The long-term objectives of this grant are to increase understanding of the mechanisms whereby cultured cells and tumors change genetic properties by the process of gene amplification, predominately with studies on resistance to methotrexate. The studies to be undertaken include: 1) an analysis of the nucleotide sequences constituting promoter regions of the dihydrofolate reductase gene, and studies on the synthesis and metabolism of the dihydrofolate reductase mRNA in the cell cycle; 2) an analysis of the propensity of normal, malignant, and metastatic breast epithelium to undergo spontaneous or induced amplification of the dihydrofolate reductase gene as detected by use of the fluorescence activated cell sorter and molecular biology technology; 3) an analysis of resistance to methotrexate that is associated with an unstable phenotype and increased ability to bind methotrexate at 4 degrees, and which has the properties of an altered transport of methotrexate, employing a combination of techniques to characterize the putative overproduced protein and its gene; 4) an attempt to generate resistance to radiomimetic drugs in such a fashion as to select for cells in which there is an increased capability for DNA recombination which results in the resistance phenotype by a gene amplification mechanism.

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National Cancer Institute (NCI)
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Experimental Therapeutics Subcommittee 1 (ET)
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Stanford University
Schools of Arts and Sciences
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de Graaf, D; Sharma, R C; Mechetner, E B et al. (1996) P-glycoprotein confers methotrexate resistance in 3T6 cells with deficient carrier-mediated methotrexate uptake. Proc Natl Acad Sci U S A 93:1238-42
Yin, D X; Schimke, R T (1996) Inhibition of apoptosis by overexpressing Bcl-2 enhances gene amplification by a mechanism independent of aphidicolin pretreatment. Proc Natl Acad Sci U S A 93:3394-8
Urbani, L; Sherwood, S W; Schimke, R T (1995) Dissociation of nuclear and cytoplasmic cell cycle progression by drugs employed in cell synchronization. Exp Cell Res 219:159-68
Sherwood, S W; Schimke, R T (1995) Cell cycle analysis of apoptosis using flow cytometry. Methods Cell Biol 46:77-97
Yin, D X; Schimke, R T (1995) BCL-2 expression delays drug-induced apoptosis but does not increase clonogenic survival after drug treatment in HeLa cells. Cancer Res 55:4922-8
Sherwood, S W; Rush, D F; Kung, A L et al. (1994) Cyclin B1 expression in HeLa S3 cells studied by flow cytometry. Exp Cell Res 211:275-81
Sharma, R C; Schimke, R T (1994) The propensity for gene amplification: a comparison of protocols, cell lines, and selection agents. Mutat Res 304:243-60
Sherwood, S W; Kung, A L; Roitelman, J et al. (1993) In vivo inhibition of cyclin B degradation and induction of cell-cycle arrest in mammalian cells by the neutral cysteine protease inhibitor N-acetylleucylleucylnorleucinal. Proc Natl Acad Sci U S A 90:3353-7
Inoue, S; Sharma, R C; Schimke, R T et al. (1993) Cellular detoxification of tripeptidyl aldehydes by an aldo-keto reductase. J Biol Chem 268:5894-8
Sharma, R C; Murphy, A J; DeWald, M G et al. (1993) A rapid procedure for isolation of RNA-free genomic DNA from mammalian cells. Biotechniques 14:176-8

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