Up-regulation of growth-inhibitory or tumor suppressive genes by overexpression of C/EBPα or C/EBPβ in ras- transformed NIH3T3 cells

CCAAT/Enhancer-binding proteins (C/EBPs) are DNA-binding proteins that regulate cell-specific growth and differentiation. Here, we have generated ras-transformed NIH 3T3 cells stably expressing C/EBPα or C/EBPβ. In cells overexpressing C/EBPα and C/EBPβ, growth arrest, changes in morphology and differentiation were induced. cDNA microarray analysis followed by RNA blot analysis identified genes that are possibly involved in growth arrest and/or differentiation of cells in response to C/EBPα and C/EBPβ transfection. Our results demonstrate that C/EBPα and C/EBPβ cause growth-inhibitory effects, which may be mediated by up-regulated proteins such as amyloid precursor protein and proteoglycans. Abbreviations: ADM: adriamycin, APP: amyloid precursor protein, C/EBP: CCATT/enhancer binding protein, DMEM: Dulbecco’s modified Eagle’s minimum essential medium, Oxct: 3’-oxoacid CoA transferase, ras-NIH: activated Ha-ras-transformed NIH3T3 mouse fibroblasts, SePP: selenoprotein P Introduction Transcription factors of the CCAAT/Enhancer-binding protein (C/EBP) family are highly conserved leucine-zipper DNA-binding proteins that are expressed in a variety of tissues and cell types. The six mammalian C/EBPs (α, β, δ, γ, ε, CHOP) bind to DNA and facilitate the activation or repression of gene transcription [1-3]. Replacement of the basic region of C/EBP family members with that of other C/ EBPs leads to alterations in DNA-binding specificity for the resultant homoor heterodimer fusion proteins [4]. C/EBPα is highly expressed in liver, fat, lung, peripheral leucocytes, epidermis, intestine, and skeletal muscle [5] C/EBPα has been implicated in the regulation of tissue-specific gene expression and differentiation of adipocytes and hepatocytes [6], as well as negative regulation of cell growth [7]. The importance of C/EBPα in the regulation of growth arrest and differentiation is exemplified by human acute myeloid leukemia [8]. Dominant-negative mutations in C/EBPα are thought to results in a block of the differentiation of granulocytic blasts and have implicated C/EBPα as a tumor suppressor gene [2,9]. These findings suggest that expression of the different C/EBP protein isoforms is tightly regulated and that the ratio of isoforms is important in controlling proliferation and differentiation. Deregulated expression of truncated C/EBP and C/EBP isoforms interferes with terminal differentiation and induces cell transformation in 3T3-L1 adipocytes [10]. Hence, regulation of the translation initiation that determines the ratio of C/EBP isoforms has a crucial role in the control of cell proliferation and differentiation in cells expressing C/EBPα and C/EBPβ. C/EBP-deficient animals exhibit an absence of subcutaneous white adipose tissue and granulocytes, and typically die within a few hours of birth due to hypoglycemia [11], demonstrating that this transcription factor plays a key role in tissue-specific gene expression and differentiation. Both C/EBPβ and C/EBPα are involved in regulating the proliferation and differentiation of adipocytes and hepatocytes [6]. C/EBPβ also plays an essential role in ovarian granulosa cell biology and development and differentiation of the mammary gland [12]. Thus, disruption of signaling through C/EBPβ appears to contribute to malignant transformation. At very early stages of liver proliferation initiated by partial hepatectomy, the transcription factor C/EBPβ is activated mainly through posttranslational modifications [13]. Lamb et al. have identified a cyclin-dependent kinase (CDK)-independent mechanism by which the interaction of cyclin D1 with the transcription factor C/EBPβ may mediate tumorigenesis [14]. It has been suggested that C/EBPβ acts as a constitutive repressor of cyclin D1 targets and that cyclin D1 antagonizes this repressor function. C/EBPδ may also be involved in the acute-phase response and appears to be required for the G0 growth arrest of epithelial cells in mammary tissues [15]. Studies in recent years have pinpointed several potential mechanisms for C/EBPα-mediated cell growth arrest. It has been suggested that C/EBPα expression in human fibrosarcoma cell line leads to the induction of p21, an inhibitor of CDK, promoter activity, and the accumulation of p21 protein. Further study has indicated the potential ability of C/EBPα to stabilize p21 protein and thereby halt cell-cycle progression [16]. Iakova and colleagues have presented Correspondence to: Takaki Hiwasa, Department of Biochemistry and Genetics, Graduate School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8670, Japan, Tel: +81-43-226-2541, Fax: +81-43-26-2037; E-mail: [email protected]