Heterogeneous cell-cycle behavior in response to UVB irradiation by a population of single cancer cells visualized by time-lapse FUCCI imaging

Abstract

The present study analyzed the heterogeneous cell-cycle dependence and fate of single cancer cells in a population treated with UVB using a fluorescence ubiquitination-based cell-cycle (FUCCI) imaging system. HeLa cells expressing FUCCI were irradiated by 100 or 200 J/m² UVB. Modulation of the cell-cycle and apoptosis were observed by time-lapse confocal microscopy imaging every 30 min for 72 h. Correlation between cell survival and factors including cell-cycle phase at the time of the irradiation of UVB, mitosis and the G₁/S transition were analyzed using the Kaplan–Meier method along with the log rank test. Time-lapse FUCCI imaging of HeLa cells demonstrated that UVB irradiation induced cell-cycle arrest in S/G₂/M phase in the majority of the cells. The cells irradiated by 100 or 200 J/m² UVB during G₀/G₁ phase had a higher survival rate than the cells irradiated during S/G₂/M phase. A minority of cells could escape S/G₂/M arrest and undergo mitosis which significantly correlated with decreased survival of the cells. In contrast, G₁/S transition significantly correlated with increased survival of the cells after UVB irradiation. UVB at 200 J/m² resulted in a greater number of apoptotic cells.

Keywords:

• apoptosis, cell-cycle
• FUCCI
• heterogeneity
• HeLa
• mitosis, survival analysis
• time-lapse imaging
• UVB

Introduction

Tumor heterogeneity is a major problem for therapy and can result in chemosensitive and chemoresistant subpopulations within a single tumor whose interaction can result in unexpected responses. Shifts in the ratios within a tumor subpopulations can be detrimental to therapy.¹

Using an imageable cell colony-forming assay (iCFA), 2.6% of cells treated with a thymidylate synthetase inhibitor were shown to maintain a colony growth rate similar to that of untreated control colonies. Such minority resistant population could account for tumor recurrence.²

Slocum et al.³ determined single cell responses to a thymidylate synthetase inhibitor.^4,5 Although immediate loss of cell division occurred in 74% of treated cells, 6% of the cells could divide more than 2 times with the more proliferative sister cell sibling producing up to 73 times more cells than the other sister in the treated population. This is a striking example of heterogeneity in sister cells after mitosis.

Sakaue-Sawano et al.⁶ have utilized oscillating proteins linked to spectrally-distinct fluorescent proteins that specifically label cell-cycle phases in living cells in a system termed FUCCI (fluorescent ubiquitination-based cell-cycle indicator). In a previous study, we utilized the FUCCI imaging system to investigate heterogeneity of response to chemotherapy at the single cancer-cell level. Time-lapse FUCCI imaging demonstrated that both doxorubicin (DOX) and cisplatinum (CDDP) could induce cell-cycle arrest in S/G₂/M in almost all the cells, but a subpopulation of the cells could escape the block and undergo mitosis. The subpopulation which went through mitosis subsequently underwent apoptosis while the cells arrested in S/G₂/M survived.⁷

We previously compared the DNA damage repair (DDR) response of pancreatic cancer cells after UVB or UVC irradiation. The UV-induced DNA damage repair was imaged by focus formation of green fluorescent protein (GFP) fused to the DDR-related chromatin-binding protein 53BP1 in MiaPaCa-2 human pancreatic cancer cells growing in 3D Gelfoam® histoculture and in tumors grown in nude mice. 53BP1-GFP forms foci during DNA damage repair. Our results demonstrated that UVB has greater tissue penetration than UVC because of its longer wavelength and has clinical potential for eradicating superficial cancer.⁸

In the present study, the modulation of the cell-cycle phase and apoptosis due to UVB irradiation were observed on HeLa cells by using the FUCCI imaging system to determine the correlation between cell-cycle progression and UVB sensitivity.

Results and Discussion

Time-lapse imaging of cell-cycle after low-dose UVB irradiation

To analyze the correlation between cell-cycle phase and sensitivity to UVB, HeLa-FUCCI cells were individually numbered. Mitosis, transition from G₁ to S phase, and apoptosis were observed in the individual cells after 100 J/m² UVB irradiation (Table 1, Figs. 1, 2A–C; Videos S2, 3). Time-lapse FUCCI imaging of HeLa cells demonstrated that UVB irradiation induced cell-cycle arrest in S/G₂/M phase within 24 h (Fig. 2D), and that a fraction of the cells irradiated by UVB escaped from cell-cycle arrest (Fig. 2D).

Figure 1. UVB irradiation and time-lapse FUCCI imaging of cell-cycle progression. HeLa-FUCCI cells were cultured in 35 mm dishes for 24 h. The cells were irradiated with 100 or 200 J/m² UVB using a Benchtop 3UV transilluminator (UVP, LLC, Upland, CA) with an emission peak at 302 nm. After irradiation, cell-cycle progression, as well as mitotsis and apoptosis were observed every 30 min for 72 h by FV1000 confocal microscopy (Olympus). The UVB dose was measured with a UVX Radiometer (UVP). After UVB irradiation, cell-cycle progression and apoptosis were observed every 30 min for 72 h using the FV1000. Scanning and image acquisition were controlled by FluoView software (Olympus).

Figure 2. Single-cell time-lapse imaging in HeLa-FUCCI cells after irradiation with 100 J/m² UVB. (A) Individualization of cancer cells. Each cell was individualized by numbering. The cell-cycle phase of each cell was observed every 30 min for 72 hours by confocal imaging. (B) Time-lapse imaging of the cell-cycle and apoptosis after irradiation with 100 J/m² UVB. (C) Apoptosis after irradiation with 100 J/m² UVB. The cells circled with white dotted lines entered apoptosis after mitosis. (D) Distribution of cell-cycle phase after irradiation with 100 J/m² UVB.

Figure 3. Survival analysis of individual cells after irradiation with 100 J/m² UVB. (A) Kaplan-Meier survival curve for G₀/G₁ phase cells and S/G₂/M phase cells at the onset of UVB irradiation. (B) Kaplan-Meier survival curve for the cells entering mitosis within 24 h after UVB irradiation compared to non-mitotic cells. (C) Kaplan-Meier survival curve for the cells which transitioned from G₁ phase to S phase within 24 h after UVB irradiation, compared with cells without G₁/S transition.

Table 1. Time-lapse cell-cycle imaging of individual HeLa-FUCCI cells after UVB irradiation

	UVB irradiation (doses)
Condition	100 J/m^2	200 J/m^2
Cell-cycle phase at the beginning of time-lapse imaging
G0/G1 phase	101 (43.2 %)	163 (55.4%)
S/G2/M phase	133 (56.8 %)	131 (44.6%)
G1/S transition within 24 h after UVB irradiation
yes	128 (54.7 %)	165 (56.1 %)
no	106 (45.3 %)	129 (43.9 %)
Mitosis during time-lapse imaging
yes	68 (29.1 %)	92 (31.3 %)
no	166 (70.9 %)	202 (68.7 %)
Apoptosis
yes	110 (47.0%)	222 (75.5%)
no	124 (53.0%)	72 (24.5%)

To determine the correlation between the cell-cycle phase when UVB irradiation was initiated and sensitivity to UVB, the cells were divided into 2 groups according to the cell-cycle phase at the onset of time-lapse imaging: Green and yellow fluorescence defined S/G₂/M phase, and red fluorescence defined G₀/G₁ phase in the FUCCI imaging system. Survival analysis of the HeLa-FUCCI cells revealed that the initiation of irradiation of 100 J/m² UVB during S/G₂/M phase significantly decreased the survival of the HeLa-FUCCI cells compared to initiation of irradiation in G₀/G₁ phase (P = 0.026) (Fig. 3A). To determine the correlation between mitosis and survival of the cells, the cells were divided into 2 groups according to the presence or absence of mitosis within 24 h after irradiation of 100 J/m² UVB: Survival analysis demonstrated that mitosis significantly decreased the survival of the cells after irradiation with 100 J/m² UVB (P = 0.019) (Fig. 3B).

To determine the correlation between G₁/S transition and survival, the cells were divided into 2 groups: the cells which were in G₁/S transition within 24 h after the irradiation of 100 J/m² UVB and cells which were not. G₁/S transition significantly correlated with increased survival of the cells after irradiation with 100 J/m² UVB (P < 0.001) (Fig. 3C).

Time-lapse imaging of cell-cycle progression after high-dose UVB irradiation

Time-lapse imaging of HeLa-FUCCI cells after UVB irradiation demonstrated that more than 90% of the cells underwent cell-cycle arrest in S/G₂/M phase within 24 h after 200 J/m² UVB irradiation (Table 1, Fig. 4A–D, Videos S4, 5A–C). The cell-cycle arrest in S/G₂/M phase continued until 36 h in more than 80% of the cells (Fig. 4D).

Figure 4. Single cell time-lapse imaging of HeLa-FUCCI cells after irradiation with 200 J/m² UVB. (A) Individualization of cancer cells. Each cell was individualized by numbering. The cell-cycle phase of each cell was observed every 30 min for 72 hours by confocal microscopy imaging. (B) Time-lapse imaging of the cell-cycle phase and apoptosis after irradiation with 200 J/m² UVB. (C) Apoptosis after irradiation with 200 J/m² UVB. Cell 96 entered apoptosis after mitosis. Cell 97 cell became apoptotic without mitosis. (D) Distribution of cell-cycle phase after irradiation with 200 J/m² UVB.

Figure 5. Survival analysis of individual cells after irradiation with 200 J/m² UVB. (A) Kaplan-Meier survival curve for G₀/G₁ and S/G₂/M cells at the onset of UVB irradiation. (B) Kaplan-Meier survival curve for cells which entered mitosis within 24 h after irradiation with UVB compared to non-mitotic cells. (C) Kaplan-Meier survival curve for the cells which transitioned from G₁ to S phase within 24 h after irradiation with UVB, compared to cells without G₁/S transition.

Survival analysis of the HeLa-FUCCI cells after 200 J/m² UVB irradiation demonstrated that cells irradiated during S/G₂/M phase are more sensitive than G₀/G₁ phase cells (P < 0.001, Fig. 5A). Mitosis within 24 h after 200 J/m² UVB irradiation significantly correlated with decreased survival of the cells (P < 0.001, Fig. 5B). Transition from G₁ to S phase within 24 h after the irradiation significantly increased the survival of the cells (P < 0.001, Fig. 5C). UVB irradiation at 200 J/m² increased apoptosis of HeLa cells compared to 100 J/m² (Table 1).

In our previous study, single cell time-lapse FUCCI imaging enabled observation of the heterogeneous effect of chemotherapy on cell-cycle progression of single cancer cells.⁷

In the present study, single-cell time-lapse FUCCI imaging of HeLa cells showed heterogeneous responses to UVB including cell-cycle arrest, escape from the arrest, mitosis and apoptosis in individual cells. The cell-cycle arrest after 200 J/m² UVB irradiation lasted longer compared with the cells irradiated by 100 J/m² UVB.

The present study also showed that mitosis has significant correlation with decreased survival of the cells after UVB irradiation, and that G₁/S transition has significant association with increased survival of the cells after the UVB irradiation. Furthermore, survival analyses of the HeLa-FUCCI cells revealed that cells in S/G₂/M phase cells had higher sensitivity to UVB than G₀/G₁ phase cells.

Our study is the first report which determined the correlation between sensitivity to UVB and cell-cycle progression by using survival analyses for individual cells whose cell-cycle phase was determined by FUCCI imaging.

The cell-cycle effect and dependence of UVB irradiation described in the present report can be synergistically used with previously-developed tumor-targeting strategies.^9-16

Materials and Methods

Establishment of HeLa cells stably transfected with FUCCI plasmids

The FUCCI system was used to visualize the cell-cycle phase in individual HeLa cells.⁶ Plasmids expressing mKO2-hCdt1 (green-yellow fluorescent protein) or mAG-hGem (orange-red fluorescent protein) were obtained from the Medical & Biological Laboratory (Nagoya, Japan). Plasmids expressing mKO2-hCdt1 were transfected into HeLa cells using Lipofectamine™ LTX (Invitrogen, Carlsbad, CA). The cells were sorted by green-yellow (S, G₂, and M phase) using a FACS-Aria cell sorter (Becton Dickinson). The first-step-sorted green-fluorescent cells were then re-transfected with mAG-hGem (orange-red) and then sorted by orange fluorescence.^7,17-21

Confocal imaging of cell-cycle behavior

After UVB irradiations, cell-cycle progression, mitosis and apoptosis were observed every 30 min for 72 h using the FluoView FV1000 confocal laser microscope (Olympus Corp., Tokyo, Japan) to determine the correlation between cell-cycle progression, mitosis and apoptosis induced by UVB. Scanning and image acquisition were controlled by FluoView software (Olympus).²²

Cellular irradiation with UVB

HeLa-FUCCI cells were cultured in high-glucose DMEM (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS) (Sigma-Aldrich) at 37°C with 95% air and 5% CO₂ for 24 h. The cells were irradiated with UVB from the bottom of a chamber using a Benchtop 3UV transilluminator (UVP, LLC, Upland, CA) with an emission peak at 302 nm.⁸ The UVB dose was measured with a UVX Radiometer (UVP, LLC, Upland, CA).

Statistical analysis

The survival period of each cell was defined as the time from initial imaging to the time of the appearance of apoptosis. Survival was calculated using the Kaplan–Meier method along with the log rank test. Statistical significance was defined as P < 0.05. Statistical analyses were performed using EZR statistical software (Saitama Medical Center, Jichi Medical University, Saitama, Japan).

Dedication

This study dedicated to the memory of Dr. AR Moossa.

Disclosure of Potential Conflicts of Interest

SM, SY, HK, MY, MT, TM, KH, NY, and RMH are or were unsalaried associates of AntiCancer Inc. There are no other competing financial interests.

Supplemental Material

Supplemental data for this article can be accessed on the publisher's website