ARMENDÁRIZ ET AL. Biol Res 39, 2006, 125-142 125 Biol Res 39: 125-142, 2006 BR Gene expression profiling in wild-type and metallothionein mutant fibroblast cell lines ÁNGELA D ARMENDÁRIZ1, FELIPE OLIVARES2, RODRIGO PULGAR2, ALEX LOGUINOV1, VERÓNICA CAMBIAZO2, CHRISTOPHER D VULPE1 and MAURICIO GONZÁLEZ2 1 Department of Nutritional Science and Toxicology, University of California, Berkeley, Berkeley, CA 94720 2 Laboratorio de Bioinformática y Expresión Génica. Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile, Santiago, Santiago, Chile ABSTRACT The role of metallothioneins (MT) in copper homeostasis is of great interest, as it appears to be partially responsible for the regulation of intracellular copper levels during adaptation to extracellular excess of the metal. To further investigate a possible role of MTs in copper metabolism, a genomics approach was utilized to evaluate the role of MT on gene expression. Microarray analysis was used to examine the effects of copper overload in fibroblast cells from normal and MT I and II double knock-out mice (MT-/-). As a first step, we compared genes that were significantly upregulated in wild-type and MT-/- cells exposed to copper. Even though wild-type and mutant cells are undistinguishable in terms of their morphological features and rates of growth, our results show that MT-/- cells do not respond with induction of typical markers of cellular stress under copper excess conditions, as observed in the wild-type cell line, suggesting that the transcription initiation rate or the mRNA stability of stress genes is affected when there is an alteration in the copper store capacity. The functional classification of other up-regulated genes in both cell lines indicates that a large proportion (>80%) belong to two major categories: 1) metabolism; and 2) cellular physiological processes, suggesting that at the transcriptional level copper overload induces the expression of genes associated with diverse molecular functions. These results open the possibility to understand how copper homeostasis is being coordinated with other metabolic pathways. Key terms: copper homeostasis, metallothionein, microarray INTRODUCTION 2004; Leonard et al., 2004). To avoid Copper is an essential nutrient that is a metal-induced toxicity, most organisms structural part in some proteins and part of have developed several cellular the electron transfer system in many redox- mechanisms of protection. Three general enzymes involved in processes such as mechanisms, which typically work in respiration, iron metabolism, and combination for effective detoxification, neurotransmitter biosynthesis (Uauy et al., include: reduction of metal uptake; 1999). Although essential metals are enhanced metal export; and metal normally present in trace amounts in the sequestration mechanisms (Dameron and cell, their levels can increase following Harrison, 1998). The third mechanism, the environmental or nutritional changes. Metal intracellular chelation or sequestration of overload can be toxic to the cell, causing a metals into less reactive complexes or range of effects and leading to cell death organelles to limit their toxicity, is a when concentrations are extremely high commonly used mechanism. In mammalian (Pulido and Parrish, 2003; Oteíza et al., systems, excess metals, in particular Corresponding author: Mauricio González, Laboratorio de Bioinformática y Expresión Génica, INTA, Universidad de Chile, Macul 5540, Macul, Santiago, Chile, Tel.: (56-2) 978-1440, Fax: 56-2-221-4030, E-mail: [email protected] Received: February 15, 2005. Accepted June, 25 2005. 126 ARMENDÁRIZ ET AL. Biol Res 39, 2006, 125-142 copper, are partially detoxified by 1996; Kelly and Palmiter, 1996; Liu et al., sequestration in the metal-binding 1999; Rojas and Klaassen, 1999; Liu et al., metallothioneins (Dameron and Harrison, 2000; Qu et al., 2002). 1998; Bremner and Beattie, 1990; The role of MT in copper homeostasis is Nordberg, 1989). of great interest, as it appears to be Metallothioneins (MTs) are a class of responsible for the regulation of low molecular weight, intracellular and intracellular copper levels during adaptation cysteine-rich proteins that have high to extracellular copper excess. It has been affinity for metal ions (Cousins, 1983; Park shown that MT protects cells against et al., 2001; Coyle et al., 2002). MT gene elevated levels of extracellular copper sequences are highly conserved in a wide (Freedman and Peisach, 1989; Kawai et al., range of species from bacteria to humans, 2000). As copper toxicity involves its which is a suggestive feature of a protein ability to catalyze the generation of free with a high biological importance. MTs radicals and/or to directly interact with have unique structural characteristics for essential biomolecules, copper sequestering their potent metal-binding and redox by MT is of critical importance for cell capabilities. Although the members of this protection. Besides serving to detoxify family were discovered nearly 40 years ago, excess copper, MT may play a role in a primary role has not been identified, and normal copper metabolism, although new functions continue to be discovered currently this role is unknown. (Palmiter, 1998). Currently, MTs are known In a recent study, we demonstrated to be involved in metal ion homeostasis and notable changes in copper metabolism in a detoxification, protection against oxidative MT I/II mutant (MT-/-) fibroblast cell line damage, cell proliferation and apoptosis, (Tapia et al., 2004). We showed that MT-/- chemoresistance, and radiotherapy cells were more sensitive to increasing resistance (Palmiter, 1998; Coyle et al., amounts of copper in the media than wild- 2002). MT expression has been implicated type cells exposed to the same as a transient response to any form of stress concentrations. Also, by measuring or injury and may provide cytoprotective intracellular copper levels and by conducting action. Four major MT isoforms – MT I, uptake studies, we demonstrated that MT II, MT III, and MT IV – have been although both mutant and wild type cell lines identified in mammals (Coyle et al., 2002; accumulate copper upon treatment with Theocharis et al., 2003). MT I and MT II copper, the MT-/- cells took up considerably are the two major forms in mammals; they less copper than the wild-type cells. are expressed in most tissues and stages of Interestingly, in this condition, we observed development. The analysis of mice with that mutant cells died with lower altered gene expression of MT I and II has intracellular copper content (2-fold lower), enhanced our understanding of the supporting a protective role for MT in the multifaceted role of MT. MT I/II double response to Cu excess. These results knockout (MT-/-) mice demonstrated that permitted us to deduce that copper toxicity MT expression is not essential for the does not rely on the increased amount of normal development, growth, or intracellular metal, but rather on the cellular reproductive capacity of these mice ability to manage the metal, therefore, in the (Klaassen and Liu, 1998). These mutant molecular interactions that copper mice and the cell lines derived from them establishes inside the cell. In this context, have been used extensively in recent years we showed that in the absence of MTs, the to investigate the role of MT. Most results capacity of copper to induce gene expression show that animals or cells lacking MT I/II of MT, SOD1 and its chaperone, Ccs, is lost. are more sensitive to a wide range of This observation could partially explain the stressors, such as oxidative stress; excess increased vulnerability of MT null cells to metals, such as cadmium, lead, copper and lower intracellular copper concentrations and zinc; and infectious and inflammatory suggest that MT may be a modulator of gene agents (Zheng et al., 1996; Kelly et al., expression (Tapia et al., 2004). ARMENDÁRIZ ET AL. Biol Res 39, 2006, 125-142 127 In the present study, we show that wild- figure 2 legend. After the treatment, cells type and mutant cells are undistinguishable were processed for Cu, Zn and Fe in terms of their morphological features and quantification. To evaluate cell viability rates of growth and define a minimal under copper treatment, cells were grown in copper dose that provided a significant 24-well cell culture plates (Nunc) and increment in the copper content of both cell relative survival of the treated cells was lines. Using these conditions, we applied a evaluated using 3-[4,5-dimethylthiazol-2- genomic approach to further investigate a yl]-2,5- diphenyltetrazolium bromide possible role of MTs as part of a pathway (MTT, Sigma) reduction assays as that induces gene expression in response to described (Denizot and Lang, 1986). The copper. As a first step in exploring the viability percentage was determined by effects that loss of this important copper comparing the measurements of the average sequestering protein had on gene absorbance from a given treatment group expression, we carried out microarray with that obtained from a reference sample analysis to identify genes significantly up- of the same cell line treated for identical regulated in wild-type and MT-/- cells time with a control medium (100% of exposed to an excess of copper and to viability). To determine the population determine which genes failed to be doubling time, the cell number was counted upregulated in MT mutant cells as under a microscope daily for at least 5 days compared to wild-type cells. The results of using a hemocytometer and the trypan blue these analyses are presented here. (0.4%) exclusion method. Immunofluorescence staining MATERIALS AND METHODS Cells were washed three times with Cell lines and treatments phosphate buffered saline (PBS), fixed for 10 min in 3.7% formaldehyde in PBS and The cell lines used in the present study permeabilized for 5 min with 0.2% triton X- correspond to wild type and MT I/II mutant 100 in 3.7% formaldehyde. The fixed cells (MT-/-) fibroblasts obtained by were re-hydrated with Tris buffered saline trypsinization of mouse embryos from day (TBS) and incubated for 1 h in blocking 11 of gestation and immortalized with solution (3% BSA in TBS).