The ets gene family of transcription factors has been widely implicated in a variety of human tumors. PEA3, the founding member of the PEA3 subfamily of ets genes is overexpressed in a mouse model of mammary tumorigenesis as well as in primary human breast tumors. PEA3 deregulation in these tumors is thought to increase the metastatic potential of mammary tumors by increasing the expression of various matrix metalloproteinases. The identification of other ets gene's expression using a mouse model of HER2/Neu-induced mammary tumorigenesis would provide insight into the mechanisms behind these mammary tumors. Degenerate RT-PCR analysis was used to screen for expression of all known ets genes in these tumors. A large spectrum of ets genes was identified as being expressed in these tumors. Quantitative analyses including semi-quantitative RT-PCR and ribonuclease protection assays, indicate that the PEA3 subfamily of ets genes, including PEA3, ERM and ER81, as being overexpressed in these tumors, while other ets genes, Ets-1, Ets-2 and GABP(alpha) were not. These results imply a specific role for the PEA3 subfamily in this model of mammary tumorigenesis and isolate this subfamily of ets genes as a possible therapeutic target. / Thesis / Master of Science (MS)

Mouse mammary tumor virus (MMTV) is a betaretrovirus that causes mammary tumors in mice. MMTV is the only known complex murine retrovirus and encodes Rem, an HIV-1 Rev-like protein. Rem is a 301-amino-acid (33 kDa) protein that is cotranslationally targeted to the ER, where the first 98 amino acids constitute the signal peptide (SP). The SP is cleaved and retrotranslocated to the cytoplasm prior to nuclear entry. In this thesis, the results show that the presence of a leucine at position 71 allows more efficient cleavage of SP and increases Rem activity. Further, in Rem-transfected cells, the majority of SP appears in the nuclear fraction, consistent with fluorescent microscopy data. The C-terminal fragment of Rem (RemCT) is glycosylated in the ER and, although glycosylation sites are present outside the SP, mutations of both these sites abolish SP activity in a reporter assay. Indirect evidence suggests that unglycosylated RemCT is degraded by the proteasome, whereas glycosylated RemCT is likely secreted out of the cell. A variant of MMTV (TBLV) that lacks functional Sag and RemCT has been prepared and will be studied in mice to elucidate the role of RemCT in vivo. Development of an antibody to RemCT will allow tracking of the protein in virus-producing cells. Although there are many other similarities between complex retroviruses like HIV-1 and MMTV, current evidence suggests that Rem lacks an HIV Tat-like transactivator function. / text

A avaliação de linfonodos é parte importante do estadiamento das neoplasias mamárias, muito comum em cadelas, podendo auxiliar no estabelecimento do prognóstico e na escolha da melhor conduta terapêutica. Neste trabalho, dentre as várias técnicas de exame que permitem a avaliação de linfonodos, foi utilizada a ultra-sonografia. A avaliação ultra-sonográfica em modo B possibilitou avaliar tamanho, contorno, borda, forma, arquitetura, ecotextura e ecogenicidade do parênquima dos linfonodos e, em modo Doppler, a quantidade e distribuição dos seus vasos internos. Os objetivos deste trabalho foram identificar as características ultra-sonográficas mais importantes utilizadas para classificar os linfonodos em metastáticos e não-metastáticos, estabelecer elementos de confiabilidade do ultra-som como ferramenta para diferenciar linfonodos metastáticos de não-metastáticos e estabelecer procedimentos que permitam ao exame ultra-sonográfico de linfonodos, para essa classificação, ser reprodutível. Foram examinados 63 linfonodos inguinais superficiais de 30 cadelas com tumor em pelo menos uma das mamas inguinais ou abdominais caudais. Após o exame, cada linfonodo foi classificado como metastático ou não-metastático. A impressão diagnóstica do exame ultra-sonográfico foi associada aos resultados do exame histopatológico dos linfonodos obtendo-se uma taxa de concordância de 92,5%, índice de sensibilidade de 94,1%, índice de especificidade de 92%, valor preditivo positivo de 0,8 e valor preditivo negativo de 0,9787. As principais características ultra-sonográficas que classificaram um linfonodo inguinal superficial como metastático foram o contorno irregular, forma arredondada com razão C/E menor que dois, arquitetura desorganizada, ecotextura homogênea ou heterogênea, ecogenicidade alterada, vascularização elevada e de localização periférica. Quanto aos linfonodos não-metastáticos, as principais características ultra-sonográficas identificadas foram contorno regular, forma ovalada com razão C/E maior que dois, arquitetura preservada, ecotextura preservada, ecogenicidade normal, vascularização discreta a pequena e de localização hilar. O exame ultra-sonográfico dos linfonodos regionais constitui importante ferramenta na detecção de metástase e sugere-se incluí-lo como rotina do estadiamento de neoplasias mamárias em cadelas. / The evaluation of lymph nodes is an important part of the staging in case of mammary tumors, which are very common in female dogs. It can help in establishing a prognosis and chosing the best therapeutic procedures. In this study we used ultrasonography to assess lymph nodes within a variety of imaging techniques. B mode ultrasonography was used to provide information on size, edge, nodal borders, shape, architecture, echotexture and echogenivity of lymph nodes and Doppler ultrasonography to evaluate the quantity and distribution of the intranodal vessels. The objectives of this work were to identify which features in an ultrasound image can be associated to the metastatic or non-metastatic character of a lymph node; to establish the accuracy of the ultrasound as a tool to differentiate between metastatic and non-metastatic behavior and to establish procedures that permit the reproducibility of the results. Sixty three lymph nodes in thirty female dogs with mammary tumors were examined. We chose female dogs that had involvement of at least one of the inguinal or caudal abdominal mammary glands. Ultrasound imaging and histopathological exams were performed for each lymph node. Based on its ultrasound imaging, each lymph node was classified as metastatic or non-metastatic. When the ultrasound diagnostic was matched to the results of the histopathologic exam it was found that the classification of the lymph nodes using ultrasound imaging was correct in 92.5% of the cases, with sensibility of 94.1%, specificity of 92%, positive predictive value of 0.8 and negative predictive value of 0.9787. The main features in an ultrasound image associated with a metastatic superficial inguinal lymph node were irregular edge, round shape with long to short axis ratio lower than 2, loss of internal structure, homogeneous or heterogeneous echotexture, alteration of the echogenicity and high and peripheral vascularity. Relating to non metastatic lymph nodes, the main ultrasonographic characteristics were regular edge, oval shape with long to short axis ratio higher than 2, maintenance of internal structure, normal echo genetics and low hilar vascularity. The ultrasound imaging of regional lymph nodes is an important tool to detect metastasis and it is suggested that it becomes a regular procedure in the staging of canine mammary tumors.

Not available / text

Vitamin E is a group of naturally occurring fat soluble compounds which consists of eight distinct forms of tocopherols and tocotrienols. Although a well-defined physiological function of vitamin E is as an antioxidant, beneficial effects of individual vitamin E compounds on chronic human diseases such as cancer need to be better understood. Studies in this dissertation investigated potential application of gamma-tocopherol (gamma-T), gamma-tocotrienol (gamma-T3) or synthetic derivatives of tocotrienols as anticancer agents in comparison to alpha-tocopherol (alpha-T), its redox-silent acetic acid derivative (alpha-TEA) or alpha-tocotrienol (alpha-T3). Redox-silent derivatives of alpha- and gamma-T3; namely alpha-T3EA and gamma-T3EA exhibited potent anti-proliferative and proapoptotic activities in a murine mammary cancer cell line as well as in human breast cancer cell lines. Moreover, studies using human vascular endothelial cells in cell culture showed that the tocotrienol derivatives exhibited strong antiangiogenic activities which were markedly improved over those of the parent compounds. An antitumor efficacy study using the 66cl-4-GFP syngeneic mouse mammary tumor model showed that each tocotrienol derivative, when delivered in the diet, significantly suppressed mammary tumor growth; however serum and tissue concentrations of these novel compounds were lower than those of alpha-TEA, suggesting that the next generation of vitamin E derivatives will need to be modified to improve bioavailability. On the other hand, some natural-source vitamin E forms, especially gamma-forms, display anticancer activities without any chemical modification in both in vitro cell culture studies and in vivo animal models. Dietary delivery of gamma-T3 suppressed tumor growth in a syngeneic implantation mouse mammary cancer model by inhibiting cell proliferation and inducing apoptosis. Cell culture studies using human breast cancer cells showed that gamma-T3 triggered apoptosis by inducing endoplasmic reticulum (ER)-stress mediated by acid sphingomyelinase (ASMase) action. Activation of stress-activated mitogen-activated protein kinases (MAPKs), JNK and p38, was associated with gamma-T3-induced ER stress followed by upregulation of extrinsic death receptor-5 (DR5) expression in a CHOP transcription factor dependent manner. Gamma-T also triggered extrinsic apoptosis signaling by increasing DR5 mRNA, protein and cell surface expression levels followed by mitochondria-dependent apoptotic signaling. In agreement with in vitro studies, gamma-T delivered in the diet suppressed the tumor growth of MDA-MB-231-GFP human breast cancer cells in a xenograft model but the antitumor activity of gamma-T was hampered by co-administration of alpha-T. The preferential tissue retention of alpha-T over gamma-T could be overcome by use of sesamin, a dietary source of human cytochrome P450 inhibitor. Based on data presented, gamma-T and gamma-T3 show preclinical potential for cancer treatment either as single agents or in combination with other agents. / text

The specific objective of this study was to determine if Fgf-3 gene expression is mediated by dietary fatty acids and to confirm mouse mammary tumor virus infection. It is well known that dietary linoleic acid enhances growth and dietary stearic acid inhibits growth of mammary tumors. Tumor RNA was extracted from female strain A/St mice fed one of four diets. A radioactively labeled anti-sense RNA probe was generated, invitro, from isolated and purified pFgf-3c (int-2c clone contained in the vector pSP65). The Fgf-3c probe was hybridized to extracted tumor RNA using the ribonuclease protection assay.Electron microscopy confirmed MMTV infection by visualization of type A and B particles in tumor tissue. Expression of Fgf-3c, qualified by RNase protection assay, ranged from 0.02 to 5.89 (relative band density) in all of the diet groups. A positive association between Fgf-3c expression and weight was observed among the tumors of the SA-1 diet (R = 0.947). The SF, SF-1, and PA experimental diets, individually, did not appear to show strong correlation with respect to tumor size. Fgf-3 expression was less in small tumors (<275 mg) and enhanced in large tumors (>275 mg) (p<0.05). / Department of Biology

The Met receptor tyrosine kinase (RTK) is expressed in the mammary gland under both normal and neoplastic conditions. Overexpression of the Met receptor is found in 15--20% of human breast cancers and is correlated with shortened disease-free interval and overall survival. In order to explore the role of dysregulated Met receptor signaling on the development of mammary tumors I have characterized a transgenic mouse model that expresses either wild type or a dysregulated Met receptor in the mammary epithelium under the control of the mouse mammary tumor virus promoter/enhancer (MMTV-Met). The Met receptor variants contained a mutation that results in decreased receptor ubiquitination and prolonged receptor signaling (Y1003F) or an activating mutation that was originally observed in patients with papillary renal carcinoma (M1250T) or both mutations (YF/MT). In vitro and in vivo transformation assays demonstrated that each mutation singly is weakly transforming, however, there was an additive effect on transformation when both mutations were present. This additive effect was observed in the transgenic mice where multiparous MMTV-Met-YF/MT mice developed tumors earlier and with much greater penetrance than did mice expressing either of the single mutants. This provides the first in vivo model that demonstrates a role for ubiquitination in suppression of transforming activity of an RTK. MMTV-Met-YF/MT tumors displayed a range of histological phenotypes but were mainly comprised of luminal lineage cells. Notably, MMTV-Met-M1250T tumors contained cells from both the basal and luminal populations, suggesting transformation of a progenitor cell. Progenitor cell transformation in RTK transgenic mouse models is uncommon and highlights distinct signaling differences and potentially lineage specificity of the two Met mutants. / Through assays of overexpression in vivo and inhibition in vitro, Met receptor signaling has been correlated with the development of the mammary gland. To examine the effects of loss of Met receptor signaling on mammary gland development I have utilized the Cre/LoxP1 recombination system to knock-out the Met receptor from the mammary epithelium. Mammary-specific Cre recombinase efficiently excised floxed DNA as visualized by activation of a beta-galactosidase reporter In Met+/+ glands, however, few beta-galactosidase positive cells are retained In the Mefl/fl glands and an intermediate number are retained in the Met fl/+ glands. This indicates that Met-null cells are selected against and supports a role for Met in the development of the mammary gland.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references.