Selenium is an essential micronutrient in the diet of humans and other mammals. Many health benefits have been attributed to selenium including preventing cancer and heart disease and the progression of acquired immunodeficiency syndrome (AIDS) in human immunodeficiency virus (HIV)-positive patients. Numerous human clinical trails have been undertaken in recent years to assess the role of this element in cancer prevention, delaying the progression of AIDS, etc., at a cost of billions of dollars, but little is known about how selenium acts at the molecular level in mammals to exert these many health benefits. We proposed that the health benefits of selenium are due largely to its presence in selenoproteins as the selenium-containing amino acid, selenocysteine (Sec). Our program therefore focuses on: 1) developing mouse models to assess the role of selenium and selenoproteins in cancer prevention and development, 2) characterizing and elucidating the function of various selenoproteins and their roles in cancer prevention and development, and 3) identifying the means by which Sec is biosynthesized and incorporated into protein. The project discussed herein examines our research on the development of various mouse models for determining the role of selenium in cancer and development. In the past year, we have focused on completing all of our studies on characterizing our mouse models (see below). We previously initiated in vivo carcinogenesis studies involving the selenoprotein 15 (Sep15) knockout mice to examine our in vitro findings elucidating the role of this selenoproteins in cancer. We examined the formation of chemically-induced aberrant crypt foci (ACF) in the colon of Sep15 mice compared to heterozygous and wild type litter mate controls and the influence of dietary selenium on the formation of ACF. Our results demonstrated that Sep15 knockout mice are protected against ACF formation, which seemed independent of dietary selenium. In addition, it has been previously reported by our group that selective removal of the selenocysteine tRNA gene (Trsp) from mouse endothelial cells is embryonic lethal. In order to study the importance of gluthathione peroxidase gene (gpx4) and thioredoxin reductase 1 (tr1) in the development of endothelial cells, we generated endothelial specific knockout mice encoding the targeted loss of either the gpx4 or tr1 gene. gpx4 endothelial knockout mice are embryonic lethal, showing a phenotype that recapitulate almost all the changes previously reported on Trsp endothelial knockout embryo. Briefly, both embryos (trsp and gpx4 endothelial knockout) showed marked central nervous system abnormalities with extensive necrotic areas in the brain. Also both of them presented more nucleated erythrocytes in the blood than the control embryos. However, the thymus of trsp endothelial knockout embryos was either not present or disorganized whereas the thymus of gpx4 embryos showed no difference with the thymus of the control embryos. tr1 endothelial knockout is not lethal, but the thymus in the E 18 days embryos is disorganized and the lymphocytes are less mature than in the thymus of control mice. We are now examining whether the adult TR1 endothelial knockout mice have problems in their thymus or other immune-related tissues compared to control mice with a fully functional TR1 gene. We have also examined the effect acetaminophen (APAP) on mice encoding a TR1 knockout in liver as APAP hepatotoxicity (APAP-H) is the most common cause of drug-induced liver failure in the United States. Since the depletion of glutathione plays a major role in APAP liver damage and much of the underlying metabolism of GSH in liver damage has been elucidated, the role of TR1, also a major antioxidant in mammalian cells, in APAP-H, and the interrelationship of this selenoenzyme with the glutathione system were examined. Liver-specific TR1 knockout mice (tr1KOliv) were treated with a hepatotoxic dose of APAP. Surprisingly, tr1KOliv mice were resistant to APAP toxicity as histology and liver enzymes used for demonstrating hepatotoxicity were not significantly different from untreated mice. Control, treated mice, however, showed extensive liver damage. The results strongly suggest that tr1KOliv mice are primed for xenobiotic detoxification primarily through NRF2 activation. The findings provide new insights into APAP-induced liver damage and suggest new therapeutic targets for prevention and/or amelioration of drug-induced liver damage. Furthermore, to examine the role of TR1 in liver cancer, liver TR1 knockout mice were exposed to the liver carcinogen, diethylnitrosamine, (DEN). We found that liver-specific TR1 knockout mice developed tumors (hepatic adenomas, hepatic carcinomas and hepatocholangiocellular adenomas) much more readily than control mice. 16 of 18 knockout mice developed tumors compared to 2 of 19 of control mice. In addition, we found an upregulation of Nrf2-modulated genes in the TR1 deficient livers, including several of the glutathione transferases and the selenoprotein, glutathione peroxidase 2 (GPx2), which was even further up-regulated in tumors. The role of GPx2 in liver tumors is currently being evaluated in vitro using a human liver carcinoma cell line (Hep G2). We have previously shown that selenoproteins play a role in proper macrophage function by targeting the removal of trsp, and thus all selenoproteins, in macrophage. In addition, we have found that TR1 is up-regulated in macrophage upon stimulation with lipopolysaccharide (LPS) and that this stimulation is dependent upon p38 signaling. We have also begun examining the role of TR1 in macrophage function by developing and characterizing a macrophage-specific TR1 knockout mouse. Although this mouse has no overt phenotype, we are currently accessing possible alterations in thioredoxin-dependent inflammatory signaling. In the past year, we completed our studies on conditional knockout of GPx4 and TR1 in epidermal cells in skin. Both these proteins are important regulators of cellular ROS levels and knocking them out results in embryonic lethality. Hence, to elucidate the in vivo role of these proteins in skin, we generated conditional knockout mouse models to examine the role of GPx4 and TR1 in skin function and development. Though no obvious phenotypic changes were observed for targeted removal of TR1 in skin, spatio-temporal disruption of GPx4 in keratinocytes modulated hair follicle development in vivo and keratinocyte proliferation in vitro through induction of COX-2 expression. This ablation generated progeny with an altered phenotype, with the variations being more evident in early stages of hair follicle morphogenesis, substantiating the importance of GPx4 in hair follicle development. Interestingly, these studies show for the first time that in keratinocytes, the lack of GPx4 is compensated by GPx1 and TR1, establishing the importance of GPx4 as an antioxidant in skin. We have established a breast TR1 conditional knockout mouse model using loxP-Cre technology and are expanding the population to elucidate the role of TR1 in breast cancer. We will compare the incidence of breast cancer in TR1 breast knockout and control mice to assess whether TR1 has a role as an anti- or pro-cancer gene in the process of this malignancy. We will examine the effect of TNF-alpha on breast tumor-bearing control and TR1 knockout mice to elucidate the involvement of TR1 in the cancer apoptotic pathway. We also will introduce sodium selenite into the diet of breast tumor-bearing mice to determine the highly specific cytotoxic effect of selenite on TR1 deficient cancer cells in an in vivo mouse model.
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