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Alcohol Dehydrogenase I is Present in Normal Human Mammary Tissue and Absent in Breast Cancer: Implications for Breast Carcinogenesis. Trudy A. Atkins, M.S. Supervisor of Science and G & T Education East Brunswick Public Schools December 20, 2011. Alcohol Dehydrogenase.
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Alcohol Dehydrogenase I is Present in Normal Human Mammary Tissue and Absent in Breast Cancer: Implications for Breast Carcinogenesis Trudy A. Atkins, M.S. Supervisor of Science and G & T Education East Brunswick Public Schools December 20, 2011
Alcohol Dehydrogenase Alcohol Dehydrogenase Retinol Retinal NAD+ NADH NADP+ NADPH NAD+ Aldhehyde Dehydrogenase NADH Retinoic Acid
Functions of Retinol • Vision • Bone Growth • Cell Division • Cell Differentiation • Epi / Endothelial Maintenance
Retinoic Acid Receptor – RAR Retinoid X Receptors – RXR RAR/RAR Homodimer (Chambon – 1987) RXR/RXR Homodimer (Manglesdorf – 1990) RAR/RXR Heterodimer Nuclear Regulation by Retinoic Acid
Alcohol Dehydrogenase Isozymes • Five Classes – I, II, III, IV, V • Dimeric • Cytoplasmic • Zinc-dependant • Chromosome 4q22-24
ADH I • Dimeric – Composed of identical or non-identical polypeptide chains alpha, beta, gamma. • Each polypeptide chain is 374 amino acids long • 40 kD • Two domains: catalytic and coenzyme binding • Functions in ethanol oxidation in liver, retinol oxidation in epithelium, oxidation of 3β-hydroxysteroids
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.
Breast Anatomy • Lobules – Epithelium with surrounding myoepithelium • Ducts - Epithelium • Stroma – Connective
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 cancer • Immunocytochemistry was used to determine the localization of ADH I in epithelial, myoepithelial, and stromal cells in normal and malignant tissue.
Results • Western Blot • Enzyme Assay • Immunocytochemistry
Initial ADH I Western Blot CA = Cancerous tissue N = Normal F = Fibrocystic Tissue
ADH I with GAP (GTPase Activating Protein) as a 5 µg loading control
Details of Western Blot • ADH I – 40 kDa • Asterisks indicate protein from “cancer” samples • Note far right asterisk-lack of 40 kDa band with abundance of GAP
Enzyme Assay • Normal versus cancer oxidation of ethyl alcohol by ADH I. • Duplicate samples with or without 4-methylpyrazole (4-MP) a known inhibitor of ADH 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.
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.
Normal Tissue Immunocytochemistry Note organized ducts and strong ADH I staining
Normal ductal epithelium - showing high immunoreactivity with minimally reactive stromal cells highlighted Bar = 5 µm
Normal Duct Bar = 10µm
Type 3 lobules, parous tissue Bar = 10µm
TDLU Bar = 10 µm
Normal 2o Ab control Bar = 10 µm
Malignant Tissue Immunocytochemistry Note loss of immunoreactivity and lack of proper ductal orientation
Loss of ADH I staining Bar = 10 µm
Cancer – Minimal ADH I immunoreactivity Bar = 10µm
Weak positive cells surrounding areas of no ADH I immunoreactive cells Bar = 10 µm
Increased magnification of previous slide Bar = 2 µm
Increased Magnification20x vs 100x Bar= 10 µm Bar = 2 µm
Overall heterogeneity of ADH I immunoreactivity Bar = 10 µm
Weak ADH I immunoreactivity seen in a morphologically “normal” duct Bar = 5µm
Cancerous tissue – note ducts lacking expression 10x and 40x Bar = 20 µm Bar = 5 µm
Increased magnification 100x Bar = 2 µm
Normal vs. Cancer20x Bar = 10 µm
References Chambon, P. (1996). A decade of molecular biology of retinoic acid receptors. FASEB. J. 10:940-954. 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, 12221-12227. Hurley, T., Bosron, W., Stone, C., and L. Amzel. (1997). Structure of three human beta alcohol dehydrogenase variants. J. Mol. Biol. 239:415-429. 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. 83:835-839. Svensson, JBC 274:29712.
Questions? Thanks!