RESUMEN
This study investigated whether micron-sized microspheres can be used as dispersed scaffolds where anchorage-dependent cells can proliferate and survive in suspension culture. Aggregates of murine 3T3 cells in a non-adherent plate cultured remained viable for more than 2 weeks by the presence of 0.5 mg/ml fibroin microspheres. A nucleoside incorporation assay confirmed the proliferation of 3T3 cells in the aggregates only when cultured with microspheres. Under these conditions, the glucose consumption rate of 3T3 cells increased to 66.5 nmol day-1 cell-1. Histological analysis demonstrated that the intercellular space of cell aggregates was larger in cultures supplemented with 0.5 mg/ml microspheres than in non-supplemented cultures. The cell aggregates with microspheres also exhibited a reduced arrest in G1 phase. Transmission electron microscopy verified the presence of microspheres in the space between cells in aggregates. Fibroin microspheres maintained the viability and proliferability of 3T3 cells cultured under non-adherent conditions and thus can be used to develop viable suspensions of anchorage-dependent cells.
RESUMEN
Epithelial cells respond to their physical neighborhood with mechano-sensitive behaviors required for development and tissue maintenance. These include anchorage dependence, matrix stiffness-dependent proliferation, contact inhibition of proliferation and migration, and collective migration that balances cell crawling with the maintenance of cell junctions. While required for development and tissue repair, these coordinated responses to the microenvironment also contribute to cancer metastasis. Predictive models of the signaling networks that coordinate these behaviors are critical in controlling cell behavior to halt disease. Here we propose a Boolean regulatory network model that synthesizes mechanosensitive signaling that links anchorage to a matrix of varying stiffness and cell density sensing to contact inhibition, proliferation, migration, and apoptosis. Our model can reproduce anchorage dependence and anoikis, detachment-induced cytokinesis errors, the effect of matrix stiffness on proliferation, and contact inhibition of proliferation and migration by two mechanisms that converge on the YAP transcription factor. In addition, we offer testable predictions related to cell cycle-dependent anoikis sensitivity, the molecular requirements for abolishing contact inhibition, and substrate stiffness dependent expression of the catalytic subunit of PI3K. Moreover, our model predicts heterogeneity in migratory vs. non-migratory phenotypes in sub-confluent monolayers, and co-inhibition but semi-independent induction of proliferation vs. migration as a function of cell density and mitogenic stimulation. Our model serves as a stepping-stone towards modeling mechanosensitive routes to the epithelial to mesenchymal transition, capturing the effects of the mesenchymal state on anoikis resistance, and understanding the balance between migration versus proliferation at each stage of the epithelial to mesenchymal transition.
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BACKGROUND: Hormone therapy and chemotherapy are not effective for castrate-resistant prostate cancer, thus development of novel treatment strategies is required. Gene therapy involving transient high-copy transfection of interleukin (IL)-24 with an adenoviral vector can exert antitumor activity; however, the effects of stable IL-24 transfection are not fully understood. The aim of this study was to investigate the effects of IL-24 overexpression in prostate cancer cells, in vitro. MATERIALS AND METHODS: DU145 cells were transfected the IL-24 gene using a retroviral vector. Apoptosis induction was investigated by the cell death detection ELISA, and the gene expression was analyzed by real time RT-PCR. RESULTS: IL-24 transduction suppressed the growth of prostate cancer and induced tumor cell apoptosis. In addition, up-regulation of epithelial markers and down-regulation of mesenchymal markers were noted, suggesting that tumor aggressiveness was reduced. CONCLUSION: Introduction of IL-24 displays antitumor activity both by induction of apoptosis and regulation of anchorage dependence.
Asunto(s)
Interleucinas/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Apoptosis , Proliferación Celular , Humanos , Masculino , Transducción GenéticaRESUMEN
Cancer cells have an elevated methionine (MET) requirement compared to normal cells and are termed MET dependent. Cancer cells were isolated in MET-restricted (MR) medium that reverted from MET dependence to MET independence. Increased MET biosynthesis was not a prerequisite for reversion to MET independence, indicating that MET dependence was not due to reduced endogenous MET synthesis. MET-independent revertants of cancer cells concomitantly reverted for some of the other properties associated with malignancy: Of the 13 MET-independent revertants isolated 5 showed increased anchorage dependence as reflected by reduced cloning efficiencies in methylcellulose; 8 showed an increased serum requirement for optimal growth; 8 showed decreased cell density in medium containing high serum; and 3 altered their cell morphology significantly. Eight of the 13 revertants have increased chromosome numbers. Thus, by selecting for MET independence, it is possible to obtain heterogeneous reduced-malignancy revertants, indicating further a relationship between altered MET metabolism and other fundamental properties of oncogenic transformation.
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Neoplasias/patología , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Humanos , Metionina , Metilación , Neoplasias/enzimología , RatasRESUMEN
Adhesion of epithelial cell to each other and to extracellular matrix, as well as cell migration ability and cytoskeleton organization undergo significant alterations in the course of neoplastic transformation, but regulatory mechanisms involved in these processes are not fully understood. Here, we studied the role of a Rho GAP protein GRAF1 (GTPase Regulator Associated with Focal adhesion kinase-1) in the regulation of the epithelial phenotype in cells of breast derived, non-malignant, MCF10A cell line. GRAF1 was shown to be localized to cell-cell junctions, and its depletion resulted in accelerated cell migration velocity, elongation of the cells and cell colonies, impaired monolayer integrity and significant disruption of desmosomes with a loss of associated keratin filaments. These processes were accompanied by formation of larger focal adhesions, an increased number of contractile actin stress fibers, reduction in epithelial markers and increase in mesenchymal markers such as epithelial-mesenchymal transition (EMT)-specific transcription factors Snail-1 and Snail-2, as well as N-cadherin, and vimentin. Moreover, unlike control cells, GRAF1 knocked-down cells demonstrated anchorage-independent growth in soft agar. GRAF1 expression in several highly invasive breast cancer cell lines was low, as compared to the non-malignant MCF10A cells, while overexpressing of GRAF1 in the malignant BT-549 cell line led to a decrease of mesenchymal markers, especially the Snail-1 and 2. Altogether, our analysis suggests that GRAF1 plays a role in the maintenance of normal epithelial phenotype and its depletion leads to an EMT-like process that might be involved in neoplastic transformation.