Summary. Upon injury to a vessel wall the exposure of subendothelial collagen results in the activation of platelets. Platelet activation culminates in shape change, aggregation, release of granule contents and generation of lipid mediators. These secreted and generated mediators trigger a positive feedback mechanism potentiating the platelet activation induced by physiological agonists such as collagen and thrombin. Adenine nucleotides, adenosine diphosphate (ADP) and adenosine triphosphate (ATP), released from damaged cells and that are secreted from platelet-dense granules, contribute to the positive feedback mechanism by acting through nucleotide receptors on the platelet surface. ADP acts through two G protein-coupled receptors, the G qcoupled P2Y 1 receptor, and the G i -coupled P2Y 12 receptor. ATP, on the other hand, acts through the ligand-gated channel P2X 1 . Stimulation of platelets by ADP leads to shape change, aggregation and thromboxane A 2 generation. ADP-induced dense granule release depends on generated thromboxane A 2 . Furthermore, costimulation of both P2Y 1 and P2Y 12 receptors is required for ADP-induced platelet aggregation. ATP stimulation of P2X 1 is involved in platelet shape change and helps to amplify platelet responses mediated by agonists such as collagen. Activation of each of these nucleotide receptors results in unique signal transduction pathways that are important in the regulation of thrombosis and hemostasis.

Robust in vitro lung models are required for risk assessment to measure key events leading to respiratory diseases. Primary normal human bronchial epithelial cells (NHBE) represent a good lung model but obtaining well-differentiated 3D cultures can be challenging. Here, we evaluated the ability to expand primary NHBE cells in different culture conditions while maintaining their 3D culture characteristics such as ciliated and goblet cells, and ion channel function. Differentiated cultures were optimally obtained with PneumaCult-Ex Plus (expansion medium)/PneumaCult-ALI (differentiation medium). Primary cells passaged up to four times maintained airway epithelial characteristics as evidenced by ciliated pseudostratified columnar epithelium with goblet cells, trans-epithelial electrical resistance (TEER) (>400 Ohms.cm2), and cystic fibrosis transmembrane conductance regulator-mediated short-circuit currents (>3 µA/cm2). No change in ciliary beat frequency (CBF) or airway surface liquid (ASL) meniscus length was observed up to passage six. For the first time, this study demonstrates that CFTR ion channel function and normal epithelial phenotypic characteristics are maintained in passaged primary NHBE cells. Furthermore, this study highlights the criticality of evaluating expansion and differentiation conditions for achieving optimal phenotypic and functional endpoints (CBF, ASL, ion channel function, presence of differentiated cells, TEER) when developing in vitro lung models.

Synthesis of certain members of the tropomyosin family of microfilament-associated proteins is suppressed in fibroblasts neoplastically transformed by a number of retroviral oncogenes, by transforming growth factor a, and by chemical mutagens. To test whether tropomyosin suppression is a required event in neoplastic transformation, expression of one of two suppressed tropomyosins in NIH 3T3 mouse cells transformed by the ras oncogene was restored by retrovirally mediated cDNA transfer. Cells expressing the inserted cDNA showed partial restoration of microfflament bundle formation (which is typicafly deranged in transformed cells) together with increased cytoplasmic spreading. More importantly, they lost anchorage-independent growth capability, and the onset of tumor growth in athymic mice was delayed. When tumors arose they no longer expressed the inserted cDNA. These observations support the conclusion that tropomyosin suppression is a necessary event for the expression of components of the transformed phenotype, particularly with respect to anchorage-independent growth and tumorigenesis, which correlate closely with neoplastic potential. This potentially reversible requirement may link different initial events produced by a variety of oncogenic modalities to a common pathway leading to neoplastic growth.Normal cells may be transformed in culture to the neoplastic state by any of a number of retroviral or activated oncogenes alone or in combination (1-4), by transforming growth factors (5, 6), or by chemical mutagens (7,8). Transformation of fibroblasts by many of these modalities is commonly accompanied by reduced synthesis of various actin-binding proteins, of which tropomyosins (TMs) (9-14), vinculin (15), gelsolin (16), and a-actinin (17) are examples. Consistent loss of expression of specific TM isoforms has also been found in cells cultured from human breast cancer (18). TMs and the other proteins named are essential structural and functional components of the actin-microfilament system of the cell (19-24), which has long been known to be severely disrupted in transformed cells (25,26).In the present study, to clarify whether TM suppression is a necessary event in the causal chain leading to neoplastic transformation, we have examined the effect of overcoming the deficiency of TM synthesis in retrovirally transformed cells (DT cells) through expression of a cDNA encoding the TM1 isoform. DT cells are NIH 3T3 mouse fibroblasts transformed by the v-Ki-ras oncogene and contain two integrated copies of the oncogene (27). They are highly tumorigenic in athymic mice, manifest anchorage-independent growth, and rarely produce spontaneous revertants. Of the five major TM isoforms expressed in NIH 3T3 cells, TM1 is suppressed by 50% and TM2 is almost absent in DT cells, whereas expression of the low molecular weight isoforms TM4 and TM5 is unaltered (9, 10). Nontransformed revertants of DT induced by chemical mutagens reexpress TM1The publication costs of this article were defrayed in part by page charge pa...

Suppression of tropomyosins (TMs), a family of actinbinding, microfilament-associated proteins, is a prominent feature of many transformed cells. Yet it is unclear whether downregulation of TMs occur in human tumors. We have investigated the expression of tropomyosin-1 (TM1) in human breast carcinoma tissues by in situ hybridization and immunofluorescence. TM1 mRNA and protein are readily detectable in normal mammary tissue. In contrast, TM1 expression is abolished in the primary human breast tumors. Expression of other TM isoforms, however, is variable among the tumors. The consistent and profound downregulation of TM1 suggests that TM1 may be a novel and useful biomarker of mammary neoplasms. These data also support the hypothesis that suppression of TM1 expression during the malignant conversion of mammary epithelium as a contributing factor of breast cancer. In support of this hypothesis, we show that the ability to suppress malignant growth properties of breast cancer cells is specific to TM1 isoform. Investigations into the mechanisms of TM1-induced tumor suppression reveal that TM1 induces anoikis (detachment induced apoptosis) in breast cancer cells. Downregulation of TM1 in breast tumors may destabilize microfilament architecture and confer resistance to anoikis, which facilitates survival of neoplastic cells outside the normal microenvironment and promote malignant growth.

Two most common properties of malignant cells are the presence of aberrant actin cytoskeleton and resistance to anoikis. Suppression of several key cytoskeletal proteins, including tropomyosin-1 (TM1), during neoplastic transformation is hypothesized to contribute to the altered cytoskeleton and neoplastic phenotype. Using TM1 as a paradigm, we have shown that cytoskeletal proteins induce anoikis in breast cancer (MCF-7 and MDA MB 231) cells. Here, we have tested the hypothesis that TM1-mediated cytoskeletal changes regulate integrin activity and the sensitivity to anoikis. TM1 expression in MDA MB 231 cells promotes the assembly of stress fibers, induces rapid anoikis via caspase-dependent pathways involving the release of cytochrome c. Further, TM1 inhibits binding of MDA MB 231 cells to collagen I, but promotes adhesion to laminin. Inhibition of Rho kinase disrupts TM1-mediated cytoskeletal reorganization and adhesion to the extracellular matrix components, whereas the parental cells attach to collagen I, spread and form extensive actin meshwork in the presence of Rho kinase inhibitor, underscoring the differences in parental and TM1-transduced breast cancer cells. Further, treatment with the cytoskeletal disrupting drugs rescues the cells from TM1-induced anoikis. These new findings demonstrate that the aberrant cytoskeleton contributes to neoplastic transformation by conferring resistance to anoikis. Restoration of stress fiber network through enhanced expression of key cytoskeletal proteins may modulate the activity of focal adhesions and sensitize the neoplastic cells to anoikis.