1987 — 1989 |
Asch, Bonnie B. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Keratins in Functional Differentiation of Mammary Cells @ Roswell Park Cancer Institute
Evidence from in vivo and in vitro studies has shown that functional differentiation (i.e., synthesis and secretion of milk) of mouse mammary epithelial cells (MMEC) in response to lactogenic hormones is profoundly influenced by that extracellular matrix (ECM). Recent experiments in our laboratory indicate that the keratin composition of normal MMEC can also be modulated by the ECM, whereas preneoplastic and neoplastic MMEC may be insensitive to this stimulus. The purpose of the proposed research is to test the hypothesis that the ability of MMEC to perform mammary-specific differentiated function is coordinately expressed with a particular set of keratins and that both of these activities are subject to control by the ECM. MMEC cultured on different types of ECM will be used in experiments to address the following questions: 1) Is the set of keratins expressed by normal MMEC in vivo a requisite for obtaining synthesis and/or secretion of the full complement of milk components? 2) Do any culture systems which support certain aspects of differentiated function allow the cells to retain their exact in vivo pattern of keratins? 3) If coordinate expression of functional ability and a particular set of keratins exists, what is the role of keratin filaments in this activity? 4) If the distribution and arrangement of keratin filaments in MMEC is disrupted, what is the effect on synthesis and secretion of milk components? 5) Are mammary preneoplastic and/or tumor cells constrained to an aberrant pattern of keratin expression and unable to respond to modulation by the ECM? 6) Can the latter cells induced to produce/secrete milk components under in vivo or culture conditions that are permissive for normal MMEC? Synthesis and secretion of milk components and expression of keratins will be analyzed by biochemical assays, immunocytochemistry, and electron microscopy. Experiments on functions of keratin filaments will involve the use of acrylamide to perturb the arrangement and distribution of the filaments. Results of these studies should provide new information of ECM control of differentiated function in mammary cells, modulation of keratin expression by the ECM, the role of keratin filaments in cells performing an epithelial-specific function, and defects in differentiation associated with malignant progression of mammary cells.
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0.906 |
1987 — 1993 |
Asch, Bonnie B. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R55Activity Code Description: Undocumented code - click on the grant title for more information. |
Markers of Mammary Cell Differentiation and Neoplasia @ Roswell Park Cancer Institute
Mouse mammary tumorigenesis is a multistep process involving at least two major events: the transformation of normal cells to preneoplastic cells and the transformation of preneoplastic cells to tumor cells. Markers capable of detecting cells in different stages of the preneoplastic state would be valuable tools for dissecting and defining the underlying sequential events in neoplastic progression in mammary epithelium. The most thoroughly studied preneoplastic lesion is the hyperplastic alveolar nodule (HAN). We have recently prepared antibodies against 3 cytoskeletal components that may represent such markers in mouse mammary epithelial cells: a 46kd keratin, recognized by a monospecific antiserum, that is found in some HAN and all mouse mammary carcinomas in vivo; a perinuclear structure (PNS), visualized by a rat monoclonal antibody designated PNS-Ab, that is present in cells of all mammary tumors and at least one type of HAN; and an apparent keratin epitope, detected by a rat monoclonal antibody designated MKE- Ab, that is found in all normal and some HAN cells but not in tumor cells. The purpose of the proposed research is to analyze the relationship of expression of these 3 components to mammary tumorigenesis more thoroughly and to determine if the presence of any of these components can be used in identifying cells in different steps of the progression of HAN to malignancy. The antibodies will be used in immunocytochemistry in three types of experiments involving mouse mammary tissues: 1) to follow the emergence of cells expressing the 46kd keratin and the PNS while losing the MKE-Ab determinant during the development of primary HAN and tumors in mammary tumorigenesis of BALB/cfC3H mice; 2) to examine cells of HAN with high and low tumor potentials for expression of the 3 components at different times during the latency period of tumor development and determine if the appearance of any of them correlates with tumor incidence; and 3) to investigate the effect of increasing the tumor potential of an HAN on expression of the 3 components. Additional experiments will determine if treatment of normal mouse mammary epithelial cells in culture with a carcinogen, e.g. DMBA, results in the appearance of cells expressing the PNS and if similar treatment of HAN cells induces a loss of the MKE-Ab determinant. If such cells are found, they will be tested for altered growth and histogenic potential in vivo by transplantation experiments.
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0.906 |
1989 |
Asch, Bonnie B. |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Confocal Fluorescence Imaging System @ Roswell Park Cancer Institute
A confocal fluorescence imaging system, including a laser-based scanner attached to an epifluorescence microscope, control computer with 80386 processor for efficient image analysis, dual detector channels, motorized focus control, transmitted light detector, photo record system, Micro SEMPER software package, and an external image archiving storage component is requested. The use of the instrument is to be shared among researchers in several departments at Roswell Park Memorial Institute. The projects requiring this equipment include, but are not limited to, studies of the cytoskeleton and organelles in mammary epithelial cells; localization of oncogene proteins; lymphocyte membranes, receptors and cytoskeleton; molecular organization and fusion of membranes; effects of drugs on normal and tumor cell growth and differentiation; tumor cell heterogeneity; subcellular abnormalities in mutant mice; tumor cell structure, function, and growth, drug resistance in human tumors; and localization of specific proteins in mouse embryos.
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0.906 |
1994 — 1997 |
Asch, Bonnie B. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Endogenous Retrotransposons in Mammary Carcinogenesis @ Roswell Park Cancer Institute
Breast cancer evolves from a complex, multistep process involving an accumulation of defects in gene structure and/or expression. Using the mouse model of chemically-induced mammary carcinogenesis, our research has three long-term objectives: l) defining the steps required for neoplastic progression of mouse mammary epithelial cells (MMEC) to malignancy, 2) identifying the factors and events responsible for advancement from one stage to the next, and 3) developing markers to detect cells in different stages of progression. We have recently shown that many preneoplastic and malignant MMEC overexpress the endogenous retrotransposons, intracisternal A particles (IAPs) and murine leukemia virus-related elements (MLVEs), compared to normal cells. Retrotransposons are mobile elements in mouse DNA capable of contributing to neoplastic transformation by transposing and causing insertional mutagenesis, which can alter gene expression and increase genetic instability of the host genome. The hypothesis of the proposed research is that amplified expression with subsequent transposition of MLVE sequences are key events in at least one pathway of mouse mammary carcinogenesis induced by 7,12-dimethylbenzanthracene (DMBA). To investigate the possible role of MLVEs in development of mammary cancer, the proposed research will use three approaches: 1) mouse mammary carcinogenesis induced by DMBA will be analyzed to determine how frequently and when altered expression of MLVEs occurs during neoplastic progression, the identity of the cells affected, and which class(es) of MLVEs is involved; 2) a probe specific for the main class of MLVEs expressed in DMBA tumors will be used to determine if any MLVE copies are located in new sites of the host genome and if so, what effect this has had on any nearby genes; and 3) a cDNA library prepared from a DMBA tumor with high levels of MLVE RNA will be screened for cDNAs corresponding to cellular transcripts which either originate in or are polyadenylated by a MLVE LTR. These studies will include the use of polymerase chain reaction, Northern and Southern analyses, in situ hybridization, and DNA sequencing. As ten percent of the human genome consists of transposable elements, several of which have been implicated in mutagenizing human DNA, these elements might be operative in human, as well as mouse, breast cancer. In fact, oncogene activation due to transposition and insertional mutation by a retroelement has already been documented in a human breast ductal carcinoma.
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0.906 |
1994 — 1995 |
Asch, Bonnie B. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Marker of Mammary Cell Differentiation/Neopl @ Roswell Park Cancer Institute
The evolution of breast cancer is a complex, multistep process. Using the mouse model of mammary carcinogenesis, our research has three long-term objectives: 1) defining the steps involved in neoplastic progression of mouse mammary epithelial cells (MMEC) to malignancy, 2) identifying the factors and underlying events responsible for advancement from one stage to the next, and 3) developing markers to detect cells in different stages of progression. Most of these steps probably involve alterations in gene structure and/or expression. We have recently shown that many preneoplastic and malignant MMEC have marked elevation in expression of the endogenous retrotransposons, intracisternal A particles (IAPs), compared to normal cells. In contrast, expression of other endogenous retrotransposons is unchanged, substantially decreased, or only occassionally increased. IAPs are mobile elements in mouse DNA with the potential for contributing to neoplastic transformation by their ability to transpose and cause insertional mutagenesis, thereby increasing genetic instability of the host genome. The hypothesis of the proposed research is that amplified expression with subsequent transposition of IAP sequences are key events in at least one pathway of mouse mammary carcinogenesis, i.e. induction by high doses of 7,12-dimethylbenzanthracene (DMBA). To investigate the possible role of IAPs in development of mammary cancer, the proposed research will use three approaches: 1) mouse mammary carcinogenesis induced by DMBA will be analyzed to define when altered expression of IAP sequences begins during neoplastic progression, the identity of the cells affected, and which class(es) of IAP elements is involved; 2) an oligonucleotide probe specific for the main class of IAP element expressed in DMBA tumors will be used to determine if any IAP copies are located in new sites of the host genome; 3) the effects of IAP transposition on MMEC will be investigated by transfecting them with an IAP element linked to a selectable marker that is expressed only after transposition occurs. After selection, cells in which the marked element has transposed will be isolated, characterized, and tested for altered growth properties, including tumorigenicity in vivo. The major methodologies for these studies will include the use of cDNA probes and restriction nucleases in Northern and Southern blotting, in situ hybridization, cell culture procedures, transfection techniques, and nude mice for testing tumorigenicity. As ten percent of the human genome consists of transposable elements, several of which have been implicated in mutagenizing human DNA, these elements might be operative in human, as well as mouse, breast cancer. In fact, oncogene activation due to transposition and insertional mutagenesis by one such element has already been documented in a human breast ductal carcinoma.
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0.906 |