Abstract
BAG-1 is an anti-apoptotic protein that interacts with a variety of cellular molecules to inhibit apoptosis. The mechanisms by which BAG-1 interacts with other proteins to inhibit apoptosis have been extensively explored. However, it is currently unknown how BAG-1 expression is regulated at the molecular level, especially in cancer cells. Here we reported to clone a novel down-regulated BAG-1 expression gene named FLJ20420 using hBAG-1 promoter as a probe to screen Human Hela 5′ cDNA library by Southernwestern blot. The FLJ20420 gene encodes a ∼26-kDa protein that is localized in both the cytoplasm and nucleus. We proved that FLJ20420 protein can specially bind hBAG-1 promoter region by EMSA in vivo and ChIP assay in vivo. Northern blot analysis revealed a low level of FLJ20420 transcriptional expression in normal human tissues (i.e., brain, placenta, lung, liver, kidney, pancreas and cervix), except for heart and skeletal muscles, which showed higher levels. Furthermore, enhanced FLJ20420 expression was observed in tumor cell lines (i.e., MDA468, BT-20, MCF-7, C33A, HeLa and Caski). Knockdown of endogenous FLJ20420 expression significantly increased BAG-1 expression in A549 and L9981 cells, and also significantly enhanced their sensitivity to cisplatin-induced apoptosis. A microarray assay of the FLJ20420 siRNA –transfectants showed altered expression of 505 known genes, including 272 upregulated and 233 downregulated genes. Finally, our gene array studies in lung cancer tissue samples revealed a significant increase in FLJ20420 expression in primary lung cancer relative to the paired normal lung tissue controls (p = 0.0006). The increased expression of FLJ20420 corresponded to a significant decrease in BAG-1 protein expression in the primary lung cancers, relative to the paired normal lung tissue controls (p = 0.0001). Taken together, our experiments suggest that FLJ20420 functions as a down-regulator of BAG-1 expression. Its abnormal expression may be involved in the oncogenesis of human malignancies such as lung cancer.
Highlights
BAG-1 is a multifunctional protein that plays important roles in apoptosis, cell survival, transcription, cell motility and proliferation
A search of the National Center for Biotechnology Information (NCBI) database revealed that one of the clones was identical to the Homo sapiens cDNA termed FLJ20420 (GI: 7020507)
To understand the molecular regulation of BAG-1 expression in human cancer, we have identified a cDNA that encodes a novel BAG-1 transcription factor, termed FLJ20420, using a protein-DNA fragment interaction cloning technique
Summary
BAG-1 is a multifunctional protein that plays important roles in apoptosis, cell survival, transcription, cell motility and proliferation. BAG-1 expression is often altered in various human malignancies, especially in human breast cancer, lung cancer and cervical cancer [1,2]. BAG-1 expression has been associated with the prognosis of a variety of human malignancies, such as breast cancer and lung cancer [3,4,5]. The proteins Hsc, Hsp, Bcl-2 and RAF-1 kinase, as well as nuclear hormone receptors and subunits of the ubiquitination-proteasome system, are all known BAG-1- interacting partners. BAG-1 interacts with Hsp via a C-terminal BAG domain which allows it to facilitate the nucleotide exchange [6,7]. Many functions of BAG-1 in cell apoptosis and cell survival are dependent on the BAG domain
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