Abstract

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.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA016318-11
Application #
3164381
Study Section
Experimental Therapeutics Subcommittee 2 (ET)
Project Start
1977-05-01
Project End
1990-04-30
Budget Start
1985-05-01
Budget End
1986-04-30
Support Year
11
Fiscal Year
1985
Total Cost
Indirect Cost

  Institution

Name
Stanford University
Department
Type
Schools of Arts and Sciences
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305

  Publications

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

Showing the most recent 10 out of 34 publications