Sunday, October 16, 2005 3:00 PM-5:00 PM Exhibit Hall

(PP063) Analysis of CD59 mutants in CHO AL cells by flow cytometry after gamma radiation or EMS treatment.

Ross, Carley1, Keysar, Stephen1, French, C. Tenley2, Fox, Michael1, 2, 3, 1 Cell and Molecular Biology Program, Fort Collins, CO, USA2 Cytomation, GTX, Fort Collins, CO, USA3 Department of Environmental and Radiological Health Sciences, Fort Collins, CO, USA

ABSTRACT- Background: A sensitive mammalian cell mutation assay was developed previously using a Chinese hamster ovary cell line (CHO AL) that stably incorporates human chromosome 11. The assay measures mutations in the CD59 gene on chromosome 11 which we detect by flow cytometry using monoclonal antibodies against CD59. Mutants are characterized by very low fluorescence when cells are bound with antibodies against CD59. However, we also observed stable intermediate expression of CD59. Methods: CHO AL cells were treated with radiation or ethyl methane sulfonate (EMS), then grown for various times for mutant expression. Cells were labeled with monoclonal antibodies against CD59 and analyzed by flow cytometry. Mutants were cloned by sorting cells from six different regions of varying fluorescence CD59 intensity, then analyzing each region for growth, survival and mutant expression, and stability of the population. To roughly determine the extent of DNA damage, mutants were analyzed by multicolor monoclonal antibodies to CD44, CD151 and CD59 on the flow cytometer. Results: Six days after 4 Gy of radiation the mutant yield was 1.7% and mutants were sorted from six different regions. Cells grew at similar rates from all six regions. Cell survival increased from 40% in region 1 to 70% in region 6. Mutant expression was entirely negative for regions 1-5 but wild-type in region 6. Cells mutated at CD59 were frequently also mutated at CD44 or CD151. Cells treated with 8 mM EMS had a maximum mutant yield of 1.7% on day 9 and mutants were sorted from six regions. Cell survival was about 30% in each region and the cell doubling time was reduced to 18 hr in the mutant regions compared to 13 hr in controls. Stable mutant populations with intermediate expression of CD59 were obtained from the sorted regions. Multicolor flow analysis gave a mixture of CD151 and CD44 mutants in the CD59 intermediate region. Conclusions: EMS and radiation, a point mutagen and a clastogen, gave similar mutant yields using the flow cytometry mutation assay, but exhibit different types of mutants, including mutants with stable intermediate expression after EMS treatment but not radiation.

Key words: Mammalian mutation assay, flow cytometry mutation assay, FCMA, Chinese hamster ovary human-hamster hybrid AL cell line, gamma radiation, ems, ethyl methanesulfonate

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2005 RRS