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1 December 2000 Computerized Video Time-Lapse Analysis of Apoptosis of REC:Myc Cells X-Irradiated in Different Phases of the Cell Cycle
Helen B. Forrester, Norman Albright, C. Clifton Ling, William C. Dewey
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Forrester, H. B., Albright, N., Ling, C. C. and Dewey, W. C. Computerized Video Time-Lapse Analysis of Apoptosis of REC:Myc Cells X-Irradiated in Different Phases of the Cell Cycle.

Asynchronous rat embryo cells expressing Myc were followed in 50 fields by computerized video time lapse (CVTL) for three to four cycles before irradiation (4 Gy) and then for 6–7 days thereafter. Pedigrees were constructed for single cells that had been irradiated in different parts of the cycle, i.e. at different times after they were born. Over 95% of the cell death occurred by postmitotic apoptosis after the cells and their progeny had divided from one to six times. The duration of the process of apoptosis once it was initiated was independent of the phase in which the cell was irradiated. Cell death was defined as cessation of movement, typically 20–60 min after the cell rounded with membrane blebbing, but membrane rupture did not occur until 5 to 40 h later. The times to apoptosis and the number of divisions after irradiation were less for cells irradiated late in the cycle. Cells irradiated in G1 phase divided one to six times and survived 40–120 h before undergoing apoptosis compared to only one to two times and 5–40 h for cells irradiated in G2 phase. The only cells that died without dividing after irradiation were irradiated in mid to late S phase. Essentially the same results were observed for a dose of 9.5 Gy, although the progeny died sooner and after fewer divisions than after 4 Gy. Regardless of the phase in which they were irradiated, the cells underwent apoptosis from 2 to 150 h after their last division. Therefore, the postmitotic apoptosis did not occur in a predictable or programmed manner, although apoptosis was associated with lengthening of both the generation time and the duration of mitosis immediately prior to the death of the daughter cells. After the non-clonogenic cells divided and yielded progeny entering the first generation after irradiation with 4 Gy, 60% of the progeny either had micronuclei or were sisters of cells that had micronuclei, compared to none of the progeny of clonogenic cells having micronuclei in generation 1. However, another 20% of the non-clonogenic cells had progeny with micronuclei appearing first in generation 2 or 3. As a result, 80% of the non-clonogenic cells had progeny with micronuclei. Furthermore, cells with micronuclei were more likely to die during the generation in which the micronuclei were observed than cells not having micronuclei. Also, micronuclei were occasionally observed in the progeny from clonogenic cells in later generations at about the same time that lethal sectoring was observed. Thus cell death was associated with formation of micronuclei. Most importantly, cells irradiated in late S or G2 phase were more radiosensitive than cells irradiated in G1 phase for both loss of clonogenic survival and the time of death and number of divisions completed after irradiation. Finally, the cumulative percentage of apoptosis scored in whole populations of asynchronous or synchronous populations, without distinguishing between the progeny of individually irradiated cells, underestimates the true amount of apoptosis that occurs in cells that undergo postmitotic apoptosis after irradiation. Scoring cell death in whole populations of cells gives erroneous results since both clonogenic and non-clonogenic cells are dividing as non-clonogenic cells are undergoing apoptosis over a period of many days.

Helen B. Forrester, Norman Albright, C. Clifton Ling, and William C. Dewey "Computerized Video Time-Lapse Analysis of Apoptosis of REC:Myc Cells X-Irradiated in Different Phases of the Cell Cycle," Radiation Research 154(6), 625-639, (1 December 2000).[0625:CVTLAO]2.0.CO;2
Received: 25 February 2000; Accepted: 1 August 2000; Published: 1 December 2000
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