Changes in gene expression profiles of human fibroblasts in response to sodium arsenite treatment

Ling-Huei Yih1, Konan Peck1 and Te-Chang Lee1,2,3

1 Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, Republic of China and
2 Institute of Pharmacology, School of Life Science, National Yang-Ming University, Taipei 112, Taiwan, R.O.C.


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Arsenic compounds are widely distributed and arsenic ingestion is associated with many human diseases, including blackfoot disease, atherosclerosis, and cancers. However, the underlying mechanism of arsenic toxicity is not understood. In human fibroblast cells (HFW), arsenite is known to induce oxidative damage, chromosome aberrations, cell cycle arrest, and aneuploidy, and the manifestation of these cellular responses is dependent on changes in gene expression which can be analyzed using the cDNA microarray technique. In this study, cDNA microarray membranes with 568 human genes were used to examine mRNA profile changes in HFW cells treated for 0 to 24 h with 5 µM sodium arsenite. On the basis of the mean value for three independent experiments, 133 target genes were selected for a 2 x 3 self-organizing map cluster analysis; 94 were found to be induced by arsenite treatment, whereas 39 were repressed. These genes were categorized as signal transduction, transcriptional regulation, cell cycle control, stress responses, proteolytic enzymes, and miscellaneous. Significant changes in the signaling-related and transcriptional regulation genes indicated that arsenite induces complex toxicopathological injury.

Abbreviations: EST, expressed-sequence tag; HFW, human fibroblasts; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Arsenic compounds are ubiquitous in the environment. Arsenic ingestion, usually through drinking arsenic-contaminated well water, is associated with an increased incidence of a variety of human diseases, such as blackfoot disease, atherosclerosis, diabetics, and cancers (1–3). However, the underlying mechanisms involved in the arsenic-induced pathological changes are largely unknown. In human populations chronically exposed to arsenic, peripheral lymphocytes show an increased frequency of sister chromatid exchange, chromosomal aberration, and micronuclei (4–6). An association between an increased frequency of chromosomal aberration in peripheral lymphocytes and an increased risk of cancer has been demonstrated in a blackfoot disease endemic area (7). The induction of cytogenetic changes by arsenic is supported by the results of numerous in vitro studies (4,8,9). In addition, arsenic treatment results in cell cycle arrest (10), mitosis disturbance (10,11), aneuploidy (10), cytoskeleton disruption (12,13), and apoptosis (14,15). It is therefore apparent that arsenic exposure causes pleiotropic injuries and leads to chromosomal alterations.

Recently, evidence has accumulated showing that reactive oxygen or nitrogen species are involved in arsenic-induced cellular injury (16–18). Cellular responses to injuries or stresses are complicated, but are important in protecting the cell against deleterious effects (19). In response to physiological injury or chemical stress, a variety of cellular defense and repair pathways are activated to maintain cellular integrity or remove seriously damaged cells; one example is the activation of p53 and NF{kappa}B as an early response to ionizing radiation (20,21). At relatively high doses and a short exposure time, arsenite stimulates JNK activity by inhibiting a JNK phosphatase (22) or enhancing the activity of MEKKs (23) and activates ERK via the Ras, Raf, and MEK signaling cascade (24). Arsenite activates the mitogenic ERK cascade primarily via a Ras-dependent pathway, possibly mediated by EGFR and Shc (25), whereas it activates the stress-activated p38 cascade via a Ras-independent mechanism (26). Recent reports have shown that arsenite exposure induces stress protein expression through various signaling cascades (27,28). In contrast, high levels of arsenite inhibit cellular responses to a variety of stimuli, such as inflammatory cytokines (29), growth factors (30,31), and lipopolysaccharide (32). Thus, the response to arsenite exposure is complex.

Monitoring the expression pattern of a panel of genes may help to reveal the role of genes in the response to physical or chemical stress and to elucidate the mechanism(s) involved (33). The recently developed cDNA microarray technology is an effective tool for simultaneously analyzing the expression of a large number of genes (34–36) and this approach has been used to show that arsenic exposure induces changes in the expression of genes associated with various aspects of cellular biochemistry and physiology in in vitro and in vivo systems (37–39). We have previously demonstrated that arsenite treatment can induce DNA damage, p53 accumulation, mitotic disturbance, and chromosomal loss in the human skin fibroblast cells, HFW (9,40). In the present study, we examined expression profiles in these cells in response to arsenic exposure using a colorimetric cDNA microarray method (41). Our results indicate that arsenite triggers a series of signaling cascades responsible for arsenite-induced insults or stress.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture
HFW cells, derived from human newborn foreskin, were kindly provided by Dr W.N.Wen (National Taiwan University, Taipei) and were routinely maintained in Dulbecco's modified Eagle medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 0.37% sodium bicarbonate, and antibiotics (complete medium) at 37°C in an incubator with water-saturated air/10% CO2 (42). HFW cells are normal human fibroblast cells manifested a normal and stable karyotype and a definite lifespan (10). They were also used in several other studies on metal toxicity (9,40,43–45).

Arsenite treatment
The inorganic trivalent arsenic compound, sodium arsenite (Merck, Darmstadt, Germany), was dissolved in distilled water immediately prior to use. Logarithmically growing cells were treated for 0, 1, 2, 4, 8, 12, 16, and 24 h with 5 µM sodium arsenite in complete medium (10). Cellular RNAs were harvested at equally subconfluent states in all treatments.

Preparation of the cDNA microarray
A 5.5 x 5.5 mm nylon membrane carrying 576 cDNA probes was prepared from IMAGE consortium human EST clones as previously described (41). Eight of the probes, derived from plant genes, served as controls, while the rest were human genes and included signaling-related genes (40%), transcription regulatory genes (15%), and cell cycle regulatory genes (8%). The remaining 33% of the probes covered a wide range of functions, such as proteolytic activity, stress responsiveness, cytoskeletal proteins, and cell–cell adhesion. Approximately 4% of the probes were of unknown identity and are labeled as expressed-sequence tag (EST). The signaling-related genes included cytokines, growth factors, receptors, protein kinases, phosphatases, and genes involved in signal transduction, while the transcription regulatory genes consisted mainly of transcription factors and a small number of splicing regulatory genes. Detailed information on these probe clones can be found on the anonymous ftp server, ftp://genestamp.ibms.sinica.edu.tw/arrayinfo/arsenite.

mRNA and cDNA target preparation and Southern hybridization
After arsenite treatment, total cellular RNA was extracted using Tri-reagent (Molecular Research Center, Cincinnati, OH) and mRNA subsequently isolated using Oligotex-dT resin (Qiagen, Hilden, Germany). Biotin-labeled cDNA targets were prepared by reverse transcription of mRNA. In brief, 1 µg of mRNA was reverse transcribed to cDNA using 200 units of MMLV reverse transcriptase in 50 µl of solution containing 6 mM random primers (GIBCO), 0.5 mM each of dATP, dCTP, and dGTP, 40 mM dTTP, 40 mM biotin-16-dUTP (Roche Diagnostic, Mannheim, Germany), 10 mM dithiothreitol, and 0.5 units/ml of RNase inhibitor (GIBCO). The reaction mixture was incubated at 25°C for 10 min, then at 42°C for 90 min, and the reaction terminated by heating at 99°C for 5 min. The remaining RNA was digested by addition of 5.5 µl of 3 N NaOH and incubation at 55°C for 30 min. After neutralization with 5.5 µl of 3 N acetic acid, the cDNA targets were precipitated by addition of 50 µl of 7.5 M ammonia acetate, 20 mg of carrier linear polyacrylamide, 375 µl of absolute ethanol, and water to a total volume of 525 µl. The biotin-labeled cDNA targets were dissolved in an appropriate amount of hybridization buffer and incubated for 16 h at 65°C with pre-hybridization treated arrays, as previously described (41). The arrays were then thoroughly washed twice for 5 min at room temperature with 2x SSC containing 0.1% sodium dodecyl sulfate (SDS), then three times for 15 min at 65°C with 0.1x SSC containing 0.1% SDS.

Colorimetric detection and image analysis.
After thorough washes, the arrays were blocked by incubation for 1 h at room temperature with 1% blocking solution (Roche Diagnostic) containing 2% dextran sulfate, then rinsed with Tris-buffered saline (10 mM Tris–HCl, pH 7.4, 150 mM NaCl). For color development, the arrays were incubated with ß-galactosidase-conjugated streptavidin (1:700 dilution in 1'TBS, GIBCO) for 1 h at room temperature, washed three times with TBS, then incubated for 30 min at 37°C with TBS containing 1.2 mM X-gal, 1 mM MgCl2, 3 mM K2Fe(CN)6, and 3 mM K4Fe(CN)6. Color development was terminated by addition of phosphate-buffered saline containing 20 mM disodium ethylenediaminetetraacetate. The intensity of spots on arrays was determined by digitizing the arrays using a flatbed scanner at 3048 dots per inch (dpi, optical resolution) and further analyzed using an in-house-produced computer program (41).

Northern blot analysis
To confirm the differential gene expression identified by microarray analysis, several genes were chosen for further analysis using the northern blot technique. Polyadenylated RNAs were prepared as described above. One µg of polyadenylated RNA was loaded into each slot, separated by electrophoresis through 1% agarose formaldehyde gels, and blotted onto a nylon membrane for 15–17 h by capillary blotting using 20x SSC. The RNAs were then fixed on the nylon membrane by UV-crosslinking (0.12 J/cm2). CDNA probes were prepared by RT-PCR of specific genes from total RNA in the presence of digoxigenin-11-dUTP (Roche Diagnostic) using primers selected by the program `xprimer' (Virtual Genome Center). Hybridization and immunological detection of digoxigenin-labeled cDNA were performed by a modification of the method described by Engler-Blum et al. (46). Digoxigenin-labeled cDNA probes were immunologically detected by incubating the membrane with alkaline phosphatase-conjugated anti-digoxigenin antibodies (Roche Diagnostic). After thorough washing with 0.1 M maleic acid, 3 M NaCl, and 0.3% Tween 20, pH 8.0, chemiluminescent substrates of alkaline phosphatase (CDP-Star, Roche Diagnostic) were applied to the membrane and the level of specific gene expression recorded by exposing the membrane for 3–15 min to X-ray film (Eastman Kodak Company, Rochester, NY).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Temporal expression profiles in arsenite-treated HFW cells
We have previously shown that, after treatment with 5 µM arsenite for up to 24 h, HFW cells remain viable and maintain their membrane integrity, but show a significant increase in chromatid breaks, micronuclei, and DNA strand breaks (9,10,40). To detect changes in expression profile in arsenite-treated HFW cells, a cDNA microarray containing 568 human cDNA clones was used to examine the expression profile following treatment for 0, 1, 2, 4, 8, 12, 16, and 24 h with 5 µM sodium arsenite. Polyadenylated RNAs were isolated, labeled with biotin-conjugated dUTP, hybridized to the cDNA microarrays, and stained with ß-galactosidase-conjugated streptavidin (Figure 1Go). The color intensity, reflecting the expression level of each individual gene, was analyzed using a computer program written in-house (41). For analysis, the intensity of each gene was averaged from three independent experiments. To study genes showing temporal, but significant, profile changes, we chose those which showed at least a 1.5-fold difference in expression in the presence of arsenite compared to controls at a minimum of two time-points. Using these criteria, 133 cDNA EST clones were chosen for cluster analysis using the GENECLUSTER program (47), which is a computer package to produce and display self-organizing maps (SOM). On the basis of similar expression patterns, the differentially expressed genes were grouped into six clusters by the algorithm of a 3 x 2 SOM (Figure 2Go). The SOM is one of many mathematical techniques that have been developed for identifying underlying patterns in complex data. An SOM has a set of nodes with a simple topology (such as a 3 x 2 grid) and a distance function on the nodes. By iterative adjusting each data point to the node, SOM thus imposes structure on data with neighboring nodes tending to define related clusters.



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Fig. 1. Representative images of colorimetric cDNA microarrays. Expression patterns in HFW cells treated with 5 µM arsenite for 0, 1, 2, 4, 8, 12, 16, or 24 h. cDNA microarrays with 568 human cDNA clones were hybridized with biotin-labeled cDNA targets from untreated or 5 µM arsenite-treated HFW cells as described in Materials and methods. Images were digitized using a flat-bed scanner at 3048 dots per inch optical resolution. The arrows and arrow heads indicate the spots corresponding, respectively, to HSPA1 (T50400) and EGR1 (H42051).

 


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Fig. 2. Temporal expression profiles of 133 genes in arsenite-treated HFW cells grouped into six clusters (c1–c6). A total of 133 genes were selected by the criteria described above and the similarity in expression levels was calculated using the program GENECLUSTER. The thick lines and dots are the average expression levels for each gene cluster, while the thin lines indicate the standard error for each time-point. The number in the bottom right corner of each panel represents the number of clones in the cluster.

 
Clusters 1 and 2 contained, respectively, 25 and 24 genes that tended to show a high (cluster 1) or moderate (cluster 2) increase in expression at all time-points of arsenite treatment. Cluster 3 contained 45 genes that displayed a delayed increase. Clusters 5 (14 genes) and 6 (5 genes) contained genes that showed either a slow (cluster 5) or sharp (cluster 6) decrease in expression on treatment with arsenite. The 20 genes in cluster 4 showed an immediate drop on exposure to arsenite, but their expression levels quickly recovered, then fluctuated. Of these 133 EST clones, the 112 with known gene function were categorized as signaling molecules, transcription regulators, cell cycle regulators, stress-responsive molecules, or miscellaneous. As summarized in Table IGo, 46 signaling-related genes and 24 transcription regulatory genes were differentially expressed in arsenite-treated cells as compared to untreated controls. These signaling-related genes were involved in various cellular pathways, such as cell survival, cell death, and cell cycle regulation. The significant changes seen in the expression of these genes demonstrate that arsenite induces complex toxicopathological injury. To simplify the description of each gene, the gene symbols recommended in the UniGene database at the National Center for Biotechnology Information, USA, were used throughout this paper. The genes that showed differential expression are listed in Table IGo.


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Table I. List of differentially expressed genes in arsenite-treated HFW cellsa
 
The altered expression of several arsenite-responsive genes, i.e. HSPA1, p450 oxidoreductase (POR), CCNG1, DDIT3/GADD153, GADD45A, PLAU, and EGR1, was confirmed in independent experiments by Northern blot analysis, the arsenite-inducible HSPA1 and HO-1 genes and the arsenite-suppressed NM23 gene being included as controls (Figure 3Go).



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Fig. 3. Northern blot analysis confirming that several genes show altered expression following arsenite treatment of HFW cells. HFW cells were treated with 5 µM arsenite for 0, 1, 6, or 16 h, then polyadenylated RNAs were prepared and subjected to Northern analysis with each probe as described in Materials and methods. All blots were then reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control.

 
Genes elevated by arsenite in HFW cells
The genes in cluster 1 were significantly elevated after 2 h treatment with arsenite and their expression increased substantially during arsenite treatment, while the genes in cluster 2 showed an immediate, but moderate, increase on arsenite treatment. Of the 49 genes in clusters 1 and 2, 16 could be classified as signaling-related genes and included receptors (IFNGR1 and TNFRSF6), tyrosine kinases (PTK2, KIT and ABL1), and RAS signaling-related genes (NRAS, RASA1, and RAB5A) (Table IGo). The transcription factor genes in clusters 1 and 2, such as HSF2, ZFP36, MYCL1, MYBL2, WT1, ATF2, SP3, and TCF12, were, in general, immediate response genes controlling the expression of a unique set of genes (Table IGo). These results indicated that arsenite-induced stress can activate certain signaling pathways and stimulate the expression of a different set of genes via the activation of a variety of transcription factors. In addition to signaling-related and transcription regulatory genes, several stress-responsive genes, such as HSPD1, DDIT3, TOP2A, and N-acetyltransferase (NAT1), were found in cluster 1 (Table IGo).

The genes in cluster 3 were late-responsive genes, their expression not being evident until the late stage of arsenite treatment (16–24 h). Several cell cycle control genes, including CCNG1, CCNF, CDC2, CDC25A, CDKN2A (p16), and CDKN3, were found in cluster 3 (Table IGo). This finding is consistent with our previous results that arsenite treatment gradually causes G2/M arrest in HFW cells (40). Furthermore, the transcription factors found in cluster 3, such as TCF6L1, GTF2IP1, VBP1, and MAD, were general regulators of gene transcription.

In clusters 1–3, arsenite treatment also enhanced the expression of several genes coding for proteins with proteolytic activity, including CFLAR, USP4, USP8, CASP10, and XK (a zinc endopeptidase), and the expression of several stress-responsive genes (Table IGo).

Genes reduced by arsenite in HFW cells
Suppression of the expression of genes in cluster 6 was rapid, but was more gradual for genes in cluster 5. The majority of genes (9) in clusters 5 and 6 were mainly involved in signaling pathways, encoding either growth factors or cytokines. In addition, expression of the transcription factors, JUNB and PML, was significantly suppressed by arsenite treatment. Although EGR1 was classified in cluster 6, its expression was transiently enhanced 1 h after arsenite treatment, indicating an immediate response to the physiological stress induced by arsenite. The expression kinetics of EGR1 was confirmed by northern blot analysis (Figure 3Go).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have previously reported that exposure of HFW cells to 5 µM arsenite results in oxidative stress, DNA damage, chromosomal aberrations, cell cycle arrest, and aneuploidy (10,43,48). All of these phenotypic changes are complicated biologic events. For example, we recently demonstrated that treatment of HFW cells with 5 µM sodium arsenite resulted in DNA strand breaks, accumulation of p53, p21, and MDM2, and G2/M arrest and that arsenite-induced p53 accumulation is possibly mediated via an ATM-dependent pathway (40). In addition, arsenite treatment induces the expression of a set of stress proteins (49,50). These studies suggest that a complicated signaling network is turned on in response to the stress or damage induced by arsenite. To gain more insights into the epigenetic response of a cell stressed by arsenite exposure, cDNA microarray technique was adopted to examine the temporal changes in gene expression levels in arsenite-treated HFW cells. In the present study, 55% of the DNA targets used in our array was signaling-related or transcription regulatory genes and the results show that a variety of signaling-regulated genes, transcription regulatory genes, and stress-responsive genes were either elevated or reduced by arsenite treatment.

The increased expression of a variety of transcription factors indicates that arsenite treatment results in a complex change in expression profile. Several of these transcription factor target genes are involved in growth control. In cluster 1, oncogenes, such as MYCL1 and MYBL2, and the tumor suppressor gene, WT1, are well known transcription factors and their target genes are mainly involved in cell growth, apoptosis, and metabolism (51–53). Transcription factor, Sp3, one of the Sp family which binds and acts through GC and/or GT boxes of genes involved in cell cycle regulation and hormonal activation (54), mediates the expression of p21/WAF1/Cip1 (a cyclin-dependent kinase inhibitor) induced by histone deacetylase inhibitors (55). In contrast, expression of PML and JUNB was suppressed by arsenite treatment. PML, also a tumor suppressor gene, is essential for the proper formation of the nuclear body and serves as a co-activator or co-repressor of transcription (56). PML-dependent transcriptional pathways are, in general, involved in cellular differentiation, apoptosis, growth inhibition, and the immune response (56). JUNB is a member of the AP-1 transcription factor family, but shows decreased homodimerization and a 10-fold weaker DNA-binding activity than c-Jun (57). A recent report showed that JUNB acts as a negative regulator of cell proliferation by activation of p16INK4a (CDKN2A) expression (58). Each transcription factor controls the expression of a set of genes. Our present results, showing that a complex network of signaling molecules and transcription regulators is involved in determining how a cell responds to insults induced by arsenite, support the idea that arsenite treatment causes pleiotropic insults.

Several transcription regulatory proteins activated by arsenite are involved in cellular stress responses. For example, transcription factor ATF-2, a member of the ATF/cAMP-response element-binding protein family, is involved in cellular stress responses (59). HSTF2 does not respond to classical cellular stresses, but it activates the transcription of heat shock proteins in response to hemin, an inducer of erythroid differentiation (60,61). Elevated expression of HSTF2 also activates the genes involved in differentiation and embryogenesis (61). In addition, a variety of signaling cascades are mediators of chemical and physiological stress. In the present study, several cell death-related genes, such as TNFRSF6, FADD, MCL1, and BCL2A1, were elevated, whereas the expression of several growth factors and cytokines, such as SCYA2, PML, PGF, IL6, GRO1, and GRO2, was decreased. However, low micromolar arsenite treatment increased the mRNA expression and secretion of keratinocyte growth factors, including granulocyte/macrophage colony-stimulating factor, transforming growth factor and tumor necrosis factor and stimulated human keratinocyte cell proliferation (62,63). These results indicate that signaling networks must be tuned to a mobilized state to face the stress or damage induced by arsenite in different cells.

In addition to transcription factors, arsenite also enhanced the expression of SMARCA1 (SWI/SNF) which regulates gene transcription by altering chromatin structure (64). Interestingly, mutation of SWI/SNF impairs the ability of cells to activate the endogenous stress response gene, hsp70, in response to arsenite (65). VBP1, the gene product of the von Hippel–Lindau tumor suppressor gene, is not a transcription factor, but is a component of ubiquitin ligase, which controls the stability of the hypoxia-inducible factor (HIF transcription factor) (66). The expression of SRPK1, an arginine/serine protein kinase which is involved in mRNA splicing (67), was also activated by arsenite. In response to arsenite-induced cellular injury, regulation of gene expression is altered not only via activation or repression of transcription factors, but also by changes in chromatin structure or mRNA splicing.

Interestingly, several of the transcription factors affected by arsenite were zinc finger proteins, i.e. WT1 (53), ZFP36 (68), SP3 (69), RNF5 (70), and EGR1 (71). In several cell systems, arsenite is reported to induce expression of the gene coding for metallothionein (72,73), a zinc storage protein (74) which plays an essential role in zinc homeostasis and the regulation of zinc finger proteins (75). Zinc pretreatment protects mice against arsenite toxicity (76) and an epidemiological study has suggested that zinc deficiency results in increased vascular disease on exposure to arsenic (77). However, this hypothesis has not been proven and the question of how arsenic interacts with zinc in vivo is presently unclear and is an interesting toxicological issue warranting further study.

EGR1 showed a unique expression profile in arsenite-treated HFW cells. As shown by microarray and Northern blot analysis, expression of EGR1 was rapidly and transiently activated, then suppressed in response to arsenite exposure. A similar EGR1 expression pattern has been observed in X-ray irradiated cells (78,79), in an immortalized human urothelial cell line treated with arsenite (80), and in mouse skin treated topically with 12-O-tetradecanoylphorbol 13-acetate (81). EGR1 has been identified as an immediate early gene in response to diverse stimuli, including mitogenic signals, oxidative stress, and genotoxic stress (82,83). The expression of POR, which is targeted by EGR1 (84), was enhanced by arsenite. Since EGR1, a pleiotropic mediator of inducible gene expression (85), is involved in normal development and differentiation and in a number of pathological settings (82), a clear understanding of how EGR1 is activated and suppressed by arsenite would be a great help in understanding the epigenetic effects of arsenite.

The cell cycle is regulated by engine kinases and regulatory cyclins (86). To avoid the reproduction of genetically-damaged cells, the cell cycle is also strictly controlled by several sets of checkpoint genes (87). The expression of several cell cycle control genes was enhanced by treatment of HFW cells with arsenite; these included cyclins (CCNB1, CCNG1, and CCNF), a cell cycle-dependent-kinase (CDC2), cell cycle-dependent kinase inhibitors (CDKN2A and CDKN3), and cell cycle checkpoint genes (CLK1 and RAD9) (Table IGo). These cell cycle control genes were mainly found in cluster 3, i.e., those showing a delayed response to arsenite injury. Both positive and negative regulators of cell cycle control were activated by arsenite, indicating that arsenite treatment causes cell cycle dysregulation, which is known to play an important role in neoplastic transformation. In our previous studies, we have demonstrated that 5 µM arsenite induces DNA damage and G2 arrest and leads to chromosomal loss in HFW cells (10,40).

On our array, 13 out of 24 stress-responsive genes were activated by arsenite treatment (Table IGo); several, including HSPA, HSPD, DDIT3/GADD153 (80,88), and MYC (89), were already known to show increased expression in arsenite-treated cells. Expression of stress proteins is a cellular defense mechanism against chemical or physiological stress, such as protein denaturation or oxidant injury (88,90). Of these genes, DDIT3/GADD153, a member of the CCAAT/enhancer binding protein family (91), is undetectable in untreated cells, but its expression is markedly increased in cells exposed to genotoxic or endoplasmic reticulum stress agents (92) and it is therefore closely associated with cell death or cell regeneration (93,94). In addition, we observed increased expression of TOP2A in arsenite-treated cells; such an increase in TOP2A expression has also been noted in the liver in an arsenic-exposed population in Guizhou, China (38). TOP2A, an important enzyme involved in DNA replication and repair processes (95), is repressed at the G0/G1 stage and activated exclusively at the G2/M stage (96). The present study indicates that arsenite-induced DNA damage might cause cellular responses, such as cell cycle arrest and DNA repair, by the switching on of a complicated signaling network involving transcriptional and translational regulation of p53, DDIT3/GADD153, and TOP2A. The arsenite-induced enhanced gene expression of DDIT3/GADD153, GADD45, and TOP2A confirmed the DNA damaging activity of arsenite noted in many cell types (17,40,48). Arsenite treatment also induced expression of several cellular defense proteins, such as NAT1, GLRX, PFDN4, and POR. NAT1 and POR are enzymes involved in the detoxification of various carcinogens (97,98). GLRX, a thiotransferase, also known as glutaredoxin, has been shown to be a ligand of arsenate reductase in Escherichia coli (99). Prefoldin 4 (PFDN4) is a chaperone which delivers unfolded proteins to cytosolic chaperonin (100)

Proteolytic activity plays important roles in a variety of cellular processes, including growth, differentiation, stress response, and death (101). We have previously reported that cell death-associated cathepsin-like protease activity is induced by arsenite in Chinese hamster ovary cells (102). In HFW cells, arsenite activated the expression of CASP8 and CASP10, which are involved in the progression of apoptosis. The expression of CASPs in response to arsenite exposure is apparently cell type-dependent. For example, expression of CASP8 is increased in acute arsenic-treated leukemia cells (103), that of CASP1 is increased in the livers of arsenite-exposed mice (39), and that of CASP 9 and 10 was increased in the liver in an arsenic-exposed human population (38). However, apoptosis is not seen in HFW cells treated with arsenite (40). In fact, in the present study, both apoptotic-related genes (TNFRSF6 (104) and FADD (105)) and anti-apoptotic genes (TANK (106), MCL1, and BCL2A1 (107)) were activated by arsenite treatment. The effect of arsenite treatment on the induction of cell death is therefore complex.

Conjugation to the small eukaryotic protein, ubiquitin, can functionally modify or target proteins for degradation by the proteasome. Removal of the ubiquitin modification, or deubiquitination, is carried out by ubiquitin-specific proteases and is an important mechanism regulating this pathway (108). Arsenite treatment enhanced the expression of the ubiquitin-specific proteases, USP4 and USP8, indicating that arsenite might interfere with the normal regulation of protein metabolism. Moreover, expression of XK (Kell blood group precursor), a zinc endopeptidase involved in the maturation of the vasoconstrictor, endothelin 3 (109), was also increased in arsenite-treated HFW cells, and endothelin 3 overexpression has been noted in liver biopsies from an arsenic-exposed human population from Guizhou, China (38). A gene homologous to rat fau gene, a tumor suppressor gene which contains ubiquitin-like region fused to S30 ribosomal protein, was identified in an arsenite-resistant clone derived from Chinese hamster V79 cells indicating that the ubiquitin system might also be involved in arsenic resistance (110).

Methylation of inorganic arsenic is possibly a major way for arsenic extrusion (111). Since both DNA and arsenic methylation share with the same methyl donor, S-adenosylmethionine, altered DNA methylation patterns, either hypermethylation or hypomethylation, were observed in arsenite-treated cells (112–114). Zhong and Mass (114) therefore hypothesized that DNA methylation imbalance could conceivably disrupt appropriate gene expression in arsenite-exposed cells. DNA methylation patterns are in general established during DNA replication, that is, a methylated double stranded DNA needs two sequential DNA replications to become a completely demethylated DNA. The arsenite-induced alteration in DNA methylation status occurred in cells chronically exposed to arsenite (2–4 weeks) (112–114). In the present study, whether the changes in gene expression levels are due to the interference of DNA methylation by a 24-h treatment with 5 µM arsenite remains to be clarified. However, we found several transcriptional regulatory genes were elevated indicating that arsenite may trigger the activation of responsive signaling cascades, and hence elevate the gene expression.

We have previously demonstrated that arsenite treatment induced mitosis disturbance and aneuploidy in HFW cells (10). In addition, several colonies survived after treatment with 5 µM arsenite for 24 h were isolated and kept growing in culture. As shown in our previous report (10), the averaged lifespan (number of population doubling) of clones from untreated control and arsenite-treated cultures was 44 ± 11 and 86 ± 18, respectively, indicating that HFW cells survived from arsenite injury have a prolonged lifespan as compared to those of clones isolated from untreated control. Increasing the chance of mitogenesis is crucial for cancer development (115). Our present study demonstrated that 5 µM arsenite induced changes in the expression of genes involved in transcriptional control, protein metabolism, cell cycle regulation, and signaling molecules in these cells. These data not only provide transcriptional profiles which can be coordinated with the previously reported cellular responses induced by arsenite, but also provide new directions for the detailed investigation of arsenic toxicity and carcinogenicity.


    Notes
 
3 To whom correspondence should be addressed Email: bmtcl{at}ibms.sinica.edu.tw. Back


    Acknowledgments
 
The authors thank Mr Wei-Chen Kao for making the arrays. This work was supported by grants from the Academia Sinica and the National Science Council (NSC 88–2318-B001–003-M51 and NSC 89–2318-B001–020-M51), Republic of China.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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