Connectin: cell surface protein that binds both laminin and actin.

A purified cell surface receptor protein for laminin (Mr = 70,000) isolated from mouse fibrosarcoma cells binds to actin with specificity and high affinity. This binding was demonstrated both by cosedimentation of the receptor with actin and binding of the receptor to actin immobilized on nitrocellulose filters. Specificity was demonstrated by displacement of 35S-labeled receptor by unlabeled receptor. Scatchard analysis of receptor binding to actin yielded a Kd of 6 X 10(-7) M. The receptor was observed to reduce the viscosity of actin filaments. It also caused the formation of bundles of parallel filaments. This observation and the stoichiometry of binding suggest that the receptor binds along the sides of actin filaments. Based on the ability of this receptor to bind both extracellular laminin and intracellular actin, we have named this protein "connectin." Connectin may be an example of a transmembrane protein that is capable of mediating the interaction of a cell with its extracellular matrix.     

Survival of Mammary Stem Cells in Suspension Culture: Implications for Stem Cell Biology and Neoplasia

There is increasing evidence that a variety of neoplasms including breast cancer may result from transformation of normal stem and progenitor cells. In the past, isolation and characterization of mammary stem cells has been limited by the lack of suitable culture systems able to maintain these cells in an undifferentiated state in vitro. We have recently described a culture system in which human mammary stem and progenitor cells are able to survive in suspension and produce spherical colonies composed of both stem and progenitor cells. Recent observation that adult stem cells from other tissues may also retain the capacity for growth under anchorage independent conditions suggests a common underlying mechanism. We propose that this mechanism involves the interaction between the canonical Wnt signal pathway and E-cadherin. The Wnt pathway has been implicated in normal stem cell self-renewal in vivo. Furthermore, there is evidence that deregulation of this pathway in the mammary gland and other organs may play a key role in carcinogenesis. Thus, the development of in vitro suspension culture systems not only provides an important new tool for the study of mammary cell biology, but also may have important implications for understanding key molecular pathways in both normal and neoplastic stem cells.     

Anticipating words and their gender: an event-related brain potential study of semantic integration, gender expectancy, and gender agreement in Spanish sentence reading

Recent studies indicate that the human brain attends to and uses grammatical gender cues during sentence comprehension. Here, we examine the nature and time course of the effect of gender on word-by-word sentence reading. Event-related brain potentials were recorded to an article and noun, while native Spanish speakers read medium- to high-constraint Spanish sentences for comprehension. The noun either fit the sentence meaning or not, and matched the preceding article in gender or not; in addition, the preceding article was either expected or unexpected based on prior sentence context. Semantically anomalous nouns elicited an N400. Gender-disagreeing nouns elicited a posterior late positivity (P600), replicating previous findings for words. Gender agreement and semantic congruity interacted in both the N400 window--with a larger negativity frontally for double violations--and the P600 window--with a larger positivity for semantic anomalies, relative to the prestimulus baseline. Finally, unexpected articles elicited an enhanced positivity (500-700 msec post onset) relative to expected articles. Overall, our data indicate that readers anticipate and attend to the gender of both articles and nouns, and use gender in real time to maintain agreement and to build sentence meaning.     

Attachment of neuroblastoma cells to extracellular matrix: correlation with metastatic capacity

Extracellular matrix (ECM) serves important attachment functions during organogenesis in mammalian species. When layered on a plastic surface, ECM extracted from the rat lung, liver, or kidney enhances the attachment of C1300 murine neuroblastoma cells to that surface. Enhanced attachment to ECM by these cells correlates with their potential to form metastatic deposits in vivo. Conversely, neuroblastoma cells selected for increased metastatic potential by in vivo passaging demonstrate enhanced attachment to organ-derived ECM. However, although ECM provides attachment sites for these murine neuroblastoma cells, the attachment is not preferential for any of the organ ECMs tested (lung, liver, kidney). Histopathologic examination of the murine liver, lungs, and kidneys performed 20 to 22 days after intravenous inoculation of C1300 cells reveals notable metastatic seeding in each of these organs, but the liver clearly exhibits a greater degree of replacement by tumor metastases than the lungs or the kidneys. Therefore, the data from the attachment assays, coupled with the histopathologic findings obtained after tumor inoculation, suggest that although the ability to attach to ECM correlates with metastatic potential, additional factors are important in determining the preferential pattern of metastatic disease observed in murine neuroblastoma.     

Targeting breast stem cells with the cancer preventive compounds curcumin and piperine

The cancer stem cell hypothesis asserts that malignancies arise in tissue stem and/or progenitor cells through the dysregulation or acquisition of self-renewal. In order to determine whether the dietary polyphenols, curcumin, and piperine are able to modulate the self-renewal of normal and malignant breast stem cells, we examined the effects of these compounds on mammosphere formation, expression of the breast stem cell marker aldehyde dehydrogenase (ALDH), and Wnt signaling. Mammosphere formation assays were performed after curcumin, piperine, and control treatment in unsorted normal breast epithelial cells and normal stem and early progenitor cells, selected by ALDH positivity. Wnt signaling was examined using a Topflash assay. Both curcumin and piperine inhibited mammosphere formation, serial passaging, and percent of ALDH+ cells by 50% at 5 μM and completely at 10 μM concentration in normal and malignant breast cells. There was no effect on cellular differentiation. Wnt signaling was inhibited by both curcumin and piperine by 50% at 5 μM and completely at 10 μM. Curcumin and piperine separately, and in combination, inhibit breast stem cell self-renewal but do not cause toxicity to differentiated cells. These compounds could be potential cancer preventive agents. Mammosphere formation assays may be a quantifiable biomarker to assess cancer preventive agent efficacy and Wnt signaling assessment can be a mechanistic biomarker for use in human clinical trials.     

Fetal expression of hemonectin: an extracellular matrix hematopoietic cytoadhesion molecule

Hemonectin, a component of bone marrow extracellular matrix, is a lineage- and organ-specific attachment molecule for cells of the granulocytic lineage. We hypothesized that hemonectin is an important marker of fetal granulopoiesis that is developmentally regulated during the ontogeny of the hematopoietic system. Murine hematopoiesis originates in the yolk sac and subsequently appears in the liver, spleen, and bone marrow. Using an affinity-purified polyclonal antibody to purified hemonectin as a probe of developing hematopoietic organs, we observe that hemonectin is coordinately expressed at developmental stages of the mouse in those tissues that are supporting hematopoiesis. Multiparameter flow cytometric analysis reveals that approximately 7% of fetal liver cells express hemonectin by day 13 of gestation, and that 32% of the cells are positive by day 19. Additionally, restricted hemonectin expression is noted in other tissues (cartilage, skin, developing bone, and capillary endothelial cells), suggesting that this molecule subserves other developmental functions and/or belongs to a previously unrecognized family of molecules.     

Extracellular matrix promotes the growth and differentiation of murine hematopoietic cells in vitro.

We have developed a long-term culture system in which murine marrow cells are cultured on a complex extracellular matrix (ECM) that is derived from marrow and extracted with guanidine hydrochloride and dithiothreitol. Marrow cultures were established with fresh murine marrow cells and recharged at 2 wk (week 0). Phase microscopy showed a dramatically increased adherent cell layer development on ECM compared with controls within a week after recharge. By electron microscopy, this adherent layer was composed of numerous reticular cells apparently attached to the ECM which extended cytoplasmic projections to the surrounding hematopoietic cells. Adherent cellularity on ECM-coated dishes increased to 30 times the control values by week 2. Cumulative suspension cells on ECM dishes were eight times controls. ECM influenced both hematopoietic progenitor cell proliferation and differentiation. Adherent colony-forming unit-granulocyte/macrophage and colony-forming unit-megakaryocyte were greater than 30 and 15 times the control values, respectively, by week 2 (P less than or equal to 0.05). There were more mature granulocytic and megakaryocytic cells in ECM-coated dishes than in controls at all time points. This new culture system directly demonstrates that ECM is an important component of the hematopoietic microenvironment.