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Di-N-Octylphthalate Acts as a Proliferative Agent in Murine Cell Hepatocytes by Regulating the Levels of Pro-Apoptotic ProteinsHepatocellular carcinoma (HCC) is the fifth most common cancer in the US. Its development is thought to be associated with inactivation of tumor suppressors by methylation. Di-n-octylphthalate (DNOP), a common plasticizer, is believed to cause hepatic pre-neoplastic lesions. Because a number of tumor suppressors are shown to be not expressed in HCC, our goal was to identify tumor suppressor genes methylated upon treatment with 0.1 % DNOP at 24, 48, 72 h in mouse hepatocytes cell line AML-12 and isolated primary cultured mouse hepatocytes. None of tumor suppressors experienced a change in the methylation status in presence of DNOP. Because we found that DNOP causes an increase in cell proliferation, we studied whether the effect is paralleled to a suppression of apoptosis. We found that DNOP causes a decrease in pro-apoptotic proteins and no change in anti-apoptotic proteins. We studied the physiological effects of DNOP in mouse liver. Mice were treated with 0.1 % DNOP for a month. DNOP caused a decrease in bile secretion and an increase in the hepatic levels of bile acids and glutathione. Avoiding the use of DNOP as a plasticizer in products for human consumption can reduce the incidence of diseases related to its hepatotoxicity.
Purification of Recombinant Human Histone Methyltransferases for Inhibition StudiesResistance to cellular apoptotic pathways is a major contributing factor in the metastasis of cancer. One such cell-intrinsic pathway which induces programmed cell death is ligand cell surface death receptors, including Fas protein. In primary colorectal cancer, Fas expression is often diminished. The silencing of Fas expression is perhaps a mechanism by which human colorectal cancer cells evade apoptosis. The decrease in Fas expression levels is associated with hypermethylation of histone 3 lysine 9 catalyzed by histone methyltransferases (HMTases) such as SUV39H1 and SUV39H2. Because of the role of methylation in silencing Fas expression, HMTase inhibitors represent potential anti-cancer agents. Verticillin A, a natural compound extracted from wild mushroom, is a broad-based HMTase inhibitor and has been shown to cause toxicity in mice. Our goal in this study is to identify specific SUV39H1 and SUV39H2 inhibitors that are less toxic than Verticillin A. To investigate this, human SUV39H1 was sub-cloned into an expression vector, transformed into an E.coli expression strain and purified using affinity chromatography. Gel electrophoresis and Western blot analysis confirmed the presence of partially purified human SUV39H1. The next phase of the project will involve testing the activity of the purified protein in an in vitro assay.