It is proposed to exploit a powerful and novel new method of selecting genes whose expression is altered in breast cancer. This method, invented by Dr. Arthur Pardee is called Differential Display (DD). DD and the even newer method, called Subtractive Display (SD) to be developed will be applied in my laboratory to the identification and selection of both TSGs (tumor suppressor genes) and oncogenes, on the basis of differential expression at the mRNA level. With this method virtually every expressed gene in a cell population can be displayed on a sequencing gel after PCR amplification. By comparing the banding patterns of mRNAs from normal and tumor cells, displayed on adjacent lanes of the gel, it is evident by eye which bands show differential expression. Two, three, or four cell lines can readily be compared simultaneously. Over-expressed bands can be recovered directly from the gel, further amplified by PCR, verified by Northern analysis, and sequenced to provide a partial sequence for identification in GenBank. Selected genes will be fully sequenced to obtain full-length cDNAs for analysis by transfection of candidate TSGs into tumor cells and candidate oncogenes into normal cells. This methodology will initially be applied to normal and tumor-derived mammary epithelial cells developed in my laboratory. The differentially expressed genes from these comparisons will provide a library for further investigation. A subset will be selected by criteria including consistent expression changes in a small test series of cell lines and primary invasive tumors detected at the mRNA level, and utilized for antibody production to examine expression at the protein level in an expanded set of neoplastic and pre-neoplastic tissues. Subsequently, with the development of a miniaturized procedure, tumor cells from staged surgical or fine needle biopsy specimens (Smith/Thor ROI) will be utilized directly to identify selected differentially expressed genes in primary tissues, and in micro metastases. Thus one focus of this application is to utilize the Pardee methodology to recover, clone, and sequence genes whose expression is altered in the early stages of breast cancer, using tissue-derived cells directly. Since some, presumably most of these genes will already be present in our library derived from normal and tumor cells grown in culture, the tissue studies will in addition validate use of cell cultures for functional studies of these genes. Initial screening of candidate TSGs and oncogenes will be carriend out by partial sequence analysis to identify known genes, relatives of known genes, as well as novel unknown genes. Selected genes will be screened by treatment of tumor cells with antisense oligonucleotides to look for growth inhibition in the set of potential oncogenes, overexpressed in tumor cells, and by treatment of normal cells with antisense to look for loss of the serum requirement for growth or by other growth proliferation assays.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA061253-03
Application #
2101985
Study Section
Special Emphasis Panel (SRC (56))
Project Start
1993-07-01
Project End
1997-06-30
Budget Start
1995-07-01
Budget End
1996-06-30
Support Year
3
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Dana-Farber Cancer Institute
Department
Type
DUNS #
149617367
City
Boston
State
MA
Country
United States
Zip Code
02215
Lamb, Justin; Ramaswamy, Sridhar; Ford, Heide L et al. (2003) A mechanism of cyclin D1 action encoded in the patterns of gene expression in human cancer. Cell 114:323-34
Fournier, Marcia V; Martin, Katherine J; Graner, Edgard et al. (2003) Sensitive assays for detection of lung cancer. Molecular markers in blood samples. Methods Mol Med 75:239-50
Sotiropoulou, Georgia; Kono, Mari; Anisowicz, Anthony et al. (2002) Identification and functional characterization of a human GalNAc [alpha]2,6-sialyltransferase with altered expression in breast cancer. Mol Med 8:42-55
Biswas, D K; Cruz, A; Pettit, N et al. (2001) A therapeutic target for hormone-independent estrogen receptor-positive breast cancers. Mol Med 7:59-67
Martin, K J; Graner, E; Li, Y et al. (2001) High-sensitivity array analysis of gene expression for the early detection of disseminated breast tumor cells in peripheral blood. Proc Natl Acad Sci U S A 98:2646-51
Fournier, M V; Martin, K J; Pardee, A B (2001) Solid tumor cancer markers and applications to steroid hormone research. Methods Mol Biol 176:329-40
Biswas, D K; Cruz, A P; Gansberger, E et al. (2000) Epidermal growth factor-induced nuclear factor kappa B activation: A major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells. Proc Natl Acad Sci U S A 97:8542-7
Huang, L; Sowa, Y; Sakai, T et al. (2000) Activation of the p21WAF1/CIP1 promoter independent of p53 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through the Sp1 sites. Oncogene 19:5712-9
Biswas, D K; Cruz, A P; Pardee, A B (2000) Detection of the level of estrogen receptor and functional variants in human breast cancers by novel assays. Biotechniques 29:1056-60, 1062-4
Li, Y; Li, C J; Yu, D et al. (2000) Potent induction of apoptosis by beta-lapachone in human multiple myeloma cell lines and patient cells. Mol Med 6:1008-15

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