Mutagenesis, Vol. 15, No. 6, 531-535,
November 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Variability of G2 checkpoint sensitivity to low doses of X-rays (2 cGy): correlation with G2 chromatid aberrations but not with an adaptive response
Department of Genetics and Molecular Biology, University `La Sapienza', P. le A.Moro 5, 00185 Rome, Italy
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Using human lymphocytes from a group of 20 donors, we investigated (i) the X-ray-induced adaptive response (AR) after four different conditioning treatments of 1, 2, 4 and 6 cGy, (ii) chromosomal sensitivity to X-irradiation during G2 and (iii) the G2/M checkpoint response. An AR was found in 11 of the 18 donors (~60%). No correlation was found between the presence of AR and G2 chromosomal radiosensitivity, or with donor sensitivity to the activation of the G2/M checkpoint by 30 cGy or a dose as low as 2 cGy. The AR was not related to any particular conditioning treatment, but was consistently present or absent in any one donor under all conditioning treatments used. As far as chromosomal breakage and induced mitotic delay in G2 are concerned, large variability between individuals was observed, together with a close correlation in the same donor between mitotic delay induced by a low dose (2 cGy) and the frequency of aberrations.
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Introduction |
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Cells pretreated with low-dose radiation (<10 cGy) (conditioning treatment) can become resistant to a second high dose (challenge treatment). This phenomenon is called the adaptive response (AR). It has been observed in many biological systems, both in vitro and in vivo, using different protocols and different endpoints (for reviews, see Wolff and Olivieri, 1996; Mitchel et al., 1997). Induction of the AR appears to be influenced by several factors. In human lymphocytes, the source is particularly important. In some donors, the AR is absent (for reviews, see Raaphorst and Boyden, 1999; Oliveira et al., 2000). This is reproducible in experiments performed at different times, several months apart, so the absence of AR may be genetically determined constitution. However, `non-responding' donors may occasionally display an AR (Olivieri et al., 1994
). In our protocol, in which G2 chromatid aberrations were investigated, inter-donor variability may be due to the fact that the conditioning treatment modifies the mitotic delay induced by the challenge dose in different ways depending on the donor. This has been repeatedly observed by us (Salone et al. 1996a, b; this study). However, the effect is not associated with the presence or absence of an AR, and indirect evidence suggests that the AR is not due to any greater mitotic delay in conditioned cells (Wolff, 1996). The variability in AR may be accounted for by variation in the sensitivity of the various donors to any effects that may be induced by a low dose of ionizing radiation (Salone et al., 1996b). Several such inducible effects have been described in the last few years (see Marples et al., 1997), including activation of checkpoints in the cell cycle. In the present work we tested the sensitivity of various donors to activation of the G2/M checkpoint by a low dose, seeking a possible relationship with the presence of an AR in the same donor. To be certain that we had identified variation in donor sensitivity to low doses, we used a series of different conditioning treatments. The different donors displayed a wide range of variability in the activation of the G2/M checkpoint by a low dose, when it was administered either in S phase or in G2. Sensitivity to G2/M checkpoint activation was not related to presence of the AR. On the other hand, we did observe a significant correlation between sensitivity to G2/M checkpoint activation by a low dose and sensitivity to the induction of chromatid aberrations in G2.
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Materials and methods |
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Donors
Donors were identified by a number; those with numbers of <20 had participated in previous studies of ours (in which they were identified by the same number), while donors 21–34 participated only in the present study. All donors were apparently in good health. Donors 3–5, 8, 13, 20–25 and 33 were males; donors 1, 6, 7, 26–32 and 34 were females. Donors 20, 24, 26, 27 and 30 were aged 20–30 years; donors 7, 21–23, 25, 28, 29, 31, 33 and 34 were aged 30–40 years; and donors 1, 3–6, 8, 13 and 32 were aged 40–60 years. Donors 1, 8, 13 and 32 were smokers. Too few subjects were studied to allow any definitive conclusions to be drawn concerning the influence of gender, age or smoking on our results.
Cell culture and irradiation
Whole blood (0.5 ml) was added to 4.5 ml of RPMI 1640 medium without fetal calf serum (Wolff et al., 1984), with 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 2% phytohaemagglutinin M (PHA). Irradiation was carried out with 200 kVp X-rays (Gilardoni MGL 200/8D, 0.2 mm Cu filtration, 6 mA, 0.20 Gy/min). Two parallel cultures were set up for each point examined. In some experiments, [3H]thymidine (dThd) (3.7 x 103 Bq/ml, sp. act. 2.5 x 1011 Bq/mmol) was added at different times before fixation; autoradiograms of the slides were prepared by standard methods.
Experimental scheme
For practical reasons, the experiments were performed in batches of three to six donors. In the series in which the AR was investigated, cultured human lymphocytes were exposed to conditioning treatments and subsequently challenged with high doses of X-rays. The cells were scored to see whether earlier exposure reduced the number of chromatid and isochromatid breaks induced by the challenging doses. A few chromatid exchanges (<10 per 100 metaphases) were recorded but not included in the analysis.
In these experiments, the results of which are presented in Table I, the conditioning treatment was administered 36 h after stimulation with PHA. In all other experiments, the conditioning treatment was administered 20 h after stimulation with PHA.
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In the experiments set out in Tables I and II
, parallel cultures were used to study the percentage of labelled mitoses after irradiation by adding [3H]dThd soon after X-ray treatment at 44 h; cells were fixed 3.5, 4.25, 5 and 5.75 h after X-ray treatment. The cultures used to study the aberrations were used also to determine mitotic index (MI). These cultures were irradiated 44 h after stimulation with PHA. Thirty minutes after irradiation with 30 cGy (or 15 min after irradiation with 2 cGy), 0.1 ml colcemid (final concentration 2 x 10–7 M) was added to each culture; fixation was performed in accordance with standard procedures, 90 min later in the 30 cGy series or 75 min later in the 2 cGy series.
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Results |
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The results of experiments in which lymphocytes were exposed to 30 cGy after 44 h either with or without a previous conditioning treatment (2 cGy) are presented in Table I
. The frequency of breaks in these two series (i. e. with or without conditioning) were compared to see if there was an AR. In this and the following tables, only the frequency of induced chromatid breaks, from which the frequency of spontaneous breaks [% = (2.6 ± 1.3)%, range 0.5–3.5%] was subtracted, is presented for each donor. The results show that an AR was present in three of the six donors. In parallel cultures previously exposed to the two treatment types, [3H]dThd was added soon after X-ray treatment at 44 h; cells were fixed 3.5, 4.25, 5 or 5.75 h after irradiation and the addition of [3H]dThd. These cultures were scored for the presence of labelled metaphases. When compared with the control, the sum of these four fixation times tells us the extent to which the various treatments have modified the time that the cells in S phase at 44 h take to reach mitosis and thus to what extent their G2 is modified. It was confirmed that the 2 cGy treatment alone, given in S phase can, in some cases (see donor 20), significantly extend G2 and increase the extensions induced by 30 cGy treatment. As expected, 30 cGy given immediately before the addition of [3H]dThd produced a significant extension of G2 in all donors. There was variation between the six donors studied in terms of induction of chromatid breakage, labelling index and presence of an AR. No correlation was found between these variables. Nevertheless, the fact that donor 20 showed not only a low number of breaks but also an AR and the greatest reduction in labelling index led us to repeat the experiment with six other donors (including donor 20) in order to investigate whether there was a relationship. In this case, in addition to treatments to evaluate the AR, we also investigated G2/M checkpoint activation after 2 and 30 cGy. This was done by both MI and labelling index, as in the preceding experiment. In these and subsequent experiments, the low dose of 2 cGy was used both at 20 h to induce an AR and, in other cultures, at 44 h in order to study the effect on breakage and G2/M checkpoint activation.
The data shown in Table II indicate that there was no correlation between the presence of an AR and a greater or lesser sensitivity to G2/M checkpoint activation. The enhanced sensitivity to the activation of this checkpoint, as indicated by the reduction in MI induced by 2 cGy (but not by 30 cGy), seems, however, to correlate with a lower breakage frequency, both after 2 cGy (r = 0.83, P < 0.05) and 30 cGy (r = 0.91, P < 0.02). The behaviour of donor 20 is a good example. As in the preceding experiment and in other unpublished work, this donor always displayed a strongly reduced MI and a low breakage frequency. In this connection, our findings support those of other laboratories (Scott et al., 1999): if a series of experiments is taken into consideration, there was less intra-individual variability in the number of chromatid breaks than inter-individual variability. It is also interesting to note that, in this experiment, donor 20 did not display any AR. Thus there was no consistency in the presence or absence of AR in a given individual.
In view of these results, we decided to extend the study of the donor samples in which MI reduction and the frequency of aberrations after 2 cGy and 30 cGy given 2 h before fixation were assessed; these samples were studied as in the preceding experiment. As well as parallel cultures before treatment with 30 cGy, four different conditioning treatments were performed, one each at 1, 2, 4 and 6 cGy. The results of the experiments are presented in Table III. Induction of the AR in any given donor does not seem to depend on conditioning dose; each donor was AR-competent or not regardless of the conditioning treatment used, as is shown, for instance, by donors 1 or 27 or 30, who each displayed the same response (absence of interaction, AR or synergy) for the four conditioning treatments given. Note that the results presented in Table III show only the differences significant for AR and synergy; the unreported values, although not significant, display the same trend as the ones reported here. It was observed also that in these donors, the presence or absence of AR did not correlate with sensitivity to the induction of breakage or with sensitivity to the activation of the G2/M checkpoint by a low or a high dose. In the case of induced breakage and mitotic delay after 2 or 30 cGy, after summing the data of the 20 donors presented in Tables II and III, we observed a good correlation between breaks induced by 2 or 30 cGy with mitotic delay induced by 2 cGy, although not with that induced by 30 cGy (Table IV). In agreement with these data, each donor displayed a positive correlation between breaks induced by a low and a high dose (r = 0.68, P < 0.001), but no correlation between the mitotic delays induced in the same donor by the two doses.
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Discussion |
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The reasons for the variability in AR observed in the lymphocytes of human donors remains to be elucidated, probably because the underlying mechanisms of AR are still not fully understood. At the molecular level we know that the conditioning treatment (Yanase et al., 1999
; Ye et al., 1999) and, more generally, ionizing radiation (Prasad et al., 1995; Sadekova et al., 1997; Amundson et al., 1999; Balcer-Kubiczek et al., 1999) affect the expression of many genes. The resulting cascade of events appears to be extremely complex (Weichselbaum et al., 1991; Ryabchenko et al., 1998; Suzuki et al., 1998; Bravard et al., 1999) and may modify in various ways the subsequent cell response to ionizing radiation (Le et al., 1998; Zhang et al., 1998; Cregan et al., 1999; Hendrikse et al., 2000). The complexity of the interactions underlying the endpoint studied here, namely chromatid breaks, means that it is difficult to isolate the cause of the absence of AR in some individuals. Since the conditioning treatment was performed using a low dose of X-rays, and since there was a high degree of variability in the induction of mitotic delay following low doses, we postulated that the AR and an individual donor's sensitivity to checkpoint induction, as indicated by mitotic delay in G2 due to low doses, are the expression of that donor's sensitivity to effects inducible by low doses. The lack of correlation between these phenomena, as revealed by the results of the present work, leads to the conclusion that this hypothesis can be rejected. Another possible explanation of AR variability could be linked to the fact that the pathway leading to the AR is easily saturated (Pieper et al., 1999) by an excess of conditioning dose (Shadley et al., 1987; Sasaki, 1995) or by other internal and environmental conditioning stimuli. This is more likely in certain donors at certain times of their lives. Under these conditions, the AR may be absent and the conditioning stimulus may even lead to synergy (Olivieri et al., 1994). We are currently checking the validity of this hypothesis.
The activation of cell cycle checkpoints by ionizing radiation or other treatments is one of the most extensively studied inducible effects (for review, see Hwang and Muschel, 1998). Considerable interest has been focused in particular on the G2/M checkpoint. Many mutagenesis studies carried out in recent years have referred to the chromosomal aberrations induced in the G2 phase, mainly in human lymphocytes. Inter-individual variations in radiosensitivity during this phase (Natarajan et al., 1982) have been found in those with cancer-prone syndromes or who are prone to breast cancer (Parshad et al., 1983; Sanford et al., 1989, 1990; Scott et al. 1994, 1996, 1999). Variations in chromatid damage in G2 have often been related to the degree of mitotic delay (Scott and Zampetti-Bosseler, 1980; Nagasawa et al., 1994; Schwartz et al., 1996). Mitotic delay could be used by the cell to repair the damage produced: the greater the delay, the more damage can be repaired and the less chromatid damage will then be observed (Scott and Zampetti-Bosseler, 1980; Painter and Young, 1980; Khilman et al., 1982; Olivieri and Micheli, 1983). The data presented relate to this hypothesis. The observed chromatid damage was inversely proportional to the mitotic delay induced by a low dose, but not to that induced by a comparatively high dose given during the same phase of the cell cycle. The extent of the mitotic delay induced by the high dose was not related to that induced by the low dose. This could be because, at the higher dose, the effect becomes saturated, or because the mitotic delay mechanisms differ, at least in part, between the high and low doses. For example, there could be differences in the sensors that activate the cascade of events leading to mitotic delay (Kaufmann and Kies, 1998; Vigh et al., 1998). With 2 cGy or even smaller doses, we observed a mitotic delay, which is unlikely to be due to induced DNA damage (O'Connell et al., 2000), which probably triggers the mitotic delay observed at higher doses. In either case, as far as mitotic delay is concerned, the low dose seems to be more effective for detecting differences in radiosensitivity. Conceivably, donors that are highly sensitive to activation of the G2/M checkpoint by a low dose could ultimately suffer less chromatid damage. This phenomenon is probably specific to G2 chromatid damage and could account for the absence of a link between G2 chromatid damage and G1 chromosomal damage (Scott et al., 1999), observed also by us in the chromosomal aberrations induced in the 20 donors (unpublished data).
In recent work to investigate chromosomal aberrations induced by ionizing radiation in four donors, Palitti et al. (1999) claimed that `the heterogeneous G2-phase chromosome radiosensitivity, usually found in different normal donors, is caused by the analysis of different cell populations rather than reflecting intrinsic differences in radiosensitivity'. We feel that this possibility does not apply to our experiments, as cells were collected only in the first 90–120 min after irradiation. This yielded a comparatively homogeneous sample of cells that, in the various labelling experiments, were all found to be unlabelled and had presumably proceeded beyond the transition point. The claims of Palitti et al. may, however, apply to cell samples fixed at a later time and subject to other confounding factors. Nevertheless, it must be borne in mind that variable inter-individual sensitivity is probably a real factor, as has been shown in research using other endpoints and by the repeatability of the results for the same donor (Odagiri et al., 1997; Brooks, 1999; Scott et al., 1999). It would be interesting to investigate whether differing sensitivity to the induction of mitotic delay is the main cause of the variations observed in G2 radiosensitivity as evaluated by chromatid damage.
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Acknowledgments |
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This work was partially supported by 40% and 60% MURST grants.
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1 To whom correspondence should be addressed. E-mail: olivieri{at}axcasp.caspur.it
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References |
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Received on July 10, 2000; accepted on August 15, 2000.
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