Cyclooxygenase-2 induction by arsenite through the IKK[beta]/NF[kappa]B pathway exerts an antiapoptotic effect in mouse epidermal Cl41 cells.

BACKGROUND: Arsenic contamination has become a major public health concern worldwide. Epidemiologic data show that long-term arsenic exposure results in the risk of skin cancer. However, the mechanisms underlying carcinogenic effects of arsenite on skin remain to be studied.

OBJECTIVES: In the present study we evaluated cyclooxygenase-2 (COX-2) expression, the signaling pathways leading to COX-2 induction, and its antiapoptotic function in the response to arsenite exposure in mouse epidermal JB6 Cl41 cells.

METHODS: We used the luciferase reporter assay and Western blots to determine COX-2 induction by arsenite. We utilized dominant negative mutant, genetic knockout, gene knockdown, and gene overexpression approaches to elucidate the signaling pathway involved in COX-2 induction and its protective effect on cell apoptosis.

RESULTS: The induction of COX-2 by arsenite was inhibited in Cl41 cells transfected with IKK[beta]-KM, a dominant mutant inhibitor of k[beta] (Ik[beta]) kinase (IKK[beta]), and in IKK[beta]-knockout (IKK[[beta].sup.-/-]) mouse embryonic fibroblasts (MEFs). IKK[beta]/nuclear factor [kappa]B (NF[kappa]B) pathway-mediated COX-2 induction exerted an antiapoptotic effect on the cells exposed to arsenite because cell apoptosis was significantly enhanced in the Cl41 cells transfected with IKK[beta]-KM or COX-2 small interference RNA (siCOX-2). In addition, IKK[[beta].sup.-/-] MEFs stably transfected with COX-2 showed more resistance to arsenite-induced apoptosis compared with the same control vector-transfected cells.

CONCLUSIONS: These results demonstrate that arsenite exposure can induce COX-2 expression through the IKK[beta]/NF[kappa]B pathway, which thereby exerts an antiapoptotic effect in response to arsenite. In light of the importance of apoptosis evasion during carcinogenesis, we anticipate that COX-2 induction may be at least partially responsible for the carcinogenic effect of arsenite on skin.

KEY WORDS: apoptosis, arsenite, carcinogenesis, COX-2, NF[kappa]B. Environ Health Perspect 115:513-518 (2007). doi:10.1289/ehp.9588 available via http://dx.doi.org/ [Online 14 December 2006]

**********

Arsenic contamination has become a major public health concern worldwide, especially in Asia. Epidemiologic data show that long-term arsenic exposure results in the risk of various cancers [Bettley and O'Shea 1975; International Agency for Research on Cancer (IARC) 1980; Landolph 1994; Nriagu 2002], especially in the lung and skin via inhalation and ingestion (Landolph 1994). High arsenic concentrations in drinking water (0.35-1.14 mg/L) caused increased risks of cancer of the skin, bladder, kidney, lung, and colon (National Research Council 1999). The skin cancers associated with arsenite exposure include Bowen's disease (carcinoma in situ), basal cell carcinoma, and squamous cell carcinoma (Tseng et al. 1968; Yu et al. 2006).

The mouse skin model of multistage carcinogenesis has demonstrated that cancer development results from the coordination of genetic mutation and alterations of epigenetic factors, including the machineries regulating cell proliferation and apoptosis (Hecker 1987; Zoumpourlis et al. 2003). Acquiring the capacity to evade apoptosis is a hallmark of most cancers (Hanahan and Weinberg 2000). Under normal circumstances, DNA-damaged or mutated cells are eliminated by apoptosis. Acquired resistance to apoptosis is a critical molecular event during carcinogenesis, and disruption of apoptosis has been shown to play a major role in tumor formation and malignant progression (Hanahan and Weinberg 2000; Hickman 2002). Whereas the induction of cell proliferation by arsenite has been extensively studied, the events implicated in regulating the apoptosis of skin cells exposed to arsenite remain largely unknown.

Cyclooxygenase (COX), the rate-limiting enzyme in the conversion of arachidonic acid to prostanoids (Sheng et al. 2001; Smith et al. 1996), exists as two distinct isoforms (Feng et al. 1993). COX-2 is an inducible immediate-early gene. Its expression is low or non-detectable in most tissues, but it can be readily induced in response to cell activation by cytokines, growth factors, and tumor promoters (Feng et al. 1993; Smith et al. 1996). Increasing evidence indicates that COX-2 is related to skin cancer development. Mice deficient in COX-2 develop 75% fewer tumors than their wild type littermates when subjected to a 9,10-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate two-stage chemical carcinogenesis protocol (Tiano et al. 2002). Moreover, oral administration of specific COX-2 inhibitors is effective in reducing ultraviolet-B-induced skin carcinogenesis by up to 90% (Fischer et al. 1999). Although the exact mechanisms remain to be extensively investigated, COX-2 is thought to contribute to carcinogenesis mainly by promoting cell proliferation and antagonizing cell apoptosis (Krysan et al. 2005; Tsujii and DuBois 1995; Wang et al. 2005).

The role of COX-2 in apoptosis resistance and carcinogenesis suggests that COX-2 may be involved in the regulation of apoptosis of skin cells exposed to arsenite. Therefore, in the present study we examined the effect of arsenite exposure on COX-2 expression in mouse epidermal JB6 Cl41 cells, and we further investigated the role of COX-2 in apoptosis resistance during arsenite exposure. The results showed that exposure to arsenite caused significant COX-2 expression through the inhibitor of [kappa][beta] (I[kappa][beta]) kinase (IKK[beta])/nuclear factor [kappa]B (NF[kappa]B) pathway, which thereby played an important role in antagonizing the apoptosis induced by arsenite. These results suggest that COX-2 induction in arsenite-exposed skin cells may facilitate skin cancer development by conferring an apoptosis resistance and supporting the survival of the cells with genetic alterations that are usually eliminated by apoptosis.

Materials and Methods

Cell culture. Mouse epidermal JB6 Cl41 cells and their stable transfectants were cultured in Eagle's minimal essential medium (MEM; Calbiochem, San Diego, CA) supplemented with 5% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 2 mM L-glutamine (Life Technologies, Inc. Rockville, MD) at 37[degrees]C in a humidified atmosphere with 5% C[O.sub.2] in the air. To investigate the potential contribution of the NF[kappa]B transcription factor to COX-2 transcriptional induction by arsenite, we used COX-2-luciferase (COX-2-Luc) reporter containing full length (-1432/+59) or a mutation of the NF[kappa]B binding sites (-223/-214) of human COX-2 gene promoter linked to the luciferase (Subbaramaiah et al. 2001; Yan et al. 2000) and/or with IKK[beta]-KM as described previously (Ouyang et al. 2006). Wild-type and IKK[beta] knockout (IKK[[beta].sup.-/-]) mouse embryonic fibroblasts (MEFs) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 1% penicillin/streptomycin, and 2 mM L-glutamine.

Construction of the siRNA vector. The specific small-interference RNA (siRNA)-targeted mouse COX-2 was designed using the siRNA converter of Ambion Inc. (2006a) according to the gene sequence in GenBank (mouse NM-011198, National Center for Biotechnology Information 2006b) and guidelines for siRNA (Ambion Inc. 2006b); the siRNA was synthesized by Invitrogen (Carlsbad, CA). The target sequence for mouse COX-2 was 5'-AGACAGATCATAAGCGAGGA-3'. The siRNA sequence was controlled via BLAST search (National Center for Biotechnology Information 2006a) and did not show any homology to other known genes. The siRNA was then inserted into pSuppressor vector and verified by DNA sequencing. The siRNA vector was designated as siCOX-2.

Stable transfection. Cl41 cells were transfected with either siCOX-2 or small-interference-green fluorescent protein. IKK[[beta].sup.-/-] MEFs were transfected with COX-2 expression vector, which was a gift from K. Subbaramaiah (Weill Medical College of Cornell University, New York, NY). The transfection was performed by Lipofectamine 2000 reagent (Gibco BRL, Rockville, MD) according to the manufacturer's instructions. Briefly, the cells were cultured in a 6-well plate to 85-90% confluence. Five micrograms of plasmid DNA was mixed with 10 [micro]L Lipofectamine 2000 reagent and then used to transfect each well in the absence of serum. After 4-6 hr, the medium was replaced with 5% FBS MEM for Cl41 cells or 10% FBS DMEM for MEFs. Approximately 36-48 hr after the beginning of the transfection, the cells were cultured with medium containing 500 [micro]g/mL G418 (Gibco BRL). After selection for 28-45 days with G418, the stable transfectants were identified by Western blot. Stable transfectants, Cl41-mock, Cl41-siCOX-2, IKK[[beta].sup.-/-](vector), and IKK[[beta].sup.-/-](COX-2) were established and cultured in G418-free medium for at least two passages before each experiment.

COX-2 expression assay. We cultured 2 x [10.sup.5] Cl41 cells, IKK[[beta].sup.-/-] MEFs, and their transfectants in each well of 6-well plates to 70-80% confluence. After exposure to arsenite for indicated times, the cells were washed once with ice-cold phosphate-buffered saline (PBS) and then extracted with sodium dodecyl sulfate (SDS) sample buffer. The cell extracts (with GAPDH used as a control for protein loading) were separated on polyacrylamide-SDS gels, transferred, and probed with a rabbit-specific antibody against COX-2 (Cayman Chemical, Ann Arbor, MI). The protein band, specifically bound to the primary antibody, was detected using an anti-rabbit IgG-alkaline phosphatase-linked antibody and an enhanced chemifluorescence Western blotting system (Amersham Biosciences, Piscataway, NJ).

Cell apoptosis analysis by flow cytometry. Cells (2 x [10.sup.5]) were seeded into each well of 6-well plates and cultured to 70-80% confluence. After exposure to arsenite, the cells were harvested and fixed with 3 mL ice-cold 80% ethanol overnight. The fixed cells were washed twice with PBS and then suspended in propidium iodide (PI) staining solution (50 [micro]g/mL PI, 10 mg/mL RNase A) (Sigma Chemical, St. Louis, MO) for at least 1 hr at 4[degrees]C. Cell apoptosis was determined by flow cytometry using the Epics XL FACS and EXPO 32 software (Beckman Coulter, Miami, FL) as described previously (Ouyang et al. 2006).

TUNEL assay. We performed the TUNEL assay using an in situ cell death detection kit (Roche Applied Science, Indianapolis, IN) following the kit instructions. Briefly, the exposed cells were fixed by 4% polyparaformaldehyde solution in PBS for 24 hr at room temperature. After rinsing with PBS, the cells were resuspended in a solution with 0.1% Triton X-100 and 0.1% sodium citrate for 5 min to increase permeability of the cell membrane, and then incubated with 50 [micro]L TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT) and fluorescein isothiocyanate-deoxyuridine triphosphate (FIT-CdUTP) for 60 min at 37[degrees]C. After washing, the label incorporated at the damaged sites of the DNA was visualized by flow cytometry using the Epics XL FACS and EXPO 32 software.

Results

Arsenite exposure induced COX-2 expression in Cl41 cells through the IKK[beta]/NF[kappa]B pathway. Previous studies demonstrated that arsenite exerts its carcinogenic effect mainly by activating signal pathways and inducing gene expression involved in the regulation of cell proliferation and apoptosis (Huang et al. 2004; Pi et al. 2005; Rossman 2003; Yang and Frenkel 2002). COX-2, a key inducible enzyme in the biosynthesis of prostaglandins, has been related to inflammation, apoptosis, and carcinogenesis (Liu et al. 1998; Tsujii and DuBois 1995; Tsujii et al. 1997, 1998). To determine whether COX-2 is also involved in cell response to arsenite exposure, we examined COX-2 induction by arsenite in mouse epidermal Cl41 cells. As determined by Western blot analysis (Figure 1A), arsenite exposure caused a significant elevation of COX-2 protein level. Moreover, Cl41 cells exposed to arsenite for 12 hr showed a marked induction of COX-2 transcription in the gene reporter assay (Figure 1B).

The promoter region of the COX-2 gene contains a canonical TATA box and multiple putative transcriptional regulatory elements, including NF[kappa]B, which has been indicated to be activated in Cl41 cells by arsenite exposure (Li et al. 2004). We investigated the potential contribution of the NF[kappa]B transcription factor to COX-2 transcriptional induction by arsenite using the COX-2-Luc reporter containing full length (-1432/+59) or mutant NF[kappa]B binding sites (-223/-214) of the COX-2 gene promoter. As shown in Figure 1C, deletion of NF[kappa]B binding sites impaired arsenite-induced COX-2 transcriptional induction. Moreover, the stable transfectants of Cl41 cells harboring IKK[beta]-KM, a dominant mutant of IKK[beta] (Ouyang et al. 2006), and IKK[[beta].sup.-/-] MEFs were used to further confirm the requirement of the IKK[beta]/NF[kappa]B pathway for the induction of COX-2 by arsenite. Arsenite-induced COX-2 expression was dramatically inhibited in the IKK[beta]-KM-transfected Cl41 cells, as well as in IKK[[beta].sup.-/-] MEFs, when compared with control vector-transfected Cl41 cells or wild-type MEFs (Figure 1D, E). The basal level of COX-2 varied at different time points, which might be due to cell cycle progression (Figure 1D, E). Collectively, these results indicate that arsenite can induce COX-2 expression at both protein and transcription levels via an IKK[beta]/NF[kappa]B-dependent pathway, suggesting that COX-2 is involved in cell response to arsenite exposure.

COX-2 induction through the IKK[beta]/NF[kappa]B pathway exerted an antiapoptotic effect on cells exposed to arsenite. In view of the importance of COX-2 in the regulation of cell apoptotic response in some cells, we proposed that the induction of COX-2 may also be implicated in the regulation of cell apoptosis upon arsenite exposure. Based on the above results that the IKK[beta]/NF[kappa]B pathway was required for COX-2 induction in the cells exposed to arsenite, we examined the apoptosis of Cl41 cells transfected with IKK[beta]-KM after the exposure to arsenite. The results obtained from microscopic observation of cell morphology (Figure 2A), DNA content analysis by PI staining followed by flow cytometry analysis (Figure 2B), and DNA fragment detection by TUNEL assay followed by flow cytometry analysis (Figure 2C) showed that the transfection of IKK[beta]-KM made Cl41 cells much more sensitive to apoptotic induction by arsenite.

To confirm the importance of COX-2 in the regulation of apoptotic response after arsenite exposure, we pretreated Cl41 cells with NS398, an inhibitor of COX-2, and found that it significantly sensitized the cells to arsenite-induced cell apoptosis (Figure 3A, B), suggesting that COX-2 may be the mediator responsible for the antiapoptotic effect. This notion was further confirmed by the finding that knockdown of endogenous COX-2 expression by its specific siRNA rendered Cl41 cells much more susceptible to cell apoptotic induction by arsenite (Figure 3C, D).

The role of COX-2 induction in protecting cells from apoptosis after arsenite exposure was further verified by the finding that over-expression of COX-2 in IKK[[beta].sup.-/-] MEFs made the cells much more resistant to arsenite-induced apoptosis (Figure 4). Collectively, these results demonstrate that COX-2 induction through the IKK[beta]/NF[kappa]B pathway can protect arsenite-exposed cells from apoptosis.

Discussion

Arsenite is a well-documented skin carcinogen (Landolph 1994; Nriagu 2002); skin lesions, including skin cancers, are characteristic of exposure to arsenite in drinking water (Haque et al. 2003). Given the low genotoxic activity, arsenite is thought to exert its carcinogenic effect mainly through inducing activation of signal pathways, which thereby affects the expression of genes involved in regulating the machineries of the cell cycle and apoptosis (Huang et al. 2004; Pi et al. 2005; Rossman 2003; Yang and Frenkel 2002). In the present study, we have addressed the events involved in the regulation of apoptosis of cells exposed to arsenite, and demonstrated that induction of COX-2 expression through the IKK[beta]/NF[kappa]B pathway plays a role in antagonizing cell apoptosis caused by arsenite in mouse epidermal Cl41 cells.

The effect of arsenite on COX-2 expression depends on cell type and arsenite dosage. Arsenite stimulates COX-2 expression in endothelial cells through activating IKK/NF[kappa]B and extracellular signal-regulated kinases, respectively (Trouba and Germolec 2004; Tsai et al. 2002), whereas in a recent study, Ding et al. (2006) found that arsenite induces COX-2 expression in human bronchial epithelial Beas-2B cells through NFAT (nuclear factor of activated T cells) rather than NF[kappa]B and activator protein-1. Arsenite has been demonstrated to repress constitutive activation of NF[kappa]B and COX-2 expression in human acute myeloid leukemia (HL-60) cells (Han et al. 2005), and pretreatment of arsenite attenuates benzo[[alpha]]pyrene cytotoxicity in a human lung adenocarcinoma cells by decreasing cyclooxygenase-2 levels (Ho and Lee 2002). In the present study, we provide the first evidence that arsenite can induce COX-2 expression through the IKK[beta]/NF[kappa]B pathway in mouse epidermal Cl41 cells.

Although the detailed mechanisms underlying tumorigenesis remain largely undefined, it is generally accepted that apoptosis evasion is one of the hallmarks during cancer development (Hanahan and Weinberg 2000). Apoptosis plays a major role in developmental biology, cellular population dynamics, and disease states. Apoptosis typically occurs when cellular genetic damage exceeds the repair capacity. The suppression of apoptosis, in the face of significant genetic damage, could facilitate accumulation of aberrant cells and may be a critical step in the pathogenesis of malignancy (Abrams 2002; Johnstone et al. 2002; Zornig et al. 2001). As a sensor of cellular stress, p53 is a critical initiator of the apoptotic pathway (Lowe and Lin 2000). p53 protein accumulates in cells under stress, which thereby promotes apoptosis mainly by activating the expression of proapoptotic Bcl-2 family members (e.g., Bax, Bak, PUMA, Noxa) and repressing antiapoptotic Bcl-2 (B-cell leukemia) proteins (Bcl-2, Bcl-XL) and inhibitor of apoptosis protein (survivin) (Bartke et al. 2001; Hoffman et al. 2002; Ryan et al. 2001; Wu et al. 2001). The elimination of these damaged cells through apoptosis maintains genomic stability and prevents tumorigenesis. Because p53 mediates cell apoptosis and growth arrest, p53 mutation is responsible for > 50% of cancer development in humans. In the present study, we demonstrated that COX-2 plays an important role in antagonizing cell apoptosis induced by arsenite in mouse epidermal cells. Although a large body of evidence indicates the importance of COX-2 in the regulation of cell apoptosis, the mechanisms are not well-defined. Nonetheless, there is evidence supporting that COX-2 may interfere with p53-mediated cell apoptosis (Han et al. 2002) and regulate mitochondrial-triggered cell apoptosis (Sun et al. 2002). Although the exact mechanisms require further investigation, the antiapoptotic effect of COX-2 observed in the present study may provide more strategies with COX-2 as the target for skin cancer prevention and skin cancer therapy, especially in those countries with high arsenite contamination in drinking water.

It is notable that the contributions of the IKKs/NF[kappa]B pathway to carcinogen-induced skin cancer remain controversial. IKK[alpha] has been demonstrated to be an inhibitory factor for the proliferation of skin epidermis (Hu et al. 1999, 2001; Li et al. 1999) and over-expression of active p50 and p65 NF[kappa]B subunits in transgenic epithelium-produced hypoplasia and growth inhibition (Seitz et al. 1998). However, it has been reported that the deletion of IKK[beta] does not affect the proliferation of skin epidermis (Pasparakis et al. 2002); I[kappa]B[alpha] deficiency results in a sustained NF[kappa]B response and severe widespread dermatitis characterized by epidermal hyperplasia in mice (Klement et al. 1996). Budunova et al. (1999) demonstrated that epidermal inflammation and hyperplasia play a critical role in skin tumor promotion, and NF[kappa]B is one of the well-known mediators of these effects. Substances such as phorbol ester and okadaic acid, which are promoters of skin cancer, are also strong inducers of the NF[kappa]B response in keratinocytes (Budunova et al. 1999). In the present study, we demonstrated that the IKK[beta]/NF[kappa]B pathway is required for COX-2 induction by arsenite, suggesting that the IKK[beta]/NF[kappa]B pathway may contribute to arsenite-induced carcinogenesis by protecting cells from apoptosis through inducing COX-2 expression. Interestingly, we also found that apoptosis of IKK[[beta].sup.-/-] MEFs induced by arsenite is affected largely by cell density. High density of IKK[[beta].sup.-/-] MEFs shows much lower susceptibility to arsenite-induced apoptosis (Song et al. 2006). The mechanisms are now under investigation in our laboratory.

In summary, we have demonstrated that exposure of the cells to arsenite causes a significant COX-2 expression in an IKK[beta]/NF[kappa]B-dependent manner, which thereby plays an important role in antagonizing apoptosis induced by arsenite. These results suggest that arsenite, as a carcinogen, may facilitate skin cancer development by supporting the survival of the cells with genetic alterations, which is usually eliminated by apoptosis. Thus, inhibition of COX-2 may be a promising approach for skin cancer prevention in those countries with severe arsenite pollution in drinking water.

REFERENCES

Abrams JM. 2002. Competition and compensation: coupled to death in development and cancer. Cell 110(4):403-406.

Ambion Inc. 2006a. siRNA Target Finder. Available: http://www.ambion.com/techlib/misc/siRNA_finder.html [accessed 15 February 2007].

Ambion Inc. 2006b. siRNA Design Guidelines. Available: http://www.ambion.com/techlib/tb/tb_506.html [accessed 15 February 2007].

Bartke T, Siegmund D, Peters N, Reichwein M, Henkler F, Scheurich P, et al. 2001. p53 upregulates cFLIP, inhibits transcription of NF-[kappa]B-regulated genes and induces caspase-8-independent cell death in DLD-1 cells. Oncogene 20(5):571-580.

Bettley FR, O'Shea JA. 1975. The absorption of arsenic and its relation to carcinoma. Br J Dermatol 92(5):563-568.

Budunova IV, Perez P, Vaden VR, Spiegelman VS, Slaga TJ, Jorcano JL. 1999. Increased expression of p50-NF-[kappa]B and constitutive activation of NF-[kappa]B transcription factors during mouse skin carcinogenesis. Oncogene 18(52):7423-7431.

Ding J, Li J, Xue C, Wu K, Ouyang W, Zhang D, et al. 2006. Cyclooxygenase-2 Induction by arsenite is through a nuclear factor of activated T-cell-dependent pathway and plays an antiapoptotic role in Beas-2B cells. J Biol Chem 281(34):24405-24413.

Feng L, Sun W, Xia Y, Tang WW, Chanmugam P, Soyoola E, et al. 1993. Cloning two isoforms of rat cyclooxygenase: differential regulation of their expression. Arch Biochem Biophys 307(2):361-368.

Fischer SM, Lo HH, Gordon GB, Seibert K, Kelloff G, Lubet RA, et al. 1999. Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, and indomethacin against ultraviolet light-induced skin carcinogenesis. Mol Carcinog 25(4):231-240.

Han JA, Kim JI, Ongusaha PP, Hwang DH, Ballou LR, Mahale A, et al. 2002. P53-mediated induction of Cox-2 counteracts p53- or genotoxic stress-induced apoptosis. Embo J 21(21):5635-5644.

Han SS, Kim K, Hahm ER, Park CH, Kimler BF, Lee SJ, et al. 2005. Arsenic trioxide represses constitutive activation of NF-[kappa]B and COX-2 expression in human acute myeloid leukemia, HL-60. J Cell Biochem 94(4):695-707.

Hanahan D, Weinberg RA. 2000. The hallmarks of cancer. Cell 100(1):57-70.

Haque R, Mazumder DN, Samanta S, Ghosh N, Kalman D, Smith MM, et al. 2003. Arsenic in drinking water and skin lesions: dose-response data from West Bengal, India. Epidemiology 14(2):174-182.

Hecker E. 1987. Three stage carcinogenesis in mouse skin--recent results and present status of an advanced model system of chemical carcinogenesis. Toxicol Pathol 15(2):245-258.

Hickman JA. 2002. Apoptosis and tumourigenesis. Curr Opin Genet Dev 12(1):67-72.

Ho IC, Lee TC. 2002. Arsenite pretreatment attenuates benzo[a]-pyrene cytotoxicity in a human lung adenocarcinoma cell line by decreasing cyclooxygenase-2 levels. J Toxicol Environ Health A 65(3-4):245-263.

Hoffman WH, Biade S, Zilfou JT, Chen J, Murphy M. 2002. Transcriptional repression of the anti-apoptotic survivin gene by wild type p53. J Biol Chem 277(5):3247-3257.

Hu Y, Baud V, Delhase M, Zhang P, Deerinck T, Ellisman M, et al. 1999. Abnormal morphogenesis but intact IKK activation in mice lacking the IKK[alpha] subunit of I[kappa]B kinase. Science 284(5412):316-320.

Hu Y, Baud V, Oga T, Kim KI, Yoshida K, Karin M. 2001. IKK[alpha] controls formation of the epidermis independently of NF-[kappa]B. Nature 410(6829):710-714.

Huang C, Ke Q, Costa M, Shi X. 2004. Molecular mechanisms of arsenic carcinogenesis. Mol Cell Biochem 255(1-2):57-66.

IARC (International Agency for Research on Cancer). 1980. Some Metals and Metallic Compounds. IARC Monogr Eval Carcinog Risk Chem Man 23:1-438.

Johnstone RW, Ruefli AA, Lowe SW. 2002. Apoptosis: a link between cancer genetics and chemotherapy. Cell 108(2):153-164.

Klement JF, Rice NR, Car BD, Abbondanzo SJ, Powers GD, Bhatt PH, et al. 1996. I[kappa]B[alpha] deficiency results in a sustained NF-[kappa]B response and severe widespread dermatitis in mice. Mol Cell Biol 16(5):2341-2349.

Krysan K, Reckamp KL, Dalwadi H, Sharma S, Rozengurt E, Dohadwala M, et al. 2005. Prostaglandin [E.sub.2] activates mitogen-activated protein kinase/Erk pathway signaling and cell proliferation in non-small cell lung cancer cells in an epidermal growth factor receptor-independent manner. Cancer Res 65(14):6275-6281.

Landolph JR. 1994. Molecular mechanisms of transformation of C3H/10T1/2 C1 8 mouse embryo cells and diploid human fibroblasts by carcinogenic metal compounds. Environ Health Perspect 102(suppl 3):119-125.

Li J, Chen H, Ke Q, Feng Z, Tang MS, Liu B, et al. 2004. Differential effects of polycyclic aromatic hydrocarbons on transactivation of AP-1 and NF-[kappa]B in mouse epidermal cl41 cells. Mol Carcinog 40(2):104-115.

Li Q, Lu Q, Hwang JY, Buscher D, Lee KF, Izpisua-Belmonte JC, et al. 1999. IKK1-deficient mice exhibit abnormal development of skin and skeleton. Genes Dev 13(10):1322-1328.

Liu XH, Yao S, Kirschenbaum A, Levine AC. 1998. NS398, a selective cyclooxygenase-2 inhibitor, induces apoptosis and down-regulates bcl-2 expression in LNCaP cells. Cancer Res 58(19):4245-4249.

Lowe SW, Lin AW. 2000. Apoptosis in cancer. Carcinogenesis 21(3):485-495.

National Center for Biotechnology Information. 2006a. BLAST (Basic Local Alignment Search Tool). Available: http://www.ncbi.nlm.nih.gov/BLAST/ [accessed 15 February 2007].

National Center for Biotechnology Information. 2006b. GenBank. Available: http://www.ncbi.nlm.nih.gov/Genbank/index.html [accessed 15 February 2007].

National Research Council. 1999. Arsenic in Drinking Water. Washington, DC:National Academy Press.

Nriagu JO. 2002. Environmental Chemistry of Arsenic. New York:Dekker.

Ouyang W, Li J, Ma Q, Huang C. 2006. Essential roles of PI-3K/Akt/IKK[beta]/NF[kappa]B pathway in cyclin D1 induction by arsenite in JB6 Cl41 cells. Carcinogenesis 27(4):864-873.

Pasparakis M, Courtois G, Hafner M, Schmidt-Supprian M, Nenci A, Toksoy A, et al. 2002. TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2. Nature 417(6891):861-866.

Pi J, He Y, Bortner C, Huang J, Liu J, Zhou T, et al. 2005. Low level, long-term inorganic arsenite exposure causes generalized resistance to apoptosis in cultured human keratinocytes: potential role in skin co-carcinogenesis. Int J Cancer 116(1):20-26.

Rossman TG. 2003. Mechanism of arsenic carcinogenesis: an integrated approach. Mutat Res 533(1-2):37-65.

Ryan KM, Phillips AC, Vousden KH. 2001. Regulation and function of the p53 tumor suppressor protein. Curr Opin Cell Biol 13(3):332-337.

Seitz CS, Lin Q, Deng H, Khavari PA. 1998. Alterations in NF-[kappa]B function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-[kappa]B. Proc Natl Acad Sci USA 95(5):2307-2312.

Sheng H, Shao J, Washington MK, DuBois RN. 2001. Prostaglandin [E.sub.2] increases growth and motility of colorectal carcinoma cells. J Biol Chem 276(21):18075-18081.

Smith WL, Garavito RM, DeWitt DL. 1996. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J Biol Chem 271(52):33157-33160.

Song L, Li J, Zhang D, Liu ZG, Ye J, Zhan Q, et al. 2006. IKK[beta] programs to turn on the GADD45[alpha]-MKK4-JNK apoptotic cascade specifically via p50 NF-[kappa]B in arsenite response. J Cell Biol 175(4):607-617.

Subbaramaiah K, Bulic P, Lin Y, Dannenberg AJ, Pasco DS. 2001. Development and use of a gene promoter-based screen to identify novel inhibitors of cyclooxygenase-2 transcription. J Biomol Screen 6(2):101-110.

Sun Y, Tang XM, Half E, Kuo MT, Sinicrope FA. 2002. Cyclooxygenase-2 overexpression reduces apoptotic susceptibility by inhibiting the cytochrome c-dependent apoptotic pathway in human colon cancer cells. Cancer Res 62(21):6323-6328.

Tiano HF, Loftin CD, Akunda J, Lee CA, Spalding J, Sessoms A, et al. 2002. Deficiency of either cyclooxygenase (COX)-1 or COX-2 alters epidermal differentiation and reduces mouse skin tumorigenesis. Cancer Res 62(12):3395-3401.

Trouba KJ, Germolec DR. 2004. Micromolar concentrations of sodium arsenite induce cyclooxygenase-2 expression and stimulate p42/44 mitogen-activated protein kinase phosphorylation in normal human epidermal keratinocytes. Toxicol Sci 79(2):248-257.

Tsai SH, Liang YC, Chen L, Ho FM, Hsieh MS, Lin JK. 2002. Arsenite stimulates cyclooxygenase-2 expression through activating I[kappa]B kinase and nuclear factor [kappa]B in primary and ECV304 endothelial cells. J Cell Biochem 84(4):750-758.

Tseng WP, Chu HM, How SW, Fong JM, Lin CS, Yeh S. 1968. Prevalence of skin cancer in an endemic area of chronic arsenicism in Taiwan. J Natl Cancer Inst 40(3):453-463.

Tsujii M, DuBois RN. 1995. Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 83(3):493-501.

Tsujii M, Kawano S, DuBois RN. 1997. Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proc Natl Acad Sci USA 94(7):3336-3340.

Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, DuBois RN. 1998. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 93(5):705-716.

Wang D, Buchanan FG, Wang H, Dey SK, DuBois RN. 2005. Prostaglandin [E.sub.2] enhances intestinal adenoma growth via activation of the Ras-mitogen-activated protein kinase cascade. Cancer Res 65(5):1822-1829.

Wu Y, Mehew JW, Heckman CA, Arcinas M, Boxer LM. 2001. Negative regulation of bcl-2 expression by p53 in hematopoietic cells. Oncogene 20(2):240-251.

Yan Z, Subbaramaiah K, Camilli T, Zhang F, Tanabe T, McCaffrey TA, et al. 2000. Benzo[a]pyrene induces the transcription of cyclooxygenase-2 in vascular smooth muscle cells. Evidence for the involvement of extracellular signal-regulated kinase and NF-[kappa]B. J Biol Chem 275(7):4949-4955.

Yang C, Frenkel K. 2002. Arsenic-mediated cellular signal transduction, transcription factor activation, and aberrant gene expression: implications in carcinogenesis. J Environ Pathol Toxicol Oncol 21(4):331-342.

Yu HS, Liao WT, Chai CY. 2006. Arsenic carcinogenesis in the skin. J Biomed Sci 13(5):657-666.

Zornig M, Hueber A, Baum W, Evan G. 2001. Apoptosis regulators and their role in tumorigenesis. Biochim Biophys Acta 1551(2):F1-37.

Zoumpourlis V, Solakidi S, Papathoma A, Papaevangeliou D. 2003. Alterations in signal transduction pathways implicated in tumour progression during multistage mouse skin carcinogenesis. Carcinogenesis 24(7):1159-1165.

Weiming Ouyang, Dongyun Zhang, Qian Ma, Jingxia Li, and Chuanshu Huang

Nelson Institute of Environmental Medicine, New York University School of Medicine, Tuxedo, New York, USA

Address correspondence to C. Huang, Nelson Institute of Environmental Medicine, New York University School of Medicine, 57 Old Forge Rd., Tuxedo, NY 10987 USA. Telephone: [phone]. Fax: [phone]. E-mail: [e-mail]

This work was supported in part by grants R01 CA094964, R01 CA112557, and R01 CA103180 from the National Cancer Institute, National Institutes of Health (NIH), and grant R01 ES012451 and R01 ES000260 from the National Institute of Environmental Health Sciences, NIH.

The authors declare they have no competing financial interests.

Received 8 August 2006; accepted 14 December 2006.