The identification of Mouse Mammary Tumour Virus (MMTV) as the causal factor for breast cancer in mice, initiated investigation into a viral cause for human breast cancer. MMTV-like virus has been detected in human breast cancers, lymphomas and primary biliary cirrhosis (PBC), suggesting the virus is not restricted to human breast cancers. We hypothesized that the virus is detected in human tissues influenced by steroid hormones. We detected a region of the envelope (env) gene of MMTV-like virus in 53/210 (25%) of liver disease, 4/21 (19%) of hepatocellular carcinoma (HCC), 14/89 (16%) of ovarian cancer, 53/147 (36%) of prostate cancer, 5/50 (10%) of endometrial cancer and 13/141 (9%) of skin cancer samples but not in lung cancers (0/51). Viral env DNA was also detected in 4/81 (5%) of placentae and 5/90 (6%) of breast milk cells from healthy women whilst viral env RNA was detected in 2/90 (2%) of breast milk supernatants and (0/81) placentae. Immunohistochemistry staining for the presence of estrogen receptor alpha (ER-??) and progesterone receptor (PgR) demonstrated a significant association between ER-??/PgR and MMTV-like virus in human ovarian, prostate, endometrial and skin cancers though no significant association was observed between ER-??/PgR and the virus in liver tissues. We were also unable to demonstrate a significant association between accumulation of p53 tumour suppressor protein and MMTV-like virus in liver disease and HCC. Despite the demonstration of viral env integration in genomic DNA from human placentae using Southern Blots, other regions of the virus were not detected following PCR attempts with published primer sets. This study adds to the current knowledge of distribution of MMTV-like virus in humans. The detection of the virus in hormonally influenced human tissues (positive for ER-?? or PgR) indicates an association between MMTV-like virus and steroid hormones in some human tissues. The detection of the virus in placentae and breast milk also suggests potential routes of transmission of the virus in humans. Although the exact role of the virus in these tissues is not known, the presence of the virus together with other genetic alterations and/or the influence of steroid hormones could be involved in the transformation of various human tissues (i.e.pathogenesis).

Modification of proteins by lipid structure is relatively common post-translational modification that affects the properties of proteins directly and has a forthright effect on the binding of modified proteins to cell membranes. Some lipoproteins play key role in various pathological processes. Before the proteomic analysis of these proteins, the sample of interest is digested using a protease. The resulting hydrolysate contains both unmodified peptides and peptides bearing a lipid modification. During the subsequent chromatographic separation, the lipopeptides differ significantly from the unmodified peptides. For this reason, the analysis of lipopeptides, lipoproteins respectively, is problematic in terms of separation and detection. The subject of the study of this diploma thesis was the optimization of the method of lipoprotein analysis using liquid chromatography and mass spectrometry. The procedures were tested on lipoprotein Cya A (bifunctional adenylate-cyclase toxin from Bordetella pertussis) and MMTV (matrix protein of mouse mammary tumour virus). First, various sample preparation procedures involving proteolytic cleavage were tested. When enzymatic digestion using trypsin on filter (eFASP) was used, the lipid modification was detected with high degree of reliability. In the next step,...

In der vorliegenden Arbeit wurde die Präsentationsfunktion der LEW-Ratten-MHC-Klasse-II-Moleküle RT1B und RT1D für verschiedene Super- und Peptidantigene sowie die Generierung gemischter MHC-Klasse-II-Isotypen in der LEW-Ratte untersucht. Sag sind Proteine bakterieller und viraler Herkunft und führen nach Bindung an MHC-Klasse-II-Moleküle durch Interaktion mit dem TZR-Vb-Teil zu einer von der TZR-Spezifität unabhängigen Aktivierung von T-Zellen, die bis zu 30 % der Gesamt-T-Zellpopulation eines Organismus erfassen kann. Die dadurch bedingte Mediatorenfreisetzung aus T- oder konsekutiv aktivierten Zellen ist einerseits für die Entwicklung bestimmter akuter Krankheitsbilder wie des toxischen Schocksyndroms, Gastroenteritiden u. a. verantwortlich, kann aber auch potentiell zu einer Aktivierung autoreaktiver T-Zellen und der Entstehung von Autoimmunkrankheiten beitragen. Zur Untersuchung der LEW-MHC-Klasse-II-Charakteristika wurden zunächst mittels retroviralen Gentransfers RT1B- und RT1D-Gene in L929-Zellen übertragen und die Oberflächenexpression durch die mAK Ox6 und 14-4-4S nachgewiesen. Anschließend erfolgte der Nachweis einer Sag-Präsentation durch Stimulation des LEW-Vb8.2-TZH 53/4 durch die bakteriellen Sag SEB, SEC1-3, MAS und YPM und von aus Lymphknoten isolierten LEW-T-Zellen durch SEC1, MAS und YPM, die beide mit der durch eine HLA-DR1-positive Zelllinie getragenen Aktivierung verglichen wurden. Beide Experimente ergaben für die Sag des primär humanpathogen Staph. aureus eine weitaus stärkere Reaktivität in Anwesenheit humaner gegenüber LEW-MHC-Klasse-II-Molekülen bei RT1B-dominierter Antwort innerhalb der präsentatorischen Rattenmoleküle. Für SEB ergaben sich zusätzlich Hinweise auf eine MHC-Klasse-II-unabhängige Aktivierung. Das von Yersinia pseudotuberculosis produzierte Sag YPM wurde ebenfalls wesentlich besser von humanen als LEW-MHC-Klasse-II-Molekülen präsentiert, zeigte allerdings nur geringe Unterschiede zwischen RT1B und RT1D. Für das aus Nagern isolierte Sag von Mykoplasma arthritidis MAS konnte eine präferentielle Bindung an RT1D mit HLA-DR1-ähnlicher Stimulationskapazität nachgewiesen werden. Darüber hinaus wurden die generierten Zelllinien auf Präsentation der definierten Antigene L.casein und gpMBP gegenüber reaktiven TZH getestet. Dabei konnte die RT1D-restringierte Antwort von Klon19 auf L.casein und die RT1B-restringierte Antwort von 53/4 auf gpMBP bestätigt werden. Ebenfalls wurden die erstellten Transfektanten mit einem viralen Sag der Maus, dem vSag7-Gen des mtv7, transfiziert und auf Stimulation des TZH RG17 und von LEW-Lymphozyten getestet. Dabei zeigte sich eine geringe Reaktivität gegenüber den erstellten Transfektanten, die RT1B-dominiert war. Gleichzeitig ergaben sich in der Auswertung Hinweise für einen vSag7-Transfer von MHC-Klasse-II-negativen Produzenten auf MHC-Klasse-II-positive Rattenzellen, die durch weitere Experimente inklusive eines In-vivo-Tests bestätigt werden konnten. In der Generierung gemischter Isotypen aus MHC-Klasse-II-Einzelkettengenen der LEW-Ratte konnte gezeigt werde, dass die Übertragung der RT1DaBb-Genkombination mit Hilfe eines retroviralen Gentransfers auf P80- und L929-Zellen nicht zu einer sicher detektierbaren Oberflächenexpression führte, auch nicht bei Koübertragung der invarianten Kette der Maus. Durch einen Western Blot unter reduzierenden Bedingungen konnte eine bezüglich Molekulargewicht und Quantität zu einem regulären RT1B-Molekül differente RT1Bb-Einzelkette in der Kombination RT1DaBb nachgewiesen werden. / In the work at hand the presenting function of LEW rat MHC class II molecules RT1B and RT1D for various superantigens and antigens as well as the generation of mixed MHC class II isotypes in the LEW rat have been investigated. Superantigens are proteins of bacterial and viral origin, which lead to a TCR-specificity-independent activation of up to 30 % of the individual's T-cell population by interacting with the Vb part of the T-cell receptor after having bound to an MHC class II molecule. The release of mediators by T- and consecutively activated cells causes on the one hand development of acute diseases like toxic shock syndrome, gastroenteritis and other, but can also potentially activate autoreactive T-cells and lead to autoimmune diseases. In order to examine characteristics of LEW MHC class II molecules, first RT1B and RT1D chain genes were transferred into L929 cells via a retroviral transfection system and surface expression was demonstrated by using the monoclonal antibodies Ox6 and 14-4-4S. Successively, superantigen presentation was verified by stimulation of the LEW Vb8.2+ T-cell hybridoma 53/4 by bacterial superantigens SEB, SEC1-3, MAS and YPM and of LEW T-cells isolated from lymph nodes by SEC1, MAS and YPM. Both results were compared to activation in context of an HLA-DR1+ cell line. Experiments showed a much higher reactivity for the superantigens of human pathogen staph. aureus in presence of human versus LEW MHC class II molecules and an RT1B dominated answer amongst LEW presentatory molecules. Additionally, clues for MHC class II independent activation were found in case of SEB. The superantigen of yersinia pseudotuberculosis YPM was also much better presented by human than LEW MHC class II molecules while showing little differences between RT1B and RT1D. MAS bound preferentially to RT1D and equal stimulative capacity compared to HLA-DR1 could be detected. Accessorily, generated cell lines were analysed for presentation of peptide antigens L.casein and gpMBP towards reactive T-cell hybridomas, in which RT1D-restricted answer of Klon19 to L.casein and RT1B-restricted answer of 53/4 to gpMBP could be confirmed. Also generated cell lines were transfected with a viral mouse superantigen, the vsag7 gene of mtv7, and tested for stimulation of the RG17 T-cell hybridoma and LEW lymphocytes. Low reactivity towards transfected cell lines was detected, which was dominated by RT1B. Additionally, evidence for a transfer of vsag7 from MHC class II- producers to MHC class II+ rat cells could be found, which was enhanced by additional experiments including in vivo testing. Attempting to create mixed isotypes consisting of LEW rat MHC class II chains, it was demonstrated, that transferring the gene combination RT1DaBb via retroviral gene transfer into P80 and L929 cell lines resulted in no certain surface expression, also not under cotransfection of these cells with mouse invariant chain. Using western blot method under reducing conditions, a RT1Bb single chain different to the one of the regular RT1B molecule concerning molecular weight and quantitiy could be detected in the combination RT1DaBb.

The adult prevalence of obesity in the United States exceeds 30% and obesity is associated with increased cancer risk and poor prognosis, including postmenopausal breast cancer. p53 is a tumor suppressor gene that responds to diverse cellular stress including DNA damage, oxidative stress and hypoxia. p53 is mutated in most human cancers, including postmenopausal breast cancer, and is involved in the regulation of lipogenic enzymes. However, the links between p53 and obesity in postmenopausal breast cancer are poorly understood. Here we test the hypothesis that the effect of obesity on mammary tumor growth is impacted by p53 status. The aim of this study was to determine how p53-deficient mammary tumor cells (relative to p53 wild-type cells) respond to obesity-driven tumor growth. To test this hypothesis, we used ovariectomized (OVX) C57BL/6 mice randomized to a control diet (n=40) or a diet-induced obesity (DIO) regimen (n=40) for 10 weeks. At the time, DIO mice were approximately 40% heavier (p<0.001) and had 45% greater adiposity (p<0.001) than control mice. Mice were then injected (in the 4th mammary fat pad) with either p53-deficient (p53+/-) or p53 wild-type (p53+/+) MMTV-Wnt-1 mammary tumor cells. Mice were monitored for tumor growth, killed when moribund, and tumors were collected at study end point. We found an interaction between diet and p53 status, with p53+/+ Wnt-1 tumors grown in DIO mice developing the more aggressive morphology compared to p53+/+ Wnt tumors in control mice while the observation was not seen in p53+/- Wnt tumors. From histopathological analysis we also discovered that the DIO regimen promotes local invasion of mammary tumor cells and alters the morphology of MMTV-Wnt-1 p53+/+ mammary tumors. Specifically, p53+/+ Wnt tumors grown in DIO mice displayed disorganized ductal structures characteristic of p53+/- tumors grown in control mice, and DIO exacerbated this aberrant morphology in p53+/- Wnt tumors. Moreover, immunohistological analyses showed that DIO reduces p53 protein expression while elevating Ki-67 expression only in the p53+/+ Wnt mammary tumors. These results suggest that p53 and DIO have interactive effects on mammary tumor growth, as p53+/+ Wnt tumors growing in DIO mice resulted in higher tumor grade similar to p53+/- Wnt tumors. / text

Estrogen therapy (ET) is most effective to reduce menopausal symptoms and prevent other disorders associated with estrogen deficiency. However, Women's Health Initiative studies found that hormone therapy (HT) containing estrogen plus progestogen, but not estrogen-alone increases breast cancer (BC) risk. To prevent the increase in BC risk and yet relieve menopausal symptoms, a novel HT with 17β-estradiol (E2) for symptom relief, progesterone (P4) for uterine protection and melatonin (Mel) for both BC and uterine protection was designed. Inclusion of Mel was postulated to offer uterine protection with lower P4 dose and protect against BC. The goal of this study was to assess the efficacy of E2, P4 and Mel Therapy (EPMT) on mammary cancer (MC) and uterine protection in MMTV-Neu mouse model that mimics HER2 BC. Starting at 2 months age, female mice received Mel in drinking water at night to supplement endogenous Mel surge; while E2 and P4 Therapy (EPT) was provided continuously in diet until 14 months with weekly MC onset and growth monitoring. Normal mammary, uterus and mammary tumors harvested by month 14 were analyzed for potential mechanisms. The results from this study revealed that EPMT delayed tumor onset leading to a decrease in MC incidence. In addition, mice in the EPMT group had no increase in relative uterine weight as opposite to an increase of this parameter in EPT group versus control. The percent tumor-bearing mice with gross metastatic lung lesions were reduced in Mel, EPT and EPMT groups. Mel receptor, estrogen receptor (ER) and progesterone receptor (PR) expression revealed that all tissues examined have Mel receptors. However, ER and PR expression varied. In normal mammary tissue, both ERα and PR were detected by immunohistochemistry. However, no ERα and PR were detected in mammary tumors of same mice. In uterus, mice given Mel or EPMT had significant decreases in PR expression but no change in ERα expression compared to control suggesting that Mel-mediated inhibition of ER binding to estrogen response elements may be involved in the down regulation of uterine PRs. Overall, this study reveal that EPMT prevents mammary cancer and may protect against uterotrophy. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Pharmacology / PhD; / Dissertation;

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

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Estrogen therapy (ET) is most effective to reduce menopausal symptoms and prevent other disorders associated with estrogen deficiency. However, Women's Health Initiative studies found that hormone therapy (HT) containing estrogen plus progestogen, but not estrogen-alone increases breast cancer (BC) risk. To prevent the increase in BC risk and yet relieve menopausal symptoms, a novel HT with 17&#946;-estradiol (E2) for symptom relief, progesterone (P4) for uterine protection and melatonin (Mel) for both BC and uterine protection was designed. Inclusion of Mel was postulated to offer uterine protection with lower P4 dose and protect against BC. The goal of this study was to assess the efficacy of E2, P4 and Mel Therapy (EPMT) on mammary cancer (MC) and uterine protection in MMTV-Neu mouse model that mimics HER2 BC. Starting at 2 months age, female mice received Mel in drinking water at night to supplement endogenous Mel surge; while E2 and P4 Therapy (EPT) was provided continuously in diet until 14 months with weekly MC onset and growth monitoring. Normal mammary, uterus and mammary tumors harvested by month 14 were analyzed for potential mechanisms. The results from this study revealed that EPMT delayed tumor onset leading to a decrease in MC incidence. In addition, mice in the EPMT group had no increase in relative uterine weight as opposite to an increase of this parameter in EPT group versus control. The percent tumor-bearing mice with gross metastatic lung lesions were reduced in Mel, EPT and EPMT groups. Mel receptor, estrogen receptor (ER) and progesterone receptor (PR) expression revealed that all tissues examined have Mel receptors. However, ER and PR expression varied. In normal mammary tissue, both ER&#945; and PR were detected by immunohistochemistry. However, no ER&#945; and PR were detected in mammary tumors of same mice. In uterus, mice given Mel or EPMT had significant decreases in PR expression but no change in ER&#945; expression compared to control suggesting that Mel-mediated inhibition of ER binding to estrogen response elements may be involved in the down regulation of uterine PRs. Overall, this study reveal that EPMT prevents mammary cancer and may protect against uterotrophy. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Pharmacology / PhD; / Dissertation;

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成2年11月22日 竹中徳哉氏の博士論文として提出された