Genetically Modified Autoactivated Cells Expressing Intracellular Forms of GM‐CSF as a Model for Regulated Administration of Cytokines
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The application of cytokines for immunotherapy is frequently hampered by undesirable side effects. To avoid systemic effects, cytokines can be directly expressed in the target cells by using gene transfer. However, the uncontrolled cellular secretion of cytokines could still exert some undesirable bystander effects. Therefore, it is important to develop additional methods for a more restricted administration of cytokines. Recently, using the murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), we have demonstrated that cytokines can be targeted to different subcellular compartments as stable and biologically active proteins. This model could be used as a method of highly restricted administration of cytokines. Here, as model for the proof of principle, we have used a cell line (DA-3) strictly dependent on mGM-CSF for growth and demonstrated that these cells acquired autonomous growth after gene modification with plasmids encoding either extracellular or intracellular forms of mGM-CSF. Cell lines expressing secreted forms of mGM-CSF displayed the highest rates of autonomous growth and released substantial amounts of mGM-CSF. However, cell lines expressing intracellular forms of mGM-CSF also acquired autonomous growth induced by a mechanism of restricted autocrine stimulation and did not release detectable mGM-CSF to the extracellular medium. Cocultivation experiments of DA-3 cell lines expressing intracellular mGM-CSF with unmodified cells showed that there was no activation of the bystander cells. Taken together, these results support the concept that genes encoding intracellular cytokines may be used to provide the desired effect of cytokines on the target cells while avoiding the side effects of their uncontrolled secretion.Cite
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Autocrine ligands regulate important cell behavioral functions in both physiological and pathological conditions. Binding of these ligands to cell-surface receptors involves more subtle considerations than that of exogenous (endocrine or paracrine) ligands. Autocrine secretion leads to a release of molecules in the local microenvironment proximal to the cell surface, thus allowing interaction with receptors to compete directly with diffusive loss to the bulk extracellular medium. Complications in autocrine systems due to this binding vs. transport competition arise in at least three aspects: (i) experimental measurement of autocrine ligand secretion rates is compromised; (ii) kinetics of autocrine ligand binding to cell-surface receptors are difficult to follow; and (iii) inhibition by exogenous blockers of autocrine ligand binding to cell receptors is problematic. At the heart of all these complications is the need to determine the fractional distribution of the secreted autocrine ligand between cell-surface receptor capture and diffusive loss to the bulk media. In this paper we offer a theoretical treatment of this problem using Brownian dynamics simulation techniques to calculate the capture probability of the cell receptors for the autocrine ligand. A major result is that the capture probability is significantly lower than the predicted by the Berg–Purcell steady-state diffusion approach. Another is that the capture probability is essentially independent of release location. Implications of these results for the complications found in autocrine systems are discussed.
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Abstract Gastrin is transcriptionally responsive to EGF stimulation (Merchant et al., 1991, Mol. Cell. Biol., 11 :2686–2696). Consequently, we hypothesized that previously recognized gastrin autocrine loops (Hoosein et al., 1990, Exp. Cell. Res., 186 :15–21), might be controlled by autocrine TGFα in human colon carcinoma cells. Therefore, we examined the interaction between these two autocrine growth factors in two colon carcinoma cell lines which utlizie TGFα. The FET cell line requires exogenous TGFα/EGF for optimal growth and has a classical TGFα autocrine loop which is disrupted by TGFα or epidermal growth factor receptor (EGFr) antibodies. The HCT 116 cell line is not dependent on exogenous TGFα/EGF and exhibits a nonclassical TGFα autocrine loop which is not disrupted by neutralizing antibodies to either TGFα itself or the EGFr. Basal gastrin mRNA production is significantly higher in HCT 116 than FET as measured by RNase protection assay. In the FET cells, exogenous EGF stimulates gastrin mRNA production but not in HCT 116. When the TGFα autocrine loop in HCT 116 is disrupted by constitutive expression of antisense TGFα mRNA, the gastrin mRNA level is significantly repressed. In xenografts derived from these antisense clones, TGFα reverted to high expression, and the gastrin mRNA level was again increased. This interaction between the strong TGFα loop in HCT 116 and the gastrin autocrine loop may confer a growth advantage to these colon cells. Such interactions between growth factors may promote enhanced tumorigenicity to transformed cells with these strong, nonclassical autocrine loops. © 1995 Wiley‐Liss, Inc.
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Autocrine regulation is defined as a mechanism of self-control in growth and differentiation; this mode of regulation among histologically homologous cells is mediated humorally. Autocrine mechanisms involve: 1. Autonomously controlled production and secretion of autocrine mediators. 2. Distribution of autocrine mediators among cells. 3. Expression by cells of functional receptors for autocrine mediators. 4. Transduction and intracellular integration of signals mediated by autocrine mediators. 5. Growth response. 6. Maintenance of autonomous control of growth and/or differentiation state in the progeny Biochemical and biological evidence for most of these steps in various transformed cells makes it possible to analyze autocrine control as a multifaceted process. This process depends on tumor cellularity and histoarchitecture, on time and on external influences on secretion of autocrine mediators (e.g., estrogens in estrogen-dependent breast cancer). We review the quantitative aspects of experimental evidence for autocrine control in tumors and examine the phenomenological and some mechanistic concepts in creating integrative, quantitative, and experimentally verifiable mathematical models of autocrine regulation.
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Autocrine ligands are important regulators of many normal tissues and have been implicated in a number of disease states, including cancer. However, because by definition autocrine ligands are synthesized, secreted, and bound to cell receptors within an intrinsically self-contained “loop,” standard pharmacological approaches cannot be used to investigate relationships between ligand/receptor binding and consequent cellular responses. We demonstrate here a new approach for measurement of autocrine ligand binding to cells, using a microphysiometer assay originally developed for investigating cell responses to exogenous ligands. This technique permits quantitative measurements of autocrine responses on the time scale of receptor binding and internalization, thus allowing investigation of the role of receptor trafficking and dynamics in cellular responses. We used this technique to investigate autocrine signaling through the epidermal growth factor receptor by transforming growth factor alpha (TGFα) and found that anti-receptor antibodies are far more effective than anti-ligand antibodies in inhibiting autocrine signaling. This result indicates that autocrine-based signals can operate in a spatially restricted, local manner and thus provide cells with information on their local microenvironment.
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Autocrine signaling is defined as the production and secretion of an extracellular mediator by a cell followed by the binding of that mediator to receptors on the same cell to initiate signaling. Autocrine stimulation often operates in autocrine loops, a type of interaction, in which a cell produces a mediator, for which it has receptors, that upon activation promotes expression of the same mediator, allowing the cell to repeatedly autostimulate itself (positive feedback) or balance its expression via regulation of a second factor that provides negative feedback. Autocrine signaling loops with positive or negative feedback are an important feature in cancer, where they enable context-dependent cell signaling in the regulation of growth, survival, and cell motility. A growth factor that is intimately involved in tumor development and progression and often produced by the cancer cells in an autocrine manner is transforming growth factor-β (TGF-β). This review surveys the many observations of autocrine TGF-β signaling in tumor biology, including data from cell culture and animal models as well as from patients. We also provide the reader with a critical discussion on the various experimental approaches employed to identify and prove the involvement of autocrine TGF-β in a given cellular response.
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We have exploited a discrepancy in the oncogenic potential of autocrine and exogenous human growth hormone (hGH) in an attempt to identify molecules that could potentially be involved in oncogenic transformation of the human mammary epithelial cell. Microarray analysis of 19,000 human genes identified a subset of 305 genes in a human mammary carcinoma cell line that were remarkably different in their response to autocrine and exogenous hGH. Autocrine and exogenous hGH also regulated 167 common genes. Semiquantitative reverse transcription-PCR confirmed differential regulation of genes by either autocrine or exogenous hGH. Functional analysis of one of the identified autocrine hGH-regulated genes, TFF3, determined that its expression is sufficient to support anchorage-independent growth of human mammary carcinoma cells. Small interfering RNA-mediated knockdown of TFF3 concordantly abrogated anchorage-independent growth of mammary carcinoma cells and abrogated the ability of autocrine hGH to stimulate oncogenic transformation of immortalized human mammary epithelial cells. Further functional characterization of the identified subset of specifically autocrine hGH regulated genes will delineate additional novel oncogenes for the human mammary epithelial cell.
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