Regulation of RASSF1A in nasopharyngeal cells and its response to UV irradiation Vien H.Y. Lee a , Billy K.C. Chow a , K.W. Lo b , Lillian S.N. Chow b , Cornelia Man c , S.W. Tsao c , Leo T.O. Lee a, ⁎ a School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, China b Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China c Department of Anatomy, The University of Hong Kong, Pokfulam Road, Hong Kong, China abstract article info Article history: Received 26 March 2009 Received in revised form 4 May 2009 Accepted 12 May 2009 Available online 18 May 2009 Received by P. Deininger Keywords: Ras-Association Domain Family 1 A (RASSF1A) Sp-proteins Nasopharyngeal carcinoma Promoter RASSF1A, which is frequently found inactivated in human cancers, is revealed as a tumor suppressor gene in nasopharyngeal carcinoma (NPC). Using RASSF1A-expressing (NP69 and HK-1) and non-RASSF1A-expressing (C666-1) cell models, the transcriptional regulation of RASSF1A was studied. By deletion analysis of 3.1 kb of 5′ flanking region, the core promoter of RASSF1A was identified in the region between -431 and -1 upstream of the translation start site. Sequence analysis of this core promoter revealed several putative transcription factor binding sties. Using NP69 cells and by block replacement mutagenesis, the presence of three functional GC-boxes were identified, to which by competitive and supershift electrophoretic mobility shift assays (EMSA), the in vitro bindings of Sp1 and Sp3 were suggested. The in vivo functions of Sp-proteins in regulating RASSF1A gene were then investigated by overexpression studies; among the tested Sp-proteins, Sp1 or Sp3, but not Sp4, was able to augment promoter activities. More interestingly, co-expression of Sp1 and Sp3 could synergistically enhance RASSF1A promoter function. UV irradiation induces oxidation stresses and hence is routinely used to investigate expressions of oncogenes and tumor suppressors. In this report, upon UV irradiation, the RASSF1A promoter activity and endogenous transcript levels were found to be reduced. By chromatin immunoprecipitation (ChIP) and EMSA, we demonstrated that the binding of Sp1 and Sp3 onto -431 to -202 were significantly reduced after UV irradiation. This UV-mediated effect on RASSF1A promoter, as shown by specific inhibitors that interrupt cellular pathways, is MEK1-, but not JNK-dependent. In summary, our data provided a simple model to explain the potential development of NPC, via silencing of the tumor suppressor RASSF1A by reduced bindings of activators Sp1 and Sp3 onto the GC-boxes in the core promoter of the gene. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Ras-association domain family 1 protein (RASSF1) belongs to a family of proteins that are characterized by the presence of a Ras- association domain at their C-termini. The gene possesses eight exons and codes for eight isoforms (A to H) (Donninger et al., 2005). The isoforms A and C are the 2 major transcripts that are derived from alternative splicing and different promoter usage, while other isoforms are tissue-specific splice variants (Dammann et al., 2000). The RASSF1 gene contains two CpG islands in exon 1α and exon 2γ. Transcriptional silencing caused by hypermethylation of CpG island(s) within the promoter region is a common mechanism that leads to the inactivation of tumor suppressor genes. As a potential tumor suppressor, RASSF1A has frequently been reported to be epigenetically inactivated by hypermethylation of CpG islands in xenografts, cell lines and primary nasopharyngeal carcinoma (NPC) as well as a broad range of human cancers (Lo et al., 2001). Oxidative stresses like UV irradiation may play an important role in carcinogenesis. UVB can initiate and promote exposed keratinocytes towards a malignant phenotype (Cooper et al., 2005). Exposure of mammalian cells to UV light has been reported to elicit various cellular responses (Assefa et al., 2005; Kaina, 2003). UV light could directly induce DNA base changes, DNA strand breaks, as well as DNA–DNA and DNA–protein cross-linking (Ghosh et al., 1993). In addition, it can modulate the expression of genes by activating UV-responsive transcrip- tion factors such as AP-1, NF-κB, ERK and JNK to phosphorylate Sp1 and Sp3 proteins (Chen et al., 1998; Bender et al., 1998; Chu and Ferro, 2006). RASSF1A is recognized as a critical tumor suppressor gene involved in development of cancers, however, our understanding on the transcriptional regulation of this gene is still unclear. In this report, we studied the transcriptional regulation of the RASSF1A gene and we found that the GC-boxes residing in the core promoter are regulated by Sp-proteins. To better understand the mechanisms leading to RASSF1A silencing, we studied the effects of UV irradiation on Gene 443 (2009) 55–63 Abbreviations: ChIP, chromatin immunoprecipitation assays; EBV, Epstein–Barr virus; EMSA, electrophoretic mobility shift assays; EP4, prostaglandin E receptor 4; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MEK, mitogen- activated protein kinase; NPC, Nasopharyngeal carcinoma; RASSF1, Ras-Association Domain Family 1; RKIP, Raf kinase inhibitor protein; ROS, reactive oxidative species; VEGF-A, vascular endothelial growth factor-A. ⁎ Corresponding author. Tel.: +852 2299 0845; fax: +852 2559 9114. E-mail address: ltolee2@hkucc.hku.hk (L.T.O. Lee). 0378-1119/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2009.05.003 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene