Temporo-spatial cell-cycle kinetics in HeLa cells irradiated by Ir-192 high dose-rate remote afterloading system (HDR-RALS)
© The Author(s). 2016
Received: 1 March 2016
Accepted: 15 July 2016
Published: 29 July 2016
Intracavitary irradiation plays a pivotal role in definitive radiotherapy for cervical cancer, and the Ir-192 high dose-rate remote afterloading system (HDR-RALS) is often used for this purpose. Under this condition, tumor tissues receive remarkably different absorption doses, with a steep gradient, depending on distance from the radiation source. To obtain temporo-spatial information regarding cell-cycle kinetics in cervical cancer following irradiation by Ir-192 HDR-RALS, we examined HeLa cells expressing the fluorescence ubiquitination-based cell cycle indicator (Fucci), which allowed us to visualize cell-cycle progression.
HeLa-Fucci cells, which emit red and green fluorescence in G1 and S/G2/M phases, respectively, were grown on 35-mm dishes and irradiated by Ir-192 HDR-RALS under normoxic and hypoxic conditions. A 6 French (Fr) catheter was used as an applicator. A radiation dose of 6 Gy was prescribed at hypothetical treatment point A, located 20 mm from the radiation source. Changes in Fucci fluorescence after irradiation were visualized for cells from 5 to 20 mm from the Ir-192 source. Several indices, including first green phase duration after irradiation (FGPD), were measured by analysis of time-lapse images.
Cells located 5 to 20 mm from the Ir-192 source became green, reflecting arrest in G2, in a similar manner up to 12 h after irradiation; at more distant positions, however, cells were gradually released from the G2 arrest and became red. This could be explained by the observation that the FGPD was longer for cells closer to the radiation source. Detailed observation revealed that FGPD was significantly longer in cells irradiated in the green phase than in the red phase at positions closer to the Ir-192 source. Unexpectedly, the FGPD was significantly longer after irradiation under hypoxia than normoxia, due in large part to the elongation of FGPD in cells irradiated in the red phase.
Using HeLa-Fucci cells, we obtained the first temporo-spatial information about cell-cycle kinetics following irradiation by Ir-192 HDR-RALS. Our findings suggest that the potentially surviving hypoxic cells, especially those arising from positions around point A, exhibit different cell-cycle kinetics from normoxic cells destined to be eradicated.
A combination of external and intracavitary irradiation is used as a definitive radiotherapy for treatment of cervical cancer [1–3]. Intracavitary irradiation using a high dose-rate radiation source, such as Ir-192, has been applied in the form of a remote afterloading system (RALS) [2, 3]. The prescribed dose is usually ~6 Gy per fraction at point A, 2 cm from the center axis of the uterus . Although the dose at this point is considered to be an index of tumor dose, it is widely recognized that tumor tissues receive remarkably different doses, with a steep gradient, depending on distance from the radiation source. We reasoned that radioresponses of tumor cells, including cell-cycle kinetics, following such an intracavitary irradiation should differ markedly depending on distance from the radiation source.
Following DNA damage, cell-cycle progression stops at the G1/S and G2/M checkpoints . The former checkpoint is dependent on p53 function, whereas the latter is not [6, 7]. Therefore, tumor cells with p53 gene mutations or human papilloma virus (HPV) infection only activate the G2/M checkpoint following irradiation, resulting in G2 arrest. Flow-cytometric analysis using DNA content as a marker has been used to detect radiation-induced G2 arrest ; however, because this method requires preparation of single cells and fixation, spatial information is lost, and temporal information must be obtained from different cell populations.
To address these issues, we used the fluorescent ubiquitination-based cell cycle indicator (Fucci) system, in which cells emit red and green fluorescence in G1 and S/G2/M phases, respectively. The Fucci system allowed us to visualize cell-cycle progression in HeLa cells, which lack p53 function due to HPV infection . In a previous study using time-lapse imaging, we showed that the fluorescence kinetics in HeLa-Fucci cells perfectly reflect radiation-induced G2 arrest kinetics [10, 11]. In this study, taking further advantage of this system, we attempted to visualize cell-cycle kinetics in vitro, as a function of the distance from the radiation source, after irradiation with an Ir-192 HDR-RALS under normoxic and hypoxic conditions. Furthermore, we determined the effect of cell-cycle phase at irradiation under both conditions.
Cell line and culture conditions
HeLa cells expressing the Fucci system (HeLa-Fucci) were provided by the RIKEN BRC through the National Bio-Resource Project of MEXT, Japan. Cells were maintained in DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10 % fetal bovine serum at 37 °C in a 5 % CO2 humidified atmosphere.
Irradiation by Ir-192 HDR-RALS
Irradiation under hypoxic conditions
Using the BIONIX-1 hypoxic culture kit (Sugiyama-Gen, Tokyo, Japan), HeLa-Fucci cells were made hypoxic (pO2 < 0.1 %) as described previously  and irradiated as described above. Briefly, 24 h after cells (5 × 104 cells) were seeded on a 35-mm glass-bottom dish, the medium was replaced by 300 μl of fresh growth medium to quickly equilibrate the gas and liquid phases. The dish was placed in an AnaeroPouch (Mitsubishi Gas Chemical, Tokyo, Japan) along with an AnaeroPack-Anaero 5 % oxygen absorber (Mitsubishi Gas Chemical) and an OXY-1 oxygen monitor (JIKCO, Tokyo, Japan), and the pouch was sealed. Irradiation was performed 2 h after verifying an oxygen monitor reading of 0 %, which confirms pO2 < 0.1 % in the gas phase, to ensure equilibration between the gas and liquid phases. The oxygen enhancement ratio (OER) obtained from dose–cell survival curves (at a surviving fraction of 0.1) was ~2.1. Time-lapse imaging was performed under normoxic conditions.
Acquisition of low-power images
For acquisition of low-power images, cells grown on dishes were fixed in 4 % paraformaldehyde 12, 24, 48, or 72 h after irradiation under normoxic conditions. A BIOREVO BZ-9000 fluorescence microscope (KEYENCE, Osaka, Japan) was equipped with a low-power objective lens (×10), and fluorescence images were acquired in a field measuring 15 mm × 3 mm in order to include cells located 5 to 20 mm from the radiation source.
Time-lapse imaging after irradiation
Time-lapse imaging was performed immediately after irradiation. Fluorescence images were acquired on a BIOREVO BZ-9000 fluorescence microscope (KEYENCE) equipped with a high-power objective lens (×40) in an incubation chamber kept at 37 °C in a 5 % CO2 humidified atmosphere. Fluorescence images were taken every 2 h until 72 h after irradiation. The field size was 700 μm (length) × 500 μm (width); at least three fields were collected from a total of about 90 cells at each point 5–20 mm from the radiation source. Several parameters were analyzed: the first green phase duration following irradiation (FGPD), reflecting the duration of G2 arrest; the number of entries into M phase; the proportion of cells undergoing mitotic catastrophe; and cell-cycle phase at the time of cell death.
Dose–cell survival curves
Dose–cell survival curves were obtained from colony forming assays. Exponentially growing HeLa-Fucci cells were irradiated with varying doses under normoxia or hypoxia, generated as described above, using an RX-650 Cabinet X-radiator system (Faxitron, Lincolnshire, IL, USA) at a dose rate of 0.8 Gy/min (130 kVp, 5 mA, 0.5 mm Al filtration). After irradiation, an appropriate number of cells were seeded in 60-mm dishes and incubated for 10 days. Clonogenic survival was determined by counting crystal violet–stained colonies consisting of more than 50 cells.
Cell sorting and flow-cytometric analysis
Cell sorting and flow-cytometric analysis was performed as described previously . Briefly, exponentially growing HeLa-Fucci cells were sorted on a MoFlo XDP (Beckman Coulter, Brea, CA, USA) according to their Fucci fluorescence. The red and green fractions were irradiated at a dose of 10 Gy, and 30 min or 3 h after irradiation, the cells were fixed in 4 % paraformaldehyde for 30 min. After staining with an anti-phospho-histone H2AX (Ser139) antibody conjugated with Alexa Fluor 647 (1:50; Cell Signaling, Danvers, MA), each sample was analyzed on a FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) using the FlowJo software (Tree Star, Ashland, OR, USA).
Mann–Whitney U test or chi-square test was used for statistical determinations. P values < 0.05 were considered statistically significant.
Dose distribution under our experimental conditions
The experimental conditions are outlined in Fig. 1a. Simulation of dose distribution by the treatment planning system, which is actually used in the clinical setting in our hospital, is depicted in Fig. 1b by some iso-dose curves (left panel). The dose absorption on the X-axis measured by a TLD plate as a function of distance from the radiation source is shown in Fig. 1b (right panel). The actual absorption dose at 5, 10, 15, and 20 mm was 24, 14, 9, and 6 Gy, respectively.
Temporo-spatial cell-cycle kinetics in low-power field images
Closer analysis using time-lapse imaging of HeLa-Fucci cells 5 or 20 mm from the radiation source
Differential responses in cells irradiated in green and red phases
Responses in cells after irradiation under hypoxic conditions
When hypoxic cells that were red or green at the time of irradiation were analyzed separately, the same dependence on distance was observed again. Unexpectedly, we found that the elongation after irradiation under hypoxia observed in Fig. 9b was mainly due to cells irradiated in red phase (Fig. 9c). Interestingly, after irradiation under hypoxia, the dependence on cell-cycle phase observed under normoxia was lost at 5 mm, whereas the opposite dependence was observed at 20 mm, where cells irradiated in red phase exhibited a longer FGPD than those irradiated in green phase (Fig. 9c). Taken together, these data show that potentially surviving hypoxic cells especially arising from positions around point A exhibited G2 arrest kinetics distinct from those of normoxic cells destined to be eradicated.
Using the Fucci system, we were able for the first time to simultaneously visualize the cell-cycle kinetics in HeLa cells at different distances (5–20 mm) relative to the radiation source following irradiation by Ir-192 HDR-RALS. Our major findings are summarized as follows; 1) cells in this area exhibited similar G2 arrest kinetics up to 12 h after irradiation; 2) more distant cells gradually began to be released from G2 arrest; 3) cells at 5 mm had a significantly longer FGPD than those at 20 mm; 4) under normoxic conditions, cells irradiated in the green phase had a longer FGPD than those irradiated in red phase, and 5) under hypoxic conditions, cells exhibited the opposite dependence on cell-cycle phase at the time of irradiation, especially in cells arising from positions around point A.
The reason that cells within the observed area accumulated in the green phase with similar kinetics up to 12 h is that the time required to reach G2 arrest is not influenced by the degree of DNA damage. In the Fucci system, cells in S and G2 phases exhibit the same green fluorescence; therefore, we can say that red phase duration was not increased even very close to the radiation source. Presumably, at greater distances (>20 mm), the degree of accumulation in green phase gradually decreased. Distance exerted a clear and significant influence on FGPD. This observation indicates that the duration of G2 arrest was strongly dependent on distance from the Ir-192 source, demonstrating that it was primarily influenced by absorption dose. Previous work showed that the duration of G2 arrest is proportional to absorption dose [15, 18]; however, in this study, the use of HeLa-Fucci cells allowed us to clearly temporo-spatially visualize this phenomenon, even in the steep dose gradient of an Ir-192 HDR-RALS.
We can discuss the difference in FGPD from the viewpoint of distance from the radiation source, i.e., the absorption dose. We speculated that this could be attributed to the degree of DNA damage, which is proportional to the absorption dose; however, it should be noted that under normoxia, FGPD at 5 mm was remarkably affected by cell-cycle phase (i.e., green vs red) at the time of irradiation. Indeed, the FGPD was much longer when cells were irradiated in green phase. To explain the phenomenon, we must consider other secondary factors. DNA double-strand breaks (DSBs) are thought to be the most crucial determinants of the DNA damage response, including cell-cycle checkpoints [19–21]. DSBs are repaired through two distinct pathways, non-homologous end joining (NHEJ) and homologous recombination. The former occurs in all cell-cycle phases, whereas the latter is restricted to S and G2 phases . Therefore, in the Fucci system, only NHEJ occurs in red phase, and both pathways are activated in green phase. Karanam et al. reported that when cells are irradiated in G1 phase, DSB repair via the NHEJ pathway is completed within a short period of time, whereas when cells are irradiated in S/G2 phases, it takes longer to perform repair because more time is required to choose one of the two pathways, leading to an elongated G2 arrest . As a matter of fact, our preliminary study showed that quantitative flow-cytometric analysis of γH2AX, a marker of DSBs, after cell sorting revealed a more rapid reduction of the mean fluorescence intensity after irradiation (30 min → 3 h) in cells irradiated in red phase (mean ± SD: 739 ± 26 → 380 ± 14) than in cells irradiated in green phase (1703 ± 95 → 2044 ± 36). Collectively, we can say that the time required for DSB repair is proportional to the number of DSBs induced by irradiation, which in turn corresponds to the absorption dose; this explains our observation that FGPD depended on absorption dose. However, FGPD could be secondarily affected by the DSB repair rate, which is determined by whether cells are irradiated in G1 or S/G2 phase.
Considering that the radiation doses given under normoxic conditions described above ultimately eradicate most HeLa-Fucci cells (Fig. 8), one could argue that our findings are not clinically interesting. Given that hypoxic cells exist within solid tumors, such surviving cells could contribute to recurrence after irradiation. Indeed, many studies have shown that cervical cancers with higher hypoxic fractions are more likely to recur after radiotherapy [16, 17]. Under our experimental conditions, many hypoxic cells around point A, receiving ~6 Gy, were likely to survive (surviving fraction, 0.4–0.5; Fig. 8). Therefore, we next examined cell-cycle kinetics after irradiation under hypoxic conditions. Our results were unexpected because the dependence on cell-cycle phase at the time of irradiation, as reflected in elongation of FGPD, differed between cells irradiated under normoxia and hypoxia, especially around point A. Despite some controversial reports , it is generally accepted that under hypoxia, DSB yield after irradiation is lower and its repair is slower . If G2 arrest was determined only by DSB-associated events, then the elongation of G2 arrest could be explained by the fact that DSB repair is slower after irradiation under hypoxia. Furthermore, DSB generation due to reoxygenation after hypoxia might also be involved [14, 26]. However, our observation that G2 arrest is enhanced when cells were irradiated in red phase could not be explained by the notion proposed by Karanam et al. , as described above. The yield of DNA-protein cross-linking is enhanced by irradiation under hypoxia, and such cross-links are repaired much more slowly than DSBs . Although the dependence on cell-cycle phase at the time of irradiation remains largely unclear, other types of DNA damage that preferentially occur after irradiation under hypoxia might influence G2 arrest. Taken together, the results of our temporo-spatial study suggest that cells with a high possibility of recurrence, especially those arising from hypoxic tumor cells around point A, exhibit different G2 arrest kinetics than those arising from normoxic cells destined to be eradicated.
There are two types of hypoxia within solid tumors; acute and chronic. The former is the result of the perfusion limit of tumor vessels, caused by their repeated occlusion and re-opening, whereas the latter is the result of the diffusion limit and reoxygenation could occur after irradiation . Our experimental hypoxic conditions may not exactly reflect the quite complicated hypoxia/ reoxygenation kinetics in vivo; however, the present study would shed additional light on the effect of irradiation under hypoxia on the DNA damage response.
We analyzed cell-cycle kinetics of HeLa-Fucci cells irradiated in monolayer cultures by Ir-192 HDR-RALS. Our findings visualized G2 arrest kinetics which depend on distance from the source and cell-cycle phase at the time of irradiation. The latter dependence differed between irradiation under nomoxia and hypoxia, and the potentially surviving hypoxic cells exhibited unique G2 arrest kinetics. This study may be insufficient to predict actual events in a more complex clinical setting. Further studies using in vivo solid tumors are required to simulate the real radioresponse following irradiation with Ir-192 HDR-RALS.
DSB, DNA double strand break; FGPD, first green phase duration; Fucci, fluorescence ubiquitination-based cell cycle indicator; HDR-RALS, high dose-rate remote afterloading system; HPV, human papilloma virus; NHEJ, non-homologous end joining; TLD, thermoluminescent dosimetry
The authors thank Dr. A. Miyawaki and Dr. A. Sakaue-Sawano for their approval to obtain HeLa cells expressing the Fucci probes through RIKEN BRC. We also thank Mr. Koji Sasamori and Ms. Asami Shimizu for their assistance with irradiation by Ir-192 HDR-RALS.
This study was supported in part by Grants-in-Aid for Scientific Research from MEXT (26861569, 26293399, and 25670796) and Japan mHDR Research Fund.
Availability of data and materials
Additional file was included, which contains Additional file 1: Table S1, Additional file 2: Table S2, Additional file 3: Table S3 and Additional file 4: Table S4, raw data for Figs. 5, 7, 8, and 9 to reconstruct each graph.
TA carried out cell cultures, irradiation, time-lapse imaging, and pedigree analysis, and drafted the manuscript. AK participated in coordination of experiments, carried out the statistical analysis, and helped to draft the manuscript. TG carried out flow cytometric analysis. RY and KS helped to draft the manuscript. MM conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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