Alcohol Dehydrogenase I is Alcohol Dehydrogenase I is Present in Normal Human Present in Normal Human

Mammary Tissue and Absent Mammary Tissue and Absent in Breast Cancer: in Breast Cancer:

Implications for Breast Implications for Breast CarcinogenesisCarcinogenesis

Trudy A. Atkins, M.S.Trudy A. Atkins, M.S.Supervisor of Science and G & T EducationSupervisor of Science and G & T Education

East Brunswick Public SchoolsEast Brunswick Public Schools

December 20, 2011December 20, 2011

Retinol

Alcohol Dehydrogenase

Retinal

A

ldheh

yde

Dehyd

rogen

ase

Retinoic Acid

NAD+ NADH

NADPHNADP+

NAD+

NADH

Alcohol Dehydrogenase

ββ-Carotene Retinol Retinoic -Carotene Retinol Retinoic AcidAcid

Functions of RetinolFunctions of Retinol

Vision Vision Bone GrowthBone Growth Cell DivisionCell Division Cell DifferentiationCell Differentiation Epi / Endothelial Maintenance Epi / Endothelial Maintenance

Nuclear Regulation by Retinoic Nuclear Regulation by Retinoic AcidAcid

Retinoic Acid Retinoic Acid Receptor – RARReceptor – RAR

Retinoid X Retinoid X Receptors – RXRReceptors – RXR

RAR/RAR RAR/RAR HomodimerHomodimer

(Chambon – 1987)(Chambon – 1987) RXR/RXR RXR/RXR

HomodimerHomodimer

(Manglesdorf – (Manglesdorf – 1990)1990)

RAR/RXR RAR/RXR HeterodimerHeterodimer

Retinoid SignalingRetinoid Signaling

Alcohol Dehydrogenase Alcohol Dehydrogenase IsozymesIsozymes

Five Classes – Five Classes – II, II, III, , II, III, IVIV, V, V DimericDimeric CytoplasmicCytoplasmic Zinc-dependantZinc-dependant Chromosome 4q22-24Chromosome 4q22-24

ADH IADH I

Dimeric – Composed of identical or non-identical Dimeric – Composed of identical or non-identical polypeptide chains alpha, beta, gamma.polypeptide chains alpha, beta, gamma.

Each polypeptide chain is 374 amino acids longEach polypeptide chain is 374 amino acids long 40 kD40 kD Two domains: catalytic and coenzyme bindingTwo domains: catalytic and coenzyme binding Functions in ethanol oxidation in liver, retinol Functions in ethanol oxidation in liver, retinol

oxidation in epithelium, oxidation of oxidation in epithelium, oxidation of

33ββ-hydroxysteroids-hydroxysteroids

Space Filling Model of ADH ISpace Filling Model of ADH I

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

ADH IV – PurpleADH IV – PurpleADH I Beta - MustardADH I Beta - Mustard

ADH FamiliesADH Families

Associated Tumor Suppressor Associated Tumor Suppressor GenesGenes

p53p53 – Induces apoptosis by inducing IGFR I – Induces apoptosis by inducing IGFR I and its binding protein IGF-BP3. RA and its binding protein IGF-BP3. RA increases IGF-BP3 thereby increasing increases IGF-BP3 thereby increasing apoptosis in MDA-MB231 breast cancer apoptosis in MDA-MB231 breast cancer cell lines.cell lines.

c-mycc-myc – Over expressed in cancer cells. – Over expressed in cancer cells. RA acts to down-regulate this gene in MCF-RA acts to down-regulate this gene in MCF-7 breast cancer cell lines.7 breast cancer cell lines.

Breast AnatomyBreast Anatomy

Lobules – Lobules – Epithelium with Epithelium with surrounding surrounding myoepitheliummyoepithelium

Ducts - EpitheliumDucts - Epithelium Stroma – Stroma –

Connective Connective

Specific Aims:Specific Aims:

Western Blotting was used to determine if Western Blotting was used to determine if ADH I was located in normal breast ADH I was located in normal breast epithelium.epithelium.

Enzyme assays were performed in order to Enzyme assays were performed in order to determine the presence of ADH I in normal determine the presence of ADH I in normal breast epithelium and in breast cancerbreast epithelium and in breast cancer

Immunocytochemistry was used to determine Immunocytochemistry was used to determine the localization of ADH I in epithelial, the localization of ADH I in epithelial, myoepithelial, and stromal cells in normal and myoepithelial, and stromal cells in normal and malignant tissue.malignant tissue.

ResultsResults

Western BlotWestern Blot

Enzyme AssayEnzyme Assay

ImmunocytochemistryImmunocytochemistry

Initial ADH I Western BlotInitial ADH I Western Blot

CA = Cancerous tissue

N = Normal

F = Fibrocystic Tissue

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

loading control loading control

Details of Western BlotDetails of Western Blot

ADH I – 40 kDaADH I – 40 kDa Asterisks indicate Asterisks indicate

protein from protein from “cancer” samples“cancer” samples

Note far right Note far right asterisk-lack of 40 asterisk-lack of 40 kDa band with kDa band with abundance of GAP abundance of GAP

Enzyme AssayEnzyme Assay Normal versus cancer oxidation of ethyl Normal versus cancer oxidation of ethyl

alcohol 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 Change in absorbance was monitored on a

dual beam spectrophotometer against a dual beam spectrophotometer against a reaction containing no substrate.reaction containing no substrate.

Enzyme activity is expressed as mIU/min/mg Enzyme activity is expressed as mIU/min/mg protein.protein.

N.D. indicates no detectable activity.N.D. indicates no detectable activity.

Enzyme Assay Enzyme Assay Normal vs. CancerNormal vs. Cancer

Normal Breast Breast Cancer

4-MP 4-MP Patient # Without With

Δ % Inhibition

Patient # Without With

Δ % Inhibition

N1 3.89 0.56 3.33 86 CA1 0.76 0.68 0.08 11

N2 4.02 1.02 3.00 75 CA2 N.D. N.D. - -

N3 4.89 N.D. 4.80 98 CA3 N.D. N.D. - -

N4 9.17 0.08 8.92 99 CA4 N.D. N.D. - -

•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 Normal Tissue ImmunocytochemistryImmunocytochemistry

Note organized ducts and Note organized ducts and strong ADH I stainingstrong ADH I staining

Normal ductal epithelium - showing Normal ductal epithelium - showing high immunoreactivity with minimally high immunoreactivity with minimally

reactive stromal cells highlightedreactive stromal cells highlighted

Bar = 5 µm

Normal DuctNormal Duct

Bar = 10µm

Type 3 lobules, parous Type 3 lobules, parous tissuetissue

Bar = 10µm

TDLUTDLU

Bar = 10 µm

Normal 2Normal 2o o Ab controlAb control

Bar = 10 µm

Malignant Tissue Malignant Tissue ImmunocytochemistryImmunocytochemistry

Note loss of immunoreactivity Note loss of immunoreactivity and lack of proper ductal and lack of proper ductal

orientationorientation

Loss of ADH I stainingLoss of ADH I staining

Bar = 10 µm

Cancer – Minimal ADH I Cancer – Minimal ADH I immunoreactivity immunoreactivity

Bar = 10µm

Weak positive cells surrounding Weak positive cells surrounding areas of no ADH I immunoreactive areas of no ADH I immunoreactive

cellscells

Bar = 10 µm

Increased magnification of Increased magnification of previous slideprevious slide

Bar = 2 µm

Increased MagnificationIncreased Magnification20x vs 100x 20x vs 100x

Bar = 10 µm

Bar = 2 µm

Overall heterogeneity of ADH I Overall heterogeneity of ADH I immunoreactivity immunoreactivity

Bar = 10 µm

Weak ADH I immunoreactivity Weak ADH I immunoreactivity seen in a morphologically seen in a morphologically

“normal” duct“normal” duct

Bar = 5µm

Cancerous tissue – note ducts Cancerous tissue – note ducts lacking expression lacking expression

10x and 40x10x and 40x

Bar = 5 µmBar = 20 µm

Increased magnification Increased magnification 100x100x

Bar = 2 µm

Normal vs. CancerNormal vs. Cancer20x20x

Bar = 10 µm

ReferencesReferencesChambon, 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.

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