Identification and in silico characterization of a novel gene: TPA induced trans-membrane protein Chung-Yip Chan 1 , Mohammad R. Salabat 1 , Xian-Zhong Ding, David L. Kelly, Mark S. Talamonti, Richard H. Bell Jr., Thomas E. Adrian * Department of Surgery and Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA Received 22 December 2004 Abstract 12-O-Tetradecanoylphorbol-13-acetate (TPA) is a potent tumor promoter with wide ranging, diverse, and sometimes opposite cel- lular effects. Using oligonucleotide microarray analysis, we have identified a novel gene that is upregulated following treatment with TPA in the pancreatic cancer cell line CD18. Real-time PCR validated the microarray results in CD18 and HeLa cells, and showed that upregulation of the gene is time- and concentration-dependent. In silico analysis showed the gene product to be a single-pass transmembrane protein of 217 residues that is localized to the endoplasmic reticulum, thus the name TPA induced trans-membrane protein (TTMP). A luciferase reporter assay demonstrated that upregulation of TTMP by TPA is triggered at the promoter level. Published by Elsevier Inc. Keywords: 12-O-Tetradecanoylphorbol-13-acetate; Protein kinase C; TTMP; Novel protein; Microarray; Real-time PCR; In silico analysis; Luciferase assay 12-O-Tetradecanoylphorbol-13-acetate (TPA) is a potent tumor promoter with wide-ranging and diverse cellular effects. Protein kinase C (PKC) is thought to play a major role in tumor promotion/progression in tis- sues because it is the major cellular receptor for tumor- promoting phorbol esters [1,2]. In cultured cells, phorbol esters influence cellular growth, morphologic features, transformation, and gene expression [3]. The complexity of phorbol ester actions is probably related to the presence of multiple phorbol ester/DAG recep- tors, which include not only PKC isozymes but also other classes of receptors like the a- and b-chimaerin iso- forms, Unc-13/Munc13 isoforms, and Ras-GRP [4]. In most cases, at least five or more PKC isozymes are present in a single cell and have overlapping or opposite functions. Activation of PKCs by phorbol esters pro- motes early phases of mitogenesis, as suggested by the involvement of PKCs in growth factor actions, mito- gen-activated protein kinase (MAPK) activation, and expression of early response genes [5]. Conversely, TPA may also inhibit cell growth or induce apoptosis in several cell types [6–8]. PKC isozymes operate as reg- ulators of the cell-cycle both during G 1 /S progression and G 2 /M transition [9]. A bimodal regulation of G 1 progression has been observed in some cell lines [10]. Overexpression of active form of PKCs was seen to block the normal phosphorylation of the retinoblastoma (Rb) protein in quiescent cultures of NIH3T3 cells that had been stimulated to enter the cell cycle. This delayed expression of cyclin-dependent inhibitors p21 and p27, and led to a reduced expression of cyclin E or cyclin A [11–13]. Several studies have suggested that Cdc2, the ki- nase involved in G 2 /M transition, as well as Cdc25 phos- phatase are also PKC targets. Cell cycle arrest has been 0006-291X/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.bbrc.2005.02.037 * Corresponding author. Fax: +1 312 503 3491. E-mail address: tadrian@northwestern.edu (T.E. Adrian). 1 These authors contributed equally to this work. www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 329 (2005) 755–764 BBRC