The Toxicology Forum

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The Toxicology Forum Aspen, Colorado July 12, 2005
Raymond W. Tennant, Ph.D. Director, National
Center for Toxicogenomics National Institute of
Environmental Health Sciences
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The v-Ha-ras Tg.AC Mouse
zeta globin promotor
v-Ha-Ras
SV40 Poly A
EcoR1
Hinbll/BamHI
XbaI
DraI
EcoRI
Phenotype induced papilloma expression

• v-Ha-ras (mutations in codons 12 and 59)
• Zeta-globin promoter
• Genetically initiated
• Recapitulates multi-stage model
• No need for initiation with carcinogen
• Tumor development is synchronous
• Skin tumor development well described

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Experimental Design
Day 0 Epidermal Abrasion
Day 3, 5, 9, 18, 30 RNA extraction from skin
Tg.AC
Competitive hybridization to the Agilent mouse
platform
FVB
Analysis of ras/tumor specific gene expression
changes
Before
After
Day 30
Tg.AC
FVB
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A Prototype for Analysis of Microarray Gene
Expression Data Facilitated Discovery Strategy
Identification of significant Differentially
Expressed Genes (DEG)
Tennant, 2005
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Agilent
2 arrays per sample
Sample collection
Image Feature Extraction LOWESS
normalization logRatio, stat, p value
Independent evaluation
Filtering 95 CI, dye flip D.E. genes
Histopathology
Unsupervised clustering
EASE
Group Behavior Biological processes enrichment
analysis
Expression Profiles
Supervised grouping
PDQ_MED EASE
Independent Verification On focus genes
Literature Text mining
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development (47, Axin2, Cd24a, Dmrt1, Etv6, Fst,
Hmox1, Klf3, Mafb, Plxnb3, Tacc3,
) morphogenesis (31, Hoxa9, Idb1, Mea1, Pax5,
Prss18, Mapk12, Nfkb2, Serpine1, ) organogenesis
(29, Cyr61, Enah, Ext1, Gjb6, Gp38, Hod, Hoxa9,
Rhoa, Pitx1, Tbx4, ) histogenesis (6, Ext1,
Krt1-14, Rbp2, Sprr2g, Sprr2j, Sprr2k) ectoderm
development (5, Krt1-14, Rbp2, Sprr2g, Sprr2j,
Sprr2k) embryonic development (Cyr61, Enah, Ext1,
Gsc, Cyr61, Mafb, Tbx4, Thbd) metabolism (141,
Acly, Ada, Adam8, Alox12, Arg1, Bcl3, Bfsp1,
Car2, Cd24a, Chd1, ) lipid metabolism (20,
Alox12, Gba, Acate2, Acly, Gyk, Hmgcr, Hsd11b1,
Lrrc8, Plaa, ) fatty acid metabolism (6, Alox12,
Acate2, Ptgds2, Ptges, Ptgs2, Tpi) icosanoid
metabolism (3, Ptgs2, Ptgds2, Ptges) prostanoid
metabolism (3, Ptgs2, Ptgds2, Ptges) prostaglandi
n metabolism (3, Ptgs2, Ptgds2,
Ptges) carbohydrate metabolism (17, Acyl, Eno1,
Gapds, Gyk, Has3, Impa1, Ldh1, Man2c1 Pgam1,
) carbohydrate catabolism (5, Eno1, Gapds, Ldh1,
Pgam1, Tpi) monosaccharide metabolism (7, Eno1,
Gapds, Impa1, Ldh1, Pgam1, Pmm2, Tpi) hexose
catabolism (5, Eno1, Gapds, Ldh1, Pgam1,
Tpi) glucose catabolism (5, Eno1, Gapds, Ldh1,
Pgam1, Tpi) Glycolysis (5, Eno1, Gapds, Ldh1,
Pgam1, Tpi) alcohol metabolism (10, Eno1, Gapds,
Gyk, Hmgcr, Hsd17b7, Impa1, Ldh1, Pgam1, Pmm2,
Tpi) coenzyme and prosthetic group metabolism
(8, Acate2, Acly, Atp6v1e1, Gsr, Gsta1, Hmox1,
) biosynthesis (25, Acly, Ada, Alox12,
Atp6v1e1, Ext1, Gamt, Gucy2c, Hmgcr, Hsd17b7,
Mocs1, ) organic acid biosynthesis (5, Alox12,
Acly, Ptgds2, Ptgs2, Tpi) carboxylic acid
biosynthesis (5, Alox12, Acly, Ptgds2, Ptgs2,
Tpi) catabolism (26, Adam8, Arg1, Blmh, Capn2,
Casp14, Eno1, Entpd4, Mmp13, Npepps,
) regulation of catabolism (4, Serpinb12,
Serpinb1a, Serpinb1b, Serpinb4) regulation of
protein catabolism (3, Serpinb1a, Serpinb1b,
Serpinb12) cell communication (54, Adora1,
Axin2, Chrng, Col17a1, Cxcl5, Dapk3, Ngfb, Dsc1,
Fgfbp1, ) signal transduction (33, Adam8,
Adora1, Axin2, Cxcl5, Dapk3, Dtr, Ghrl, Hras1,
Il6ra, Klrd1, ) cell surface receptor linked
signal transduction (16, Adam8, Adora1, Axin2,
Hras1, Dtr, ) integrin-mediated signaling
pathway (5, Adam8, Itga6, Itgb1, Itgb4,
Rhoa) cell adhesion (20, Adam8, Col17a1, Dsc1,
Fn1, Klra7, Lama3, Lamc2, Klrd1, Pcdhb4, ) cell
organization and biogenesis (34, Apoe, Arpm2,
Cbx2, Cd2, Dncl2b, Hip1, Krt1-1, Tekt2, Pxn,
) cytoplasm organization and biogenesis (29,
Apoe, Arpm2, Cd2, Dncl2b, Krt1-1, Krt1-2, Myh1,
) organelle organization and biogenesis (29,
Apoe, Arpm2, Krt1-1, Cd2, Stmn1, Tubb4,
) cytoskeleton organization and biogenesis (28,
Apoe, Krt1-1, Myh1, Ophn1, Pdlim3,
) development (69, Apoe, Cd2, Clu, Crym, Dct,
Dlx4, Dmd, Egr1, Etv4, Expi, Fhl1, Fzd8, Msx2,
) morphogenesis (52, Apoe, Asb1, Bai1, Cd2,
Col18a1, Crip2, Crym, Dmd, Kit, Lef1, S100a1,
) Wnt receptor signaling pathway (7, Fzd8, Lef1,
Nkd2, Six3, Wnt2b, Wnt5b, Wnt6) organogenesis
(49, Apoe, Bai1, Cd2, Dmd, Dscr1l1, Egr1, Fhl1,
Foxc1, Gdf8, Hoxc13, ) muscle development (12,
Dmd, Fhl1, Gdf8, Myh1, Myh4, Myh7, Myh8, Myl1,
Mylpf, ) neurogenesis (17, Apoe, Crym, Dscr1l1,
Egr2, Lhx2, Mt3, Nr4a2, Olig1, Ptpn11,
Slit2) central nervous system development (8,
Apoe, Lhx2, Six3, Snca, Tagln3, Titf1,
) Metabolism (150, Aldh6a1, Abl1, Adss, Alox15,
Apoe, Cds2, Ddr1, Car6, Cd2, Eno3, ) organic
acid metabolism (21, Aldh6a1, Alox15, Arg2,
Bbox1, Crot, Cte1, Dct, Farsla, Glul, Pklr,
) carboxylic acid metabolism (21, Aldh6a1,
Alox15, Arg2, Bbox1, Crot, Cte1, Dct, Pklr,
Prodh, ) amine metabolism (16, Aldh6a1, Arg2,
Bbox1, Ctps, Dct, Prodh, Nin, Nnmt, Nr4a2,
Ppfibp2, Si, ) amino acid metabolism (13,
Aldh6a1, Arg2, Ctps, Dct, Farsla, Glul, Prodh,
Si, Slc22a17, Tyrp1, ) amino acid and derivative
metabolism (16, Aldh6a1, Arg2, Bbox1, Ctps, Dct,
Glul, Nin, Nnmt, Tyrp1, ) pigment metabolism (3,
Dct, Si, Tyrp1) melanin metabolism (3, Dct, Si,
Tyrp1) melanin biosynthesis (3, Dct, Si,
Tyrp1) melanin biosynthesis from tyrosine (3,
Dct, Si, Tyrp1) cell motility (13, Apoe, Dmd,
Dnali1, Etv4, Myh1, Myh4, Myh7, Myh8, Pxn, Scn4a,
Slit2, Tm4sf6, Tpm1) muscle contraction (7, Dmd,
Myh1, Myh4, Myh7, Myh8, Scn4a, Tpm1) striated
muscle contraction (4, Myh1, Myh4, Myh7,
Myh8) response to temperature (5, Hspa1b,
Krtap5-1, Thea, 4833428E21Rik, A030005K14Rik)
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FVB/N (HE)
Stefin A1
Day 5
Day 9
Day 18
Early papilloma (Tg.AC)
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Comparison of Day 30 TSDE Genes with SAGE Data
(Rundhaug et al.)
Krt2-6b 66 15.9 Keratin complex 2, basic, gene
6b Krt2-6b 10.6 15.9 Keratin complex 2, basic,
gene 6b Sprrl1 28.2 40 Small proline-rich-like 1
(Sprrl 1) Sprrl1 28.2 23.7 Small
proline-rich-like 1 (Sprrl 1) Sprr1b 30.5 33 Small
proline-rich protein 1B (cornifin B
Sprr1b) Sprr2h 4.2 18 Small proline-rich protein
2H (Sprr2h) Sprr2f 23.4 15 Small proline-rich
protein 2F (Sprr2f) Sprr2e 17.6 14 Small
proline-rich protein 2E (Sprr2e) Sprr2a 20.9 11 Sm
all proline-rich protein 2A (Sprr2a) Sprr2g 6.8 10
Small proline-rich protein 2G (Sprr2g) Sprr2d 10.
5 9.5 Small proline-rich protein 2D
(Sprr2d) Krt1-16 9.2 6.9 Keratin 16
(K16) Krt2-1 2.1 -3.7 Keratin 1
(K1) Hif1a 2.0 12 Hypoxia inducible factor 1, a
subunit (HIF-1a) Hod 2.3 -5.8 Homeodomain only
protein (transcriptional repressor) Casp14 3.3 -8.
5 Caspase-14 Stfa1 2.4 19 Stefin A1 (cystatin
family cysteine protease inhibitor) Pp11r 3.0 12
Placental protein 11 related (probable serine
protease) Mmp13 3.5 11 Matrix metalloproteinase-13
(MMP-13 collagenase-3) Stfa3 2.1 3.4 Stefin A3
(cystatin family cysteine protease
inhibitor) Dcn -2.9 18 Decorin S100a8 10.8 38 S100
calcium binding protein A8 (calgranulin A
Mrp-8) S100a9 14.3 9.9 S100 calcium binding
protein A9 (calgranulin B Mrp-14) Gprc5b -2.0 -14
G protein-coupled receptor, family C, group 5,
member B Tm4sf8 2.2 11 Transmembrane 4
superfamily member 8 (tetraspanin-3) Gjb2 1.9 8.5
Connexin26 (gap junction membrane channel protein
b2) Actb 3.7 4.3 b-actin Ptges 4.5 10 Prostaglandi
n E synthase Gsto1 11.0 12 Glutathione
S-transferase omega 1 (GST-O1) Aldh3a2 -3.2 -1 Alc
ohol dehyrogenase family 3, subfamily A2
(NAD-dependent) Hrnr -11 9.3 Hornerin
NOTE Fold changes are from microarray data for
TSDE genes at day 30 and SAGE data of Squamous
Cell Carcinoma (SCC) and normal skin. Genes that
have the same direction of change between TS day
30 and SCC are highlighted in bold font.
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• Summary
• The Tg.AC Mouse Model can be induced to provide
  synchronous skin tumor development
• The molecular pathogenesis of the neoplastic
  process can be analyzed by microarray technology
  and bioinformatics
• Process clustering can be used to provide
  functional annotation and to identify potentially
  novel genes and processes
• The expression profiles of non-annotated genes,
  when compared to similar known genes, provide
  functional support to their provisional
  annotation derived from sequence alignment,
  chromosomal location, and transcription factor
  binding sites
• Analysis of neoplastic processes may provide
  potential targets for intervention or prevention
  of cancer morbidity

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