Dennis J. Thiele

Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals

Cisplatin is a chemotherapeutic drug used to treat a variety of cancers. Both intrinsic and acquired resistance to cisplatin, as well as toxicity, limit its effectiveness. Molecular mechanisms that underlie cisplatin resistance are poorly understood. Here we demonstrate that deletion of the yeast CTR1 gene, which encodes a high-affinity copper transporter, results in increased cisplatin resistance and reduced intracellular accumulation of cisplatin. Copper, which causes degradation and internalization of Ctr1 protein (Ctr1p), enhances survival of wild-type yeast cells exposed to cisplatin and reduces cellular accumulation of the drug. Cisplatin also causes degradation and delocalization of Ctr1p and interferes with copper uptake in wild-type yeast cells. Mouse cell lines lacking one or both mouse Ctr1 (mCtr1) alleles exhibit increased cisplatin resistance and decreased cisplatin accumulation in parallel with mCtr1 gene dosage. We propose that cisplatin uptake is mediated by the copper transporter Ctr1p in yeast and mammals. The link between Ctr1p and cisplatin transport may explain some cases of cisplatin resistance in humans and suggests ways of modulating sensitivity and toxicity to this important anticancer drug.

Mechanisms for copper acquisition, distribution and regulation

Copper (Cu) is a redox-active metal ion essential for most aerobic organisms. Cu serves as a catalytic and structural cofactor for enzymes that function in energy generation, iron acquisition, oxygen transport, cellular metabolism, peptide hormone maturation, blood clotting, signal transduction and a host of other processes. The inability to control Cu balance is associated with genetic diseases of overload and deficiency and has recently been tied to neurodegenerative disorders and fungal virulence. The essential nature of Cu, the existence of human genetic disorders of Cu metabolism and the potential impact of Cu deposition in the environment have been driving forces for detailed investigations in microbial and eukaryotic model systems. Here we review recent advances in the identification and function of cellular and systemic molecules that drive Cu accumulation, distribution and sensing.

Molecular mechanisms of copper uptake and distribution.

In the past few years, exciting advances have been made toward understanding how copper is transported into and distributed to cupro-proteins within cells. Recent work has identified high-affinity copper transporters at the plasma membrane in a number of organisms. The elucidation of the three-dimensional structure of copper chaperones and target cupro-proteins has shown that highly specific interactions between homologous domains foster copper transfer between conserved copper ligands, and facilitate a detailed understanding of vectorial copper-transfer reactions. Furthermore, the recent generation of mouse-knockout models, deficient in a high-affinity copper transporter, or in copper chaperones, has demonstrated the importance of copper uptake and targeted distribution in both predicted and fascinating unanticipated ways in growth and development.

Genome-Wide Analysis of the Biology of Stress Responses through Heat Shock Transcription Factor

ABSTRACT Heat shock transcription factor (HSF) and the promoter heat shock element (HSE) are among the most highly conserved transcriptional regulatory elements in nature. HSF mediates the transcriptional response of eukaryotic cells to heat, infection and inflammation, pharmacological agents, and other stresses. While HSF is essential for cell viability in Saccharomyces cerevisiae, oogenesis and early development in Drosophila melanogaster, extended life span in Caenorhabditis elegans, and extraembryonic development and stress resistance in mammals, little is known about its full range of biological target genes. We used whole-genome analyses to identify virtually all of the direct transcriptional targets of yeast HSF, representing nearly 3% of the genomic loci. The majority of the identified loci are heat-inducibly bound by yeast HSF, and the target genes encode proteins that have a broad range of biological functions including protein folding and degradation, energy generation, protein trafficking, maintenance of cell integrity, small molecule transport, cell signaling, and transcription. This genome-wide identification of HSF target genes provides novel insights into the role of HSF in growth, development, disease, and aging and in the complex metabolic reprogramming that occurs in all cells in response to stress.

Essential role for mammalian copper transporter Ctr1 in copper homeostasis and embryonic development

The trace metal copper (Cu) plays an essential role in biology as a cofactor for many enzymes that include Cu, Zn superoxide dismutase, cytochrome oxidase, ceruloplasmin, lysyl oxidase, and dopamine β-hydroxylase. Consequently, Cu transport at the cell surface and the delivery of Cu to intracellular compartments are critical events for a wide variety of biological processes. The components that orchestrate intracellular Cu trafficking and their roles in Cu homeostasis have been elucidated by the studies of model microorganisms and by the characterizations of molecular basis of Cu-related genetic diseases, including Menkes disease and Wilson disease. However, little is known about the mechanisms for Cu uptake at the plasma membrane and the consequences of defects in this process in mammals. Here, we show that the mouse Ctr1 gene encodes a component of the Cu transport machinery and that mice heterozygous for Ctr1 exhibit tissue-specific defects in copper accumulation and in the activities of copper-dependent enzymes. Mice completely deficient for Ctr1 exhibit profound growth and developmental defects and die in utero in mid-gestation. These results demonstrate a crucial role for Cu acquisition through the Ctr1 transporter for mammalian Cu homeostasis and embryonic development.

ACE1 regulates expression of the Saccharomyces cerevisiae metallothionein gene

Copper resistance in Saccharomyces cerevisiae is mediated, in large part, by the CUP1 locus, which encodes a low-molecular-weight, cysteine-rich metal-binding protein. Expression of the CUP1 gene is regulated at the level of transcriptional induction in response to high environmental copper levels. This report describes the isolation of a yeast mutant, ace1-1, which is defective in the activation of CUP1 expression upon exposure to exogenous copper. The ace1-1 mutation is recessive and lies in a genetic element that encodes a trans-acting CUP1 regulatory factor. The wild-type ACE1 gene was isolated by in vivo complementation and restores copper inducibility of CUP1 expression and copper resistance to the otherwise copper-sensitive ace1-1 mutant. Linkage analysis and gene deletion experiments verified that this gene represents the authentic ACE1 locus. ACE1 maps to the left arm of chromosome VII, 9 centimorgans centromere distal to lys5. The ACE1 gene appears to play a direct or indirect positive role in activation of CUP1 expression in response to elevated copper concentrations.

COPPER-SPECIFIC TRANSCRIPTIONAL REPRESSION OF YEAST GENES ENCODING CRITICAL COMPONENTS IN THE COPPER TRANSPORT PATHWAY

Copper is an essential micronutrient that is toxic in excess. To maintain an adequate yet non-toxic concentration of copper, cells possess several modes of control. One involves copper uptake mediated by genes encoding proteins that play key roles in high affinity copper transport. These include the FRE1-encoded Cu2+/Fe3+ reductase and the CTR1and CTR3-encoded membrane-associated copper transport proteins. Each of these genes is transcriptionally regulated as a function of copper availability: repressed when cells are grown in the presence of copper and highly activated during copper starvation. Our data demonstrate that repression of CTR3 transcription is exquisitely copper-sensitive and specific. Although copper repressesCTR3 gene expression at picomolar metal concentrations, cadmium and mercury down-regulate CTR3 expression only at concentrations 3 orders magnitude greater. Furthermore, copper-starvation rapidly and potently induces CTR3 gene expression. We demonstrate that the CTR1, CTR3, and FRE1 genes involved in high affinity copper uptake share a common promoter element, TTTGCTC, which is necessary for both copper repression and copper-starvation activation of gene expression. Furthermore, the Mac1p is essential for down- or up-regulation of the copper-transport genes. In vivo footprinting studies reveal that the cis-acting element, termed CuRE (copper-response element), is occupied under copper-starvation and accessible to DNA modifying agents in response to copper repression, and that this regulated occupancy requires a functional MAC1 gene. Therefore, yeast cells coordinately express genes involved in high affinity copper transport through the action of a common signaling pathway.

New Roles for Copper Metabolism in Cell Proliferation, Signaling, and Disease

Over the past 2 decades, the molecular mechanisms by which cells acquire, distribute, and utilize copper have been under intense investigation. Significant progress has been made in the identification of genes encoding copper homeostasis proteins and in fundamental aspects of their structure, function, and mechanisms of action in copper balance. A number of more comprehensive reviews of the field with respect to the genetics, structure, function, and physiology of copper metabolism have recently appeared elsewhere (1–4). Here, we review general mechanisms for eukaryotic copper metabolism at the cellular level in the context of recent discoveries in the field, identifying potential new functions for copper and copper metabolism proteins in cell signaling, gene expression, tumor cell metastasis, and resistance to anti-neoplastic drugs.

Isolation of a murine copper transporter gene, tissue specific expression and functional complementation of a yeast copper transport mutant

A polymerase chain reaction (PCR)-based strategy was used to isolate a mouse cDNA (mCtr1) encoding a Cu transport protein. The deduced mCtr1 protein sequence exhibits 92% identity to human Ctr1, and has structural features in common with known high affinity Cu transporters from yeast. The expression of mouse Ctr1 functionally complements bakers yeast cells defective in high affinity Cu transport. Characterization of the mCtr1 genomic clone showed that the mCtr1 coding sequence is encompassed within four exons and that the mCtr1 locus maps to chromosome band 4C1-2. RNA blotting analysis demonstrated that mCtr1 is ubiquitously expressed, with high levels in liver and kidney, and early in embryonic development. Steady state mammalian Ctr1 mRNA levels were not changed in response to cellular Cu availability, which is distinct from the highly Cu-regulated transcription of genes encoding yeast high affinity Cu transporters. These studies provide fundamental information for further investigations on the function and regulation of Ctr1 in Cu acquisition in mammals.

Characterization of mouse embryonic cells deficient in the ctr1 high affinity copper transporter. Identification of a Ctr1-independent copper transport system.

The trace metal copper is an essential cofactor for a number of enzymes that have critical roles in biological processes, but it is highly toxic when allowed to accumulate in excess of cellular needs. Consequently, homeostatic copper metabolism is maintained by molecules involved in copper uptake, distribution, excretion, and incorporation into copper-requiring enzymes. Previously, we reported that overexpression of the human or mouse Ctr1 copper transporter stimulates copper uptake in mammalian cells, and deletion of one Ctr1 allele in mice gives rise to tissue-specific defects in copper accumulation and in the activities of copper-dependent enzymes. To investigate the physiological roles for mammalian Ctr1 protein in cellular copper metabolism, we characterized wild type, Ctr1 heterozygous, and Ctr1 homozygous knock-out cells isolated from embryos obtained by the inter-cross of Ctr1 heterozygous mice. Ctr1-deficient mouse embryonic cells are viable but exhibit significant defects in copper uptake and accumulation and in copper-dependent enzyme activities. Interestingly, Ctr1-deficient cells exhibit ∼30% residual copper transport activity that is saturable, with a K m of ∼10 μm, with biochemical features distinct from that of Ctr1. These observations demonstrate that, although Ctr1 is critical for both cellular copper uptake and embryonic development, mammals possess additional biochemically distinct functional copper transport activities.