Cyclin D1 Induction through IKB Kinase B/Nuclear Factor-KB Pathway Is Responsible for Arsenite-Induced Increased Cell Cycle G1-S Phase Transition in Human Keratinocytes

Environmental and occupational exposure to arsenite is associated with an increased risk of human cancers, including skin, urinary bladder, and respiratory tract cancers. Although much evidence suggests that alterations in cell cycle machinery are implicated in the carcinogenic effect of arsenite, the molecular mechanisms underlying the cell cycle alterations are largely unknown. In the present study, we observed that exposure of human keratinocyte HaCat cells to arsenite resulted in the promotion of cell cycle progression, especially G1-S transition. Further studies found that arsenite exposure was able to induce cyclin D1 expression. The induction of cyclin D1 by arsenite required nuclear factor-KB (NF-KB) activation, because the inhibition of IKB phosphorylation by overexpression of the dominant-negative mutant, IKKB-KM, impaired arsenite-induced cyclin D1 expression and G1-S transition. The requirement of IKB kinase B (IKKB) for cyclin D1 induction was further confirmed by the findings that arsenite-induced cyclin D1 expression was totally blocked in IKKB knockout (IKKB / ) mouse embryo fibroblasts. In addition, knockdown of cyclin D1 expression using cyclin D1–specific small interference RNA significantly blocked arsenite-induced cell cycle progression in HaCat cells. Taken together, our results show that arsenite-induced cell cycle from G1 to S phase transition is through IKKB/NF-KB/cyclin D1–dependent pathway. (Cancer Res 2005; 65(20): 9287-93) Introduction Arsenite (trivalent arsenic, As) is introduced into the environment during energy production based on coal, oil shale, and geothermal sources. Available epidemiologic 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). Many cases of skin cancer have been documented in people exposed to arsenite through medical or other occupational exposures (5). Based on these facts, the IARC (1980 and 1987) and the U.S. Environmental Protection Agency (1988) classified inorganic arsenic as a human carcinogen. Several hypotheses have been proposed to describe the mechanisms of arsenite-induced carcinogenesis in the previous studies (6–12). It has been suggested that arsenic induces chromosome aberration and sister chromatid exchange may be involved in arsenite-induced carcinogenesis (9, 10). Zhao et al. (11) reported that arsenic may act as a carcinogen by inducing DNA hypomethylation, which in turn facilitates aberrant gene expression. However, more and more evidence indicates that the effect of arsenite on modulating the signaling pathways and genes expression responsible for cell cycle machinery may play a more important role in the carcinogenesis than classic genotoxic and mutagenic mechanisms, which are believed to be associated with the carcinogenesis of other metals such as cadmium or chromium (13, 14). It is widely accepted that alterations of gene expression that drive uncontrolled cell cycle progression are requisite events during tumorigenesis (15–17). Cyclin D1, acting as a sensor in response to extracellular changes, can be induced by growth factors and stress (18–20). Aberrant cyclin D1 expression has been observed early in carcinogenesis (21–23), and overexpression of cyclin D1 was reported in several human cancers, including uterine cervix (24), ovary (25), breast (26), urinary bladder (27), endometrium (28), and skin (29) cancers. Moreover, antisense to cyclin D1 was reported to inhibit the growth and tumorigenicity of human colon cancer cells and induce apoptosis in human squamous carcinomas (30, 31). Rossman et al. reported that exposure of human fibroblasts to arsenite results in the induction of cyclin D1 expression (32). Nonetheless, the effect of arsenite on cell cycle progression remains obscure. Because human skin is a major target of arsenite, it would be interesting to know whether cyclin D1 is induced by arsenite in human keratinocytes. If it is, what are the signaling pathways responsible for this induction and what are their roles in alternations of cell cycles caused by arsenite exposure? Consequently, we here addressed these questions in human keratinocyte HaCat cells. Materials and Methods Cell culture and reagents. Human keratinocytic HaCat cells and their stable transfectants, wild-type (WT) and InB kinase h (IKKh)–deficient (IKKh / ) mouse embryo fibroblasts were cultured in monolayers at 37jC, 5% CO2 using DMEM containing 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, and 25 Ag of gentamicin/mL. The cultures were detached with trypsin and transferred to new 75-cm culture flasks (Fisher, Pittsburgh, PA) from one to three times per week. FBS was purchased from Life Technologies, Inc. (Gaithersburg, MD); DMEM was from Calbiochem (San Diego, CA); luciferase assay substrate was from Promega (Madison, WI). As was purchased from Aldrich (Milwaukee, WI). The cyclin D1 promoterdriven luciferase reporter (cyclin D1 Luc) was from Dr. Anil Rustgi (Gastroenterology Division, University of Pennsylvania, Philadelphia, PA). Note: Z-G. Liu is currently at the Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892. A.K. Rustgi is currently at Division of Gastroenterology, Departments of Medicine and Genetics, Abramson Cancer Center, University of Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104. Requests for reprints: Chuanshu Huang, Nelson Institute of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987. Phone: 845-731-3519; Fax: 845-351-2320; E-mail: [email protected]. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-0469 www.aacrjournals.org 9287 Cancer Res 2005; 65: (20). October 15, 2005 Research Article American Association for Cancer Research Copyright © 2005 on February 21, 2013 cancerres.aacrjournals.org Downloaded from DOI:10.1158/0008-5472.CAN-05-0469