Protein Expression Profiles in the Epidermis of ... - ACS Publications

Protein Expression Profiles in the Epidermis of Cyclooxygenase-2 Transgenic Mice by 2-Dimensional Gel Electrophoresis and Mass Spectrometry Jianjun Shen,† Amy Pavone,† Carol Mikulec,† Sean C. Hensley,† Angelina Traner,† Thom K. Chang,† Maria D. Person,‡ and Susan M. Fischer*,† The University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas, and CRED/ICMB Analytical Core, College of Pharmacy, The University of Texas at Austin, Austin, Texas Received August 17, 2006

Exposure of murine skin to tumor-promoting agents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) causes up-regulation of cyclooxygenase-2 (COX-2) and increased prostaglandin (PG) synthesis. Pharmacological inhibition of COX-2 significantly reduces skin tumor development. However, we previously demonstrated that K14.COX-2 transgenic (TG) mice that overexpressed COX-2 in the epidermis were unexpectedly resistant to tumor development under the classical 7,12-dimethylbenz[a]anthracene-TPA protocol. In the present study, we employed a proteomic approach of 2-dimensional gel electrophoresis (2-DE) and mass spectrometry to profile differentially expressed proteins in the epidermis of K14.COX-2 TG and wild-type control mice. Various 2-DE approaches were used to identify the maximum number of differentially expressed proteins: 20 for untreated samples, 3 for acetonetreated samples, and 22 for TPA-treated samples. These proteins include 14-3-3 sigma, numerous actin fragments, actin filament related proteins cofilin-1 and destrin, galectin-3, galectin-7, prohibitin, S100A6, S100A9, and many others. The differential expression of galectin-3, galectin-7, S100A9 was validated by Western blot analysis and/or immunohistochemical analysis. The current data suggest that some of the differentially expressed proteins might increase apoptosis and cell cycle arrest, which, in turn, may provide insight into the role of COX-2 in skin tumorigenesis. Keywords: cyclooxygenase-2 • proteomics • galectin • S100 calcium binding protein • epidermis

Introduction The two isoforms of cyclooxygenase (COX), COX-1 and COX2, are the key enzymes that convert arachidonic acid to prostaglandin (PG). Whereas COX-1 is constitutively expressed in most tissues, COX-2 expression is not detectable in most normal epithelial tissues, but it can be induced by mitogenic and/or inflammatory stimuli, such as cytokines, growth factors, serum, and phorbol esters.1-3 Increased expression of COX-2 has been described in many premalignant and malignant tissues in a number of organ systems.4 Human cancers associated with overexpression of COX-2 include colorectal,5,6 breast,7 prostate,8 and pancreatic9,10 cancer. Also, recent evidence from human skin cancers and experimental animal models suggests that COX-2 expression and increased PGE2 production play a critical role in the development of skin cancer.11 Lee and colleagues recently reviewed the toxicological and pharmacological implications of COX-2 expression in the skin.11 In human skin, COX-2 is normally expressed in suprabasal keratinocytes, * To whom correspondence should be addressed. Dr. Susan M. Fischer, Department of Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX 78957; E-mail: [email protected]; Fax: +1-512-237-9566. † The University of Texas M. D. Anderson Cancer Center. ‡ The University of Texas at Austin. 10.1021/pr060418h CCC: $37.00

 2007 American Chemical Society

although at very low levels.12,13 Furthermore, investigators have found COX-2 expression to be rapidly upregulated by acute exposure to UV-B radiation13 and highly elevated in squamous cell carcinomas.14 COX-2 is not normally expressed in murine epidermis, but its expression can be induced by acute or chronic exposure to UV-B radiation12,15 or chemical tumor promoters, such as 12-O-tetradecanoyl phorbol-13-acetate (TPA). Overexpression of COX-2 has also been observed in benign papillomas and squamous cell carcinomas.14 Topical application or oral administration of the selective COX-2 inhibitors SC-58125 in NMRI mice and celecoxib in SKH-1 hairless mice resulted in suppressed papilloma development and a reduction in the number and size of tumors, respectively.16-20 A number of genetic mouse models have been developed to examine the mechanisms by which COX-2 expression and PG production affect tumor development. In one study, the human COX-2 gene was overexpressed in female mouse mammary glands under the control of the mouse mammary tumor virus promoter.21 Mammary tumors spontaneously developed in these transgenic (TG) mice at a high frequency after several pregnancies. Another study directed COX-2 expression to murine skin under the control of the bovine keratin-5 promoter.22 Although tumors did not develop spontaneously in these TG mice, the mice did present with epidermal hyperplasia Journal of Proteome Research 2007, 6, 273-286

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research articles and dysplasia, a sign of premalignant lesions, indicating that COX-2 overexpression alone is not sufficient to cause tumor development in murine skin. In a third study, mice deficient in either COX-1 or COX-2 exhibited reduced skin tumorigenesis, most likely because of premature terminal differentiation.23 These genetic models provide direct experimental evidence linking COX-2 with carcinogenesis, especially in the skin. In our laboratory, we generated TG mice that overexpressed COX-2 under the control of the keratin 14 promoter, referred to as K14.COX-2 mice.24 The response of these mice to skin carcinogenesis protocols depends on the particular agents used. For example, administration of a single low dose of 7,12dimethylbenz[a]anthracene (DMBA) resulted in significantly more tumors in TG than in wild-type mice (manuscript submitted). However, quite unexpectedly, the TG mice were found to be resistant to tumor development under the classical DMBA-TPA protocol,24 although they had significantly more tumors when the non-phorbol ester tumor promoter anthralin was administered (unpublished data). In the present study, we employed a proteomic approach consisting of protein separation by two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF MS or MALDI-TOF/TOF MS/MS analysis, and a database search of a nonredundant protein database, to identify proteins that are differentially expressed in K14.COX-2 and wild-type mice under the classical DMBA-TPA protocol.

Materials and Methods TG Mouse Production and Treatment. K14.COX-2 TG mice of the FVB strain were generated as described by Bol et al.24 A single topical application of 200 µL of acetone (vehicle) or 2.5 µg of TPA in acetone was carried out. The mice were killed 6 h later. This treatment schedule was based on previous studies showing that a 6 h treatment with TPA elicited the overexpression of a variety of genes that regulate proliferation and inflammation.17,25,26 Preparation of Epidermal Lysates. Epidermal lysates from TG and age-matched non-TG mice were prepared as described previously27 with minor modifications. Briefly, the epidermis was removed by scraping on ice, suspended in a modified RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 0.66 µg/mL aprotinin, 0.5 µg/mL leupeptin, 1 µg/mL pepstatin, 1 mM Na3VO4, 1 mM NaF), and sonicated three times in 5-s bursts. The cell lysates were centrifuged at 14 000 × g for 15 min at 4 °C. The supernatants were collected, and their protein concentrations were measured using the Bradford assay (Bio-Rad Laboratories, Hercules, CA). Two-Dimensional Gel Electrophoresis (2-DE). 2-DE was performed as described previously28 with minor modifications. Briefly, 150 µg of protein was precipitated from the RIPA buffer using a Perfect-FOCUS kit (Geno Technology, Inc., St. Louis, MO). Solubilization of the protein pellet in a rehydration buffer, first-dimensional isoelectric focusing on the PROTEAN IEF cell, and second-dimensional SDS-PAGE separation with a precast Criterion 8-16% gradient gel in a Criterion Cell apparatus were all performed according to the manufacturer’s protocols (BioRad Laboratories). Immobilized pH gradient strips with pH ranges of 4-7, 5-8, and 7-10, respectively, were employed. SDS-polyacrylamide gels were fixed, stained with SYPRO Ruby (Bio-Rad Laboratories) overnight, and destained for 4 h. SYPRO Ruby-stained gel images were captured on a Typhoon 9410 Variable Mode Imager (GE HealthCare, Piscataway, NJ). 274

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For the CyDye labeled 2D difference gel electrophoresis (DIGE) approach, the protein lysates in the modified RIPA buffer were initially concentrated using Microcon YM-10 spin columns (Millipore, Bedford, MA), and then buffer exchanged with a new lysis buffer suitable for labeling (7 M urea, 2 M thiourea, 4% CHAPS, and 30 mM Tris). The pH of each such sample was then adjusted to 8.5 using 30 mM NaOH and its protein concentration was measured with the Bradford assay (Bio-Rad Laboratories). Fifty micrograms of protein from each pooled sample was labeled with Cy5 for TG, Cy3 for wild type, and Cy2 for the internal control (25 µg of each from TG and wild type). Samples were finally combined and separated on a single 2-D gel for each sample set as described above. Duplicate 2-D gels were run for each set of samples to ensure reproducibility. The individual 2-D gel images of Cy2, Cy3, and Cy5 were captured using mutually exclusive excitation/emission wavelengths of 489/506, 550/570, and 649/670, respectively, on the Typhoon 9410 Imager (GE HealthCare). 2-DE Image Data Analysis. Analysis of SYPRO Ruby-stained 2-D gel images was performed as described previously.28 Briefly, integrated signal intensities were analyzed quantitatively using the Bio-Rad PDQuest 2-D gel image analysis software program. For 2D DIGE gels, pairwise comparisons of TG and wild-type protein abundance were carried out with the GE Healthcare DeCyder Differential In-gel Analysis (DIA) module against a mixed WT/TG internal standard. Volume ratios were generated by comparing Cy5-labeled TG to Cy3-labeled WT spot volumes after normalization against Cy2-labeled internal standard spot volumes. A 2-fold threshold with 2 S.D. of 1.8 was used as a selection criterion. Differential expression of protein spots indicated by either PDQuest or DeCyder software was then visually verified to eliminate artifacts erroneously identified by the software programs. Spots that differed consistently in both software analysis and visual observation were excised manually and subjected to in-gel tryptic digestion with sequencing-grade trypsin (Promega, Madison, WI), based on a previously described procedure.29 Statistical Analysis. No statistical analysis was available for our regular SYPRO Ruby-stained 2-DE images because of limited sample numbers and the number of repeats performed. For the 2D DIGE gels, Biological Variation Analysis (BVA) was used to perform inter-gel analyses of duplicate samples using spot data generated by DIA. Average volume ratios and paired Student’s t test p-values were calculated in BVA by importing and matching spot maps generated in DIA. A p-value e 0.05 for protein abundance difference is generally considered to be statistically significant. Protein Identification. Tryptic digests were analyzed using a Voyager-DE PRO MALDI-TOF mass spectrometer as described previously30 or a 4700 Proteomics Analyzer MALDITOF/TOF (Applied Biosystems, Foster City, CA). Samples were dried to < 5 µL in a SpeedVac (ThermoSavant, Holbrook, NY), desalted with a Ziptip µ-C18 pipet tip (Millipore, Billerica, MA), and eluted with 1.5 µL of matrix into two or three spots on the MALDI target. The matrix was a saturated solution of R-cyano4-hydroxycinnamic acid in 50% acetonitrile/0.1% trifluoroacetic acid diluted in half with a solvent. Calibration Mixture 1 or 4700 mix (Applied Biosystems) was prepared according to the manufacturer’s recommendations and diluted in half with a solvent. For proteins identified using the MALDI-TOF/TOF, both MS and MS/MS spectra were acquired automatically using 4000 Series Explorer V 3.0 RC1. MS spectra were acquired with 2000

Protein Expression Profiles in COX-2 Transgenic Mice

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Figure 1. Representative 2-D gel images of the epidermal lysates of K14.COX-2 TG and wild-type mice. The pH range of 4-7 for all three treatment groups is shown here. (A and B) Mice that did not undergo treatment with differentially expressed proteins marked. The protein identifications for the numbered spots are listed in Table 1. The other 2-D gels of pH ranges 5-8 and 7-10 were run but are not shown here. (C and D) Wild-type and K14.COX-2 TG mice were treated with TPA, respectively. (E and F) Wild-type and K14.COX-2 TG mice were treated with acetone, respectively. Two-D gels of pH 5-8 were run, but not shown for both TPA- and acetone-treatment groups, whereas no gel was run for pH 7-10 for these groups. WT, wild-type and TG, transgenic.

laser shots over the mass range 700-4000 and calibrated internally on trypsin autolysis peaks. An interpretation method selected the top 20 MS signals with minimum S/N 20 for MS/ MS fragmentation after exclusion of matrix, trypsin and keratin peaks. Up to 3750 shots were acquired for MS/MS. Additional peak processing and database search were performed using GPS Explorer v3.5. MASCOT V2.0 http://www.matrixscience.com/ was used for the database search with the same parameters as before with these exceptions: 30 ppm MS mass tolerance over the mass range 700-4000, 0.2 Da MS/MS fragment mass tolerance, up to 100 peaks with minimum S/N 15 selected for MS, and up to 65 fragment ions with minimum S/N 3. Samples were searched against both the Swiss-Prot (Dec. 30, 2005) and the mammalian subset of the Trembl (Dec. 6,

2005) databases. The search output combines the scores from MS search and the MS/MS search using a probabilistic MOWSE algorithm. The MASCOT score is defined as -10*logP, where P is the probability that the observed match is a random event. A score of 66 corresponds to P < 0.05 for this data set, and is chosen as the cutoff for a significant hit. The top ranking mouse hit is reported for the search. Western Blot Analysis. For 1-D immunoblotting, protein extracts (50 µg) from TG and wild-type mouse epidermis were run on a 15% SDS-PAGE gel. For 2-DE Western blotting, protein extracts (150 µg) were separated on 2-D gels with a pH of 3-10 to cover the maximum pH range. The separated proteins were then transferred onto PVDF membranes. Antibodies specific to caspase-3 (BIOMOL International, Plymouth Meeting, PA), Journal of Proteome Research • Vol. 6, No. 1, 2007 275

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Table 1. Identification of Differentially Expressed Proteins between K14.COX2 TG and Wild-Type Mice Without Treatment theor/observed protein spot #

1 2 3 4 5

ID

14-3-3 protein sigma (Stratifin) odorant binding protein-1a S100A6 (Calcyclin) apolipoprotein A-1 precursor (Apo-AI) adenylate kinase isoenzyme 1 (ATP-AMP transphosphorylase) fatty acid-binding protein, epidermal galectin-7 variant superoxide dismutase [Cu-Zn] cathepsin D HSP60 β-enolase 3 moesin serum albumin precursor transitional endoplasmic reticulum ATPase aldehyde dehydrogenase 2, mitochondrial creatine kinase, M type carbonic anhydrase 3 hemoglobin β-1 peptidylprolyl cis-trans isomerase A galectin-3

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Swiss protein accession #

fold change (TG/WT)

# peptides matched

% coverage

O70456 P97336 P14069 Q00623 Q9R0Y5

0.2 7.1 0.4 0.2 0.6

4/20 2/3 1 8/20 5/20

16 10 9 28 37

Q05816 Q9CRB1 P08228 P18242 P63038 P21550 P26041 P07724 Q01853 P47738 P07310 P16015 P02088 P17742 P16110

0.5 3.5 1.7 2.6 0.5 1.8 4.0 Saturated 0.4 0.2 0.2 0.4 2.7 1.7 4.3

2/8 7/20 3/4 3/5 4/5 4/5 3/4 10/20 9/12 5/20 11/20 5/20 7/20 6/20 6/20

17 50 24 7 9 12 6 20 10 10 36 19 66 33 24

PSD # ions matcheda

10/12 9/11

12/16 15/16 11/14 11/14

pI

MW

4.75/4.61 5.24/5.26 5.3/5.04 5.65/5.52 5.67/5.55

27.7/27.7 16.8/16.8 10.1/10 30.6/24.5 21.5/21.5

6.14/6.17 6.15/6.70 6.03/6.39 6.71/5.90 5.91/5.52 6.58/6.63 6.11/6.48 5.75/5.81 5.14/5.12 7.53/6.41 6.58/7.00 6.49/6.94 7.26/7.81 7.73/6.23 8.91/9.73

15.1/15 15.2/15 15.8/16 45/37 60.9/60 69.7/42 66.5/67 68.7/58 89.3/89 56.5/53 43/43 29.2/29 15.7/15 18.0/62 27.5/27.5

a For samples with fewer than four peptides matched to one protein, at least one ion from the MS spectrum was subjected to manual post-source decay (PSD) analysis for protein identification by peptide fragmentation.

galectin-3 (Affinity BioReagents, Golden, CO), and galectin-7 (Bethyl Laboratories, Inc., Montgomery, TX) were used and diluted at 1:500,1:2000, and 1:40 000, respectively. Ten micrograms of protein extract was used for 1-D immunoblotting with the rabbit anti-galectin-7 antibody (Bethyl Laboratories, Inc.). The relative intensities of the protein bands in the TG and wildtype mouse samples were measured with a Kodak Image Station 440CF and quantitatively analyzed using the Kodak 1D image analysis software program (Eastman Kodak Company, Rochester, NY). Immunohistochemical (IHC) Analysis. IHC analysis was performed as described previously24 with minor modifications. The primary antibodies against S100A8, S100A9 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and galectin-7 (Bethyl Laboratories, Inc.) were diluted at 1:250, 1:250, and 1:10 000, respectively. The biotinylated secondary antibody was used at a dilution of 1:250. Measurement of Apoptotic Cells. Groups of three each TG and wild-type mice were treated as follows: (a) topically with acetone, (b) topically with 2.5 µg of TPA, (c) irradiated with 220 mJ/cm2 UV as described previously17 and topically with acetone, or (d) irradiated with UV and topically with TPA. All of the mice were killed 24 h after treatment, and three sections of skin from each mouse were fixed in formalin prior to being embedded in paraffin. Four-micron sections were cut and immunohistochemically stained for active caspase-3 (1:1000; R&D Systems, Minneapolis, MN). The average number of positive cells per 40× field was determined by manual counting of at least 20 fields per mouse.

Results No Treatment Group. Epidermis was harvested from two K14.COX-2 TG and two sex- and age-matched wild-type control mice from each treatment group (no treatment, treatment with TPA, and treatment with acetone) as described in the Materials and Methods. The untreated protein samples were run on three immobilized pH gradient strips-one each for the pH ranges 276


4-7, 5-8, and 7-10-to obtain better resolution of the separated proteins. These samples were run twice at each pH range to verify reproducibility. Overall, all of the 2-D gels were quite consistent when compared by visual examination and using the PDQuest 2-D gel image analysis software program. The top two panels of Figure 1 (A and B) show two representative 2-D gel images between pH 4 and 7 of the epidermal lysates of untreated TG mice and wild-type control mice. The other 2-D gel images of pH 5-8 and 7-10 are not shown. In total, 20 protein spots were determined to be differentially expressed and manually excised for in-gel digestion. Among these protein spots, 20 distinct proteins were identified by MALDI-TOF MS analysis and a search of the nonredundant protein database (Table 1). As shown in Table 1, the proteins observed to be upregulated in untreated K14.COX-2 TG mice included odorant binding protein-1a, galectin-3, moesin, galectin-7, hemoglobin β-1, cathepsin D, superoxide dismutase [Cu/Zn], β-enolase 3, and peptidylprolyl cis-trans isomerase A. Those that were downregulated in the TG mice included adenylate kinase, fatty acid binding protein (epidermal), HSP60, transitional endoplasmic reticulum ATPase, carbonic anhydrase 3, calcyclin/S100A6, creatine kinase (muscle form), apolipoprotein A-1, aldehyde dehydrogenase, and 14-3-3 sigma. Acetone Treatment Group. For comparison, the middle two panels of Figure 1 (C and D) show two 2-D gel images of the epidermal lysates of acetone-treated K14.COX-2 TG and wildtype control mice. Two-D gel images of pH 5-8 are not shown, whereas no gel was run for pH 7-10 for this treatment groups. In total, PDQuest 2-D gel image analysis identified eight protein spots as differentially expressed. Among these protein spots, three distinct proteins were identified, by MALDI-TOF MS analysis and database search, as five distinct actin fragments, Rho GDP-dissociation inhibitor 2, and ATP synthase beta chain, mitochondrial precursor (as shown in Table 2). The most striking result was identification of five different actin fragment spots (spots #1-5 in Table 2). Figure 2 shows

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Table 2. Identification of Differentially Expressed Proteins between Acetone-Treated K14.COX2 TG and Wild-Type Mice theor/observed protein spot #

1 2 3 4 5 6 7 8

ID

actinb actinb actinb actinb actinb rho GDPdissociation inhibitor 2 (Rho GDI 2) ATP synthase beta chain, mitochondrial precursor ATP synthase beta chain, mitochondrial precursor

accession #

fold change (TG/WT)

# peptides matched

% coverage

P60710 P60710 P60710 P60710 P60710 Q61599

TG ) 0 TG ) 0 0.22 TG ) 0 TG ) 0 TG ) 0

2/6 4/6 4/7 5/10 4/5 2/8

5 14 14 17 14 14

P56480

WT ) 0

12/20

P56480

WT ) 0

12/20

PSD # ions matcheda

Mowse

pI

MW

8 29 29 103 29

5.29/4.9 5.29/5.2 5.29/5.5 5.29/5.6 5.29/5.4 4.97/4.9

42/13 42/22 42/26 42/25 42/22 23/23

31

6.63E+03

5.19/5.0

56/48

31

1.25E+04

5.19/5.1

56/48

37/39 5/7

a For samples with fewer than four peptides matched to one protein, at least one ion from the MS spectrum was subjected to manual post-source decay (PSD) analysis for protein identification by peptide fragmentation or peptides matched to previous identification of a protein with the same MW and pI. b Actin, β, and γ are not distinguished. Protein spots #1 and 4 have one more peptide for β and γ than R.

tryptic peptide encased by a dash-dot line from residues 94-112, in spot #3. A C-terminal fragment was also detected, at a different observed molecular weight of 25 kDa. Similar results were seen for alpha actin, although fewer peptides were detected (see Figure 2B).

Figure 2. Protein sequences of mouse cytoplasmic-1 (beta) actin (A) and skeletal muscle (alpha-1) actin (B). Multiple fragment spots were seen for actin. The peptides encased by dashed lines were detected in the spot for the 13 kDa N-terminal fragment, seen in spot #1 of Table 2. The peptides encased by solid lines were seen in the 22 kDa central fragment, spots #2 and 5 of Table 2. A central fragment of larger size, 26 kDa, contained the same peptides, along with the semi-tryptic peptide encased dash-dot lines from residues 94-112, in spot #3 of Table 2. Two C-terminal fragments were detected, at different gel molecular weights. A 27 kDa fragment in spot #4 of Table 3 contained the peptides encased solid, dash-dot and dotted lines. A smaller 25 kDa fragment contained the same peptides, but not the peptide encased dash-dot lines, spot #4 of Table 2. Similar results were seen for alpha actin, though less peptides were detected (see Figure 2B).

peptides detected in these actin fragment spots. The peptides encased by dashed lines in Figure 2A were detected in the spots for the 13 kDa N-terminal fragment, seen in spot #1. The peptides encased by solid lines were seen in the 22 kDa central fragment, spots #2 and 5. Another central fragment of larger size, 26 kDa, contained the same peptides, along with the semi-

TPA Treatment Group. Because we were most interested in differentially expressed proteins between the epidermis of the K14.COX-2 TG mice and wild-type control mice in the TPAtreated group, these epidermal protein lysates were separated on 2-DE gels using three slightly different approaches. The first approach was the same as described for the acetone treatment group, as shown in the bottom two panels of Figure 1 (E and F). The second 2-DE approach was to use CyDye labeling to separate both wild-type and TG epidermal proteins in a single gel to avoid gel-to-gel variation. The last approach was to first partially remove an abundant protein, albumin, and then separate proteins using the CyDye labeling method. Figure 3A shows a representative 2-D gel image of the CyDye labeled experiment. Figure 3B shows two separated 2-D gel images obtained from the Cy5 and Cy3 channels individually imaged using mutually exclusive wavelengths as previously described.32 Combining these three approaches, a total of 32 protein spots were identified by the PDQuest and DeCyder software programs as differentially expressed between the epidermis of TPAtreated K14.COX-2 TG mice and wild-type mice. Among these protein spots, 22 distinct proteins were identified (Table 3). As shown in Table 3, the 16 proteins observed to be upregulated in TPA-treated K14.COX-2 TG mice included 143-3 σ (Figure 3C), two actin fragments, 10 kDa heat shock protein (Hsp10) (Figure 3C), both epidermal and adipocyte fatty acid binding proteins (Figure 3C), acyl-CoA-binding protein (ACBP), guanine nucleotide binding protein β subunit 2, cell division control protein 42 homolog, cofilin-1, destrin, caspase14 precursor, eukaryotic translation initiation factor 5A, cathepsin B precursor, prohibitin, and proteasome activator complex subunit 2. The two actin fragments identified here were specific to TPA treatment and different from the five actin fragments in either pI or MW values as described in the acetone treatment group. One of the two fragments, spot #4 in Table 3, was a C-terminal fragment of cytoplasmic actin. It contains the peptides shown in the solid, dash-dot and dotted lines of Figure 2A. The six proteins that were downregulated in the epidermis of TPA-treated TG mice (as shown in Table 3) included hemopexin, serotransferrin, β-enolase, S100A9 (Figure 3C), Journal of Proteome Research • Vol. 6, No. 1, 2007 277

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Figure 3. 2-D DIGE analysis of wild-type and K14.COX-2 transgenic with either TPA- or acetone-treatment. The pH range of 3-10 was run for all CyDye labeled 2-D gels. (A) Representative composite, overlaid 2-D gel image of CyDye labeled samples of 50 µg of TG (Cy5), 50 µg of wild-type (Cy3), and the internal control (Cy2) of 25 µg of each from transgenic and wild-type mice. (B) Cy3 (wild-type) and Cy5 (transgenic) 2-D DIGE images of wild-type and K14.COX-2 transgenic with TPA-treatment. (C) Cropped 2-D gel images of a selected number of proteins identified as differentially expressed between epidermis of wild-type and K14.COX-2 transgenic with TPAtreatment. Protein IDs are as indicated. The left panel of each group was Cy3 of wild-type mice, and the right panel was Cy5 of K14.COX-2 transgenic mice. (D) Graphic comparisons of pixel volume ratio of these selected proteins in (C). Protein IDs are as labeled. 278


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Table 3. Identification of Differentially Expressed Proteins between TPA-Treated K14.COX2 TG and Wild-Type Mice theor/observed protein spot #

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

ID

14-3-3 σ 14-3-3 σ R actin actin, cytoplasmic 10 kDa heat shock protein, mitochondrial (Hsp10) fatty acid-binding protein, epidermal (E-FABP) E-FABP E-FABP fatty acid-binding protein, adipocyte hemopexin precursor hemopexin precursor hemopexin precursor hemopexin precursor serotransferrin precursor serotransferrin precursor β enolase acyl-CoA-binding protein (ACBP) guanine nucleotide binding protein beta subunit 2 - like 1 cell division control protein 42 homolog cofilin-1 destrin (actindepolymerization factor) S100A9 (Calgranulin B) apolipoprotein A-II precursor (Apo-AII) serine protease inhibitor A3K precursor (Contrapsin) serine protease inhibitor A3K precursor serine protease inhibitor A3K precursor (Contrapsin) serine protease inhibitor A3K precursor caspase-14 precursor eukaryotic translation initiation factor 5A (eIF-5A) cathepsin B precursor prohibitin (B-cell receptor associated protein 32) (BAP 32) proteasome activator complex subunit 2

accession #

fold change (TG/WT)

t-test p-valuea

O70456 O70456 P68033 P60710 Q64433

2.1 2.2 2.0 3.3 2.3

0.0053 0.0048 0.025

Q05816

# peptides matched

% coverage

0.00073

8/20 8/20 4/20 6/14 3/3

35 34 13 17 19

2.5

0.00048

4/20

Q05816 Q05816 P04117

2.6 2.7 2.8

0.0016 2.40E-05 0.0013

Q91X72 Q91X72 Q91X72 Q91X72 Q921I1 Q921I1 P21550 P31786 P68040

-2.2 -2.8 -2.9 -2.8 -2.7 -2.3 -2.5 2.5 2.0

P60766

PSD # ions matchedb

Mowse (or Mascot score)c

pI

MW

126 126 141 359 17.3

4.75/4.4 4.75/4.5 5.24/4.9 5.29/5.7 8.17/5.4

28/18 28/18 42/14 42/27 11/9

30

65.0

6.18/5

15/12

4/20 6/20 3/3

23 41 17

22.0 69.0 44.0

6.18/6.2 6.18/5.4 8.55/4.8

15/12 15/12 15/11

0.018 0.0053 0.0012 0.085 0.0081 0.0012 0.0039 0.006 0.0052

6/20 9/20 8/20 4/20 11/20 12/20 3/5 3/3 8/20

14 26 24 7 20 22 10 37 23

305 3.22E+04 1.23E+04 56.0 1970 4.27E+04 33.0 109 479

7.92/7.1 7.92/7.3 7.92/7.6 7.92/7.8 6.94/7.4 6.94/8 6.81/8.1 8.78/6.2 7.6/8.1

51/50 51/50 51/50 51/50 77/62 77/62 47/38 10/9 35/26

2.5

0.0051

1/1

6

5.77/7.3

21/18

P18760 Q9R0P5

2.6 2.6

0.019 0.042

6/6 5/7

35 32

76.0 39.0

8.27/8.9 8.19/8.8

18/17 18/16

P31725 P09813

-2.0 -2.0

0.0043 0.023

5/8 2/9

37 18

89.0

6.72/7.3 6.57/4.4

13/12 11/9

P07759

-2.5

18

632c

5.05/4.6

47/125

P07759

-2.9

7/20

22

214

5.05/4.65

47/125

P07759

-2.9

7/20

22

214

5.05/4.75

47/125

P07759

-2.5

20

813c

5.05/4.8

47/125

O89094 P63242

Wt)0 Wt)0

20 9

775c 227c

4.8/4.8 5.1/5.2

30/31 17/17

P10605 P67778

2.3 3.3

5 13

104c 754c

5.6/5.3 5.6/5.6

38/26 30/26

P97372

3.3

14

306c

5.6/5.6

27/26

14/14

10/11 12/12

9/10

a p-value was only available for the protein spots identified from 2-D gel image analysis using the CyDye labeled 2-D gels and DeCyder software. b For samples with fewer than four peptides matched to one protein, at least one ion from the MS spectrum was subjected to manual post-source decay (PSD) analysis for protein identification by peptide fragmentation. c MS + MS/MS identification. MASCOT score is combined MS + MS/MS score. Score 66 corresponds to p < 0.05.

apolipoprotein A-II precursor (Figure 3C), and serine protease inhibitor (serpin) A3K precursor. The two panels of Figure 3D further illustrate the quantitative views of up-regulation of 143-3 sigma, E-FABP and HSP10, and down-regulation of ApoAII and S100A9 in the epidermis of TPA-treated TG mice as compared to WT mice. Among the proteins identified in Table 3, one unusual observation was that the four 125 kDa spots (spots #24-27) were identified as serpin A3K, a 47 kDa serine protease inhibitor. No other protein was found in these spots, though homologous serpins were also found via the database search (data not shown). Because they did not have any unique peptides with MS/MS, it is unlikely that these other serpins were present in the spots. Also, serpin A3K is a potential

glycoprotein, but 100 kDa of sugars are not likely to produce a well-defined spot on the gel. The protein acts by forming a covalent bond to a protease, but no other protease was detected in the digests, and all the major ions were accounted for as serpin A3K. Another observation was that both prohibitin and proteasome activator complex subunit 2 were initially identified from a single spot. Careful examination of the 2D gel revealed that there were in fact two closely overlapping spots. Their respective theoretical and observed molecular weights and pI’s are very similar, resulting in two partially overlapping spots. Validation Studies. It is essential to validate differentially expressed proteins using other methods such as Western blot and/or IHC analysis. IHC approach is especially powerful, not Journal of Proteome Research • Vol. 6, No. 1, 2007 279

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Figure 4. Validation of differential expression of a few selected proteins. (A) Confirmation of COX-2 overexpression in K14.COX-2 TG mice and COX-2 induction by TPA-treatment in both K14.COX-2 TG and wild-type mice. (B) 1-D Western blot analyses of galectin-3 in epidermis of K14.COX-2 TG and wild-type mice. (C) Traditional 1-D Western blot analysis of capase-3 as a loading control. (D) Cropped 2-D gel images of galectin-3 of epidermal protein lysates of K14.COX-2 TG and wild-type mice that received no treatment. The images were cropped from 2-D gels run between the pH range of 7-10. (E) 2-D Western blot analyses of galectin-3 of all three-treatment groups in epidermis of K14.COX-2 TG and wild-type mice. For 2-D Western blotting, protein extracts (150 µg) were separated on 2-D PAGE gels with a pH range of 3-10 to cover the maximum pH. (F) Cropped 2-D gel images of galectin-7 of all three-treatment groups in epidermis of K14.COX-2 TG and wild-type mice. (G) 2-D Western blot analyses of galectin-7 of all three-treatment groups in epidermis of K14.COX-2 TG and wild-type mice.

only to validate the differential expression, but also to demonstrate protein cellular localization and rule out potential contamination. COX-2. Western blot analysis was used to confirm that the K14.COX-2 TG mice had overexpression of COX-2 protein. Figure 4A clearly demonstrates that COX-2 was indeed overexpressed in these mice. Treatment with TPA further increased the expression of this protein in these mice and slightly induced COX-2 expression in wild-type control mice. However, this COX-2 overexpression in TG mice was not detected by SYPRO Ruby stained 2D gels. This could have resulted from a lower detection limit of SYPRO Ruby stain, which is about 1000-fold less sensitive than immunoblotting. COX-2 protein may also not resolved well on 2D gels. Galectin-3. Protein spot #20 in Table 1 (Figure 4D) was identified as galectin-3. It had a higher expression level at that 280


specific spot in TG mice when compared with its expression in untreated wild-type mice. The images of Figure 4D were cropped from pH range of 7-10. For the acetone or TPA treated epidermal samples, 2-D gels were not run between this particular pH range. However, 1-D immunoblotting demonstrated that the galectin-3 level was roughly equal in untreated TG mice and wild-type control mice (Figure 4B). This can be explained by the fact that individual protein spots were compared in a 2-D gel experiment, whereas the total amount of protein in each sample was compared by 1-D Western blot analysis. In the TPA-treated group, galectin-3 expression increased in wild-type mice, whereas it remained the same in TG mice. However, in the acetone-treated group, the level of galectin-3 expression was the same in both TG and wild-type mice. In addition, 2-D immunoblotting was performed to examine whether galectin-3 existed as a single spot or as


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Figure 5. Immunohistochemical analysis of three selected proteins. (A) Galectin-7 of both acetone- and 1x TPA-treated K14.COX-2 TG and wild-type mice as indicated. Galectin-7 had a much higher protein level in epidermis of 1× TPA-treated TG mice than that of WT control mice. (B) S100A9 of acetone-, 1× TPA-, and 4× TPA-treated K14.COX-2 TG and wild-type mice. S100A9 was not expressed in epidermis of acetone-treated TG and WT mice. 1× TPA treatment slightly induced S100A9 protein expression in epidermis of both TG and WT mice. Because IHC was not sensitive enough to distinguish differences in S100A9 between TG and WT mice at the very low protein levels, a 4× TPA treatment protocol was conducted, which resulted in a much higher S100A9 protein expression in epidermis of WT mice than TG mice. (C) S100A8 of acetone-, 1× TPA-, and 4× TPA-treated K14.COX-2 TG and wild-type mice. Likewise, S100A8 had a much higher protein expression in epidermis of WT mice than TG mice from 4× TPA-treatment.

multiple spots. The overall spot pattern shown in Figure 4E was very complex in both wild-type and TG mice in all three treatment groups, indicating possible modification of galectin-

3, involving charge-dependent changes. Nevertheless, the results of 2-D Western blot analysis confirmed those of 1-D immunoblotting, demonstrating that the level of galectin-3 Journal of Proteome Research • Vol. 6, No. 1, 2007 281

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Discussion

Figure 6. Measurement of apoptotic cells with the use of an anticaspase-3 antibody.

expression was higher in TPA-treated wild-type mice than in TPA-treated TG mice. Galectin-7. Protein spot #7 in Table 1 and Figure 1A and 1B was identified as galectin-7. This protein was expressed in TG mouse epidermis at a level about two to three times higher than that in age-matched wild-type control mouse epidermis before and after TPA treatment. Figure 4F shows an enlarged area of the gel containing galectin-7. 2-D immunoblotting of galectin-7 was performed both with and without treatment with TPA as well as with treatment with acetone as a control to determine whether galectin-7 exists as a single spot or multiple spots in TG and wild-type mice. This revealed that galectin-7 ran as a single protein spot at its native pI and that its relative intensity was consistent with that of 2-D gel experiments for each sample (Figure 4G). The results of an IHC experiment for galectin-7, shown in Figure 5A, demonstrated that the level of galectin-7 was higher in the epidermis of TPA-treated TG mice compared to that of wild type controls. This IHC experiment further confirmed that galectin-7 protein was indeed expressed in the epidermis of both TG and wild type mice. S100 Proteins. Figure 5B showed the results of an IHC experiment for S100A9. This protein was clearly not expressed in the epidermis of acetone-treated TG and wild-type mice. It was slightly induced by a single TPA treatment in the epidermis of both TG and wild-type mice, to a higher level in TG than in wild-type mice, which was consistent with the results of S100A9 from 2-D gel experiments. Because the protein level of S100A9 was so low, a further experiment using 4 TPA treatments resulted in much higher S100A9 protein expression in both TG and wild-type mice, to a much greater extent in epidermis of wild-type mice than TG mice. Because S100A8 and S100A9 usually form a heterodimeric complex to interact with their target proteins, S100A8 protein expression was also examined and had a similar protein expression pattern as S100A9, as shown in Figure 5C. Preliminary Functional Study of Apoptosis. Apoptosis was analyzed in both wild-type and TG mice. Figure 6 shows that with and without TPA treatment, neither wild-type nor TG mice had caspase-3-positive cells. UV irradiation resulted in more caspase-3-positive cells in TG mice than in wild-type mice (p ) 0.001). Combined UV irradiation and TPA treatment further induced apoptotic cells, slightly more so in TG mice than in wild-type mice (p ) 0.003). 282


In the classical initiation/promotion (TPA) model of skin tumorigenesis, TPA causes the induction of COX-2 and increased papilloma formation. In a previous study, we generated K14.COX-2 TG mice that overexpressed COX-2 in the epidermis.24 Although these TG mice developed significantly more tumors than wild-type controls with the administration of either a single low dose of DMBA or the non-phorbol ester tumor promoter anthralin (manuscript submitted), they were unexpectedly resistant to tumor development under the classical DMBA-TPA protocol.24 Because COX-2 induction is required for papilloma formation, it is not clear why forced expression of COX-2 would reduce papilloma formation. The present study was designed to identify differences at the protein level between K14.COX-2 TG mice and their wild-type controls that could begin to elucidate this unexpected phenomenon. Twenty proteins were identified as differentially expressed in the epidermis of untreated K14.COX-2 TG mice compared to that of wild-type control mice (as shown in Table 1). Three distinct proteins were identified as differentially expressed between acetone-treated K14.COX-2 TG mice and wild-type controls (Table 2). Twenty-two proteins were identified as differentially expressed for the TPA-treated samples (as shown in Table 3). Because it is not possible to discuss the potential roles of all the proteins identified, we will focus our discussion on a few selected groups of proteins. In the present study, the expression of galectin-7 was greater in TG mice than in wild-type mice prior to treatment as well as with treatment with TPA or acetone. Two studies demonstrated that galectin-7 exerts a proapoptotic effect in response to exposure to UV radiation or to upregulated expression of p53.33-34 More recently, Kuwabara et al.35 reported that galectin-7 expression enhances mitochondrial cytochrome c release. Furthermore, the absence of and/or decreased expression of galectin-7 has been observed in squamous carcinoma cell lines and carcinomas.36 These studies point to a proapoptotic function for galectin-7. Thus the higher galectin-7 levels in TG mice may be linked to their reduced sensitivity to TPA promotion. On the other hand, expression of galectin-3 was greater in wild-type mice than in TG mice following TPA treatment. Galectin-3 exerts an antiapoptotic effect by controlling cell growth.37-38 Yang et al.39 demonstrated that galectin-3 and Bcl-2 form heterodimers in vitro. Therefore, they suggested that galectin-3 regulates cell growth and antiapoptotic activity through a Bcl-2-related pathway. Furthermore, investigatiors have suggested that phosphorylation of galectin-3 acts as an “on/off switch” for its sugar-binding function,40 is required for its antiapoptotic function,41 and may govern its subcellular localization.42 As shown in Figure 5E, galectin-3 was modified in both TG and wild-type mice. Therefore, downregulation of galectin-3 by overexpression of COX-2, in conjunction with possible modifications such as phosphorylation, may play a role in rendering TG mice more susceptible to apoptosis compared to wild-type mice. Taken together, the differential expression of the proapoptotic protein galectin-7 and antiapoptotic protein galectin-3 in TG and wild-type mice suggests that the unexpected resistance of K14.COX-2 TG mice to tumor development under the classical DMBA-TPA protocol might have resulted from greater apoptotic potential in these mice. Consistent with our previous study,24 we were not able to detect apoptotic cells up to 48 h


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Figure 7. Speculated protein interaction network leading to the resistance of K14.COX-2 TG mice to tumor promotion by TPA treatment. A part of this figure is modified from Figure 3 of a publication by Huang, C.-M. et al.78 with permission. The arrows denote the upregulation and/or activation of proteins or pathways. The T-shaped symbols denote the down-regulation and/or inactivation of proteins or pathways.

after TPA treatment in TG mice, using the standard terminal deoxynucleotidyl transferase-mediated biotin dUTP nick-end labeling (TUNEL) assay (data not shown). However, in vivo apoptosis normally involves only 3-5% of cells, and the fast turnover of this low number of apoptotic cells places them below the detection limit with the TUNEL assay. To overcome this problem, a second agent, UV irradiation, can be used in combination with TPA treatment to enhance apoptosis. UV irradiation was previously demonstrated to induce an increase in the number of apoptotic cells in the epidermis of wild-type mice when compared with that in BK5.IGF-1 TG mice, whereas no difference in the epidermal apoptotic index between these two types of mice was found before UV exposure.27 In the present study, UV irradiation alone induced more caspase 3-positive apoptotic cells in TG mice than in wild-type mice. Combined treatment with TPA and UV irradiation further increased the number of apoptotic cells in TG mice, indicating that apoptosis may indeed be involved in the resistance of these mice to tumor promotion by TPA. 2-D gel electrophoresis/MALDI-TOF was reported as a very effective approach to identify protein fragments and domains.30 Employing this approach, the present study identified several actin fragments. Although absent in K14.COX-2 TG mice that received either acetone or TPA treatment, five different actin fragments were identified in the epidermis of wild-type control mice as acetone-treatment specific under the same conditions. In contrast, two other actin fragments were identified in the epidermis of K14.COX-2 TG mice as TPA-treatment specific. From the MALDI spectra of these fragments, we can deduce that the fragments are produced by cleavage of the actin protein in at least 2 places. One cleavage point is likely between L93 and R94, thus generating the non-tryptic peptide 94-112. The second cleavage occurs between residues 112 and 196, and the final cleavage is between 334 and 359. Previously, actin proteolysis by active caspase-3 and fractin, a 32 kDa actin fragment, were shown to play a role in the apoptosis of P19 mouse embryonal carcinoma cell line.43 The functionality of

the actin fragments identified in the present study needs further investigation. Interestingly, cofilin-1 and destrin, two actin depolymerizing factor (ADF)/cofilin family members, were identified as being more abundant (2.6-fold for both proteins, p 0.019 and 0.042, respectively) in the epidermis of TPA-treated K14.COX-2 TG mice. They are highly homologous actin-regulating proteins with distinct functions.44 Destrin depolymerizes actin filaments by interacting with the actin monomers and removing them from the filaments,45 whereas cofilin-1 controls actin polymerization and depolymerization reversibly in a pH-dependent manner.46 Altered cofilin expression has been associated with several diseases, including ovarian cancer.47-48 In human epidermis, these two proteins were activated following UV irradiation.49 It is possible that the higher protein levels of cofilin-1 and destrin in epidermis of TPA-treated K14.COX-2 TG mice may be associated with the fewer actin fragments identified in these mice. S100 proteins belong to a family of 21 low molecular weight calcium binding proteins.50-51 These proteins were implicated in various biological functions in a calcium-dependent manner, such as enzyme activity, protein phosphorylation, calcium homeostasis, cell growth and differentiation.52-53 Of the two S100 proteins identified in the present study, S100A6 was downregulated 2.5-fold in the epidermis of untreated K14.COX-2 TG compared to that of wild-type control mice. Several previous studies showed that S100A6 expression was increased in many types of cancers.54-56 The higher expression of S100A6 was also suggested to be associated with the extent of colorectal adenocarcinoma invasion/metastasis in yet another study.57 Although the biological function of S100A6 remains unknown, its decreased protein level in epidermis of K14.COX-2 TG, in contrast to its overexpression in many tumors, correlates with the resistance of these mice to TPA tumor promotion. On the other hand, consistent with the previous findings of minimal expression of S100A8 and S100A9 in normal epidermis53 and TPA-induced mRNA expression of S100A8 and Journal of Proteome Research • Vol. 6, No. 1, 2007 283

research articles S100A9 in murine epithelial cells,58 the present study showed that, in epidermis of K14.COX-2 TG and wild-type control mice that received either no treatment or acetone treatment, S100A9 protein was either not expressed or was expressed at levels below the detection limit. In the TPA-treated samples, S100A9 expression was induced, to a greater extent in the epidermis of wild-type controls. Both S100A8 and S100A9 proteins were reported to be overexpressed in human gastric cancers and malignant tissues of colorectal carcinoma.59-60 In another study, these two S100 proteins, together with S100A11, were identified as colon tumor-specific protein markers.61 The S100A8/S100A9 complex was implicated in fatty acid transport and superoxide production due to its ability to bind arachidonic acid.62-63 The S100A8/S100A9 complex was also reported to bind and potentiate NADPH oxidase activation in the presence of arachidonic acid.64-66 As a result, this interaction increased generation of oxidative stress during the respiratory burst of phagocytes or, possibly, in epithelial cells. Thus, the lower level of S100A8 and S100A9 proteins in the epidermis of TPA-treated TG mice compared to that of wild type controls could correlate with decreased oxidative stress and resistance to tumor development in these TG mice. 14-3-3 sigma protein was expressed at about a 2-fold higher level in the epidermis of TPA-treated TG mice compared to wild-type controls (Table 3). It belongs to a family of small and highly conserved signal transduction mediators with diverse activities. 14-3-3 sigma was identified as an epithelial and keratinocyte specific protein and reported to be downregulated in many human cancers and/or their cell lines.67-71 Hypermethylation in 5′ CpG islands of the 14-3-3 sigma gene was most likely responsible for the decreased expression of this gene in these cancers.71-72 In contrast, Yang et al. demonstrated that increased expression of 14-3-3 sigma suppressed tumorigenicity by regulating p53 activity.73 Therefore, increased expression of 14-3-3 sigma protein in the epidermis of TPA-treated TG mice might partly contribute to the resistance of these mice to tumor promotion by TPA. Prohibitin protein was expressed at about a 3-fold higher level in the epidermis of TPA-treated TG mice compared to that of wild-type controls. This protein was proposed to be a potential tumor suppressor, as it was reported to interact with the retinoblastoma tumor suppressor (Rb) protein and regulate E2F to prevent cell proliferation.74-75 These observations suggest that increased expression of prohibitin might be partly responsible for the resistance of the TG mice to tumor promotion by TPA. In summary, using a 2-DE-based proteomic approach, we identified 43 distinct proteins that were differentially expressed in K14.COX-2 TG mice compared to wild-type controls. Although our current method was only able to identify relatively abundant proteins in mouse epidermis, our data demonstrate the feasibility of studying an increasing number of animal models utilized in cancer research by a more global approach. More importantly, our analysis of the abundant proteins suggests that the differential expression of galectin-3 and galectin-7, calcium binding proteins S100A6 and S100A9, 143-3 sigma, prohibitin, and perhaps the actin filament interacting proteins cofilin and destrin, and their related pathways, likely contribute to the resistance of K14.COX-2 TG mice to tumor promotion by TPA. These presumed protein interaction networks are presented in a schematic illustration in Figure 7. This was further supported by the observation that the combined treatment with UV radiation and TPA resulted in more 284


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apoptotic cells in TG mice than in wild-type controls. Additional studies are needed to determine how these identified proteins play a role in skin tumorigenesis. Abbreviations: BVA, Biological Variation Analysis; COX-2, cyclooxygenase-2; 2-DE, two-dimensional gel electrophoresis; 2D DIGE, two-dimensional difference gel electrophoresis; DIA, Differential In-gel Analysis; DMBA, 7,12-dimethylbenz[a]anthracene; HSP, heat shock protein; IHC, immunohistochemical; moesin, membrane-organizing extension spike protein; NCBI, National Center for Biotechnology Information; PG, prostaglandin; SOD1, Cu/Zn superoxide dismutase; TG, transgenic; TPA, 12-O-tetradecanoyl phorbol-13-acetate; WT, wild-type.

Acknowledgment. We thank Dr. Jun-Wei Liu for our discussion of apoptosis, Lisa Schroeder for help with in-gel tryptic digestion, Dr. Michael MacLeod and Kelli Kochan for reading and commenting on the manuscript, Donald R. Norwood for editorial assistance, Vanessa Edwards for preparation of the manuscript, and Joi Holcomb and Christine Brown for help with the illustrations. This work was supported by grants CA100140, CA16672, and ES07784. References (1) Xie, W.; Herschman, H. R. v-src induces prostaglandin synthase 2 gene expression by activation of the c-Jun N-terminal kinase and the c-Jun transcription factor. J. Biol. Chem. 1995, 270 (46), 27622-27628. (2) Smith, W. L.; DeWitt, D. L.; Garavito, R. M. Cyclooxygenases: structural, cellular, and molecular biology. Annu. Rev. Biochem. 2000, 69 145-182. (3) Muller-Decker, K.; Scholz, K.; Marks, F.; Furstenberger, G. Differential expression of prostaglandin H synthase isozymes during multistage carcinogenesis in mouse epidermis. Mol. Carcinog. 1995, 12 (1), 31-41. (4) Subbaramaiah, K.; Dannenberg, A. J. Cyclooxygenase 2: a molecular target for cancer prevention and treatment. Trends Pharmacol. Sci. 2003, 24 (2), 96-102. (5) Sano, H.; Kawahito, Y.; Wilder, R. L.; Hashiramoto, A.; Mukai, S.; Asai, K.; Kimura, S.; Kato, H.; Kondo, M.; Hla, T. Expression of cyclooxygenase-1 and -2 in human colorectal cancer. Cancer Res. 1995, 55 (17), 3785-3789. (6) Gustafson-Svard, C.; Lilja, I.; Hallbook, O.; Sjodahl, R. Cyclooxygenase-1 and cyclooxygenase-2 gene expression in human colorectal adenocarcinomas and in azoxymethane induced colonic tumours in rats. Gut 1996, 38 (1), 79-84. (7) Half, E.; Tang, X. M.; Gwyn, K.; Sahin, A.; Wathen, K.; Sinicrope, F. A. Cyclooxygenase-2 expression in human breast cancers and adjacent ductal carcinoma in situ. Cancer Res. 2002, 62 (6), 16761681. (8) Yoshimura, R.; Sano, H.; Masuda, C.; Kawamura, M.; Tsubouchi, Y.; Chargui, J.; Yoshimura, N.; Hla, T.; Wada, S. Expression of cyclooxygenase-2 in prostate carcinoma. Cancer 2000, 89 (3), 589-596. (9) Tucker, O. N.; Dannenberg, A. J.; Yang, E. K.; Zhang, F.; Teng, L.; Daly, J. M.; Soslow, R. A.; Masferrer, J. L.; Woerner, B. M.; Koki, A. T.; Fahey, T. J., 3rd. Cyclooxygenase-2 expression is upregulated in human pancreatic cancer. Cancer Res. 1999, 59 (5), 987-990. (10) Kokawa, A.; Kondo, H.; Gotoda, T.; Ono, H.; Saito, D.; Nakadaira, S.; Kosuge, T.; Yoshida, S. Increased expression of cyclooxygenase-2 in human pancreatic neoplasms and potential for chemoprevention by cyclooxygenase inhibitors. Cancer 2001, 91 (2), 333-338. (11) Lee, J. L.; Mukhtar, H.; Bickers, D. R.; Kopelovich, L.; Athar, M. Cyclooxygenases in the skin: pharmacological and toxicological implications. Toxicol. Appl. Pharmacol. 2003, 192 (3), 294-306. (12) Leong, J.; Hughes-Fulford, M.; Rakhlin, N.; Habib, A.; Maclouf, J.; Goldyne, M. E. Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation. Exp. Cell Res. 1996, 224 (1), 79-87. (13) Buckman, S. Y.; Gresham, A.; Hale, P.; Hruza, G.; Anast, J.; Masferrer, J.; Pentland, A. P. COX-2 expression is induced by UVB exposure in human skin: implications for the development of skin cancer. Carcinogenesis 1998, 19 (5), 723-729.

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