Gene expression profiling of copper-resistant Caco-2 clones.

The Caco-2 cell line is composed of a heterogeneous mix of cells; isolation of individual clonal populations from this mix allows for specific mechanisms and phenotypes to be further explored. Previously we exposed Caco-2 cells to inorganic copper sulphate or organic copper proteinate to generate resistant variant populations. Here we describe the isolation and characterisation of clonal subpopulations from these resistant variants to organic (clone Or1, Or2, Or3) or inorganic (clone In1 and In2) copper. The clones show considerable homogeneity in response to Cu-induced toxicity and heterogeneity in morphology with variations in level of cross-resistance to other metals and doxorubicin. Population growth was reduced for Cu-resistant clones In2 and Or3 in selective pressure relative to parental Caco-2 cells. Gene expression analysis identified 4026 total (2102 unique and 1924 shared) differentially expressed genes including those involved in the MAP Kinase and Rap1 signalling pathways, and in the focal adhesion and ECM-receptor contact pathways. Gene expression changes common to all clones included upregulation of ANXA13 and GPx2. Our analysis additionally identified differential expression of multiple genes specific to copper proteinate exposure (including overexpressed UPK1B) in isolated clones Or1, Or2 and Or3 and CuSO4 exposure (including decreased AIFM2 expression) in isolated clones In1 and In2. The adaptive transcriptional responses established in this study indicate a cohort of genes, which may be involved in copper resistance regulation and chronic copper exposure.

Characterisation and proteomic profiling of continuously exposed Cu-resistant variants of the Caco-2 cell line.

Studies in hepatic systems identify multiple factors involved in the generation of copper resistance. As the intestine is the route of exposure to dietary copper, we wanted to understand how intestinal cells overcome the toxic effects of high copper and what mechanisms of resistance develop. Using the intestinal cell line Caco-2, resistance was developed by serial subculture in 50 µM copper in inorganic (CuSO4) or organic (Cu proteinate) forms. Caco-2 variants exhibited resistance to copper and retained the non-monotonic dose response while displaying stable phenotypes following repeated subculture in the absence of copper. Phenotypic changes on exposure to copper in parental Caco-2 cells included significantly increased total protein yield, ROS, SOD, metallothionein expression, GSH and total glutathione. These phenotypic changes were not replicated in resistant variants on a per cell basis. Quantitative label-free LC-MS/MS proteomic analysis identified 1113 differentially expressed proteins (DEPs) between parental Caco-2 and resistant cells. With some exceptions, most of the DEPs were overexpressed to a low level around 2-fold suggesting resistance was supported by multiple small changes in protein expression. These variants may be a useful tool in studying the toxicity of stress responses in further Cu-related studies.

Acute exposure to organic and inorganic sources of copper: Differential response in intestinal cell lines.

SCOPE: Copper supplementation in nutrition has evolved from using inorganic mineral salts to organically chelated minerals but with limited knowledge of the impact at the cellular level. METHODS: Here, the impact of inorganic and organic nutrient forms (glycinate, organic acid, and proteinate) of copper on the cellular level is investigated on intestinal cell lines, HT29 and Caco-2, after a 2-hr acute exposure to copper compounds and following a 10-hr recovery. RESULTS: Following the 10-hr recovery, increases were observed in proteins involved in metal binding (metallothioneins) and antioxidant response (sulfiredoxin 1 and heme oxygenase 1), and global proteomic analysis suggested recruitment of the unfolded protein response and proteosomal overloading. Copper organic acid chelate, the only treatment to show striking and sustained reactive oxygen species generation, had the greatest impact on ubiquitinated proteins, reduced autophagy, and increased aggresome formation, reducing growth in both cell lines. The least effect was noted in copper proteinate with negligible impact on aggresome formation or extended growth for either cell line. CONCLUSION: The type and source of copper can impact significantly at the cellular level.

Unexpected fluctuations of trace element levels in cell culture medium in vitro: caveat emptor.

Copper is an essential trace element micronutrient in human and animal nutrition. Trace amounts present even in ultrapure water, serum and other cell culture medium components are sufficient to support the health requirements of most cell types in culture. Analysis of a variety of different types of basal media from a number of different suppliers revealed large fluctuations in the levels of copper, and also of other micronutrients including zinc, iron, selenium and cobalt. Investigations on proliferating Caco-2 cells revealed reductions in growth with increasing copper concentrations within the range seen in the commercial media and changes in expression of apoptosis- and autophagy-related proteins were noted. Even at concentrations of 1 µM CuSO4 where there was no significant change in cell growth, there was a significant decrease in procaspase-3 expression. These results stress the importance of batch testing of basal media when undertaking trace metal research since the baseline levels may vary. Batch variation of serum is well established but our results suggest that batch variation of the media may also be important.