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Carcinogenesis Advance Access originally published online on December 29, 2005
Carcinogenesis 2006 27(4):864-873; doi:10.1093/carcin/bgi321
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Essential roles of PI-3K/Akt/IKKß/NF{kappa}B pathway in cyclin D1 induction by arsenite in JB6 Cl41 cells

Weiming Ouyang 1, Jingxia Li 1, Qian Ma and Chuanshu Huang *

Nelson Institute of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987, USA

* To whom correspondence should be addressed. Tel: +1 845 731 3519; Fax: +1 845 351 2320; Email: chuanshu{at}env.med.nyu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Skin is a major target of carcinogenic trivalent arsenic (arsenite, As3+). It has been thought that cell proliferation is one of the central events involved in the carcinogenic effect of arsenite. Cyclin D1, a nuclear protein playing a pivotal role in cell proliferation and cell cycle transition from G1 to S phases, has been reported to be induced in human fibroblast by arsenite via uncertain molecular mechanisms. In the present study, the potential roles of PI-3K/Akt/IKKß/NF{kappa}B signal pathway in cyclin D1 induction by arsenite were addressed in mouse epidermal Cl41 cells. We found that exposure of Cl41 cells to arsenite was able to induce cell proliferation, activate PI-3K->Akt/p70S6k signal pathway and increase cyclin D1 expression at both transcription and protein levels. Pre-treatment of Cl41 cells with PI-3K inhibitor, wortmannin, significantly inhibited the phosphorylation of Akt and p70S6k and thereby dramatically impaired the cyclin D1 induction by arsenite, implicating the importance of the PI-3K signal pathway in the cyclin D1 induction by arsenite. Furthermore, inhibition of PI-3K/Akt by overexpression of {Delta}p85 or DN-Akt blocked arsenite-induced IKK phosphorylation, I{kappa}B{alpha} degradation and cyclin D1 expression, indicating that IKK/NF{kappa}B is the downstream transducer of arsenite-triggered PI-3K/Akt cascade. Moreover, inhibition of IKKß/NF{kappa}B signal pathway by overexpression of its dominant negative mutant, IKKß-KM, also significantly blocked arsenite-induced cyclin D1 expression. Overall, arsenite exposure triggered PI-3K/Akt/IKKß/NF{kappa}B signal cascade which in turn plays essential roles in inducing cyclin D1 expression.

Abbreviations: APC, human adenomatous polyposis coli gene; CDKs, cyclin-dependent kinases; DMSO, dimethyl sulfoxide; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; GSK3ß, glycogen synthase kinase-3ß; IKK, I{kappa}B kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEM, Eagle's minimal essential medium; mTOR, mammalian target of rapamycin; NF{kappa}B, nuclear factor-{kappa}B; PI-3K, phosphotidylinositol-3 kinase; PKC{alpha}, protein kinase C{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Arsenite is introduced into the environment during energy production based on coal, oil shale and geothermal sources (1). Once in the environment, arsenite represents a potential health hazard of unknown magnitude. Available epidemiological data have shown that exposure to arsenite is associated with increased risks of human cancer of the skin, respiratory tract, hematopoietic system and urinary bladder (1–4). Epidemiological investigations also indicated that long-term arsenic exposure results in promotion of carcinogenesis, especially in lung and skin via inhalation and ingestion (3). Many cases of skin cancer have been documented in people exposed to arsenite through medical or other occupational exposures (2,4). It has also been reported that high arsenic levels in drinking water (0.35–1.14 mg/l) increased the risks of cancer of skin, bladder, kidney, lung and colon (5). Based on these facts, the International Agency for Research on Cancer (IARC, 1980, 1987) and the US Environmental Protection Agency (EPA, 1988) classified inorganic arsenic as a known human carcinogen.

As a carcinogen, arsenite was demonstrated to induce cell proliferation both in cell culture model and animal model (6–13). In cell culture studies, arsenite increases cell proliferation in human keratinocytes (6–8) and enhances the mitogenic effect of suboptimal serum concentrations on quiescent C3H10T1/2 cells (9). In animal studies, low doses of arsenite cause hyperplasia in the urinary bladder epithelium and in skin (8,10–12), and mice exposed to arsenite in drinking water show an increased epidermal thickness and an increased fraction of epidermal cells expressing proliferating cell nuclear antigen (PCNA) as comparison with control mice (13). It is believed that alterations of gene expression that drive uncontrolled cell cycle progression are associated with the aberrant proliferation during tumorigenesis (14,15). Normal eukaryotic cells progress through a well-defined cell cycle consisting of four distinct stages, G1, S, G2 and M. The cell cycle progression is mainly controlled by several key checkpoints, including G1/S checkpoint, S-phase DNA damage checkpoint and G2/M spindle integrity checkpoint. Previous studies demonstrated that faulty G1/S control caused by activation of many oncogenes or inactivation of tumor suppressor genes plays a critical role in tumorigenesis. Cyclin D1 is one of the key regulators of G1/S transition (14–16). Previous studies revealed that antisense to cyclin D1 was able to inhibit the growth and tumorigenicity of human colon cancer cells and induce apoptosis in human squamous carcinomas (17,18). Increasing evidence indicated that the ability of arsenite to modulate the signaling pathways and genes expression responsible for cell growth may play a more important role in its carcinogenesis (19,20). Rossman et al. (21) reported that exposure of human fibroblasts to arsenic can induce cyclin D1 expression; however, it remains unknown whether arsenite exposure is able to cause the increase in cyclin D1 expression in skin cells which are the major target of arsenite; moreover, the molecular mechanisms which mediate this induction remain to be elucidated. Because cell proliferation is believed to be a central event of arsenite skin carcinogenic effect, investigation on the signal pathways leading to cyclin D1 induction may shed some light on the unraveling of the molecular mechanisms, and thereby provide some clues to prevent arsenite-induced carcinogenesis. So we addressed these questions in the present study in mouse epidermal Cl41 cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Reagents and plasmids
Fetal bovine serum (FBS) was purchased from Life Technologies, Inc. (Gaithersburg, MD); MEM (minimal essential medium) was from Calbiochem (San Diego, CA); Arsenite (As3+) was purchased from Aldrich (Milwaukee, WI). The luciferase assay substrate was purchased from Promega (Madison, WI). Wortmannin, PI-3K inhibitor and rapamycin, an mTOR/p70S6k pathway inhibitor, were purchased from Calbiochem (La Jolla, CA). Antibodies specific targeting phospho-Akt (Thr308), phospho-Akt (Ser473), Akt, phospho-p70S6k (Thr389), phospho-p70S6k (Thr421/Ser424), p70S6k, phospho-IKK{alpha}/ß, I{kappa}B{alpha} and cyclin D1 were purchased from Cell Signaling Technology (Berverly, MA). Antibody against GAPDH was from Abcam (Cambridge, MA). The cytomegalovirus-neo vector plasmid, the Akt mutant plasmid (SR{alpha}-Akt-T308A/S473A) and the dominant negative mutant PI-3K plasmid ({Delta}p85), were as described previously (22–25). The dominant negative form of IKKß (IKKß-KM, K44A), which is a gift from Dr. Hiroyasu Nakano (Juntendo University, Japan). The cyclin D1 promoter-driven luciferase reporter (cyclin D1 Luc) construct was obtained by inserting a 1.23 kb EcoR I-Pvu II fragment of the cyclin D1 gene promoter, which contains the cyclin D1 promoter sequence from –1095 to +135 relative to the translation initiation site, into the pA3LUC vector as described previously (26,27).

Cell culture
Mouse epidermal JB6 Cl41 cells and their stable transfectants were cultured in monolayers at 37°C, 5% CO2 using Eagle's MEM containing 5% FBS, 2 mM L-glutamine and 25 µg of gentamicin/ml. The cultures were detached with trypsin and transferred to new 75-cm2 culture flasks (Fisher, Pittsburgh, PA) from once to thrice per week.

Cell proliferation assay
Confluent monolayers of Cl41 transfectants were trypsinized and 1 x 103 viable cells suspended in 100 µl MEM supplemented with 5% FBS were added to each well of 96-well plates. The plates were incubated at 37°C in a humidified atmosphere of 5% CO2. Twelve hours later, the cells were exposed to arsenite, and then lysed with 50 µl lysis buffer. The proliferation of the cells was measured using CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, Madison, WI) with a luminometer (Wallac 1420 Victor2 multipliable counter system). The results are expressed as luciferase activity relative to control medium (proliferation index).

Cell cycle assay
Cl41 cells (2 x 105) were seeded into each well of 6-well plates. After the confluence reached 70–80%, the cells were exposed to 5 µM of arsenite for 48 h, and then harvested and fixed with 3 ml of ice-cold ethanol 80% overnight. The fixed cells was then centrifuged (3000 rpm, 3 min), suspended in lysis buffer (100 mM sodium citrate and 0.1% Triton X-100) and incubated for 15 min at room temperature. The cells were incubated with RNAse A (10 mg/ml) (Sigma Chemical, St. Louis, MO) for 10 min at room temperature and DNA was stained with propidium iodide (50 µg/ml) for at least 1 h at 4°C. The cell proportion in sub-G1 and S phases was determined by flow cytometry using a Epics XL FACS (Beckamn Coulter) and EXPO 32 software (27).

Stable transfection
Cl41 cells were transfected with cyclin D1 luciferase gene reporter, NF{kappa}B luciferase gene reporter, the dominant negative mutant of PI-3K regulatory subunit p85 ({Delta}p85), the dominant negative mutant of Akt (DN-Akt), the dominant negative mutant of IKKß (IKKß-KM) and mock vector according to the manual of Lipofectamine 2000 reagent. Briefly, Cl41 cells were cultured in 6-well plates to 85–90% confluence. Five micrograms of plasmid DNA in combination with 1 µg of CMV-neo vector for co-transfection, were mixed with 10 µl of Lipofectamine 2000 reagent, and used to transfect each well in the absence of serum. After 6 h, the medium was replaced with 5% FBS MEM. Approximately 30–36 h after the beginning of the transfection, the cells were digested with 0.033% trypsin, and the cell suspensions were plated onto 75-ml culture flasks and cultured for 24–28 days with G418 selection (500 µg/ml). Stable transfectant was established and cultured in G418-free MEM for at least two passages before each experiment.

Gene reporter assay
Confluent monolayers of Cl41 transfectants were trypsinized, and 8 x 103 viable cells suspended in 100 µl MEM supplemented with 5% FBS were added to each well of 96-well plates. The plates were incubated at 37°C in a humidified atmosphere of 5% CO2. After the cell density reached 80–90%, the cell culture medium was replaced with an equal volume of MEM supplemented with 0.1% FBS and 2 mM L-glutamine. 12 h later, the cells were exposed to arsenite for cyclin D1 induction or NF{kappa}B activation. After 12-h culture, the cells were lysed with 50 µl lysis buffer, and luciferase activity was measured using Promega Luciferase assay reagent with a luminometer (Wallac 1420 Victor2 multipliable counter system). The results are expressed as cyclin D1 luciferase activity relative to control medium (relative cyclin D1 induction) or NF{kappa}B activity relative to control medium (relative NF{kappa}B activation) (22).

PI-3 kinase assay
PI-3 kinase activities were assayed as described in our previous reports (23,24). Cells were cultured in monolayers in 100 mm dishes using normal culture medium. The media were replaced with 0.1% FBS MEM containing 2 mM L-glutamine and 25 µg of gentamicin/ml after the cell density reached 70–80%. Forty-five hours later, the cells were incubated with fresh serum-free MEM for 3–4 h at 37°C. Arsenite was then added to cell cultures for PI-3K induction. The cells were washed once with ice-cold PBS and lysed in 400 µl of lysis buffer/plate [20 mM Tris (pH 8), 137 mM NaCl, 1 mM MgCl2, 10% glycerol, 1% NP-40, 1 mM DTT, 0.4 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride]. The lysates were centrifuged and the supernatants incubated at 4°C with 40 µl of agarose beads (conjugated previously with the monoclonal antiphosphotyrosine antibody Py20) overnight. Beads were washed twice with each of the following buffers: (i) PBS with 1% NP-40, 1 mM DTT; (ii) 0.1 M Tris (pH 7.6), 0.5 M LiCl, l mM DTT; and (iii) 10 mM Tris (pH 7.6), 0.1 M NaCl, 1 mM DTT. Beads were incubated for 5 min on ice in 20 µl of buffer 3 and then 20 µl of 0.5 mg/ml phosphatidylinositol [sonicated previously in 50 mM HEPES (pH 7.6), 1 mM EGTA, 1 mM NaH2PO4] were added. After 5 min at room temperature, 10 µl of the reaction buffer were added [50 mM MgCl2, 100 mM HEPES (pH 7.6), 250 µM ATP containing 5 µCi of [{gamma}-32P]ATP], and beads were incubated for an additional 15 min. The reactions were stopped by the addition of 15 µl of 4 N HCl and 130 µl of chloroform–methanol (1 : 1). After vortexing for 30 s, the solutions, 30 µl from the phospholipid-containing chloroform phase, were spotted onto TLC plates coated with silica gel H containing 1.3% potassium oxalate and 2 mM EDTA applied in H2O/methanol (3 : 2). The plates were heated at 110°C for at least 3 h before use. The plates were then placed in tanks containing chloroform–methanol–NH4OH/H2O (600 : 470 : 20 : 113) for 40–50 min until the solvent reached the top of the plates. The plates were dried at room temperature and autoradiographed.

Western blot
Cl41 cells (3 x 104) were cultured in each well of 6-well plates to 70–80% confluence with normal culture medium. The cell culture medium was replaced with 0.1% FBS MEM with 2 mM L-glutamine and 25 µg of gentamicin and cultured for 33 h. The cells were incubated in serum-free MEM for 3–4 h at 37°C. After exposure to arsenite, the cells were washed once with ice-cold PBS and then extracted with SDS-sample buffer. The cell extracts were separated on polyacrylamide-SDS gels, transferred, and probed with each of the antibodies against phosphospecific Akt (Thr308), phosphospecific Akt (Ser473), Akt, phosphospecific p70S6k (Thr389), phosphospecific p70S6k (Ser421/Ser424), p70S6k, phosphospecific IKK{alpha}/ß, and I{kappa}B. The protein bands specifically bound to the primary antibodies were detected using an anti-rabbit IgG alkaline phosphatase-linked secondary antibody and an ECF western blot system (Amersham, Piscataway, NJ) (28).

Cyclin D1 expression assay
Cl41 cells and their transfectants (3 x 104) were cultured in each well of 6-well plates to 90% confluence. After exposure to arsenite for different time points as indicated in the figure legends, the cells were washed once with ice-cold PBS and then extracted with SDS-sample buffer. The cell extracts were separated on polyacrylamide-SDS gels, transferred, and probed with antibodies against cyclin D1 or GADPH. The protein bands specifically bound to the primary antibodies were detected as mentioned above.

Statistical analysis
The significance of the difference between treated and untreated groups were determined with the Student's t-test. The results are expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Arsenite exposure induces cell proliferation and cyclin D1 expression in mouse epidermal Cl41 cells
Arsenite was reported to be able to induce cell proliferation both in cell culture model and animal model (6–13). Here we found that exposure of mouse epidermal Cl41 cells which are wildly used as a cell model for the study of tumor promotion, to arsenite also caused significant increase in cell proliferation (Figure 1A). The cell proliferation is strictly controlled by a set of proteins including cyclins. It is demonstrated that cyclin D1 plays a crucial role in controlling cell proliferation (29). Alteration in its expression is believed to associate with tumorigenesis (30). It has been reported that cyclin D1 can be induced by arsenite exposure in normal human fibroblasts (21). In this study, we examined whether cyclin D1 can also be induced by arsenite in Cl41 cells. The results showed that exposure of Cl41 cells to 5 µM of arsenite, a concentration showing no observed toxicity to the cells (Figure 1B and C), resulted in a marked cyclin D1 induction at protein level (Figure 1D) and significant elevation of cell proportion in S phase (Figure 1C). In contrast, other metals including vanadate, cadmium and nickel chloride showed no effect on cyclin D1 expression (Figure 1D). In cyclin D1-luciferase reporter assay, arsenite exposure also induced an increase in cyclin D1 transcription, with over 3-fold induction at 12 h after 2 µM arsenite exposure (Figure 1e). These results demonstrate that arsenite exposure is able to induce cyclin D1 transcription and protein expression in mouse epidermal Cl41 cells.


Figure 1
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  		Fig. 1. Cell proliferation and cyclin D1 expression induced by arsenite in mouse epidermal Cl41 cells. (A) 1 x 103 of viable cells were seeded into each well of 96-well plates. After being cultured at 37°C in a humidified atmosphere of 5% CO2 for 12 h, the cells were exposed to 1.25 µM of arsenite for 72 h. The proliferation of the cells was measured using CellTiter-Glo® Luminescent Cell Viability Assay kit. The results are expressed as luciferase activity relative to control medium (proliferation index). The symbol (*) indicates a significant increase from medium control (P < 0.01). (B and C) 2 x 105 of Cl41 cyclin D1-Luc mass1 cells were seeded into each well of 6-well plates. After being cultured at 37°C overnight, the cells were treated for 24 h with 5 µM of arsenite. The photos for the cell morphology were taken under microscope (B); and the cell apoptosis (cell sub-G1 phase) and S phase were analyzed by propidium iodide staining with Flow Cytometry (C). (D) 2 x 105 of Cl41 cyclin D1-Luc mass1 cells were seeded into each well of 6-well plates. After being cultured at 37°C overnight, the cells were treated for 24 h with 5 µM of arsenite, 100 µM of vanadate, 2 µM of cadmium and 1 mM of NiCl2, respectively. The cells were then washed once with ice-cold PBS and extracted with SDS-sample buffer. Western blot was carried out as described in the Materials and methods section. GAPDH was used as a control for protein loading. (E) 8 x 103 of Cl41 cyclin D1-Luc mass1 cells were seeded into each well of 96-well plates. After being cultured at 37°C overnight, the cells were treated with various concentrations of arsenite as indicated for 12 h. The luciferase activity was then measured and the results are presented as cyclin D1 luciferase activity relative to control. Each bar indicates the mean ± SD of three replicate assay wells.

 
PI-3K-AKT/p70S6K pathway is involved in cyclin D1 induction by arsenite exposure
Numerous studies have demonstrated that PI-3K signal pathway may play a crucial role in eukaryotic cells by activating a set of transcription factors, including CREB and NF{kappa}B, and in turn modulating the expression of genes involved in cell proliferation, survival and malignant transformation (31,32). Here we found that exposure of Cl41 cells to arsenite led to PI-3K activation (Figure 2A), and increase in phosphorylation of Akt (Figure 2B) and p70S6K in dose-dependent manner (Figure 2C). Pretreatment of cells with wortmannin, a chemical inhibitor of PI-3K, resulted in a dramatic inhibition of the phosphorylation of Akt and p70S6K (Figure 2D). Furthermore, inhibition of PI-3K by pre-treatment of Cl41 cells with wortmannin resulted in a significant reduction in arsenite-induced cyclin D1 expression, whereas inhibition of p70S6K pathway by pre-treatment of cells with rapamycin, a chemical inhibitor of mTOR-p70S6K pathway, did not show any inhibitory effect on cyclin D1 induction by arsenite (Figure 2E and F). These results demonstrate that activation of PI-3K/Akt pathway may be implicated in cyclin D1 induction by arsenite in Cl41 cells.


Figure 2
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  		Fig. 2. Activation of PI-3K signaling pathway and its role in cyclin D1 induction by arsenite. 2 x 105 of Cl41 cells were seeded into each well of 100 mm dishes (A) or 6-well plates (B–F) and cultured in 5% FBS MEM at 37°C. After the cell density reached 80–90% (A), the cells were exposed to 5 µM of arsenite for 20 min and then extracted. PI-3K activity was measured as described in the Materials and methods section; (B and C) the cells were exposed to various concentrations of arsenite as indicated for 1 h; (D) the cells were pretreated with various concentrations of wortmannin for 30 min and exposed to 5 µM of arsenite for 1 h; (E and F) the cells were pretreated with wortmannin (100 nM) or rapamycin (20 nM) for 30 min and then exposed to 5 µM of arsenite for 12 h (E) or different time period as indicated (F). The cells were washed once with ice-cold PBS and extracted with SDS-sample buffer. Western blot was carried out as described in the Materials and methods section. GAPDH was used as a control for protein loading.

 
PI-3K/AKT mediates arsenite-induced cyclin D1 expression via IKKß/NF{kappa}B in Cl41 cells
The downstream of PI-3K may include Akt (33,34), mTOR/p70S6K (35), IKK/NF{kappa}B (36), MEK/ERKs/AP-1 (37) and GSK3ß/ß-catanin (38) pathways. The above result with utilization of rapamycin indicated that mTOR/p70S6K did not contribute to PI-3K-mediated cyclin D1 induction by arsenite, even though its activation is mediated by PI-3K. The above results using wortmannin also suggested that arsenite-induced Akt activation may be mediated by PI-3K activation. To test whether Akt is the downstream target of PI-3K, we determined the effect of dominant negative PI-3K mutant ({Delta}p85) on Akt phosphorylation induced by arsenite. As shown in Figure 3A, {Delta}p85 transfection led to impairment of arsenite-induced Akt phosphorylation, suggesting that Akt activation is mediated by PI-3K. To further unravel the downstream kinase of PI-3K/Akt responsible for cyclin D1 induction by arsenite, we addressed IKKß/NF{kappa}B pathway which has been reported to be transactivated by arsenite and is believed to associate with tumor promotion effect of arsenite (39). NF{kappa}B is expressed in an inactive form in most cells, composed of NF{kappa}B p50 and Rel A p65 subunits and bound to an inhibitory protein, I{kappa}B{alpha} (40). In response to stress, cytokine, or growth factor, I{kappa}B{alpha} is phosphorylated by IKK, then ubiquitinated and degraded by the proteasome, enabling nuclear localization and binding of p50/p65 to the promoter region of target genes (40). Cyclin D1 promoter region includes NF{kappa}B binding sites which have been indicated to regulate cyclin D1 expression in Swiss 3T3 cells (41). To test the hypothesis that IKK/NF{kappa}B is the downstream of PI-3K/Akt cascade, we established Cl41-DN-Akt stable transfectants, and analyzed IKK phosphorylation and I{kappa}B{alpha} degradation after exposure of the transfectants to arsenite. The results demonstrated that arsenite exposure was able to induce phosphorylation of IKK and degradation of I{kappa}B{alpha} in Cl41 cells, which were blocked by {Delta}p85 and DN-Akt transfections (Figure 3A), indicating that IKK/NF{kappa}B does act as the downstream transducer of arsenite-triggered PI-3K/Akt cascade. Moreover, arsenite-induced cyclin D1 expression was also blocked in Cl41-{Delta}p85 and Cl41-DN-Akt stable transfectants (Figure 3B). It may be notable that the transfection of {Delta}p85 blocked arsenite-induced cyclin D1 expression more effectively than the transfection of DN-Akt, which may be explained by that endogenous PI-3K kinase can be more effectively inhibited by its dominant negative mutant as compared with Akt kinase, or that there are some signal pathways other than Akt can mediate the signal cascade from PI-3K to cyclin D1 expression. To further confirm the involvement of IKK/NF{kappa}B in arsenite-induced cyclin D1 expression, we examined the effect of arsenite on NF{kappa}B transactivation in Cl41 cells by gene reporter assay. The result indicated that exposure of Cl41 cells to arsenite markedly induced NF{kappa}B activation which was dramatically inhibited by the co-transfection of IKKß-KM, the dominant negative mutant of IKKß (Figure 4A and B). Moreover, the overexpression of IKKß-KM inhibited arsenite-induced cyclin D1 expression dramatically at both transcription and protein levels (Figure 4C–E). It may be noted that cyclin D1 induction decreased when the dosage of arsenite is higher than 4 µM in the luciferase reporter assay (Figure 4D), whereas 5 µM of arsenite seems to cause the maximal cyclin D1 induction in western blot assay (Figure 4E). This inconsistent induction between transcription and protein levels may be due to that high dosage of arsenite may also affect the degradation of cyclin D1 which facilitates cyclin D1 accumulation. Overall, our data strongly demonstrate that induction of cyclin D1 expression by arsenite in Cl41 cells requires PI-3K/Akt/IKKß/NF{kappa}B pathway.


Figure 3
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  		Fig. 3. The role of PI-3K/AKT in the activation of IKKß/NF{kappa}B and cyclin D1 expression induced by arsenite. 2 x 105 of Cl41-mock vector, Cl41-{Delta}p85 and Cl41-DN-Akt cells were seeded into each well of 6-well plates and cultured in 5% FBS MEM at 37°C. After the cell density reached 80–90%, the cells were exposed to various concentrations of arsenite as indicated for 1 h (A) or 24 h (B). The cells were then extracted with SDS-sample buffer. Western blot was carried out using specific antibodies as described in the Materials and methods section.

 

Figure 4
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  		Fig. 4. Involvement of IKKß/NF{kappa}B in arsenite-induced cyclin D1 expression. (A) Cl41 NF{kappa}B/vector control mass1 cells and stable co-transfectant of Cl41 NF{kappa}B/IKKß-KM mass1 cells were extracted with SDS-sample buffer. The cell extracts were analyzed by western blot with specific anti-FLAG antibody (M2) or GAPDH antibody. (B) 8 x 103 of Cl41 NF{kappa}B/control mass1 cells and stable co-transfectant of Cl41 NF{kappa}B/IKKß-KM mass1 cells were seeded into each well of 96-well plates. After being cultured at 37°C overnight, the cells were treated with 5 µM of arsenite for 12 h. The luciferase activity was then measured and the results are presented as NF{kappa}B luciferase activity relative to control. Each bar indicates the mean and SD of three replicate assay wells. The symbol (*) indicates a significant difference between IKKß-KM- and control vector-transfected cells (P < 0.01). (C) Cl41 cyclin D1-2/control mass1 cells and stable co-transfectant of Cl41 cyclin D1-2/IKKß-KM mass1 cells were extracted with SDS-sample buffer. The cell extracts were analyzed by western blot with specific anti-FLAG antibody (M2). (D) 8 x 103 of Cl41 cyclin D1-2/control mass1 cells and stable co-transfectant of Cl41 cyclin D1-2/IKKß-KM mass1 were seeded into each well of 96-well plates. After being cultured at 37°C overnight, the cells were treated with various concentrations of arsenite as indicated for 12 h. The luciferase activity was then measured and the results are presented as cyclin D1 luciferase activity relative to control. Each bar indicates the mean and SD of three replicate assay wells. (E) 2 x 105 of Cl41 cyclin D1-2/control mass1 and Cl41 cyclin D1–2/IKKß-KM mass1 cells were seeded into each well of 6-well plates and cultured in 5% FBS MEM at 37°C. After the cell density reached 80–90%, the cells were exposed to various concentrations of arsenite as indicated for 24 h. The cells were then extracted with SDS-sample buffer. Western blot was carried out as described in the Materials and methods section. GAPDH was used as protein loading control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Arsenite is a well-documented skin carcinogen (1,3). Numerous studies have demonstrated that exposure to arsenite can induce cell proliferation and cell transformation. It is believed that modulation on cell cycle machinery is associated with arsenite-induced carcinogenesis although the precise mechanisms remain to be fully elucidated. D type cyclins (particularly cyclin D1) play a crucial role in controlling the checkpoint of G0/G1 to S phase transition (16), and are implicated in cell proliferation and tumorigenesis (42). Cyclin D1 expression varies in abundance during the cell cycle, and its protein level is mainly controlled by the rate of cyclin D1 gene transcription, which is regulated by multiple transcription factors. The cyclin D1 promoter region contains multiple cis-elements, includes binding sites for NF{kappa}B (32,41,43–45), AP-1 (46,47), STAT (48), E2F/DP (49), ATF/CREB (47,50–52) and sp-1/sp-3 (52,53). These cis-elements are potentially important for transcriptional activation of the cyclin D1 gene. In fact, it was reported that NF{kappa}B binding sites in the cyclin promoter were implicated in transcriptional activation of the gene (32,41,43–45). Angiotensin II induced activation of the cyclin D1 promoter through the AP-1 binding site (46,47). It was also reported that STAT-binding sites were implicated in cytokine-induced transcriptional activation of the cyclin D1 gene in hematopoietic cells (48). E2F-1 mediated a Neu-signaling cascade to cyclin D1 induction (49). The ATF/CREB-binding site was also demonstrated to mediate the expression of the cyclin D1 gene (47,50–52). pp60v-src induced transcriptional activation of the cyclin D1 gene mediated by the ATF/CREB site (47). Moreover, it was found that estrogen-induced activation of the cyclin D1 gene depended on the ATF/CREB site in which ATF-2 and c-Jun formed heterodimers (51). Whereas in vascular endothelial cells, activation of the cyclin D1 promoter was largely mediated by the ATF/CREB and SP1 sites (52). These results suggest that the transcriptional activation of the cyclin D1 gene may occur in a cell type- and mitogen-specific manner.

In the present study, we demonstrated that exposure of mouse epidermal Cl41 cells to arsenite led to activation of PI-3K/Akt which thereby induce cyclin D1 expression via its downstream IKK/NF{kappa}B signal pathway. Inhibitions of either PI-3K/Akt or IKKß dramatically impaired cyclin D1 induction by arsenite. In our previous study, arsenite was shown to be able to induce cyclin D1 expression and thereby cause the progression of G1/S transition in human keratinocytes (27), which also requires PI-3K/Akt activation demonstrated in our recent studies (Ouyang et al. unpublished data). Although most previous studies on NF{kappa}B activation through Akt/IKK signaling have focused their attention on the IKK{alpha}, and it has been shown that Akt directly phosphorylates the IKK{alpha} under stimulation by TNF, EGF, insulin-like growth factor-I and platelet-derived growth factor, there are some studies that most recently demonstrated that IKKß is also a substrate of Akt in HTLV-1-transformed cells (54), and in breast cells when treated by FGF-2 (55). The results in this study suggest that IKKß may also be the substrate of Akt in arsenite-exposed Cl41 cells. In addition, the effect of arsenite on NF{kappa}B activation has been indicated to be time-, dose- and cell type-dependent (56). Whereas high concentrations of arsenic (>10 µM) generally inhibit NF{kappa}B which was recently demonstrated to be caused by reaction with the critical cysteine in the activation loop of IKKs (56), low and non-cytotoxic concentrations of arsenic (1–10 µM) usually activate this transcription factor. In the current and our previous studies, arsenite was demonstrated to induce NF{kappa}B activation in mouse epidermal JB6 cell model and human keratinocytes (27).

However, it is interesting that the contributions of 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 (57–59); and overexpression of active p50 and p65 NF{kappa}B subunits in transgenic epithelium produced hypoplasia and growth inhibition (60). But it has been reported that the deletion of IKKß did not affect the proliferation of skin epidermis (61); I{kappa}B{alpha} deficiency results in a sustained NF{kappa}B response and severe widespread dermatitis characterized by epidermal hyperplasia in mice (62); and NF{kappa}B is a key mediator of integrin ß4-induced epidermal growth (63). Although it is claimed that some carcinogens may exert their carcinogenic effect by inhibiting IKK{alpha} activation, and thereby promote the proliferation of skin epidermis, while a growing body of evidence indicates NF{kappa}B might be involved in keratinocyte transformation and skin carcinogenesis (64,65). It has been 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 (64). Substances like phorbol ester and okadaic acid, which are promoters of skin cancer, are also strong inducers of an NF-{kappa}B response in keratinocytes (64). Furthermore, a Ha-ras oncogene point mutation is found in over 90% of mouse skin tumor, in which NF-{kappa}B activation has been observed (64). In the Ha-ras point mutation mouse model, it has also been shown that while in normal epidermis NF-{kappa}B was expressed in the cytoplasm of basal cells, p50 and p52 are elevated in squamous cell carcinoma as well as in skin papillomas (65).

In addition to IKK/NF{kappa}B (36), mTOR (35), MEK/ERKs/AP-1 (37) and GSK3ß/ß-catanin (38) pathways are also the downstreams of PI-3K/Akt cascade. In this study, inhibition of mTOR by rapamycin did not show any inhibitory effect on the cyclin D1 induction by arsenite, suggesting that mTOR may not be the downstream transducer of PI-3K/Akt cascade in arsenite-induced cyclin D1 expression. GSK3ß has been reported to play a very important role in the negative-regulation of cyclin D1 level at both transcription and protein levels (38,50,66). It has been demonstrated that GSK3ß can phospholate cyclin D1 and thereby facilitate its degradation (38,66). In addition, GSK3ß is able to phosphorylate ß-catanin and retain it in cytoplasm with APC (38). Phosphorylation of GSK3ß by Akt leads to its inactivation, and in turn dephosphorylation of ß-catanin, which enables its translocation into nuclear and activation of its target genes expression including cyclin D1 (38,50,67). Actually, we have observed that ß-catanin can be activated after arsenite exposure. Its role in cyclin D1 induction by arsenite is currently under investigation. In addition, we found that exposure of Cl41 cells to arsenite can also induce the activation of other transcription factors, such as AP-1 (68). It remains to be explored if these transcription factors are also involved in cyclin D1 induction by arsenite.

It has been well-accepted that alterations in the cell cycle machinery at checkpoints are associated with cell proliferation, tumor development (29,30,69). The re-entry of cell cycle of the quiescent cells from G0 to G1 phase and the progression of G1/S transition in actively proliferating cells are regulated by a set of cyclins (particularly cyclin D) via regulating the activity of the cyclin-dependent kinases (CDK4 and CDK6) (16,42,70,71). Aberrant cyclin D1 expression has been observed early in carcinogenesis (72–74), and overexpression of cyclin D1 was reported in several human cancers, including uterine cervix (75), ovary (76), breast (77), urinary bladder (78) and endometrium cancers (79). Moreover, antisense to cyclin D1 was reported to inhibit the growth and tumorigenicity of human colon and human lung cancer cells (17,18). Here, we demonstrated that exposure of mouse epidermal Cl41 cells to arsenite was able to induce cyclin D1 expression through PI-3K/Akt/IKK/NF{kappa}B pathway, which may play a key role in arsenite-induced cell proliferation. In fact, we found that cyclin D1 induction is critical for arsenite-induced cell cycle progression from G0/G1 phase to S phase in human keratinocyte (27). The exact role of the increased cyclin D1 in arsenite-induced proliferation and cell transformation is currently also under investigation in our laboratory.

In summary, our studies demonstrated that exposure of mouse epidermal Cl41 cells to arsenite leads to activation of PI-3K/Akt/IKK/NF{kappa}B signal pathway which thereby induce cyclin D1 expression. Because of the important role of cyclin D1 in controlling cell cycle progression, its induction may associate arsenite-induced cell proliferation and transformation. Thus, our present studies provide novel information for understanding the molecular mechanisms involved in cyclin D1 expression and carcinogenic effect by arsenite in its major target tissue of skin.


   Notes
 
1 These authors contributed equally to this work. Back


   Acknowledgments
 
We thank Dr Hiroyasu Nakano (Juntendo University, Japan) for the gift of IKKß-KM expression vector, and Dr Rustgi (University of Pennsylvania, USA) for cyclin D1 luciferase gene reporter. This work was supported in part by grants from NIH/NCI (R01 CA094964, R01 CA112557 and R01 CA103180) and NIH/NIEHS (R01 ES012451 ES010344 and ES000260).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 25, 2005; revised September 28, 2005; accepted December 17, 2005.


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