Objective To identify the proximal promoter of down-expression of growth arrest DNA damage-inducible gene 46 β(GADD45β)and to evaluate the influence of hydroxyurea on HCC cell lines.Methods The proximal promoter fragments(-618/-314)were synthesized in vitro and cloned into pGL3 basic luciferase expression plasmid,then transfected into HepG2 by electroporation.The promoter regions were identified in reference of TRANSFAC database.Following hydroxyurea administration,quantitative real-time PCR was employed to validate the expression changes of GADD45β.The effect of hydroxyurea on promoter activity was further focused on.The influence of cell growth and the expression of Caspase-8,Caspase-9 and Caspase-3 were further determined.Results Using the luciferase assay,several transcription factor binding sites were identified in the -528)and one E2F-1(-470/-436).After hydroxyurea administration,the increasing expression of GADD45β was marked in a dose-dependent manner.The luciferase assay results also demonstrated hydroxyurea.The colony formation and DNA syntheses were inhibited apparently and hydroxyurea could trigger the apoptosis rapidly following treatment.Conclusion Hhydroxyurea could induce the expression of GADD45β,which is specifically down-regulated in HCC.The possible mechanism may be the regulation of transcription factors in GADD45β proximal promoter. Key words: Carcinoma hepatocellular; Hydroxyurea; GADD45β; Promoter