The overall objective of this proposal is to develop a flow cytometry- based assay utilizing optimized flow cytometry instrumentation and cultured mammalian cells that can be routinely employed by pharmaceutical and other companies to measure the mutagenic potential of chemicals and drugs. The assay is based on a Chinese hamster ovary- human hybrid cell line, CHO AL, which contains human chromosome 11. The principal target for mutation is the gene for the CD59 antigen which is encoded on chromosome 11. The hypotheses are(1) that mutations in chromosome 11 can be detected accurately, sensitively and rapidly by flow cytometry measurement of the loss or presence of surface antigens encoded by chromosome 11 and (2) that this mutation assay system can be developed so that it could be used routinely to determine the genotoxicity of physical and chemical agents.
Specific Aim 1 is to build a relatively inexpensive benchtop flow cytometer optimized for analysis of antigen loss in mammalian cells.
Specific Aim 2 is to validate the flow cytometry assay for detecting mutations in CHO AL cells by measuring the mutation dose response from a panel of 25 physical and chemical agents at a range of doses. Independent studies will be done at an off-site laboratory.
Specific Aim 3 is to extend the assay to include the analysis of intragenic and chromosomal mutations by measuring the presence or absence of multiple antibodies bound to different cell surface antigens encoded by separate genes on human chromosome 11. The development of this assay system would allow for rapid, accurate, relatively cheap but informative screening of pharmaceuticals for genotoxicity and substantially reduce the cost of carrying out mutation analysis.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
Project #
5R44CA091566-03
Application #
6626024
Study Section
Special Emphasis Panel (ZRG1-SSS-U (10))
Program Officer
Daschner, Phillip J
Project Start
2000-09-18
Project End
2006-03-31
Budget Start
2003-05-23
Budget End
2006-03-31
Support Year
3
Fiscal Year
2003
Total Cost
$382,607
Indirect Cost
Name
Cytomation Gtx, Inc.
Department
Type
DUNS #
City
Fort Collins
State
CO
Country
United States
Zip Code
80525
Keysar, Stephen B; Trncic, Nadira; Larue, Susan M et al. (2010) Hypoxia/reoxygenation-induced mutations in mammalian cells detected by the flow cytometry mutation assay and characterized by mutant spectrum. Radiat Res 173:21-6
Keysar, Stephen B; Fox, Michael H (2009) EMS mutant spectra generated by multi-parameter flow cytometry. Mutat Res 671:6-12
Keysar, Stephen B; Fox, Michael H (2009) Kinetics of CHO A L mutant expression after treatment with gamma radiation, EMS, and asbestos. Cytometry A 75:412-9
Ross, Carley D; Fox, Michael H (2008) Multiparameter analysis of CHO AL mutant populations sorted on CD59 expression after gamma irradiation. Radiat Res 170:628-37
Ross, Carley D; French, C Tenley; Keysar, Stephen B et al. (2007) Mutant spectra of irradiated CHO AL cells determined with multiple markers analyzed by flow cytometry. Mutat Res 624:61-70
French, C Tenley; Ross, Carley D; Keysar, Stephen B et al. (2006) Comparison of the mutagenic potential of 17 physical and chemical agents analyzed by the flow cytometry mutation assay. Mutat Res 602:14-25
Ross, Carley D; Lim, Chang-Uk; Fox, Michael H (2005) Assay to measure CD59 mutations in CHO A(L) cells using flow cytometry. Cytometry A 66:85-90