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A 1.4-year-old virgin female brown-hooded fancy rat presented for abdominal distention, jaundice, and dyspnea. At physical examination, a firm mass was palpable in the caudoventral abdomen as well as multiple small nodular masses associated with the abdominal viscera. At necropsy, in addition to a large mass replacing the left ovary and myriad nodules studding the peritoneal surface, there was 31 ml of abdominal effusion. By cytology, the abdominal fluid contained numerous pleomorphic vacuolated tumor cells surrounding globular pale eosinophilic to amphophilic acellular material that was strongly periodic acid–Schiff positive. Histologically, the tumor was biphasic with abundant acellular hyaline matrix that was also periodic acid–Schiff positive.

A 7-mo-old female New Zealand white rabbit presented with hemorrhage of the gingiva surrounding a loose lower right incisor. Antemortem conventional radiographs revealed only a small bone fragment adjacent to the left mandible's body. In light of a provisional diagnosis of mandibular fracture, the rabbit was euthanized. Postmortem radiographs of the disarticulated mandible demonstrated mandibular symphyseal fracture and comminuted fracture of the ramus and body of the left mandible. According to histopathology, the left submandibular salivary gland had necrotizing sialometaplasia, a nonneoplastic condition of the salivary glands that is caused by ischemic infarction. Although rabbits have been used as animal models of mandibular fracture and necrotizing sialometaplasia, no nonexperimental case of such conditions had been reported previously.

Endoluminal infusion and incubation of elastase with or without collagenase into the rabbit common carotid artery is an established model of arterial saccular aneurysm. The model mimics naturally occurring human cerebral aneurysms in many ways, including histologic and morphologic characteristics, hemodynamic pressures, and shear stresses. However, complications have been associated with the model. Here, we report 2 complications: 1) the first known case of iatrogenic laryngeal hemiplegia in a rabbit; and 2) histopathologically confirmed iatrogenic hippocampal and cerebellar infarcts (stroke). Finally, we present and review data from current literature on the morbidity and mortality associated with this model.

The mechanisms that can account for the remarkable mammary carcinogenicity of the environmental pollutant 6-nitrochrysene (6-NC) in the rat remain elusive. In our previous studies, we identified several 6-NC-derived DNA adducts in the rat mammary gland; one major adduct was derived from (±)-trans-1,2-dihydroxy-1,2-dihydro-6-nitrochrysene (1, 2-DHD-6-NC). In the present study, we resolved the racemic (±)-1, 2-DHD-6-NC into (−)-[R,R]- and (+)-[S,S] -1,2-DHD-6-NC and compared their in vivo mutagenicity and carcinogenicity in the mammary glands of female transgenic (BigBlue F344 × Sprague-Dawley)F1 rats harboring lacI/cII and Sprague-Dawley rats, respectively. Both [R,R]- and [S,S]-isomers exerted similar mutagenicity and carcinogenicity but were less potent than 6-NC. Additional in vivo and in vitro studies were then performed to explore possible mechanisms that can explain the higher potency of 6-NC than 1, 2-DHD-6-NC. Using ELISA, we found that neither 6-NC nor 1, 2-DHD-6-NC increased the levels of several inflammatory cytokines in plasma obtained from rats 24 h after treatment. In MCF-7 cells, as determined by immunoblotting, the effects of 6-NC and 1, 2-DHD-6-NC on protein expression (p53, Akt, p38, JNK, c-myc, bcl-2, PCNA, and ERβ) were comparable; however, the expressions of AhR and ERα proteins were decreased by 6-NC but not 1, 2-DHD-6-NC. The expression of both receptors was decreased in mammary tissues of rats treated with 6-NC. Our findings suggest that the differential effects of 6-NC and 1, 2-DHD-6-NC on AhR and ERα could potentially account for the higher carcinogenicity of 6-NC in the rat mammary gland.

Malignant transformation by oncogenes requires additional genetic/epigenetic changes to overcome enhanced susceptibility to apoptosis. In the present study, we report that Bif-1 (Sh3glb1), a gene encoding a membrane curvature–driving endophilin protein, is a haploinsufficient tumor suppressor that plays a key role in the prevention of chromosomal instability and suppresses the acquisition of apoptosis resistance during Myc-driven lymphomagenesis. Although a large portion of Bif-1–deficient mice harboring an Eμ-Myc transgene displayed embryonic lethality, allelic loss of Bif-1 dramatically accelerated the onset of Myc-induced lymphoma. At the premalignant stage, hemizygous deletion of Bif-1 resulted in an increase in mitochondrial mass, accumulation of DNA damage, and up-regulation of the antiapoptotic protein Mcl-1. Consistently, allelic loss of Bif-1 suppressed the activation of caspase-3 in Myc-induced lymphoma cells. Moreover, we found that Bif-1 is indispensable for the autophagy-dependent clearance of damaged mitochondria (mitophagy), because loss of Bif-1 resulted in the accumulation of endoplasmic reticulum–associated immature autophagosomes and suppressed the maturation of autophagosomes. The results of the present study indicate that Bif-1 haploinsufficiency attenuates mitophagy and results in the promotion of chromosomal instability, which enables tumor cells to efficiently bypass the oncogenic/metabolic pressures for apoptosis. .

We have established a clinically relevant murine model to reveal tumor-induced immunotoerance in hepatocellular carcinoma (HCC). Critical factors are targeted to develop immune-based therapies for HCC control. Intrasplenic (ISPL) inoculation of oncogenic hepatocytes and intraperitoneal (IP) injection of carbon tetrachloride (CCl4) are combined to induce progressive HCCs in fibrotic livers of immunocompetent mice. We characterize the features of this model, examine tumor-antigen-specific (TAS) immunity during tumor initiation and progression, and identify the critical factors in tumor-induced immune tolerance. The established murine model recapitulates human HCC and reflects its typical features. TAS CD8+ T cells initially maintain a naive phenotype and function in early-stage tumor-bearing mice, then become profound exhaustion with the tumor progression to the advanced stage. The deep immunosuppression is associated with the significant upregulation of Programmed cell death protein 1(PD-1), Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and the increase of Tregs. While the changes in Tregs and PD-1 are systemic, they are particularly pronounced in the tumor microenvironment. Sunitinib-mediated reduction of Treg together with antibody-mediated blockade of PD-1 synergistically suppress tumor growth and activate anti-tumor immunity. Our data provide evidence that oncogenic hepatocytes escape immune surveillance during tumor initiation via immune ignorance, while later-stage established HCCs evade immune destruction via tumor-induced immunotolerance. Synergy of sunitinib and anti-PD-1 Ab generates favorable effect on suppressing tumor growth and activating anti-tumor immunity. This combinational strategy has high translational potential in HCC treatment. Citation Format: Dai Liu, Guangfu Li, Timothy Cooper, Eric Kimchi, Xiaoqiang Qi, Ningfei Li, Don Rockey, Todd Schell, Kevin Staveley-O’Carroll. Development of a successful combination therapy for hepatocellular cancer by targeting Treg and PD-1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4004.

Tobacco smoking is an etiological factor of ovarian cacner; however, the mechanisms remain largely undefined. Therefore, as an initial investigation, we examined the carcinogenicity and DNA adducts formation in the ovary of mice treated with DB[a,l]P, a tobacco smoke constituent and environmental pollutant. Ovarian tumors in B6C3F1 mice were induced by direct application of DB[a,l]P (24, 12, 6, and 3 nmol/mouse, three times a week for 38 weeks) into the oral cavity of mice. At 6 nmol, DB[a,l]P induced the highest total ovarian tumor incidence (79%), but the incidence of malignancy was only 15%. However, at the dose of 12 nmol, the total ovarian tumor incidence was 75%, and the incidence of malignancy was 65%. In addition to ovarian tumors, at the dose of 24 nmol, DB[a,l]P induced lesions in sites distal from the ovaries including the skin, mammary, lung, and oral tissues, which were rare at doses lower than 24 nmol. Another bioassay was conducted to detect and quantify DNA adducts induced by DB[a,l]P (24 nmol, three times a week for 5 weeks) in the ovary at 48 h and 1, 2, and 4 weeks after the last administration of DB[a,l]P. DNA was isolated, and the dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE)-DNA adducts were analyzed by a LC-MS/MS method. DB[a,l]P resulted in the formation of (-)-anti-cis-DB[a,l]PDE-dA and (-)-anti-trans-DB[a,l]PDE-dA adducts, which were 0.8 and 1.6 fmol/10(6) dA, respectively, in ovaries of mice within 48 h, and the level of adducts decreased over a week. Our results indicated that DB[a,l]P can be metabolized to form (-)-anti-DB[a,l]PDE; the latter may, in part, account for DB[a,l]P-induced ovarian cancer. This animal model should assist to better understand the mechanisms, account for the induction of ovarian cancer by tobacco carcinogens, and facilitate the development of chemopreventive agents against ovarian cancer.

In these experiments, we tested the hypothesis that inhibition of the estrogen receptor (ER) with Tamoxifen and activation of PPARγ with fish oil (FO) rich in omega-3 (n-3; known PPAR agonists) inhibit the development of hormone-independent breast cancer in view of the known crosstalk between the ER and PPARγ pathways. We selected the polyoma middle T transgenic mouse model, since in this system the development of ER− tumors is preceded by ER positive preneoplastic lesions. Tamoxifen admixed with a 20% corn oil (CO) modified AIN-76A diet delayed mammary carcinogenesis and inhibited tumor multiplicity, volume, and weight in a dose-dependent (1, 10, and 100 ppm) fashion. Administration of increasing concentrations of FO in the diet (5%, 10%, and 17%) did not affect any of the tumor parameters. Combined administration of different doses of Tamoxifen and FO delayed carcinogenesis and suppressed tumor multiplicity and volume to the same extent as Tamoxifen alone. Mice fed 10% FO exhibited the expected increase in n-3/n-6 ratio in plasma and tumor based on diet analysis. Further increase in the n-3/n-6 ratio was not observed in mice fed the 17% FO diet. FO reduced tissue levels of arachidonic acid and its metabolite PGF-2α. Our results support the role of ER expression by preneoplastic lesions in the development of hormone-independent tumors and consequently the importance of including ER targeting in combination with mechanistically based novel chemopreventive agents.

Epidemiologic studies on the protective role of omega-3 fatty acids (n:3) on breast cancer prevention remain inconclusive but studies in preclinical models provide more positive outcome. However, the mechanisms accounting for the protective effect of n:3 are not defined. In the present study, conducted in the N-methyl-N-nitrosourea-induced rat mammary carcinogenesis model, we examined the effects of n:3 individually and in combination with the anti-estrogen Tamoxifen (Tam) on a comprehensive panel of systemic and preneoplastic mammary gland restricted biomarkers which may be critical in the progression to invasive cancer. We observed that fish oil (FO) rich diets significantly reduced Ki67 expression in hyperplastic lesions, while cleaved caspase-3 expression was not affected. Dietary FO and/or Tam did not have major effects on systemic oxidative stress biomarkers, based on oxidative damage to DNA measured as 8-hydroxy-2-deoxyguanosine (8-OH-dG) and lipid peroxidation assessed as thiobarbituric acid reactive substances (TBARS). Tissue levels of 8-isoprostane, on the other hand, were markedly reduced (p

Cancer of the oral cavity is a serious disease, affecting about 30,000 individuals in US annually. There are several animal models of oral cancer, but each has certain disadvantages. As a new model, we investigated whether topical application of the tobacco smoke carcinogen, dibenzo[a,l]pyrene (DB[a,l]P) is mutagenic and carcinogenic in the oral cavity of the B6C3F1 lacI and B6C3F1 mouse, respectively. B6C3F1 lacI mice received DB[a,l]P (0, 3, 6, 12 nmol) 3× per week. B6C3F1 mice received the same doses and also 24 nmol. At 38 weeks mutagenesis was measured in oral tissues in lacI mice. For the high dose group, the mutant fraction (MF) in upper mucosa and tongue increased about twofold relative to that in vehicle-alone. The increases were statistically significant. The mutational profile in the DB[a,l]P-induced mutants was compared with that induced by benzo[a]pyrene (BaP) in oral tissue. BaP is mutagenic in many tissues when administered by gavage. The mutational profile for DB[a,l]P was more similar to that reported for p53 mutations in head and neck cancers than was that of BaP. At 47 weeks, oral squamous cell carcinomas (OSCC) were found in 31% of the high-dose B6C3F1 group. Elevations of p53 and COX-2 protein were observed in tumor and dysplastic tissue. As DB[a,l]P induces mutations and tumors in the oral cavity, and has a mutational profile in oral tissue similar to that found in p53 in human OSCC, the treatment protocol described here may represent a new and relevant model for cancer of the oral cavity.

Vascular Ehlers-Danlos syndrome is a rare genetic disorder resulting from mutations in the α-1 chain of type III collagen ( COL3A1) and manifesting as tissue fragility with spontaneous rupture of the bowel, gravid uterus, or large or medium arteries. The heterozygous Col3a1 knockout mouse was investigated as a model for this disease. The collagen content in the abdominal aorta of heterozygotes was reduced, and functional testing revealed diminishing wall strength of the aorta in these mice. Colons were grossly and histologically normal, but reduced strength and increased compliance of the wall were found in heterozygotes via pressure testing. Although mice demonstrated no life-threatening clinical signs or gross lesions of vascular subtype Ehlers-Danlos syndrome type IV, thorough histological examination of the aorta of heterozygous mice revealed the presence of a spectrum of lesions similar to those observed in human patients. Lesions increased in number and severity with age (0/5 [0%] in 2-month-old males vs 9/9 [100%] in 14-month-old males, P < .05) and were more common in male than female mice (23/26 [88.5%] vs 14/30 [46.7%] in 9- to 21-month-old animals, P < .05). Haploinsufficiency for Col3a1 in mice recapitulates features of vascular Ehlers-Danlos syndrome in humans and can be used as an experimental model.

Utilization of finfish and aquatic invertebrates in biomedical research and as environmental sentinels has grown dramatically in recent decades. Likewise the aquaculture of finfish and invertebrates has expanded rapidly worldwide as populations of some aquatic food species and threatened or endangered aquatic species have plummeted due to overharvesting or habitat degradation. This increasing intensive culture and use of aquatic species has heightened the importance of maintaining a sophisticated understanding of pathology of various organ systems of these diverse species. Yet, except for selected species long cultivated in aquaculture, pathology databases and the workforce of highly trained pathologists lag behind those available for most laboratory animals and domestic mammalian and avian species. Several factors must change to maximize the use, understanding, and protection of important aquatic species: 1) improvements in databases of abnormalities across species; 2) standardization of diagnostic criteria for proliferative and nonproliferative lesions; and 3) more uniform and rigorous training in aquatic morphologic pathology.

Because of their close phylogenic relationship with humans, the use of non-human primates (NHP) as experimental subjects has a long history in biomedical research. Although research topics have shifted focus and species used have changed, NHP remain vital as models in basic and applied research. While there is a wealth of information available on the spontaneous lesions of NHP, most of this information is fragmented, dated, or narrow in focus, often limited to single case reports. This review attempts to integrate this information to illustrate and enumerate the spectrum of spontaneous pathology of the reproductive tract and mammary gland of NHP. Although not the focus of this review, steroid-related changes are inextricably linked to these tissues, and brief consideration is given to this subject as well.

Meeting abstracts Adoptive T cell therapy (ACT) can promote dramatic regressions of established cancer, yet durable clinical responses are observed in only a subset of patients. ACT provides the opportunity to alter the immune suppressive host environment associated with cancer prior to

International audience; Background: Acute hydrogen sulfide (H2S) poisoning produces a coma, the outcome of which ranges from full recovery to severe neurological deficits. The aim of our study was to 1- describe the immediate and long-term neurological effects following H2S-induced coma in un-anesthetized rats, and 2- determine the potential benefit of methylene blue (MB), a compound we previously found to counteract acute sulfide cardiac toxicity.Methods: NaHS was administered IP in un-sedated rats to produce a coma (n = 34). One minute into coma, the rats received MB (4 mg/kg IV) or saline. The surviving rats were followed clinically and assigned to Morris water maze (MWM) and open field testing then sacrificed at day 7.Results: Sixty percent of the non-treated comatose rats died by pulseless electrical activity. Nine percent recovered with neurological deficits requiring euthanasia, their brain examination revealed major neuronal necrosis of the superficial and middle layers of the cerebral cortex and the posterior thalamus, with variable necrosis of the caudate putamen, but no lesions of the hippocampus or the cerebellum, in contrast to the typical distribution of post-ischemic lesions. The remaining animals displayed, on average, a significantly less effective search strategy than the control rats (n = 21) during MWM testing. Meanwhile, 75% of rats that received MB survived and could perform the MWM test (P

Background Papillomavirus disease and associated cancers remain a significant health burden in much of the world. The current protective vaccines, Gardasil and Cervarix, are expensive and not readily available to the underprivileged. In addition, the vaccines have not gained wide acceptance in the United States nor do they provide therapeutic value. Papillomaviruses are strictly species specific and thus human viruses cannot be studied in an animal host. An appropriate model for mucosal disease has long been sought. We chose to investigate whether the newly discovered mouse papillomavirus, MmuPV1, could infect mucosal tissues in Foxn1nu/Foxn1nu mice. Methods The vaginal and anal canals of Foxn1nu/Foxn1nu mice were gently abraded using Nonoxynol-9 and “Doctor’s BrushPicks” and MmuPV1 was delivered into the vaginal tract or the anal canal. Results Productive vaginal, cervical and anal infections developed in all mice. Vaginal/cervical infections could be monitored by vaginal lavage. Dysplasias were evident in all animals. Conclusions Anogenital tissues of a common laboratory mouse can be infected with a papillomavirus unique to that animal. This observation will pave the way for fundamental virological and immunological studies that have been challenging to carry out heretofore due to lack of a suitable model system.

Six common marmosets from a colony of 50 died over a period of 3 weeks, with the predominant finding of gram-negative bacterial septicemia. Four of these animals died peracutely; the other two were found when they were moribund, and they subsequently died despite clinical intervention. Gram-negative bacterial rods were present in the blood vessels of stained tissues from five of the six marmosets. Three marmosets also had severe fibrinopurulent peritonitis. In addition, one of the marmosets with peritonitis also had purulent mesenteric lymphadenitis with large colonies of gram-negative bacterial rods within dialated colonic crypts. Klebsiella pneumoniae was isolated from multiple organs in three of the marmosets. Clinical evaluation of the entire colony identified four marmosets with anorexia, nasopharyngeal discharge and diarrhea. These marmosets were treated with enrofloxacin immediately, and they responded well. K. pneumonia could not be cultured from nasal or fecal samples obtained from the colony animals. Because of the peracute nature of the disease, animals often die before exhibiting clinical symptoms, and antibiotics are seldom helpful. In this outbreak we saw both of the major forms of Klebsiella infection in common marmosets: the peracute form with bacteremia and minimal inflammatory reaction around blood vessels, and the chronic form with bacteremia, fibrinopurulent peritonitis, and mesenteric lymphadenitis.

Despite the identification of several preventive agents and strategies, prevention of lung cancer, especially in smokers who are at high risk, is still largely unattained. 1,4-Phenylenebis(methylene)selenocyanate (p-XSC) has been shown to inhibit tobacco carcinogen NNK induced lung cancer development in several animal models. It metabolizes through the formation of active bis-selenol (p-XSeH) along with the release of poisonous hydrogen cyanide (HCN). Nevertheless, the HCN released upon metabolism of p-XSC to form active metabolite p-XSeH, pose a serious challenge its clinical use in a chemopreventive set up where a continuous intake is required for healthy individuals over a longer period of time. Recently, we developed a hybrid agent, p-XS-Asp, linking p-XSe- to commonly used non-steroidal anti-inflammatory drug, aspirin (Asp), which has been shown to be preventive of lung, and colorectal cancer. We hypothesized that p-XS-Asp would cleave in vivo to release the active p-XSeH, not releasing undesired HCN but the aspirin, thus making the compound less toxic and more potent than p-XSC or aspirin alone. Our studies have shown p-XS-Asp to be orally bioavailable and a highly effective lung cancer chemopreventive agent both in vitro and in animal studies. Elemental selenium (Se) analysis of plasma, lung, and liver tissue in orally fed mice showed that the level of Se significantly higher for p-XS-Asp than p-XSC, denoting a better bioavailability profile for p-XS-Asp. Dietary p-XS-Asp inhibited both O-6 methyl guanine and pyridoxobutyl (pob) DNA adducts, in lung and liver of A/J mice, more effectively than p-XSC. Particularly, in the lung, the inhibition of O-6 methyl guanine adducts, which are critical for A/J mouse lung tumor development, were more than 2 times higher than p-XSC. In a NNK-induced lung cancer A/J mouse model, p-XS-Asp at doses of 15 ppm and 7.5 ppm Se, showed a significantly marked decrease in percentage of lung tumor incidence of 50% and 87%, as compared to p-XSC (79% and 100%), respectively; NNK-control showed an 100% tumor incidence. In addition, the multiplicity for p-XS-Asp was 0.87 and 1.93 tumors/mouse as compared with NNK-control (11.53 tumors/mouse) and p-XSC (1.66 and 4.10 tumors/mouse, respectively) at the two doses tested. Notably, blood and tissue analyses showed no signs of systemic toxicity for the p-XS-Asp fed group. In conclusion, p-XS-Asp, is less toxic and more effective chemopreventive agent than p-XSC and is a promising candidate to future clinical evaluation. Citation Format: Daniel Plano, Cesar Aliaga, Manoj K. Pandey, Arunangshu Das, Timothy K. Cooper, Shantu Amin, Arun K. Sharma. Pre-clinical chemopreventive efficacy of a novel hybrid p-XSC-aspirin compound in a NNK-induced A/J mouse lung cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2139. doi:10.1158/1538-7445.AM2014-2139

Adoptive T cell transfer following whole body irradiation (WBI) can induce dramatic tumor regression in a subset of metastatic melanoma patients. We previously found that this approach also eliminates established autochthonous SV40 T-antigen induced murine brain tumors. However, the broader utility of this immunotherapy approach has not been systematically evaluated in solid tumors of different tissue origin. Here, we assessed the efficacy of applying WBI with adoptive T cell transfer to an SV40 T-antigen driven murine model of prostate cancer (TRAMP) previously shown to rapidly tolerize tumor-specific CD8+ T cells. TRAMP mice bearing established prostate tumors were treated with 475cGy WBI followed by adoptive transfer of T-antigen-specific T cell receptor transgenic CD8+ T cells. Accumulation, phenotype and function of the transferred T cells were monitored by flow cytometry, while therapeutic impact on mouse prostate tumors was assessed by histological scoring. Transferred T cells proliferated in both irradiated and non-irradiated TRAMP mice, but accumulated to significantly higher levels and persisted longer in the secondary lymphoid organs and prostate of irradiated mice. WBI also significantly increased the proportion of T cells that produced the effector molecule granzyme B. Prostate-infiltrating T cells in both treatment groups exhibited an effector differentiated phenotype, although neither developed a significant population of IFNγ-producing cells. Gross observations revealed that the urogenital tract in irradiated TRAMP mice was significantly smaller than that of non-irradiated mice two weeks after transfer, corresponding with a modest but statistically significant drop in pathological score. By three weeks after transfer, when prostate-infiltrating T cells had dramatically contracted in irradiated TRAMP mice, cumulative histological scores were generally equivalent although only mice in the non-irradiated treatment group had progressed to adenocarcinoma. Our results suggest that the addition of irradiation extends the persistence of functional tumor-specific T cells in the highly tolerogenic microenvironment of the TRAMP prostate. T cell accumulation was associated with a short-term delay in progression of established tumors. Citation Format: Lindsay Ward-Kavanagh, Timothy Cooper, Todd D. Schell. Whole body irradiation extends the duration of antitumor immunity toward established prostate tumors by adoptively transferred CD8+ T cells in TRAMP mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3960. doi:10.1158/1538-7445.AM2013-3960

Ovarian cancer, a highly metastatic disease, is the fifth leading cause of cancer-related deaths in women. Chickens are widely used as a model for human ovarian cancer as they spontaneously develop epithelial ovarian tumors similar to humans. The cellular and molecular biology of chicken ovarian cancer (COVCAR) cells, however, have not been studied. Our objectives were to culture COVCAR cells and to characterize their invasiveness and expression of genes and proteins associated with ovarian cancer. COVCAR cell lines (n = 13) were successfully maintained in culture for up to19 passages, cryopreserved and found to be viable upon thawing and replating. E-cadherin, cytokeratin and α-smooth muscle actin were localized in COVCAR cells by immunostaining. COVCAR cells were found to be invasive in extracellular matrix and exhibited anchorage-independent growth forming colonies, acini and tube-like structures in soft agar. Using RT-PCR, COVCAR cells were found to express E-cadherin, N-cadherin, cytokeratin, vimentin, mesothelin, EpCAM, steroidogenic enzymes/proteins, inhibin subunits-α, βA, βB, anti-müllerian hormone, estrogen receptor [ER]-α, ER-β, progesterone receptor, androgen receptor, and activin receptors. Quantitative PCR analysis revealed greater N-cadherin, vimentin, and VEGF mRNA levels and lesser cytokeratin mRNA levels in COVCAR cells as compared with normal ovarian surface epithelial (NOSE) cells, which was suggestive of epithelial-mesenchymal transformation. Western blotting analyses revealed significantly greater E-cadherin levels in COVCAR cell lines compared with NOSE cells. Furthermore, cancerous ovaries and COVCAR cell lines expressed higher levels of an E-cadherin cleavage product when compared to normal ovaries and NOSE cells, respectively. Cancerous ovaries were found to express significantly higher ovalbumin levels whereas COVCAR cell lines did not express ovalbumin thus suggesting that the latter did not originate from oviduct. Taken together, COVCAR cell lines are likely to improve our understanding of the cellular and molecular biology of ovarian tumors and its metastasis.

In our previous proteomic study aimed at developing biomarkers for early detection of lung cancer, we identified several proteins that were differentially expressed in lung adenocarcinoma in A/J mice treated with the tobacco-specific nitrosamine NNK and were further modulated by the synthetic selenium chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC). Unfortunately, many potential biomarkers described in previous reports, including our own, are based on comparative analysis between normal and end-stage lung tumors, and thus, can be considered as a consequence of tumor development rather than a cause of cancer induction. Therefore, in the present study we determined the effect of p-XSC and the naturally occurring chemopreventive agent myo-inositol (MI) on protein profiling during different stages of NNK-induced lung tumorigenesis; we used the proteomic approach isobaric Tags for Relative and Absolute Quantitation (iTRAQ). Four groups of mice were included in this study: vehicle, NNK, NNK+p-XSC, and NNK+MI; ten mice were sacrificed at 3 and 9 weeks after the first dose of NNK and additional mice will be sacrificed after 18, 24, and 31 weeks. Histopathology of the lungs showed airway and alveolar epithelial hyperplasia in all groups after 3 and 9 weeks, as well as airway epithelial atypia; however, no clear difference was observed. Equal amounts of total lung protein from five-mice/group were pooled and labeled with one of the isobaric tags 113-119, or 121. Mass spectrometry analysis led to identification of 790 proteins. After 3 and 9 weeks, 11 and 13 proteins, respectively, were significantly modulated in the lungs of mice treated with NNK vs. vehicle-treated mice; of these proteins, filamin-A was down-regulated, while clathrin heavy chain 1 was up-regulated after 3 and 9 weeks in NNK-treated mice; administration of either p-XSC or MI in NNK-treated mice had no apparent affect. After 3 weeks, the 60S ribosomal protein L3 was significantly down-regulated in mice treated with NNK alone, but was up-regulated in mice treated with NNK+p-XSC or MI; after 9 weeks no apparent change was associated with this protein for all groups. After 3 and 9 weeks, using immunoblot analysis, NNK had no apparent effect on 14-3-3 σ protein expression; however, it was increased in lungs of mice treated with NNK+p-XSC and not NNK+MI. The results of this study show that certain proteins in the lung are modulated after 3 and 9 weeks of NNK administration; the impact of p-XSC on NNK-induced protein alteration appears to vary from that observed with MI. Further validation of the proteins identified in this study and proteomic analysis after 18, 24, and 31 weeks of the bioassay will provide better insight into the selection of candidate protein biomarkers for lung cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1852. doi:10.1158/1538-7445.AM2011-1852

Our laboratories are interested in testing the value of combining omega-3 fatty acids and antiestrogens as a safe and effective chemopreventive strategy for breast cancer. We have shown that a fish oil rich diet (FO) (17% FO + 3% corn oil [CO]) enhances the chemopreventive action of Tamoxifen (Tam) in a prepubertal model of N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis (A. Manni, et al., Cancer Prev Res 3:322, 2010). To gain insight into mechanisms of prevention and the importance of different ratios of n-3:n-6 fatty acids in inhibiting carcinogenesis, we tested the effects of two FO rich diets (17% FO + 3% CO and 10% FO + 10% CO) individually and in combination with Tam on the histologic progression of preneoplastic lesions, indices of proliferation and apoptosis, as well as expression of multiple mammary gland specific and systemic biomarkers of oxidative stress in carcinogen treated rats. Following MNU administration at 21 days of age, separate groups of rats within each experimental arm were sacrificed 24 hours, 21 days, 28 days and 35 days prior to the development of palpable tumors. Increasing the amount of FO in the diet progressively increased the n-3:n-6 ratio in the plasma from 0.13 (20% CO) to 0.38 (10% FO) and 0.96 (17% FO). Although the FO rich diets significantly reduced Ki67 expression in hyperplastic lesions in a dose-dependent fashion (17% FO > 10% FO), neither the diets nor Tam decreased the development of preneoplastic lesions indicating that the inhibitory effect on carcinogenesis results from blocking the transition from hyperplasia to adenocarcinoma, consistent with our recent published study. Dietary FO and/or Tam did not have major effects on systemic oxidative stress biomarkers (plasma protein carbonyls and blood protein bound GSH) or mammary specific biomarkers of oxidative stress, based on oxidative damage to DNA measured as 8-hydroxy-2’-deoxyguanosine (8-OH-dG) and lipid peroxidation assessed as thiobarbituric-acid reactive substances (TBARS). On the other hand, tissue levels of 8-isoprostanes, prostaglandin-like compounds formed by non-enzymatic oxidation of arachidonic acid in the cell membrane, were markedly reduced by both FO rich diets at all time points (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5571. doi:10.1158/1538-7445.AM2011-5571

There are currently no known effective regiments able to prevent the development of estrogen-receptor (ER) negative breast cancer in humans. We hypothesize that ER, frequently upregulated in preneoplastic mammary lesions, may contribute to the development of hormone-independent tumors through crosstalk with multiple cellular oncogenic pathways. Therefore, a multi-targeted approach is likely to be necessary to prevent ER negative tumors. In our previous study, we tested this hypothesis in CD rats and we showed that omega-3 rich fish oil (FO) potentiated the suppressive effect of Tamoxifen (Tam) on mammary carcinogenesis (A. Manni, et al. Cancer Prev Res 3:322, 2010). In the present investigations, we tested this hypothesis in polyoma middle T transgenic mice, since in this experimental system the development of ER negative tumors is preceded by preneoplastic lesions overexpressing functional ER, as evidenced by the concomitant upregulation of progesterone receptors which are products of estrogen action. Administration of increasing concentrations of Tam (1 ppm, 10 ppm, and 100 ppm) admixed with 20% corn oil (CO) modified AIN-76A diet, starting at 3 weeks of age, significantly delayed mammary carcinogenesis, inhibited tumor multiplicity by ∼50%, tumor volume by ∼90%, and tumor weight by ∼70% in a dose dependent fashion. At termination (week 12), however, tumor incidence was nearly 100% in all experimental groups. When Tam treatment was started after tumors had developed, no inhibition of tumor growth was observed, a finding consistent with the absence of ER in invasive adenocarcinomas in this experimental model. Administration of increasing concentrations of FO in the diet (5% FO + 15% CO; 10% FO + 10% CO, 17% FO + 3% CO) started at 3 weeks of age did not affect any of the tumor parameters compared to the control 20% CO diet. Combined administration of suboptimal (1 ppm) and optimal (100 ppm) doses of Tam with 17% FO and 10% FO respectively, delayed carcinogenesis and suppressed tumor multiplicity and volume to the same extent as observed with Tam alone. Therefore, the results indicate that, under these experimental conditions, FO failed to potentiate the chemopreventive action of Tam. Our results, however, provide support for the role of ER expression by the preneoplastic lesions in the development of hormone independent tumors and, consequently, for the importance of including ER targeting in combined chemoprevention strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3710. doi:10.1158/1538-7445.AM2011-3710

Ovarian cancer ranks second among gynecologic cancers, and it is the fifth leading cause of cancer deaths among women; 13,850 deaths are expected in 2010. There is usually no obvious symptoms of early stage ovarian cancer; therefore only 15% of ovarian cancers are diagnosed in the localized stage, and the five-year survival rate for ovarian cancer patients is 46%. A positive relation of mucinous ovarian tumors to tobacco smoking had been reported; furthermore, an association was shown between mucinous ovarian cancer with ex-smokers as well. The mechanisms that can account for the role of tobacco smoking on this disease remain unknown. Currently, there is no preclinical animal model that can accurately reflect the ovarian cancer resulting from chronic exposure to chemical carcinogens present in cigarette smoke. A previous report showed that dibenzo[a,l]pyrene (DB[a,l]P) can induce ovarian cancer in mice. Therefore, in this study we examined the carcinogenicity and DNA adducts formation in the ovary of mice treated with DB[a,l]P. In carcinogenesis study, ovarian tumors in B6C3F1 mice were induced by administering DB[a,l]P into the oral cavity of mice. Several groups of B6C3F1 mice (20/group) received various doses of DB[a,l]P (24, 12, 6, and 3 nmol/mouse), and additional groups received DMSO 3 times a week for 38 weeks or were left untreated as controls. Mice were sacrificed 4 weeks after the last carcinogen administration. A short-term bioassay was also conducted to detect and quantify DNA-adducts induced by DB[a,l]P as a function of time in the ovary; mice were administered orally (24 nmol) 3 times a week for 5 weeks. Ovaries were removed from mice 48 h, 1, 2 and 4 weeks after the last dose. DNA were isolated and hydrolyzed enzymatically, and the DB[a,l]PDE-DNA adducts were analyzed by our newly developed LC-MS/MS method. At the dose of 6 nmol in carcinogenesis study, we observed the highest total ovarian tumor incidence (79%), but the incidence of malignancy was only 15%. However, at the dose of 12 nmol, the total tumor incidence was 75%, and the incidence of malignant granulosa cell tumor was 65%. In addition to ovarian tumors, we also observed lesions in sites distal from the ovaries including the skin, mammary, lung, and oral tissues at the dose of 24 nmol, which were rare in other dosing groups. Treatment of DB[a,l]P in mice resulted in the formation of (−)-anti-cis-DB[a,l]PDE-dA and (−)-anti-trans-DB[a,l]PDE-dA adducts, which were 1.6 and 3.1 fmol/µg DNA respectively in ovaries of mice within 48 hours, and the level of adducts decreased dramatically over a week. Our results indicated that DB[a,l]P can be activated to form (−)-anti-DB[a,l]PDE; the latter may account for DB[a,l]P-induced ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1333. doi:10.1158/1538-7445.AM2011-1333

The annual incidence of cancer of the oral cavity and pharynx in the US is about 30,000 cases, similar to that for leukemia and pancreas, and resulting in a 5 yr survival rate of about 50%. Current models utilizing 4-nitroquinoline-N-oxide in rats and mice or 7,12-dimethylbenzanthracene in the hamster cheek pouch, suffer from relevancy of these synthetic carcinogens and/or the target site. One 2-year feeding study reported that dietary administration of the tobacco smoke carcinogen, benzo(a)pyrene (BaP) leads to tumors at several sites including tongue, but only at very high doses, and mainly in forestomach (Culp, S.J., et al., Carcinogenesis, 9, 117-124, 1998). Here as a potential new model, we investigated whether the more powerful tobacco smoke carcinogen, dibenzo[a,l]pyrene (DB[a,l]P) is mutagenic and carcinogenic in the oral cavity of the B6C3F1 lacI and B6C3F1 mouse resp. upon topical application. Four groups of B6C3F1 lacI mice (6/group) received DB[a,l]P (12, 6, and 3 nmol) or vehicle 3x per week. B6C3F1 mice (20/group) received the same treatment, with an additional 24 nmol group. At 38 weeks the lacI mice were euthanized and DNA was isolated from tongue and upper oral mucosa. For the 12 nmol dose group, the mutant fraction (MF) in the cII gene in upper mucosa and tongue increased about 2-fold relative to that in vehicle-alone (5.2 +/- 0.8 and 4.6 +/- 1.2 vs 2.7 +/- 1.3 and 2.6 +/- 1.2) resp. in units of mutants/105 pfu. The increases were statistically significant (ρ = 0.023 and 0.016) for upper mucosa and tongue resp. The MF's at lower doses were non-significantly elevated. The mutational profile in the DB[a,l]P-induced mutants was compared with that induced by BaP in upper mucosa. BaP was previously shown to be mutagenic in many tissues including oral tissues when administered by gavage (Guttenplan, J.B, et al., Mutation Res., 559,199-210, 2004). Only 4% of BaP-induced mutations were at AT base pairs vs. 31% for DB[a,l]P; consistent with a report of the comparative mutational profile of BaP and DB[a,l]P in lung (Leavitt, S.A., et al., Mutagenesis 23, 445-450, 2008). The fraction of mutations at AT base pairs for DB[a,l]P is very similar to that observed in p53 mutations in the human oral cavity. One yr after the start of treatments, B6C3F1 mice were euthanized and oral squamous cell carcinomas (OSCC) were found in 31% of the high-dose group. Tumors were located in several sites in the oral cavity, although not in tongue. Immunohistochemical analyses revealed that p53 protein was overexpressed in malignant tissues, but not controls. The facts that DB[a,l]P induces mutations and tumors in the oral cavity, and has a mutational profile in oral tissue similar to that found in p53 in human OSCC, indicate DB[a,l]P administration to the mouse oral cavity will likely provide the basis for a new and more relevant model for cancer of the oral cavity than existing models. Supported by NCI grant no. R01 CA 100924 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1968.