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Repair, repopulation and cell cycle redistribution in rat foot skin
Radiotherapy and Oncology 46 (1998) 193–199
Repair, repopulation and cell cycle redistribution in rat foot skin Mohi Rezvani a ,*, John W. Hopewell a, Gerard M. Morris a, Dilys Wilding a, Elizabeth Whitehouse a, Mike E.C. Robbins b, Mario J.F. Cortina-Borja c a
Research Institute, University of Oxford, Churchill Hospital, Oxford, UK Radiation Research Laboratory, University of Iowa, Iowa City, IA, USA c Department of Statistics, University of Oxford, Oxford, UK
b
Received 3 June 1996; revised version received 21 May 1997; accepted 12 June 1997
Abstract The influence of the phenomena of the repair of sublethal damage, repopulation and the role of the reassortment of surviving clonogenic target cells within the cell cycle have been examined in the foot skin of rats using a series of split dose experiments. The dose-related incidence of moist desquamation was used as an end-point. Initially the iso-effect dose for moist desquamation (ED50) increased with an increasing time interval (1–22 h) between two equal fractions. This effect was attributed to the well established phenomenon of the repair of sublethal damage. This appeared to be maximal with a 22 h gap between fractions. A further increase in the time interval, from 2–7 days, between two equal fractions resulted in a decrease in the ED50 value for moist desquamation. The phenomenon is most likely to be explained by a shortening of the cell cycle time in surviving epithelial target cells as repopulation first initiated. With intervals between two fractions of greater than 10 days the ED50 for moist desquamation again increased. This is likely to represent an increase in the number of epidermal target cells (repopulation). Further evidence for the effect of a reassortment of cells in the cell cycle has come from another study in which a half-tolerance priming dose of 16.8 Gy was followed by three daily fractions starting 48 or 125 h after the priming dose. The ED50 for moist desquamation based on the total fractionated dose (three fractions) was significantly lower (P , 0.05) after the longer time interval, i.e. fractions given on days 5, 6 and 7 after the primary dose. These findings were supported by the results of a cell proliferation kinetic study and jointly question the validity of a frequently made assumption of equal biological effect per fraction in a prolonged fractionated irradiation schedule. 1998 Elsevier Science Ireland Ltd. Keywords: Repair; Repopulation; Cell cycle; Redistribution; Fractionation; Radiation
1. Introduction In mathematical models developed to describe the response of tissues to fractionated irradiation it is assumed that an equal biological effect is produced by each dose fraction [3,24,25]. However, it is well recognised that cells do vary in their radiosensitivity with respect to their position in the cell cycle [22]. Thus, when a heterogeneous population of cells is subjected to split dose irradiation both in vivo and in vitro [4,5] the most sensitive cells are reduced in number by the first fraction. The surviving cells form a more homogenous population, which consists mainly of the more radioresistant cells. Therefore, the average sensitivity
* Corresponding author.
of the population of cells appears to decrease. As the time interval between two fractions increases and cells move through the cell cycle, the cell population becomes more heterogeneous and the average sensitivity of the cells will increase. These phenomena, together with the repair of sublethal damage, cell cycle block, accelerated repopulation and other perturbations in the cell cycle induced by radiation, make the process of the recovery of tissues very complex during and after fractionated irradiation. For human tissues it is assumed that repair of sublethal damage is, for practical purposes, complete within 24 h. It is also assumed that, for the majority of human tissues, accelerated proliferation does not start until a few weeks after the start of fractionated irradiation. Therefore, it has been assumed that as long as the interfraction interval is long enough to allow for the complete repair of sublethal damage
0167-8140/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0167-8140 (97 )0 0114-X
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and that the overall treatment time is shorter than the time required for the onset of accelerated repopulation, the radiosensitivity of cells will not vary significantly. Thus, in mathematical models [3,24,25] developed to describe the response of tissues to fractionated irradiation, it is assumed that an equal biological effect is produced by each dose fraction. Ruifrok et al. [20] reported that 3 Gy fractions applied with a 48 h interfraction interval were more damaging to mouse skin than 3 Gy fractions applied with 6 or 24 h interfraction intervals. Following a priming dose, a higher sensitivity was also reported by the same authors after 14– 18 days as compared with 1–10 days. In pig skin a marked shortening of the turnover time of basal cells was reported from approximately 14 days after a single approximate halftolerance dose (15–20 Gy) or at intervals .14 days after daily (five per week) irradiation with doses of ~2 Gy. This precedes microscopic evidence for the development of cell colonies and the subsequent rapid repopulation of the epidermis [17]. The suggestion of enhanced radiosensitivity when the cell cycle time is first shortened, prior to accelerated repopulation, was supported by the results of fractionated dose studies [10]. In this experiment pig skin was irradiated with 20 or 28 fractions from 90Sr/90Y b-rays. The ED50 was significantly lower for treatments given in longer than 14 days as compared with the same treatments given within 14 days. This indicated an increased sensitivity over the time period associated with the initial shortening of the cell cycle time prior to the recognition of accelerated repopulation. The purpose of the present study was to determine if a similar effect could be demonstrated in another system, albeit over a different time scale, consistent with the cell proliferation kinetics of that particular system and whether the general assumption of an equal effect per fraction was valid. In rat skin there are marked age-related changes in the cell proliferation kinetics of the epidermis, particularly between young (7-week-old) and fully mature (52-weekold) animals. Moreover, in young adults (14-week-old) frequently used for radiobiological studies, a mixed population of cells with varying kinetics has been reported [18]. This does not stabilise until animals are approximately 26 weeks of age [16]. Thus, the foot skin of adult 26-week-old rats was used in this study.
2. Materials and methods 2.1. Split dose studies Mature (26-week-old) female Sprague–Dawley rats were used in this study. The animals were housed in groups, three per cage, and received standard pellet diet food and water ad libitum. Both hind feet of each rat were irradiated under anaesthesia with a range of doses of 60Co g-rays, at a dose-rate of ~1.3 Gy/min. Initially, animals were anaesthe-
tised in a perspex box flushed with oxygen and 2–3% halothane. Pre-anaesthetised rats were then positioned in a perspex irradiation jig and anaesthesia was maintained by continuous flushing with oxygen and 1–1.5% halothane at a rate of 2 l/min. The foot to be irradiated was positioned in a slot in a circular perspex holder (1 cm thick, 11 cm in diameter) located at the centre of the jig. Rats were positioned radially around this central perspex portion of the jig. Nine animals were irradiated at each time. Irradiation schedules involved two equal dose fractions, separated by intervals of 1–22 h or 4–14 days. In a separate study, a halftolerance dose of 16.8 Gy (based on the results of studies with two fractions/22 h) was followed after an interval of 48 or 125 h by a range of total doses given as three equal daily fractions. The feet were examined three times a week for the appearance of moist desquamation between 10 and 20 days after the end of irradiation. Nine animals were used per dose point. Quantal data for the incidence of moist desquamation were analysed by probit analysis [6] and ED50 (±SE) values, the dose required to produce moist desquamation in 50% of irradiated feet, were obtained for each irradiation schedule. Data analysis was carried out by the S-Plus statistical package [28]. 2.2. Cell kinetics studies The left hind foot of ~26-week-old female Sprague– Dawley rats was irradiated under chloral hydrate/air anaesthesia with a single dose of 22.5 Gy of 250 kV X-rays (dose rate 1.1 Gy/min). A total of 32 animals were used. At intervals of 2–7, 9 and 12 days after irradiation, groups of four rats were injected intraperitoneally with a single pulse of tritium-labelled thymidine (3H-TdR) diluted in 1 ml of normal saline (5.60 kBq/g body weight; specific activity 2 GBq/mmol). Rats were always injected at 0900–1000 h and killed 40 min after the injection of 3H-TdR. Skin on the dorsal surface of the left foot (irradiated) and right foot (control) was removed and fixed in Bouin’s fluid for 4 h, after which it was dehydrated through graded alcohols, cleared in chloroform and embedded in paraplast. Histological sections, 5 mm thick, were cut perpendicular to the skin surface. Slides, pre-stained with Mayer’s haematoxylin, were dipped in Ilford K2 photographic emulsion (diluted 1:1 with distilled water) at 45°C. Autoradiographs were exposed for 5 weeks at 4°C and then developed using Kodak D19 developer and fixed in 10% sodium thiosulphate. DPX was used as the mounting medium. Autoradiographs were examined to determine the number of labelled basal cells in the epidermis. The basal cell labelling index (LI) was determined by counting cells along a minimum of 10 mm of basement membrane. The LI was calculated according to the following standard equation: LI = NL =NT where NL is the number of labelled basal cells and NT is the total number of cells counted in the basal layer.
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3. Results
Table 1
3.1. Split dose studies
Variation in ED50 ± SE, iso-effective doses, for moist desquamation of the skin of rat foot after irradiation with two equal fractions of 60Co g-rays with varying time intervals
The dose-related incidence of moist desquamation from the split dose studies involving varying time intervals up to 22 h between the two fractions is shown in Fig. 1a. Increasing the time interval between the two fractions resulted in the dose-effect curves being shifted to the right. The ED50 increased, reaching a value of 33.33 ± 0.36 Gy with an interval of 22 h between fractions compared with 25.46 ± 0.46 Gy for a single dose. With time intervals of 2 and 7 days between two fractions the dose-effect curves shifted back slightly to the left, indicating a decrease in the ED50 values (Fig. 1b). For time intervals .7 days between the two fractions the dose-effect curves were again shifted to the right indicating an increase in the ED50 values. The ED50 ± SE values for moist desquamation of the skin of rat foot after irradiation with different time intervals between two equal fractions are given in Table 1. The analysis of variance (ANOVA) showed that the differences between
Fig. 1. Dose-related changes in the incidence of moist desquamation in the foot skin of rats irradiated with 60Co g-rays with varying time intervals between two equal doses. (a) Single dose (A), 1 h (W), 2 h (K), 4 h (S), 12 h ([) and 22 h (L); (b) 2 days (W), 4 days (A), 5.21 days (K), 7 days (L), 10 days (S), 12 days ([) and 14 days ( d ). Error bars indicate ±SE.
Time interval
ED50 ± SE (Gy)
Single dose 1h 2h 4h 12 h 22 h 2 days 4 days 5.21 days 7 days 10 days 12 days 14 days
25.46 27.18 27.89 29.78 31.41 33.33 32.71 32.15 31.21 31.82 34.53 36.45 38.22
± ± ± ± ± ± ± ± ± ± ± ± ±
0.46 0.43 0.35 0.42 0.21 0.36 0.19 0.40 0.22 0.19 0.44 0.50 0.62
these ED50 values were highly significant (likelihood ratio statistic of 737.22, P , 0.001). This variability in the ED50 values with the time interval between two equal fractions is shown in Fig. 2. There was an initial rise in ED50 values followed by a decrease before a final rise with intervals of ≥10 days between the two equal fractions. These results suggested that radiation dose fractions given between 4 and 8 days might have a greater effect in comparison to those given between 1 and 3 days after an initial dose. The analysis of variance (ANOVA) showed that the difference between the values of 33.33 ± 0.36 and 31.21 ± 0.22 Gy, the ED50 values for 22 h and 5.21 days time intervals, respectively, was statistically significant (likelihood ratio statistic of 25.37, P , 0.00l). In a separate study, a priming dose of 16.8 Gy (an approximate half-tolerance dose) was followed by three equal daily fractions starting at either 48 h (2 days) or 125 h (5.2 days) after the priming dose. The results are shown in Fig. 3. The ED50 value for moist desquamation after irradiation with
Fig. 2. Variation in ED50 values (±SE) with changes in the time interval between two equal dose fractions.
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Fig. 3. Dose-related changes in the incidence of moist desquamation in the foot skin of rats after irradiation with a priming (half-tolerance) dose of 16.8 Gy followed by three equal daily fractions starting at 48 h (O) or 125 h (B) after the primary dose. Error bars indicate ± SE. ED50 values were 22.52 ± 0.58 and 20.08 ± 0.91 Gy, respectively.
three fractions starting 5.21 days after the priming dose was 20.08 ± 0.91 Gy. This was significantly (likelihood ratio statistic of 5.1, P = 0 01) lower than the value of 22.52 ± 0.58 Gy, the ED50 value after irradiation with only a 48 h gap between the first of three fractions and the priming dose. 3.2. Cell kinetic studies There was an initial inhibition of DNA synthesis after irradiation with a single X-ray dose of 22.5 Gy. At 2 days after irradiation the LI was 0.1 ± 0.1% compared with 7.9 ± 0.3% prior to the start of irradiation (Fig. 4). The average LI subsequently began to increase and reached levels comparable to pre-treatment values at 5 days after irradiation. Thereafter, the value of the LI continued to increase progressively and at 7 days after irradiation was 12.8 ± 0.6%. However, on day 9 the value of the average LI value had declined slightly to 9.5 ± 0.4%. By day 12 after irradiation two distinct types of epidermis were discernible, described as regenerating or degenerating. The LI in the apparently regenerating epidermis was considerably elevated above control levels at 29.9 ± 6.8%, whereas the LI in a radiation damaged epidermis was significantly lower at 8.5 ± 1.1%, similar to that in the unirradiated epidermis. Cell colonies were evident in histological sections by 12 days after irradiation. These were identified as cords of 10 or more cells exhibiting no morphological evidence of radiation damage. The labelling index in these cell colonies was 50.05 ± 5.2%.
4. Discussion The influence of the phenomena of the repair of sublethal damage, repopulation and the role of the progression and reassortment of surviving clonogenic target cells within the cell cycle have been examined in the skin of rats using a series of split dose experiments. Mature rats were used throughout this study because the cell kinetic parameters of the skin of the foot were known to be stable over that age range [18]. This is important in studies designed to examine the possible effects of perturbations in the cell cycle on the radiation sensitivity of the epidermis. All experiments were initiated at 0900–1000 h to minimise the potential effects of diurnal variation on radiosensitivity. In the present series of split dose studies the ED50 value for moist desquamation increased initially as the time interval between fractions was increased from 1 to 22 h. This progressive increase in the ED50 value was due to the repair of sublethal damage which was apparently maximal at 22 h in these studies. Following the repair of sublethal damage there was a subsequent decrease in the ED50 value between 2 and 7 days before a final rise in the ED50 value, probably due to proliferation around 9 days after irradiation. This finding is consistent with the results of cell kinetic studies in this model. Irradiation was with a single X-ray dose of 22.5 Gy. This dose was sufficiently high to produce a significant modulatory effect on the cell population dynamics of the epidermis, without complete sterilisation of the stem cell compartment. In this model a single X-ray dose of ≥30
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Gy is required for sterilisation of all stem cells within the irradiated field [8]. There was a marked reduction in the labelling index of basal cells (cell cycle delay) for the first 2 days after irradiation. This was followed by a rapid rise in the labelling index to above the control level at ≥6 days after irradiation. This suggests a shortening of the cell cycle time. Cell cycle delay is one of the initial effects of radiation on proliferating cells. This radiation-induced delay in cell cycle progression is due to a temporary arrest of cells in the G1/S or G2 phases of the cell cycle [15] and its duration is related to the radiation dose and the cell population doubling time. Single dose photon exposure of 1 Gy induces a cell cycle delay approximately equal to 6–10% of the basal cell layer doubling time in the epidermis of the mouse [9], pig [1,17] and human [19]. Cell cycle delay causes an accumulation of cells at the G1/S and G2/M boundaries of the cell cycle. When the epithelial cells are released from the cell cycle block the process of compensatory repopulation starts. This process is associated with a rise in the mitotic or cell labelling indices compared with normal proliferating cells. In rodent skin repopulation of epithelium starts between 7 and 16 days after single X-ray doses of 9–20 Gy [5,9] in the same order of magnitude as used in the present study. Once initiated, repopulation is associated with a shortening of the cell cycle time in surviving clonogenic cells and the rate of recovery in cell number is dependent on the total radiation dose [16]. The shortening of the cell cycle time is likely to render the tissue more radiosensitive. A shorter cell cycle time has also been used as an explanation for the differential radiosensitivity of younger rats in comparison with older rats of the same strain [8]. The skin of young (7-week-old) rats was more radiosensitive than that of the skin of mature (52week-old) rats to X-rays. The cell cycle time of the epidermal cells of the foot of mature rats was 53.9 ± 5.3 h, which was considerably longer than 30.1 ± 1.3 h in younger rats (Table 2). This is due to changes in the length of the G1 phase of the cell cycle which was reduced from 31.2 ± 3.5 h in mature rats to 15.0 ± 0.8 h in 7-week-old rats. There was no significant difference in the duration of the most sensitive phases of the cell cycle (G2 + M) between the young and old rats. This indicates that the epidermal cells of the foot of mature rats are radioresistant because a larger
Table 2 Cell kinetic parameters for the epidermis of the skin of the foot of rats of 7 and 52 weeks of age Cell cycle phase duration (h) Age (weeks)
Ts
TG2 + M
TG1
Tc
7 52
12.0 ± 3.0 19.0 ± 4.7
3.1 ± 0.3 3.7 ± 1.3
15.0 ± 0.8 31.2 ± 3.5
30.1 ± 1.3 53.9 ± 5.3
Values of Ts (duration of DNA synthesis), TG2 + M (duration of G2 plus M phases), TG1 (duration of G1 phase) and Tc (cell cycle time) are expressed as mean values ± SE (from Morris et al. [18]).
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Fig. 4. Time-related changes in the mean labelling index of cells of the basal layer of the epidermis in the foot skin of rats after irradiation with a single dose of 22.5 Gy of X-rays (X). At 12 days after irradiation regions of regenerating (O) and degenerating epidermis (B) in addition to cell colonies (♦) could be identified. The labelling index for each of the regions was evaluated separately. Error bars indicate ±SE.
proportion of the target cells are in the radioresistant G1 phase of the cell cycle. The reduction in the ED50 value for moist desquamation at around 4–5 days reported in the present study is also likely to reflect a shorter duration of the G1 phase of the cell cycle time of epidermal cells. This would render the cells more radiosensitive and thus smaller ED50 values. However, at longer interfraction times, the balance tips towards repopulation and the ED50 values increase again (Fig. 2). This view is supported by the results of the cell kinetic study (Fig. 4) which demonstrated that the labelling index rises sharply at these longer time intervals. The phenomenon of enhanced radiosensitivity at certain interfraction intervals has also been seen in mouse [5] and pig skin [27] as shown in Fig. 5a,b. These results show a remarkably similar pattern to those observed in the present study, albeit over a different time scale. The sensitive phase in mouse skin appears to occur at around 6–7 days while the sensitive phase in pig skin, a cell population with a slower turnover, appears to occur at around 14–15 days. Both Emery et al. [5] and van den Aardweg et al. [27] failed to comment on this phenomenon, perhaps because it was not a current view at that time. This enhanced radiosensitivity, probably due to shortening of the cell cycle time, in simple split dose studies is supported by the results of fractionated irradiation studies. In the present studies there was a reduction in the ED50 value for moist desquamation for three dose fractions delivered between 5 and 7 days after an appropriate half-tolerance priming dose in comparison to those delivered between 2 and 4 days after the priming dose. Similar findings, sensitisation over the 14–18 day period after priming doses of 20 and 30 Gy, have been reported using a mouse skin model [20]. This 14–18 day period is much longer than the sensitive period of 4–8 days observed in the present study. However, Ruifrok et al. [20] concluded that proliferation started about 13 days after the start of treatment in their mouse
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basal cells from the epidermis of breast cancer patients after accelerated radiotherapy as compared with conventional (2 Gy/day, 25 fractions in 5 weeks) fractionated treatment. The accelerated fractionation schedule consisted of 2 × 2 Gy/ day, 5 days/week to a total of 50 Gy in 2.5 weeks. For the initial 21 days period, the rate of cell loss from the basal layer was similar for both fractionated irradiation schedules. However, the accelerated fractionation schedule resulted in a considerably reduced rate of loss of cells from the basal cell layer of the epidermis between days 21 and 42. This somewhat unexpected result, a more severe response to the conventional schedule, is possibly due to the fact that irradiation in the accelerated regime is completed prior to any shortening of the cell cycle in surviving target cells. With the conventional schedule irradiation will continue over a period when the cell cycle is likely to be shorter and hence cells will be more radiosensitive. However, due to the absence of mitotic or labelling index data, it was not possible to determine the overall duration of the cell cycle delay period which would also influence the rate of cell loss. The results of a randomised clinical trial of continuous hyperfractionated accelerated radiotherapy treatment (CHART) has also showed that skin reactions were less severe in CHART patients than those treated with conventional regimes [21]. The results of the present study are of considerable clinical significance, particularly with the increased interest in the use of modified dose-fractionation schedules in radiotherapy. In addition, these findings might influence the current thinking about dose-fractionation relationships where an equal biological effect per fraction is assumed and used by modellers who wish to mathematically represent the dose–time relationships in fractionated radiotherapy. Fig. 5. Variation in isoeffective dose with changes in the time interval between two equal dose fractions in (a) mouse skin and (b) pig skin (redrawn from Emery et al. [5] and van den Aardweg et al. [27], respectively).
model. Similarly, Tsang et al. [26] suggested that clonogen doubling time does not begin to shorten in mouse skin until about 12 days after the start of fractionated radiotherapy. This might explain why the previous rodent studies [12], which employed an overall time of less than 7 days, failed to demonstrate a significant change in effect per fraction. Although not directly comparable with the present studies, the effect of cell cycle changes due to the partial synchronisation of the cell population has been noted by other studies both in vitro [13,23] and in vivo [2,5,11,14,29]. This kind of radiosensitivity was attributed to the arrival of a group of synchronised cells in the G2/M phase of the cell cycle. This phenomena is discussed in detail by Hahnfeldt and Hlatky [7]. These authors have shown mathematically that variation with the time of resensitisation due to redistribution is not monotonic but oscillatory. The present conclusion might be used to provide a more informed explanation of recent clinical studies. Nyman and Turreson [19] reported a distinct change in the rate of loss of
Acknowledgements The authors are grateful to F.W. Dickinson and N. Hubbard for their day-to-day care of the animals. The technical assistance of Mr J.H. Wilkinson is gratefully acknowledged.
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Repair, repopulation and cell cycle redistribution in rat foot skin Mohi Rezvani a ,*, John W. Hopewell a, Gerard M. Morris a, Dilys Wilding a, Elizabeth Whitehouse a, Mike E.C. Robbins b, Mario J.F. Cortina-Borja c a
Research Institute, University of Oxford, Churchill Hospital, Oxford, UK Radiation Research Laboratory, University of Iowa, Iowa City, IA, USA c Department of Statistics, University of Oxford, Oxford, UK
b
Received 3 June 1996; revised version received 21 May 1997; accepted 12 June 1997
Abstract The influence of the phenomena of the repair of sublethal damage, repopulation and the role of the reassortment of surviving clonogenic target cells within the cell cycle have been examined in the foot skin of rats using a series of split dose experiments. The dose-related incidence of moist desquamation was used as an end-point. Initially the iso-effect dose for moist desquamation (ED50) increased with an increasing time interval (1–22 h) between two equal fractions. This effect was attributed to the well established phenomenon of the repair of sublethal damage. This appeared to be maximal with a 22 h gap between fractions. A further increase in the time interval, from 2–7 days, between two equal fractions resulted in a decrease in the ED50 value for moist desquamation. The phenomenon is most likely to be explained by a shortening of the cell cycle time in surviving epithelial target cells as repopulation first initiated. With intervals between two fractions of greater than 10 days the ED50 for moist desquamation again increased. This is likely to represent an increase in the number of epidermal target cells (repopulation). Further evidence for the effect of a reassortment of cells in the cell cycle has come from another study in which a half-tolerance priming dose of 16.8 Gy was followed by three daily fractions starting 48 or 125 h after the priming dose. The ED50 for moist desquamation based on the total fractionated dose (three fractions) was significantly lower (P , 0.05) after the longer time interval, i.e. fractions given on days 5, 6 and 7 after the primary dose. These findings were supported by the results of a cell proliferation kinetic study and jointly question the validity of a frequently made assumption of equal biological effect per fraction in a prolonged fractionated irradiation schedule. 1998 Elsevier Science Ireland Ltd. Keywords: Repair; Repopulation; Cell cycle; Redistribution; Fractionation; Radiation
1. Introduction In mathematical models developed to describe the response of tissues to fractionated irradiation it is assumed that an equal biological effect is produced by each dose fraction [3,24,25]. However, it is well recognised that cells do vary in their radiosensitivity with respect to their position in the cell cycle [22]. Thus, when a heterogeneous population of cells is subjected to split dose irradiation both in vivo and in vitro [4,5] the most sensitive cells are reduced in number by the first fraction. The surviving cells form a more homogenous population, which consists mainly of the more radioresistant cells. Therefore, the average sensitivity
* Corresponding author.
of the population of cells appears to decrease. As the time interval between two fractions increases and cells move through the cell cycle, the cell population becomes more heterogeneous and the average sensitivity of the cells will increase. These phenomena, together with the repair of sublethal damage, cell cycle block, accelerated repopulation and other perturbations in the cell cycle induced by radiation, make the process of the recovery of tissues very complex during and after fractionated irradiation. For human tissues it is assumed that repair of sublethal damage is, for practical purposes, complete within 24 h. It is also assumed that, for the majority of human tissues, accelerated proliferation does not start until a few weeks after the start of fractionated irradiation. Therefore, it has been assumed that as long as the interfraction interval is long enough to allow for the complete repair of sublethal damage
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and that the overall treatment time is shorter than the time required for the onset of accelerated repopulation, the radiosensitivity of cells will not vary significantly. Thus, in mathematical models [3,24,25] developed to describe the response of tissues to fractionated irradiation, it is assumed that an equal biological effect is produced by each dose fraction. Ruifrok et al. [20] reported that 3 Gy fractions applied with a 48 h interfraction interval were more damaging to mouse skin than 3 Gy fractions applied with 6 or 24 h interfraction intervals. Following a priming dose, a higher sensitivity was also reported by the same authors after 14– 18 days as compared with 1–10 days. In pig skin a marked shortening of the turnover time of basal cells was reported from approximately 14 days after a single approximate halftolerance dose (15–20 Gy) or at intervals .14 days after daily (five per week) irradiation with doses of ~2 Gy. This precedes microscopic evidence for the development of cell colonies and the subsequent rapid repopulation of the epidermis [17]. The suggestion of enhanced radiosensitivity when the cell cycle time is first shortened, prior to accelerated repopulation, was supported by the results of fractionated dose studies [10]. In this experiment pig skin was irradiated with 20 or 28 fractions from 90Sr/90Y b-rays. The ED50 was significantly lower for treatments given in longer than 14 days as compared with the same treatments given within 14 days. This indicated an increased sensitivity over the time period associated with the initial shortening of the cell cycle time prior to the recognition of accelerated repopulation. The purpose of the present study was to determine if a similar effect could be demonstrated in another system, albeit over a different time scale, consistent with the cell proliferation kinetics of that particular system and whether the general assumption of an equal effect per fraction was valid. In rat skin there are marked age-related changes in the cell proliferation kinetics of the epidermis, particularly between young (7-week-old) and fully mature (52-weekold) animals. Moreover, in young adults (14-week-old) frequently used for radiobiological studies, a mixed population of cells with varying kinetics has been reported [18]. This does not stabilise until animals are approximately 26 weeks of age [16]. Thus, the foot skin of adult 26-week-old rats was used in this study.
2. Materials and methods 2.1. Split dose studies Mature (26-week-old) female Sprague–Dawley rats were used in this study. The animals were housed in groups, three per cage, and received standard pellet diet food and water ad libitum. Both hind feet of each rat were irradiated under anaesthesia with a range of doses of 60Co g-rays, at a dose-rate of ~1.3 Gy/min. Initially, animals were anaesthe-
tised in a perspex box flushed with oxygen and 2–3% halothane. Pre-anaesthetised rats were then positioned in a perspex irradiation jig and anaesthesia was maintained by continuous flushing with oxygen and 1–1.5% halothane at a rate of 2 l/min. The foot to be irradiated was positioned in a slot in a circular perspex holder (1 cm thick, 11 cm in diameter) located at the centre of the jig. Rats were positioned radially around this central perspex portion of the jig. Nine animals were irradiated at each time. Irradiation schedules involved two equal dose fractions, separated by intervals of 1–22 h or 4–14 days. In a separate study, a halftolerance dose of 16.8 Gy (based on the results of studies with two fractions/22 h) was followed after an interval of 48 or 125 h by a range of total doses given as three equal daily fractions. The feet were examined three times a week for the appearance of moist desquamation between 10 and 20 days after the end of irradiation. Nine animals were used per dose point. Quantal data for the incidence of moist desquamation were analysed by probit analysis [6] and ED50 (±SE) values, the dose required to produce moist desquamation in 50% of irradiated feet, were obtained for each irradiation schedule. Data analysis was carried out by the S-Plus statistical package [28]. 2.2. Cell kinetics studies The left hind foot of ~26-week-old female Sprague– Dawley rats was irradiated under chloral hydrate/air anaesthesia with a single dose of 22.5 Gy of 250 kV X-rays (dose rate 1.1 Gy/min). A total of 32 animals were used. At intervals of 2–7, 9 and 12 days after irradiation, groups of four rats were injected intraperitoneally with a single pulse of tritium-labelled thymidine (3H-TdR) diluted in 1 ml of normal saline (5.60 kBq/g body weight; specific activity 2 GBq/mmol). Rats were always injected at 0900–1000 h and killed 40 min after the injection of 3H-TdR. Skin on the dorsal surface of the left foot (irradiated) and right foot (control) was removed and fixed in Bouin’s fluid for 4 h, after which it was dehydrated through graded alcohols, cleared in chloroform and embedded in paraplast. Histological sections, 5 mm thick, were cut perpendicular to the skin surface. Slides, pre-stained with Mayer’s haematoxylin, were dipped in Ilford K2 photographic emulsion (diluted 1:1 with distilled water) at 45°C. Autoradiographs were exposed for 5 weeks at 4°C and then developed using Kodak D19 developer and fixed in 10% sodium thiosulphate. DPX was used as the mounting medium. Autoradiographs were examined to determine the number of labelled basal cells in the epidermis. The basal cell labelling index (LI) was determined by counting cells along a minimum of 10 mm of basement membrane. The LI was calculated according to the following standard equation: LI = NL =NT where NL is the number of labelled basal cells and NT is the total number of cells counted in the basal layer.
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3. Results
Table 1
3.1. Split dose studies
Variation in ED50 ± SE, iso-effective doses, for moist desquamation of the skin of rat foot after irradiation with two equal fractions of 60Co g-rays with varying time intervals
The dose-related incidence of moist desquamation from the split dose studies involving varying time intervals up to 22 h between the two fractions is shown in Fig. 1a. Increasing the time interval between the two fractions resulted in the dose-effect curves being shifted to the right. The ED50 increased, reaching a value of 33.33 ± 0.36 Gy with an interval of 22 h between fractions compared with 25.46 ± 0.46 Gy for a single dose. With time intervals of 2 and 7 days between two fractions the dose-effect curves shifted back slightly to the left, indicating a decrease in the ED50 values (Fig. 1b). For time intervals .7 days between the two fractions the dose-effect curves were again shifted to the right indicating an increase in the ED50 values. The ED50 ± SE values for moist desquamation of the skin of rat foot after irradiation with different time intervals between two equal fractions are given in Table 1. The analysis of variance (ANOVA) showed that the differences between
Fig. 1. Dose-related changes in the incidence of moist desquamation in the foot skin of rats irradiated with 60Co g-rays with varying time intervals between two equal doses. (a) Single dose (A), 1 h (W), 2 h (K), 4 h (S), 12 h ([) and 22 h (L); (b) 2 days (W), 4 days (A), 5.21 days (K), 7 days (L), 10 days (S), 12 days ([) and 14 days ( d ). Error bars indicate ±SE.
Time interval
ED50 ± SE (Gy)
Single dose 1h 2h 4h 12 h 22 h 2 days 4 days 5.21 days 7 days 10 days 12 days 14 days
25.46 27.18 27.89 29.78 31.41 33.33 32.71 32.15 31.21 31.82 34.53 36.45 38.22
± ± ± ± ± ± ± ± ± ± ± ± ±
0.46 0.43 0.35 0.42 0.21 0.36 0.19 0.40 0.22 0.19 0.44 0.50 0.62
these ED50 values were highly significant (likelihood ratio statistic of 737.22, P , 0.001). This variability in the ED50 values with the time interval between two equal fractions is shown in Fig. 2. There was an initial rise in ED50 values followed by a decrease before a final rise with intervals of ≥10 days between the two equal fractions. These results suggested that radiation dose fractions given between 4 and 8 days might have a greater effect in comparison to those given between 1 and 3 days after an initial dose. The analysis of variance (ANOVA) showed that the difference between the values of 33.33 ± 0.36 and 31.21 ± 0.22 Gy, the ED50 values for 22 h and 5.21 days time intervals, respectively, was statistically significant (likelihood ratio statistic of 25.37, P , 0.00l). In a separate study, a priming dose of 16.8 Gy (an approximate half-tolerance dose) was followed by three equal daily fractions starting at either 48 h (2 days) or 125 h (5.2 days) after the priming dose. The results are shown in Fig. 3. The ED50 value for moist desquamation after irradiation with
Fig. 2. Variation in ED50 values (±SE) with changes in the time interval between two equal dose fractions.
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Fig. 3. Dose-related changes in the incidence of moist desquamation in the foot skin of rats after irradiation with a priming (half-tolerance) dose of 16.8 Gy followed by three equal daily fractions starting at 48 h (O) or 125 h (B) after the primary dose. Error bars indicate ± SE. ED50 values were 22.52 ± 0.58 and 20.08 ± 0.91 Gy, respectively.
three fractions starting 5.21 days after the priming dose was 20.08 ± 0.91 Gy. This was significantly (likelihood ratio statistic of 5.1, P = 0 01) lower than the value of 22.52 ± 0.58 Gy, the ED50 value after irradiation with only a 48 h gap between the first of three fractions and the priming dose. 3.2. Cell kinetic studies There was an initial inhibition of DNA synthesis after irradiation with a single X-ray dose of 22.5 Gy. At 2 days after irradiation the LI was 0.1 ± 0.1% compared with 7.9 ± 0.3% prior to the start of irradiation (Fig. 4). The average LI subsequently began to increase and reached levels comparable to pre-treatment values at 5 days after irradiation. Thereafter, the value of the LI continued to increase progressively and at 7 days after irradiation was 12.8 ± 0.6%. However, on day 9 the value of the average LI value had declined slightly to 9.5 ± 0.4%. By day 12 after irradiation two distinct types of epidermis were discernible, described as regenerating or degenerating. The LI in the apparently regenerating epidermis was considerably elevated above control levels at 29.9 ± 6.8%, whereas the LI in a radiation damaged epidermis was significantly lower at 8.5 ± 1.1%, similar to that in the unirradiated epidermis. Cell colonies were evident in histological sections by 12 days after irradiation. These were identified as cords of 10 or more cells exhibiting no morphological evidence of radiation damage. The labelling index in these cell colonies was 50.05 ± 5.2%.
4. Discussion The influence of the phenomena of the repair of sublethal damage, repopulation and the role of the progression and reassortment of surviving clonogenic target cells within the cell cycle have been examined in the skin of rats using a series of split dose experiments. Mature rats were used throughout this study because the cell kinetic parameters of the skin of the foot were known to be stable over that age range [18]. This is important in studies designed to examine the possible effects of perturbations in the cell cycle on the radiation sensitivity of the epidermis. All experiments were initiated at 0900–1000 h to minimise the potential effects of diurnal variation on radiosensitivity. In the present series of split dose studies the ED50 value for moist desquamation increased initially as the time interval between fractions was increased from 1 to 22 h. This progressive increase in the ED50 value was due to the repair of sublethal damage which was apparently maximal at 22 h in these studies. Following the repair of sublethal damage there was a subsequent decrease in the ED50 value between 2 and 7 days before a final rise in the ED50 value, probably due to proliferation around 9 days after irradiation. This finding is consistent with the results of cell kinetic studies in this model. Irradiation was with a single X-ray dose of 22.5 Gy. This dose was sufficiently high to produce a significant modulatory effect on the cell population dynamics of the epidermis, without complete sterilisation of the stem cell compartment. In this model a single X-ray dose of ≥30
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Gy is required for sterilisation of all stem cells within the irradiated field [8]. There was a marked reduction in the labelling index of basal cells (cell cycle delay) for the first 2 days after irradiation. This was followed by a rapid rise in the labelling index to above the control level at ≥6 days after irradiation. This suggests a shortening of the cell cycle time. Cell cycle delay is one of the initial effects of radiation on proliferating cells. This radiation-induced delay in cell cycle progression is due to a temporary arrest of cells in the G1/S or G2 phases of the cell cycle [15] and its duration is related to the radiation dose and the cell population doubling time. Single dose photon exposure of 1 Gy induces a cell cycle delay approximately equal to 6–10% of the basal cell layer doubling time in the epidermis of the mouse [9], pig [1,17] and human [19]. Cell cycle delay causes an accumulation of cells at the G1/S and G2/M boundaries of the cell cycle. When the epithelial cells are released from the cell cycle block the process of compensatory repopulation starts. This process is associated with a rise in the mitotic or cell labelling indices compared with normal proliferating cells. In rodent skin repopulation of epithelium starts between 7 and 16 days after single X-ray doses of 9–20 Gy [5,9] in the same order of magnitude as used in the present study. Once initiated, repopulation is associated with a shortening of the cell cycle time in surviving clonogenic cells and the rate of recovery in cell number is dependent on the total radiation dose [16]. The shortening of the cell cycle time is likely to render the tissue more radiosensitive. A shorter cell cycle time has also been used as an explanation for the differential radiosensitivity of younger rats in comparison with older rats of the same strain [8]. The skin of young (7-week-old) rats was more radiosensitive than that of the skin of mature (52week-old) rats to X-rays. The cell cycle time of the epidermal cells of the foot of mature rats was 53.9 ± 5.3 h, which was considerably longer than 30.1 ± 1.3 h in younger rats (Table 2). This is due to changes in the length of the G1 phase of the cell cycle which was reduced from 31.2 ± 3.5 h in mature rats to 15.0 ± 0.8 h in 7-week-old rats. There was no significant difference in the duration of the most sensitive phases of the cell cycle (G2 + M) between the young and old rats. This indicates that the epidermal cells of the foot of mature rats are radioresistant because a larger
Table 2 Cell kinetic parameters for the epidermis of the skin of the foot of rats of 7 and 52 weeks of age Cell cycle phase duration (h) Age (weeks)
Ts
TG2 + M
TG1
Tc
7 52
12.0 ± 3.0 19.0 ± 4.7
3.1 ± 0.3 3.7 ± 1.3
15.0 ± 0.8 31.2 ± 3.5
30.1 ± 1.3 53.9 ± 5.3
Values of Ts (duration of DNA synthesis), TG2 + M (duration of G2 plus M phases), TG1 (duration of G1 phase) and Tc (cell cycle time) are expressed as mean values ± SE (from Morris et al. [18]).
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Fig. 4. Time-related changes in the mean labelling index of cells of the basal layer of the epidermis in the foot skin of rats after irradiation with a single dose of 22.5 Gy of X-rays (X). At 12 days after irradiation regions of regenerating (O) and degenerating epidermis (B) in addition to cell colonies (♦) could be identified. The labelling index for each of the regions was evaluated separately. Error bars indicate ±SE.
proportion of the target cells are in the radioresistant G1 phase of the cell cycle. The reduction in the ED50 value for moist desquamation at around 4–5 days reported in the present study is also likely to reflect a shorter duration of the G1 phase of the cell cycle time of epidermal cells. This would render the cells more radiosensitive and thus smaller ED50 values. However, at longer interfraction times, the balance tips towards repopulation and the ED50 values increase again (Fig. 2). This view is supported by the results of the cell kinetic study (Fig. 4) which demonstrated that the labelling index rises sharply at these longer time intervals. The phenomenon of enhanced radiosensitivity at certain interfraction intervals has also been seen in mouse [5] and pig skin [27] as shown in Fig. 5a,b. These results show a remarkably similar pattern to those observed in the present study, albeit over a different time scale. The sensitive phase in mouse skin appears to occur at around 6–7 days while the sensitive phase in pig skin, a cell population with a slower turnover, appears to occur at around 14–15 days. Both Emery et al. [5] and van den Aardweg et al. [27] failed to comment on this phenomenon, perhaps because it was not a current view at that time. This enhanced radiosensitivity, probably due to shortening of the cell cycle time, in simple split dose studies is supported by the results of fractionated irradiation studies. In the present studies there was a reduction in the ED50 value for moist desquamation for three dose fractions delivered between 5 and 7 days after an appropriate half-tolerance priming dose in comparison to those delivered between 2 and 4 days after the priming dose. Similar findings, sensitisation over the 14–18 day period after priming doses of 20 and 30 Gy, have been reported using a mouse skin model [20]. This 14–18 day period is much longer than the sensitive period of 4–8 days observed in the present study. However, Ruifrok et al. [20] concluded that proliferation started about 13 days after the start of treatment in their mouse
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basal cells from the epidermis of breast cancer patients after accelerated radiotherapy as compared with conventional (2 Gy/day, 25 fractions in 5 weeks) fractionated treatment. The accelerated fractionation schedule consisted of 2 × 2 Gy/ day, 5 days/week to a total of 50 Gy in 2.5 weeks. For the initial 21 days period, the rate of cell loss from the basal layer was similar for both fractionated irradiation schedules. However, the accelerated fractionation schedule resulted in a considerably reduced rate of loss of cells from the basal cell layer of the epidermis between days 21 and 42. This somewhat unexpected result, a more severe response to the conventional schedule, is possibly due to the fact that irradiation in the accelerated regime is completed prior to any shortening of the cell cycle in surviving target cells. With the conventional schedule irradiation will continue over a period when the cell cycle is likely to be shorter and hence cells will be more radiosensitive. However, due to the absence of mitotic or labelling index data, it was not possible to determine the overall duration of the cell cycle delay period which would also influence the rate of cell loss. The results of a randomised clinical trial of continuous hyperfractionated accelerated radiotherapy treatment (CHART) has also showed that skin reactions were less severe in CHART patients than those treated with conventional regimes [21]. The results of the present study are of considerable clinical significance, particularly with the increased interest in the use of modified dose-fractionation schedules in radiotherapy. In addition, these findings might influence the current thinking about dose-fractionation relationships where an equal biological effect per fraction is assumed and used by modellers who wish to mathematically represent the dose–time relationships in fractionated radiotherapy. Fig. 5. Variation in isoeffective dose with changes in the time interval between two equal dose fractions in (a) mouse skin and (b) pig skin (redrawn from Emery et al. [5] and van den Aardweg et al. [27], respectively).
model. Similarly, Tsang et al. [26] suggested that clonogen doubling time does not begin to shorten in mouse skin until about 12 days after the start of fractionated radiotherapy. This might explain why the previous rodent studies [12], which employed an overall time of less than 7 days, failed to demonstrate a significant change in effect per fraction. Although not directly comparable with the present studies, the effect of cell cycle changes due to the partial synchronisation of the cell population has been noted by other studies both in vitro [13,23] and in vivo [2,5,11,14,29]. This kind of radiosensitivity was attributed to the arrival of a group of synchronised cells in the G2/M phase of the cell cycle. This phenomena is discussed in detail by Hahnfeldt and Hlatky [7]. These authors have shown mathematically that variation with the time of resensitisation due to redistribution is not monotonic but oscillatory. The present conclusion might be used to provide a more informed explanation of recent clinical studies. Nyman and Turreson [19] reported a distinct change in the rate of loss of
Acknowledgements The authors are grateful to F.W. Dickinson and N. Hubbard for their day-to-day care of the animals. The technical assistance of Mr J.H. Wilkinson is gratefully acknowledged.
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