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

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 – 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.
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 Magnification 20x 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. 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!