• Cloning of Human Suv39h1 for Inhibition Studies

      Jahan, Asmat; Shaikh, Zahid; Department of Chemistry and Physics (2016-03)
      Cancer is one of the leading causes of death in the Western world and is characterized by abnormal cell growth. Despite the advancement in genetic engineering, there is not a significant amount of change in the rate of morbidity associated with the disease. For human colorectal cancers (CRC), early diagnosis and treatment is important for survival; if the cancer has metasta- sized to the liver, the survival rates are poor. Hence, improvements must be made to the existing therapies for metastatic human CRC. Applying knowledge of molecular mechanisms to modify and control the outgrowth of cells is the key to introducing new therapies. Fas is a receptor protein involved in apoptosis or, more commonly known as, “cell death.” Human carcinoma cells lower the expression of Fas on the cell surface. This lowering of expression levels of Fas is correlated with increased levels of histone H3 lysine 9 trimethylation (H3K9me). SUV39H1 is a histone lysine methyltransferase which catalyzes H3K9me. The goal of this study is to identify novel SUV39H1-specific inhibitors to decrease the level of methylation at the Fas promoter. Thus, to initiate our study, we have cloned the gene for human SUV39H1 into the expression vector pET-21c(+). Upon transforming the recombinant DNA into an expression strain of E. coli such as BL21, we will overexpress and purify SUV39H1 using affinity chromatography. Ultimately, the activity of SUV39H1 will be tested in the presence of various inhibitors.
    • Cloning, Purification, and Inhibition of the SUV39H2 Histone Methyl Transferase

      Jones, Preston Dimitri; Department of Chemistry and Physics (2016-03)
      The most significant challenge in treating human colorectal cancer (CRC) is metastasis of the cancer. Although the study of colorectal cancer metastasis is still elementary, it is known that silencing of Fas expression is a hallmark of human colorectal cancer metastasis. The Fas protein is a member of the tumor necrosis factor (TNF)- a receptor superfamily that plays a major role in the regulation of programmed cell death. The interaction of Fas with its ligand, Fas L, triggers a signal cascade that ends in apoptosis. Silencing of Fas expression thus allows cancer cells to evade cell death triggered by the immune system. In human CRC patients, the Fas promoter is characterized by high H3K9 trimethylation (H3K9me3). This methylation is primarily caused by histone methyl transferases such as SUV39H2. We hypothesize that inhibiting H3K9 trimethylation is a potential anti-cancer strategy. To study this possibility, the gene for human SUV39H2 was cloned into pET21 and the protein was overexpressed in E.coli BL21- DE3 cells. The activity of the purified protein will be tested against various inhibitors. Funding Source: National Science Foundation