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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: To visualize the cell-cycle phase, we employed the fluorescent ubiquitination-based cell cycle indicator (Fucci).The results revealed that endoreduplication rarely occurs in this cell line under the conditions we studied.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

Cell-cycle kinetics in p53-functional cells following irradiation.(A) Order of progression to M phase in red and green cells in non-irradiated BJ1-hTERT-Fucci cells. Mitotic, red, and green cells at the start of observation were monitored until the next entry into M phase. (B) Pedigree analysis on Fucci fluorescence in irradiated BJ1-hTERT-Fucci cells. Time-lapse imaging was performed following 5 Gy irradiation. Each color represents the same as described in Fig 1A and 1D except that the white box represents early G1 phase without any fluorescence after mitotic skipping.
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pone.0128090.g008: Cell-cycle kinetics in p53-functional cells following irradiation.(A) Order of progression to M phase in red and green cells in non-irradiated BJ1-hTERT-Fucci cells. Mitotic, red, and green cells at the start of observation were monitored until the next entry into M phase. (B) Pedigree analysis on Fucci fluorescence in irradiated BJ1-hTERT-Fucci cells. Time-lapse imaging was performed following 5 Gy irradiation. Each color represents the same as described in Fig 1A and 1D except that the white box represents early G1 phase without any fluorescence after mitotic skipping.

Mentions: p53 in HeLa cells has been inactivated by human papilloma virus (HPV). Next, we attempted to determine whether cells irradiated in G1 phase catch up with cells irradiated in S phase only when p53 is deficient. For this purpose, p53 was expressed in HeLa-Fucci cells via adenovirus infection. Above multiplicity of infection (MOI) of 60, most cells exhibited apoptosis (data not shown). With MOI < 60, percentages of cells expressing p53 was very low, ~5%, by immunostaining (Fig. A in S2 File). In the latter condition, we observed that a very small number of cells, corresponding to the frequency of p53 expression, exhibited prolongation of red phase and higher red fluorescence (Fig. B in S2 File), which was not observed in uninfected cells. Therefore, we speculated that cells carrying a wild-type p53 gene with Fucci probes may exhibit elongation of red phase, representing G1 arrest. Because appropriate regulation of p53 expression was very difficult in this system, we performed further analysis using BJ1-hTERT-Fucci cells, which were established from h-TERT–immortalized normal human diploid foreskin fibroblasts (BJ-hTERT) with wild-type p53 function [27]. In an unirradiated condition, green cells entered M phase first, whereas red cells entered M phase after almost all the green cells had done so (Fig 8A), like Fucci-HeLa cells (Fig 1D). After 5 Gy irradiation, most cells irradiated in red phase exhibited a remarkable elongation of red phase, and cells irradiated in green phase turned red before the irradiated red cells entered M phase (or underwent mitotic skipping). Thus, the catching up of red cells with green cells, which was characteristic of HeLa-Fucci cells after irradiation, was unlikely to occur in cells carrying a functional p53 gene.


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)

Cell-cycle kinetics in p53-functional cells following irradiation.(A) Order of progression to M phase in red and green cells in non-irradiated BJ1-hTERT-Fucci cells. Mitotic, red, and green cells at the start of observation were monitored until the next entry into M phase. (B) Pedigree analysis on Fucci fluorescence in irradiated BJ1-hTERT-Fucci cells. Time-lapse imaging was performed following 5 Gy irradiation. Each color represents the same as described in Fig 1A and 1D except that the white box represents early G1 phase without any fluorescence after mitotic skipping.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4472673&req=5

pone.0128090.g008: Cell-cycle kinetics in p53-functional cells following irradiation.(A) Order of progression to M phase in red and green cells in non-irradiated BJ1-hTERT-Fucci cells. Mitotic, red, and green cells at the start of observation were monitored until the next entry into M phase. (B) Pedigree analysis on Fucci fluorescence in irradiated BJ1-hTERT-Fucci cells. Time-lapse imaging was performed following 5 Gy irradiation. Each color represents the same as described in Fig 1A and 1D except that the white box represents early G1 phase without any fluorescence after mitotic skipping.
Mentions: p53 in HeLa cells has been inactivated by human papilloma virus (HPV). Next, we attempted to determine whether cells irradiated in G1 phase catch up with cells irradiated in S phase only when p53 is deficient. For this purpose, p53 was expressed in HeLa-Fucci cells via adenovirus infection. Above multiplicity of infection (MOI) of 60, most cells exhibited apoptosis (data not shown). With MOI < 60, percentages of cells expressing p53 was very low, ~5%, by immunostaining (Fig. A in S2 File). In the latter condition, we observed that a very small number of cells, corresponding to the frequency of p53 expression, exhibited prolongation of red phase and higher red fluorescence (Fig. B in S2 File), which was not observed in uninfected cells. Therefore, we speculated that cells carrying a wild-type p53 gene with Fucci probes may exhibit elongation of red phase, representing G1 arrest. Because appropriate regulation of p53 expression was very difficult in this system, we performed further analysis using BJ1-hTERT-Fucci cells, which were established from h-TERT–immortalized normal human diploid foreskin fibroblasts (BJ-hTERT) with wild-type p53 function [27]. In an unirradiated condition, green cells entered M phase first, whereas red cells entered M phase after almost all the green cells had done so (Fig 8A), like Fucci-HeLa cells (Fig 1D). After 5 Gy irradiation, most cells irradiated in red phase exhibited a remarkable elongation of red phase, and cells irradiated in green phase turned red before the irradiated red cells entered M phase (or underwent mitotic skipping). Thus, the catching up of red cells with green cells, which was characteristic of HeLa-Fucci cells after irradiation, was unlikely to occur in cells carrying a functional p53 gene.

Bottom Line: To visualize the cell-cycle phase, we employed the fluorescent ubiquitination-based cell cycle indicator (Fucci).The results revealed that endoreduplication rarely occurs in this cell line under the conditions we studied.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