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

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. Alcohol Dehydrogenase Alcohol Dehydrogenase Retinol Retinal NAD+ NADH NADP+ NADPH NAD+ Aldhehyde Dehydrogenase NADH Retinoic Acid

3. β-Carotene Retinol Retinoic Acid

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

5. 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

6. Retinoid Signaling

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

8. 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

9. Space Filling Model of ADH I

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

11. ADH IV – PurpleADH 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. • 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 - Epithelium • Stroma – 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 cancer • 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 • Enzyme Assay • 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 • Asterisks indicate protein from “cancer” samples • Note 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 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.

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

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 Bar = 10 µm

29. Normal 2o 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 Magnification20x vs 100x Bar= 10 µm Bar = 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 = 20 µm Bar = 5 µm

39. Increased magnification 100x Bar = 2 µm

40. Normal vs. Cancer20x Bar = 10 µm

41. 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.

42. Questions? Thanks!