NCM460, a normal human colon mucosal epithelial cell line

Moyer, Mary Pat; Manzano, L.A.; Merriman, Ronald L.; Stauffer, Jay S.; Tanzer, Lee R.

doi:10.1007/bf02722955

Cited by 209 publications

(191 citation statements)

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“…We chose this normal non-transformed human colonic epithelial cell line because these cells possess many of the characteristics of the native colonocytes (24,25). Our results showed that uptake of folic acid was temperature-dependent and occurred with minimal metabolic alterations to the transported substrate.…”

Section: Discussionmentioning

confidence: 99%

“…The human normal colon epithelial cell line NCM460 was propagated to maintain its colonocyte features (24) in the culture medium M3:10 TM (INCELL Corp., San Antonio, TX). The M3:10 TM medium is M3 TM base medium supplemented with 10% (v/v) fetal bovine serum and antibiotics and contains many growth factors and nutrients, some of which have been described elsewhere (26 -28).…”

Section: Methodsmentioning

confidence: 99%

“…A colonic cell line that possesses a folate uptake mechanism that is similar to that of the native colonocyte would be an ideal model system to address this issue. The NCM460 cells (a normal, non-transformed epithelial cell line derived from the human transverse colonic mucosa) (24) were chosen as a model system because they possess char-acteristics similar to those of normal colonic epithelia (24,25). In this report, we demonstrated that the mechanism of folate uptake is similar to that of the native colonic tissue and that the intracellular regulation of folate uptake is mediated by cAMP and PTK-mediated pathways.…”

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confidence: 99%

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A Protein-tyrosine Kinase-regulated, pH-dependent, Carrier-mediated Uptake System for Folate in Human Normal Colonic Epithelial Cell Line NCM460

Kumar

Moyer

Dudeja

et al. 1997

Journal of Biological Chemistry

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A significant proportion of the bacterially synthesized folate in the large intestine exists in the form of folate monoglutamate. Recent studies in our laboratory using human colonic apical membrane vesicles have shown the existence of an efficient carrier-mediated system for folate uptake. Nothing, however, is known about the cellular regulation of the colonic uptake process. In this study, we used a recently established human normal colonic epithelial cell line NCM460 to address this issue. Uptake of folic acid by NCM460 cells was: 1) linear with time for 4 min of incubation and occurred with minimal metabolic alterations, 2) temperature-and pH-(but not Na ؉ ) dependent, 3) saturable as a function of concentration (apparent K m of 1.4 M), 4) inhibited by structural analogs and anion transport inhibitors, and 5) energydependent. These characteristics of folic acid uptake by NCM460 cells are similar to those seen with apical membrane vesicles derived from human native colonic tissue. Using these cells, we found that protein kinase Cand Ca 2؉ /calmodulin-mediated pathways have no role in regulating folic acid uptake. On the other hand, cAMP (through a mechanism independent of protein kinase A) and protein-tyrosine kinase-mediated pathways were found to play a role in the regulation of folic acid uptake by these cells. These results establish the suitability of NCM460 cells as an in vitro model system for investigating the details of the mechanism of colonic folate uptake and its regulation. Folic acid uptake by these cells appears to involve a carrier-mediated system, which is temperature-, pH-, and energy-dependent and appears to be under the regulation of cAMP and protein tyrosine kinase.Folate is an essential micronutrient, which acts as a coenzyme in the synthesis of DNA and RNA and the interconversion and degradation of several amino acids (1-4). An adequate supply of folate is therefore necessary for normal cellular function, growth, and development. Folate deficiency has been suggested as one of the most common vitamin deficiencies in the Western Hemisphere (5, 6). Humans and other mammals cannot synthesize folate and rely on exogenous sources to meet their metabolic requirements. Folates are presented to the host from the diet and are also synthesized in the large intestine by normal microflora. The mechanism of absorption of dietary folate has been intensively examined over the past two decades at the tissue, cellular, subcellular, and more recently, molecular levels (6 -15). Absorption of dietary folate has been shown to occur mainly in the proximal small intestine and involves a specialized, carrier-mediated system (6 -12).As to the bacterially synthesized folate in the large intestine, a significant amount of that folate exists in the monoglutamate, i.e. the absorbable form. Using [ 3 H]p-aminobenzoic acid to label the newly synthesized folate by the intestinal flora, Rong et al. (16) have shown that a portion of this folate is indeed absorbed by the rat and is incorporated into its various tissues. ...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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A Protein-tyrosine Kinase-regulated, pH-dependent, Carrier-mediated Uptake System for Folate in Human Normal Colonic Epithelial Cell Line NCM460

Kumar

Moyer

Dudeja

et al. 1997

Journal of Biological Chemistry

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“…The NCM460 cell line is a human colonic epithelial cell line that was originally derived from the normal mucosa of a 68-year-old Hispanic male [7]. Cells were grown in M3:10™ media (INCELL, San Antonio, TX) and maintained at 37°C in a humidified, 5% CO 2 environment with media renewal at two-day intervals.…”

Section: Cell Culture Of Colonocytesmentioning

confidence: 99%

Carrageenan-induced NFκB activation depends on distinct pathways mediated by reactive oxygen species and Hsp27 or by Bcl10

Bhattacharyya

Dudeja

Tobacman

2008

Biochimica et Biophysica Acta (BBA) - General Subjects

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Carrageenans are highly sulfated polysaccharides that are widely used as food additives due to their ability to improve food texture. They are also widely recognized for their ability to induce inflammation in animal models of colitis. Recently, we reported that carrageenan (CGN) activated a pathway of innate immunity in human colonic epithelial cells mediated by Bcl10 (B-cell CLL/ lymphoma 10). However, increases in phospho-IκBα and Interleukin-8 (IL-8) were not completely inhibited by silencing Bcl10, suggesting that CGN also influenced another mechanism, or mechanisms, of inflammation. In this report, we demonstrate that CGN increases production of reactive oxygen species (ROS) in human colonic epithelial cells. The combination of ROS quenching by the free radical scavenger Tempol and of Bcl10 silencing by siRNA completely inhibited the CGN-induced increases in nuclear NFκB (p65), phospho-IκBα, and secretion of IL-8. The CGN-induced increase in ROS was associated with declines in phosphorylation of MAPK 12 (p38γ), MAPK 13 (p38δ), and heat-shock protein (Hsp) 27. The CGN-induced decline in phospho-Hsp27 was reversed by co-administration of Tempol (100nM), but unaffected by silencing Bcl10. Since Hsp27 phosphorylation is inversely associated with phosphorylation of the IκBα kinase (IKK) signalosome, CGN exposure appears to affect the IKK signalosome by both the catalytic component, mediated by ROS-phospho-Hsp27, and the regulatory component, mediated by Bcl10 interaction with IKKγ (Nemo). Hence, the CGN-activated inflammatory cascades related to innate immunity and to generation of ROS may be integrated at the level of the IKK signalosome.

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“…To do this, we first evaluated ROCK-I and -II expressions in non-malignant (NCM460 and IEC-6) and malignant (Caco-2 E, SW620, and HCT-116) intestinal epithelial cell lines. NCM460 cells are a non-malignant human colonic epithelial cell line 16 and IEC-6 cells ROCK-II, colon cancer, and invadopodiaare a non-malignant rat small intestinal cell line. While IEC-6 cells are not colonic in origin, we used them in addition to NCM460 cells so that we could validate our observations about ROCK-II in at least two non-malignant cell lines.…”

Section: Rock-ii Expression Not Significantly Impacted By Intestinal mentioning

confidence: 99%

ROCK-II mediates colon cancer invasion via regulation of MMP-2 and MMP-13 at the site of invadopodia as revealed by multiphoton imaging

Vishnubhotla

Sun

Huq

et al. 2007

Laboratory Investigation

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The ROCK-II isoform of Rho's downstream effector, Rho kinase, has been linked with greater invasion and metastasis in solid tumors. We have previously shown that ROCK-II is overexpressed at the advancing edge of colon cancers. The mechanism whereby ROCK-II contributes invasion, particularly in the setting of colon cancer, remains to be elucidated fully. To better understand its contribution, we evaluated ROCK-II expression in both non-malignant (NCM460 and IEC-6) and malignant (Caco-2 E, SW620, and HCT-116) intestinal epithelial cell lines grown in type I collagen scaffolds. Using multiphoton microscopy, we observed that ROCK-II localized to the actin cytoskeleton in non-malignant cells but localized to the cell periphery as focal collections with an absence of adjacent collagen in all colon cancer cell lines. By transmission electron microscopy, these collections corresponded with finger-like projections previously described as invadopodia. Immunogold staining with cortactin, matrix metalloprotease (MMP)-2, -9, and -13 confirmed that these were indeed invadopodia. To further link ROCK-II to colon cancer invasion, we treated non-malignant and malignant intestinal epithelial cell lines with ROCK-II siRNA and evaluated depth of invasion, proliferation, and MMP-2, -9, and -13 activities. The most striking effect was seen in the highly tumorigenic cell lines, SW620 and HCT-116, wherein ROCK-II knockdown resulted in a two-fold or more reduction in invasion. This reduction in invasion was not due to a decrease in cell proliferation, as a significant reduction in proliferation was only observed in the two non-malignant intestinal cell lines. Finally, both MMP-2 and -13 activities were significantly decreased in all colon cancer cell lines. Taken together, these data suggest for the first time that ROCK-II is a critical mediator of colon cancer cell invasion through its modulation of MMP-2 and -13 at the site of invadopodia but regulates proliferation in non-malignant intestinal cells.

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NCM460, a normal human colon mucosal epithelial cell line

Cited by 209 publications

References 6 publications

A Protein-tyrosine Kinase-regulated, pH-dependent, Carrier-mediated Uptake System for Folate in Human Normal Colonic Epithelial Cell Line NCM460

A Protein-tyrosine Kinase-regulated, pH-dependent, Carrier-mediated Uptake System for Folate in Human Normal Colonic Epithelial Cell Line NCM460

Carrageenan-induced NFκB activation depends on distinct pathways mediated by reactive oxygen species and Hsp27 or by Bcl10

ROCK-II mediates colon cancer invasion via regulation of MMP-2 and MMP-13 at the site of invadopodia as revealed by multiphoton imaging

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