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Potentially lethal damage repair and its inhibitory effect of caffeine in two yolk sac tumor cell lines with different radiosensitivities
Cancer Letters 147 (1999) 199±206 www.elsevier.com/locate/canlet
Potentially lethal damage repair and its inhibitory effect of caffeine in two yolk sac tumor cell lines with different radiosensitivities Tetsuo Akimoto*, Norio Mitsuhashi, Hiroko Matsumoto, Hideyuki Sakurai, Katsuya Maebayashi, Keiko Higuchi, Miwako Nozaki, Hideo Niibe Department of Radiology and Radiation Oncology, Gunma University School of Medicine, 3-39-22, Showa-machi, Maebashi, Gunma 371-8511, Japan Received 27 May 1999; received in revised form 3 August 1999; accepted 4 August 1999
Abstract Purpose: In order to investigate the role of potentially lethal damage repair (PLDR) in cellular radiosensitivity, PLDR and its inhibitory effect by caffeine was examined. In addition, cell cycle distribution was also examined. Materials and methods: Two rat yolk sac tumor cell lines, NMT-1 and NMT-1R, with different radiosensitivities in vitro were used. The capacity for PLDR was examined using con¯uent-phase cells, and evaluated by calculating the recovery ratio. Inhibitory effect of caffeine on PLDR was examined with doses of 1, 5 and 10 mM. Results: The capacity of PLDR in two cell lines re¯ected radiosensitivity. The recovery ratio after irradiation of 5 Gy was 2.8 in the radiosensitive NMT-1 and 5.2 in the radioresistant NMT-1R, and recovery reached its peak level at 6 h in both cell lines. The degree of inhibition of PLDR was weaker in NMT-1R than that in NMT-1 at the same dose level, and was correlated with reduction of G2-arrested cells by caffeine. Conclusions: The results of this study suggest that the capacity of PLDR may be one of the determinant factors for radiosensitivity in the two cell lines used, and the inhibitory effect of caffeine on PLDR was in part attributable to the modi®cation of the cell cycle progression. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Radiation; Potentially lethal damage repair; Caffeine; Cell cycle
1. Introduction Advances in radiation biology have revealed the mechanism of radiosensitivity, and many intrinsic and extrinsic factors including a tumor suppresser gene, cell cycle related-proteins, growth factors and the capacity for radiation-induced damage repair have been proposed [1±3]. Especially in radiation-induced DNA damage repair, two types of repair processes, i.e. sublethal damage repair (SLDR) and potentially * Corresponding author. Tel.: 181-027-220-8383; fax: 181027-220-8397. E-mail address: [email protected] (T. Akimoto)
lethal damage repair (PLDR), are well known [4,5], and the degree of PLDR is closely associated with intrinsic radiosensitivity of malignant human tumors [6±8]. In vivo solid tumors usually contain a greater proportion of quiescent, non-cycling cells than established cell lines in vitro. This population is generally more radioresistant due to the high capacity for recovery or repopulation after DNA damage, thus the enhancement of cell-killing in non-cycling cells is clinically bene®cial. In an in vitro cell system, PLDR has been de®ned as an increased surviving fraction in the cells plated immediately after radiation compared with that in cells whose plating is delayed.
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00308-0
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T. Akimoto et al. / Cancer Letters 147 (1999) 199±206
Con¯uent-phase cultures in vitro contain a larger number of quiescent cells than exponentially growing cells, and the arrest of cell cycle progression allows cells to repair DNA damage. Several investigators reported that PLDR correlated well with the repair of double strand breaks [9] and inhibition of PLDR increases radiosensitivity [5]. We have already reported the establishment of a radioresistant rat yolk sac tumor cell line, NMT-1R by repeated irradiation of a radiosensitive line, NMT1 [10,11]. These cell lines would be a unique and good model to investigate the mechanism or determinant factors for radioresistance. A difference in the frequency of apoptosis induction and morphological change after radiation in these cell lines was reported previously by Mitsuhashi et al. [12,13] and the sensitivities for cisplatin and taxol were also examined [14,15], but the impact of recovery capacity on cellular radiosensitivity has not yet been examined. In this report, we investigated how the degree of PLDR correlates with radiosensitivities of both cell lines. In addition, the inhibitory effects of caffeine was also examined. Caffeine potentiates cytotoxicity of anti-cancer drugs and/or radiation [16] by way of the modi®cation of cell cycle progression or alteration of DNA conformation [17±19]. A characteristic pharmacological effect of caffeine on the cell cycle is abrogation of G2 arrest induced by DNA damaging agents [20], and a recent investigation revealed that caffeine acts at the G2-M checkpoint by altering phosphorylation of p34 Cdc2 or cyclin B1 expression [17,21]. Considering the fact that radiation usually in¯uence cell cycle checkpoint and induces G2 arrest via p53-dependent or -independent pathways, one may speculate that cell cycle progression may also be involved in the mechanism of inhibition of PLDR by caffeine. Therefore, cell cycle distribution was also examined.
2. Materials and methods 2.1. Cells and cell cultures Two rat yolk sac tumor cell lines which derived from the same origin and showed different radiosensitivity were used in this study. The characteristics of these two cell lines have already been reported
[10,11]. Brie¯y, NMT-1 cells are 1.7 times more radiosensitive than NMT-1R, as estimated from their D0 values. p53 status as assessed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was wild type in NMT-1 and mutant in NMT-1R [22]. The cells were maintained in RPMI1640 supplemented with 10% heat-inactivated fetal calf serum at 378C in a humidi®ed atmosphere of 5% CO2 and 95% air. 2.2. Radiation method and caffeine treatment Con¯uent cells, which were seeded into 60 mm diameter petri dishes with 5 ml medium, were irradiated with a Hitachi (MBR1505R) X-ray machine that was operated at 140 kV, 4.5 mA with a 0.5 mm Al ®ltration and FSD of 30 cm with a dose-rate of 1.11 Gy/min. Radiation was performed in air at room temperature. Caffeine was purchased from Sigma Chemicals (Tokyo, Japan), and used for experiment by dissolving in warmed (608C) medium. Caffeine was incubated with cells for 1 h immediately after radiation at 378C in a humidi®ed atmosphere of 5% CO2 and 95% air with ®nal concentrations of 1, 5 and 10 mM. After incubation, the medium containing caffeine was removed and new prewarmed medium was put into the dishes after washing twice with complete medium. 2.3. Evaluation of PLDR The PLDR in an in vitro system has been de®ned as follows. After single-irradiation of con¯uent cells, the cells were either immediately subcultured into 60 mm petri dishes (immediate plating) or were placed for optimal time in a incubator (incubation time) to allow cells to repair radiation damage and were then subcultured (delayed plating). An increase in the surviving fraction (SF) of delayed plating compared with that of immediate plating implies PLDR. Firstly, we examined the PLDR in NMT-1 and NMT-1R after irradiation with various single doses at a ®xed incubation time (6 h). The change in recovery capacity according to the incubation time (3, 6 and 9 h) was then examined. Surviving fractions were determined by clonogenic assay. Colonies were ®xed and stained
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A ®nal calculated value less than 1 implies that caffeine inhibited PLDR which was observed after radiation alone. 2.4. Cell cycle analysis The cell cycle distribution was examined as follows. After trypsinization of the cells, single cells were washed twice with phosphate-buffered saline, ®xed with 70% ethanol and stained with propidium iodide for 30 min at room temperature. The samples were then analyzed using ¯ow cytometry (FACScan, Becton-Dickinson, Mountain View, CA). Cell-cycle distribution was determined by analyzing the DNA histogram. 2.5. Statistical analysis Each data point was based on at least three independent experiments. Statistical analyses were performed by Student's t-test.
Fig. 1. PLDR in both cell lines. (a), NMT-1; (b), NMT-1R. Con¯uent-phase cells were irradiated with 1, 3, 5 and 7 Gy, and subcultured immediately after radiation (closed circle, X) or 6 h after postirradiation incubation (open circle: W).
with crystal violet (2% in methanol) at least 14 days after subculture for counting. The recovery ratio was de®ned as follows when the cells were treated by radiation alone. In order to evaluate the inhibitory effect by caffeine on PLDR, cell toxicity by caffeine alone was normalized by the almost same method NG et al. [23] reported. The SF after treatment with combined radiation (RT) and caffeine is divided by the SF after treatment with radiation alone, and then the calculated result is divided by the SF after treatment with radiation with incubation time of 6 h.
Fig. 2. Recovery ratio as a function of incubation time. Con¯uentphase cells were irradiated with 5 Gy, and subcultured at 3, 6 and 9 h after post-irradiation incubation. Closed circle (X), NMT-l; open square (A): NMT-1R. Vertical bars represent the standard deviation.
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T. Akimoto et al. / Cancer Letters 147 (1999) 199±206 Table 2 Cell cycle distribution of NMT-1 and NMT-1R before subculture Cell line
Treatment
Cell cycle distribution (%) G1
S
G2 1 M
NMT-1
Control a 5 Gy b Caffeine c
55 53 59
18 22 20
27 25 21
NMT-1R
Control a 5 Gy b Caffeine c
58 57 59
21 28 23
21 15 18
a
Con¯uent phase cells with no treatment. Con¯uent phase cells were irradiated with a dose of 5 Gy. After 6h incubation (before subculture), the cell cycle distribution was analyzed by the method described in Materials and methods (see Section 2.5). c Con¯uent phase cells were incubated with caffeine for 1 h immediately after radiation. After removing and washing caffeine, cells were incubated for 5 h and the cell cycle analyzed. b
Fig. 3. Dose-response curves for caffeine. Closed circle (X), NMT1; open circle (W), NMT-1R. Con¯uent-phase cells were exposed to caffeine for 1 h. After removing medium containing caffeine and washing with fresh medium, the cells were incubated for 5 h at 378C in a humidi®ed atmosphere of 5% CO2 and 95% air and subcultured. Surviving fractions were determined by clonogenic assay.
3. Results
time after irradiation, PLDR was already observed at 3 h, and reached its peak at 6 h (Fig. 2).
3.1. Capacity of PLDR
3.2. Inhibitory effect of caffeine on PLDR
As shown in Fig. 1, PLDR was observed in both cell lines. The recovery ratio at 5 Gy was 2:8 ^ 0:8 in NMT-1 and 5:2 ^ 1:1 in NMT-1R, respectively, and was consistent with the difference in radiosensitivity of the two cell lines The difference was statistically signi®cant (P , 0:05). According to the incubation
Table 1 Inhibitory effect of caffeine on PLDR in NMT-1 and NMT-1R a Concentration of caffeine (mM)
1 5 10 a
Recovery ratio b NMT-1
NMT-1R
0.72 0.52 0.23
1.15 0.88 0.47
Con¯uent-phase cells were incubated with caffeine for 1 h after irradiation of 5 Gy. The cells were subcultured 6 h after irradiation for clonogenic assay. Recovery ratios were calculated by the method described in Materials and methods (see Section 2.3). b Recovery ratio with less than 1 implies that PLDR was inhibited by caffeine.
Fig. 3 shows the dose-response curves of the con¯uent-phase of NMT-1 and NMT-1R treated with caffeine alone. The radiosensitive NMT-1 showed a slightly higher sensitivity than the radioresistant NMT-1R for each drug, but caffeine alone showed weak cytotoxic effect, and surviving fractions were 0.91 at 1 mM, 0.85 at 5 mM and 0.80 at 10 mM in NMT-1, while those in NMT-1R were 0.94 at 1 mM, 0.89 at 5 mM and 0.84 at 10 mM. Table 1 demonstrated recovery ratio of NMT-1 and NMT-1R after treatment with radiation and caffeine. The recovery ratios of radiosensitive NMT-1 were less than 1 at the all dose levels of caffeine, which means that caffeine inhibited PLDR completely in NMT-1. The recovery ratios of radioresistant NMT1R were slightly greater than those of NMT-1, and caffeine with a dose of 1 mM was not able to inhibit PLDR. 3.3. Alteration of cell cycle distribution after treatment Table 2 demonstrates the distribution of the cell
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Fig. 4. Changes in the cell cycle distribution after radiation (5 Gy). (a), NMT-1; (b), NMT-1R. Con¯uent-phase cells were irradiated with a dose of 5 Gy. After 6 h of post-irradiation incubation time, cells were subcultured. In order to evaluate alteration of cell cycle after subculture, cell cycle distribution was examined 6, 12 and 24 h after subculture. Vertical bars represent standard deviation.
Fig. 5. Changes in the cell cycle distribution following radiation in combination with caffeine with doses of 1 or 5 mM in NMT-1. (a), 1 mM caffeine; (b), 5 mM caffeine. Con¯uent-phase cells were irradiated with a dose of 5 Gy, followed by 1 h exposure to caffeine. Cell cycle distribution was analyzed at the indicated time after subculture. Vertical bars represent the standard deviation.
cycle before subculture. Radiation either alone or in combination with caffeine with a dose of 5 mM did not affect cell cycle during incubation time. There was not an apparent difference in the cell cycle distribution between NMT-1 and NMT-1R. After subculture, an increase in G2M phase cells
(G2 arrest) in both cell lines was observed 24 h after irradiation (Fig. 4a,b). Accumulation of G2-arrested cells was more prominent in NMT-1 than NMT-1R (54:1 ^ 3:5% in NMT-1 and 43:5 ^ 2:1% in NMT1R), and the difference in proportion of the cells was statistically signi®cant (P , 0:05). When caffeine
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4. Discussion
Fig. 6. Changes in the cell cycle distribution following radiation in combination with caffeine with doses of 1 or 5 mM in NMT-1R. (a), 1 mM of caffeine; (b), 5 mM of caffeine.
was applied in combination with radiation, proportion of G2-arrested cells observed after radiation alone was decreased with a statistically signi®cance (abrogation of G2 arrest) (Figs. 5 and 6). This phenomenon was observed in the both cell lines when caffeine concentration was 5 mM, but caffeine with a dose of 1 mM did not abrogate G2-arrest in NMT-1R (Fig. 6a) while abrogated in NMT-1 (Fig. 5a). The relationship between the dose of caffeine and degree of abrogation of G2 arrest showed the similar tendency of inhibitory effect of caffeine on PLDR.
The cell lines used in this study were derived from the same origin, and they may have similar tumor characteristics. Thus these would be a good model to investigate determinant factors for radiosensitivity. This study demonstrated that PLDR was observed in both NMT-1 and NMT-1R and their capacities were consistent with a difference in the radiosensitivities of two rat yolk sac tumor cell lines. i.e. the radioresistant NMT-1R showed higher capacity of PLDR than the radiosensitive NMT-1. The frequency of apoptosis induction, which has been a major determinant factor for cellular radiosensitivity, is different in these cell lines [12,13]. Radford et al. [24] recently reported that DNA repair capability in¯uences susceptibility to induction of apoptosis, therefore higher proportion of unrepairable DNA damage in NMT-1 than NMT1R may cause different apoptosis induction in addition to the difference of p53 status. The results of cell cycle analysis also indicated that the proportion of G2arrested cells induced by radiation alone was larger in the radiosensitive NMT-1 than in the radioresistant NMT-1R. As the magnitude of G2 delay has been shown to re¯ect the degree of radiation-induced DNA damage and cellular radiosensitivity [25], it may be speculated that NMT-1R had less residual DNA damage due to higher PLDR than NMT-1. The relationship between p53 function and repair capacity, has proved controversial. The tumors with wild type p53 have larger capacity of DNA damage repair through the mismatch repair and the nucleotide excision repair than tumors with mutant p53 [26±28], while the cell lines with p53 mutations showed higher SLDR than those with no mutations [29]. Thus, further investigations are needed to clarify relationship between p53 status and exact repair capacity in these cell lines. Although many investigators have reported the inhibition of PLDR by drugs using an in vitro PLDR system [23,30,31], a de®nite role of cell cycle progression in inhibition of PLDR is still unclear. In this study, caffeine inhibited PLDR in both cell lines at the dose level which showed weak cytotoxicity. Caffeine has inhibitory effect on DNA damage repair through inhibition of repair enzyme, but the pharmacological effect of caffeine on cell cycle is also considered to play an important role in the inhibition of
T. Akimoto et al. / Cancer Letters 147 (1999) 199±206
PLDR. Radiation and/or caffeine did not affect cell cycle distribution before subculture, but alteration and/or progression of cell cycle was observed after subculture. Especially, accumulation of G2-arrested cells which observed after radiation was reduced when caffeine was incubated after radiation. The degree of reduction in G2-arrested cells correlated with inhibitory effect of caffeine on PLDR (Fig. 6), i.e. 5 mM caffeine reduced radiation-induced G2 arrest, whereas 1 mM caffeine which showed no inhibitory effect on PLDR, did not. The shortening of radiation-induced G2 arrest which results in insuf®cient time for DNA repair has been shown to be the main mechanism in the sensitizing effect of caffeine in exponentially growing cells, and this mechanism may also be responsible for the inhibitory effect on PLDR using con¯uent phase cells [31]. Taken together, this study indicated that PLDR using in vitro system may be in¯uenced by not only repair capacity during incubation time, but also cell cycle progression after subculture. From the clinical and therapeutic standpoint, inhibition of PLDR will be one of the targets in order to gain the therapeutic effect. In vivo solid tumors usually show heterogeneity and have various conditions including a quiescent cell population, a portion of acidic pH and a hypoxic fraction. These microenvironments cause enhancement of PLDR [32±34]. Reduction or inhibition of PLDR, therefore, appears to be important method in clinical treatment to improve radiosensitivity. In conclusion, the present study showed that capacity for PLDR was associated with radiosensitivities of rat yolk sac tumor cell lines derived from the same origin, and an inhibition of PLDR by caffeine is in part attributed to the control or modi®cation of the cell cycle progression. Acknowledgements This study was supported in part by Grants-in-Aid for Scienti®c Research from the Ministry of Education, Science and Culture of Japan. References [1] J.H. Peacock, J.J. Eady, S. Edwards, A. Hoimers, T.J. Macmil-
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Potentially lethal damage repair and its inhibitory effect of caffeine in two yolk sac tumor cell lines with different radiosensitivities Tetsuo Akimoto*, Norio Mitsuhashi, Hiroko Matsumoto, Hideyuki Sakurai, Katsuya Maebayashi, Keiko Higuchi, Miwako Nozaki, Hideo Niibe Department of Radiology and Radiation Oncology, Gunma University School of Medicine, 3-39-22, Showa-machi, Maebashi, Gunma 371-8511, Japan Received 27 May 1999; received in revised form 3 August 1999; accepted 4 August 1999
Abstract Purpose: In order to investigate the role of potentially lethal damage repair (PLDR) in cellular radiosensitivity, PLDR and its inhibitory effect by caffeine was examined. In addition, cell cycle distribution was also examined. Materials and methods: Two rat yolk sac tumor cell lines, NMT-1 and NMT-1R, with different radiosensitivities in vitro were used. The capacity for PLDR was examined using con¯uent-phase cells, and evaluated by calculating the recovery ratio. Inhibitory effect of caffeine on PLDR was examined with doses of 1, 5 and 10 mM. Results: The capacity of PLDR in two cell lines re¯ected radiosensitivity. The recovery ratio after irradiation of 5 Gy was 2.8 in the radiosensitive NMT-1 and 5.2 in the radioresistant NMT-1R, and recovery reached its peak level at 6 h in both cell lines. The degree of inhibition of PLDR was weaker in NMT-1R than that in NMT-1 at the same dose level, and was correlated with reduction of G2-arrested cells by caffeine. Conclusions: The results of this study suggest that the capacity of PLDR may be one of the determinant factors for radiosensitivity in the two cell lines used, and the inhibitory effect of caffeine on PLDR was in part attributable to the modi®cation of the cell cycle progression. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Radiation; Potentially lethal damage repair; Caffeine; Cell cycle
1. Introduction Advances in radiation biology have revealed the mechanism of radiosensitivity, and many intrinsic and extrinsic factors including a tumor suppresser gene, cell cycle related-proteins, growth factors and the capacity for radiation-induced damage repair have been proposed [1±3]. Especially in radiation-induced DNA damage repair, two types of repair processes, i.e. sublethal damage repair (SLDR) and potentially * Corresponding author. Tel.: 181-027-220-8383; fax: 181027-220-8397. E-mail address: [email protected] (T. Akimoto)
lethal damage repair (PLDR), are well known [4,5], and the degree of PLDR is closely associated with intrinsic radiosensitivity of malignant human tumors [6±8]. In vivo solid tumors usually contain a greater proportion of quiescent, non-cycling cells than established cell lines in vitro. This population is generally more radioresistant due to the high capacity for recovery or repopulation after DNA damage, thus the enhancement of cell-killing in non-cycling cells is clinically bene®cial. In an in vitro cell system, PLDR has been de®ned as an increased surviving fraction in the cells plated immediately after radiation compared with that in cells whose plating is delayed.
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00308-0
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T. Akimoto et al. / Cancer Letters 147 (1999) 199±206
Con¯uent-phase cultures in vitro contain a larger number of quiescent cells than exponentially growing cells, and the arrest of cell cycle progression allows cells to repair DNA damage. Several investigators reported that PLDR correlated well with the repair of double strand breaks [9] and inhibition of PLDR increases radiosensitivity [5]. We have already reported the establishment of a radioresistant rat yolk sac tumor cell line, NMT-1R by repeated irradiation of a radiosensitive line, NMT1 [10,11]. These cell lines would be a unique and good model to investigate the mechanism or determinant factors for radioresistance. A difference in the frequency of apoptosis induction and morphological change after radiation in these cell lines was reported previously by Mitsuhashi et al. [12,13] and the sensitivities for cisplatin and taxol were also examined [14,15], but the impact of recovery capacity on cellular radiosensitivity has not yet been examined. In this report, we investigated how the degree of PLDR correlates with radiosensitivities of both cell lines. In addition, the inhibitory effects of caffeine was also examined. Caffeine potentiates cytotoxicity of anti-cancer drugs and/or radiation [16] by way of the modi®cation of cell cycle progression or alteration of DNA conformation [17±19]. A characteristic pharmacological effect of caffeine on the cell cycle is abrogation of G2 arrest induced by DNA damaging agents [20], and a recent investigation revealed that caffeine acts at the G2-M checkpoint by altering phosphorylation of p34 Cdc2 or cyclin B1 expression [17,21]. Considering the fact that radiation usually in¯uence cell cycle checkpoint and induces G2 arrest via p53-dependent or -independent pathways, one may speculate that cell cycle progression may also be involved in the mechanism of inhibition of PLDR by caffeine. Therefore, cell cycle distribution was also examined.
2. Materials and methods 2.1. Cells and cell cultures Two rat yolk sac tumor cell lines which derived from the same origin and showed different radiosensitivity were used in this study. The characteristics of these two cell lines have already been reported
[10,11]. Brie¯y, NMT-1 cells are 1.7 times more radiosensitive than NMT-1R, as estimated from their D0 values. p53 status as assessed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was wild type in NMT-1 and mutant in NMT-1R [22]. The cells were maintained in RPMI1640 supplemented with 10% heat-inactivated fetal calf serum at 378C in a humidi®ed atmosphere of 5% CO2 and 95% air. 2.2. Radiation method and caffeine treatment Con¯uent cells, which were seeded into 60 mm diameter petri dishes with 5 ml medium, were irradiated with a Hitachi (MBR1505R) X-ray machine that was operated at 140 kV, 4.5 mA with a 0.5 mm Al ®ltration and FSD of 30 cm with a dose-rate of 1.11 Gy/min. Radiation was performed in air at room temperature. Caffeine was purchased from Sigma Chemicals (Tokyo, Japan), and used for experiment by dissolving in warmed (608C) medium. Caffeine was incubated with cells for 1 h immediately after radiation at 378C in a humidi®ed atmosphere of 5% CO2 and 95% air with ®nal concentrations of 1, 5 and 10 mM. After incubation, the medium containing caffeine was removed and new prewarmed medium was put into the dishes after washing twice with complete medium. 2.3. Evaluation of PLDR The PLDR in an in vitro system has been de®ned as follows. After single-irradiation of con¯uent cells, the cells were either immediately subcultured into 60 mm petri dishes (immediate plating) or were placed for optimal time in a incubator (incubation time) to allow cells to repair radiation damage and were then subcultured (delayed plating). An increase in the surviving fraction (SF) of delayed plating compared with that of immediate plating implies PLDR. Firstly, we examined the PLDR in NMT-1 and NMT-1R after irradiation with various single doses at a ®xed incubation time (6 h). The change in recovery capacity according to the incubation time (3, 6 and 9 h) was then examined. Surviving fractions were determined by clonogenic assay. Colonies were ®xed and stained
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A ®nal calculated value less than 1 implies that caffeine inhibited PLDR which was observed after radiation alone. 2.4. Cell cycle analysis The cell cycle distribution was examined as follows. After trypsinization of the cells, single cells were washed twice with phosphate-buffered saline, ®xed with 70% ethanol and stained with propidium iodide for 30 min at room temperature. The samples were then analyzed using ¯ow cytometry (FACScan, Becton-Dickinson, Mountain View, CA). Cell-cycle distribution was determined by analyzing the DNA histogram. 2.5. Statistical analysis Each data point was based on at least three independent experiments. Statistical analyses were performed by Student's t-test.
Fig. 1. PLDR in both cell lines. (a), NMT-1; (b), NMT-1R. Con¯uent-phase cells were irradiated with 1, 3, 5 and 7 Gy, and subcultured immediately after radiation (closed circle, X) or 6 h after postirradiation incubation (open circle: W).
with crystal violet (2% in methanol) at least 14 days after subculture for counting. The recovery ratio was de®ned as follows when the cells were treated by radiation alone. In order to evaluate the inhibitory effect by caffeine on PLDR, cell toxicity by caffeine alone was normalized by the almost same method NG et al. [23] reported. The SF after treatment with combined radiation (RT) and caffeine is divided by the SF after treatment with radiation alone, and then the calculated result is divided by the SF after treatment with radiation with incubation time of 6 h.
Fig. 2. Recovery ratio as a function of incubation time. Con¯uentphase cells were irradiated with 5 Gy, and subcultured at 3, 6 and 9 h after post-irradiation incubation. Closed circle (X), NMT-l; open square (A): NMT-1R. Vertical bars represent the standard deviation.
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T. Akimoto et al. / Cancer Letters 147 (1999) 199±206 Table 2 Cell cycle distribution of NMT-1 and NMT-1R before subculture Cell line
Treatment
Cell cycle distribution (%) G1
S
G2 1 M
NMT-1
Control a 5 Gy b Caffeine c
55 53 59
18 22 20
27 25 21
NMT-1R
Control a 5 Gy b Caffeine c
58 57 59
21 28 23
21 15 18
a
Con¯uent phase cells with no treatment. Con¯uent phase cells were irradiated with a dose of 5 Gy. After 6h incubation (before subculture), the cell cycle distribution was analyzed by the method described in Materials and methods (see Section 2.5). c Con¯uent phase cells were incubated with caffeine for 1 h immediately after radiation. After removing and washing caffeine, cells were incubated for 5 h and the cell cycle analyzed. b
Fig. 3. Dose-response curves for caffeine. Closed circle (X), NMT1; open circle (W), NMT-1R. Con¯uent-phase cells were exposed to caffeine for 1 h. After removing medium containing caffeine and washing with fresh medium, the cells were incubated for 5 h at 378C in a humidi®ed atmosphere of 5% CO2 and 95% air and subcultured. Surviving fractions were determined by clonogenic assay.
3. Results
time after irradiation, PLDR was already observed at 3 h, and reached its peak at 6 h (Fig. 2).
3.1. Capacity of PLDR
3.2. Inhibitory effect of caffeine on PLDR
As shown in Fig. 1, PLDR was observed in both cell lines. The recovery ratio at 5 Gy was 2:8 ^ 0:8 in NMT-1 and 5:2 ^ 1:1 in NMT-1R, respectively, and was consistent with the difference in radiosensitivity of the two cell lines The difference was statistically signi®cant (P , 0:05). According to the incubation
Table 1 Inhibitory effect of caffeine on PLDR in NMT-1 and NMT-1R a Concentration of caffeine (mM)
1 5 10 a
Recovery ratio b NMT-1
NMT-1R
0.72 0.52 0.23
1.15 0.88 0.47
Con¯uent-phase cells were incubated with caffeine for 1 h after irradiation of 5 Gy. The cells were subcultured 6 h after irradiation for clonogenic assay. Recovery ratios were calculated by the method described in Materials and methods (see Section 2.3). b Recovery ratio with less than 1 implies that PLDR was inhibited by caffeine.
Fig. 3 shows the dose-response curves of the con¯uent-phase of NMT-1 and NMT-1R treated with caffeine alone. The radiosensitive NMT-1 showed a slightly higher sensitivity than the radioresistant NMT-1R for each drug, but caffeine alone showed weak cytotoxic effect, and surviving fractions were 0.91 at 1 mM, 0.85 at 5 mM and 0.80 at 10 mM in NMT-1, while those in NMT-1R were 0.94 at 1 mM, 0.89 at 5 mM and 0.84 at 10 mM. Table 1 demonstrated recovery ratio of NMT-1 and NMT-1R after treatment with radiation and caffeine. The recovery ratios of radiosensitive NMT-1 were less than 1 at the all dose levels of caffeine, which means that caffeine inhibited PLDR completely in NMT-1. The recovery ratios of radioresistant NMT1R were slightly greater than those of NMT-1, and caffeine with a dose of 1 mM was not able to inhibit PLDR. 3.3. Alteration of cell cycle distribution after treatment Table 2 demonstrates the distribution of the cell
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Fig. 4. Changes in the cell cycle distribution after radiation (5 Gy). (a), NMT-1; (b), NMT-1R. Con¯uent-phase cells were irradiated with a dose of 5 Gy. After 6 h of post-irradiation incubation time, cells were subcultured. In order to evaluate alteration of cell cycle after subculture, cell cycle distribution was examined 6, 12 and 24 h after subculture. Vertical bars represent standard deviation.
Fig. 5. Changes in the cell cycle distribution following radiation in combination with caffeine with doses of 1 or 5 mM in NMT-1. (a), 1 mM caffeine; (b), 5 mM caffeine. Con¯uent-phase cells were irradiated with a dose of 5 Gy, followed by 1 h exposure to caffeine. Cell cycle distribution was analyzed at the indicated time after subculture. Vertical bars represent the standard deviation.
cycle before subculture. Radiation either alone or in combination with caffeine with a dose of 5 mM did not affect cell cycle during incubation time. There was not an apparent difference in the cell cycle distribution between NMT-1 and NMT-1R. After subculture, an increase in G2M phase cells
(G2 arrest) in both cell lines was observed 24 h after irradiation (Fig. 4a,b). Accumulation of G2-arrested cells was more prominent in NMT-1 than NMT-1R (54:1 ^ 3:5% in NMT-1 and 43:5 ^ 2:1% in NMT1R), and the difference in proportion of the cells was statistically signi®cant (P , 0:05). When caffeine
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4. Discussion
Fig. 6. Changes in the cell cycle distribution following radiation in combination with caffeine with doses of 1 or 5 mM in NMT-1R. (a), 1 mM of caffeine; (b), 5 mM of caffeine.
was applied in combination with radiation, proportion of G2-arrested cells observed after radiation alone was decreased with a statistically signi®cance (abrogation of G2 arrest) (Figs. 5 and 6). This phenomenon was observed in the both cell lines when caffeine concentration was 5 mM, but caffeine with a dose of 1 mM did not abrogate G2-arrest in NMT-1R (Fig. 6a) while abrogated in NMT-1 (Fig. 5a). The relationship between the dose of caffeine and degree of abrogation of G2 arrest showed the similar tendency of inhibitory effect of caffeine on PLDR.
The cell lines used in this study were derived from the same origin, and they may have similar tumor characteristics. Thus these would be a good model to investigate determinant factors for radiosensitivity. This study demonstrated that PLDR was observed in both NMT-1 and NMT-1R and their capacities were consistent with a difference in the radiosensitivities of two rat yolk sac tumor cell lines. i.e. the radioresistant NMT-1R showed higher capacity of PLDR than the radiosensitive NMT-1. The frequency of apoptosis induction, which has been a major determinant factor for cellular radiosensitivity, is different in these cell lines [12,13]. Radford et al. [24] recently reported that DNA repair capability in¯uences susceptibility to induction of apoptosis, therefore higher proportion of unrepairable DNA damage in NMT-1 than NMT1R may cause different apoptosis induction in addition to the difference of p53 status. The results of cell cycle analysis also indicated that the proportion of G2arrested cells induced by radiation alone was larger in the radiosensitive NMT-1 than in the radioresistant NMT-1R. As the magnitude of G2 delay has been shown to re¯ect the degree of radiation-induced DNA damage and cellular radiosensitivity [25], it may be speculated that NMT-1R had less residual DNA damage due to higher PLDR than NMT-1. The relationship between p53 function and repair capacity, has proved controversial. The tumors with wild type p53 have larger capacity of DNA damage repair through the mismatch repair and the nucleotide excision repair than tumors with mutant p53 [26±28], while the cell lines with p53 mutations showed higher SLDR than those with no mutations [29]. Thus, further investigations are needed to clarify relationship between p53 status and exact repair capacity in these cell lines. Although many investigators have reported the inhibition of PLDR by drugs using an in vitro PLDR system [23,30,31], a de®nite role of cell cycle progression in inhibition of PLDR is still unclear. In this study, caffeine inhibited PLDR in both cell lines at the dose level which showed weak cytotoxicity. Caffeine has inhibitory effect on DNA damage repair through inhibition of repair enzyme, but the pharmacological effect of caffeine on cell cycle is also considered to play an important role in the inhibition of
T. Akimoto et al. / Cancer Letters 147 (1999) 199±206
PLDR. Radiation and/or caffeine did not affect cell cycle distribution before subculture, but alteration and/or progression of cell cycle was observed after subculture. Especially, accumulation of G2-arrested cells which observed after radiation was reduced when caffeine was incubated after radiation. The degree of reduction in G2-arrested cells correlated with inhibitory effect of caffeine on PLDR (Fig. 6), i.e. 5 mM caffeine reduced radiation-induced G2 arrest, whereas 1 mM caffeine which showed no inhibitory effect on PLDR, did not. The shortening of radiation-induced G2 arrest which results in insuf®cient time for DNA repair has been shown to be the main mechanism in the sensitizing effect of caffeine in exponentially growing cells, and this mechanism may also be responsible for the inhibitory effect on PLDR using con¯uent phase cells [31]. Taken together, this study indicated that PLDR using in vitro system may be in¯uenced by not only repair capacity during incubation time, but also cell cycle progression after subculture. From the clinical and therapeutic standpoint, inhibition of PLDR will be one of the targets in order to gain the therapeutic effect. In vivo solid tumors usually show heterogeneity and have various conditions including a quiescent cell population, a portion of acidic pH and a hypoxic fraction. These microenvironments cause enhancement of PLDR [32±34]. Reduction or inhibition of PLDR, therefore, appears to be important method in clinical treatment to improve radiosensitivity. In conclusion, the present study showed that capacity for PLDR was associated with radiosensitivities of rat yolk sac tumor cell lines derived from the same origin, and an inhibition of PLDR by caffeine is in part attributed to the control or modi®cation of the cell cycle progression. Acknowledgements This study was supported in part by Grants-in-Aid for Scienti®c Research from the Ministry of Education, Science and Culture of Japan. References [1] J.H. Peacock, J.J. Eady, S. Edwards, A. Hoimers, T.J. Macmil-
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