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Effect of X-Irradiation at Different Stages in the Cell Cycle on Individual Cell-Based Kinetics in an Asynchronous Cell Population.

Tsuchida E, Kaida A, Pratama E, Ikeda MA, Suzuki K, Harada K, Miura M - PLoS ONE (2015)

Bottom Line: Using an asynchronously growing cell population, we investigated how X-irradiation at different stages of the cell cycle influences individual cell-based kinetics.To visualize the cell-cycle phase, we employed the fluorescent ubiquitination-based cell cycle indicator (Fucci).The value was the largest when cells were irradiated in mid or late S phase and the smallest when they were irradiated in G1 phase.

View Article: PubMed Central - PubMed

Affiliation: Section of Oral Radiation Oncology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan; Section of Maxillofacial Surgery, Department of Maxillofacial and Neck Reconstruction, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan.

ABSTRACT
Using an asynchronously growing cell population, we investigated how X-irradiation at different stages of the cell cycle influences individual cell-based kinetics. To visualize the cell-cycle phase, we employed the fluorescent ubiquitination-based cell cycle indicator (Fucci). After 5 Gy irradiation, HeLa cells no longer entered M phase in an order determined by their previous stage of the cell cycle, primarily because green phase (S and G2) was less prolonged in cells irradiated during the red phase (G1) than in those irradiated during the green phase. Furthermore, prolongation of the green phase in cells irradiated during the red phase gradually increased as the irradiation timing approached late G1 phase. The results revealed that endoreduplication rarely occurs in this cell line under the conditions we studied. We next established a method for classifying the green phase into early S, mid S, late S, and G2 phases at the time of irradiation, and then attempted to estimate the duration of G2 arrest based on certain assumptions. The value was the largest when cells were irradiated in mid or late S phase and the smallest when they were irradiated in G1 phase. In this study, by closely following individual cells irradiated at different cell-cycle phases, we revealed for the first time the unique cell-cycle kinetics in HeLa cells that follow irradiation.

No MeSH data available.


Related in: MedlinePlus

Estimation of G2 arrest durations in cells irradiated in G1, early/mid/late S, and G2 phases.(A) Pedigree analysis of cells irradiated in green phase. a: Distribution of total green-phase durations in cells irradiated in each phase, sorted according to green fluorescence intensities. Two straight lines represent the remaining S-phase durations at irradiation (maximal 7 h for the leftmost cell in early S phase) (lower dashed line) and plus elongation of S phase (maximal 2 h for the leftmost cell in early S phase) (upper dashed line) in each phase. b: Distribution of G2-arrest durations after subtraction of the corresponding S phase and its elongation from the left panel a. (B) Comparisons of G2-arrest durations in cells irradiated in each phase. Data are represented as box-and-whisker plots as shown in Fig 3E. Cell number in each sub-phase is equivalent to that in Fig 5C. *, p < 0.05; **, p < 0.01 by Mann–Whitney U test.
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pone.0128090.g007: Estimation of G2 arrest durations in cells irradiated in G1, early/mid/late S, and G2 phases.(A) Pedigree analysis of cells irradiated in green phase. a: Distribution of total green-phase durations in cells irradiated in each phase, sorted according to green fluorescence intensities. Two straight lines represent the remaining S-phase durations at irradiation (maximal 7 h for the leftmost cell in early S phase) (lower dashed line) and plus elongation of S phase (maximal 2 h for the leftmost cell in early S phase) (upper dashed line) in each phase. b: Distribution of G2-arrest durations after subtraction of the corresponding S phase and its elongation from the left panel a. (B) Comparisons of G2-arrest durations in cells irradiated in each phase. Data are represented as box-and-whisker plots as shown in Fig 3E. Cell number in each sub-phase is equivalent to that in Fig 5C. *, p < 0.05; **, p < 0.01 by Mann–Whitney U test.

Mentions: Based on the criteria described above, cells irradiated in green phase were sub-divided into early/mid/late S and G2 phases, and then sorted in the order of green fluorescence intensity within each phase (Fig 7Aa). Using thymidine-blocked early S phase–synchronized cells, we found that S-phase cells exhibited a roughly 2-h delay of S-phase progression (S3 Fig). The results of previous studies indicate that this phenomenon is the result of S-phase checkpoint activation [30]. To obtain the duration of G2 phase including G2 arrest, S-phase elongation in addition to the remaining S-phase duration at irradiation had to be subtracted from the green-phase duration. Two straight lines were assumed to reflect the remaining S-phase duration at irradiation (lower dashed line) plus its elongation (upper dashed line), depending on the positions of S phase at irradiation in Fig 7Aa. Each value on the upper line was then subtracted from each corresponding green-phase duration for cells in S phase at irradiation. The subtracted values were redrawn and are shown in Fig 7Ab. For cells irradiated in G1 phase, S phase was assumed not to be elongated [14]. Finally, each duration in G2 arrest was compared among cells irradiated in G1, early/mid/late S, and G2 phases. G2 arrest for cells irradiated in G1 phase was the shortest among cells irradiated in all examined phases; on the other hand, when cells were irradiated in mid or late S phase, G2 arrest was the longest (Fig 7B and S4 Fig).


Effect of X-Irradiation at Different Stages in the Cell Cycle on Individual Cell-Based Kinetics in an Asynchronous Cell Population.

Tsuchida E, Kaida A, Pratama E, Ikeda MA, Suzuki K, Harada K, Miura M - PLoS ONE (2015)

Estimation of G2 arrest durations in cells irradiated in G1, early/mid/late S, and G2 phases.(A) Pedigree analysis of cells irradiated in green phase. a: Distribution of total green-phase durations in cells irradiated in each phase, sorted according to green fluorescence intensities. Two straight lines represent the remaining S-phase durations at irradiation (maximal 7 h for the leftmost cell in early S phase) (lower dashed line) and plus elongation of S phase (maximal 2 h for the leftmost cell in early S phase) (upper dashed line) in each phase. b: Distribution of G2-arrest durations after subtraction of the corresponding S phase and its elongation from the left panel a. (B) Comparisons of G2-arrest durations in cells irradiated in each phase. Data are represented as box-and-whisker plots as shown in Fig 3E. Cell number in each sub-phase is equivalent to that in Fig 5C. *, p < 0.05; **, p < 0.01 by Mann–Whitney U test.
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Related In: Results  -  Collection

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pone.0128090.g007: Estimation of G2 arrest durations in cells irradiated in G1, early/mid/late S, and G2 phases.(A) Pedigree analysis of cells irradiated in green phase. a: Distribution of total green-phase durations in cells irradiated in each phase, sorted according to green fluorescence intensities. Two straight lines represent the remaining S-phase durations at irradiation (maximal 7 h for the leftmost cell in early S phase) (lower dashed line) and plus elongation of S phase (maximal 2 h for the leftmost cell in early S phase) (upper dashed line) in each phase. b: Distribution of G2-arrest durations after subtraction of the corresponding S phase and its elongation from the left panel a. (B) Comparisons of G2-arrest durations in cells irradiated in each phase. Data are represented as box-and-whisker plots as shown in Fig 3E. Cell number in each sub-phase is equivalent to that in Fig 5C. *, p < 0.05; **, p < 0.01 by Mann–Whitney U test.
Mentions: Based on the criteria described above, cells irradiated in green phase were sub-divided into early/mid/late S and G2 phases, and then sorted in the order of green fluorescence intensity within each phase (Fig 7Aa). Using thymidine-blocked early S phase–synchronized cells, we found that S-phase cells exhibited a roughly 2-h delay of S-phase progression (S3 Fig). The results of previous studies indicate that this phenomenon is the result of S-phase checkpoint activation [30]. To obtain the duration of G2 phase including G2 arrest, S-phase elongation in addition to the remaining S-phase duration at irradiation had to be subtracted from the green-phase duration. Two straight lines were assumed to reflect the remaining S-phase duration at irradiation (lower dashed line) plus its elongation (upper dashed line), depending on the positions of S phase at irradiation in Fig 7Aa. Each value on the upper line was then subtracted from each corresponding green-phase duration for cells in S phase at irradiation. The subtracted values were redrawn and are shown in Fig 7Ab. For cells irradiated in G1 phase, S phase was assumed not to be elongated [14]. Finally, each duration in G2 arrest was compared among cells irradiated in G1, early/mid/late S, and G2 phases. G2 arrest for cells irradiated in G1 phase was the shortest among cells irradiated in all examined phases; on the other hand, when cells were irradiated in mid or late S phase, G2 arrest was the longest (Fig 7B and S4 Fig).

Bottom Line: Using an asynchronously growing cell population, we investigated how X-irradiation at different stages of the cell cycle influences individual cell-based kinetics.To visualize the cell-cycle phase, we employed the fluorescent ubiquitination-based cell cycle indicator (Fucci).The value was the largest when cells were irradiated in mid or late S phase and the smallest when they were irradiated in G1 phase.

View Article: PubMed Central - PubMed

Affiliation: Section of Oral Radiation Oncology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan; Section of Maxillofacial Surgery, Department of Maxillofacial and Neck Reconstruction, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan.

ABSTRACT
Using an asynchronously growing cell population, we investigated how X-irradiation at different stages of the cell cycle influences individual cell-based kinetics. To visualize the cell-cycle phase, we employed the fluorescent ubiquitination-based cell cycle indicator (Fucci). After 5 Gy irradiation, HeLa cells no longer entered M phase in an order determined by their previous stage of the cell cycle, primarily because green phase (S and G2) was less prolonged in cells irradiated during the red phase (G1) than in those irradiated during the green phase. Furthermore, prolongation of the green phase in cells irradiated during the red phase gradually increased as the irradiation timing approached late G1 phase. The results revealed that endoreduplication rarely occurs in this cell line under the conditions we studied. We next established a method for classifying the green phase into early S, mid S, late S, and G2 phases at the time of irradiation, and then attempted to estimate the duration of G2 arrest based on certain assumptions. The value was the largest when cells were irradiated in mid or late S phase and the smallest when they were irradiated in G1 phase. In this study, by closely following individual cells irradiated at different cell-cycle phases, we revealed for the first time the unique cell-cycle kinetics in HeLa cells that follow irradiation.

No MeSH data available.


Related in: MedlinePlus