Plastic Substratum Research Articles

Reversible and irreversible differentiation of cardiac fibroblasts

AimsDifferentiation of cardiac fibroblasts (Fbs) into myofibroblasts (MyoFbs) is responsible for connective tissue build-up in myocardial remodelling. We examined MyoFb differentiation and reversibility.Methods and resultsAdult rat cardiac Fbs were cultured on a plastic substratum providing mechanical stress, with conditions to obtain different levels of Fb differentiation. Fb spontaneously differentiated to proliferating MyoFb (p-MyoFb) with stress fibre formation decorated with alpha-smooth muscle actin (α-SMA). Transforming growth factor-β1 (TGF-β1) promoted differentiation into α-SMA-positive MyoFb showing near the absence of proliferation, i.e. non-p-MyoFb. SD-208, a TGF-β-receptor-I (TGF-β-RI) kinase blocker, inhibited p-MyoFb differentiation as shown by stress fibre absence, low α-SMA expression, and high proliferation levels. Fb seeded in collagen matrices induced no contraction, whereas p-MyoFb and non-p-MyoFb induced 2.5- and four-fold contraction. Fb produced little collagen but high levels of interleukin-10. Non-p-MyoFb had high collagen production and high monocyte chemoattractant protein-1 and tissue inhibitor of metalloproteinases-1 levels. Transcriptome analysis indicated differential activation of gene networks related to differentiation of MyoFb (e.g. paxilin and PAK) and reduced proliferation of non-p-MyoFb (e.g. cyclins and cell cycle regulation). Dedifferentiation of p-MyoFb with stress fibre de-polymerization, but not of non-p-MyoFb, was induced by SD-208 despite maintained stress. Stress fibre de-polymerization could also be induced by mechanical strain release in p-MyoFb and non-p-MyoFb (2-day cultures in unrestrained 3-D collagen matrices). Only p-MyoFb showed true dedifferentiation after long-term 3-D cultures.ConclusionsFb, p-MyoFb, and non-p-MyoFb have a distinct gene expression, ultrastructural, and functional profile. Both reduction in mechanical strain and TGF-β-RI kinase inhibition can reverse p-MyoFb differentiation but not non-p-MyoFb.

Comparative profiling of miRNA expression of lung adenocarcinoma cells in two-dimensional and three-dimensional cultures

Three-dimensional organotypic culture using reconstituted basement membrane matrix (rBM 3-D) is an invaluable tool to characterize morphogenesis of epithelial cells and to elucidate the tumor-modulating actions of extracellular matrix. microRNAs (miRNA) are a novel class of tumor modulating genes. A substantial amount of investigation of miRNAs in cancer is carried out using monolayer 2-D culture on plastic substratum, which lacks a consideration of the matrix-mediated regulation of miRNAs. In the current study we compared the expression of miRNAs in rBM 3-D and 2-D cultures of two lung adenocarcinoma cell lines. Our findings revealed a profound difference in miRNA profiles between 2-D and rBM 3-D cultures of lung adenocarcinoma cells. The rBM 3-D culture-specific miRNA profile was highlighted with higher expression of the tumor suppressive miRNAs (i.e., miR-200 family) and lower expression of the oncogenic miRNAs (i.e., miR-17–92 cluster and miR-21) than that of 2-D culture. Moreover, the expression pattern of miR-17, miR-21, and miR-200a in rBM 3-D culture correlated with the expression of their targets and acinar morphogenesis, a differentiation behavior of lung epithelial cells in rBM 3-D culture. Over-expression of miR-21 suppressed its target PTEN and disrupted acinar morphogenesis. In summary, we provide the first miRNA profile of lung adenocarcinoma cells in rBM 3-D culture with respect to acinar morphogenesis. These results indicate that rBM 3-D culture is essential to a comprehensive understanding of the miRNA biology in lung epithelial cells pertinent to lung adenocarcinoma.

Establishment and characterization of a buffalo (Bubalus bubalis) mammary epithelial cell line.

BackgroundThe objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions.MethodologyBuffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot.Principal FindingsThe established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of β-casein, κ-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n = 50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence.ConclusionsWe have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions.

Constructing Three-Dimensional Models to Study Mammary Gland Branching Morphogenesis and Functional Differentiation

Tissue organogenesis is directed by both intercellular interactions and communication with the surrounding microenvironment. When cells are cultured on two-dimensional plastic substrata (2D), important signals controlling programs of cell proliferation, metabolism, differentiation and death responsible for the formation of correct tissue-specific architecture and function are lost. Designing three-dimensional (3D), physiologically relevant culture models, we can recapitulate some crucial aspects of the dynamic and reciprocal signaling necessary for establishing and maintaining tissue specific morphogenic programs. Here we briefly describe the details of robust methods for culturing mouse primary mammary organoids in 3D gels of different extracellular matrices and describe techniques for analyzing the resulting structures. These designer microenvironments are useful for both understanding branching morphogenesis and signaling integrations, but also for analysis of individual susceptibilities and drug testing.

The microRNA expression associated with morphogenesis of breast cancer cells in three-dimensional organotypic culture

Three-dimensional organotypic culture using reconstituted basement membrane matrix Matrigel (rBM 3-D) is an indispensable tool to characterize morphogenesis of mammary epithelial cells and to elucidate the tumor-modulating actions of extracellular matrix (ECM). microRNAs (miRNAs) are a novel class of oncogenes and tumor suppressors. The majority of our current knowledge of miRNA expression and function in cancer cells is derived from monolayer 2-D culture on plastic substratum, which lacks consideration of the influence of ECM-mediated morphogenesis on miRNAs. In the present study, we compared the expression of miRNAs in rBM 3-D and 2-D cultures of the non-invasive MCF-7 and the invasive MDA-MB231 cells. Our findings revealed a profound difference in miRNA profiles between 2-D and rBM 3-D cultures within each cell type. Moreover, rBM 3-D culture exhibited greater discrimination in miRNA profiles between MCF-7 and MDA-MB231 cells than 2-D culture. The disparate miRNA profiles correlated with distinct mass morphogenesis of MCF-7 and invasive stellate morphogenesis of MDA-MB231 cells in rBM 3-D culture. Supplementation of the tumor promoting type I collagen in rBM 3-D culture substantially altered the miRNA signature of mass morphologenesis of MCF-7 cells in rBM 3-D culture. Overexpression of the differentially expressed miR-200 family member miR429 in MDA-MB231 cells attenuated their invasive stellate morphogenesis in rBM 3-D culture. In summary, we provide the first miRNA signatures of morphogenesis of human breast cancer cells in rBM 3-D culture and warrant further utilization of rBM 3-D culture in investigation of miRNAs in breast cancer.

Defective signaling in a spontaneous transformation model of rat prostate epithelial cells

We describe an in vitro model of spontaneous transformation using normal rat prostate epithelial cells (PEC) that culminates at high passage (p>85) in anchorage independent growth when plated on soft agar. High passage PEC also formed large tumors in immunodeficient mice. We identified a small subpopulation of cells derived from the high passage anchorage independent cells that migrate through soft agar and expand on the underlying plastic substratum. In addition, several antibodies generated against rat liver epithelial cells of ductal origin that react strongly with rat prostate cells were examined. OC.2 was expressed by a low percentage of low and high passage PEC but was expressed by 100% of freshly isolated soft agar invasive (SAI) cells. Conversely, OC.4 and OC.5 were expressed by a high percentage of low and high passage PEC but were absent in SAI cells. Tumorigenicity experiments demonstrated that the SAI subpopulation was considerably more tumorigenic than high passage cells, evidenced by decreased latency period and increased tumor size. Immunoblot analysis identified alterations in signaling proteins belonging to EGFR and JAK/STAT pathways between low pass, high pass and SAI cells. We are investigating the role of these pathways on the phenotype of the 3 PEC models by examining anchorage independent growth, invasion, and tumorigenicity. (Supported by COBRE grant 1P20RR17695)

Coral larvae settle at a higher frequency on red surfaces

Although chemical cues serve as the primary determinants of larval settlement and metamorphosis, light is also known to influence the behavior and the settlement of coral planulae. For example, Porites astreoides planulae settle preferentially on unconditioned red substrata. In order to test whether this behavior was a response to color and whether other species also demonstrate color preference, settlement choice experiments were conducted with P. astreoides and Acropora palmata. In these experiments, larvae were offered various types of plastic substrata representing three to seven different color choices. Both species consistently settled on red (or red and orange) substrata at a higher frequency than other colors. In one experiment, P. astreoides settled on 100% of red, plastic cable ties but failed to settle on green or white substrata. In a second experiment, 24% of larvae settled on red buttons, more than settled on six other colors combined. A. palmata settled on 80% of red and of orange cables ties but failed to settle on blue in one experiment and settled on a greater proportion of red acrylic squares than on four other colors or limestone controls in a second experiment. The consistency of the response across a variety of plastic materials suggests the response is related to long-wavelength photosensitivity. Fluorescence and reflectance spectra of experimental substrata demonstrated that the preferred substrata had spectra dominated by wavelengths greater than 550 nm with little or no reflection or emission of shorter wavelengths. These results suggest that some species of coral larvae may use spectral cues for fine-scale habitat selection during settlement. This behavior may be an adaptation to promote settlement in crustose coralline algae (CCA)-dominated habitats facilitating juvenile survival.

Characteristics and EGFP Expression of Goat Mammary Gland Epithelial Cells

The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing.

Initial Development of Marine Assemblages on Woody and Plastic (Polypropylene) Substrata

Initial Development of Marine Assemblages on Woody and Plastic (Polypropylene) Substrata

Role of Proteins and Water in the Initial Attachment of Mammalian Cells to Biomedical Surfaces: A Review

Anchorage-dependent mammalian cells are typically grown in vitro on hydrophilic glass and plastic substrata in a medium supplemented with 5–20% v/v blood-serum proteins. Inoculated single cells gravitate from suspension to within close proximity of substrata surfaces whereupon initial contact and attachment occurs followed by progressive cell adhesion, spreading, and ultimately proliferation. A critical examination of the role of proteins and water in the initial attachment phase concludes that the cell attachment phase is not mediated by biological recognition of surface-adsorbed ligands by cell membrane receptors as frequently depicted in various textbook explanations of cell adhesion. This conclusion is based on extensive experimental evidence showing that blood proteins do not adsorb on hydrophilic surfaces that are most conducive to cell growth but do adsorb on hydrophobic surfaces that are not conducive to cell growth. As a consequence, the conventional idea that initial cell attachment is mediated by various adhesin factors adsorbed from serum-protein solutions is viewed as untenable. Rather, it is concluded that the initial contact-and-attachment of cells to hydrophilic surfaces is controlled by physicochemical interactions unrelated to biological recognition. The general physics of these interactions is known but an adequate descriptive theory that can be tested against experimentally measured cell adhesion kinetics has yet to be developed. The role of these physicochemical interactions in stimulating biological machinery within cells to fully adhere and proliferate on surfaces of biotechnical interest is unknown but is of great significance to the science underlying various biomedical and biotechnical applications of materials.

Laminin‐1 induces E‐cadherin expression in 3‐dimensional cultured breast cancer cells by inhibiting DNA methyltransferase 1 and reversing promoter methylation status

This study determines the role of laminin-1 in promoting metastatic colonization during breast cancer. For this purpose, human mammary epithelial cell lines representing normal (MCF-10A), adenocarcinoma (MCF-7), and malignant carcinoma (MDA-MB-231) were propagated in 3-dimensional cultures composed of laminin-1, collagen I, or mixtures of the two, and analyzed by Western blot, immunocytochemistry, semiquantitative reverse transcription polymerase chain reaction, and methylation-specific PCR. Here we demonstrate that laminin-1 decreases methylation of the E-cadherin promoter, resulting in increased mRNA and protein expression for malignant mammary epithelial cells. This decreased methylation is associated with dramatic changes in the cellular and structural morphology as well as a 70-fold decrease in DNA methyltransferase 1 (DNMT1) and a 6-fold decrease in cadherin 11 protein expression. To control for specificity of laminin-1 interactions, cells were also cultured on 2-dimensional plastic substrata and collagen I hydrogels for analysis, and the MCF-10A and MCF-7 were used as nonmalignant controls. Using a 3-dimensional model, we present evidence that laminin-1 is capable of inducing epigenetic change by inhibiting expression of DNMT1 and preventing methylation of the E-cadherin promoter, resulting in E-cadherin expression and the formation of cell-cell bonds in malignant carcinoma.

Topographical control of prostate cancer cell migration

Tumour cells can efficiently respond to numerous factors affecting their motility. However, the role of substrata topography in the regulation of cancer cell motility has been quantitatively studied in only a few cases. We demonstrated that human (DU-145) and rat (MAT-LyLu and AT-2) prostate cancer cells are efficiently contact guided by underlying normal cells when invading surrounding tissues and forming metastases. Prostate cancer cells moving on the surface of fibroblasts displayed significantly greater cell displacement than those moving on plastic substrata. This effect was correlated with the polarization (contact guidance) and increased speed of cell movements. We subsequently verified the hypothesis that the observed contact guidance of prostate cancer cells migrating on the surface of fibroblasts results from their reaction to the microtopography of normal cells. The responses of cells to multiple grooved substrata of a size corresponding to the dimensions of a compact monolayer culture of human skin fibroblasts were studied, and the migration of prostate cancer cells appeared to be efficiently affected by topographical features of the substratum. In contrast to random movement under control conditions, all investigated prostate cancer cell lines grown on patterned substrata migrated mainly along artificial grooves and covered, as a result of contact guidance, a longer distance than cells on plain substrata. Moreover, the reaction to microtopography was correlated with the metastatic activity of prostate cancer cells. In conclusion, our results show that grooved substrata have a substantial effect on prostate cancer migration. Since all types of tissue show some kind of patterning and alignment, topographic factors may be crucial for the effective migration of prostate cancer cells during the metastatic process.

Bombyx moriSilk Fibroin Membranes as Potential Substrata for Epithelial Constructs Used in the Management of Ocular Surface Disorders

Membranes were prepared from fibroin, a protein isolated from the domesticated silkworm (Bombyx mori) silk, and evaluated as a potential substratum for corneal limbal epithelial cells. These membranes (i.e., B. mori silk fibroin [BMSF] membranes) were cast from dialyzed solutions of fibroin protein (4% w/v) dispensed into 35-mm-diameter culture dishes and dried at room temperature (23-24 degrees C). The resulting material was transparent, easy to handle, and supported levels of human limbal epithelial (HLE) cell growth comparable to that observed on tissue culture plastic. Remarkably, these results were obtained utilizing a commercial serum-free medium (CnT-20) designed for the ex vivo expansion of corneal epithelial progenitor cells. The potential benefits of serum proteins on this culture system were examined through addition of fetal bovine serum (FBS) either to fibroin stocks prior to membrane casting or by supplementation of the CnT-20 medium. Membranes cast in the presence of FBS displayed increasing opacity and induced little change in HLE growth. Supplementation of CnT-20 medium with FBS deterred cell growth on all substrata, including tissue culture plastic control substrata. The remarkable properties of BMSF membranes demonstrated under serum-free conditions warrant investigation of this material as a substratum in the creation of tissue-engineered constructs for the restoration of diseased or damaged ocular surface.

Cell shape regulates global histone acetylation in human mammary epithelial cells

Extracellular matrix (ECM) regulates cell morphology and gene expression in vivo; these relationships are maintained in three-dimensional (3D) cultures of mammary epithelial cells. In the presence of laminin-rich ECM (lrECM), mammary epithelial cells round up and undergo global histone deacetylation, a process critical for their functional differentiation. However, it remains unclear whether lrECM-dependent cell rounding and global histone deacetylation are indeed part of a common physical–biochemical pathway. Using 3D cultures as well as nonadhesive and micropatterned substrata, here we showed that the cell ‘rounding’ caused by lrECM was sufficient to induce deacetylation of histones H3 and H4 in the absence of biochemical cues. Microarray and confocal analysis demonstrated that this deacetylation in 3D culture is associated with a global increase in chromatin condensation and a reduction in gene expression. Whereas cells cultured on plastic substrata formed prominent stress fibers, cells grown in 3D lrECM or on micropatterns lacked these structures. Disruption of the actin cytoskeleton with cytochalasin D phenocopied the lrECM-induced cell rounding and histone deacetylation. These results reveal a novel link between ECM-controlled cell shape and chromatin structure and suggest that this link is mediated by changes in the actin cytoskeleton.

Three-dimensional culture models of normal and malignant breast epithelial cells

Extracellular matrix is a key regulator of normal homeostasis and tissue phenotype. Important signals are lost when cells are cultured ex vivo on two-dimensional plastic substrata. Many of these crucial microenvironmental cues may be restored using three-dimensional (3D) cultures of laminin-rich extracellular matrix (lrECM). These 3D culture assays allow phenotypic discrimination between nonmalignant and malignant mammary cells, as the former grown in a 3D context form polarized, growth-arrested acinus-like colonies whereas the latter form disorganized, proliferative and nonpolar colonies. Signaling pathways that function in parallel in cells cultured on plastic become reciprocally integrated when the cells are exposed to basement membrane-like gels. Appropriate 3D culture thus provides a more physiologically relevant approach to the analysis of gene function and cell phenotype ex vivo. We describe here a robust and generalized method for the culturing of various human breast cell lines in three dimensions and describe the preparation of cellular extracts from these cultures for molecular analyses. The procedure below describes the 3D 'embedded' assay, in which cells are cultured embedded in an lrECM gel (Fig. 1). By lrECM, we refer to the solubilized extract derived from the Engelbreth-Holm-Swarm mouse sarcoma cells. For a discussion of user options regarding 3D matrices, see Box 1. Alternatively, the 3D 'on-top' assay, in which cells are cultured on top of a thin lrECM gel overlaid with a dilute solution of lrECM, may be used as described in Box 2 (Fig. 1 and Fig. 2).

PRL3 Promotes Cell Invasion and Proliferation by Down-regulation of Csk Leading to Src Activation

Phosphatase of regenerating liver 3 (PRL3) is overexpressed in a variety of tumors, and high levels of PRL3 expression are associated with tumorigenesis and metastasis. Consistent with an oncogenic role for PRL3, we show that ectopic PRL3 expression promotes cell proliferation and invasion. However, little is known about the molecular basis for PRL3 function. Obtaining this knowledge is vital for understanding PRL3-mediated disease processes and for the development of novel anticancer therapies targeted to PRL3. Here we report that up-regulation of PRL3 activates the Src kinase, which initiates a number of signal pathways culminating in the phosphorylation of ERK1/2, STAT3, and p130(Cas). The activation of these pathways likely contributes to the increased cell growth and motility of PRL3 cells. We provide evidence that PRL3 induces Src activation through down-regulation of Csk, a negative regulator of Src. Importantly, Src activation and Csk down-regulation are also observed in colon cancer cells expressing a higher level of PRL3. Thus, we have revealed a biochemical mechanism for the PRL3-mediated cell invasion and proliferation in which elevated PRL3 expression causes a reduction in Csk level, leading to Src activation.

RCE1 Corneal Epithelial Cell Line: Its Variability on Phenotype Expression and Differential Response to Growth Factors

By serial transfer of rabbit corneal epithelial cells, the spontaneous RCE1 cell line was previously established. These cells mimic the stage-dependent differentiation of the corresponding cell type. RCE1 cells were cultured either on plastic culture dishes or on collagen rafts to compare the epithelial ultrastructure after growth on these substrata. Phenotypic variability was studied after subcloning of cells. The differentiation ability of each subclone was determined by Western blot with antibodies against the differentiation-linked keratin pair K3/K12 and by measuring LDH activity and LDH isozymes in cytosolic extracts. The proliferative response of RCE1 cells to EGF, TGFalpha, amphiregulin, bFGF or IL-6 was determined under serum-free culture conditions. Cells grown on collagen rafts formed 5- to 7-layered epithelia with characteristics closer to those found in normal corneal epithelium than cells cultivated on plastic substrata, which formed 3- to 5-layered epithelia. Subcloning experiments demonstrated that every proliferative cell is able to grow and constitute stratified epithelia expressing K3/K12 keratins. LDH levels in RCE1 epithelia were similar to those of cultured or freshly harvested corneal epithelia; however, they showed a slightly altered LDH isozyme set, with prevalence of LDH-3 isoform. Whereas EGF and TGF-alpha were equipotent, amphiregulin elicited a 4-fold lower proliferative response. Also, bFGF was 10-fold less mitogenic than EGF, and IL-6 had the lowest effect with an ED(50) 20-fold lower than EGF. The results demonstrate that every RCE1 proliferative cell has the ability to generate epithelial sheets. We conclude that EGF and TGF-alpha are the major effectors of RCE1 cell proliferation.

Establishment and characterization of a spontaneously immortalized porcine mammary epithelial cell line

We have established a spontaneously immortalized porcine mammary epithelial cell line (SI-PMEC) from the mammary gland of a lactating sow and maintained it long-term in culture by continuous subculturing. SI-PMEC cells were maintained for more than 8 months (70 passages) in DMEM/F12 medium supplemented with 10% fetal calf serum, insulin, and hydrocortisone without obvious signs of senescence. When grown at low density on a plastic substratum, SI-PMEC cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobblestone morphology of epithelial cells. The subcultured SI-PMEC cells appeared to proliferate without changes in morphology or growth pattern, with an estimated population doubling time of 20-22 h. With increasing density, SI-PMEC cells organized into lumen-like structures with elongated cells at the margins. SI-PMEC cells from stocks frozen at Passage 30 were subcultured up to 20 times without changes in cell viability, proliferation rate, or morphology. Furthermore, SI-PMEC cells remained immunopositive to an antibody against cytokeratin AE3 and immunonegative to an antibody against a human fibroblast antigen. The SI-PMEC cells could form functional structures resembling ducts, lateral-buds, and alveoli in a Matrigel matrix-dependent manner in vitro. When grown on the Matrigel and stimulated by prolactin, the cells differentiated and formed mammary gland structures and strongly expressed transcripts encoding the milk proteins alpha-lactalbumin, beta-casein, and beta-lactoglobulin. Our results indicate that the SI-PMEC cell line can be subpassaged many times and still form functional differentiated secretory structures. To our knowledge, this is the first report of an immortalized mammary epithelial cell line from pig.

Competition between bound and free peptides in an ELISA-based procedure that assays peptides derived from protein digests

BackgroundWe describe an ELISA-based method that can be used to identify and quantitate proteins in biological samples. In this method, peptides in solution, derived from proteolytic digests of the sample, compete with substrate-attached synthetic peptides for antibodies, also in solution, generated against the chosen peptides. The peptides used for the ELISA are chosen on the basis of their being (i) products of the proteolytic (e.g. tryptic) digestion of the protein to be identified and (ii) unique to the target protein, as far as one can know from the published sequences.ResultsIn this paper we describe the competition assay and we define the optimal conditions for the most effective assay. We have performed an analysis of the kinetics of interaction between the four components of the assay: the plastic substratum to which the peptide is bound, the bound peptide itself, the competing added peptide, and the antibody that is specific for the peptide and we compare the results of theoretical simulations to the actual data in some model systems.ConclusionThe data suggest that the peptides bind to the plastic substratum in more than one conformation and that, once bound, the peptide displays different affinities for the antibody, depending on how it has bound to the plate

Contact stimulation of prostate cancer cell migration: the role of gap junctional coupling and migration stimulated by heterotypic cell‐to‐cell contacts in determination of the metastatic phenotype of Dunning rat prostate cancer cells

Motile activity of tumour cells is regarded as a critical factor determining their metastatic potential. We have shown previously that contrary to the majority of normal cells, homotypic contacts between some tumour cells, among them low metastatic (AT-2) and highly metastatic (MAT-LyLu) rat prostate cancer cells, increase the speed of their movements. The aim of the present study was to determine the effect of heterotypic cell-to-cell contacts on the migration of rat prostate cancer cells differing in metastatic potential, and to correlate it with the intensity of homo- and heterologous gap junctional coupling. MAT-LyLu and AT-2 cells moving on the surface of fibroblasts displayed significantly greater cell displacement than those moving on plastic substrata. This effect correlated with the polarization (contact guidance) and increased speed of cell movements. However, in contrast with the migration on plastic substrata, where MAT-LyLu cells displayed considerably higher motility than AT-2 cells, no differences between both cell lines were observed on the surface of fibroblasts. On the other hand, in contrast with AT-2, Mat-LyLu cells displayed extensive homologous coupling mediated by connexin43 and were able to couple with normal fibroblasts. Heterotypic contacts between migrating prostatic cancer cells and normal fibroblasts can strongly stimulate their migration during invasion; however, this effect does not correlate with the gap junctional coupling between cancer cells and normal fibroblasts.