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 irradi ation (4 Gy) and then for 6-7 days thereafter. Pedigrees were constructed f or 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 occ urred by postmitotic apoptosis after the cells and their progeny had divide d from one to six times. The duration of the process of apoptosis once it w as 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 occu r 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 G(1) phase divided one to six times and survived 40-120 h bef ore undergoing apoptosis compared to only one to two times and 5-40 h for c ells irradiated in G(2) phase. The only cells that died without dividing af ter irradiation were irradiated in mid to late S phase. Essentially the sam e results were observed for a dose of 9.5 Gy, although the progeny died soo ner and after fewer divisions than after 4 Gy. Regardless of the phase in w hich they were irradiated, the cells underwent apoptosis from 2 to 150 h af ter their last division. Therefore, the postmitotic apoptosis did not occur in a predictable or programmed manner, although apoptosis was associated w ith lengthening of both the generation time and the duration of mitosis imm ediately prior to the death of the daughter cells. After the non-clonogenic cells divided and yielded progeny entering the first generation after irra diation with 4 Gy, 60% of the progeny either had micronuclei or were sister s of cells that had micronuclei, compared to none of the progeny of clonoge nic cells having micronuclei in generation 1. However, another 20% of the n on-clonogenic cells had progeny with micronuclei appearing first in generat ion 2 or 3. As a result, 80% of the non-clonogenic cells had progeny with m icronuclei. Furthermore, cells with micronuclei were more likely to die dur ing the generation in which the micronuclei were observed than cells not ha ving micronuclei. Also, micronuclei were occasionally observed in the proge ny from clonogenic cells in later generations at about the same time that l ethal sectoring was observed. Thus cell death was associated with formation of micronuclei. Most importantly, cells irradiated in late S or G(2) phase were more radiosensitive than cells irradiated in G(1) phase for both loss of clonogenic survival and the time of death and number of divisions compl eted after irradiation. Finally, the cumulative percentage of apoptosis sco red in whole populations of asynchronous or synchronous populations, withou t distinguishing between the progeny of individually irradiated cells, unde restimates the true amount of apoptosis that occurs in cells that undergo p ostmitotic apoptosis after irradiation. Scoring cell death in whole populat ions of cells gives erroneous results since both clonogenic and non-clonoge nic cells are dividing as non-clonogenic cells are undergoing apoptosis ove r a period of many days. (C) 2000 by Radiation Research Society.