Establishment and characterization of SV40 large T antigen-immortalized cell lines derived from fetal bovine brain tissues after prolonged cryopreservation

Bovine brain cell lines with specific characteristics are useful in vitro experimental systems for molecular and cellular investigation of the interactions between bovine specific neuropathogenic agents and the host. Here, we established two novel cell lines from cultures of cryopreserved fetal bovine brain tissue by the transfection of SV40 large T antigen. Both cell lines showed cobblestone morphology in DMEM/F12 medium supplemented with 10% fetal bovine serum, epidermal growth factor and basic fibroblast growth factor. They were immunostained with endothelial marker, Von Willebrand Factor. Endothelial properties, such as capillary-like tube formation on matrigel and the incorporation of DiI-AcLDL were confirmed with these cells. Removal of growth factors increased the number of cells expressing alpha-smooth muscle actin, suggesting the potential of these cell lines to differentiate into smooth muscle cells. This study suggests an efficient protocol to immortalize brain endothelial cell lines from fetal bovine brain tissue culture.     

Cellular lifespan and senescence: a complex balance between multiple cellular pathways

The study of cellular senescence and proliferative lifespan is becoming increasingly important because of the promises of autologous cell therapy, the need for model systems for tissue disease and the implication of senescent cell phenotypes in organismal disease states such as sarcopenia, diabetes and various cancers, among others. Here, we explain the concepts of proliferative cellular lifespan and cellular senescence, and we present factors that have been shown to mediate cellular lifespan positively or negatively. We review much recent literature and present potential molecular mechanisms by which lifespan mediation occurs, drawing from the fields of telomere biology, metabolism, NAD⁺ and sirtuin biology, growth factor signaling and oxygen and antioxidants. We conclude that cellular lifespan and senescence are complex concepts that are governed by multiple independent and interdependent pathways, and that greater understanding of these pathways, their interactions and their convergence upon specific cellular phenotypes may lead to viable therapies for tissue regeneration and treatment of age‐related pathologies, which are caused by or exacerbated by senescent cells in vivo.     

The Role of Endocytosis during Morphogenetic Signaling

Morphogens are signaling molecules that are secreted by a localized source and spread in a target tissue where they are involved in the regulation of growth and patterning. Both the activity of morphogenetic signaling and the kinetics of ligand spreading in a tissue depend on endocytosis and intracellular trafficking. Here, we review quantitative approaches to study how large-scale morphogen profiles and signals emerge in a tissue from cellular trafficking processes and endocytic pathways. Starting from the kinetics of endosomal networks, we discuss the role of cellular trafficking and receptor dynamics in the formation of morphogen gradients. These morphogen gradients scale during growth, which implies that overall tissue size influences cellular trafficking kinetics. Finally, we discuss how such morphogen profiles can be used to control tissue growth. We emphasize the role of theory in efforts to bridge between scales.A fundamental challenge in biology is to understand how morphologies and complex patterns form in multicellular systems by the collective organization of many cells. Cells divide and undergo apoptosis, and they communicate via signaling pathways that use molecules as information carriers. In tissues, large-scale patterns of gene expression emerge from the coordinated signaling activity and response of many cells. The establishment of such patterns is often guided by long-range concentration profiles of morphogens. Cell divisions and cell rearrangements must be coordinated over large distances to achieve specific tissue sizes and shapes. To unravel how molecular processes and interactions can eventually be responsible for the formation of structures and patterns in tissues during development, it is important to study processes at different scales and understand how different levels of organization are connected. Such an approach becomes strongest if it involves a combination of quantitative experimental studies with theory.In the present article, we discuss several such approaches on different scales with a particular emphasis on theory. Starting from the kinetic and dynamic properties of endosomal networks inside a cell, we discuss transport processes in a tissue that can be related to kinetic trafficking parameters. Such transport processes are then responsible for the formation of graded morphogen concentration profiles. To permit scalable patterns in tissues of different sizes, it has been suggested that morphogen gradients scale during growth. This can be achieved on the tissue level by feedback systems that are sensitive to tissue size and regulate, for example, morphogen degradation. Finally, morphogen gradients that scale with tissue size can provide a system to robustly organize cell division in a large tissue and generate homogeneous growth. Theory can play an important role to bridge scales and understand how molecular and cellular processes can control pattern formation and tissue growth on larger scales.Morphogens are signaling molecules that are secreted in specific regions of developing tissues and can induce signaling activity far from their source. They typically form graded concentration profiles and therefore endow cells with positional information (cells can obtain information about their position in a tissue). Thus, they can guide cells to differentiate into complex morphological patterns. Morphogens also control cell growth and cell division. Because they control both patterning and growth, they may play a key role to coordinate these two processes. Such coordination is important because the size of morphological patterns must adjust during growth, whereas growth influences such patterns. A well-studied morphogen is Decapentaplegic (Dpp), which controls morphogenesis in the imaginal wing disc of developing Drosophila. Consequently, mutations in Dpp or defects in the trafficking pathways that control its graded concentration profiles and signaling affect the formation and structure of the adult wing.The study of morphogens was traditionally approached from a genetic perspective: Which gene products behave like morphogens? Which mutants affect patterning and growth? The realization that morphogens typically operate by a gradient of concentration raised the question of how morphogen gradients are generated. It became clear that the cellular trafficking of morphogens is a key issue for the generation of morphogen profiles. Morphogens are secreted ligands that bind receptors in the plasma membrane. The secretion of the ligands and the concentrations of receptor, ligand, and receptor/ligand complex at the plasma membrane are governed by their trafficking in the cell by vesicular transport. In particular, it was shown that trafficking through the endocytic pathway has an important impact on the formation of morphogen gradients (reviewed in Gonzalez-Gaitan 2003; see Bökel and Brand 2014). This is, to a large extent, how the cells respond to morphogens and contribute to set their local concentrations. To understand functions of morphogens in a tissue, we need to study how the gradient is formed. This, in turn, requires insights into morphogen trafficking through the endocytic pathway. The problem of morphogen behavior, therefore, becomes a problem spanning several levels of complexity: the organ level, the tissue level, the cell level, the organelle level, and the molecular level. Theoretical approaches motivated by physics combined with quantitative experimental approaches provide an ideal framework to understand how these different levels of complexity are intertwined.Two recent discoveries highlighted such integration. (1) The observation that profiles of the morphogen Dpp scale during growth, which implies that the rate of Dpp degradation mediated by the endocytic pathway of each of the cells in the tissue depends on the size of the overall tissue. This suggests that two levels of complexity are linked because cellular trafficking receives cues about the global tissue size. (2) As a result of the changes of the degradation rate that leads to gradient scaling, cells receive an increasing level of signaling. This, in turn, can be used by the cells to decide when to divide. This regulation again involves two levels of complexity because regulation at the endocytic pathway determines the growth properties of the tissue and, ultimately, its final size.In the following, we discuss quantitative approaches to study cellular signaling processes on different scales. Here, the aim is to understand how patterns on large scales can emerge during development from molecular processes and signaling pathways that involve endocytosis and cellular trafficking. We begin by describing trafficking of ligands in the endocytic pathway. We then consider the situation of a morphogen ligand and its impact in gradient formation. Subsequently, we discuss how gradient scaling might be realized. Finally, we discuss how such scaling processes play an important role in the regulation of morphogenetic growth.     

Autocrine, paracrine and juxtacrine signaling by EGFR ligands

Receptor and cytoplasmic protein tyrosine kinases play prominent roles in the control of a range of cellular processes during embryonic development and in the regulation of many metabolic and physiological processes in a variety of tissues and organs. The epidermal growth factor receptor (EGFR) is a well-known and versatile signal transducer that has been highly conserved during evolution. It functions in a wide range of cellular processes, including cell fate determination, proliferation, cell migration and apoptosis. The number of ligands that can activate the EGF receptor has increased during evolution. These ligands are synthesized as membrane-anchored precursor forms that are later shed by metalloproteinase-dependent cleavage to generate soluble ligands. In certain circumstances the membrane anchored isoforms as well as soluble growth factors may also act as biologically active ligands; therefore depending on the circumstances these ligands may induce juxtacrine, autocrine, paracrine and/or endocrine signaling. In this review, we discuss the different ways that EGFR ligands can activate the receptor and the possible biological implications.     

Hypoxia increases Sca-1/CD44 co-expression in murine mesenchymal stem cells and enhances their adipogenic differentiation potential

The emergence of multicellular animals could only take place once evolution had produced molecular mechanisms for cell adhesion and communication. Today, all metazoans express integrin-type adhesion receptors and receptors for growth factors. Integrins recognize extracellular matrix proteins and respective receptors on other cells and, following ligand binding, can activate the same cellular signaling pathways that are regulated by growth factor receptors. Recent reports have indicated that the two receptor systems also collaborate in many other ways. Here, we review the present information concerning the role of integrins as assisting growth factor receptors and the interplay between the receptors in cell signaling and in the orchestration of receptor recycling.     

PTB or not to be: promiscuous, tolerant and Bizarro domains come of age

PTB domains are protein modules that usually interact with the cytoplasmic tail of a wide variety of growth factor receptors. In so doing, they mediate the transduction of extracellular information to specific downstream targets within the cell that ultimately determine the fate of a number of important biological processes such as cell growth and differentiation, cell cycle regulation and apoptosis. Recent structural and functional studies of PTB domains from a variety of cellular proteins have begun to shed light on the molecular mechanisms of action of these important protein modules. In the present review, we provide an account of such studies and suggest that PTB domains can be subdivided into three distinct categories on the basis of their topological differences. We also discuss the various mechanisms employed by the PTB domains in recognition of a diverse set of ligands without a consensus sequence. Finally, we discuss the role of molecular plasticity as a possible determinant of functional versatility of PTB domains.     

TACE cleavage of proamphiregulin regulates GPCR-induced proliferation and motility of cancer cells

Communication between G protein-coupled receptor (GPCR) and epidermal growth factor receptor (EGFR) signalling systems involves cell surface proteolysis of EGF-like precursors. The underlying mechanisms of EGFR signal transactivation pathways, however, are largely unknown. We demonstrate that in squamous cell carcinoma cells, stimulation with the GPCR agonists LPA or carbachol specifically results in metalloprotease cleavage and release of amphiregulin (AR). Moreover, AR gene silencing by siRNA or inhibition of AR biological activity by neutralizing antibodies and heparin prevents GPCR-induced EGFR tyrosine phosphorylation, downstream mitogenic signalling events, cell proliferation, migration and activation of the survival mediator Akt/PKB. Therefore, despite some functional redundancy among EGF family ligands, the present study reveals a distinct and essential role for AR in GPCR-triggered cellular responses. Furthermore, we present evidence that blockade of the metalloprotease-disintegrin tumour necrosis factor-alpha-converting enzyme (TACE) by the tissue inhibitor of metalloprotease-3, a dominant-negative TACE mutant or RNA interference suppresses GPCR-stimulated AR release, EGFR activation and downstream events. Thus, TACE can function as an effector of GPCR-mediated signalling and represents a key element of the cellular receptor cross-talk network.     

Spatial range of autocrine signaling: modeling and computational analysis

Autocrine loops formed by growth factors and their receptors have been identified in a large number of developmental, physiological, and pathological contexts. In general, the spatially distributed and recursive nature of autocrine signaling systems makes their experimental analysis, and often even their detection, very difficult. Here, we combine Brownian motion theory, Monte Carlo simulations, and reaction-diffusion models to analyze the spatial operation of autocrine loops. Within this modeling framework, the ability of autocrine cells to recapture the endogenous ligand and the distances traveled by autocrine ligands are explicitly related to ligand diffusion coefficients, density of surface receptors, ligand secretion rate, and rate constants of ligand binding and endocytic internalization. Applying our models to study autocrine loops in the epidermal growth factor receptor system, we find that autocrine loops can be highly localized--even at the level of a single cell. We demonstrate how the variations in molecular and cellular parameters may "tune" the spatial range of autocrine signals over several orders of magnitude: from microns to millimeters. We argue that this versatile regulation of the spatial range of autocrine signaling enables autocrine cells to perceive a broad spectrum of environmental information.     

Epidermal growth factor (EGF)- and scatter factor/hepatocyte growth factor (SF/HGF)-mediated keratinocyte migration is coincident with induction of matrix metalloproteinase (MMP)-9

Receptor tyrosine kinases are key regulators of cellular function including cell growth, differentiation, migration, and morphogenesis. Disruptions of receptor tyrosine kinase signaling pathways are often associated with changes in cellular proliferative capacity and tumorigenesis. Both receptor-specific and cell type-specific factors may contribute to the ultimate cellular responses observed after receptor activation. In this regard, we find that both normal keratinocytes and their tumorigenic counterparts display differential responses to activation of receptor tyrosine kinases. Multiple ligands were mitogenic for keratinocytes, but only epidermal growth factor (EGF), transforming growth factor α (TGFα), and scatter factor/hepatocyte growth factor (SF/HGF) promoted cell motility as assessed by colony dispersion (scattering) and in vitro reepithelialization. Interestingly, growth factor specificity for motility coincided with ligand-mediated cell invasion through a reconstituted basement membrane and induction of the 92-kDa metalloproteinase (MMP-9) activity as determined by gelatin zymogram analysis. Inhibitors of MMP activity or addition of an MMP-9 neutralizing antibody resulted in the loss of growth factor-induced colony dispersion, suggesting a functional role for MMP-9 induction during this response. Coordinate regulation of MMP-9 induction and the migratory response are likely to contribute to the enhanced invasive potential observed in response to EGF and SF/HGF. Our findings suggest that alternate receptor-mediated signaling pathways leading to differences in gene expression may be involved in complex cellular responses such as colony dispersion or invasion. J. Cell. Physiol. 176:255–265, 1998. © 1998 Wiley-Liss, Inc.     

A cellular automaton model for tumour growth in inhomogeneous environment

Most of the existing mathematical models for tumour growth and tumour-induced angiogenesis neglect blood flow. This is an important factor on which both nutrient and metabolite supply depend. In this paper we aim to address this shortcoming by developing a mathematical model which shows how blood flow and red blood cell heterogeneity influence the growth of systems of normal and cancerous cells. The model is developed in two stages. First we determine the distribution of oxygen in a native vascular network, incorporating into our model features of blood flow and vascular dynamics such as structural adaptation, complex rheology and red blood cell circulation. Once we have calculated the oxygen distribution, we then study the dynamics of a colony of normal and cancerous cells, placed in such a heterogeneous environment. During this second stage, we assume that the vascular network does not evolve and is independent of the dynamics of the surrounding tissue. The cells are considered as elements of a cellular automaton, whose evolution rules are inspired by the different behaviour of normal and cancer cells. Our aim is to show that blood flow and red blood cell heterogeneity play major roles in the development of such colonies, even when the red blood cells are flowing through the vasculature of normal, healthy tissue.     

Targeting the Sheddase Activity of ADAM17 by an Anti-ADAM17 Antibody D1(A12) Inhibits Head and Neck Squamous Cell Carcinoma Cell Proliferation and Motility via Blockage of Bradykinin Induced HERs Transactivation

A disintegrin and metalloproteinase 17 (ADAM17) regulates key cellular processes including proliferation and migration through the shedding of a diverse array of substrates such as epidermal growth factor receptor (EGFR) ligands. ADAM17 is implicated in the pathogenesis of many diseases including rheumatoid arthritis and cancers such as head and neck squamous cell carcinoma (HNSCC). As a central mediator of cellular events, overexpressed EGFR is a validated molecular target in HNSCC. However, EGFR inhibition constantly leads to tumour resistance. One possible mechanism of resistance is the activation of alternative EGFR family receptors and downstream pathways via the release of their ligands. Here, we report that treating human HNSCC cells in vitro with a human anti-ADAM17 inhibitory antibody, D1(A12), suppresses proliferation and motility in the absence or presence of the EGFR tyrosine kinase inhibitor (TKI) gefitinib. Treatment with D1(A12) decreases both the endogenous and the bradykinin (BK)-stimulated shedding of HER ligands, accompanied by a reduction in the phosphorylation of HER receptors and downstream signalling pathways including STAT3, AKT and ERK. Knockdown of ADAM17, but not ADAM10, also suppresses HNSCC cell proliferation and migration. Furthermore, we show that heregulin (HRG) and heparin-binding epidermal growth factor like growth factor (HB-EGF) predominantly participate in proliferation and migration, respectively. Taken together, these results demonstrate that D1(A12)-mediated inhibition of cell proliferation, motility, phosphorylation of HER receptors and downstream signalling is achieved via reduced shedding of ADAM17 ligands. These findings underscore the importance of ADAM17 and suggest that D1(A12) might be an effective targeted agent for treating EGFR TKI-resistant HNSCC.     

Mitogenic action of LPA in prostate

The lipid growth factor lysophosphatidic acid (LPA) elicits multiple cellular responses, including cell growth and survival. LPA acts upon target cells by activating its cognate receptors, which belong to the G protein-coupled endothelial differentiation gene (EDG) family. To date, three known LPA receptors, termed LPA1, LPA2 and LPA3, have been molecularly characterized and cloned. Here, we review recent data describing the molecular steps involved in the LPA receptor-mediated activation of mitogenic extracellular signal-regulated kinase (ERK) pathway in prostate cancer. Induction of ERK by LPA proceeds via Gbetagamma-dependent activation of tyrosine kinases, including the epidermal growth factor (EGF) receptor and c-Src. Further, LPA-induced ERK activation involves matrix metalloproteinases (MMPs), which cause the release of active EGFR ligands. Finally, we present data demonstrating a correlation between the mitogenic effects of LPA and expression of the lp(A1) gene in the prostate cancer cells.     

EGF and PDGF receptor tyrosine kinases as therapeutic targets for chronic lung diseases

Cell-surface receptor tyrosine kinases play pivotal roles in development, tissue repair, and normal cellular homeostasis. Aberrant expression or signaling patterns of these kinases has also been linked to the progression of a diversity of diseases, including cancer, atherosclerosis, asthma, and fibrosis. Two major families of receptor tyrosine kinases, the epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) families, have received a great deal of attention as potential therapeutic targets for pulmonary diseases, as these receptors have been shown to play key roles in chronic tissue remodeling in asthma, bronchitis, and pulmonary fibrosis. The EGFR system on epithelial cells and underlying mesenchymal cells (fibroblasts, myofibroblasts, and smooth muscle cells) drives numerous phenotypic changes during the progression of these pulmonary diseases, including epithelial cell mucous cell metaplasia and mesenchymal cell hyperplasia, differentiation, and extracellular matrix production. The PDGFR system, located primarily on mesenchymal cells, transduces signals for cell survival, growth and chemotaxis. The variety of EGFR and PDGFR ligands produced by the airway epithelium or adjacent mesenchymal cells allows for intimate epithelial-mesenchymal cell communication. A full understanding of the complex mechanisms involving these receptors and ligands should lead to therapeutic strategies for the treatment of a wide range of fibroproliferative lung diseases.     

In vitro culture: an epigenetic challenge for plants

In vitro plant cell and tissue culture techniques are the basis of many micropropagation and breeding programs for scientific research. Plant tissue culture (PTC) involves organogenesis and embryogenesis, and the outcome depends on the different conditions to which the tissue is exposed. PTC is a stressful environment—high relative humidity, low ventilation rate, high concentrations of plant growth regulators, and low light availability—for plants that need to rapidly change their molecular regulation in order to respond fast and efficiently during cell division and growth. For instance, somatic embryogenesis (SE), which plays an important role in plant multiplication, requires complex cellular, biochemical and molecular processes for embryo formation and development. New data has come out about a connection between plant morphogenesis and epigenetics. Epigenetics is a very sensitive regulatory mechanism, which in most of cases is affected by the environment. Although it is known that, under plant morphogenesis, the genome has little or no change, DNA methylation and histone modifications are very susceptible to those in vitro environmental conditions. In the present review, we highlight the most used in vitro systems such as organogenesis and SE in plants and discuss how epigenetics plays a pivotal role in the phenotype outcome. Furthermore, we discuss the big role that the small RNAs have during cell division and propagation and propose different challenges and opportunities to study epigenetics in plant cell tissue and organ cultures.     

Biogenesis and function of fibrillin assemblies

Fibrillin-1 and fibrillin-2 are large cysteine-rich glycoproteins that serve two key physiological functions: as supporting structures that impart tissue integrity and as regulators of signaling events that instruct cell performance. The structural role of fibrillins is exerted through the temporal and hierarchical assembly of microfibrils and elastic fibers, whereas the instructive role reflects the ability of fibrillins to sequester transforming growth factor β (TGFβ) and bone morphogenetic protein (BMP) complexes in the extracellular matrix. Characterization of fibrillin mutations in human patients and in genetically engineered mice has demonstrated that perturbation of either function manifests in disease. More generally, these studies have indicated that fibrillins are integral components of a broader biological network of extracellular, cell surface, and signaling molecules that orchestrate morphogenetic and homeostatic programs in multiple organ systems. They have also suggested that the relative composition of fibrillin-rich microfibrils imparts contextual specificity to TGFβ and BMP signaling by concentrating the ligands locally so as to regulate cell differentiation within a spatial context during organ formation (positive regulation) and by restricting their bioavailability so as to modulate cell performance in a timely fashion during tissue remodeling/repair (negative regulation). Correlative evidence suggests functional coupling of the cell-directed assembly of microfibrils and targeting of TGFβ and BMP complexes to fibrillins. Hence, the emerging view is that fibrillin-rich microfibrils are molecular integrators of structural and instructive signals, with TGFβ and BMPs as the nodal points that convert extracellular inputs into discrete and context-dependent cellular responses.     

Comparison of molecular mechanisms mediating cell contact phenomena in model developmental systems: an exploration of universality

Are there universal molecular mechanisms associated with cell contact phenomena during metazoan ontogenesis? Comparison of adhesion systems in disparate model systems indicates the existence of unifying principles. Requirements for multicellularity are (a) the construction of three‐dimensional structures involving a crucial balance between adhesiveness and motility; and (b) the establishment of integration at molecular, cellular, tissue, and organismal levels of organization. Mechanisms for (i) cell–cell and cell–substrate adhesion, (if) cell movement, (Hi) cell‐cell communication, (iv) cellular responses, (v) regulation of these processes, and (vi) their integration with patterning, growth, and other developmental processes are all crucial to metazoan development, and must have been present for the emergence and radiation of Metazoa. The principal unifying themes of this review are the dynamics and regulation of cell contact phenomena. Our knowledge of the dynamic molecular mechanisms underlying cell contact phenomena remains fragmentary. Here we examine the molecular bases of cell contact phenomena using extant model developmental systems (representing a wide range of phyla) including the simplest i.e. sponges, and the eukaryotic protist Dictyostelium discoideum, the more complex Drosophila melanogaster, and vertebrate systems. We discuss cell contact phenomena in a broad developmental context. The molecular language of cell contact phenomena is complex; it involves a plethora of structurally and functionally diverse molecules, and diverse modes of intermolecular interactions mediated by protein and/or carbohydrate moieties. Reasons for this are presumably the necessity for a high degree of specificity of inter‐molecular interactions, the requirement for a multitude of different signals, and the apparent requirement for an increasingly large repertoire of cell contact molecules in more complex developmental systems, such as the developing vertebrate nervous system. However, comparison of molecular models for dynamic adhesion in sponges and in vertebrates indicates that, in spite of significant differences in the details of the way specific cell–cell adhesion is mediated, similar principles are involved in the mechanisms employed by members of disparate phyla. Universal requirements are likely to include (a) rapidly reversible intermolecular interactions; (b) low‐affinity intermolecular interactions with fast on–off rates; (c) the compounding of multiple intermolecular interactions; (d) associated regulatory signalling systems. The apparent widespread employment of molecular mechanisms involving cadherin‐like cell adhesion molecules suggests the fundamental importance of cadherin function during development, particularly in epithelial morphogenesis, cell sorting, and segregation of cells.     

Molecular interactions of the IGF system

The insulin-like growth factor (IGF) system is a complex network of two soluble ligands; several cell surface transmembrane receptors and six soluble high-affinity binding-proteins. The IGF system is essential for normal embryonic and postnatal growth, and plays an important role in the function of a healthy immune system, lymphopoiesis, myogenesis and bone growth among other physiological functions. Deregulation of the IGF system leads to stimulation of cancer cell growth and survival. In order to manipulate the IGF system in the treatment of certain disorders, we must understand the protein-protein interactions at a molecular level. The complex molecular interactions of the ligands and receptors of the IGF system underlie all the biological actions mentioned above and will be the focus of this review.     

An integrated agent-mathematical model of the effect of intercellular signalling via the epidermal growth factor receptor on cell proliferation

We have previously developed Epitheliome, a software agent representation of the growth and repair characteristics of epithelial cell populations, where cell behaviour is governed by a number of simple rules. In this paper, we describe how this model has been extended to incorporate an example of a molecular 'mechanism' behind a rule-in this case, how signalling by both endogenous and exogenous ligands of the epidermal growth factor receptor (EGFR) can impact on the proliferation of cell agents. We have developed a mathematical model representing release of endogenous ligand by cells, three-dimensional diffusion of the secreted molecules through a volume of cell culture medium, ligand-receptor binding, and bound receptor internalization and trafficking. Information relating to quantities of molecular species associated with each cell agent is frequently exchanged between the agent and signalling models, and the ratio of bound to free receptors determines cell cycle progression and hence the proliferative behaviour of the cell agents. We have applied this integrated model to examine the effect of plating density on tissue growth via autocrine/paracrine signalling. This predicts that cell growth is dependent on the concentration of exogenous ligand, but where this is limited, then growth becomes dependent on cell density and the availability of endogenous ligand. We have further modified the calcium concentration of the medium to modulate the formation of intercellular bonds between cells and shown that the increased propensity for cells to form colonies in physiological calcium does not result in significantly different patterns of receptor occupancy. In conclusion, our approach demonstrates that by combining agent-based and mathematical modelling paradigms, it is possible to probe the complex feedback relationship between the behaviour of individual cells and their interaction with one another and their environment.