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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

Establishment of a method for classifying green phase into early/mid/late S and G2 phases.(A) Experimental procedure for classification of green phase. Time-lapse imaging was performed for cells flash-labeled with EdU immediately after 5 Gy irradiation, followed by EdU staining. Time is shown as hours:minutes. (B) Classification of green-phase cells into early/mid/late S and G2 phases, according to the method described in A. For simplicity, a subset of cells in each phase is presented. To adjust the fluorescence intensities among different fields, background fluorescence intensity of each image was normalized to 1. (C) Comparison of green fluorescence intensities among sub-groups. Data are represented as box-and-whisker plots, as described in Fig 3E, from 60 cells in early S, 139 cells in mid S, 101 cells in late S, and 127 cells in G2 phase. *, p < 0.05; **, p < 0.01 by Mann–Whitney U test. (D) Validation of the methodology, using GP1 and GP2 from Fig 3D. Cell numbers in each sub-phase within GP1 (a) and GP2 (b) were determined. GP1 and GP2 consisted of 138 and 289 cells, respectively.
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pone.0128090.g006: Establishment of a method for classifying green phase into early/mid/late S and G2 phases.(A) Experimental procedure for classification of green phase. Time-lapse imaging was performed for cells flash-labeled with EdU immediately after 5 Gy irradiation, followed by EdU staining. Time is shown as hours:minutes. (B) Classification of green-phase cells into early/mid/late S and G2 phases, according to the method described in A. For simplicity, a subset of cells in each phase is presented. To adjust the fluorescence intensities among different fields, background fluorescence intensity of each image was normalized to 1. (C) Comparison of green fluorescence intensities among sub-groups. Data are represented as box-and-whisker plots, as described in Fig 3E, from 60 cells in early S, 139 cells in mid S, 101 cells in late S, and 127 cells in G2 phase. *, p < 0.05; **, p < 0.01 by Mann–Whitney U test. (D) Validation of the methodology, using GP1 and GP2 from Fig 3D. Cell numbers in each sub-phase within GP1 (a) and GP2 (b) were determined. GP1 and GP2 consisted of 138 and 289 cells, respectively.

Mentions: In general, the Fucci system is unable to distinguish S and G2 phases [24]. However, we reasoned that such a distinction would be possible if two types of information were simultaneously available: the intensity of green fluorescence, which gradually increases during the green phase due to the constitutively active promoter of Fucci probes [24], and the level of DNA synthesis activity. A simple example of the classification procedure is shown in Fig 6A. At the time of irradiation, a higher intensity of green fluorescence was observed in cell #1 than in cell #2. Immediately after irradiation, EdU was incorporated. After acquisition of time-lapse images, EdU staining was performed, and the results revealed that cell #1 [EdU(−)] and #2 [EdU(+)] were in G2 and S phases, respectively, at the time of irradiation. To apply this method to the 427 previously analyzed green-phase cells (Fig 3B), which had been already EdU-incorporated immediately after irradiation, the following steps were taken to further sub-divide S phase: 1) Green-phase cells were divided into two groups, EdU(+) (S phase) and EdU(−) (G2 phase); 2) The EdU(+) group, emitting both red and green fluorescence, was classified into early S phase, and then sorted according to the quantitative intensity of green fluorescence (ranked from low to high); 3) The remaining EdU(+) group and EdU(−) group were sorted according to the green fluorescence intensity within each group, and cells in EdU(+) group whose intensities overlapped with those in the EdU(−) group (higher intensity) were defined as late S phase; and 4) Finally, the remaining EdU(+) group was defined as mid-S phase. Collective results are shown in Fig 6B; for simplicity, a subset of cells from each sub-S and G2 phase are shown. Among all cells analyzed, the intensity levels differed significantly between each phase (Fig 6C and S2 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)

Establishment of a method for classifying green phase into early/mid/late S and G2 phases.(A) Experimental procedure for classification of green phase. Time-lapse imaging was performed for cells flash-labeled with EdU immediately after 5 Gy irradiation, followed by EdU staining. Time is shown as hours:minutes. (B) Classification of green-phase cells into early/mid/late S and G2 phases, according to the method described in A. For simplicity, a subset of cells in each phase is presented. To adjust the fluorescence intensities among different fields, background fluorescence intensity of each image was normalized to 1. (C) Comparison of green fluorescence intensities among sub-groups. Data are represented as box-and-whisker plots, as described in Fig 3E, from 60 cells in early S, 139 cells in mid S, 101 cells in late S, and 127 cells in G2 phase. *, p < 0.05; **, p < 0.01 by Mann–Whitney U test. (D) Validation of the methodology, using GP1 and GP2 from Fig 3D. Cell numbers in each sub-phase within GP1 (a) and GP2 (b) were determined. GP1 and GP2 consisted of 138 and 289 cells, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4472673&req=5

pone.0128090.g006: Establishment of a method for classifying green phase into early/mid/late S and G2 phases.(A) Experimental procedure for classification of green phase. Time-lapse imaging was performed for cells flash-labeled with EdU immediately after 5 Gy irradiation, followed by EdU staining. Time is shown as hours:minutes. (B) Classification of green-phase cells into early/mid/late S and G2 phases, according to the method described in A. For simplicity, a subset of cells in each phase is presented. To adjust the fluorescence intensities among different fields, background fluorescence intensity of each image was normalized to 1. (C) Comparison of green fluorescence intensities among sub-groups. Data are represented as box-and-whisker plots, as described in Fig 3E, from 60 cells in early S, 139 cells in mid S, 101 cells in late S, and 127 cells in G2 phase. *, p < 0.05; **, p < 0.01 by Mann–Whitney U test. (D) Validation of the methodology, using GP1 and GP2 from Fig 3D. Cell numbers in each sub-phase within GP1 (a) and GP2 (b) were determined. GP1 and GP2 consisted of 138 and 289 cells, respectively.
Mentions: In general, the Fucci system is unable to distinguish S and G2 phases [24]. However, we reasoned that such a distinction would be possible if two types of information were simultaneously available: the intensity of green fluorescence, which gradually increases during the green phase due to the constitutively active promoter of Fucci probes [24], and the level of DNA synthesis activity. A simple example of the classification procedure is shown in Fig 6A. At the time of irradiation, a higher intensity of green fluorescence was observed in cell #1 than in cell #2. Immediately after irradiation, EdU was incorporated. After acquisition of time-lapse images, EdU staining was performed, and the results revealed that cell #1 [EdU(−)] and #2 [EdU(+)] were in G2 and S phases, respectively, at the time of irradiation. To apply this method to the 427 previously analyzed green-phase cells (Fig 3B), which had been already EdU-incorporated immediately after irradiation, the following steps were taken to further sub-divide S phase: 1) Green-phase cells were divided into two groups, EdU(+) (S phase) and EdU(−) (G2 phase); 2) The EdU(+) group, emitting both red and green fluorescence, was classified into early S phase, and then sorted according to the quantitative intensity of green fluorescence (ranked from low to high); 3) The remaining EdU(+) group and EdU(−) group were sorted according to the green fluorescence intensity within each group, and cells in EdU(+) group whose intensities overlapped with those in the EdU(−) group (higher intensity) were defined as late S phase; and 4) Finally, the remaining EdU(+) group was defined as mid-S phase. Collective results are shown in Fig 6B; for simplicity, a subset of cells from each sub-S and G2 phase are shown. Among all cells analyzed, the intensity levels differed significantly between each phase (Fig 6C and S2 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