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:
1 Alcohol 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
13 Associated 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.
14 Breast Anatomy Lobules – Epithelium with surrounding myoepithelium Ducts - EpitheliumStroma – Connective
15 Specific 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.
17 Initial ADH I Western Blot CA = Cancerous tissueN = NormalF = Fibrocystic Tissue
18 ADH I with GAP (GTPase Activating Protein) as a 5 µg loading control
19 Details of Western Blot ADH I – 40 kDaAsterisks indicate protein from “cancer” samplesNote far right asterisk-lack of 40 kDa band with abundance of GAP
20 Enzyme 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.
21 Enzyme 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.
22 Normal Tissue Immunocytochemistry Note organized ducts and strong ADH I staining
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