Kinetics of CHO A L mutant expression after treatment with gamma radiation, EMS, and asbestos

Abstract

The flow cytometry mutation assay (FCMA) uses hybrid CHO A(L) cells to measure mutations of the cd59 gene located on human chromosome 11 by the absence of fluorochrome-conjugated antibody binding to the CD59 surface antigen. Mutant expression peaks between 6 and 12 days, then decreases to a stable plateau, instead of a constant mutant fraction obtained by clonogenic assays. To evaluate this variable mutant expression time, cells were treated with radiation, EMS or asbestos and cell proliferation and survival were measured at times leading up to peak mutant expression. Potential doubling time (T(pot)) values increased by at least 75% for each agent by 3 h after treatment but returned to control levels after only 3 days. Survival returned to 90% of control within a week, close to the peak expression day for all three agents. The survival of CD59(-) cells sorted on the peak day of expression was roughly half that of CD59(+) cells. Cloned EMS-treated CD59(-) cells had a doubling time of 16.7 vs. 14.1 h for CD59(+) cells. Triple mutants (CD59(-)/CD44(-)/CD90(-)) were preferentially lost from the population over time, while the proportion of CD59(-)/CD90(-) mutants increased. In conclusion, the peak day of mutant expression occurs only when cells recover from the toxic effects of the mutagen. A fraction of cells originally quantified as mutants are lost over time due to lethal deletions and slower growth.

Figure 1

Mutant fraction (mutants/1×10⁵ cells) over time for cells treated with (A) 8 mM EMS (1 experiment run in triplicate) (B) 4 Gy γ-rays (2 experiments run in triplicate) and (C) 20 µg/ml asbestos (2 experiments run in triplicate). Error bars represent the SEM. All results are corrected for background mutants which were ~0.3–0.5%

Figure 2

Potential doubling time of CHO AL cells calculated by incorporation of BrdU after treatment with (A) 8 mM EMS (2 experiments), (B) 20 µg/ml asbestos (2 experiments), and (C) percentage of cells in G2 phase after treatment with 4 Gy γ-rays (3 experiments). Error bars represent the SEM.

Figure 3

Clonogenic survival of CHO A_L cells at time points following treatment with (A) 6.5 and 8.0 mM EMS (1 experiment run in triplicate) (B) 20 µg/ml asbestos (1 experiment run in triplicate) and (C) 4 Gy γ-rays (2 experiments run in triplicate). All error bars represent the standard deviation.

Figure 4

Clonogenic survival of cells based on CD59 expression. (A) Six regions of a CD59 histogram on the peak day of mutant expression after γ radiation; (B) surviving fraction of cells from various regions (top) 6 days after γ radiation (4 experiments run in triplicate, error bars represent the SEM), (bottom) 9 days after treatment with 8 mm EMS (3 experiments run in triplicate with error bars representing the SEM).

Figure 5

Clonogenic survival of cells sorted from six CD59 negative regions of the mutant peak (Figure 4A) on day 35 after treatment with 8 mM EMS, 26 days after the peak day of mutant expression (1 experiment run in triplicate with error bars representing the standard deviation).

Figure 6

Rate at which 11 CD59⁻ mutant clonal populations were lost after being mixed at 1:1 ratios with CD59⁺ clonal populations. The line is the best-fit exponential (y=50e^−0.1955t) for the loss of cells from the mixed population.

Figure 7

Bivariate histogram gated on CD59⁻ cells (Regions 1–2 in Fig 4) then analyzed for CD44 and CD90 expression 9 days after treatment with EMS.

Figure 8

Fraction of CD59 negative cells scored as (A) only CD59⁻, (B) CD59⁻ and CD44⁻, (C) CD59⁻ and CD90⁻ and (D) CD59−, CD44⁻ and CD90⁻. Data were obtained from histograms similar to that shown in Figure 7. Two independent experiments run in triplicate with error bars representing the SEM.