G₂ checkpoint control and G₂ chromosomal radiosensitivity in cancer survivors and their families

Abstract

Significant inter-individual variation in G₂ chromosomal radiosensitivity, measured as radiation-induced chromatid-type aberrations in the subsequent metaphase, has been reported in peripheral blood lymphocytes of both healthy individuals and a range of cancer patients. One possible explanation for this variation is that it is driven, at least in part, by the efficiency of G₂–M checkpoint control. The hypothesis tested in the current analysis is that increased G₂ chromosomal radiosensitivity is facilitated by a less efficient G₂–M checkpoint. The study groups comprised 23 childhood and adolescent cancer survivors, their 23 partners and 38 of their offspring (Group 1) and 29 childhood and young adult cancer survivors (Group 2). Following exposure to 0.5 Gy of 300 kV X-rays, lymphocyte cultures were assessed for both G₂ checkpoint delay and G₂ chromosomal radiosensitivity. In Group 1, the extent of G₂ checkpoint delay was measured by mitotic inhibition. No statistically significant differences in G₂ checkpoint delay were observed between the cancer survivors (P = 0.660) or offspring (P = 0.171) and the partner control group nor was there any significant relationship between G₂ checkpoint delay and G₂ chromosomal radiosensitivity in the cancer survivors (P = 0.751), the partners (P = 0.634), the offspring (P = 0.824) or Group 1 taken as a whole (P = 0.379). For Group 2, G₂ checkpoint delay was assessed with an assay utilising premature chromosome condensation to distinguish cell cycle stage. No significant relationship between G₂ checkpoint delay and G₂ chromosomal radiosensitivity was found (P = 0.284). Thus, this study does not support a relationship between G₂–M checkpoint efficiency and variation in G₂ chromosomal radiosensitivity.

Introduction

Ionising radiation delivered in the G₂ phase of the cell cycle can cause a transient ataxia telangiectasia mutated (ATM)-dependent cell cycle arrest, which allows time for DNA repair and prevents the progression of damaged cells from the G₂ phase into mitosis (1). Studies of radiation-induced mitotic inhibition have revealed that cell cycle arrest in G₂ is much less pronounced in cells from patients with the inherited cancer-prone syndrome ataxia-telangiectasia (AT) than in normal cells (2,3). Less G₂ arrest has also been postulated to contribute towards the enhanced G₂ chromosomal radiosensitivity seen in cells from some patients with sporadic cancer, e.g. breast cancer (4).

The premature chromosome condensation (PCC) technique enables categorisation of each cell cycle phase by the visualisation of distinct chromosome morphologies and has been applied to the study of cell cycle kinetics following irradiation. An early application of the PCC technique revealed that arrested cells in G₂ repair many of their DNA breaks before mitosis (5), indicating that one purpose of G₂ checkpoint delay is to allow additional time for the repair of DNA damage. More recently, the PCC technique has been improved (6) and combined with the G₂ radiosensitivity assay (7,8). A comparison of radiation-induced chromatid aberration levels in both normal and AT lymphocytes and their lymphoblastoid derivatives, before and after G₂ to mitosis transition, provided direct evidence that activation of the ATM-dependent G₂ checkpoint following irradiation is a key event in the repair of damage prior to progression to metaphase (7). Moreover, abrogation of the G₂ checkpoint in normal cells by pre-irradiation treatment with caffeine (7) or the benzene metabolite hydroquinone (9) was found to result in enhanced levels of radiation-induced G₂ chromosome damage progressing to metaphase.

G₂ chromosomal radiosensitivity has been postulated as a marker for cancer predisposition with many studies concluding that enhanced radiosensitivity may be a marker of low-penetrance cancer predisposition genes involved with the recognition and repair of DNA damage (10–14). Previously we reported on the relationship between G₂ chromosomal radiosensitivity and early onset cancer in a population of Danish childhood and adolescent cancer survivors and their families (15) and this study has now been extended (16). Here, we examine the relationship between G₂ chromosomal radiosensitivity and efficacy of the G₂ checkpoint in the same study groups and test the hypothesis that an increase in G₂ chromosomal radiosensitivity is facilitated by a less efficient G₂–M checkpoint. We present previously unreported mitotic inhibition data for the cancer families of the initial study (15) and the results of applying the PCC methodology to study G₂ checkpoint efficacy in survivors of cancer in the extended study (16).

Materials and methods

Sample collection

Peripheral blood samples were collected from Danish survivors of childhood and young adult cancer who had been treated with radiotherapy, their partners and their offspring. This work formed part of a large multinational collaboration between research groups in the USA, UK, Denmark and Finland utilising epidemiology and genetic techniques to study germ cell mutagenesis and cancer susceptibility (17) (http://www.gcct.org, last accessed October 20, 2010). Approval for the study was obtained from the Danish Scientific Ethical Committee and the Danish Data Protection Agency. All cancer patients were selected from the Danish survivor cohort, which was derived from the files of the nation-wide population-based Danish Cancer Registry. Full details of patient selection, blood sampling and cancer diagnosis have been reported (15,16). Two groups provided material for the analyses reported here. Group 1 comprised 28 cancer survivors and their families who provided blood samples between March 2002 and April 2003 (15). As described previously (15), only 23 of the 28 Danish cancer survivor families proved suitable for G₂ chromosomal radiosensitivity analysis and these 23 survivors, their 23 partners and 38 offspring were subsequently analysed for radiation-induced mitotic inhibition. Group 2 comprised 30 cancer survivors, who provided samples between June and December 2006 (16). One of these samples failed to culture successfully, leaving 29 samples, which were analysed for G₂ chromosomal radiosensitivity (16) and also for radiation-induced G₂ delay using the PCC technique.

The G₂ chromosomal radiosensitivity assay

The G₂ chromosomal radiosensitivity results for both study groups have been reported (15,16). The assay was performed according to the method previously described (18), which was based on modified versions of earlier published techniques (12,19). Briefly, whole blood samples were cultured for 72 h and irradiated with 0.5 Gy of 300 kV X-rays. Following a 30-min recovery period, 0.2 ml colcemid (10 μg/ml) was added for 60 min prior to plunging into ice and harvesting. Metaphase slides were prepared according to standard procedures and stained with Giemsa. One hundred well-spread metaphases were analysed and the total number of chromatid gaps and breaks was determined to give the G₂ aberration yield. Studies using the G₂ assay in our laboratory have shown the spontaneous aberration yield to be at a minimal level (<1.0 aberration per 100 cells). On this basis, and due to the additional time required to score control samples, it was deemed unnecessary to determine spontaneous aberration yields. Thus, the G₂ aberration yields reported for Group 2 (16) were determined from irradiated samples only (i.e. without subtraction of the spontaneous score derived from the control sample). Aberration yields reported for Group 1 initially included subtraction of control yields (15), but for the present study, the total irradiated G₂ score is reported in order to be comparable with the subsequent study (16).

Mitotic inhibition

Mitotic indices were determined by calculating the percentage of mitotic cells per 1000 unselected mononuclear cells using the irradiated and non-irradiated slides from the G₂ assay. Mitotic inhibition was then calculated as the percentage reduction in mitotic index in irradiated compared with non-irradiated samples. The relative decrease in the mitotic index following irradiation represents the extent of G₂ checkpoint delay.

Assessment of G₂ checkpoint delay using the PCC technique

Two additional cultures were set up in parallel to those for the G₂ chromosomal radiosensitivity assay, one of which was irradiated, with the other being the sham-irradiated control. At 30-min post-irradiation, 0.1 ml colcemid (10 μg/ml) was added and then at 60-min post-irradiation, 5 μl of calyculin A (to a final concentration of 50 nM) was added to induce PCC and cultures harvested as for the G₂ chromosomal radiosensitivity assay.

Following Giemsa staining, cells in G₂ and metaphase could be distinguished by their distinct chromosome morphologies (6,7,9,20). G₂ phase PCC cells contain bivalent condensed chromosomes, which are similar in shape to metaphase chromosomes. However, the key difference is that the two sister chromatids have no visible centromeric region conferring a distinctive morphology that is easily distinguished from metaphase spreads. For each sample, a combined total of at least 500 G₂ and metaphase cells were recorded and G₂ checkpoint delay was determined by subtracting the proportion of G₂ cells in the sham-irradiated culture from the proportion in the irradiated culture. This value was used as a measure of the extent of G₂ checkpoint delay. A positive value indicates an increase in G₂ cells after irradiation, which in turn represents an increase in G₂ checkpoint delay.

Results

For Group 1, mean and median mitotic inhibition data are presented in Table I. One-way analysis of variance of the mitotic inhibition values for cancer survivors, partners and offspring revealed no statistically significant differences (P = 0.212). Comparison of median values using the Mann–Whitney U-test also revealed no statistically significant differences between the cancer survivors (P = 0.660) or offspring (P = 0.171) and the partner control group (Table I). When making gender comparisons, no statistically significant differences in mitotic inhibition values were observed between male and female survivors (P = 0.560), partners (P = 0.065) or offspring (P = 0.193). In addition, there was no statistically significant correlation between mitotic inhibition and age when survivors (P = 0.559), partners (P = 0.237) and offspring (P = 0.510) were considered as separate groups. A scatter plot showing the correlation between the previously derived radiation-induced G₂ aberration yields (15) and the corresponding percentage mitotic inhibition is illustrated in Figure 1. Spearman’s rank correlation analysis revealed that there were no statistically significant relationships between mitotic inhibition and G₂ aberration yield in cancer survivors (P = 0.751), partner controls (P = 0.634), offspring (P = 0.824) or when all three groups were considered together (P = 0.379).

Table I.

Mitotic inhibition in 23 childhood and adolescent cancer survivors, their 23 partners and 38 offspring (Group 1)

Donor group	Number	Mean age (years) (range)	Mean mitotic inhibition (%) ± SD	Median mitotic inhibition (%) (range)
Survivors
Males	8	33.5 (31.0–36.0)	81.1 ± 19.7	86.3 (33.3–94.7)
Females	15	32.9 (25.0–37.0)	79.0 ± 17.5	83.3 (33.3–100.0)
Total	23	33.1 (25.0–37.0)	79.7 ± 17.9	84.6 (33.3–100.0)
Partners
Males	15	34.6 (26.0–43.0)	84.1 ± 9.1	87.5 (66.7–93.1)
Females	8	34 (31.0–36.0)	66.0 ± 29.8	78.2 (0.0–91.7)
Total	23	34.4 (26.0–43.0)	77.8 ± 20.3	82.4 (0.0–93.1)
Offspring
Males	22	4.7 (0.3–10)	86.7 ± 11.3	87.9 (60.0–100.0)
Females	16	6.7 (0.6–14)	82.3 ± 10.4	82.8 (55.6–96.7)
Total	38	5.5 (0.3–14)	84.8 ± 11.0	86.1 (55.6–100.0)

SD, Standard deviation.

Fig. 1.

Correlation between radiation-induced mitotic inhibition and G₂ aberration yield in Group 1. Mitotic inhibition is calculated as the percentage reduction in mitotic index in irradiated compared with non-irradiated samples.

For Group 2, which comprised only cancer survivors, G₂ checkpoint delay is presented in Table II. A scatter plot of the G₂ aberration yields observed at metaphase and the corresponding G₂ checkpoint delay measured by PCC analysis is presented in Figure 2. No statistically significant relationship was observed between G₂ checkpoint delay and G₂ aberration yield (P = 0.284). Furthermore, there was no significant gender effect upon the extent of G₂ checkpoint delay (Table II) (P = 0.479) neither was there any significant correlation between G₂ checkpoint delay and age (P = 0.767).

Table II.

G₂ checkpoint delay in 29 childhood and young adult cancer survivors (Group 2)

Donor group	Number	Mean age (years) (range)	G2 delaya (range)
Males	17	50.5 (38.0–68.0)	0.038 (−0.29 to 0.39)
Females	12	47.0 (32.0–62.0)	0.075 (−0.20 to 0.33)
Total	29	49.0 (32.0–68.0)	0.053 (−0.29 to 0.39)

^aSee Materials and Methods for derivation.

Fig. 2.

Correlation between radiation-induced G₂ checkpoint delay, measured by the PCC technique, and G₂ aberration yield for Group 2. G₂ checkpoint delay is calculated by subtracting the proportion of G₂ cells in the unirradiated culture from the proportion in the irradiated culture.

Discussion

A relatively low radiation-induced mitotic inhibition value is thought to represent a deficient checkpoint in which less time is allowed for the repair of chromosome damage before the onset of mitosis and thus, higher aberration yields would be observed at metaphase (2,4,21). In lymphocytes from normal donors, an increased G₂ chromosomal radiosensitivity has been associated with a decrease in mitotic delay induced by 0.02 Gy X-rays, although no correlation was found when a dose of 0.3 Gy was used (22). The authors speculated that a saturation effect may occur at the higher dose suggesting that the 0.4–0.5 Gy X-ray dose, generally utilised in the G₂ chromosomal radiosensitivity assay, may be too high to uncover subtle differences in G₂ checkpoint delay. However, in a mixed population of breast cancer patients and normal females, an increase in mitotic delay correlated with a decrease in the level of chromatid aberration frequencies following exposure in G₂ to an acute dose of 0.5 Gy (4). In addition, this study revealed less G₂ arrest in breast cancer patients in comparison to normal females suggesting a putative G₂ checkpoint defect that may contribute to the enhanced G₂ chromosomal radiosensitivity seen in 40% of cases (4). The authors, however, suggested that only a very small proportion of radiosensitive patients have a putative G₂ checkpoint deficiency and concluded that radiation-induced mitotic inhibition and G₂-induced chromatid aberration frequency observed at the subsequent metaphase may not be causally related.

In the current study, the distributions and the median mitotic inhibition values reported for the cancer survivors, their partners and their offspring in Group 1 are broadly in line with those reported for breast cancer (4), prostate cancer (23), benign prostatic hyperplasia (23) and AT heterozygotes (24). However, Group 1 showed no association between mitotic inhibition and cancer status (Table I) nor any correlation between radiation-induced mitotic inhibition and G₂ aberration yield (Figure 1). Unlike a report by Scott et al. (4), we found no age or gender effects on mitotic inhibition in the cancer survivors or in the corresponding partner control group.

The PCC technique has revealed that elevated chromosomal radiosensitivity observed at metaphase following G₂ irradiation is directly linked to a less efficient G₂ checkpoint in AT lymphocytes (7). However, application of the PCC technique has also demonstrated that despite an increased radiation-induced G₂ delay, BRCA1 heterozygotes have an increase in chromosome aberrations at metaphase when compared to normal controls, thus suggesting that mitotic delay does not necessarily reduce the radiation-induced chromosome damage (20). In the present study, application of the PCC technique to evaluate G₂–M checkpoint efficacy in the 29 childhood and young adult cancer patients of Group 2 revealed no correlation between the extent of radiation-induced G₂ checkpoint delay and chromosomal radiosensitivity when G₂-irradiated cells were examined for aberrations at the subsequent metaphase (Figure 2). In addition, there was no suggestion that gender or age influenced the extent of the radiation-induced delay (Table II).

The reasons for the large inter-individual variation observed when applying the G₂ chromosomal radiosensitivity assay have not yet been elucidated. However, this study, using two different assays, has found no support for the suggestion that a longer G₂ delay results in less radiation-induced chromosome damage progressing to metaphase, thus indicating that inter-individual variation in G₂ chromosomal radiosensitivity is more likely to be driven by other factors, such as variation in DNA repair capability, rather than being influenced by G₂ checkpoint differences.

Funding

National Institutes of Health, USA (1 RO1 CA 104666) through Vanderbilt Medical Centre, USA. Permissions were granted from the Danish Data Protection Agency (2001-41-1113) and the Danish Scientific Ethical Committee [(KF) 01–150/01, (KF) 11–129/02, (H-KF) 01–150/01].