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Purpose: The purpose of this study is to determine whether mouse mammary tumor virus (MMTV)-associated human breast cancer has the same or similar histology to MMTV-associated mouse mammary tumors. Such associations may indicate a role for MMTV in human breast cancer. Methods: Immunohistochemical techniques (using antibodies directed against the signal peptide p14 of the envelope precursor protein of MMTV) and polymerase chain reaction (PCR) analyses were used to identify MMTV proteins and MMTV-like envelope gene sequences in a series of breast cancers from Australian women. The histological characteristics of these human breast cancer specimens were compared with MMTV positive mouse mammary tumors. The same methods were used to study benign breast tissues which 1-11 years later developed into breast cancer. Results: MMTV p14 proteins were identified in 27 (54%) of 50 human breast cancers. MMTV env gene sequences were identified by PCR in 12 (27%) of 45 human breast cancers. There was a significant correlation between the presence of MMTV (identified by p14 immunohistochemistry) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.001). There was a non-significant correlation between the presence of MMTV env gene sequences (identified by PCR) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.290). MMTV p14 proteins were identified in 7 (54%) of 13 benign breast specimens that later developed into human breast cancers. MMTV by PCR was identified in two benign specimens one of whom later developed MMTV positive breast cancer. Discussion: These observations offer evidence that MMTV may be associated with characteristic human breast cancer histology. p14-based immunohistochemistry appears to be a more reliable technique than PCR for the identification of MMTV in human breast cancer. Identification of MMTV-associated p14 proteins in benign breast tissues c

Background: Multiple oncogenic viruses including, mouse mammary tumor virus, bovine leukemia virus, human papilloma virus, and Epstein Barr virus, have been identified as separate infectious pathogens in human breast cancer. Here we demonstrate that these four viruses may be present in normal and benign breast tissues 1 to 11 years before the development of same virus breast cancer in the same patients. Methods: We combined the data we developed during investigations of the individual four oncogenic viruses and breast cancer. Patients who had benign breast biopsies 1-11 years prior to developing breast cancer were identified by pathology reports from a large Australian pathology service (Douglas Hanly Moir Pathology). Archival formalin fixed specimens from these patients were collected. The same archival specimens were used for (i) investigations of mouse mammary tumour virus (also known as human mammary tumour virus) conducted at the Icahn School of Medicine at Mount Sinai, New York and at the University of Pisa, Italy, (ii) bovine leukemia virus conducted at the University of California at Berkeley,(iii) human papilloma virus and Epstein Barr virus conducted at the University of New South Wales, Sydney, Australia. Seventeen normal breast tissues from cosmetic breast surgery conducted on Australian patients were used as controls. These patients were younger than those with benign and later breast cancer. Results: Standard and in situ polymerase chain reaction (PCR) methods were used to identify the four viruses. The detailed methods are outlined in the separate publications.: mouse mammary tumor virus, human papilloma virus and Epstein Barr virus (Infect Agent Cancer 12:1, 2017, PLoS One 12:e0179367, 2017, Front Oncol 5:277, 2015, PLoS One 7:e48788, 2012). Epstein Barr virus and human papilloma virus were identified in the same breast cancer cells by in situ PCR. Mouse mammary tumour virus was identified in 6 (24%) of 25 benign breast specimens and in 9 (36%) of 25

Background: There is substantial evidence that a virus homologous to mouse mammary tumor virus (MMTV) may have a role in human breast cancer. The present study indicates that those who developed breast cancer associated with an MMTV-like virus had this virus in their non-cancerous breast tissues years before the cancer developed. Methods: Polymerase chain reaction (PCR) techniques and sequencing were used to identify MMTV-like envelope gene sequences (MMTV-like env sequences) in Australian benign breast biopsy specimens from women who several years later developed breast cancer. Murine contamination was excluded by stringent laboratory procedures, and the absence of intracisternal A particle sequences and mitochondrial cyclooxygenase sequences. Results: MMTV-like env sequences (also called HMTV sequences to denote their source) were found in 9 of 25 breast cancer specimens (36%). Among 25 non-cancerous breast biopsies of these same patients taken 1 to 11 years earlier, six contained MMTV-like sequences (24%). Five of the six were among the nine virally-associated breast cancers. In two pairs of specimens, benign and malignant, env sequences were 97% identical. Conclusions: The identification of MMTV (MMTV-like) sequences in breast tissues prior to the development of MMTV positive breast cancer fulfills a key criterion for a possible causal role for the MMTV-like virus in human breast cancer.

Active participation of endogenous retroviruses (ERVs) in disease processes has been exemplified by the finding that the HERV (human ERV)-W envelope protein is involved in the pathogenesis of multiple sclerosis, an autoimmune disease. We also demonstrated that injury-elicited stressors alter the expression of murine ERVs (MuERVs), both murine leukemia virus-type and mouse mammary tumor virus (MMTV)-type (MMTV-MuERV). In this study, to evaluate MMTV-MuERVs' responses to stress (e.g., injury, infection)-elicited systemic glucocorticoid (GC) levels, we examined the GC-stress response of 64 MMTV-MuERV promoters isolated from the genomes of 23 mouse strains. All 64 promoters responded to treatment with a synthetic GC, dexamethasone (DEX), at a wide range from a 0.6- to 85.7-fold increase in reporter activity compared to no treatment. An analysis of the 10 lowest and 10 highest DEX responders revealed specific promoter elements exclusively present in either the three lowest or the two highest responders. Each promoter had a unique profile of transcription regulatory elements and the glucocorticoid response element (GRE) was identified in all promoters with the number of GREs ranging from 2 to 7. The three lowest DEX responders were the only promoters with two GREs. The findings from this study suggest that certain MMTV-MuERVs are more responsive to stress-elicited systemic GC elevation compared to the others. The mouse strain-specific genomic MMTV-MuERV profiles and individual MMTV-MuERVs' differential responses to GC-stress might explain, at least in part, the variable inflammatory responses to injury and/or infection, often observed among different mouse strains. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.

Complement system is a part of innate and adaptive immune system that act as a ‘first line of defense’ against infections and immune complex diseases. There are three main pathways activate the complement system; classical pathway, alternative pathway and lectin pathway. Although these three pathways are activated by different triggers and stimuli, these step-wise activations eventually lead to the cleavage of the C5 molecule and generating C5a and C5b. C5a is a 74 amino acids sequence protein fragment derived from the cleavage of complement component by protease C5-convertase. C5a was first described as a classical anaphylatoxin and a highly inflammatory peptide and have been found in the serum of patient with inflammatory disorder such as cancer. However, the possible involvement between C5a anaphylatoxin and tumour progression is not yet well understood. In this study, we assessed the expression of C5a/C5aR in the EMT6 cells and measured the expression and up-regulation of C5a/C5aR in both mRNA and protein levels. We demonstrate that the expression of C5a/C5aR is up-regulated in EMT6 cells and can be potentially use as a screening biomarker for mammary cancer. The first part of study was the determination of the expression of C5a/C5aR mouse mammary cancer cells by Immunofluorescence staining using compatible antibodies followed by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) of EMT6 cell. The study was further analyzed by treating the EMT6 cells using three types of treatment; EP54 (C5a agonist peptide), PMX205 (C5a antagonist peptide) and control drug of tamoxifen. Thus, the magnitude of the C5a/C5aR expression of the treated EMT6 cell was measured by quantitative Real-Time Polymerase Chain Reaction (qPCR). The validation assessment of C5a/C5aR as a screening biomarker was determined using cell cytotoxicity assay of MTT and enzyme-linked immunosorbent assay (ELISA) using C5a antibodies. The immunofluorescence staining of EMT6 cells indicate the prese

Complement system is a part of innate and adaptive immune system that act as a ‘first line of defense’ against infections and immune complex diseases. There are three main pathways activate the complement system; classical pathway, alternative pathway and lectin pathway. Although these three pathways are activated by different triggers and stimuli, these step-wise activations eventually lead to the cleavage of the C5 molecule and generating C5a and C5b. C5a is a 74 amino acids sequence protein fragment derived from the cleavage of complement component by protease C5-convertase. C5a was first described as a classical anaphylatoxin and a highly inflammatory peptide and have been found in the serum of patient with inflammatory disorder such as cancer. However, the possible involvement between C5a anaphylatoxin and tumour progression is not yet well understood. In this study, we assessed the expression of C5a/C5aR in the EMT6 cells and measured the expression and up-regulation of C5a/C5aR in both mRNA and protein levels. We demonstrate that the expression of C5a/C5aR is up-regulated in EMT6 cells and can be potentially use as a screening biomarker for mammary cancer. The first part of study was the determination of the expression of C5a/C5aR mouse mammary cancer cells by Immunofluorescence staining using compatible antibodies followed by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) of EMT6 cell. The study was further analyzed by treating the EMT6 cells using three types of treatment; EP54 (C5a agonist peptide), PMX205 (C5a antagonist peptide) and control drug of tamoxifen. Thus, the magnitude of the C5a/C5aR expression of the treated EMT6 cell was measured by quantitative Real-Time Polymerase Chain Reaction (qPCR). The validation assessment of C5a/C5aR as a screening biomarker was determined using cell cytotoxicity assay of MTT and enzyme-linked immunosorbent assay (ELISA) using C5a antibodies. The immunofluorescence staining of EMT6 cells indicate the prese

The “diploid” genome of retroviruses consists of two strands of RNA that are non-covalently linked as a “dimer” at their 5’ ends. The ubiquitous presence of a dimeric genome among retroviruses suggests that dimerization plays a crucial role in gRNA packaging during viral life-cycle. For almost all retroviruses, determinants of gRNA dimerization and packaging, which are for the most part physically and genetically indistinguishable, reside at the 5’ end of the gRNAs and have been shown to assume higher order structures. Employing a combination of genetic and structural prediction approaches, we have earlier shown that Mason-Pfizer monkey virus (MPMV) and mouse mammary tumor virus (MMTV) packaging determinants comprise sequences at the 5’ end of the genome, starting from R and extending into the beginning of Gag. Sequences encompassing these regions were predicted to fold into stable RNA secondary structures comprising several structural motifs. In an attempt to establish structure-function relationship of the higher order features of MPMV and MMTV packaging signal RNAs, we first validated their predicted structures employing a novel chemo-enzymatic probing strategy, selective 2’hydroxyl acylation primer extension (SHAPE). The SHAPE-analyzed structures of MPMV and MMTV packaging signal RNAs validated the major structural motifs, including U5/Gag long range interactions (LRIs), a stretch of single-stranded purine (ssPurine)-rich region, and a distinctive GC-rich palindromic (pal) helix loop. Minimum free-energy structure predictions, phylogenetic, and in silico analyses of different MPMV and MMTV strains further suggested the existence of these major structural motifs. viii To test the importance of the pal sequences in MPMV and MMTV gRNA dimerization and packaging, we introduced a series of mutations in the pal helix loops. Tests of these mutations employing in vitro and in vivo complementary approaches, phylogenetic, and structure prediction analyses revealed pal

Antiangiogenic therapy of cancers targets their blood vessels in an effort to deprive tumors of oxygen and nutrients. Results from human trials have been poorer than results from mouse testing. This disparity may be explained by more extensive coverage of vessels in human cancers, like breast cancer, by protective periendothelial mesenchymal cells (pericytes), rendering these vessels more therapy resistant. Mouse mammary tumor virus (MMTV) -induced mammary carcinomas reproduce the extensive pericyte coverage of tumor vessels seen in human breast cancers. This project will determine whether inhibiting activation of the endothelial cell Tie2 receptor reduces vessel pericyte coverage in MMTV- induced tumors and improves response to antiangiogenic agents. To do this, we are creating tumors with doxycycline-inducible expression of Tie2Ex, an inhibitor of Tie2 activation. Prior to doing this in mice that develop MMTV-induced mammary carcinomas, which will require transgenic introduction of regulatory components and the Tie2Ex gene, we are first developing and testing doxycycline-inducible Tie2Ex expression in transfectable Kl735 tumors. Progress has been made towards the creation of these tumors and testing can soon begin on the effects of Tie2Ex expression on Kl735 tumor vessel pericyte coverage and response to antiangiogenic therapy.

Estrogen provides one of the most important growth stimulators for breast epithelium. There is emerging evidence for complex interactions between estrogen and other growth factors that are important for the growth of breast cancer cells. Growth factor signalling pathways stimulate estrogen responsive genes, perhaps via the estrogen receptor. These findings open new opportunities to block breast cancer cell growth using mutant receptors that function as antagonists. In year 1 of this project, we created a series of estrogen receptor (ER) mutants that were shown to function in a dominant negative manner. In year 2, we created more potent dominant negative ER mutants and developed a more selective estrogen-responsive reporter gene. We found that estrogen and growth factor signaling pathways are separable when studied with sensitive luciferase reporter gene assay. The effects of these dominant negative mutants are being evaluated in estrogen-dependent proliferation assays were in T47D breast cancer cells. Tetracycline-inducible ER expression vectors were created to allow induction of ER mutants in stably transfected cells. Preliminary results show potent inhibition of cell growth by mutant ERs. For transgenic studies, MMTV promoter-ER expression vectors have been created. Thus we have completed each of the goals as outlined in the original proposal.

Ever since hybridoma technology was introduced by Kohler and Milstein in the 70's, numerous efforts have been undertaken to produce monoclonal antibodies (MAbs ) against mammary cancer cells. However, even to this day, all the MAbs produced still possess cross-reactivities toward other types of cancerous cells and also normal mammary cells. In this study, the breast cancer eel} line MCF-7 was used as an immunogen to raise MAb against mammary cancer cells. Fusion between lymphocytes sensitised with MCF-7 cell line and myeloma cells, SP2, was performed using 50% of polyethylene glycol (PEG). The hybridoma secreting MAb against MCF-7 cell line was selected using cell-ELISA technique. Limiting dilution of five times was performed to yield a stable hybridoma clone secreting the MAb.The selected clone, C2E7, which secreted MAb of the Igt-1 class and lamda light chains was chosen for further studies. MAb secreted by C2E7 was found to react with an antigenic determinant located in the cytoplasm of the MCF-7 cell line. Immunocytochemical studies showed that apart from the MCF-7 cell line, the anti genic determinant was also present in mammary cancer cell line of T47-D. Weak cross-reactivities were also observed against cell lines Panc-l and Ova-3. Immunohistochemical studies using the immunoperoxidase technique showed that staining occurred in the cytoplasmic region of all mammary lobular carcinoma and 90% of mammary ductal carcinoma examined. Staining was also found in 50% of mammary fibroadenoma cases studied. On the contrary, no staining of tissues was found in uterine leiomyoma, stomach showing intestinal metaplasia, cervical carcinoma, tonsillitis, neurofibroma, ductal papilloma of the breast and normal mammary tissues. Biochemical studies showed that the antigenic determinant on the MCF-7 cell line with reactivity towards MAb C2E7 was composed of endopeptide chain having arginine and lysine as the side chains, and possessed a specific conformational order which was dis

Ever since hybridoma technology was introduced by Kohler and Milstein in the 70's, numerous efforts have been undertaken to produce monoclonal antibodies (MAbs ) against mammary cancer cells. However, even to this day, all the MAbs produced still possess cross-reactivities toward other types of cancerous cells and also normal mammary cells. In this study, the breast cancer eel} line MCF-7 was used as an immunogen to raise MAb against mammary cancer cells. Fusion between lymphocytes sensitised with MCF-7 cell line and myeloma cells, SP2, was performed using 50% of polyethylene glycol (PEG). The hybridoma secreting MAb against MCF-7 cell line was selected using cell-ELISA technique. Limiting dilution of five times was performed to yield a stable hybridoma clone secreting the MAb.The selected clone, C2E7, which secreted MAb of the Igt-1 class and lamda light chains was chosen for further studies. MAb secreted by C2E7 was found to react with an antigenic determinant located in the cytoplasm of the MCF-7 cell line. Immunocytochemical studies showed that apart from the MCF-7 cell line, the anti genic determinant was also present in mammary cancer cell line of T47-D. Weak cross-reactivities were also observed against cell lines Panc-l and Ova-3. Immunohistochemical studies using the immunoperoxidase technique showed that staining occurred in the cytoplasmic region of all mammary lobular carcinoma and 90% of mammary ductal carcinoma examined. Staining was also found in 50% of mammary fibroadenoma cases studied. On the contrary, no staining of tissues was found in uterine leiomyoma, stomach showing intestinal metaplasia, cervical carcinoma, tonsillitis, neurofibroma, ductal papilloma of the breast and normal mammary tissues. Biochemical studies showed that the antigenic determinant on the MCF-7 cell line with reactivity towards MAb C2E7 was composed of endopeptide chain having arginine and lysine as the side chains, and possessed a specific conformational order which was dis