Construction and Characterization of Human Mammary Epithelial Cell Lines Containing Mutations in the P53 Or BRCAl Genes
Construction and Characterization of Human Mammary Epithelial Cell Lines Containing Mutations in the P53 Or BRCAl Genes

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Published: 1997

Total Pages: 0

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The overall goal of this project is to identify and characterize the consequences of human mammary epithelial cells (HMEC) that become deficient in normal p53 and BRCA1 gene functions. Our work during the third year of the funding period has produced good progress establishing new systems to create and characterize HMEC with an altered tumor suppressor gene activity. In collaboration with Dr. Bissell's laboratory (UC Berkeley), we have established three-dimensional culture system in our laboratory. This system will allow us to see differences of the growth properties, expression and distribution of certain cell lineage markers, morphology and behavior in between normal cells and partially transformed cells. Our recent acquisition of a Microarray Spotting and Scanning instrument enabled us the use of this robust and sensitive microarray technology to establish genetic expression profiles from any cell source of interest. We have also applied the differential display protocol to identify genes with altered expression in a model system of normal and neoplastic epithelial cells. We firmly believe that these new systems will provide us critical information to our understanding of breast carcinogenesis.

Construction and Characterization of Human Mammary Epithelial Cell Lines Containing Mutations in the P53 Or BRCA1 Genes
Construction and Characterization of Human Mammary Epithelial Cell Lines Containing Mutations in the P53 Or BRCA1 Genes

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Published: 1999

Total Pages: 0

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The overall goal of this project is to identify and characterize the consequences of human mammary epithelial cells (HMEC) that become deficient in normal p53 or BRCA-l gene functions. We have created retroviral vectors which allow us conditionally express the E6/E7 gene of human papillomavirus type 16 (HPVl6), dominant-negative p53 gene, or anti-sense BRCA-l gene. The consequences of transduction of these viral constructs into primary human mammary epithelial cells will be discovered through controlled in vitro comparisons between genetically altered derivatives and their isogenic parent cells. As we proposed in our last report, we are now focused our efforts on microarray-based comparisons to identify breast cancer related genes. During the past year we have successfully implemented the Microarray Spotting and Scanning techniques. This includes development of robust fluorescent labeling and hybridization protocols as well as the preparation and testing of over 23,000 minimally redundant cDNA target samples for deposition on the microarray slides. We have compared expression profiles from several distinct breast cell lines we had created. We believe that this system will provide us critical information to our understanding of early breast carcinogenesis.

Construction and Characterization of Human Mammary Epithelial Cell Lines Containing Mutations in P53 Or BRCA1 Genes
Construction and Characterization of Human Mammary Epithelial Cell Lines Containing Mutations in P53 Or BRCA1 Genes

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Published: 1995

Total Pages: 12

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To study the effects of inactivating mutations in the p53 and BRCA1 genes early in the breast cancer pathway, we will develop genetically defined human mammary epithelial cell (HMEC) lines by introducing heterozygous and homozygous mutations of each gene using homologous recombination. Additionally, we will construct HMEC lines deficient in p53 protein by expressing the E6 gene of human papillomavirus type 16 (HPV16), which increases the rate of degradation of the p53 protein. The consequences of these genetic changes for cell metabolism will be discovered through controlled in vitro comparisons between genetically altered derivatives and their isogenic parent cells. One level of comparison will involve observations of their growth properties, expression of certain cell lineage markers (e.g. keratins, integrins), morphology and behavior. At another level, we will take a global approach to the discovery of metabolic changes associated with genetic alterations in early tumorigenesis by constructing substraction cDNA libraries and by differential display to reveal changes in mRNA transcription that are associated with loss of activity of each of these genes. Clones showing differential expression will be sequenced in our Sequence Core Facility at the University of Utah to reveal genes potentially critical in growth control, by reference to databanks.

Genetic Construction and Molecular Characterization of Breast Cancer Precursor Cells
Genetic Construction and Molecular Characterization of Breast Cancer Precursor Cells

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Published: 1995

Total Pages: 15

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Mutations in genes involved in cell-cycle and growth regulation, such as the retinoblastoma gene (RB), are expected to have a significant role in the early stages of breast cancer. RB-deficient cells are being created by two distinct methods. First, targeting vectors, currently being constructed, will be transfected into human mammary epithelial cell lines (HMEC) in order to knock out both RB allels via homologous recombination. Second, a human papilloma virus (HPV) E6/E7 fusion construct, previously shown to specifically target the retinoblastoma protein (pRb) for degradation, will be transfected into HMEC. The E6/E7 gene fusion will be introduced into the HMEC under the control of a regulatable promoter, allowing for direct regulation of protein expression. Breast cells made deficient for pRb by these two methods will be examined for cellular and molecular alterations, such as changes in morphology and behavior, and their ability to form duct-like structures when grown on or within an extracellular matrix. Additionally, since the normal RB product has been implicated in transcriptional regulation, the effect on mRNA transcription in the RB-deficient breast cells will be assayed by Northern blot analysis and differential display.

Determining the Role of P53 Mutation in Human Breast Cancer Progression Using Recombinant Mutant
Determining the Role of P53 Mutation in Human Breast Cancer Progression Using Recombinant Mutant

Author: Damian Jerome Junk

Publisher:

Published: 2008

Total Pages: 372

ISBN-13:

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Breast cancer is the most frequently diagnosed form of cancer in women and the second leading cause of cancer-related deaths. Breast cancer is a heterogeneous disease consisting of many types of tissue neoplasia, and there appears to be no model of how a particular lesion develops into an aggressive, malignant, invasive carcinoma. Genetic mutation and aberrant epigenetic regulation are among the most common events that lead to neoplasia. In breast cancer, p53 mutation is the most common genetic defect related to a single gene. Therefore, this dissertation focuses on the mechanisms and consequences of p53 mutation during breast tumorigenesis. Genome-wide analysis of gene expression and epigenetic modifications in a panel of breast cancer cell lines suggested that p53 mutation and aberrant epigenetic silencing were cooperating mechanisms in the silencing of wild-type p53 target genes during cancer progression. Therefore, models of p53 inactivation were created in non-malignant human mammary epithelial cells to determine the role of p53 mutation on the epigenetic status of its target genes and the acquisition of malignant phenotypes. Comparisons of each model demonstrated that differing modes of p53 inactivation produced different functional consequences. Loss of wild-type p53 function alone ablated the normal cellular response to external stress stimuli, but had no affect on the expressionof genes or epigenetic status in untreated cells. Introduction of missense mutant p53 protein caused very few changes when the protein was expressed at low levels. However, accumulation of mutant p53 caused a variety of gene expression changes and interfered with endogenous wild-type p53. The accumulation of mutant p53 also caused an increase in migration and invasion of the cells that expressed it. Interestingly, epigenetic aberrations were not detected in response to any of the p53 manipulations. These data suggest that accumulation of missense mutation is particularly dangerous to normal cells. They also suggest that p53 mutation and epigenetic aberration are two distinct mechanisms, which overlap and cooperate during tumorigenesis. These data suggest that treatment strategies for human breast cancer should include modalities to target both defects for increased efficacy.

Identification and Characterization of PEA3 Ets Subfamily Target Genes in Human Mammary Epithelial Cells
Identification and Characterization of PEA3 Ets Subfamily Target Genes in Human Mammary Epithelial Cells

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Published: 2003

Total Pages: 0

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The human ETS gene family comprises 27 individual members and encodes sequence specific DNA binding proteins that regulate transcription. PEA3 is the founding member of a subfamily of ETS genes, which also include ER81 and ERM. PEA3 is overexpressed in 76% of all human breast tumors and in 93% of the HER2-positive subclass of such tumors. PEA3 is also overexpressed in most human breast tumor cell lines by comparison to non-transformed immortalized human mammary epithelial cells. Furthermore, PEA3 transcripts are elevated in the mammary tumors of three different breast cancer prone transgenic mouse strains and inhibition of the function of PEA3 (and ER81 and ERM) in mammary epithelial cells abrogates mammary oncogenesis induced by Her2. These findings imply that PEA3 and its subfamily members are required for mammary oncogenesis by regulating the expression of genes whose products play cardinal roles in breast cancer. Our objective is to identify PEA3 regulated genes in human mammary tumor cells; such genes may encode diagnostic and prognostic markers and molecular targets for drug discovery. To this end we constructed and characterized recombinant human adenoviruses encoding PEA3 or dominant-negative PEA3 and assessed their capacity to infect and regulated expression of cellular genes in untransformed and transformed human mammary epithelial cells.

ダブルクライシス
ダブルクライシス

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Published: 1965

Total Pages: 50

ISBN-13: 9780670064663

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Raised to be a thief, blind orphan Peter Nimble, age ten, steals from a mysterious stranger three pairs of magical eyes, that lead him to a hidden island where he must decide to become a hero or resume his life of crime.

Reverse Phase Protein Arrays
Reverse Phase Protein Arrays

Author: Tesshi Yamada

Publisher: Springer

Published: 2021-01-08

Total Pages: 0

ISBN-13: 9789813297579

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This book provides practical guidance on all aspects of reverse phase protein array (RPPA) technology, which permits the quantification of protein levels in cell or tissue lysates. In addition, the latest results are presented from laboratories across the world where experts are successfully running the challenging RPPA data platform. After an introductory chapter by the inventor of the RPPA technique, the demanding task of lysate preparation is explained. Subsequent chapters review printing platforms and analytical platforms and discuss antibody screening in detail. The role of RPPA data in integrative analyses is thoroughly examined, and the various applications of RPPA, for example to identify molecular targeting drugs or evaluate cancer drug efficacy, are discussed with reference to the latest knowledge. It is hoped that the book will foster the implementation of RPPA by documenting the practical details of the technology and by revealing its great potential. It will be of high value for researchers who plan to build an RPPA platform in their institute and for researchers, clinicians, and companies who use RPPA through collaboration with an existing RPPA facility.