ovalbumin and Breast-Neoplasms

Topics: Animals; Base Sequence; Breast Neoplasms; Cell Line; Chickens; Chromatin; Conalbumin; DNA; DNA-Directed RNA Polymerases; DNA, Recombinant; DNA, Viral; Enhancer Elements, Genetic; Estrogens; Gene Expression Regulation; Humans; Liver; Models, Biological; Mutation; Nucleosomes; Operon; Ovalbumin; Oviducts; Progesterone; Promoter Regions, Genetic; RNA Caps; RNA, Messenger; Simian virus 40; Transcription Factors; Transcription, Genetic

Maspin, a tumor suppressor (SERPINB5), inhibits cancer migration, invasion, and metastasis in vitro and in vivo. The tumor-suppressing effects of maspin depend in part on its ability to enhance cell adhesion to extracellular matrix. Although the molecular mechanism of maspin's action is still unclear, its functional domain is believed to be located at the reactive center loop (RCL). We have elucidated the role of maspin RCL on adhesion, migration, and invasion by transfecting the highly invasive human breast carcinoma MDA-MB-231 cell line with pcDNA3.1-His/FLAG containing wild-type maspin, ovalbumin, or maspin/ovalbumin RCL chimeric mutants in which maspin RCL is replaced by ovalbumin (MOM) and vice versa (OMO). MDA-MB-231 cells transfected with maspin- or OMO-containing recombinant expression plasmid manifested significant increase in adhesion to fibronectin and reduction in in vitro migration and invasion through Matrigel compared with mock transfection or cells transfected with ovalbumin or MOM. Proteomics analysis of maspin- or OMO-transfected MDA-MB-231 cells revealed reduction in contents of proteins known to promote cancer metastasis and those of ubiquitin-proteasome pathway, while those with tumor-suppressing properties were increased. Furthermore, MDA-MB-231 cells containing maspin or OMO transgene have significantly higher levels of ubiquitin and ubiquitinated conjugates, but reduced 20S proteasome chymotrypsin-like activity. These results clearly demonstrate that the tumor-suppressive properties of maspin reside in its RCL domain.

Topics: Adenocarcinoma; Amino Acid Motifs; Amino Acid Sequence; Breast Neoplasms; Catalytic Domain; Cell Line, Tumor; Female; Humans; In Vitro Techniques; Models, Molecular; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Proteins; Ovalbumin; Proteasome Endopeptidase Complex; Protein Conformation; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; Serpins; Transgenes; Ubiquitination

This study used chloroquine to direct radiation-induced tumor cell death pathways to harness the antitumor activity of the immune system.. Chloroquine given immediately after tumor irradiation increased the cure rate of MCaK breast cancer in C3H mice. Chloroquine blocked radiation-induced autophagy and drove MCaK cells into a more rapid apoptotic and more immunogenic form of cell death.. Chloroquine treatment made irradiated tumor vaccines superior at inducing strong interferon gamma-associated immune responses in vivo and protecting mice from further tumor challenge. In vitro, chloroquine slowed antigen uptake and degradation by dendritic cells, although T-cell stimulation was unaffected.. This study illustrates a novel approach to improve the efficacy of breast cancer radiation therapy by blocking endosomal pathways, which enhances radiation-induced cell death within the field and drives antitumor immunity to assist therapeutic cure. The study illuminates and merges seemingly disparate concepts regarding the importance of autophagy in cancer therapy.

Topics: Animals; Antigen Presentation; Apoptosis; Autophagy; Breast Neoplasms; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Survival; Chloroquine; Combined Modality Therapy; Dendritic Cells; Endosomes; Female; Immunotherapy, Active; Mice; Mice, Inbred C3H; Ovalbumin

The microenvironment drives mammary gland development and function, and may influence significantly both malignant behavior and cell growth of mammary cancer cells. By restoring context, and forcing cells to properly interpret native signals from the microenvironment, the cancer cell aberrant behavior can be quelled, and organization re-established. In order to restore functional and morphological differentiation, human mammary MCF-7 and MDA-MB-231 cancer cells were allowed to grow in a culture medium filled with a 10% of the albumen (EW, Egg White) from unfertilized chicken egg. That unique microenvironment behaves akin a 3D culture and induces MCF-7 cells to produce acini and branching duct-like structures, distinctive of mammary gland differentiation. EW-treated MDA-MB-231 cells developed buds of acini and duct-like structures. Both MCF-7 and MDA-MB-231 cells produced β-casein, a key milk component. Furthermore, E-cadherin expression was reactivated in MDA-MB-231 cells, as a consequence of the increased cdh1 expression; meanwhile β-catenin - a key cytoskeleton component - was displaced behind the inner cell membrane. Such modification hinders the epithelial-mesenchymal transition in MDA-MB-231 cells. This differentiating pathway is supported by the contemporary down-regulation of canonical pluripotency markers (Klf4, Nanog). Given that egg-conditioned medium behaves as a 3D-medium, it is likely that cancer phenotype reversion could be ascribed to the changed interactions between cells and their microenvironment.

Topics: Actins; Animals; Breast Neoplasms; Cadherins; Caseins; Cell Polarity; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Keratin-18; Kruppel-Like Factor 4; Mammary Glands, Human; MCF-7 Cells; Ovalbumin; Protein Transport; Tumor Microenvironment

Approximately one third of patients with advanced human epidermal growth factor receptor 2 (HER-2)/neu-positive breast cancer respond to trastuzumab monotherapy, a humanized anti-HER-2/neu antibody. However, de novo and acquired antibody resistance is one of the major limitations of trastuzumab therapy warranting the search for other therapeutic strategies. One of the most remarkable features of adenovirus (AdV)-based vaccine is its ability to induce exceptionally high and sustained frequencies of transgene product-specific CD8(+) T-cell responses. In this study, we constructed two recombinant AdVs (AdV(OVA) and AdV(HER-2)) expressing ovalbumin (OVA) and HER-2/neu, and assessed AdV-induced antigen-specific cellular immune responses and preventive/therapeutic antitumor immunity. We demonstrate that AdV(OVA) stimulates efficient OVA-specific CD8(+) cytotoxic T lymphocyte (CTL) and natural killer responses, leading to preventive long-term immunity against OVA-expressing BL6-10ova melanoma in wild-type C56BL/6 mice. We further demonstrate that AdV(HER-2) stimulates HER-2/neu-specific CD8(+) CTL responses, leading to a significant reduction in breast carcinogenesis in transgenic FVBneuN mice (P<0.05), but has little therapeutic effect on pre-existing Tg1-1 tumor even at early stage (15 mm(3)). In contrast, the anti-HER-2/neu antibody therapy is capable of completely inhibiting Tg1-1 tumor growth at early stage, but fails to eradicate well-established Tg1-1 breast tumor (100 mm(3)). Interestingly, a combinatorial immunotherapy of anti-HER-2/neu antibody with AdV(HER-2) vaccine was capable of curing 4 of 10 studied mice bearing well-established Tg1-1 breast tumors and significantly delaying in death of the remaining six tumor-bearing mice (P<0.05). Taken together, our results suggest an adjuvant effect of AdV(HER-2) on anti-HER-2/neu antibody therapy for well-established breast tumor in transgenic FVBneuN mice, and this combinatorial immunotherapy of trastuzumab with AdV(HER-2) vaccine may be used as a new therapeutic strategy for treatment of advanced HER-2/neu-positive breast cancer.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; CD8-Positive T-Lymphocytes; Female; Killer Cells, Natural; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Rats; Receptor, ErbB-2; Trastuzumab

Estrogen-responsive genes are regulated by altering the balance of estrogen receptor (ER) interaction with transcription activators and inhibitors. Here we examined the role of ER ligand on ER interaction with the Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF) orphan nuclear receptor. COUP-TF binding to half-site estrogen response elements (EREs) was increased by the addition of estradiol (E2) -liganded ER (E2-ER), but not by ER liganded with the antiestrogens 4-hydroxytamoxifen (4-OHT-ER) or tamoxifen aziridine (TAz-ER). ER did not bind to single half-sites. Conversely, COUP-TF enhanced the ERE binding of purified E2-ER, but did not affect TAz-ER-ERE binding. In contrast, only antiestrogens enhanced direct interaction between ER and COUP-TF as assessed by GST pull-down assays. Identical results were obtained using either purified bovine or recombinant human ERalpha. Co-immunoprecipitation assays showed that ER and COUP-TF interact in extracts from MCF-7 and ERalpha-transfected MDA-MB-231 cells. Here we document that ER ligand impacts COUP-TF-ER interaction. COUP-TF interaction is mediated by the DNA binding and ligand-binding domains of ER. We suggest that changes in ER conformation induced by DNA binding reduce ER-COUP-TF interaction. Transient transfection of human MCF-7 breast cancer cells with a COUP-TFI expression vector repressed E2-induced luciferase reporter gene expression from single or multiple tandem copies of a consensus ERE. COUP-TFI stimulated 4-OHT-induced luciferase activity from a minimal ERE. Alone, COUP-TFI increased transcription from ERE half-sites or a single ERE in a sequence-dependent manner. These data provide evidence that the ERE sequence and its immediate flanking regions influence whether COUP-TF enhances, inhibits, or has no effect on ER ligand-induced ERE reporter gene expression and that COUP-TFI activates gene transcription from ERE half-sites. We suggest that COUP-TFI plays a role in mitigating estrogen-responsive gene expression.

Topics: Animals; Binding Sites; Breast Neoplasms; Cattle; Chickens; COUP Transcription Factor I; DNA-Binding Proteins; ERRalpha Estrogen-Related Receptor; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Female; Gene Expression Regulation; Humans; Ligands; Luciferases; Nuclear Receptor Co-Repressor 2; Ovalbumin; Precipitin Tests; Proteins; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; Recombinant Proteins; Repetitive Sequences, Nucleic Acid; Repressor Proteins; Response Elements; Tamoxifen; Transcription Factors; Transcription, Genetic; Transfection; Trefoil Factor-1; Tumor Cells, Cultured; Tumor Suppressor Proteins

The vitamin hormone retinoic acid (RA) regulates many complex biological programs. The hormonal signals are mediated at the level of transcription by multiple nuclear receptors. These receptors belong to the steroid/thyroid hormone receptor superfamily that also includes a large number of orphan receptors whose biological roles have not yet been determined. Although much has been learned in recent years about RA receptor (RAR) functions, little is known about how specific RA response programs are restricted to certain tissues and cell types during development and in the adult. It has been recently shown that RAR activities are regulated by retinoid X receptors (RXR) through heterodimer formation. In an effort to isolate and further characterize nuclear receptors that modulate RAR and/or RXR activities, we have screened cDNA libraries by using a RXR alpha cDNA probe. Two clones, COUP alpha and COUP beta, identical and closely related to the orphan receptor COUP-TF, were obtained. We show that COUP proteins dramatically inhibit retinoid receptor activities on certain response elements that are activated by RAR/RXR heterodimers or RXR homodimers. COUP alpha and -beta bind strongly to these response elements, including a palindromic thyroid hormone response element and a direct repeat RA response element as well as an RXR-specific response element. In addition, we found that the previously identified COUP-TF binding site in the ovalbumin gene functions in vitro as an RA response element that is repressed in the presence of COUP. Our data suggest that the COUP receptors are a novel class of RAR and RXR regulators that can restrict RA signaling to certain elements. The COUP orphan receptors may thus play an important role in cell- or tissue-specific repression of subsets of RA-sensitive programs during development and in the adult.

Topics: Base Sequence; Binding Sites; Breast Neoplasms; Carrier Proteins; COUP Transcription Factor I; DNA; DNA-Binding Proteins; Gene Expression Regulation; Humans; Molecular Sequence Data; Ovalbumin; Promoter Regions, Genetic; Receptors, Cell Surface; Receptors, Retinoic Acid; Repetitive Sequences, Nucleic Acid; Retinoid X Receptors; Solutions; Transcription Factors; Tretinoin; Tumor Cells, Cultured

A nonspecific staining of mast cells by avidin-biotinylated peroxidase complexes (ABC) has been observed and related to an ionic binding of the basic residues of avidin (isoelectric point at pH 10) and peroxidase to the sulphate groups of heparin. This affects the correct interpretation of the results of the immuno-peroxidase ABC procedure, especially in mast cells-rich areas such as the lymphoid tissues. The spurious staining can be prevented by using the ABC solution at pH 9.4 (instead of pH 7.6 as usually recommended): this high pH does not affect the previous binding of primary antibodies nor the affinity of avidin to biotin. The modified ABC procedure provides a clear background and a sharp specific staining and can be recommended for routine use.

Topics: Animals; Antigens; Avidin; Biotin; Breast Neoplasms; Colon; Histocytochemistry; Humans; Hydrogen-Ion Concentration; Immunoenzyme Techniques; Mast Cells; Ovalbumin; Rats; Skin; Staining and Labeling

To study the regulation of expression of the chicken ovalbumin gene by steroid hormones, the entire ovalbumin gene and its flanking sequences were cloned together with the bacterial gene for xanthine-guanine phosphoribosyltransferase in plasmid pBR322. This recombinant plasmid was linearized and used to transform an estrogen-responsive breast carcinoma cell line (MCF-7) which was shown to possess estrogen receptors and to be estrogen responsive. Transformants were selected by their ability to grow in a medium containing mycophenolic acid and xanthine. The entire ovalbumin gene was integrated into high molecular weight DNA within all transformants analyzed and it retained its original sequence organization. Ovalbumin mRNA and protein were identified from these transformant cells and they were found to be indistinguishable from the authentic counterparts. An 8- to 10-fold increase in the amount of ovalbumin mRNA was observed to be present in cells cultured in 10(-8)M estradiol. We also constructed a hybrid gene containing the 5'-flanking sequence and the first exon of the ovalbumin gene which was linked to the xanthine-guanine phosphoribosyltransferase gene such that expression of this bacterial gene would be promoted and regulated by the chicken sequences. After introduction of this hybrid gene into MCF-7 cells, we observed that the survival of the transformed cells in our selection medium was highly dependent on the presence of estradiol. Our results indicated that the chicken ovalbumin sequence was expressed properly and was regulated to some extent by estradiol in this heterologous system.

Topics: Animals; Breast Neoplasms; Cell Line; Chickens; Cloning, Molecular; DNA Restriction Enzymes; DNA, Recombinant; Estradiol; Female; Genes; Humans; Nucleic Acid Hybridization; Ovalbumin; Pentosyltransferases; Plasmids; Protein Biosynthesis; RNA, Messenger

Carbohydrate residues in tissue can be localized by a very sensitive, specific, and simple technique, utilizing a biotin-labeled lectin followed by an avidin-biotin-peroxidase complex (ABC). Distribution of peanut receptors in benign and malignant tissues was found to be quite different. Normal colonic mucosa occasionally shows small dot staining in the supranuclear cytoplasm, while the tumor cells of colonic adenocarcinoma tend to show diffuse cytoplasmic or membranous staining. Normal breast ductal epithelium shows membranous staining at luminal borders, while medullary carcinoma of the breast shows distinct membranous and cytoplasmic staining. The use of this avidin-biotin-peroxidase complex technique may contribute to understanding the process of tumorigenesis as well as providing a sensitive method to determine early stages of malignant transformation.

Topics: Avidin; Biotin; Breast Neoplasms; Carbohydrates; Colonic Neoplasms; Female; Histocytochemistry; Humans; Immunoenzyme Techniques; Lectins; Ovalbumin

Topics: Animals; Biotin; Breast Neoplasms; Drug Synergism; Female; Humans; Laryngeal Neoplasms; Male; Neoplasm Metastasis; Neoplasms; Ovalbumin; Pharyngeal Neoplasms; Swine

Topics: Albumins; Animals; Body Weight; Breast Neoplasms; Caseins; Dietary Proteins; Humans; Lactalbumin; Mammary Neoplasms, Animal; Mammary Neoplasms, Experimental; Methylcholanthrene; Ovalbumin; Rats; Research; Toxicology