Presentation on theme: "Alcohol Dehydrogenase I is Present in Normal Human Mammary Tissue and Absent in Breast Cancer: Implications for Breast Carcinogenesis Trudy A. Atkins,"— Presentation transcript:
1Alcohol Dehydrogenase I is Present in Normal Human Mammary Tissue and Absent in Breast Cancer: Implications for Breast CarcinogenesisTrudy A. Atkins, M.S.Supervisor of Science and G & T EducationEast Brunswick Public SchoolsDecember 20, 2011
13Associated Tumor Suppressor Genes p53 – Induces apoptosis by inducing IGFR I and its binding protein IGF-BP3. RA increases IGF-BP3 thereby increasing apoptosis in MDA-MB231 breast cancer cell lines.c-myc – Over expressed in cancer cells. RA acts to down-regulate this gene in MCF-7 breast cancer cell lines.
15Specific Aims:Western Blotting was used to determine if ADH I was located in normal breast epithelium.Enzyme assays were performed in order to determine the presence of ADH I in normal breast epithelium and in breast cancerImmunocytochemistry was used to determine the localization of ADH I in epithelial, myoepithelial, and stromal cells in normal and malignant tissue.
17Initial ADH I Western Blot CA = Cancerous tissueN = NormalF = Fibrocystic Tissue
18ADH I with GAP (GTPase Activating Protein) as a 5 µg loading control
19Details of Western Blot ADH I – 40 kDaAsterisks indicate protein from “cancer” samplesNote far right asterisk-lack of 40 kDa band with abundance of GAP
20Enzyme Assay Normal versus cancer oxidation of ethyl alcohol by ADH I. Duplicate samples with or without4-methylpyrazole (4-MP) a known inhibitor ofADH I.Change in absorbance was monitored on a dual beam spectrophotometer against a reaction containing no substrate.Enzyme activity is expressed as mIU/min/mg protein.N.D. indicates no detectable activity.
21Enzyme Assay Normal vs. Cancer Normal – ADH Inhibition with 4-MP ranges from 75-99%Cancer – Only one sample showed any uninhibited ADH activity followed by 11% inhibition with addition of 4-MP.
22Normal Tissue Immunocytochemistry Note organized ducts and strong ADH I staining
41ReferencesChambon, P. (1996). A decade of molecular biology of retinoic acid receptors. FASEB. J. 10:Duester, G. (1996). Involvement of alcohol dehydrogenase, short-chain dehydrogenase/reductase, aldehyde dehydrogenase, and cytochrome p450 in the control of retinoid signaling by activation of retinoic acid synthesis. Biochemistry. 35,Hurley, T., Bosron, W., Stone, C., and L. Amzel. (1997). Structure of three human beta alcohol dehydrogenase variants. J. Mol. Biol. 239:Manglesdorf, D., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., and R. Evans. (1995). The nuclear receptor superfamily: The second decade. Cell. 83:Svensson, JBC 274:29712.