DNA replication in eukaryotic cells is a carefully regulated process requiring the orchestrated assembly of many proteins at origin sites, including the origin recognition complex and minichromosome maintenance (MCM) complex. The MCM complex consists of six subunits, MCM2 through MCM7 (MCM2-7), which form a hexamer. Studies in Saccharomyces cerevisiae, where MCM proteins were first identified (25), showed that each of the MCM subunits performs an essential function in the initiation and elongation of DNA replication (20,21). Genetic and biochemical studies conducted in yeast, Xenopus, Drosophila, and mammals point to probable roles of the MCM proteins in melting origin DNA and in functioning as the replicative helicase at replication forks (8,27).Biochemical analyses of the MCM complex have shown that MCM4, -6, and -7 are the most stably associated subunits, referred to as the helicase core (MCM4/6/7), with MCM2 and a dimer of MCM3 and MCM5 being more loosely associated with the core (13,23,30,36). MCM4, MCM6, and MCM7 on their own can form hexamers with weak but measurable DNA helicase activity. The addition of MCM2 to the MCM4/6/7 core complex disrupts the hexamer and inhibits DNA helicase activity (12, 23). The complete MCM2-7 complex has no detectable helicase activity in vitro (12, 23), but helicase activity has been reported for a larger complex containing MCM2-7, cdc45, and GINS (29). As expected, MCM complexes exhibit ATPase activity (6,23,35). ATPase activity has not been observed in individual MCM subunits but occurs when certain pairs of MCM proteins interact (6).Each of the six MCM subunits shares a region of homology referre...

The interaction of annexin A6 (AnxA6) with membrane phospholipids and either specific extracellular matrix (ECM) components or F-actin suggests that it may influence cellular processes associated with rapid plasma membrane reorganization such as cell adhesion and motility. Here, we examined the putative roles of AnxA6 in adhesion-related cellular processes that contribute to breast cancer progression. We show that breast cancer cells secrete annexins via the exosomal pathway and that the secreted annexins are predominantly cell surface-associated. Depletion of AnxA6 in the invasive BT-549 breast cancer cells is accompanied by enhanced anchorage-independent cell growth but cell-cell cohesion, cell adhesion/spreading onto collagen type IV or fetuin-A, cell motility and invasiveness were strongly inhibited. To explain the loss in adhesion/motility, we show that vinculin-based focal adhesions in the AnxA6-depleted BT-549 cells are elongated and randomly distributed. These focal contacts are also functionally defective because the activation of focal adhesion kinase and the phosphoinositide-3 kinase/Akt pathway were strongly inhibited while the MAP kinase pathway remained constitutively active. Compared with normal human breast tissues, reduced AnxA6 expression in breast carcinoma tissues correlates with enhanced cell proliferation. Together this suggests that reduced AnxA6 expression contributes to breast cancer progression by promoting the loss of functional cell-cell and/or cell-ECM contacts and anchorage-independent cell proliferation.

We hereby report studies that suggest a role for serum exosomes in the anchorage independent growth (AIG) of tumor cells. In AIG assays, fetal bovine serum is one of the critical ingredients. We therefore purified exosomes from fetal bovine serum and examined their potential to promote growth of breast carcinoma cells in soft agar and Matrigel after reconstituting them into growth medium (EEM). In all the assays, viable colonies were formed only in the presence of exosomes. Some of the exosomal proteins we identified, have been documented by others and could be considered exosomal markers. Labeled purified exosomes were up-taken by the tumor cells, a process that could be competed out with excess unlabeled vesicles. Our data also suggested that once endocytosed by a cell, the exosomes could be recycled back to the conditioned medium from where they can be up-taken by other cells. We also demonstrated that low concentrations of exosomes activate MAP kinases, suggesting a mechanism by which they maintain the growth of the tumor cells in soft agar. Taken together, our data demonstrate that serum exosomes form a growth promoting platform for AIG of tumor cells and may open a new vista into cancer cell growth in vivo.

The identity of the cell adhesive factors in fetal bovine serum, commonly used to supplement growth media, remains a mystery due to the plethora of serum proteins. In the present analyses, we showed that fetuin-A, whose function in cellular attachment in tissue culture has been debated for many years, is indeed a major serum cell attachment factor particularly for tumor cells. We are able to report this because of a new purification strategy that has for the first time given us a homogeneous protein band in colloidal Coomassie-stained gels that retains biological activity. The tumor cells adhered to immobilized fetuin-A and not ␣ 2 -macroglobulin, its major contaminant. The interaction of cells with fetuin-A was driven mainly by Ca 2؉ ions, and cells growing in regular medium supplemented with fetal bovine serum were just as sensitive to loss of extracellular Ca 2؉ ions as cells growing in fetuin-A. Fractionation of human serum revealed that cell attachment was confined to the fractions that had fetuin-A. Interestingly, the tumor cells also took up fetuin-A and secreted it back to the medium using an unknown mechanism that can be observed in live cells. The attachment of tumor cells to fetuin-A was accompanied by phosphatidylinositol 3-kinase/Akt activation that was down-regulated in cells that lack annexin-A6, one of the cell surface receptors for fetuin-A. Taken together, our data show the significance of fetuin-A in tumor cell growth mechanisms in vitro and open new research vistas for this protein.Serum, particularly fetal bovine serum, is widely used as a supplement in culture media that is required for growth of most cells in culture (for a review, see Ref. 1). In the absence of serum, most cells fail to adhere properly, spread, and grow on culture dishes. Serum has a plethora of adhesion (2-4) and growth factors (5). The prevailing assumption over the past two decades has been that integrins are the major cellular receptors for adhesion in cell cultures (6). Cellular adhesion to extracellular adhesion molecules such as vitronectin, fibronectin, and laminin using integrins requires the divalent ions Mg 2ϩ /Mn 2ϩ (7). In addition, cell signaling cues for spreading and growth mechanisms in anchorage-dependent cells emanate from the interaction of cells with the adhesion molecules (8). Although it is easy to define such signals in situations where cells are allowed to adhere to known purified extracellular matrix proteins such as fibronectin and collagen (9), adhesion in the presence of fetal bovine serum is complex in that any one of the myriad attachment proteins and or growth factors has the potential to mediate adhesion and growth signals.Ever since it was first purified and described, fetuin-A/ ahsg 2 was suspected of being the principal cell adhesion molecule in serum (1, 10). Fetuin-A isolated and purified from serum by the Pedersen method (hereafter referred to as Pedersen fetuin-A) demonstrated cell adhesive properties in the presence of divalent ions (11). The Pedersen fetuin-A, however, is not cons...