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in walking (60%), outdoor sports (59%) and skill-based sports (37%) than in other activities, while more women participated in walking (81%), gym- ...

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• Publié par: Sportif
• Nombre de lectures: 78
• Langue: Français

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Poster Session B

Carcinogenesis

Animal Models of Carcinogenesis and Chemoprevention

B1 Effects of freeze-dried 5% black raspberry diet on early molecular events in N-Nitrosomethylbenzylamine-induced cytotoxicity of rat esophagus.J. F. Lechner, 1 R. K. Reen, 1 A. A. Dombkowski, 2 D. Cukovic, 2 S. Salagrama, 2 G. D. Stoner 1 . 1 Ohio State University Comprehensive Cancer Center, Columbus, OH, 2 Institute of Environmental Health Sciences, Wayne State University, Detroit, MI. Our recent report (Reen RK, et al. Cancer Res 2007;67:6484-92) described 2261 changes in gene expressions in esophagi from rats that received a short-term exposure to the carcinogen, N- Nitrosomethylbenzylamine (NMBA). We also identified 1323 genes that were differentially modulated to near-normal levels of expression by co- treatment of rats with phenylethylisothiocyanate (PEITC). Herein we report a companion study wherein the rats were fed AIN-76A diet or AIN-76A diet containing 5% freeze-dried black raspberries (BRB) for three weeks. During the third week one-half of the diet-only animals and one-half of the BRB-diet animals received three s.c. doses of NMBA (0.5 mg/kg b.w.). All animals were sacrificed 24h after the last dose of NMBA. Esophagi were rapidly excised and both processed for histological grading and extracted for RNA for microarray profiling. Histopathological analysis showed that treatment of rats with BRB had a protective effect on NMBA-induced lesions in the rat esophagus. We identified 24 genes that were up- regulated by BRB alone, and although the specimens were carefully stripped of the submucosal and muscularis layers before extracting their RNA, 10 of these are associated with muscle and/or contraction processes. Twelve genes were down-regulated by the BRB diet only, and two of these, Rbbp6 and RGD1564921_predicted, were also down-regulated by PEITC alone. We found that 259 of the 2261 genes affected by NMBA were expressed at near control levels (± 33%) in the esophagi of rats treated with both 5% BRB and NMBA. Fifty of these genes were also restored to control levels of transcription by PEITC. Overall, the results of our study indicate that BRB-treatment had a genome-wide modulating effect on NMBA-caused dysregulation of gene expression, including genes involved in phase I and phase II metabolism, oxidative damage, apoptosis, cell- cycling, and angiogenesis. Supported by NCI grants CA096130 and CA103180 to GDS. B2 Preventive effects of the RXR agonists targretin and UAB-30 in transgenic mouse models of estrogen receptor (ER) negative mammary cancer.R. A. Lubet, 1 J. Ruppert, 2 D. D. Muccio, 2 M. You, 3 W. J. Brouilette, 2 R. Atigadda, 2 V. E. Steele, 1 M. Juliana, 2 C. J. Grubbs 2 . 1 National Cancer Institute, Bethesda, MD, 2 University of Alabama at Birmingham, Birmingham, AL, 3 Washington University, St. Louis, MO. Two different RXR agonists were examined for their ability to inhibit the formation of ER negative mammary cancers in transgenic mice. Targretin is clinically employed in the treatment of cutaneous lymphoma, and has as its primary side effect an increase in triglycerides levels. UAB-30 is a new RXR agonist (synthesized by modifying 9-cis-retinoic acid) that does not cause an increase in serum triglycerides. MMTV-Neu female mice were initially treated with dimethybenzanthracence (DMBA), 1.0 mg/mouse/week by gavage for 4 weeks. Beginning five days after the last DMBA treatment, diets were supplemented either with targretin (250 mg/kg diet) or UAB-30 (300 mg/kg diet). At 105 days after the last DMBA treatment, Targretin and UAB-30 reduced the multiplicity of mammary cancers by 72 and 61%, respectively (controls had a multiplicity of 5.7 cancers/mouse). Thus, both agonists were relatively effective. In addition, the preventive efficacy of Targretin (250 mg/kg diet), initiated either early (10 weeks of age) or late (24 weeks of age), was evaluated in female mice that were heterozygous both for MMTV-Neu and KO of the p53 tumor suppressor gene. Treatment with Targretin early or late reduced mammary cancer incidence by 46 and 16% and cancer multiplicity by 69 and 56%, respectively. In contrast, Targretin failed to inhibit the formation of lymphomas or sarcomas; two cancers that often develop in these

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Carcinogenesis

Animal Models of Carcinogenesis and Chemoprevention

bitransgenic mice. In summary, both agents which were previously shown to be effective in inhibiting ER positive rat mammary cancers were also effective in inhibiting ER negative mammary cancers. Furthermore, it appears that these agents are effective even if treatment is delayed to a point when mammary lesions already exist.

B3 Cross-sectional analysis of intermittent versus chronic caloric restriction in the TRAMP mouse model of prostate cancer.M. J.L. Bonorden, 1 O. P. Rogozina, 1 C. M. Kluczny, 1 M. E. Grossmann, 1 J. P. Grande, 2 M. P. Cleary 1 . 1 Hormel Institute, Austin, MN, 2 Mayo Foundation, Rochester, MN. Caloric restriction has been shown to prevent the development of cancer in rodent models. With respect to prostate cancer Lobund-Wistar rats subjected to 30% chronic caloric restriction (CCR) developed prostate adenocarcinoma at lower rates (Cancer 64: 686-690, 1989) and had increased survival and lower rates of prostatitis compared to those fed ad libitum (AL) (J Gerontol 45: B52-58, 1990). When CCR and intermittent caloric restriction (ICR) were compared in two mouse transgenic mammary tumor models, tumor incidence was dramatically decreased in ICR mice compared to CCR or ad libitum (AL) fed mice (Cancer Epidemiol Biomarkers Prev 11: 836-843,2002; Nutr Cancer 44: 162-168, 2002). Recently we found that transgenic adenocarcinoma of the mouse prostate (TRAMP) mice undergoing ICR had extended tumor latency and survival times compared to AL or CCR mice (submitted manuscript). Here, we used TRAMP mice to provide a cross-sectional perspective of the protective mechanisms of ICR vs CCR during the development of prostate cancer. Male C57BL6 TRAMP mice were assigned to 1) AL (free access to AIN-93M diet), 2) ICR (2-wk of 50% caloric restriction using AIN-93M diet with 2x protein, fat, vitamins, and minerals followed by 2-wk of 100% AL consumption of AIN-93M for each corresponding 2-wk), or 3) CCR (fed a diet mixture to match calorie and nutritent intake for each four week ICR cycle which equaled ~75% of AL consumption) groups. Protocols were initiated at 7 wk of age and mice were sacrificed at predetermined ages of 16, 18, 28, 30, 40 and 42 weeks. The two-week separation between time points in a cycle distinguished mice euthanized at the end of a restriction period (ICR-Rest) and those euthanized at the end of a refeeding period (ICR-Refed) within the ICR cohort. As expected, body weights fluctuated in response to calorie intake and were significantly different among the dietary groups (P<0.0001). Final body weights were significantly different among the groups at 16-18 (P<0.0001) and 40-42 (P<0.0054) weeks. A similar trend was found for urogenital tract weights at 16-18 (P<0.0001) and 40-42 (P<0.0003) weeks with ICR-Rest signficantly lower than AL and CCR at both time points. At 16-18 weeks, adenocarcinoma was pathologically confirmed in 0, 12.5, 24 and 33% of the ICR-Rest, ICR- Refed, AL and CCR groups, respectively. At 28-30 weeks, 86% of ICR-Rest mice had adenocarcinoma while all other groups reached 100%; by the 40-42 week time point all mice had confirmed urogenital adenocarcinoma. ICR-Rest mice had significantly lower serum leptin than AL and CCR mice at 16-18 weeks (P<0.05). In contrast ICR mice had the highest serum adiponectin levels across all ages, which reached significance at 28-30 and 40-42 weeks of age. Western blot analysis of prostate tumor tissue for cleaved PARP, PCNA, Bax, Bcl-2 and Bax: Bcl-2 ratio did not show any consistent pattern and no significant differences were found among the groups (P=0.7120, 0.1173, 0.6410, 0.5444 and 0.4698, respectively). Our results show ICR can significantly reduce urogenital tract weight and alter concentrations of serum lepin and adiponectin at various time points in the TRAMP mouse, which may, in part, explain the protective effect ICR. (Support: DAMD17-03-1-0258 and Hormel Foundation)

American Association for Cancer Research

Carcinogenesis

Animal Models of Carcinogenesis and Chemoprevention

B4 Protective effect of intermittent versus chronic calorie restriction on mammary tumor development in relationship to prospective IGF-1 serum concentrations in MMTV-TGF-αmice. O. P. Rogozina, 1 M. J.L. Bonorden, 1 E. J. Kain Quealy, 1 J. P. Grande, 2 M. P. Cleary 1 . 1 Hormel Institute, University of Minnesota, Austin, MN, 2 Mayo Foundation, Rochester, MN. Numerous studies indicate that chronic caloric restriction (CCR) provides a protective effect on chemically-induced and spontaneous mammary tumor (MT) development in rodents. Recently, intermittent caloric restriction (ICR) was reported to be more protective than CCR in transgenic mouse models that develop MTs. One growth factor considered to mediate tumorigenesis is IGF-I. Here we compare prospective serum IGF-I levels in relationship to mammary tumorigenesis for these two modes of calorie restriction. At 8 wk of age 225 MMTV-TGF-αfemale mice had ad libitum access to AIN-93M diet. At 10 wk they were assigned to AL (ad libitum AIN-93M diet), ICR (3-wk of 50% caloric restriction of AIN-93M-mod diet with 2x protein, fat, vitamins and minerals followed by 3-wk of 100% AL mice’s consumption of AIN-93M), and CCR (fed a diet to match calorie and nutrient intake for each 6-wk ICR cycle, ~ 75% of AL consumption) groups. Food intakes were determined daily. Body weights, palpation and tumor measurements were done weekly. Mice were followed until MT burden necessitated euthanasia or they reached terminal endpoints of 79 (end of restriction) or 82 (end of refeeding) weeks of age. Serum samples were obtained at euthansia and in cycles 1, 3, 5, 8, and 11 from cohorts corresponding to the 1st, 2nd and 3rd weeks of restriction and refeeding for ICR mice to relate levels of IGF-1 to the appearance of palpable MT. Cumulative food intakes for ICR and CCR mice were 24.8% and 22.6% lower, than AL mice (p<0.05). Body weights fluctuated in response to calorie intake during the study for ICR mice and there was an overall significant difference among the groups. Final body weights, mammary and internal fat pad weights of AL mice were heaviest, followed by CCR and IR-refed mice with similar weights (p>0.05) while ICR-restricted mice weights were the lowest. 68.3% of AL mice developed 1-6 MTs/mouse (grade 1-3), 36.2% of CCR mice had 1-2 MTs/mouse (grade 1-3) and only 8.2% of ICR mice developed 1 MT/mouse (grade 1-2). There were no differences for time to tumor detection and MT weights among the groups. Terminal serum IGF-1 concentration of AL mice was higher (p<0.05), than for CCR, ICR-refed, and ICR-restricted mice, which had lower (p<0.05) IGF-I than CCR and ICR-refed mice. There was a positive correlation between terminal IGF-1 and body weights (r=0.95) and mammary fad pad weights (r=0.95) for all mice. IGF-1 serum concentrations across the study were higher prior to MT detection from the earliest time point for AL and CCR mice. However, this was not found for ICR mice. These results confirm that ICR provides greater protection compared to CCR with respect to prevention of MT development. Additionally, tumor burden and aggressiveness were reduced in ICR mice. The IGF-1 axis appears to be involved in MT development but its specific role in the protective effect of calorie restriction remains to be determined. It is anticipated that ongoing mammary fat pad and MT analyses will provide additional insights into why ICR is superior to CCR with respect to prevention of mammary tumorigenesis. (Support: NIH CA 101858 and The Hormel Foundation). B5 Chemo preventive efficacy of tinospora cordifolia (a medicinal plant) against chemical induced skin papillomagenesis in mice. P. Goyal, R. Chaudhary, S. Jahan, U. Gupta. University of Rajasthan, Jaipur, Jaipur, India. Cancer chemoprevention is defined as the use of natural, synthetic, or biologic chemical agents to reverse, suppress, or prevent carcinogenic progression to invasive cancer. The success of several recent clinical trials in preventing cancer in high-risk populations suggests that chemoprevention is a rational and appealing strategy. Recently, several isolated plant products and crude extracts that have a combination of various bioactive molecules capable of cancer prevention through different mechanisms have been investigated. Tinospora cordifolia (Family- Menispermaceae) is a glabrous, succulent,

Poster Session B

climbing shrub widely used in medicine for treatment of different ailments such as jaundice, diabetes, rheumatoid arthritis, gout, skin diseases and infections. Furthermore, this plant exhibits certain mechanisms under different experimental conditions such as free radical scavenging, calcium channel blocking, inhibition of lipid peroxidation, enhancement of DNA repair and stimulation of stem cell proliferation that are required for chemoprevention. For this purpose, the inhibition of skin papillomas by root extract of this plant (TCE) has been evaluated on two-stage process of skin carcinogenesis in Swiss albino mice, induced by a single application of 7, 12-dimethylabenz (a) anthracene (100µg/100µl of acetone) and two weeks later promoted by repeated application of croton oil (1% in acetone/thrice a week) till the end of the experiment i.e 16 weeks. The tumor incidence, tumor yield, tumor burden and cumulative number of papillomas were found to be higher in the DMBA treated control as compared to TCE treated experimental animals. The difference in the values of above parameters of both the groups were statistically analyzed and found to be significant. Thus, the present study demonstrates the anti-tumor activity of this plant and its possible use in the field of chemoprevention. B6 Prevention of MAPK pathway by Brazilian medicinal plant, Tabebuia avellanedae on peroxynitrite induced carcinogenesis.H. Tokuda, 1 A. Iida 2 . 1 Kyoto Prefectural University of Medicine, Kyoto, Japan, 2 Takasaki University of Welfare and Health, Takasaki, Japan. The present study was carried out to examine the chemopreventive activity of natural Brazilian medicinal plant, Tabebuia avellanedae ext. and its components on peroxynitrite induced carcinogenesis. Tabebuia avellanedae,(TA) which is a plant that has been used for herbal medicine in South America and from Brazil to northern Argentina, is well known in traditional folk medicne used for the treatment of various disease during five hundred years. The inner bark of this plant produced in Brazil is distributed in Asia as a herb tea and healthy purpose. On the fundamental findings, several studies have suggested that these compounds, anti- oxidatives were observed the inhibitory effect against chemical carcinogenesis induced tumor initiating and promoting activity using two- stage mouse skin model. In the course of these studies, female SENCAR mouse (6 weeks of age) were treated topically with single dose of PN solution, followed by TPA twice a weekly for 20 weeks. Tumor incidence were 100% with 6 to 7 per mouse at end of experiment as positive control group. TA powder were orally fed with drinking water for only 2 weeks, before and after initiation and following promoting treatment with drinking water only, as test compounds. In our observation, TA and its components treated group cause about 60-70 % reduction in the average number of tumors per mouse after 20 weeks of experiment, respectively. Topical administration of TA and its components had much influence against PN induced expression stage. We postulate that these data suggeste possible role of a regulatory mechanism of chemopreventive activity in PN induced carcinogenesis. Employing Western blot analysis studies, we found that H-Ras, MEK-2 and p38 levels observed the effects against PN induced activation, and more detail Western blot analysis indicated a active decrease in p38 expression in the skin after TA treatment. Summary of our findings, we suggest that one target of TA and its component effects in mouse skin is the modulation or regulation of the MAPK signaling. B7 Chemoprevention of oral cancer in hamster cheek pouch by topical application of lyophilized black raspberries.B. C. Casto, B. M. Warner, T. J. Knobloch, Z. Yu, C. M. Weghorst. Ohio State University, Columbus, OH. Introduction and Purpose:Oral cancers represent 2.3% of US cancers and account for a sixth of the worldwide cancer burden. Oral cancer is directly linked to tobacco exposure and has a poor prognosis. 5- and 10- year survival rates are 59% and 48%, respectively, and have not changed dramatically over the past three decades. 20%-40% of patients will develop a locoregional recurrence or a second primary tumor. Since current treatments are relatively ineffective for long-term survival, it appears

Frontiers in Cancer Prevention Research • December 5-8, 2007 • Philadelphia, PA

121

Poster Session B

germane to develop and evaluate novel strategies for prevention of secondary tumors and inhibition of primary tumor development.. An animal model that mimics the major characteristics of human oral cancer is the hamster cheek pouch (HCP); a model has been used to evaluate chemoprevention by natural and synthetic food products. In the current study, we examined the ability of topically applied LBR to inhibit the progression of established premalignant lesions into malignant oral cancer. This method of administration mimics a delivery system that is amenable to humans for prevention of primary tumors or recurrence. Methods:Lyophilized black raspberries (LBR) were suspended to a 10% concentration in a 1:1 mixture of saliva substitute and 2% methylcellulose. Premalignant lesions were established by application of 0.2% 7,12- dimethylbenz(a)anthracene (DMBA) in DMSO to both HCPs for 6 weeks at which time premalignant lesions were present. One cheek pouch of each hamster was treated topically with 0.1 ml of LBR suspension, 3x per week, for six weeks; the contralateral pouch of the same animal was treated with 0.1 ml of the vehicle. Animals were sacrificed and examined at 12 weeks to determine the effect of berries on tumor incidence and multiplicity. To evaluate the effect of LBR on early premalignant oral lesions, treated and control cheek pouches containing carcinogen-induced lesions were harvested at 0, 2, 4, 7, 10, and 14 days after berry administration. mRNA was isolated from HCP and relative expression of cancer-relevant genes was determined using real-time PCR. Results:Treatment of hamsters with the carcinogen or berries did not affect weight gain or food consumption. LBR administration inhibited tumor formation by 37% and 42% in separate animal experiments using the same lot of berries. After 7 days of LBR treatment of HCP, Bax, Ccnb2, Cdk2na(p13-ARF), Cdk2na(p16), and p53 showed increasing expression, whereas Cdk2 and Bcl2, showed decreasing expression; the expression of c-myc and Vegf remained constant. Conclusions:These experiments provide evidence of prevention of oral cancer by a natural food product under conditions that mimic those present in former tobacco users and an assessment of the ability to modulate gene expression and inhibit oral cancer development when given in a delivery mode that is suitable for administration to the human oral mucosa. Acknowledgements.Grant support:Prevent Cancer Foundation and OSU Comprehensive Cancer Center, MCC Program. B8 Rapamycin prevents tobacco-carcinogen induced lung tumorigenesis and selectively reduces lung-associated Foxp3 positive cells.C. A. Granville, 1 J. Mariotti, 2 S. Kawabata, 1 J. Foley, 2 M. Christine Hollander, 1 W. Han, 1 A. Balogh, 1 R. Memmott, 1 J. LoPiccolo, 1 D. Liewehr, 2 S. Steinberg, 2 D. H. Fowler, 2 P. A. Dennis 1 . 1 NCI/NNMC, Bethesda, MD, 2 NCI, Bethesda, MD. Inhibitors of the serine/threonine kinase mTOR can prevent tumorigenesis in many model systems, but the use of mTOR inhibitors for cancer prevention in humans is theoretically limited by the risk of secondary malignancies, which was observed in patients receiving rapamycin as part of multi-drug immunosuppressive therapy. However, little is known about immune modulation by rapamycin as a single agent. Previously, we showed that physiologic concentrations of rapamycin inhibited lung tumorigenesis driven by a tobacco-specific carcinogen, NNK. Here we characterize immune modulation in this system. NNK alone acutely depleted multiple lineages of immune splenic cells without suppressing immune function. In contrast to these nominal systemic effects, NNK doubled the percentage of cells expressing the Treg marker Foxp3 in lung tissues prior to tumor development and caused a dose- dependent increase in tumor-infiltrating Foxp3+ cells at 12 weeks. NNK administration did not increase the overall number of CD3+ cells, suggesting that the effect of NNK was specific for Foxp3+ cells. Rapamycin rapidly decreased splenic Foxp3+/CD4+ cells, and was associated with depletion of Foxp3+ cells in lung tissues. At 12 wk, rapamycin inhibited tumorigenesis, decreased the number of tumor-associated Foxp3+ cells by 88%, modestly decreased systemic CD4+ and CD8+ cells, and did not induce an immunosuppressive Th2 phenotype. Thus, single-agent

122

Carcinogenesis

Animal Models of Carcinogenesis and Chemoprevention

rapamycin prevents lung infiltration of Foxp3+ cells that occurs early in tumorigenesis without causing profound immunosuppression. B9 Gene expression analysis of oral cancer chemoprevention by

lyophilized strawberries in DMBA-induced hamster cheek pouch.B. M. Warner, 1 B. C. Casto, 1 Z. Yu, 2 T. J. Knobloch, 1 C. M. Weghorst 1 . 1 The Ohio State University, College of Public Health and the Comprehensive Cancer Center, Columbus, OH, 2 The Ohio State University, College of Public Health, Division of Biostatistics, Columbus, OH. Oral cancers represent 2.5-3.0% of all cancers worldwide. Prognosis for oral cancers is poor with 5- and 10-year survival rates of 59% and 44%, respectively. Even with advances in treatment, these survival rates have not changed dramatically in three decades. Since current long-term treatments are relatively ineffective, it is important to develop novel strategies for the prevention of oral cancer.Chemoprevention by natural food products, e.g., black raspberries, has been demonstrated to be a viable approach for aerodigestive cancer prevention.The hamster cheek pouch (HCP) model was used to evaluate administration of lyophilized strawberries (LS) during the initiation and progression of oral cancer induced by 7,12-dimethylbenz(a)anthracene (DMBA). LS were incorporated into AIN-76A pellets at 5% and 10%, and fed to hamsters before, during, and after carcinogen treatment (Complete Chemoprevention Assay; CC), or after establishment of premalignant lesions (Post-initiation Assay; PI). Animals were sacrificed at 12 weeks and examined for total lesions, and tumor incidence and multiplicity. HCP tissues were excised and quick frozen for RNA isolation. Using Real-Time RT-PCR, we analyzed gene expression in apoptosis (Bcl2, Bax), angiogenesis (Vegfa), and proliferation (Myc, Ccnb2, Cdk2, Psmd10) pathways, as well as in select tumor suppressor genes (Cdkn2ap16, Cdkn2ap13-ARF, Trp53) in HCP tissues.LS at 5% and 10% inhibited tumor formation by 28% (p<0.05) and 41% (p<0.01) in the CC Assay, and 54% (p<0.01) and 36% (p<0.01) in the PI Assay, respectively. Total lesions (leukoplakias, papillomas, tumors) were inhibited in both assays. Histological analysis of HCPs demonstrated that 10% LS in the CC Assay inhibited moderate and severe dysplasias by 31% (p<0.01) and 34% (p<0.01), respectively. With respect to untreated HCP, DMBA-initiated HCP significantly increased Cdkn2ap16 (34-fold), Cdkn2ap13-ARF (23-fold), Bcl2 (3-fold), Ccnb2 (3-fold), Trp53 (3-fold), and Myc (1.4-fold) expression. In contrast, only Psmd10 (2-fold) demonstrated significantly decreased gene expression (p<0.000 for all genes). Conversely, with respect to DMBA-initiated HCP, DMBA + 5% LS-treatment in the PI Assay resulted in decreased Cdkn2ap16 (2-fold) and Cdkn2ap13- ARF (2-fold) expression, and increased Psmd10 (1.3-fold) expression (p<0.003 for all genes). These findings demonstrate that 5% LS in the PI Assay significantly attenuate cancer gene expression towards a more “normal” expression pattern.These experiments provide strong evidence that LS prevent oral tumorigenesis under conditions mimicking those in former tobacco users. Furthermore, this study extends our data supporting the ability of natural food products to modulate oral carcinogenesis when delivered in a mode directly amenable to the human oral cavity. Translation of preclinical oral cancer chemoprevention data into patient intervention strategies is essential. Ongoing human clinical trials targeting oral cancer prevention and recurrence will further define the utility of LS chemoprevention. B10 Rats with late stage precancerous esophageal lesions develop fewer papillomas if fed a diet of freeze-dried black raspberries.L. Wang, C. M. Rocha, N. Zikri, C. M. McIntyre, J. F. Lechner, G. D. Stoner. Ohio State University, Columbus, OH. Aim:Our lab has shown that rats treated for five weeks with NMBA develop ~ 12 tumors per esophagus 30 weeks after cessation of carcinogen. We have also shown that a diet of black raspberries (BRB) during the 30 weeks after carcinogen treatment reduces the development of tumors to ~ four per esophagus. This study was conducted to examine (1) whether BRB is effective when administered from 10 to 30 weeks after NMBA treatment, when the esophagi contain several pre-cancerous lesions, (2) alternatively, if the BRB diet was replaced at 15 weeks with control diet how many tumors per esophagus would develop.

American Association for Cancer Research

Carcinogenesis

Animal Models of Carcinogenesis and Chemoprevention

Methods:Five-week-old male Fisher-344 rats were administered an AIN-76A control diet and injected subcutaneously with 0.3 mg/kg bw NMBA three times a week for five weeks. Starting from week six (denoted as week one of prevention treatment), the rats were randomly assigned to 4 groups. The first group of the rats continuously received AIN-76A diet. The second and third groups received a diet of 10% BRB. In the third group, the BRB treatment was replaced with AIN-76A control diet after nine weeks. In the fourth group, which received AIN-76A control, the diet was switched to the BRB diet at ten weeks. At 30 weeks post-carcinogen exposure, all rats were sacrificed and tumor incidence, number and volumes were determined. Results:Esophageal tumor incidences and volumes in NMBA-treated rats were not influenced by any of the berry treatments. The NMBA-only rats had 12+ tumors per esophagus. The whole term BRB treated animals had 70% fewer tumors per esophagus. The third group of animals, that were switched to control diet at ten weeks, exhibited no significant inhibition of tumor number. However, the animals that received the BRB diet after 9 weeks showed a 60% reduction in the number of tumors per esophagus. Conclusion:Our results suggest that continuously daily consumption of BRB is the best way to the prevention of esophageal tumors in individuals at high risk. Furthermore, a BRB diet has significant therapeutic value in inhibiting conversion of late stage pre-disease into tumors. Supported by NCI grants CA096130 and CA103180 to GDS. B11 Potential of selected dietary oils in azoxymethane induced colon cancer.M. Verghese, J. A. Boateng, R. Field, V. Panala, D. Williams, L. Shackelford, D. Asiamah, L. T. Walker, A. S. Clisby. Alabama A&M University, Normal, AL. Dietary fat, depending on the source, (i.e. Animal or vegetable), quantity and fatty acid composition (saturated, monounsaturated and polyunsaturated), may have implications in colon cancer. The objective of this study was to compared the inhibitory effects of red palm oil (RPO), wheat germ oil (WGO), rice bran oil (RBO), flaxseed oil (FSO), corn oil (CO) and soybean oil (SBO) at 7% (normal fat level ) and 14% (high fat level) on Azoxymethane (AOM) induced aberrant crypt foci (ACF). We also determined the long term effects (end point tumor study (EPT)) of dietary fat from the above sources on colon cancer in F344 male rats. In the ACF study 2 groups of F344 rats (4wks old) (n=6) received AIN-93G control (C) diet containing 7% and 14% soybean oil (SBO), respectively. The remaining groups were assigned treatment diets consisting of 7% and 14% RPO, WGO, RBO, FSO and CO, respectively. The rats remained on their respective diets for 13wks. The rats in the EPT study were fed a control (AIN-93G) diet consisting with 7% SBO, while the treatment groups were fed diets containing 7% RPO, WGO, RBO, FSO and CO, respectively. At 20wk of age rats in the EPT study were switched to AIN-93M diets. All rats received 2 S/c injections of AOM at 7 and 8 wk of age @ 16 mg/kg body weight in saline. At 45 wk of age all rats were killed by CO2 asphyxiation. Total ACF in the colon of rats fed SBO, WGO, RPO, RBO, FSO and CO at 7 and 14% levels ranged from 46 to 189. In the EPT study, all the rats fed 7% SBO and CO developed tumors (100% incidence) while tumor incidence in the groups fed WGO, RBO, FSO ranged from 39% to 79%. Tumor size (mm) ranged from 1.77 to 6.50. Our results indicate that the type and constituents (such as n-3 PUFA, carotenoids, vitamin E, phytosterols) in dietary fat plays a significant role in the incidence of AOM induced colon tumors in Fisher 344 rats. Dietary fats rich in phytochemicals may have implications in colon cancer.

Poster Session B

B12 Chemopreventive potential of selected cereals on chemically induced colon cancer in a Fisher 344 rat model.J. A. Boateng, M. Verghese, V. R. Panala, R. A. Field, D. S. Williams, L. A. Shackelford, L. T. Walker, D. Asiamah, R. Sunkara. Alabama A&M University, Normal, AL. Dietary fiber has been shown to have implications in colon cancer. In this study we examined the effects of selected cereals which are significant sources of dietary fiber (wheat bran (WB), rice bran (RB), yellow corn meal (CM) and flaxseed meal (FM) on the incidence of (AOM) induced colon tumorigenesis (Aberrant crypt foci (ACF) and End point tumor(EPT) studies) in Fisher 344 male rats. Fisher 344 male rats were randomly assigned into 20 groups after a 1 wk acclimatization period. For ACF study, Fisher 344 male rats were fed AIN-93G control diet (C) (n=6) and treatment groups (n=6); WB, RB, FM and CM at 5 and 10% levels. In EPT study, 20 groups of rats (n=14) were initially assigned AIN-93G control diet (C) and treatment groups; WB, RB, FM and CM at 5 and 10% levels. At 20wk of age rats were switched to AIN-93M diets. All rats received two S/c injections of AOM at 7 and 8 wk of age @ 16 mg/kg body weight in saline. At 45 wk of age all rats were killed by CO2 asphyxiation. Total number of ACF (SEM±) in the colon ranged from 26 to 160 for groups fed C, WB, RB CM and FM. Tumor incidence (%) in C, WB, RB CM and FM ranged from a low of 31% in rats fed selected cereals to a high of 100% in control group. Tumors/ tumor bearing rats (TBR) and tumor size (mm) were significantly (p<0.05) lower in the rats fed the treatment diets (WB, RB CM and FM) compared to the control. All the rats fed cereal diets had significantly (p<0.05) higher glutathione-S-transferase (GST) activity (µmol/g protein) compared to rats fed control diets. Our results suggests that feeding wheat bran, rice bran, corn meal and flax meal which are significant sources of dietary fiber at 5 and 10% levels significantly (P<0.05) reduced the incidence of AOM induced colon tumors in Fisher 344 male rats. We conclude that the protective effects of WB, RB, CM and FM against colon tumorigenesis may possibly be attributed to the synergistic/additive actions of phytochemicals and fiber contained in these foods. Carcinogen Activation and Carcinogen-Detoxifying Enzymes B14 The association between variant alleles of GSTM1 and GSTT1 and lung cancer risk: A nested case-control study in Washington County, Maryland.T. K. Lam, 1 I. Ruczinski, 2 K. Helzlsouer, 3 Y. Shugart, 4 K. E. Li, 5 S. C. Hoffman, 6 A. J. Alberg 7 . 1 National Cancer Institute, Bethesda, MD, 2 Department of Biostatistics, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, 3 Mercy Medical Center, Baltimore, MD, 4 Department of Epidemiology, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, 5 Applied Biosystems, Foster City, CA, 6 The Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, 7 Hollings Cancer Center and Department of Biostatistics, Bioinformatics, and Epidemiology, Medical University of South Carolina, Charleston, SC. Background:GSTM1 and GSTT1 genes regulate thePolymorphic detoxification of tobacco-related carcinogens and may play a role in lung carcinogenesis. Extensive studies have investigated the association between GSTM1 and GSTT1 and lung cancer risk. However, traditional genotyping assays were unable to discriminate between homozygous wild- type +/+ and heterozygous +/0 individuals and phenotypic differences in enzymatic activity associated with these genotypes may modulate lung cancer risk. In this study, we used a recently developed genotyping assay to investigate the associations between copy number variant alleles of GSTM1 and GSTT1 and lung cancer risk.

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Methods:We conducted a case-control study nested in a well- characterized, community-based cohort. First-time diagnoses of lung cancer (n=137) were matched to cancer-free controls (n=441) on age, gender, and cigarette smoking history. DNA obtained from blood samples provided at baseline was genotyped using real-time PCR-based assay. Conditional logistic regression models were used to estimate Odds Ratios (ORs) and 95% Confidence Intervals (CIs). Results:Compared to those with the GSTM1 homozygous wild-type 0/0, the relative odds of lung cancer increased from 1.49 (95% CI: 0.66- 3.40) to 1.80 (95% CI: 0.81-4.01) for the GSTM1 +/0 and 0/0 genotypes, respectively (p-value for trend: 0.13). When stratified by smoking intensity, the risk associations were primarily concentrated among lighter smokers (OR: 3.25; 95% CI: 0.93-11.34 for≥1 variant GSTM1 alleles). The results for GSTT1 were null. Conclusions:Additional variant alleles of GSTM1 were not strongly associated with the risk of lung cancer overall, but the risks were more pronounced among lighter cigarette smokers. GSTM1 variant alleles may have a more discernable impact on lung cancer susceptibility under circumstances when the dose of exposure to carcinogens is less. The results for GSTM1 suggest that new genotyping assays that classify those heterozygous from those homozygous for variant alleles will allow the role of GSTs in lung carcinogenesis to be more precisely characterized.

B15 Differential induction of cytochrome p450 by smokeless tobacco in various organs of rats mediates genotoxic damage by modulating the pro and anti-apoptotic genes.P. K. Avti, C. Mohan Pathak, K. Vaiphei, K. Lal Khanduja. Postgraduate Institute of Medical Education and Research, Chandigarh, India. Introduction:smokeless tobacco (ST) is increasing worldThe use of wide. The use of ‘gutkha’ (a form of ST) has been classified as carcinogen to humans and may be associated with oral diseases. Apart from oral cancer, the molecular mechanisms involved in other ST-related health effects are unknown. Therefore, the effects of chronic use of aqueous extract of ‘gutkha’ on cytochrome p450 (cyp) mediated genotoxic damage in liver, lung, and kidney of male Wistar rats were studied. Materials and Methods:The experiments were performed on pathogen-free young male Wistar rats weighing 100-120 g after the study was cleared by the Institute’s Animal Ethics Committee. The aqueous extract of gutkha was lyophilized and orally administered to animals (n=7, in each group) at a low-dose (96 mg/kg b.wt/d) for 2 and 32 weeks, and at a high-dose (960 mg/kg b.wt/d) for 2 weeks. The immunohistochemical expression of Phase-I (cyp1A1, 1A2, 2E1) and Phase-II (GST-µ) enzymes; gene expression of p53, NF-κB, Bcl-2 and Bax by RT-PCR; myeloperoxidase activity, protein carbonylation, caspase-3 activity, DNA fragmentation and blood micronuclei formation (assay for chromosomal damage) were measured in lung, liver and kidney of rats. Results:Our study shows that chronic use of aqueous extract of ST induces the differential immunohistochemical expression of xenobiotic phase-I and phase-II enzymes in various organs like lung, liver and kidney of rats. Immunohistochemical studies suggest that the expression of cyp2E1 in the chronic group was significantly higher as compared to cyp 1A1 and 1A2 in the lungs and kidney. There was significantly enhanced (p<0.05) activity of myeloperoxidase, protein carbonyl content, caspase-3 activity and DNA fragmentation in the above organs. The whole blood analysis in the chronic group confirmed the formation of micronuclei. Chronic use of ST significantly enhanced the gene expression of Bax, p53 and NF-κB and decreased the expression of Bcl-2 in a differential manner in various organs. However, the changes observed were greater in lungs and kidneys as compared to liver.

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Conclusions:Chronic use of ST-induced expression of phase-I and phase-II enzymes mediates differential genotoxic damage in various organs of rats by modulating the apoptotic-antiapoptotic signaling pathways. The differential cellular damage by ST may be due to variable sensitivities and functions carried out by these organs. These changes might create a microenvironment favorable for ST-induced pathogenesis and initiate the process of carcinogenesis in different organs. Extensive research is further needed to decipher the molecular mechanisms involved in the ST-induced pathogenesis. B16 Prior oral contraceptive use and soy isoflavone exposure alter cytochrome P450s by two different mechanisms.L. M. Scott, P. M. Durant, S. Leone-Kabler, A. J. Townsend, J. Cline. Wake Forest University School of Medicine, Winston Salem, NC. Estrogen exposure and metabolism are of current interest because they may play an important role in estrogen-sensitive cancers in postmenopausal women. We investigated the effects of long-term soy isoflavone (IF) consumption or premenopausal oral contraceptive (OC) use on postmenopausal expression of cytochrome P450 (CYP) mRNA, and whether IF directly affects CYP activity. One-hundred eighty-one female cynomolgus monkeys were randomized to receive OC or placebo for 28 months premenopausally, then ovariectomized and randomized to either an IF-free or IF-containing diet for 36 months. We measured mRNA levels of CYP1A1, CYP1B1, and CYP3A4 from total RNA isolated from liver and mammary samples obtained at necropsy using real-time RT-PCR. Long- term IF consumption did not significantly alter postmenopausal mammary CYP mRNA levels, while premenopausal OC administration reduced postmenopausal mRNA levels of all three enzymes assayed (50% reduction in both CYP1A1 and CYP1B1 mRNA and 80% reduction of CYP3A4 mRNA). Neither treatment changed any CYP mRNA level within the liver. To complement our studies in monkeys and increase our mechanistic understanding of IF effects, we also measured the effect of IF on CYP activity using a fluorogenic CYP activity assay in intact V79 cells stably transfected to express one of the three aforementioned CYP enzymes. Cells were preincubated with 1, 3, 10, or 30uM genistein, daidzein, or equol (a major metabolite of daidzein) added to the medium thirty minutes prior to addition of substrate for assay of activity. Overall, the activity of each CYP enzyme was altered in a dose-dependent and isoflavone-specific manner, with genistein exhibiting the most significant reduction of CYP1A1 and CYP1B1 activities. CYP1B1 activity was the enzyme most sensitive to IF pretreatment with each isoflavone achieving greater than 50% reduction in activity (approximate IC50 values of 2.1, 5.6, and 7.9uM for genistein, equol, and daidzein respectively). In summary, long-term mRNA expression of CYP isozymes involved in steroid hormone metabolism in the mammary tissues of postmenopausal non-human primates was reduced by prior OC exposure, but not by dietary IF consumption. However, our cell culture studies indicated that soy IF altered CYP activity acutely at concentrations achievable in the tissues of individuals on a diet including significant amounts of soy isoflavones. The strong reduction of CYP1B1 activity by IF could translate to reduced amounts of 4-OHE1, a precursor to mutagenic catecholestrogen quinones, produced in the tissue, thereby reducing the risk of estrogen-induced cancers. Finally, the reduction in CYP mRNA expression by OC exposure suggests that OC may actually attenuate the risk thought to be associated with metabolism of estrogens in this pathway.

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Carcinogenesis

Carcinogen Activation and Carcinogen-detoxifying Enzymes

B17 Soy isoflavones decrease the catechol-O-methyltransferase- mediated inactivation of 4-hydroxyestradiol in cultured MCF-7 cells.J. Wagner, L. Lehmann. University of Karlsruhe, Karlsruhe, Germany. The tissue concentrations of the female sex hormone 17beta-estradiol (E2) and its reactive catechol metabolites such as 4-hydroxyestradiol (4- HO-E2) play important roles in hormonal carcinogenesis. They are influenced by the activity of local enzymes involved in the metabolic activation and inactivation of E2. In the mammary gland, catechol estrogens are predominately inactivated by catechol-O-methyltransferase (COMT). Food supplements containing the soy isoflavones genistein (GEN) and daidzein (DAI) are consumed because they are believed to protect from breast cancer; however, this proposed benefit is controversial. The aim of the present study was to investigate the influence of soy isoflavones on the gene expression and activity of COMT in cultured human mammary adenocarcinoma MCF-7 cells. Levels of COMT mRNA were determined by reverse transcription/competitive polymerase chain reaction and COMT activity was determined by HPLC analysis of the methylation products of both the model substrate quercetin and the physiological relevant substrate 4-HO-E2. Our study demonstrates for the first time that soy isoflavones at hormonally-active concentrations cause a significant reduction of both COMT mRNA levels and COMT activity as well as of the methylation of 4-HO-E2. Experiments using the estrogen receptor antagonist ICI 182,780 support a role of the estrogen receptor in the isoflavone-induced downregulation of COMT expression. Thus, this study not only demonstrates that hormonally-active concentrations of soy isoflavones inhibit the detoxification of catechols in this human breast cancer cell line, but also implies that diet might influence COMT activity to a greater extent than heretofore recognized. This research was supported by the Deutsche Forschungsgemeinschaft (grant Le1329-7-1).

B18 Metabolic activation of benzo[a]pyrene in vitro by hepatic cytochrome P450 contrasts with detoxification in vivo: Experiments with hepatic cytochrome P450 reductase null mice.D. H. Phillips, 1 M. Stiborova, 2 C. J. Henderson, 3 M. Thiemann, 4 E. Frei, 5 D. Aimova, 2 R. Singh, 6 G. Gamboa da Costa, 7 O. Schmitz, 4 P. B. Farmer, 6 C. Roland Wolf, 3 V. M. Arlt 1 . 1 Institute of Cancer Research, Sutton, United Kingdom, 2 Charles University, Prague, Czech Republic, 3 Biomedical Research Centre, Dundee, United Kingdom, 4 University of Wuppertal, Wuppertal, Germany, 5 German Cancer Research Center, Heidelberg, Germany, 6 University of Leicester, Leicester, United Kingdom, 7 National Center for Toxicological Research, Jefferson, AR. Hepatic cytochrome P450 (CYP) enzymes play a pivotal role in the metabolism of many drugs and carcinogens. Much of the work carried out on the role of hepatic CYPs in xenobiotic metabolism has been done in vitro. However, additional factors such as route of administration, absorption, renal clearance and extra-hepatic CYP expression, make it difficult to extrapolate from in-vitro data to in-vivo pharmacokinetics. Moreover, functional redundancy inevitably found in the large CYP family of isoenzymes make it difficult to determine the role of CYPs in metabolism as a whole. To overcome these limitations a mouse line, HRN (Hepatic Cytochrome P450 Reductase Null), has been developed in which cytochrome P450 oxidoreductase (POR), the unique electron donor to CYPs is deleted specifically in the liver, resulting in the loss of essentially all hepatic P450 function. CYP1A1 or CYP1B1 activate many polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP). As a result, it is widely accepted that CYP1 potentiates BaP genotoxicity. We used the HRN model to evaluate the role of hepatic versus extrahepatic metabolism and disposition of BaP. HRN and wild-type (WT) mice were treated with 125 mg/kg body weight (bw) BaP once daily for up to five days by intraperitoneal injection. DNA adduct levels measured by 32 P-postlabelling at day 1 and 5 were up to 13-fold higher in livers of HRN than in WT mice. On day 1 sequential blood samples were obtained and analysed by HPLC with fluorescence detection for BaP clearance. Pharmacokinetic analysis of BaP in blood revealed no difference in e.g. clearance, terminal half-life, and AUC in HRN relative to WT mice. When hepatic microsomal fractions from

Poster Session B

HRN and WT mice were incubated with BaP, DNA adduct formation was 7- fold higher in WT than in HRN fractions. Most of the hepatic microsomal activation of BaP in vitro was attributable to CYP1A enzyme activity. These data reveal an apparent paradox, whereby CYP enzyme activity appears to be more important for detoxification of BaP in vivo, despite being essential for its metabolic activation in vitro. B19 Metabolism of estrogens and cancer of the tongue.E. G. Shatalova, S. I. Meireles, S. L. Mosier, M. L. Clapper. Fox Chase Cancer Center, Philadelphia, PA. Background. Squamous cell carcinoma (SCC) of the tongue is the most common cancer of the oral cavity. Recently, the incidence of tongue cancer has increased dramatically in young adults, particularly in women of premenopausal age. While tobacco smoking is a major risk factor for developing tongue cancer, the potential contribution of hormonal factors to the increased susceptibility of females must be considered. We hypothesize that estrogens and products of their oxidative metabolism participate in carcinogenesis in the tongue. The goal of the present study was to determine if estrogen metabolism genes are expressed in the tongue epithelium and if their expression can be modulated by 17β- estradiol (E 2 ), the most potent estrogen. Methods. Immunohistochemical (IHC) analyses of murine tongue tissue and tissue microarrays of human head and neck specimens (31 non- neoplastic and 111 SCCs, in duplicate) were performed using antibodies against cytochrome P450 (CYP) 1B1, estrogen receptor (ER)βand E 2 . Squamous epithelial cells were isolated from frozen sections of tongue tissue by laser capture microdissection. SCC9 cells (80% confluent) were plated and treated with either vehicle (0.1% ethanol) or 10 nM E 2 for 24 hours. RNA, extracted from both LCM samples and cultured cells, was subjected to quantitative real-time PCR, performed in triplicate, using primers specific for CYP1B1, CYP1A1, CYP3A4, catechol-O- methyltransferase (COMT), sulfotransferase (SULT) 1A1, UDP- glucuronosyltransferase (UGT) 1A, and glutathione S-transferase (GST) P1. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) served as a reference gene. Resultstongues of intact female A/J mice revealed: IHC analysis of the the presence of CYP1B1 and ER within the nuclei of squamous epithelial cells. Staining of E 2 was localized to both the cytoplasm and nucleus. Evaluation of the mRNA levels of estrogen metabolism genes in the microdissected tongue epithelium of ovariectomized A/J mice revealed the expression of CYP1B1, CYP1A1, COMT, SULT1A1, UGT1A, and GSTP1. Consistent with these mouse data, staining for E 2 , ERβand CYP1B1 was detected in both non-neoplastic and neoplastic human head and neck epithelium. Comparison of the intensity of staining of each antibody in non-neoplastic and neoplastic tissue revealed a significant increase in CYP1B1 levels in SCCs as compared to non-neoplastic epithelium (p=0.001). Expression of the estrogen metabolism genes CYP1B1, CYP1A1, CYP3A4, COMT, SULT1A1, UGT1A and GSTP1 was observed in human tongue carcinoma (SCC9) cells. Treatment of SCC9 cells with E 2 resulted in induction of the expression of the CYP1B1 gene, which encodes the protein that oxidizes estrogens to mutagenic metabolites, but not COMT, a gene that encodes the major conjugating enzyme, as compared to untreated control cells. In summary, estrogen metabolism genes are expressed in both mouse and human tongue tissue. Both the increased levels of CYP1B1 in head and neck SCCs and the ability of E 2 to modulate its expression suggest that estrogen metabolism may contribute to tongue carcinogenesis.

Frontiers in Cancer Prevention Research • December 5-8, 2007 • Philadelphia, PA

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Poster Session B

DNA Damage and Repair Mechanisms

B20 Hepatitis B virus causes DNA double-strand breaks via XRCC3 down-regulation and ROS stress.H. Kang, 1 H. Kim, 1 S. Lim, 1 Y. Park, 2 Y. Park, 3 G. Jung 1 . 1 Seoul National University, Seoul, Republic of Korea, 2 Yonsei University, Seoul, Republic of Korea, 3 Hepatology Center and Laboratory of Hepatocarcinogenesis, Bundang Jesaeng General Hospital, Bundang, Republic of Korea. The hepatitis B virus (HBV) causes DNA damage such as nucleotide excision as well as various types of liver diseases. Furthermore, DNA double-strand break (DSB) is a very hazardous DNA damage that can cause carcinogenesis or apoptosis. However, the correlation between HBV and DNA DSB has been unclear thus far. We first analyzed theγH2AX foci intensity, which is known as a marker of DNA DSB formation, at the cell lines where HBV or HBx was stably expressed , and found out that DNA DSBs increased. In addition, treatment with the antioxidant, N- acetylcysteine (NAC) depleted the reactive oxygen species (ROS) level, which reduced the DNA DSBs. To find the other cause of the increase in DNA DSBs due to HBV, we analyzed the alteration of the DNA DSB repair genes expressions in HBV or HBx stable cell lines. As a result, only XRCC3 was reduced by HBV. Moreover, based on the findings that DNA DSBs increased during XRCC3 shRNA expression, we concluded that XRCC3 was an essential repair gene for repairing DNA DSBs. The level of XRCC3 expression was different for the human liver tissues depends on the presence of HBV; XRCC3 expression decreased according to the HBV titer. In conclusion, our findings suggest that HBV increases DNA DSBs according to the down-regulation of XRCC3 expression and the raising of ROS stress.

Environmental and Radiation Carcinogenesis

B21 Reaction mechanisms and products of benzo[a]pyrene and benzo[e]pyrene with oxygen and nitrogen oxides.S. Fioressi, Z. Muñz, A. Vicés. Univerisdad del Turabo, Gurabo, PR. Thermal and photochemical reactions of benzo[e]pyrene (BeP) and benzo[a]pyrene (BaP) with nitrogen oxides and oxygen that take place in polluted atmospheres could produce direct mutagenic compounds. We have determined that, under laboratory conditions, these reactions generate diones, diols, hydroxy- and nitro- derivatives of BeP and BaP. The reaction mechanism of both isomers are different, therefore the microenvironment affects BeP and BaP in different ways. BaP react photochemically with oxygen through a singlet oxygen mediated mechanism whereas BeP reacts via a radical cation intermediate. We have determined that BeP also forms a radical cation intermediate when exposed to NO 2 in the dark, in solution or adsorbed on surfaces. The radical cation then reacts with water, traces of oxygen or with nitrogen dioxide to yield stable products. Some of the products observed are the same as those formed by photoreaction of BeP in the presence of oxygen; the other products were identified as mononitro- derivatives. In the case of BaP, the products formed after reaction with NO 2 are different to those observed by irradiation under aerobic conditions. Nitro derivatives are the main products of BaP exposed to NO 2 ; the nitration of BaP is thought to occur through an electrophilic substitution. We have identified thirteen products of BeP and BaP, and at least three of them presented direct mutagenic properties. BeP is considered non mutagenic and BaP is mutagenic only after metabolic activation; therefore, reactions with oxygen and NO 2 are processes that increase the toxicity of these pollutants in the atmosphere.

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Infections and Carcinogenesis

B22 E7 oncoprotein of human papillomavirus type 16 and nuclear factor-kappaB in laryngeal cancer.G. G. Chen, A. C. Vlantis, H. C. Liu, H. Xu, M. Tong, C. A. van Hasselt. Chinese University of Hong Kong, Shatin, NT, Hong Kong. About 60% of laryngeal cancers in China and Hong Kong are positive for high risk HPV-16 and HPV-18. E7 is an oncoprotein of HPV16 and/or HPV18. Human epithelial cells that express E7 exhibit a variety of growth control defects, which benefits the extended cell life span and immortalization of cells. E7 dramatically decrease pRB levels by binding to pRb and its related family members p107 and p130. E7 can also up- regulate p21 WAF1/CIP1 (p21) which inhibits apoptosis and promotes the growth of cells. We have shown that E7 induces increased levels of NF-kB subunit p65 in the nucleus of laryngeal cancer cells. Since NF-kB is a well known survival factor, this finding indicates that activation of NF-kB may contribute to cell growth and proliferation of HPV-16-infected laryngeal cancer cells. Using immunohistochemical staining method, we also showed that p65 was increased in human laryngeal cancer tissues, compared with non-cancer tissues. Quite interestingly in the promoter region of p21, we have found two binding sites for NF-kB, indicating that p21 may be a gene regulated by NF-kB. Therefore, it appears that the loss of inhibition of proliferation and growth of E7-containing epithelial cells is not only related to inactivation of pRb but also to the activation of survival molecules such as p21. Oxidative Stress and Carcinogenesis

B23 The antioxidant effect of lovastatin on phagocyte-induced DNA damage.A. B. Weitberg. Roger Williams Medical Center, Boston University School of Medicine, Providence, RI. Lovastatin is one of a series of HMG-CoA reductase inhibitors used in the treatment of hypercholesterolemia. It may also inhibit atheroma formation by inhibiting the oxidation of low density lipoproteins. We investigated the antoxidant properties of lovastatin and its effect on phagocyte-induced genotoxicity in mammalian cells. In a cytochrome c reduction assay, using phorbol ester-induced phagocytes as a positive control, lovastatin (10 uM) in its naturally occurring lactone form, had no effect. When converted to its acid form, however, superoxide anion formation was inhibited by 36%. Similarly, lovastatin in its acid form completely inhibited hydrogen peroxide formation by stimulated phagocytes. Using the fluorometric analysis of DNA unwinding, single- strand breakage was assayed in human lung cells exposed to stimulated human phagocytes with and without lovastatin (10uM, 5 minutes). Stimulated phagocytes induced a decrease in double-stranded DNA to 48% of control values which, in the presence of lovastatin, reverted to 91% of control values. Lovastatin acts as an antoxidant by decreasing oxygen radical production by human phagocytes and may be important in abrogating the carcinogenic effect of chronic inflammation.

B24 Apple polyphenols modulate antioxidant defense in human colon cancer cells.C. Janzowski, 1 P. Bellion, 1 B. Soyalan, 1 M. Baum, 1 F. Will, 2 H. Dietrich, 2 G. Eisenbrand 1 . 1 University of Kaiserslautern, Kaiserslautern, Germany, 2 Research Institute Geisenheim, Geisenheim, Germany. Diets rich in fruits and vegetables are associated with a lower risk of tumour incidence in the intestine and other sites. This is mainly attributed to high amounts of secondary plant constituents such as polyphenols, which display a spectrum of bioactive effects, including prevention from ROS (reactive oxygen species)-mediated DNA damage, mutations and cell death. We investigated the modulation of antioxidant response element (ARE) dependent gene expression, poly-(ADP-ribosyl)-transferase (PARP1) transcription (real time TaqMan PCR) and DNA damage (comet assay) in the human colon cell lines Caco-2 and HT-29 by polyphenolic apple juice extracts (AE05: from different cider apple varieties; BA: from cider apple “Bohnapfel”). Selected phenolic constituents (chlorogenic acid, ChA;

American Association for Cancer Research

Carcinogenesis Oxidative Stress and Carcinogenesis

caffeic acid, CA) were also studied. Incubation with both AEs (50/100 µg/ml) as well as with ChA and CA (10/ 30 µM) resulted in a decrease of γ-glutamylcystein-ligase (γ-GCL), gastrointestinal glutathione peroxidase and glutathione reductase transcription in Caco-2 cells; in HT-29 cells, however,γ-GCL transcription was distinctly elevated by AE05 and CA. PARP1 mRNA transcription was significantly depleted down to 40% of DMSO control by BA (6h, 50/ 100 µg/ml) in HT-29 cells; AE05 (50/ 100 µg/ml) and ChA (30µM) caused a moderate PARP1 inhibition and CA (30 µM) was inhibitory in both cell lines. In Caco-2 cells, with both extracts/ components slight modulation of PARP1 transcription was observed; menadione induced DNA-damage was slightly decreased after 24h incubation with AE05 and BA (10 µg/ ml). Taken together, the apple juice extracts and ChA show a distinct potential to deplete PARP1 transcription, to diminish oxidative DNA- damage and partially elevateγ-GCL transcription in human colon cell lines. The results suggest that apple polyphenols can prevent oxidative cell damage by inhibiting excessive PARP1 gene expression; ChA, the major constituent of both extracts might well contribute to the effects observed with the AEs. Other extract constituents, however, might also account for the protection against ROS-mediated DNA and cell damage. Supported by Federal Ministry of Education and Research (BMBF), grant no. 01EA0501 and federal state of Rhineland-Palatinate (project A4, intestinal health and nutrition “)

B25 Initial mechanistic studies of a new antioxidant approach for cancer prevention.Y. Zhao, 1 J. Liu, 1 X. Gu, 1 J. McCord 2 . 1 LSU Health Sciences Center, Shreveport, LA, 2 University of Colorado Health Sciences Center, Denver, CO. Beneficial effects of dietary supplements for human health have been proposed for years. However, although trials of cancer prevention by antioxidant supplements have been analyzed in a number of studies, most of these results have been discouraging. It has been suggested that single ingredient is not sufficient to eliminate all of the physiological/pathological mediators during cancer development, and a combination of multiple ingredients provides a possible solution. Traditional oriental medicines from food or plant have documented activities that make them excellent candidates for this approach. Protandim, a composite of extracts of five widely studied medicinal plants (B. monniera, S. marianum, W. somnifera, green tea, and turmeric), is such a product. The possibility of this new approach for cancer prevention has been examined in the well-established skin carcinogenesis model. Cancer development is caused by multifactors including cell proliferation, apoptosis, inflammation, all of which have been clearly examined in the skin carcinogenesis model. At first, as proof of principle, mouse skin was treated with Protandim extracts and skin tissues were collected for analysis. The initial studies first demonstrated that mice on Protandim diet showed 50% reduction in tumor incidence and multiplicity. To investigate the molecular mechanism, a single application of Protandim significantly suppressed TPA-induced increases in Jun D and PCNA expression, which was further confirmed in skin epidermal JB6 (P+) cells. The results also demonstrated that Protandim inhibited mitochondrial translocation of the tumor suppressor p53 and its target, Bax, suggesting that Protandim plays an important role in regulating both cell proliferation and apoptotic cell death. In addition, the initial studies also demonstrated that TPA induced cutaneous inflammatory response, evidenced by the increases in expression of the adhesion molecules ICAM-1/VCAM-1 in both skin epidermal tissues and keratinocytes. Protandim suppressed these increases. To seek the molecular mechanisms of Protandim’s bioactivities, the effects of Protandim as a dietary supplement on reducing oxidative stress has been investigated. The studies demonstrated that Protandim diet could induce both SOD expression and activities in mouse skin, which provides an essential support for the whole studies and for the hypothesis that Protandim may reduce skin tumor formation by reducing oxidative stress and hence modulating cell proliferation, cell death and inflammation. Finally, this combination (Protandim) is suitable for translational research and may serve as a therapeutic approach for cancer prevention.

Poster Session B

B26 Differential increased expression of oxidative stress modulatory enzymes in breast cancer cells by tocotrienols. S. Elangovan, J. M. Wu. New York Medical College, Valhalla, NY. Tocotrienols, a subclass of vitamin E compounds, display potent anticancer and apoptotic activity with little or no effect on normal cell growth and function. However, little if any attempt has been made to examine its influence on carcinogenesis, which often are inhibited by protective phase II and antioxidant enzymes. Determining the chemopreventive mechanism of tocotrienols will provide essential information necessary for understanding the potential health benefits of these compounds in reducing the risk of breast cancer in women. The primary aim of this study is to test the potential chemoprotective effects of tocotrienols by studying the relationship between antioxidant-enzyme defence responses and cellular growth suppression in human MCF-7 (estrogen receptor-positive) and MDA-MB-231 (estrogen receptor-negative) breast cancer cells. Comparing the effects ofα-,γ-, andδ-tocotrienols on breast cancer cell proliferation, we observed thatγ- andδ-tocotrienols significantly induced stronger growth inhibition in both cell lines at 10 µM concentration andα-tocotrienol had no effect on the growth of breast caner cells at tested concentrations. Hence, further studies are conducted using 10 µM concentrations. The expression and activity of antioxidant defenses were assessed using Western blot analysis and biochemical assays, respectively. In MDA-MB-231 cells,δ-tocotrienol was significantly more effective in increasing the expression of thioredoxin and glutathione peroxidase thanα- orγ-tocotrienols. All three tocotrienols had no effect on the expression and activity of superoxide dismutase in both MCF-7 and MDA-MB-231 cells. In MCF-7 cells, a moderate decrease in the expression of catalase was observed byα- andγ-tocotrienol treatment, but no difference in the expression of catalase was seen in MDAMB-231 cells. In MCF-7 cells, the activity of phase 2 detoxifying enzyme, glutathione-S- transferase was significantly induced byγ- andδ-tocotrienol, but the activity or expression of quinone reductase 1 was not altered by tocotrienols. The effects of tocotrienols on Nrf2 and KEAP1 expression were also examined by Western blot analysis. In MDA-MB-231 cells, treatment withα-,γ- andδ-tocotrienol lead to 2.6-, 1.9 and 3.2-fold increase, respectively, in Nrf2 protein level with concomitant decrease in KEAP1 expression. In MCF-7 cells, no significant changes in Nrf2 and KEAP1 levels were detected. The induction of antioxidant defence responses and phase 2 enzymes is more pronounced in the estrogen receptor-negative MDA-MB-231 cells, wherein response is prominent with γ- andδ-tocotrienol treatment. These studies demonstrate for the first time that the ability of a specific form of tocotrienols selectively regulates the Nrf2-KEAP1 system, which in turn induces the expression of cytoprotective genes. (Supported by a grant BCTR0600943 from the Susan G Komen Breast Cancer Foundation)

B27 Analysis of 8-oxo-2`-deoxyguanosine on exposure of human bronchoalveolar cells to benzo[a]pyrene-7,8-dihydrodiol.D. Mangal, S. Lee, J. Park, C. Mesaros, T. M. Penning, I. A. Blair. University of Pennsylvania, Philadelphia, PA. Introduction:B[a]P is a member of a family ofBenzo[a]pyrene polycyclic aromatic hydrocarbons that are ubiquitous environmental carcinogens. B[a]P is metabolically activated to the proximate carcinogen B[a]P-7,8-dihydrodiol which is converted to a catechol by Aldo-Keto Reductases (AKRs). The catechol metabolite then undergoes two sequential 1-electron oxidations to give B[a]P-7,8-dione with the concomitant generation of reactive oxygen species (ROS). B[a]P-7,8-dione is then reduced back to the catechol establishing a futile redox cycle, which continually produces ROS that in turn can cause the formation of 8-oxo-2’- deoxyguanosine (8-oxo-dGuo) adducts in DNA. Experimental Procedure:It is difficult to quantify true cellular 8-oxo- dGuo levels due to artifact formation during isolation and hydrolysis of the DNA from the cells and tissue samples by the presence of trace amounts of transition metal ions in the buffers that are used. To avoid this problem a cold guanidine thiocyanate non-phenolic method was used to extract DNA from cells and desferal was used in Chelex-treated buffers to remove

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transition metal ions. This prevented the artifactual formation of 8-oxo- dGuo from Fenton Chemistry. Immunoaffinity purification was employed in order to remove interfering substances that co-eluted with 8-oxo-dGuo during subsequent chromatography. Stable isotope dilution liquid chromatography/electrospray ionization/multiple reaction monitoring/mass spectrometry (LC/ESI/MRM/MS) was used to provide maximal specificity and sensitivity. Human bronchoalveolar cells were treated with B[a]P-7,8- dihydrodiol and 8-oxo-dGuo present in the DNA was quantified. Results:The 8-oxo-dGuo levels were increased after B[a]P-7,8- dihydrodiol treatment compared to untreated cells and DMSO treated cells. The 8-oxo-dGuo levels were increased in dose dependent manner. However, when the cells were pre-treated with the COMT inhibitor, R041- 0961, a significant increase in 8-oxo-dGuo levels was observed. The COMT inhibitor alone had no effect on 8-oxo-dGuo formation. B[a]P-7,8-dione formation in the cells indicated that 8-oxo-dGuo was formed by the ROS generated during the two electron oxidation of the B[a]P-7,8-catechol. Conclusion:These data confirm that the new LC-MS-based method efficiently eliminated artifact formation that occurred during the isolation and hydrolysis of DNA. This made it possible to detect elevated 8-oxo- dGuo levels on exposure of H358 cells to B[a]P-7,8-dihydrodiol. The formation of B [a]P-7,8-dione in the cells also supports the proposed formation of ROS during the oxidation of the catechol metabolite. The increased level of 8-oxo-dGuo on pretreatment of the COMT inhibitor, suggests that the catechol metabolite is normally detoxified by conversion to a methyl ether derivative. Supported by NIH grants 1R01CA130038 and 5P30ES013508. Tumor Promotion and Progression B28 A potential role for Periostin in Barrett’s carcinogenesis. A. Saadi, 1 M. Das, 1 N. Clemons, 1 C. Zhang, 2 M. Ferguson, 3 G. Tokiwa, 4 K. Serikawa, 4 J. S. Hardwick, 5 H. Dai, 2 L. Carlini, 5 R. C. Fitzgerald 6 . 1 Hutchison/MRC Research Centre, Cambridge, United Kingdom, 2 Custom Analysis, Rosetta Inpharmatics, Seattle, WA, 3 Management, Merck Research laboratories, West Point, PA, 4 Gene Expression Laboratory, Rosetta Inpharmatics, LLC, Seattle, WA, 5 Oncology Molecular Profiling/Guided Solutions, Merck Research laboratories, West Point, PA, 6 Hutchison/MRC Research Centre, Cambridge, United Kingdom. Introduction:Periostin is a secreted extracellular matrix protein important in embryonic development and wound repair. It is a potential inducer of epithelial-mesenchymal transition and cell motility in normal development and cancer; although, whether it has a tumor suppressive or tumor promoting role is controversial. Overexpression of periostin mRNA was recently reported in Barrett’s (BE) and oesophageal adenocarcinoma (OAC) tissues (Hao Y, Gastroenterology 2006). Aims:1. Use gene expression data to determine whether periostin is overexpressed in the Barrett’s-dysplasia- carcinoma sequence. 2. Validate array data in independent samples at the protein level. 3. Investigate the capacity for periostin to promote invasion in vitro. Methods:RNA expression was determined using Agilent microarrays from samples representing different stages of OAC development (normal squamous-oesophagus (NE); n=6, intestinal metaplasia (IM); n=21, Low- grade-dysplasia (LGD); n=17, High-grade-dysplasia (HGD); n=13, OAC; n=8). Periostin protein expression was examined immunohistochemically on an independent data-set (abcam14041). Oesophageal stromal fibroblasts were isolated from normal (NAFs), Barrett’s (BAFs), and tumor- associated patient tissues (CAFs) and characterized using vimentin and CK8/18. Periostin expression was determined by Western blotting and QPCR in primary fibroblasts, oesophageal adenocarcinoma (SEG, FLO, OE33, BIC) and dysplastic epithelial cell lines (GohTert, ChTert, GihTert). Effects of recombinant periostin (100-400ng/ml) and addition of primary fibroblasts on FLO invasiveness and motility were investigated in vitro using matrigel and wound healing assays. Results:Periostin was significantly overexpressed in OAC compared to NE (4.36 fold, p=0.0035), IM (2.76 fold, p=0.0275), LGD (3.61 fold, p=0.0027) and HGD (14 fold, p=8.83E-08), (One-Way ANOVA). IHC

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showed that periostin protein expression was restricted to the stroma of BE and cancer tissues, with an apparent concentration at the epithelial- stromal interaction front in OAC. Purified cell types (primary stromal fibroblasts and established epithelial cell lines) were used to further characterize the cell specific expression of periostin. Apart from SEG cells, dysplastic and OAC epithelial cell lines expressed lower levels of periostin mRNA compared to oesophageal fibroblast lines derived from patients with dysplasia and cancer (2 fold, p<10 -4 , t-test). Periostin was detectable at the protein level in all cell types. Recombinant Periostin (100ng/ml) promoted invasion of FLO cells in vitro (1.7 fold. P=0.0092), but had no significant effect on cell motility. 2/3 CAFs increased invasiveness of co- cultured FLO cells compared to corresponding NAFs (p= n.s), but this was not directly attributable to periostin. Conclusion:Overexpression of periostin in primary fibroblasts and in stroma of cancer related tissues, combined with periostin-induced promotion of invasion in vitro, suggests a role for fibroblast derived periostin in cancer. The isolation of stage-specific primary stromal fibroblasts offers a valuable tool for further functional studies of stromal- epithelial interactions in Barrett’s carcinogenesis.

B29 Detecting mutation rate change in Barrett’s esophagus after treatment with NSAIDs.R. Kostadinov, 1 M. Kuhner, 2 C. Maley 3 . 1 University of Pennsylvania, Philadelphia, PA, 2 University of Washington, Seattle, WA, 3 The Wistar Institute, Philadelphia, PA. Background:To prevent or delay progression to cancer, it is vital to -assess the effect of exposures such as alcohol, tobacco smoke, or non steroidal anti-inflammatory drugs (NSAIDs) on the clonal evolution that drives neoplastic progression. NSAIDs have been shown to reduce the risk of esophageal adenocarcinoma (EA) in Barrett’s Esophagus (BE) patients (Vaughan et al., Lancet Oncol. 2005;6(12):945-52). However, the mechanism through which NSAIDs delay progression to EA is not well understood. Four parameters determine the rate of evolution of neoplastic clones: mutation rate, stem cell population size and generation time, and clonal expansion rate. We hypothesize that NSAID use slows clonal evolution by reducing mutation rate. To test this hypothesis in-vivo, Illumina SNP arrays can be used to detect loss of heterozygosity (LOH) events in frozen longitudinal biopsies from BE patients from the Seattle Barrett’s Esophagus cohort. Using coalescent theory, the observed LOH events, known sampling times and known onset of NSAIDs treatment we can estimate the rate of LOH per year, the stem cell population size, and changes in these parameters after treatment. Methods:We performed in-silico analysis of the power to detect mutation rate changes, which is critical for selecting the number of time points and biopsies per time point to assay per patient. We modified SerialSimCoal (Excoffier et.al. J. Hered., 91, 506-509) to simulate a scenario in which 4 biopsies were taken from patients every 2 years for 10 years, with NSAID use beginning in year 5, and asked whether we could reliably detect mutation rate changes of various intensities (1.1, 2, 5, 10, and 100- fold). We varied stem cell population sizes and base mutation rates over 3 orders of magnitude on DNA sequences of 8,000 loci. We used BEAST (Drummond et.al. Genetics 161, 1307-1320) to estimate population sizes and mutation rates before and after year 5. Results:For 91% (41/45) of parameter combinations, we had >80% power to detect a 1.1- to 100- fold decrease in mutation rate (paired Wilcoxon test, p<0.05). In the four worst cases, a high stem cell population size and low base mutation rate, yielded broad confidence intervals for the estimated mutation rates and resulted in >70% power to detect 1.1- to 10- fold decrease in mutation rate. Conclusion:These results allow for the design of longitudinal studies to be carried out in BE cohorts to determine the in-vivo effects of NSAIDs and other exposures. Our results suggest that assaying 24 longitudinal samples will give >80% power to detect as small as a 1.1-fold change in mutation rate associated with an intervention. We are currently adapting these methods to use SNP arrays to measure the rate of loss of heterozygosity events, number of stem cells and time since initiation in neoplastic progression.

American Association for Cancer Research

Clinical Prevention Trials

Other Carcinogenesis Research

Other Carcinogenesis Research

B30 Cancer as competing clones: Theoretical and experimental cellular sociology systems biology.C. MacAulay, S. Lam, W. Lam, M. Guillaud. BC Cancer Agency, Vancouver, British Columbia, Canada. The pre-invasive neoplastic development process is one of competing normal and molecularly evolving clonal populations. These altered competing cells are recapitulating their internal molecular dynamics and interactions at the physical level through increased growth, decreased apoptosis, resource request signaling and usage, as well as interacting with the host immune system and surrounding stroma. An in depth understanding of all these interactions is key to understanding and altering the neoplastic process for therapeutic (chemoprevention for pre-neoplastic tissue and chemotherapy for invasive cancer) purposes. We are developing a two prong approach to this problem; 1) a computational 3D static and dynamic model of tissue epithelium and stroma (based on physical and molecular interactions) and 2) an automated system for multi-colour FISH of tissue sections for the identification of genetically related clones in excised tissue samples. Our current mathematical model is based on quantitative data from the nuclear phenotypes of the cells, molecular characteristics from quantitative IHC and quantitative tissue architectural information (spatial arrangement of cells in tissue) from more than 10,000 of lung, cervix and oral sections. The automated system for multi-colour FISH is designed to scan up to 6 FISH labels automatically across an entire tissue section and analyze the results cell by cell and then by clonal neighbourhood. Several versions of clonal neigbourhood have been tested. These are derived from the Voronoi tessellation of the imaged tissue based upon the spatial arrangement of the individual cells and their FISH attributes. Initial testing of this system showed clonal populations of cells resistant to lung cancer chemotherapy based upon a specific gene copy number alteration profile, derived form array CGH data.

Clinical Prevention Trials

Breast Cancer

B32 Tamoxifen modulates tumor suppressor gene methylation in breast cells and growth factors in plasma in women at increased risk for breast cancer.C. M. Lewis, 1 D. Bu, 1 R. Ashfaq, 1 X. Xie, 1 A. Miller, 2 W. Dooley, 3 J. O’Shaughnessy, 4 B. Arun, 5 K. Hunt, 5 D. M. Euhus 1 1 UT . Southwestern Medical Center, Dallas, TX, 2 Cancer Therapy and Research Center, San Antonio, TX, 3 The University of Oklahoma Health Sciences Center, Oklahoma City, OK, 4 Baylor Sammons Cancer Center, Texas Oncology, PA, US Oncology, Dallas, TX, 5 UT MD Anderson Cancer Center, Houston, TX. [In addition to poster presentation, this proffered abstract has been selected for oral presentation in Concurrent Session 16 (page 53). For the complete abstract text, refer to abstract PR-5 in the Proffered Papers section of this Proceedings (pages 71-72).] B33 Low-dose aspirin and breast cancer risk: Results from a randomized trial.Zhang, N. R. Cook, J. E. Manson, I. Lee, J. E. Buring.S. Brigham and Women’s Hospital and Harvard Medical School, Boston, MA. The Women’s Health Study trial reported a lack of effect of low-dose aspirin (100-mg every other day) on cancer risk, including invasive breast cancer, over an average of 10 years of treatment and follow-up. Outstanding issues that have not been addressed previously for breast cancer include whether low-dose aspirin might reduce the risk in subgroups according to baseline risk factors or tumor characteristics. We randomly assigned 39,876 women aged 45 and older and who were free of cancer and cardiovascular disease to receive 100-mg of aspirin every other day (19,934 participants) or placebo (19,942 participants) from 1993 to 1996. Participants were followed through March 31, 2004. Using intent- to-treat analysis, we compared the risk for breast cancer after

Poster Session B

randomization between the groups and calculated hazard ratios and 95% confidence intervals using Cox proportional hazards regression models. Low-dose aspirin had no significant effect on the risk for total (762 vs. 779 cases, hazard ratio = 0.98; 95% confidence interval: 0.88, 1.08) or invasive breast cancers (608 vs. 622 cases, hazard ratio = 0.98; 95% confidence interval: 0.87, 1.09) compared with placebo. There was also no significant effect of low-dose aspirin on the risk for invasive breast cancers by categories of baseline risk factors for breast cancer, or by tumor characteristics at diagnosis, including tumor size, lymph node metastasis, histology, histologic grading and differentiation, or hormone receptor status. These results suggest that 100-mg aspirin taken every other day over 10 years does not have a chemopreventive effect on breast cancer. Colon and Other Gastrointestinal Cancers B34 Dietary administration of black raspberries modulates markers of oxidative stress in patients with Barrett’s esophagus. J. Fromkes, W. Frankel, G. D. Stoner, C. Hammond, L. A. Kresty. Ohio State University, Columbus, OH. Oxidative damage has been implicated in the progression and development of esophageal adenocarcinoma (EAC), a rapidly increasing and extremely deadly malignancy. Barrett’s esophagus (BE) is a premalignant condition in which the normal stratified squamous epithelium lining the esophagus is replaced by metaplastic columnar epithelium with goblet cells. Barrett’s typically develops in the background of gastroesophageal reflux disease. BE importance lies in the fact that it confers a 30-40 fold increased risk for the development of esophageal adenocarcinoma. Thus, BE patients represent a population for whom targeted chemopreventive interventions may prove particularly beneficial. Dietary administration of lyophilized black raspberries (LBR) has inhibited chemically induced oral, esophageal, and colon carcinogenesis in animal models. LBR have reduced measures of oxidative stress, decreased DNA damage, inhibited cellular proliferation rates, and reduced levels of esophageal and colon preneoplasia in pre-clinical models. These observations lead us to hypothesize that dietary administration of LBR may modulate markers of DNA or oxidative damage associated with gastroesophageal reflux disease and in turn inhibit the progression of Barrett’s esophagus. We conducted a six month chemopreventive intervention where 32 and 45 grams of LBR (female and male, respectively) were administered daily to twenty patients with BE. Tissue, blood, and urinary biomarkers were assessed pre- and post-intervention utilizing enzyme-linked immunosorbent assays to measure urinary 8-epi- prostaglandin F2α(8-Iso-PGF2α) and 8-hydroxy-2’-deoxyguanosine (8- OHdG) and immunohistochemical methods were employed to measure GSTπ, NFκB, Ki-67 and CDX2 in Barrett’s epithelium. Urinary biomarkers were corrected for urinary creatinine levels to control for potential differences in urine volumes collected between patients. Patients experienced a statistically significant decline in mean levels of 8-Iso- PGF2αfollowing 26 weeks of LBR consumption and 58% of patients experienced marked individual level declines. Mean concentrations of 8- Iso-PGF2αwere 1.59E-10, 1.38E-10, and 1.30E-10 mg/ml urine at baseline, week 12, and week 26 of study, respectively. Conversely, mean levels of urinary 8-OHdG were not significantly reduced in this patient population at 26 weeks of study. However, all patients experiencing declines in urinary 8-OHdG also had reduced 8-Iso-PGF2αlevels (r=0.82). LBR also resulted in increased expression of GSTπin Barrett’s tissues among 37% of the patients. LBR treatment did not result in significant changes in mean Ki-67 staining levels in this cohort. Only low levels of CDX2 were detectable limiting the usefulness of this marker. NFκB analysis is ongoing. Pilot study results support that daily administration of LBR for 6 months significantly reduces urinary 8-Iso-PGF2α, a marker of oxidative stress, and that LBR increases tissue levels of the phase II enzyme GSTπin a subset of BE patients. These results are encouraging and support conducting a randomized placebo controlled trail in this patient cohort to more fully assess LBR as inhibitors of esophageal adenocarcinogenesis.

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