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Effects of cadmium on DNA-(Cytosine-5) methyltransferase activity and DNA methylation status during cadmium-induced cellular transformation

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Abstract

Cadmium is a human carcinogen that likely acts via epigenetic mechanisms. Since DNA methylation alterations represent an important epigenetic event linked to cancer, the effect of cadmium on DNA methyltransferase (MeTase) activity was examined using in vitro (TRL1215 rat liver cells) and ex vivo (M.SssI DNA MeTase) systems. Cadmium effectively inhibited DNA MeTases in a manner that was noncompetitive with respect to substrate (DNA), indicating an interaction with the DNA binding domain rather than the active site. Based on these results, the effects of prolonged cadmium exposure on DNA MeTase and genomic DNA methylation in TRL1215 cells were studied. After 1 week of exposure to 0–2.5 μM cadmium, DNA MeTase activity was reduced (up to 40%) in a concentration-dependent fashion, while genomic DNA methylation showed slight but significant reductions at the two highest concentrations. After 10 weeks of exposure, the cells exhibited indications of transformation, including hyperproliferation, increased invasiveness, and decreased serum dependence. Unexpectedly, these cadmium-transformed cells exhibited significant increases in DNA methylation and DNA MeTase activity. These results indicate that, while cadmium is an effective inhibitor of DNA MeTase and initially induces DNA hypomethylation, prolonged exposure results in DNA hypermethylation and enhanced DNA MeTase activity.

Introduction

Several different metal compounds have been identified as human carcinogens, including the toxic transition metal cadmium [1], [2], [3]. Epidemiological studies have provided evidence that occupational and/or environmental exposure to cadmium is associated with human pulmonary cancers and possibly cancers in other tissues [1], [3], [4]. Chronic rodent studies are generally confirmatory of the human epidemiology of cadmium oncogenesis [1], [3], [4]. Despite this, the mechanisms underlying cadmium carcinogenesis remain poorly understood. Even though much effort has been directed toward elucidation of the molecular mechanisms that underlie the process of cadmium as a carcinogen, no single mechanism has yet been identified unequivocally and the actual means by which this metal initiates the carcinogenic process is unknown. Cadmium does not form stable adducts with DNA [5], thus eliminating this as a potential means of DNA damage leading to mutation. Furthermore, although cadmium can be genotoxic in eukaryotic cells in vitro, this typically requires levels that completely arrest cell growth [6], and how this might bear on events in vivo is unclear. There is evidence that cadmium may be indirectly genotoxic by compromising the cell’s ability to accurately replicate DNA [7], by perturbing apoptosis [8], [9], or by increasing background mutagenesis [6]. In addition, cadmium is not a directly active redox metal, a potential mechanism of oxidative DNA damage-induced genotoxicity for some heavy metals. Thus, the mechanism of cadmium may well have a nongenotoxic or epigenetic component.

In mammals, the methylation of cytosine residues in DNA by DNA (5-cytosine) methyltransferase (DNA MeTase) is the predominant postreplication base modification. The function of these methylation sites, which are frequently found in CpG dinucleotide islands of promoter regions in mammalian DNA, is related to imprinting and the regulation of gene transcription [10], [11], [12], [13], [14]. Mammalian DNA MeTases are a group of enzymes that transfer a methyl group from S-adenosylmethionine (SAM) onto the C-5 position of cytosine within the CpG dinucleotides of double-stranded DNA [15]. A prokaryotic DNA MeTase purified from Spiroplasma, M.SssI, is now widely used as a model to study the effects of DNA methylation in mammals since this bacterial enzyme recognizes the same sequence (CpG) as mammalian DNA MeTases [16], [17]. Defects in methylation, including hypomethylation and hypermethylation, of genomic DNA or particular DNA sequences have been shown to be associated with the carcinogenic process, possibly as a factor in causation that facilitates aberrant under- or overexpression of genes linked to oncogenesis [13], [18], [19], [20], [21]. Understanding what modulates changes in the DNA methylation patterns during malignant transformation is an important issue in chemical carcinogenesis, including carcinogenesis by inorganics. For instance, DNA methylation changes appear to occur with various carcinogenic inorganics, including arsenic [22] and nickel [23], and appear to be a potentially important aspect of their carcinogenic mechanism. Although, in general terms, cadmium is often a potent enzyme inhibitor [24], the effects of cadmium on DNA MeTase or DNA methylation status are unknown.

In the present study we have used rat liver epithelial cells (TRL 1215) to define the effects of cadmium in vitro on DNA MeTase activity and genomic DNA methylation status during acute exposure and chronic exposure resulting in morphological transformation. In addition, M.SssI was used in ex vivo experiments to define the effects of cadmium on DNA MeTase enzyme kinetics. Effects of cadmium on DNA methylation status may be an important aspect in aberrant gene expression and, potentially, carcinogenesis.

Section snippets

Cell culture and treatments

The TRL 1215 cell line was originally derived from the livers of 10-day-old Fisher 344 rats as previously described [25]. The cells are diploid and normally nontumorigenic. The cells were cultured in William’s E medium (Gibco-BRL Products, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cultures were maintained in a humidified atmosphere of 5% CO2/95% air at 37°C. For the in vitro acute study of the effects of

Acute effects of cadmium on DNA MeTase in vitro and ex vivo

Cadmium frequently inhibits enzymes as part of its toxicity. Thus, initial studies investigated the acute effects of cadmium on DNA MeTase activity in TRL 1215 liver cells. DNA MeTase was assessed in cell lysates by measuring the incorporation of radioactive methyl groups from [3H]-SAM (specifically labeled at the donated methyl group) onto the poly[dI · dC] · poly[dI · dC] substrate. TRL 1215 cells were exposed to 10 μM cadmium for 24 h and compared with untreated control cells. This

Discussion

Cadmium is classified as a known human carcinogen by the International Agency for Research on Cancer [1] and the National Toxicology Program [2]. Exposure to cadmium, either environmentally or occupationally, has been definitively associated with lung cancer in humans [1], [2]. In addition, there is evidence associating human cadmium exposure with cancers of the liver, prostate, kidney, hematopoietic system, and stomach [3], [4]. In rodents, cadmium has been shown to induce tumors of the lungs,

Acknowledgements

We are grateful to Dr. M. Watanabe, Mie University; Japan, for help with the cell invasion assay and to Dr. Jie Liu and Dr. Hua Chen for critical evaluation of this manuscript.

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    Current addresses: Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, 770-8514 Japan (M. Takiguchi). Neurogen Corporation, 35 NE Industrial Road, Branford, CT 06405, USA (G. Li).

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