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Investigator(s) |
Foreman JE, Sorg JM, McGinnis KS, Rigas B, Williams JL, Clapper ML, Gonzalez FJ, Peters JM. Regulation of Peroxisome Proliferator-Activated Receptor-beta/delta by the APC/beta-CATENIN Pathway and Nonsteroidal Antiinflammatory Drugs. Mol Carcinog. 2009 Oct;48(10):942-52.
Studies indicate that peroxisome proliferator-activated receptor-beta/delta (PPAR beta/delta) can either attenuate or potentiate colon cancer. One hypothesis suggests that PPAR beta/delta is upregulated by the adenomatous polyposis coli (APC)/beta-CATENIN pathway and a related hypothesis suggests that PPAR beta/delta is downregulated by nonsteroidal antiinflammatory drugs (NSAIDs). The present study examined these possibilities using in vivo and in vitro models. While APC/beta-CATENIN-dependent expression of CYCLIN D1 was observed in vivo and in vitro, expression of PPAR beta/delta was not different in colon or intestinal polyps from wild-type or Apc(min) heterozygous mice or in human colon cancer cell lines with mutations in APC and/or beta-CATENIN. No difference in the level of PPAR beta/delta was found in colon from wild-type or Apc(min) heterozygous mice following treatment with NO-donating aspirin (NO-ASA). NSAIDs inhibited cell growth in RKO (wild-type APC) and DLD1 (mutant APC) human colon cancer cell lines but expression of PPAR beta/delta was not downregulated in these cell lines in response to a broad concentration range of celecoxib, indomethacin, NS-398, or nimesulide. However, indomethacin caused an increase in PPAR beta/delta mRNA and protein that was accompanied with increased expression of a known PPAR beta/delta target gene. Interestingly, expression of PPAR alpha was also increased in the human colon cancer cell lines by several NSAIDs at the highest concentration examined. Results from these studies provide additional evidence indicating that PPAR beta/delta is not upregulated by the APC/beta-CATENIN pathway. Further, these studies suggest that increased PPAR beta/delta and/or PPAR alpha by NSAIDs in human colon cancer cell lines could contribute to the mechanisms underlying the chemopreventive effects of NSAIDs. (C) 2009 Wiley-Liss, Inc.
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Clapper
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Hensley HH, Merkel CE, Chang WC, Devarajan K, Cooper HS, Clapper ML. Endoscopic imaging and size estimation of colorectal adenomas in the multiple intestinal neoplasia mouse. Gastrointest Endosc. 2009 Mar;69(3 Suppl):742-9.
BACKGROUND: The scientific potential of animal models of carcinogenesis has not been fully realized because of our limited ability to monitor tumor growth in vivo. OBJECTIVE: To develop an endoscopy-based protocol for the accurate estimation of adenoma size in vivo from images obtained during colonoscopy. DESIGN: To compare estimates of lesion size acquired during endoscopy with those obtained from magnetic resonance imaging (MRI) and at necropsy. SETTING: A small-animal imaging facility. SUBJECTS: Adenomatous polyposis coli multiple intestinal metaplasia Fox Chase Cancer Center mice that develop multiple colorectal adenomas. METHODS: The mice received colonoscopic examination by using a rigid endoscope, and high-resolution images of colon adenomas were captured by using a charge-coupled-device camera. Lesion size was estimated by comparing the dimensions of the adenoma relative to a reference rod by using a novel geometric construction. The resulting areas were compared with estimates from MRIs and validated at necropsy. MAIN OUTCOME MEASUREMENTS: Cross-sectional area of colon adenomas. RESULTS: The cross-sectional area of 20 adenomas was measured in vivo during colonoscopy and compared with the size as measured at necropsy, which yielded a Pearson correlation coefficient of 0.94 (P = 6.52 x 10(-9)). Assessment of interoperator variability, when using measurements from 11 adenomas, yielded a Pearson correlation coefficient of 0.85 (P = 4.35 x 10(-3)) and demonstrated excellent reproducibility. LIMITATIONS: Only the distal colon could be viewed, and endoscopic measurements were 2-dimensional. CONCLUSIONS: An endoscopic method for the reliable measurement of colorectal adenomas in vivo was established. The application of this technique to mouse models of colon carcinogenesis will provide unique insight into the dynamics of adenoma growth.
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Clapper
Devarajan
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Clapper ML, Gary MA, Coudry RA, Litwin S, Chang WC, Devarajan K, Lubet RA, Cooper HS. 5-aminosalicylic acid inhibits colitis-associated colorectal dysplasias in the mouse model of azoxymethane/dextran sulfate sodium-induced colitis. Inflamm Bowel Dis. 2008 Oct;14(10):1341-7.
BACKGROUND: The impact of the antiinflammatory agent 5-aminosalicylic acid (5-ASA) on the risk for colitis-associated colorectal cancer remains controversial. The chemopreventive activity of 5-ASA was evaluated in the Swiss Webster model of azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis-associated neoplasia. METHODS: Mice were injected with AOM (7.4 mg/kg i.p.) and randomized to receive either vehicle or 5-ASA (75, 150, and 225 mg/kg) for the remainder of the study. DSS treatment began at 9 weeks of age and continued for 3 cycles. At the time of sacrifice (18 weeks of age), the entire colon and rectum were processed for histopathologic examination. RESULTS: An inverse trend was observed between dose and multiplicity of colonic dysplasias in all drug-treated groups (P = 0.03), with animals receiving 75 mg/kg 5-ASA exhibiting 56% of the number of dysplasias of the AOM/DSS controls (mean +/- SEM: 7.6 +/- 1.4 and 13.6 +/- 2.7, respectively). Administration of 75 mg/kg 5-ASA decreased both the mean multiplicity of flat dysplasias (1.8 +/- 0.4 for drug-treated versus 5.6 +/- 1.2 for AOM/DSS control) and the burden of polypoid dysplasias (tumor burden: 6.7 +/- 2.7 for drug-treated versus 14.9 +/- 3.9 units for AOM/DSS controls) significantly (P = 0.002 and 0.04, respectively). Inflammation was least severe in the 75 mg/kg group, which exhibited the fewest number of colorectal tumors. CONCLUSIONS: These data suggest that low-dose 5-ASA may be efficacious in preventing colitis-associated dysplasias and provide strong support for optimizing this therapy for the prevention of colonic neoplasms in patients with ulcerative colitis.
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Clapper
Litwin
Devarajan
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Caretti E, Devarajan K, Coudry R, Ross E, Clapper ML, Cooper HS, Bellacosa A. Comparison of RNA amplification methods and chip platforms for microarray analysis of samples processed by laser capture microdissection. J Cell Biochem. 2008 Feb;103(2):556-63.
Laser capture microdissection (LCM) permits isolation of pure cell populations from which RNA can be extracted, amplified, and subjected to microarray analysis, allowing information to be obtained on the gene expression profile of defined cell types. To avoid amplification artifacts and detect genes expressed at different levels, it is important to optimize the choice of both RNA amplification step and microarray platform. We captured by LCM the same colon cancer biopsy and conducted a cross comparison of distinct RNA amplification methods and different chip platforms. We tested two RNA amplification methods with different chemistry: the one-cycle Ovation (TM) system (NuGEN) and the two-cycle Ribo OA (TM) method (Arcturus). We also compared two different whole genome platforms, based on Affymetrix technology: the U133 plus 2.0 and the X3P array, with probe sets closer to the 3' end of transcripts. After RNA amplification, microarray analysis, and data normalization, we in!
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Clapper
Bellacosa
Devarajan
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Clapper ML, Gary MA, Coudry RA, Litwin S, Chang WC, Devarajan K, Lubet RA, Cooper HS. 5-Aminosalicylic Acid Inhibits Colitis-Associated Colorectal Dysplasias in the Mouse Model of Azoxymethane/Dextran Sulfate Sodium-Induced Colitis. Inflamm Bowel Dis. 2008 Oct;14(10):1341-7.
Background: The impact of the antiinflammatory agent 5-aminosalicylic acid (5-ASA) on the risk for colitis-associated colorectal cancer remains controversial. The chemopreventive activity of 5-ASA was evaluated in the Swiss Webster model of azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis-associated neoplasia. Methods: Mice were injected with AOM (7.4 mg/kg i.p.) and randomized to receive either vehicle or 5-ASA (75, 150, and 225 mg/kg) for the remainder of the study. DSS treatment began at 9 weeks of age and continued for 3 cycles. At the time of sacrifice (18 weeks of age), the entire colon and rectum were processed for histopathologic examination. Results: An inverse trend was observed between dose and Multiplicity of colonic dysplasias in all drug-treated groups (P = 0.03), with animals receiving 75 mg/kg 5-ASA exhibiting 56% of the number of dysplasias of the AOM/DSS controls (mean +/- SEM: 7.6 +/- 1.4 and 13.6 +/- 2.7. respectively). Administration of 75 mg/kg 5-ASA decreased both the mean multiplicity of flat dysplasias (1.8 +/- 0.4 for drug-treated versus 5.6 +/- 1.2 for AOM/DSS control) and the burden of polypoid dysplasias (tumor burden: 6.7 +/- 2.7 for drug-treated versus 14.9 +/- 3.9 units for AOM/DSS controls) significantly (P = 0.002 and 0.04. respectively). Inflammation was least severe in the 75 mg/kg group. which exhibited the fewest number of colorectal tumors. Conclusions: These data suggest that low-dose 5-ASA may be efficacious in preventing colitis-associated dysplasias and provide strong support for optimizing this therapy for the prevention of colonic neoplasms in patients with ulcerative colitis. (Inflamm Bowel Dis 2008; 14:1341-1347)
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Clapper
Litwin
Devarajan
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Aiyer HS, Vadhanam MV, Stoyanova R, Caprio GD, Clapper ML, Gupta RC. Dietary berries and ellagic acid prevent oxidative DNA damage and modulate expression of DNA repair genes. International Journal of Molecular Sciences. 2008 Mar;9(3):327-41.
DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on > 95% inhibition of 8-oxodeoxyguosine (8-oxodG) and other unidentified oxidative DNA adducts induced by 4-hydroxy-17 beta-estradiol and CuCl2 in vitro. Inhibition of the latter occurred at lower concentrations ( 10 mu M) than that for 8-oxodG ( 100 mu M). In the in vivo study, female CD-1 mice (n=6) were fed either a control diet or diet supplemented with ellagic acid ( 400 ppm) and dehydrated berries (5% w/w) with varying ellagic acid contents - blueberry ( low), strawberry ( medium) and red raspberry ( high), for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%). However, both red raspberry and ellagic acid diets showed a significant reduction of 59% ( p < 0.001) and 48% ( p < 0.01), respectively. Both diets also resulted in a 3-8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA), DNA excision repair protein (ERCC5) and DNA ligase III (DNL3). These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair.
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Clapper
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Caretti E, Devarajan K, Coudry R, Ross E, Clapper ML, Cooper HS, Bellacosa A. Comparison of RNA amplification methods and chip platforms for microarray analysis of samples processed by laser capture microdissection. J Cell Biochem. 2008 Feb 1;103(2):556-63.
Laser capture microdissection (LCM) permits isolation of pure cell populations from which RNA can be extracted, amplified, and subjected to microarray analysis, allowing information to be obtained on the gene expression profile of defined cell types. To avoid amplification artifacts and detect genes expressed at different levels, it is important to optimize the choice of both RNA amplification step and microarray platform. We captured by LCM the same colon cancer biopsy and conducted a cross comparison of distinct RNA amplification methods and different chip platforms. We tested two RNA amplification methods with different chemistry: the one-cycle Ovation system (NuGEN) and the two-cycle Ribo OA method (Arcturus). We also compared two different whole genome platforms, based on Affymetrix technology: the U133 plus 2.0 and the X3P array, with probe sets closer to the 3' end of transcripts. After RNA amplification, microarray analysis, and data normalization, we investigated reproducibility and correlation of different methods and arrays. Our results indicate that the Arcturus Ribo OA method is superior for both array choices, especially in combination with X3P arrays, showing the lowest variance and Spearman correlation of 0.986. The quicker NuGEN procedure, when coupled with X3P arrays, also yielded excellent results (correlation of 0.951). These observations will be useful for planning large-scale analyses of LCM-dissected clinical samples.
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Clapper
Bellacosa
Devarajan
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Caretti E, Devarajan K, Coudry R, Ross E, Clapper ML, Cooper HS, Bellacosa A. Comparison of RNA amplification methods and chip platforms for microarray analysis of samples processed by laser capture microdissection. J Cell Biochem. 2007;103(2):556-63.
Laser capture microdissection (LCM) permits isolation of pure cell populations from which RNA can be extd., amplified, and subjected to microarray anal., allowing information to be obtained on the gene expression profile of defined cell types. To avoid amplification artifacts and detect genes expressed at different levels, it is important to optimize the choice of both RNA amplification step and microarray platform. We captured by LCM the same colon cancer biopsy and conducted a cross comparison of distinct RNA amplification methods and different chip platforms. We tested two RNA amplification methods with different chem.: the one-cycle Ovation system (NuGEN) and the two-cycle Ribo OA method (Arcturus). We also compared two different whole genome platforms, based on Affymetrix technol.: the U133 plus 2.0 and the X3P array, with probe sets closer to the 3' end of transcripts. After RNA amplification, microarray anal., and data normalization, we investigated reproducibility and correlation of different methods and arrays. Our results indicate that the Arcturus Ribo OA method is superior for both array choices, esp. in combination with X3P arrays, showing the lowest variance and Spearman correlation of 0.986. The quicker NuGEN procedure, when coupled with X3P arrays, also yielded excellent results (correlation of 0.951). These observations will be useful for planning large-scale analyses of LCM-dissected clin. samples. [on SciFinder (R)]
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Clapper
Bellacosa
Devarajan
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Clapper ML, Cooper HS, Chang WC. Dextran sulfate sodium-induced colitis-associated neoplasia: a promising model for the development of chemopreventive interventions. Acta Pharmacologica Sinica. 2007 Sep;28(9):1450-9.
Individuals diagnosed with ulcerative colitis face a significantly increased risk of developing colorectal dysplasia and cancer during their lifetime. To date, little attention has been given to the development of a chemopreventive intervention for this high-risk population. The mouse model of dextran sulfate sodium (DSS) -induced colitis represents an excellent preclinical system in which to both characterize the molecular events required for tumor formation in the presence of inflammation and assess the ability of select agents to inhibit this process. Cyclic administration of DSS in drinking water results in the establishment of chronic colitis and the development of colorectal dysplasias and cancers with pathological features that resemble those of human colitis-associated neoplasia. The incidence and multiplicity of lesions observed varies depending on the mouse strain used (ie, Swiss Webster, C57BL/6J, CBA, ICR) and the dose (0.7%-5.0%) and schedule (1-15 cycles wit!
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Clapper
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Wong Y-N, Change W-C, Clapper M, Engstrom PE. Chemoprevention of colorectal cancer. In: Saltz LB, editor. Colorectal cancer : evidence-based chemotherapy strategies. Totowa, N.J.: Humana Press; 2007. p. 33-49.
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Clapper
Engstrom
Wong
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Patel BB, Li XM, Dixon MP, Blagoi EL, Seeholzer SH, Chen Y, Miller CG, He YA, Tetruashvily M, Chaudhry AH, Ke E, Xie J, Cooper H, Bellacosa A, Clapper ML, Boman BM, Zhang T, Litwin S, Ross EA, Conrad P, Crowell JA, Kopelovich L, Knudson A, Yeung AT. Searchable high-resolution 2D gel proteome of the human colon crypt. J Proteome Res. 2007 Jun;6(6):2232-8.
We seek alterations in protein patterns at the earliest possible step on the path to cancer, namely, in cells of the target tissue from normal persons versus the corresponding normally appearing cells from persons who are heterozygous for mutation in a tumor suppressor gene that predisposes strongly to carcinoma in that tissue. To begin a systematic comparison of the proteomes of cells from normal and from neoplastic colons, we have undertaken the isolation of human colon crypts that are derived from the normal-appearing mucosa of left (descending) colon of patients with sporadic colorectal cancer. Two-dimensional (2D) gel electrophoresis is a proteomic approach that excels in the resolution of protein isoforms. Here, we document the practicality of this approach with human samples using gels of three overlapping pH ranges. For the first time, about 800 nonredundant proteins and 900 isoforms from purified human colonic crypts were identified, permitting an assessment of the contributions of protein isoforms. These interactive, searchable, hyperlink-enabled proteome maps and gene ontology analyses will facilitate future studies to discover the earliest markers and intervention targets during progression to colon cancer.
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Yeung
Clapper
Bellacosa
Knudson
Litwin
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Chang WC, Coudry RA, Clapper ML, Zhang X, Williams KL, Spittle CS, Li T, Cooper HS. Loss of p53 enhances the induction of colitis-associated neoplasia by dextran sulfate sodium. Carcinogenesis. 2007 Nov;28(11):2375-81.
Loss of p53 function is an early event in colitis-associated neoplasia in humans. We assessed the role of p53 in a mouse model of colitis-associated neoplasia. Colitis was induced in p53-/-, p53+/- and p53+/+ mice using three or four cycles of dextran sulfate sodium (DSS) followed by 120 days of water. Mice were examined for incidence, multiplicity and types of neoplastic lesions. Lesions were examined for mutations in beta-catenin (exon 3), K-ras (codons 12/13) and p53 (exons 5-8) by sequencing and for cellular localization of beta-catenin by immunohistochemistry. The incidence of neoplastic lesions was 57, 20 and 20% in p53-/-, p53+/- and p53+/+ mice, respectively (P = 0.013). p53-/- mice had a greater number of total lesions (P < 0.0001), cancers (P = 0.001) and dysplasias (P = 0.009) per mouse than either p53+/- or p53+/+ mice. Flat lesions were associated with the p53-/- genotype, whereas polypoid lesions were associated with the p53+/- and p53+/+ genotypes (P < 0.0001). beta-Catenin mutations were present in 75% of lesions of p53+/+ mice and absent in lesions from p53-/- mice (P = 0.055). Nuclear expression of beta-catenin was seen only in polypoid lesions (91%). No K-ras or p53 mutations were detected. These data indicate that loss of p53 enhances the induction of colitis-associated neoplasia, particularly flat lesions, and dysregulation of beta-catenin signaling plays an important role in the formation of polypoid lesions in this mouse model. As observed in humans, p53 plays a protective role in colitis-associated neoplasia in the DSS model.
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Clapper
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Wong YN, Chang WC, Clapper M, Engstrom PF. Chemoprevention of colorectal cancer. In: Saltz LB, editor. Colorectal cancer : evidence-based chemotherapy strategies. Totowa, N.J.: Humana Press; 2007. p. 33-50.
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Clapper
Engstrom
Wong
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Coudry RA, Meireles SI, Stoyanova R, Cooper HS, Carpino A, Wang XQ, Engstrom PF, Clapper ML. Successful application of microarray technology to microdissected formalin-fixed, paraffin-embedded tissue. Journal of Molecular Diagnostics. 2007 Feb;9(1):70-9.
The establishment of a reliable method for using RNA from formalin-fixed, paraffin-embedded (FFPE) tissue would provide an opportunity to obtain novel gene expression data from the vast amounts of archived tissue. A custom-designed 22,000 oligonucleotide array was used in the present study to compare the gene expression profile of colonic epithelial cells isolated by laser capture microdissection from FFPE-archived samples with that of the same cell population from matched frozen samples, the preferred source of RNA. Total RNA was extracted from FFPE tissues, amplified, and labeled using the Paradise Reagent System. The quality of the input RNA was assessed by the Bioanalyzer profile, reverse transcriptase-polymerase chain reaction, and agarose gel electrophoresis. The results demonstrate that it is possible to obtain reliable microarray data from FFPE samples using RNA acquired by laser capture microdissection. The concordance between matched FFPE and frozen samples was evaluated and expressed as a Pearson's correlation coefficient, with values ranging from 0.80 to 0.97. The presence of ribosomal RNA peaks in FFPE-derived RNA was reflected by a high correlation with paired frozen samples. A set of practical recommendations for evaluating the RNA integrity and quality in FFPE samples is reported.
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Clapper
Engstrom
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Arif JM, Dresler C, Clapper ML, Gairola CG, Srinivasan C, Lubet RA, Gupta RC. Lung DNA adducts detected in human smokers are unrelated to typical polyaromatic carcinogens. Chem Res Toxicol. 2006 Feb;19(2):295-9.
Several studies have reported the presence of DNA adducts derived from benzo(a)pyrene and other polyaromatics by P-32-postlabeling/TLC by measuring diagonal radioactive zones (DRZs) in lung tissues of human smokers. However, our experimental studies in rodent models, which used modified chromatographic conditions to obtain distinct adduct spots, suggested that cigarette smoke-related lipophilic DNA adducts may not be derived from polycyclic aromatic hydrocarbons (PAHs) or aromatic amines. In the present study, we have performed similar analysis of human lung tissues to study the chemical nature of DNA adducts. Fifty human lung tissues from cancer patients (ages 42-83 years) with active, ex-, or never-smoking status were analyzed for highly lipophilic DNA adducts by nuclease P1- and n-butanol enrichment-mediated (32)p-postlabeling assay. All DNA samples yielded low to highly intense adduct DRZs when adducts were resolved by PEI-cellulose TLC in standard high-salt, high-urea s! olvents. Adduct burden ranged from 6.6 to 2930 per 10(10) nucleotides. However, when adducts were resolved in a different solvent system comprising of high-salt, high-urea in direction 3 and dilute ammonium hydroxide in direction 4, which retained adducts derived from PAHs and aromatic amines on the chromatograms, this yielded no detectable adducts from human lung DNAs. Furthermore, analysis of human lung DNAs mixed with reference adducted DNAs in multisolvent systems confirmed an absence of PAH- and aromatic amine-derived adducts in human smoker lung DNA. To determine the origin of cigarette smoke-associated DNA adducts, calf thymus DNA was incubated with formaldehyde and acetaldehyde, which are known to be present in cigarette smoke in significant quantities. Analysis of purified DNAs by P-32-postlabeling resulted in adduct DRZs in the aldehyde-modified DNAs when adducts were resolved in standard urea-containing solvents, but no adducts were detected when the ammonium hyd! roxide-based solvent was used, suggesting that even nonpolyaro! matic el ectrophiles can result in adduct DRZs on the chromatograms similar to those from PAH metabolites. Taken together, our data demonstrate that cigarette smoke-associated lung DNA adducts appear on chromatograms as DRZs, consistent with the literature, but they are not related to PAHs and aromatic amines.
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Clapper
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Yeung A, Patel B, Li XM, Paterson M, Blagoi E, Seeholzer S, Chen Y, He A, Chaudhry A, Ke E, Coundry R, Cooper H, Bellacosa A, Clapper M, Boman B, Zhang T, Ross E, Litwin S, Conrad P, Crowell J, Kopelovich L, Knudson A. Heterozygous Adenomatous Polyposis Coli mutation in human colon crypt alters the proteome. Mol Cell Proteomics. 2006 Oct;5(10):213.
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Yeung
Clapper
Bellacosa
Knudson
Litwin
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Engstrom P, Clapper M. Prevention of tobacco-related cancers. In: Kufe DW, Bast RC, Hait WN, Hong WG, Pollock RE, Weichselbaum RR, Holland J, Frei E, editors. Cancer Medicine 7. Ontario, Canada: B.C. Dekker, Inc.; 2006. p. 354-62.
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Clapper
Engstrom
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Lakshmi VM, Clapper ML, Chang WC, Zenser TV. Hemin potentiates nitric oxide-mediated nitrosation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline. Chem Res Toxicol. 2005 Mar;18(3):528-35.
Heme has been reported to be an important contributor to endogenous N-nitrosation within the colon and to the enhanced incidence of colon cancer observed with increased intake of red meat. This study uses the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) as a target to evaluate hemin potentiation of nitric oxide (NO)-mediated nitrosation. Formation of C-14-2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) was monitored by HPLC following incubation of 10 mu M IQ with the NO donor spermine NONOate (1.2 mu M NO/min) at pH 7.4 in the presence or absence of hemin. N-NO-IQ formation due to autoxidation of NO was at the limit of detection (0.1 mu M) and increased 22-fold in the presence of 10 mu M hemin and an in situ system for generating H2O2 (glucose oxidase/glucose). A linear increase in N-NO-IQ formation was observed from 1 to 10 mu M hemin. Significant nitrosamine formation occurred at fluxes of NO and H2O2 as low as 0.024 and 0.25 mu M/min, respectiv ely. Potentiation by hemin was not affected by a 400-fold excess flux of H2O2 over NO or a 4.8-fold excess flux of NO over H2O2. Reactive nitrogen species produced by hemin potentiation had a 46-fold greater affinity for IQ than those produced by autoxidation. Azide inhibited autoxidation, suggesting involvement of the nitrosonium ion, NO+. Hemin potentiation was inhibited by NADH, but not azide, suggesting oxidative nitrosylation with NO2center dot or a NO2center dot-like species. IQ and 2,3-diaminonaphthylene were much better targets for nitrosation than the secondary amine morpholine. Apc(min) mice with dextran sulfate sodium-induced colitis demonstrated increased levels of urinary nitrite and nitrate consistent with increased expression of iNOS and NO synthesis. As reported previously, identical conditions increased fecal N-nitroso compounds. Thus, hemin potentiation of NO-mediated nitrosation of heterocyclic amines provides a testable mechanism by which red meat consum ption can generate N-nitroso compounds and initiate colon cancer under inflammatory conditions, such as colitis.
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Clapper
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Raimondi S, Boffetta P, Anttila S, Brockmoller J, Butkiewicz D, Cascorbi I, Clapper ML, Dragani TA, Garte S, Gsur A, Haidinger G, Hirvonen A, Ingelman-Sundberg M, Kalina I, Lan Q, Leoni VP, Le Marchand L, London SJ, Neri M, Povey AC, Rannug A, Reszka E, Ryberg D, Risch A, Romkes M, Ruano-Ravina A, Schoket B, Spinola M, Sugimura H, Wu XF, Taioli E. Metabolic gene polymorphisms and lung cancer risk in non-smokers - An update of the GSEC study. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis. 2005 Dec 30;592(1-2):45-57.
Background: Since genetic factors may play an important role in lung cancer development at low dose carcinogen exposure, non-smokers are a good model to study genetic susceptibility and its interaction with environmental factors.Materials and methods: We evaluated the role of the metabolic gene polymorphisms CYP1A1MsPI, CYP1A11le(462) Val, GSTM1, and GSTT1 in non-smoker lung cancer patients from the International Collaborative Study on Genetic Susceptibility to Environmental Carcinogens (GSEC). Non-smokers (defined as subjects who never smoked on a regular basis) were selected from the GSEC database. We pooled the raw data from 21 case-control studies for a total of 2764 Caucasians (555 cases and 2209 controls) and 383 Asians (113 cases and 270 controls). Tests of heterogeneity and of inclusion bias were performed.Results: A significant association between lung cancer and CYP1A11le(462)Val polymorphism was observed in Caucasians (adjusted OR= 2.04, 95% Cl 1.17-3.54). GSTT1 d eletion seems to be a risk factor for lung cancer in Caucasian non smokers only when the analysis was restricted to studies including healthy controls (adjusted OR= 1.66, 95% Cl 1.12-2.46). A protective effect on lung cancer was observed with the combination of CYP1A1 wild type, GSTM1 null, and GSTT1 non-null genotypes. None of the analysed polymorphisms were associated with lung cancer in Asian non-smokers.Discussion: Our analysis confirms previous findings that CYP1A11le(462) Val polymorphism may play a role in lung carcinogenesis in Caucasian non-smokers. (c) 2005 Elsevier B.V. All rights reserved.
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Clapper
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Chang WC, Everley LC, Pfeiffer GR, Cooper HS, Barusevicius A, Clapper ML. Sulindac sulfone is most effective in modulating beta-catenin-mediated transcription in cells with mutant APC. In: TUMOR PROGRESSION AND THERAPEUTIC RESISTANCE 1059. New York Academy of Sciences; 2005. p. 41-55.
Sulindac sulfone (FGN-1, Aptosyn), a metabolite of the nonsteroidal anti-inflammatory drug sulindac, lacks cyclooxygenase inhibitory activity. Although its ability to inhibit tumorigenesis in both carcinogen-treated animals and patients with familial adenomatous polyposis has been attributed to the induction of apoptosis, its complete mechanism of action remains unclear. The purpose of the present study was to determine the ability of sulindac metabolites to regulate cellular levels of beta-catenin and downstream targets of the adenomatous polyposis coli (APC)/beta-catenin pathway in vitro. Sulindac sulfone was consistently more potent than the sulfide metabolite in all analyses, significantly decreasing the expression of total cellular beta-catenin (50% of control), pro-caspase 3 (49%), cyclin D1 (51%), and PPAR delta (65%) in SW480 cells. No significant alteration in pro-caspase 3 or beta-catenin expression was found in HCA7, LS174, or Caco-2 cells treated with sulindac su! lfone. A dose-dependent reduction in TCF-mediated transcriptional activity was also observed in SW480 cells. These data demonstrate that sulindac sulfone can modulate the APC/beta-catenin pathway in vitro and that its efficacy is dependent upon the mutational status of APC and beta-catenin.
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Clapper
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Cooper HS, Chang WC, Coudry R, Gary MA, Everley L, Spittle CS, Wang H, Litwin S, Clapper ML. Generation of a unique strain of multiple intestinal neoplasia (Apc +/Min-FCCC) mice with significantly increased numbers of colorectal adenomas. Mol Carcinog. 2005;44(1):31-41.
The relevance of the Apc+/Min mouse model in the study of human colorectal cancer remains uncertain due to the predominance of small intestinal adenomas and few, if any, colorectal adenomas. A new strain of Apc +/Min mice (Apc+/Min-FCCC) with significantly greater numbers of colorectal adenomas has been generated and characterized. Male C57BL/6J-Apc+/Min mice (the Jackson Laboratory, Bar Harbor, ME) were crossed with wild-type (Apc+/+) C57BL/ 6J females from an independent colony at this institution (offspring =Apc+/Min-FCCC) and 233 animals were evaluated over 20 generations. In order to determine the contribution of genetics to the enhanced colorectal adenoma phenotype, breeding pairs (Apc+/Min male × wild type female C57BL/6J) were purchased from the Jackson Laboratory and offspring (Apc+/Min-JAX) were maintained in our facility under identical conditions (n = 98). Animals were fed Purina Rodent chow (#5013) diet containing 5% fat. The entire intestinal tract was examined histopathologically in both strains. Both the Apc and Pla2g2a (candidate for Mom1) genes were sequenced and found to be identical for both the Apc+/Min-FCCC and Apc+/Min-JAX mouse strains. The multiplicity of colorectal adenomas in the Apc+/Min-FCCC mice was much higher than reported in the literature and significantly greater than the multiplicity of colorectal adenomas in Apc+/Min-JAX mice maintained in our facility (P = 0.01). Apc+/Min-FCCC had a significantly greater incidence of rectal prolapse (P = 0.02) and small intestinal adenocarcinomas (P = 0.001), and multiplicity of small intestinal adenocarcinomas (P = 0.001) compared to Apc+/Min-JAX mice. Male Apc+/Min-FCCC mice had significantly greater numbers of colorectal adenomas compared to female Apc+/Min-FCCC mice (P = 0.0002), as did male Apc+/Min-JAX mice vs. female Apc+/Min-JAX mice (P < 0.0001). These results allow us to conclude: (1) Apc+/Min-FCCC mice are unique in that they develop significantly greater numbers of colorectal adenomas and small intestinal cancers, and a significantly greater incidence of small intestinal cancers and rectal prolapse than Apc+/Min-JAX mice. (2) This study represents the first report of a significant gender difference in multiplicity of colorectal adenomas. (3) Differences between Apc +/Min-FCCC and Apc+/Min-JAX mice in currently undefined genetic modifiers may contribute to the enhanced colorectal phenotype. (4) The Apc+/Min-FCCC strain is highly suited for the investigation of colorectal neoplastic disease and chemoprevention studies. © 2005 Wiley-Liss, Inc.
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Clapper
Litwin
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Lessin
Clapper
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Lessin
Clapper
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Vineis P, Veglia F, Anttila S, Benhamou S, Clapper ML, Dolzan V, Ryberg D, Hirvonen A, Kremers P, Le Marchand L, Pastorelli R, Rannug A, Romkes M, Schoket B, Strange RC, Garte S, Taioli E. CYP1A1, GSTM1 and GSTT1 polymorphisms and lung cancer: a pooled analysis of gene-gene interactions. Biomarkers. 2004 May-Jun;9(3):298-305.
Gene - environment interactions have been extensively studied in lung cancer. It is likely that several genetic polymorphisms cooperate in increasing the individual risk. Therefore, the study of gene - gene interactions might be important to identify high-susceptibility subgroups. GSEC is an initiative aimed at collecting available data sets on metabolic polymorphisms and the risks of cancer at several sites and performing pooled analyses of the original data. Authors of published papers have provided original data sets. The present paper refers to gene - gene interactions in lung cancer and considers three polymorphisms in three metabolic genes: CYP1A1, GSTM1 and GSTT1. The present analyses compare the gene - gene interactions of the CYP1A1 2A, GSTM1 and GSTT1 polymorphisms from studies on lung cancer conducted in Europe and the USA between 1991 and 2000. Only Caucasians have been included. The data set includes 1466 cases and 1488 controls. The only clear-cut association was found with CYP1A1 2A. This association remained unchanged after stratification by polymorphisms in other genes (with an odds ratio [OR] of approximately 2.5), except when interaction with GSTM1 was considered. When the OR for CYP1A1 2A was stratified according to the GSTM1 genotype, the OR was increased only among the subjects who had the null ( homozygous deletion) GSTM1 genotype ( OR = 2.8, 95% CI = 0.9 - 8.4). The odds ratio for the interactive term ( CYP1A1 2A by GSTM1) in logistic regression was 2.7 (95% CI = 0.5 - 15.3). An association between lung cancer and the homozygous CYP1A1 2A genotype is confirmed. An apparent and biologically plausible interaction is suggested between this genotype and GSTM1.
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Clapper
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Clapper
Litwin
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Clapper ML, Coudry J, Chang WC. beta-catenin-mediated signaling: a molecular target for early chemopreventive intervention. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis. 2004 Nov 2;555(1-2):97-105.
Dysregulation of Wnt signaling appears to be a critical event in the formation of intestinal tumors and some other cancers. Accumulating data from preclinical studies strongly suggest that targeted disruption of P-catenin-mediated TCF signaling is a promising strategy for early chemopreventive intervention, particularly with respect to intestinal tumorigenesis. While the search for potent inhibitors is just getting underway, the ability of several synthetic and naturally occurring agents to decrease the transcriptional activity of a luciferase reporter plasmid under the control of TCF-4 regulatory elements (pTOPFLASH) has been demonstrated already. Additional enthusiasm for this approach is provided by data from several groups, which indicate that sulindac, sulindac sulfone and indomethacin can modulate the subcellular localization of beta-catenin in vivo, resulting in either decreased nuclear compartmentalization or enhanced localization of beta-catenin to the plasma membra ne. Although the mechanism by which agents disrupt beta-catenin-mediated TCF signaling remains to be elucidated, possibilities include: (1) physical inhibition of the beta-catenin/TCF complex formation, (2) upregulation of the ubiquitin-mediated proteosomal degradation of P-catenin, (3) accelerated nuclear export of beta-catenin and (4) enhanced sequestration of P-catenin by E-cadherin. The common role of P-catenin in both Wnt signaling and cell adhesion provides a unique opportunity to develop chemopreventive therapies that both prevent the development of cancer and delay tumor progression. (C) 2004 Elsevier B.V. All rights reserved.
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Clapper
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Stoyanova R, Clapper ML, Bellacosa A, Henske EP, Testa JR, Ross EA, Yeung AT, Nicolas E, Tsichlis N, Li YS, Linehan WM, Howard S, Campbell KS, Godwin AK, Boman BM, Crowell JA, Kopelovich L, Knudson AG. Altered gene expression in phenotypically normal renal cells from carriers of tumor suppressor gene mutations. Cancer Biol Ther. 2004 Dec;3(12):1313-21.
BACKGROUND: The inherently complex signaling networks of tumors result from genetic and epigenetic alterations that occur during cancer initiation and progression. METHODS: In an attempt to identify early molecular changes associated with dominantly inherited predisposition to "two-hit" renal tumors, the expression profiles of primary cultures of phenotypically normal renal epithelial cells from individuals bearing a germline mutation in either the von Hippel-Lindau (VHL) or the tuberous sclerosis complex (TSC) gene were compared to that of renal epithelial cells from control nonmutation carriers by microarray analysis. RESULTS: Reliability of the microarray data from pooled samples was confirmed by real-time RT-PCR. Principal Component Analysis revealed substantial differences in the gene expression profiles of the renal epithelial cells from VHL and TSC mutation carriers. In several instances, the microarray data confirm our present knowledge of the cellular pathways affected by biallelic VHL and TSC mutations. CONCLUSIONS: These findings demonstrate that heterozygosity for a mutant tumor suppressor gene may alter the expression profiles of phenotypically normal epithelial cells in a gene-specific manner. Detectable effects of "one-hit" represent early molecular changes in tumorigenesis that may serve as targets for chemopreventive intervention.
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Yeung
Campbell
Testa
Godwin
Henske
Clapper
Bellacosa
Knudson
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Stoyanova R, Upson JJ, Patriotis C, Ross EA, Henske EP, Datta K, Boman B, Clapper ML, Knudson AG, Bellacosa A. Use of RNA amplification in the optimal characterization of global gene expression using cDNA microarrays. J Cell Physiol. 2004 Dec;201(3):359-65.
Microarray analysis of human tissue is frequently hindered by the limited amount of RNA available. Although amplification protocols can be utilized, the relative representation of transcripts present in the starting material must remain unaltered. In this study, 200 ng of total RNA derived from cultured renal epithelial cells from tuberous sclerosis complex (TSC) carriers and control individuals was amplified by in vitro transcription with T7 RNA polymerase. The resulting Cy-labeled cDNAs (from total or amplified RNA (aRNA)) were analyzed as direct replicates and dye-flips on slides containing 10,000 human cDNAs. The Pearson correlation coefficients for the direct replicate experiments were 0.80 (20 microg total RNA), 0.85 (40 microg total RNA), and 0.93 (2 microg of aRNA). Comparisons between the array data revealed that the majority of genes expressed in total RNA (97% for 20 microg and 85% for 40 microg) were also detected in aRNA. The correlation coefficient of the expression ratios for genes detected in both total RNA (40 microg) and aRNA was 0.63. Further, Student's t-test indicated no significant difference (P = 0.83) between these ratios. These results indicate that the number of expressed genes detected with total RNA is proportional to the amount of RNA used and underscore the requirement of large amounts of total RNA for a comprehensive characterization of gene expression profiles. RNA amplification allows the detection of a large number of genes expressed in the starting RNA population without altering their relative intensities significantly. Thus, an RNA amplification step improves the quality of gene expression results obtained by microarray analysis. This study indicates that high quality microarray data can be generated from small amounts of RNA, including those extracted from limiting clinical samples and microdissected histological specimens.
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Henske
Clapper
Bellacosa
Knudson
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Hensley HH, Chang WC, Clapper ML. Detection and volume determination of colonic tumors in Min mice by magnetic resonance micro-imaging. Magn Reson Med. 2004 Sep;52(3):524-9.
We applied MRI to the in vivo detection of spontaneous colorectal tumors in a unique mouse model, the Fox Chase Cancer Center (FCCC) Apc(MIN) mouse. Unlike other Min (multiple intestinal neoplasia) strains, FCCC Apc(MIN) animals develop an appreciable number of tumors in the large intestine, which makes them an appropriate mouse model for colon cancer in humans. We describe a method for marking the colon on MRI data sets that involves a bowel-cleansing procedure and the insertion of a polyurethane tube (filled with an MRI contrast agent) fully into the colon. We found that tumors as small as 1.5 mm in diameter can be consistently identified from MRI datasets with a voxel size of 0.1 mm x 0.133 mm x 0.133 mm. Tumor volumes were determined from the MRM data sets with the use of a novel approach to planimetry in 3D data sets. We observed a correlation between tumor volume (as measured from the MRI datasets) and tumor weight of 0.942, and a P-value of 0.008, based on Spearman's test. These data show that MRI can be used to accurately monitor tumor growth in mouse models of colorectal carcinogenesis. (C) 2004 Wiley-Liss, Inc.
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Clapper
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Smits KM, Benhamou S, Garte S, Weijenberg MP, Alamanos Y, Ambrosone C, Autrup H, Autrup JL, Baranova H, Bathum L, Boffetta P, Bouchardy C, Brockmoller J, Butkiewicz D, Cascorbi I, Clapper ML, Coutelle C, Daly AK, Muzi G, Dolzan V, Duzhak TG, Farker K, Golka K, Haugen A, Hein DW, Hildesheim A, Hirvonen A, Hsieh LL, Ingelman-Sundberg M, Kalina I, Kang D, Katoh T, Kihara M, Ono-Kihara M, Kim H, Kiyohara C, Kremers P, Lazarus P, Le Marchand L, Lechner MC, London S, Manni JJ, Maugard CM, Morgan GJ, Morita S, Nazar-Stewart V, Kristensen VN, Oda Y, Parl FF, Peters WH, Rannug A, Rebbeck T, Pinto LF, Risch A, Romkes M, Salagovic J, Schoket B, Seidegard J, Shields PG, Sim E, Sinnett D, Strange RC, Stucker I, Sugimura H, To-Figueras J, Vineis P, Yu MC, Zheng W, Pedotti P, Taioli E. Association of metabolic gene polymorphisms with tobacco consumption in healthy controls. Int J Cancer. 2004 Jun;110(2):266-70.
Polymorphisms in genes that encode for metabolic enzymes have been associated with variations in enzyme activity between individuals. Such variations could be associated with differences in individual exposure to carcinogens that are metabolized by these genes. In this study, we examine the association between polymorphisms in several metabolic genes and the consumption of tobacco in a large sample of healthy individuals. The database of the International Collaborative Study on Genetic Susceptibility to Environmental Carcinogens was used. All the individuals who were controls from the case-control studies included in the data set with information on smoking habits and on genetic polymorphisms were selected (n = 20,938). Sufficient information was available on the following genes that are involved in the metabolism of tobacco smoke constituents: CYPIAI, GSTMI, GSTTI, NAT2 and GSTPI. None of the tested genes was clearly associated with smoking behavior. Information on smoking ! dose, available for a subset of subjects, showed no effect of metabolic gene polymorphisms on the amount of smoking. No association between polymorphisms in the genes studied and tobacco consumption was observed; therefore, no effect of these genes on smoking behavior should be expected. (C) 2004 Wiley-Liss, Inc.
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Clapper
Yu
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