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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,

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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 Carcinogenesis Trudy A. Atkins, M.S. Supervisor of Science and G & T Education East Brunswick Public Schools December 20, 2011

2 Retinol Alcohol Dehydrogenase Retinal Aldhehyde Dehydrogenase Retinoic Acid NAD + NADH NADPHNADP + NAD + NADH Alcohol Dehydrogenase

3 β-Carotene Retinol Retinoic Acid

4 Functions of Retinol Vision Vision Bone Growth Bone Growth Cell Division Cell Division Cell Differentiation Cell Differentiation Epi / Endothelial Maintenance Epi / Endothelial Maintenance

5 Nuclear Regulation by Retinoic Acid Retinoic Acid Receptor – RAR Retinoic Acid Receptor – RAR Retinoid X Receptors – RXR Retinoid X Receptors – RXR RAR/RAR Homodimer RAR/RAR Homodimer (Chambon – 1987) (Chambon – 1987) RXR/RXR Homodimer RXR/RXR Homodimer (Manglesdorf – 1990) (Manglesdorf – 1990) RAR/RXR Heterodimer RAR/RXR Heterodimer

6 Retinoid Signaling

7 Alcohol Dehydrogenase Isozymes Five Classes – I, II, III, IV, V Five Classes – I, II, III, IV, V Dimeric Dimeric Cytoplasmic Cytoplasmic Zinc-dependant Zinc-dependant Chromosome 4q22-24 Chromosome 4q22-24

8 ADH I Dimeric – Composed of identical or non-identical polypeptide chains alpha, beta, gamma. Dimeric – Composed of identical or non-identical polypeptide chains alpha, beta, gamma. Each polypeptide chain is 374 amino acids long Each polypeptide chain is 374 amino acids long 40 kD 40 kD Two domains: catalytic and coenzyme binding Two domains: catalytic and coenzyme binding Functions in ethanol oxidation in liver, retinol oxidation in epithelium, oxidation of Functions in ethanol oxidation in liver, retinol oxidation in epithelium, oxidation of 3β-hydroxysteroids 3β-hydroxysteroids

9 Space Filling Model of ADH I

10 ADH I (Alpha-blue / Beta 1-yellow)

11 ADH IV – Purple ADH I Beta - Mustard

12 ADH Families

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. 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. 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 Lobules – Epithelium with surrounding myoepithelium Ducts - Epithelium Ducts - Epithelium Stroma – Connective Stroma – Connective

15 Specific Aims: Western Blotting was used to determine if ADH I was located in normal breast epithelium. 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 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. Immunocytochemistry was used to determine the localization of ADH I in epithelial, myoepithelial, and stromal cells in normal and malignant tissue.

16 Results Western Blot Western Blot Enzyme Assay Enzyme Assay Immunocytochemistry Immunocytochemistry

17 Initial ADH I Western Blot CA = Cancerous tissue N = Normal F = Fibrocystic Tissue

18 ADH I with GAP (GTPase Activating Protein) as a 5 µg loading control

19 Details of Western Blot ADH I – 40 kDa ADH I – 40 kDa Asterisks indicate protein from cancer samples Asterisks indicate protein from cancer samples Note far right asterisk-lack of 40 kDa band with abundance of GAP Note far right asterisk-lack of 40 kDa band with abundance of GAP

20 Enzyme Assay Normal versus cancer oxidation of ethyl alcohol Normal versus cancer oxidation of ethyl alcohol by ADH I. by ADH I. Duplicate samples with or without Duplicate samples with or without 4-methylpyrazole (4-MP) a known inhibitor of 4-methylpyrazole (4-MP) a known inhibitor of ADH I. ADH I. Change in absorbance was monitored on a dual beam spectrophotometer against a reaction containing no substrate. 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. Enzyme activity is expressed as mIU/min/mg protein. N.D. indicates no detectable activity. 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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25 Normal ductal epithelium - showing high immunoreactivity with minimally reactive stromal cells highlighted Bar = 5 µm

26 Normal Duct Bar = 10µm

27 Type 3 lobules, parous tissue Bar = 10µm

28 TDLU

29 Normal 2 o Ab control Bar = 10 µm

30 Malignant Tissue Immunocytochemistry Note loss of immunoreactivity and lack of proper ductal orientation

31 Loss of ADH I staining Bar = 10 µm

32 Cancer – Minimal ADH I immunoreactivity Bar = 10µm

33 Weak positive cells surrounding areas of no ADH I immunoreactive cells Bar = 10 µm

34 Increased magnification of previous slide Bar = 2 µm

35 Increased Magnification 20x vs 100x Bar = 10 µmBar = 2 µm

36 Overall heterogeneity of ADH I immunoreactivity Bar = 10 µm

37 Weak ADH I immunoreactivity seen in a morphologically normal duct Bar = 5µm

38 Cancerous tissue – note ducts lacking expression 10x and 40x Bar = 5 µmBar = 20 µm

39 Increased magnification 100x Bar = 2 µm

40 Normal vs. Cancer 20x Bar = 10 µm

41 References Chambon, 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.

42 Questions? Thanks!


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