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

Our plain-English summary of Kinetics of CHO A L mutant expression after treatment with gamma radiation, EMS, and asbestos, by S.B. Keysar, M.H. Fox (Cytometry. Part a, 2009).

The study

• Authors: S.B. Keysar, M.H. Fox
• Journal: Cytometry. Part a
• Published: 2009;75(5):412-9
• Cited by: 4 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

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.

(c) 2009 International Society for Advancement of Cytometry.

In plain English

This was a laboratory study of cells, not people. They used Chinese hamster ovary cells carrying one copy of human chromosome 11. A flow cytometry test counted cells that had lost a surface marker, CD59, as mutants. Cells received gamma radiation, the chemical EMS, or chrysotile "A" Rhodesian asbestos (20 micrograms per ml for 24 hours). Mutant cells peaked 6 to 9 days after treatment, then decreased. For asbestos, the authors reported the later plateau was not significantly different from the peak. After asbestos, the cells' potential doubling time rose 75% by 6 hours but was similar to controls by day 3. Peak-day colony survival, relative to untreated controls, was reported as 97% for asbestos, 96% for EMS and 70% for radiation. Later experiments used only radiation and EMS. CD59-negative cells sorted on the peak day survived less well than other cells; those sorted 35 days after EMS survived like controls. Cloned EMS-induced CD59-negative cells grew more slowly than stock cells. The authors concluded that the post-peak drop came from loss of cells destined to die and of slowly growing mutants.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) cell culture experiment
• Who was studied: CHO AL (N) cells: Chinese hamster ovary K1 cells carrying a single copy of human chromosome 11, obtained from Colorado State University. No human subjects.
• Exposure: Chrysotile "A" Rhodesian asbestos at 20 micrograms per ml for 24 hours. For comparison, cells were also given cesium-137 gamma radiation (4 Gy) or ethyl methane sulfonate (EMS, 6.5 or 8 mM for 3 hours). Mock-treated cells served as controls.
• Main results:
  • After treatment with EMS, radiation or asbestos, the fraction of CD59 mutant cells peaked 6 to 9 days after treatment and then decreased over time.
  • The potential doubling time of asbestos-treated cells increased 75% by 6 hours after treatment. For EMS (8 mM), it more than doubled at 3 hours. Control cells had a potential doubling time of 14.1 hours.
  • By day 3 after treatment, normal cell cycling had resumed, with potential doubling time values similar to controls. This was well before the peak day of mutant expression for all three agents.
  • Clonogenic survival on the peak day of mutant expression was 96% for both 6.5 and 8.0 mM EMS, 97% for asbestos, and 70% for radiation-treated cells. These are surviving fractions of treated cells plated on that day, calculated using the plating efficiency of untreated control cells.
  • The authors stated that the plateau level, relative to the peak, was about 40% for EMS, less than 20% for gamma radiation, and not significantly different for asbestos.
  • Radiation-treated CD59-negative cells sorted on the peak day had a survival of 20% (region 1) and 50% (regions 2 to 6) of control cells. Asbestos-treated cells were not tested in this experiment.
  • CD59-negative cells sorted 35 days after treatment with 8 mM EMS had survival indistinguishable from controls.
  • Eleven CD59-negative clones from EMS-treated cells had an average doubling time of 16.7 hours, compared with 14.5 hours for stock CHO AL cells. Asbestos-treated cells were not tested in this experiment.
  • After 8 mM EMS, triple mutants (CD59, CD44 and CD90 negative) fell from 35% of CD59-negative cells on day 6 to slightly more than 20% on day 24. Over the same period, CD59/CD90-negative cells rose from 10% to nearly 60%.
• The authors' conclusion: The authors concluded that the decrease in the mutant fraction after the peak day is caused by loss of cells destined to die, which may have lost chromosome 11 entirely, and of slowly growing mutants. They also concluded that the peak day varies with the time cells need to recover from genetic insult.
• Limitations the authors state:
  • The clones used in the growth-rate experiment had been in culture for more than 30 days and were selected as the fastest-growing clones.
  • Because radiation caused a large delay in the G2 phase of the cell cycle, potential doubling time could not be measured accurately for irradiated cells.

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