Induction of cyclin D1 by submicromolar concentrations of arsenite in human epidermal keratinocytes.
Arsenic is a prevalent environmental carcinogen but arsenic is not directly mutagenic and the mechanism by which arsenite brings about oncogenic transformation is poorly understood. To gain insight into the oncogenic properties of arsenic, we studied the expression of cyclin D1 in cultured human epidermal keratinocytes treated with submicromolar concentrations of sodium arsenite. Arsenite at concentrations between 200 and 800 nM over a 3-day period brought about an increase in cell growth rate. Uptake of the vital stain, neutral red, arsenite at 200 and 400 nM concentrations brought about a parallel increase in cell viability over the same treatment period. Analysis of cell cycle parameters by flow cytometry showed that the growth stimulation was accompanied by a concomitant shift from the G1 into the S/G2 cell cycle compartment in the arsenite-treated cells. Real-time PCR analysis of cyclin D1 transcription showed that there was an induction of more than three-fold in cells exposed to 400 nM arsenite for 3 days. Quantitation of cyclin D levels in Western blots showed that arsenite treatment caused a time-dependent induction of cyclin D proteins representing an induction of about 2.0-fold after a 7 day treatment period. Electrophoretic mobility shift assays (EMSA) showed that arsenite also stimulated binding of the transcription factors, AP1 and CREBP to their respective binding motifs within 3 days. This supports a mechanism of oncogenesis based on persistent upregulation of D type cyclins leading to a concomitant loss of G1/S checkpoint control.
Hwang
Bor-Jang
BJ
Department of Chemistry, The City College of the City University of New York, Convent Avenue and 138th Street, New York, NY 10031, USA.
Utti
Charles
C
Steinberg
Mark
M
D018053:Arsenites / Q000633:toxicity*; D050882:CREB-Binding Protein / Q000378:metabolism; D002273:Carcinogens / Q000633:toxicity*; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D056741:Cyclin D; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D024202:Electrophoretic Mobility Shift Assay; D004817:Epidermis / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D017670:Sodium Compounds / Q000633:toxicity*; D013997:Time Factors; D018808:Transcription Factor AP-1 / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects*
D018053:Arsenites; D002273:Carcinogens; D056741:Cyclin D; D016213:Cyclins; D012333:RNA, Messenger; D017670:Sodium Compounds; D018808:Transcription Factor AP-1; C017947:sodium arsenite; D004247:DNA; D050882:CREB-Binding Protein; C494363:CREBBP protein, human