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HT29 Cell Line

Daniel Martínez-Maqueda, Beatriz Miralles, and Isidra Recio

Abstract The human colon adenocarcinoma cell line HT29, is not only used to study the biology of human colon cancers, but it is receiving special interest in studies focused on food digestion and bioavailability due to the ability to express characteristics of mature intestinal cells. In the differentiated phenotype, they are able to form a monolayer with tight junctions between cells and a typical apical brush border. In addition, these differentiated cells express brush-border-associated hydrolases typical of the small intestine although the enzymatic activity is lower than that found in vivo. Although they represent a valuable model due to their similarities with enterocytes of the small intestine, their limitations and the relevance to the in vivo situation are also considered in this chapter. The application of this cell line to transport studies of drugs and food compounds is illustrated, especially when the effect of the mucus layer is considered or used as co-culture in combination with Caco-2 cells. They have also been frequently used to study the intestinal immune response to bacterial infection, and microorganism survival, adhesion or invasion. Finally, the use of these cells to evaluate the effect of several food compounds and mucin secretion is summarized.

Keywords HT29 • Cell differentiation • Transport studies • Intestinal cell

Origin

The human colon adenocarcinoma cell line HT29 was isolated from a primary tumor of a 44 years old Caucasian female in 1964 by Fogh and Trempe (1975). Since then, many cell lines have been derived from human colon cancers. Initially, this cell line was used to study different aspects of the biology of human cancers. However, these cells have attracted attention due to the fact they were able to express characteristics of mature intestinal cells, such as enterocytes or mucus producing cells.

Features and Mechanisms

These cells have shown a high rate of glucose consumption and therefore, they require high glucose concentration in the medium. Under these standard growth conditions, i.e., in the presence of 25 mM glucose and 10 % serum, these cells display an undifferentiated phenotype; they grow as a multilayer of unpolarised undifferentiated cells, and functionally they do not express any typical characteristics of intestinal epithelial cells, and they express low amounts of hydrolases. Since the discovery that the replacement of glucose by galactose in the culture medium induced a reversible enterocytic differentiation (Pinto et al. 1982), HT29 cells have become a unique model for studying the molecular mechanisms of intestinal cell differentiation. When these cells are grown under appropriate culture conditions or after treatment with different inducers, like butyrate (Augeron and Laboisse 1984) or acid (Fitzgerald et al. 1997), they can be modulated to express different pathways of enterocyte differentiation. For this reason, HT29 is considered a pluripotent intestinal cell line which can be used for the study of the structural and molecular events involved in cell differentiation.

The polarised phenotype is characterised morphologically, physiologically and by biochemical markers. The differentiation is growth-related, starting after confluence (after 15 days of growth), and the cells form a monolayer with tight junctions between cells and a typical apical brush border. The brush border of these differentiated cells contains proteins which are normally present in the intestinal microvilli, such as villin. In addition, these differentiated cells express brush border-associated hydrolases typical for the small intestine, have brush border microvilli although the enzyme activities is much lower than that of the normal intestine, and do not express lactase. The maximum enzyme activities, such as sucrose-isomaltase, aminopeptidase N, dipeptidylpeptidase-IV and alkaline phosphatase, are reached after 30 days in culture. Under these conditions, p.e. growth in glucose-free medium, cell differentiation is very similar to that observed in Caco-2 cells but in HT29 the time course of the differentiation process is longer (30 days vs. 15–20 days in Caco-2); the levels of enzymatic activities are lower than in Caco-2; lactase is absent; and only 40–50 % of HT29 cells express sucrose-isomaltase (Zweibaum 1986; Zweibaum et al. 2011). However, one of the main differences between this cell line and Caco-2 is that HT29 cells can produce mucin at a relatively high level (Huet et al. 1987; Maoret et al. 1989). Stepwise adaptation of exponentially growing HT29 cells to increasing concentrations of methotrexate (MTX) (up to 10−5 mol) resulted in their transformation into mucus-secreting differentiated cells (Lesuffleur et al. 1990). As occurs under other metabolic stress conditions like glucose-deprivation, after a high rate of mortality, the resistance to MTX is associated with the cells possessing this stable differentiated mucus-secreting phenotype. Interestingly, cells adapted to low-dose MTX consist of a double population of columnar absorptive and mucus-secreting cells and, at a higher dose, cells are almost exclusively of mucus-secreting phenotype. This mucus-secreting phenotype has been used in the transport studies of different compounds, to study the mucusinducing properties of different food compounds or in studies regarding microorganisms adhesion and survival (see Sect. 11.6).

The factors secreted from cells in culture medium include metabolites, cytokines, growth factors, etc., which are known to promote cell survival. Recently, the soluble factors secreted by HT29 and other tumor cells in basal conditions and after gamma radiation have been reported. HT29 secreted pro-inflammatory cytokines, such as, tumor necrosis factor (TNF) α and interleukins (IL) 1β and IL 6; growth factors such as platelet-derived growth factor AA and transforming growth factors (TGF) α and β; chemokines such as fractalkine, IL-8, monocyte chemoattractant protein-1 and interferon-γ-induced protein 10; pro-angiogenic factors such as IL-15 and vascular endothelial growth factor; and immune-modulatory cytokines such as granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor and IL-3. It was suggested, based on previous reports on biopsy samples, that in vivo, a similar cytokine secretion profile can be observed (Desai et al. 2013).

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