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Effects of Arsenic Exposure on Oxylipid Metabolism in Human Keratinocytes
Mentor: Theresa Pedersen, Dr. John W. Newman, Dr. Robert H. Rice, Dr. Bruce D. Hammock, Department of Environmental Toxicology, Department of Entomology & UC Davis Cancer Center, UC Davis
Arsenic, a known human carcinogen, has previously been found to induce heme oxygenase-1 (HO-1) in exposed human keratinocytes. Heme is degraded by HO-1 into iron, carbon monoxide, and biliverdin. An important enzyme requiring heme is cytochrome P450. It was hypothesized that the degradation of heme in keratinocytes exposed to arsenite would decrease the cytochrome P450-dependent oxidation of linoleate and arachidonate. These oxylipid metabolites are important regulators of cellular homeostasis and inflammation. Furthermore, it was hypothesized that a change in other oxylipids of the arachidonic acid and linoleic acid cascades would be observed. Specifically, oxylipids synthesized by 5-lipoxygenase would be effected as they are known to inhibit HO-1 gene expression. Using HPLC-MS/MS, about thirty oxylipids were simultaneously quantified. Changes in lypoxygenase metabolism were observed in cell pellets, but not in the media.
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Characterization of Carcinoma Cell Lines Derived From HPV16/MMP-2 and HPV16/MMP-9 Homozygous-null Transgenic Mice
Mentor: Kerstin Dehne, and Lisa M. Coussens, Cancer Research Institute; Pathology, UC, San Francisco
Human papilloviruses (HPVs) are sexually transmitted DNA tumor viruses identified to contribute to the initiation of certain types of cancers. HPV16 oncogenes expressed under the control of human keratin-14 promoter/enhancer initiate neoplastic progression in a transgenic mouse model of squamous carcinogenesis. Matrix metalloproteinases (MMPs) are extrinsic regulators known to effect carcinogenesis and angiogenesis. These extracellular proteases are capable of degrading components of the extracellular matrix, specifically type IV collagen of basal lamina and type I collagen. Two members of the MMP family, e.g., MMP2/gelatinase A and MMP9/gelatinase B have been linked to both the initiation and metastasis of cancer cells. We have previously reported that the absence of MMP9 decreases the occurrence of carcinomas, while the tumors that do grow in the absence of MMP9 are more aggressive than with the presence of MMP9. To assess the functional significance of MMP-9 and MMP-2 on epithelial proliferation, we have derived carcinoma cell lines from HPV/MMP-2 and HPV/MMP-9 deficient transgenic mice. The aim of this project is to characterize the heterogeneity and proliferative capacity of these derived cell lines in order to study the functional significance of MMP-2 and MMP-9 during epithelial carcinogenesis. Using carcinoma-derived cell lines from HPV transgenic mice as controls, carcinoma-derived cell lines from HPV/MMP2 and HPV/MMP9 deficient mice will be characterized using immunohistochemical methodologies. Immunodetection of vimentin and/or kertin-5 antigens will be used to assess the heterogeneity of these cell lines.
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Are there other components required for Mgm1p's function in mitochondrial structure?
Mentor: Dr. Jodi Nunnari, UC Davis
We propose that like Dnm1p, a dynamin-related GTPase that mediates fission on the outer membrane, Mgm1p will be regulated by additional proteins on the inner membrane. We are conducting a novel enhancer screen to identify components that assist Mgm1p in its function to remodel the inner mitochondrial membrane for efficient mitochondrial fusion. After mutagenizing mgm1-5 cells with uv rays, we screened for mutants that enhanced the phenotype of mgm1-5 cells. In order to ascertain whether the enhancer mutation was specific to MGM1, we introduced a copy of wild type MGM1 into mutagenized cells containing putative enhancer mutations and tested whether MGM1 restored wild type growth. Next, the mutant was backcrossed to mgm1-5 to ensure that only a mutation in a single locus was conferring the enhanced phenotype. The mutant was also backcrossed to wild type to obtain haploid cells containing the enhancer mutation alone and assess its phenotype. If enhancer mutants were specific to MGM1, we proceeded to clone, identify, and characterize. The primary screen yielded four mutants. After the secondary screens, one of the mutants was discarded. The secondary screen analysis on the second mutant is forthcoming. Third and fourth mutants are being currently cloned and prepared for characterization.
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