Colony Growth Suppression by Tumor Suppressor Genes
The ability of a tumor suppressor gene to inhibit cell growth is critical for its tumor suppression. Such activity is carried out largely by inducing cell cycle arrest and apoptosis. For example, when the p53 tumor suppressor gene is activated by DNA damage (1), it inhibits the growth of tumor cells by inducing cell cycle arrest or apoptosis (2). Despite a temporal and reversible event, cell cycle arrest allows damaged DNA to be repaired and thereby prevents abnormal DNA propagation and the emergence of cancer cells. On the other hand p53-induced apoptosis is powerful and irreversible (3). When p53 is introduced into tumor cells, for example via p53 adenovirus infection, the cells undergo rapidly apoptosis (4). Conversely, loss of p53-mediated apoptosis has been implicated not only in tumor progression, but also in the drug-resistant phenotype of tumor cells (5). Thus, the tumor suppressor gene often possesses these two features. Accordingly, several techniques have been designed to analyze cell cycle arrest and apoptosis induction to characterize the growth inhibition activity of tumor suppressor genes, including flow cytometry assay (6), MTT assay (7), and clonogenic survival assay (8). Among these, the clonogenic survival assay is the most commonly used assay to assess growth inhibition in vitro. Basically, this method examines how the gene of interest affects cell growth in cultured cells. It includes three steps. The first step is to construct a mammalian expression vector that can express the gene of interest. This involves standard molecular cloning techniques. The second step introduces the expression vector into human cancer cell lines via a carrier such as lipofectin.
KeywordsTumor Suppressor Gene Inhibit Cell Growth Human Cancer Cell Line Induce Cell Cycle Arrest Ligation Reaction
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