Molecular Cloning And Characterization Of A Novel Membrane-associated Gene CT120 Implicated In Human Lung Carcinogenesis

Tumorigenesis in human often reflects numerous genetic lesions including sequence alterations (point mutations, deletions, insertions) and/or structural abnormalities (rearrangements, large-scale deletions, chromosomal amplifications), which result in the progressive transformation of normal human cells into highly malignant derivatives. In a variety of human malignancies, 17p13.3 is one of the chromosomal regions most frequently affected by allelic loss or genomic imbalances, suggesting that one or more tumor-related genes in this region may play important roles in human carcinogenesis.Within the most frequently mutated region on chromosome 17p13.3 in human cancers, a novel human plasma membrane-associated gene, named CT120, was isolated from a human kidney cDNA library using electronical cloning and RACE. The novel gene CT120 consists of 2145 base pairs and encodes a protein with 257 amino acids. Database search revealed that homologs of CT120 exist in different organisms from plant to animal kingdoms, which suggests that CT120 is a highly conserved gene during biological evolution. Different expression patterns of CT120 were observed in many different human normal tissues and in various human tumor cell lines. Transcript of CT120 was not detectable in normal lung tissue, but was abundant in SPC-A-1 (human epithelial-like lung adenocarcinoma) cell line, suggesting that CT120 maybe involve in lung cancer development. Subcellular localization analysis showed that CT120 is a novel membrane-associated protein. CT120 can interact with SLC3A2 (member 2 of solute carrier family 3) and GGTL3B (isoform of γ-glutamyltranspeptidase-like 3) in eukaryotic cells by yeast two-hybrid screen and co-immunoprecipitation assay, which suggested that CT120 may assume very essential physiological functions involved in amino acid transport and glutathione metabolism.To further study the relationships between CT120 and human lung carcinogenesis, we investigate the differential expression patterns of CT120 proteins in different lung cancer and noncancerous tissues using western blotting and immunohistochemistry assays with chicken anti-CT120 polypeptide antibody, and to study its effects on cell growth in vitro and in vivo. A polypeptide at the C-terminus of CT120 was selected by bioinformatics, then synthesized and conjugated to KLH. The chicken anti-CT120 antibody IgY was prepared with the synthesized antigen and was used to detect the different expression patterns of CT120 in various tumor cell lines and in lung cancer and noncancerous tissues. The results showed that the novel gene CT120 expresses in various tumor cell lines and expresses remarkably higher in lung cancers than in noncancerous tissues as well as normal lung tissues. Also, both of two transcripts CT120-A and CT120-B expressed in A549 and SPC-A-1 cells. Although CT120-A expressed in all of the lung cancer tissues used in this study, the expression patterns of CT120-B was quite different, The regulatory effects of CT120 on NIH3T3 and A549 cell growth were investigated through colony formation analysis, growth curve assay and tumor formation assay of transfected cells in nude mice. The results showed that it could promote the proliferation of NIH3T3 and A549 cells in vitro and in vivo. Novel gene CT120 may be a key modulator of cell proliferation closely related to huma lung carcinogenesis.To probe the roles of CT120 in promoting cell proliferation and understand the implications of the signal transduction pathways regulated by CT120 in the development of human lung cancers, we investigated the downstream signaling events regulated by CT120 using ATLAS mouse cDNA expression arrays. Among 588 known genes, total of 133 genes were upregulated or downregulated by CT120. Two major signaling pathways involved in cell proliferation, cell survival and anti-apoptosis were overexpressed and activated in response to CT120: One of the major pathways upregulated and activated was the Raf/MAPK signaling cascades. The expressions of 9 genes (Myc, A-Raf, B-Raf, Erk1, MA...