IntroductionIn cancer cells, different pathways may be activated and/or different transcription factors may be expressed, resulting in the specific expression of a repertoire of genes in distinct cell types. A good example is the urokinase-type plasminogen activator (uPA) gene, which is expressed at high basal levels in PC3 cells but is not expressed in HeLa or LNCaP cells. [1][2][3] uPA is a serine protease involved in cell migration in nonpathological (eg, tissue remodeling) and pathological (eg, tumor invasion and metastases formation) events. 4,5 Many human cancers, in fact, overexpress uPA, and reduction of the invasive and metastatic phenotype has been obtained by down-regulating the expression of the gene or by inhibiting the enzymatic activity of this protease. 4,5 The minimal promoter (MP) of the human uPA gene extends approximately 86 bp upstream of the transcription start site 1,6 and contains 5 (high, 3 ϫ GGGCGG; low, 2 ϫ GGGAGG) affinity binding sites for the Sp1-family transcription factors immediately upstream of the TATA box. This cis-acting element binds the transcription factor Sp1 in vivo in PC3 cells, which constitutively express the uPA gene. In this cell line, a uPA minimal promoterdriven reporter construct is 10-fold transcriptionally more active than in HeLa cells. 1 This result correlates with the presence of the phosphorylated form of Sp1 in PC3 cells and with the absence of the phosphorylated transcription factor 1 and the lack of transcription of the endogenous uPA gene in HeLa cells. Thus, the uPA minimal promoter plays a very important role in uPA gene expression, invasion, and metastasis formation in cancer cells.The expression of the uPA gene is inducible in several cells by stimulating the enhancer, located about 2 kb upstream of the transcription start site, 6 which binds transcription factors belonging to different families. Despite its relevance in these cells, the uPA enhancer appears to have no role (or a very modest one) in uPA expression in PC3 cells, where, instead, transcriptional activity is dependent on the binding of the transcription factor Sp1 to the minimal promoter. 1 Phosphorylation of Sp1 seems to be an absolute requirement, because HeLa cells, which express but do not phosphorylate Sp1, are unable to activate the MP.Recently, it has been shown that in a CCL39 (Chinese hamster fibroblast)-derivative cell line, which expresses a chimeric, estrogeninducible Raf:ER chimera, vascular endothelial growth factor (VEGF) expression is under the control of the p42/p44 mitogenactivated protein (MAP) kinase pathway, 28 which targets the transcription factor Sp1 and promotes its phosphorylation at residues Thr453 and Thr739. 29 The phosphorylated form of Sp1, in turn, drives transcription from the VEGF minimal promoter, which contains 2 binding sites for this transcription factor, which bracket a binding site for the transcription factor AP-2. 28 The role of VEGF-MP in the overall expression of VEGF is not known. Interestingly, the constitutive levels of VEGF and uPA mRNA i...
Cell lines have become an integral resource and tool for conducting biological experiments ever since the Hela cell line was first developed (Scherer et al. in J Exp Med 97:695–710, 1953). They not only allow detailed investigation of molecular pathways but are faster and more cost-effective than most in vivo approaches. The last decade saw many emerging model systems strengthening basic science research. However, lack of genetic and molecular tools in these newer systems pose many obstacles. Astyanax mexicanus is proving to be an interesting new model system for understanding metabolic adaptation. To further enhance the utility of this system, we developed liver-derived cell lines from both surface-dwelling and cave-dwelling morphotypes. In this study, we provide detailed methodology of the derivation process along with comprehensive biochemical and molecular characterization of the cell lines, which reflect key metabolic traits of cavefish adaptation. We anticipate these cell lines to become a useful resource for the Astyanax community as well as researchers investigating fish biology, comparative physiology, and metabolism.
The tetra fish species Astyanax mexicanus comprises two morphotypes: cavefish that live in caves and surface fish that inhabit rivers and lakes. Because cavefish have adapted to the nutrient‐poor conditions in their habitat whereas the surface fish populations can be used as a proxy for the ancestral condition, this species has become a powerful model system for understanding genetic variation underlying metabolic adaptation. The liver plays a critical role in glucose and fat metabolism in the body and hence is an important tissue for studying altered metabolism in health and disease. Cavefish morphs of A. mexicanus have been shown to develop fatty livers and exhibit massive differences in gene expression and chromatin architecture. Primary cell lines from various tissues have become invaluable tools for biochemical, toxicology, and cell biology experiments, as well as genetic and genomic analyses. To enhance the utility of the model system by enabling an expanded set of biochemical and in vitro experiments, we developed protocols for the isolation and maintenance of primary liver cells from A. mexicanus surface fish and cavefish. We also describe methods that can be used for primary cell characterization, including cloning, characterization of cell growth pattern, and lentivirus transduction. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Primary culture of liver cells Support Protocol 1: Maintenance of A. mexicanus primary liver cells Support Protocol 2: Banking of A. mexicanus primary liver cells Support Protocol 3: Recovery of A. mexicanus primary liver cells Support Protocol 4: Primary liver cell cloning Support Protocol 5: Characterization of A. mexicanus primary liver cell growth pattern Basic Protocol 2: Lentiviral transduction of A. mexicanus primary liver cells
Cell lines have become an integral resource and tool for conducting biological experiments ever since the Hela cell line was first developed (1). They not only allow detailed investigation of molecular pathways but are faster and more cost-effective than most in vivo approaches. The last decade saw many emerging model systems strengthening basic science research. However, lack of genetic and molecular tools in these newer systems pose many obstacles. Astyanax mexicanus is proving to be an interesting new model system for understanding metabolic adaptation. To further enhance the utility of this system, we developed liver-derived cell lines from both surface-dwelling and cave-dwelling morphotypes. In this study, we provide detailed methodology of the derivation process along with a comprehensive biochemical and molecular characterization of the cell lines, which reflects key metabolic traits of cavefish adaptation. We anticipate these cell lines to become a useful resource for the Astyanax community as well as researchers investigating fish biology, comparative physiology, and metabolism.
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