Skip Navigation


Mutagenesis Advance Access originally published online on March 6, 2008
Mutagenesis 2008 23(4):267-270; doi:10.1093/mutage/gen009
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
23/4/267    most recent
gen009v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Shovman, O.
Right arrow Articles by Bryant, P. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shovman, O.
Right arrow Articles by Bryant, P. E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Author 2008. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

An improved assay for radiation-induced chromatid breaks using a colcemid block and calyculin-induced PCC combination

Olga Shovman, Andrew C. Riches, Douglas Adamson1 and Peter E. Bryant*

Bute Medical School, University of St Andrews, St Andrews, KY19 9TS, UK 1Ward 32, Ninewells Teaching Hospital, University of Dundee, Dundee DD1 9SY, UK

We report on a new method for the study of radiation-induced chromatid breaks in stimulated human peripheral blood T lymphocytes, involving a combination of a 1-h colcemid block and a short (15 min) calyculin A treatment. We find that this procedure eliminates the problem of centromere splitting when calyculin A is used alone for a longer period and produces metaphase spreads with superior quality. By this procedure, the chromosomes and the chromatid breaks are expanded and thereby make for improved break scoring. In a comparison of the new technique with the conventional colcemid block method, we show a close proportionality between the frequencies of chromatid breaks scored with the two methods. The frequency of chromatid breaks with the new method was found to be significantly higher than that with colcemid alone, adding a higher sensitivity to the assay as an additional advantage.


   Introduction
Top
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
The frequency of ionizing radiation-induced chromatid breaks in metaphases of phytohaemagglutinin (PHA)-stimulated human peripheral blood T lymphocytes (PBLs) has been shown to vary widely in different apparently normal individuals when the cells are sampled at 1.5 h following exposure. This short sampling time, including a 30-min incubation at 37°C to allow any mitotic cells to re-enter the G1 phase and a 1-h treatment with colcemid, ensures that the metaphase cells sampled by the colcemid block were in the G2 phase of the cell cycle at the time of radiation exposure. Further interest in this so-called ‘G2 assay’ of chromatid breaks was generated by the finding that the frequency of chromatid breaks is highly variable between different normal individuals and also significantly higher in PBL from over 40% of breast and other sporadic cancer cases (1Go–9Go). PBL derived from several different types (but not all types) of sporadic cancer cases also showed elevated frequencies of radiation-induced chromatid breaks (5Go,10Go). Earlier reports (e.g. 3Go,11Go) had claimed that most, if not all, cancer cells would show elevated chromatid break frequencies. However, such a universal hypothesis was not substantiated by other studies (e.g. 12Go,13Go).

Elevated chromatid breakage (sometimes referred to as ‘chromatid radiosensitivity’) has been interpreted in terms of the presence of mutations in low-penetrance genes that confer both chromatid radiosensitivity and susceptibility to breast cancer (1Go,4Go,6Go). These putative susceptibility or predisposing genes may be more penetrant in individuals with early onset cancers, although the evidence for this is controversial (14Go–16Go).

The concept of low-penetrance susceptibility genes in chromatid radiosensitivity has been strengthened by the demonstration of heritability of G2 chromatid radiosensitivity among families of breast cancer sufferers (4Go,17Go). Thus, further study and understanding of the mechanisms underlying the formation of chromatid breaks from radiation-induced DNA double-strand breaks could help clarify the reasons for breast cancer susceptibility.

In all these studies, conventional colcemid-induced mitotic block was used to collect metaphases, and as a consequence, the frequency of chromatid breaks scored could have been influenced by the activation of cycle arrest at the G2 checkpoint, allowing cells time to rejoin their chromatid breaks, and so possibly introducing some uncertainty into the interpretation of elevated chromatid radiosensitivity. Controversy surrounds this hypothesis. Some studies, e.g. of tumour cell lines in vitro (18Go), show a clear inverse relationship between mitotic inhibition and chromatid break frequency, whereas other studies using PBL from normal or breast cancer patients show only a very weak or no correlation at radiation doses normally used in the ‘G2 assay’ of chromatid breaks (19Go,20Go), suggesting that while cell cycle checkpoint delay may have a small influence on G2 chromatid break scores in stimulated PBL, it is not a major determinant of chromatid break frequency.

Recently, it has become possible to use the powerful phosphatase inhibitor calyculin A to efficiently cause premature chromosome condensation (PCC) of G2 cells so that chromatid breaks can be scored more directly following irradiation without the requirement of cells to progress to ‘normal’ mitosis (21Go–27Go). Unfortunately, in lymphocytes, calyculin induces not only chromosome condensation but also causes widespread centromere splitting (e.g. 24Go,28Go) making scoring more difficult due to the lack of close proximity of the sister chromatids and consequent possible displacement or loss of some chromatid fragments.

To circumvent centromere splitting in lymphocyte PCC, the duration of calyculin treatment can be reduced, and here we describe a technique that combines colcemid block and a short duration calyculin-induced PCC treatment, which gives improved chromosome visualization wider chromatid breaks and an enhanced sensitivity, almost entirely without the unwelcome side effect of centromere splitting. We have made a comparison between the chromatid break scores with colcemid block alone and colcemid plus calyculin treatments.


   Materials and methods
Top
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Peripheral blood samples
Peripheral blood samples were obtained with informed consent and ethical approval from five normal control individuals and six breast cancer cases at Ninewells Hospital, Dundee. The blood samples were taken by venipuncture into heparinized vacutubes (Becton Dickinson, Plymouth, UK) and transported within 24 h to the Bute Medical School where experimental procedures were carried out.

Cell culture and irradiation
Blood samples were diluted 1:10 into pre-warmed RPMI medium (Gibco/Invitrogen, Paisley, UK) in 25-cm2 tissue culture flasks (Nunc, Roskilde, Denmark) and 150 µl of PHA (HA16; Remel Europe, Dartford, UK) was added. Cultures were maintained at 37°C and in 5% CO2 for 72 h. For each sample of blood, four 10 ml cultures were set up: two control cultures (sham irradiated) and two irradiated cultures (given 0.4 Gy of gamma rays in a CIS-UK gamma irradiator operating at a dose rate of ~3 Gy/min).

Colcemid and calyculin treatment
Following irradiation, cultures were allowed to incubate for 30 min at 37°C to allow any mitotic cells present during irradiation to progress into the next G1 phase. Colcemid (KaryoMax; Gibco/Invitrogen) was then added at a concentration of 0.1 µg/ml for 1 h. Calyculin A (Sigma, Basingstoke, UK; stock solution 50 µg/ml in dimethyl sulphoxide; final concentration in medium 50 ng/ml) was added to two cultures (control and irradiated) for the last 15 min of the 1-h incubation with colcemid.

Cell fixation and chromosome preparation
Following incubation, cultures were centrifuged and the medium was aspirated. Pellets were vortexed and 5 ml of hypotonic solution (75 mM KCl; 4°C) was added while vortexing and samples were then held at room temperature for 10 min before centrifuging again. Hypotonic solution was aspirated, pellets vortexed again and 5 ml of fixative (methanol:acetic acid, 3:1, made up freshly) was added during vortexing. Tubes were allowed to stand for 10 min before centrifuging again, fixative aspirated and 5 ml of fixative again added during vortexing. This procedure was repeated four times, and cells were transferred to 1.5 ml eppendorf tubes in ~0.5 ml of fixative. Ten microlitres of cell suspension was dropped on to ethanol-washed slides in a Hanabi humidifier apparatus (drying index below 8.0). Once dry, slides were stained in 5% Giemsa in Gurr's buffer (pH 6.8) for 5 min, rinsed briefly in Gurr's buffer and blotted dry on fibre-free paper.

Chromatid break scoring
Chromatid breaks were defined as any clear chromatid discontinuity (29Go). One hundred metaphases were scored for chromatid breaks using a metaphase finder (Applied Imaging, Newcastle on Tyne, UK) coupled with a Zeiss Axioplan 2 microscope using an oil immersion x63 objective.


   Results
Top
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Figure 1 (panels a and b) show examples of metaphases of irradiated cells using the colcemid plus calyculin method. It was found that the use of a combination of colcemid for 1 h, including calyculin for 15 min (Figure 1a and b), significantly reduced the centromere splitting (or loss of centromeric constriction) as observed with longer (30 min) calyculin treatment alone (Figure 1c and d). Although some chromosomes in metaphase (b) show a tendency for split centromeres, the chromatids still show centromeric constriction and remain closely adherent, thus allowing confident scoring of breaks. A further advantage of the colcemid plus calyculin method was the enlargement and clarity of breaks as compared to those seen following condensation with colcemid alone (see examples in Figure 1a and b). Since with the addition of calyculin to the colcemid block a proportion of G2 cells are condensed, the combination of the two agents substantially increases the number of scorable condensed chromosome figures.


Figure 1
View larger version (30K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Examples of PBLs chromosome spreads using (a and b) colcemid plus calyculin (15 min prior to harvesting) and using (c and d) calyculin alone for 30 min. Note the marked loss of centromeres and chromatid cohesion in cells (c) and (d), when using calyculin alone.

 
Figure 2 shows a plot of chromatid break frequencies obtained with colcemid block alone against colcemid plus calyculin for the 11 individuals (five controls and six breast cancer cases). There was a clear linear relationship between the two end-points with a high degree of correlation (r2 = 0.929). The overall mean chromatid break values for colcemid plus calyculin treatments (196 breaks/100 cells; SEM = 14) were significantly higher (P = 0.0001) than that for colcemid alone (155 breaks/100 cells; SEM = 11), thus making the combined assay more sensitive. The mean chromatid break value (Figure 3) for the colcemid block technique (126 breaks/100 cells; SEM = 9) was significantly different from that for the colcemid plus calyculin technique (152 breaks/100 cells; SEM = 9) (P = 0.005). The mean chromatid break value for breast cancer cases with the colcemid technique (178 breaks/100 cells; SEM = 4) was also significantly different from that for the colcemid plus calyculin technique (232 breaks/100 cells; SEM = 6) (P = 0.002).


Figure 2
View larger version (8K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. Frequencies of chromatid breaks in stimulated and irradiated PBLs of controls (filled circles) and breast cancer cases (open circles), scored after treatment with either colcemid as blocking agent plotted against those scored after colcemid plus calyculin treatment. Frequencies of spontaneous chromatid breaks have been subtracted. The coefficient of determination for the combined data r2 = 0.929. Error bars represent standard deviations.

 


Figure 3
View larger version (9K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. Mean values of chromatid breaks per 100 cells in cells from controls (open bars) and breast cancer cases (shaded bars) using the colemid block and colcemid plus calyculin techniques. Vertical bars represent standard errors of means. Mean values for breast cancer cases versus controls using the colcemid technique were significantly different (P = 0.006). Mean values for breast cancer cases versus controls using the colcemid plus calyculin technique were significantly different (P = 0.0009). Means for controls for the two different techniques were significantly different (P = 0.005). Means for breast cancer cases for the two different techniques were significantly different (P = 0.002).

 

   Discussion
Top
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
The new method described here using a combination of colcemid and calyculin was found to be superior to colcemid alone, in that scoring is made easier due to the enlargement of chromatid breaks. Also, the new method was found to have a higher sensitivity, giving significantly higher frequencies of chromatid breaks (Figures 2 and 3) for a standard dose of gamma rays (0.4 Gy) without a reduction in mitotic index which would be the case if dose was increased significantly to achieve the same frequency of chromatid breaks using the standard colcemid technique. Our data (Figure 2) show that the frequency of chromatid breaks in samples treated with the new method (colcemid and calyculin) are proportional to those with colcemid block alone (r2 = 0.929), and their frequency is significantly higher, presumably as a result of including prematurely condensed G2 chromosomes (PCC) in the analyses.

Calyculin is a phosphatase inhibitor that causes condensation of cells in the G2 phase of the cell cycle, removing the need for cells to progress to mitosis (21Go–26Go). Thus, the metaphases scored following combined treatment with colcemid and calyculin will comprise cells from both mitosis and from the G2 phase of the cell cycle. In a recent study (28Go) of ataxia telangiectasia (AT) cells, a combination of colcemid and calyculin was also used. However, in contrast to our procedure, these authors used a longer (30 min) calyculin treatment, purposely to yield a fraction of cells (PCC) with split centromeres that were assumed to be G2 cells. Terzoudi et al. showed that when scoring these cells with split centromeres, no enhancement of chromatid break frequency was found for AT cells as compared with normal controls.

However, for our data, the close proportionality of chromatid break frequencies between the colcemid plus calyculin procedure and the standard colcemid block technique for samples from both breast cancer cases and normal individuals (Figure 2) give us confidence to propose that the new procedure is not altering the ‘relative’ radiosensitivity of cells in the assays. The proportionality between the two different procedures also suggests that the radiosensitivity of cells as determined by the two assays is not significantly influenced by the G2 checkpoint, although some of the metaphases scored with the new calyculin procedure will arise from cells in the G2 phase. This could be a real difference between our assay and those used by others (23Go,28Go) where a longer calyculin treatment was used. It is possible that in our procedure, the short (15 min) additional calyculin treatment immediately prior to fixation tends to favour those G2 cells that are close to mitosis and initiating chromosome condensation. Hence, our technique could be preferentially sampling cells that are already beyond the G2 checkpoint. A further difference between our technique and that of Terzoudi et al. (28Go) is the radiation dose used. Terzoudi et al. used 1 Gy, a dose more than twice our standard dose of 0.4 Gy. The higher radiation dose could therefore significantly enhance the effect of the G2 checkpoint.

Thus, when coupled with the fact that we registered very few cells containing split centromeres, we suggest that our combined colcemid plus calyculin procedure has advantages over both the conventional colcemid block technique and the calyculin-alone method when scoring chromatid breaks in stimulated irradiated T lymphocytes.


   Funding
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
The authors would like to acknowledge the support of the Chief Scientist Office (Edinburgh) (CSO/BTRC Award CZB/4/510) and the Breast Cancer Campaign (Project 2006NovSP12). Conflict of interest statement: None declared.


   Notes
 
* To whom correspondence should be addressed. Tel: +44 (0)1334 463510; Fax: +44 (0)1334 463482; Email: peb{at}st-and.ac.uk


   References
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

    1. Scott D, Spreadborough A, Levine E, Roberts SA. Genetic predisposition to breast cancer. Lancet (1994) 344:1444.[Web of Science][Medline]

    2. Scott D, Barber JBP, Spreadborough AR, Burrill W, Roberts SA. Increased radiosensitivity in breast cancer patients: a comparison of two assays. Int. J. Radiat. Biol. (1999) 75:1–10.[CrossRef][Web of Science][Medline]

    3. Parshad R, Price FM, Bohr VA, Cowan R, Zujewski JKA, Sanford KK. Deficient DNA repair capacity, a predisposing factor in breast cancer. Br. J. Cancer (1996) 74:1–5.[Web of Science][Medline]

    4. Roberts SA, Spreadborough AR, Bulman B, Barber JBP, Evans DRG, Scott D. Heritability of cellular radiosensitivity: a marker of low penetrance predisposition genes in breast cancer? Am. J. Hum. Genet. (1999) 65:784–794.[CrossRef][Web of Science][Medline]

    5. Baria K, Warren C, Roberts SA, West CM, Scott D. Chromosomal radiosensitivity as a marker of predisposition to common cancers. Br. J. Cancer (2001) 84:892–896.[CrossRef][Web of Science][Medline]

    6. Riches AC, Bryant PE, Steel CM, Gleig A, Robertson AJ, Preece PE, Thompson AM. Chromosomal radiosensitivity in G2-phase lymphocytes identifies breast cancer patients with distinctive tumour characteristics. Br. J. Cancer (2001) 85:1157–1161.[CrossRef][Web of Science][Medline]

    7. Buchholz TA, Wu X. Radiation-induced chromatid breaks as a predictor of breast cancer risk. Int. J. Radiat. Oncol. Biol. Phys. (2001) 49:533–537.[CrossRef][Web of Science][Medline]

    8. Baeyens A, Broecke RVD, Makar A, Thierens H, de Ridder L, Vral A. Chromosomal radiosensitivity in breast cancer patients: influence of age of onset of the disease. Oncol. Rep. (2005) 13:347–353.[Web of Science][Medline]

    9. Smart V, Curwen GB, Whitehouse CA, Edwards A, Tawn EJ. Chromosomal radiosensitivity: a study of the chromosomal G2 assay in human blood lymphocytes indicating significant inter-individual variability. Mutat. Res. (2003) 528:105–110.[Web of Science][Medline]

    10. Terzoudi GI, Jung T, Hain J, Vrouvas J, Margaritis K, Donta-Bakoyiannis V, Makropoulos CV, Angelakis PH, Pantelias GE. Increased G2 chromosomal radiosensitivity in cancer patients: the role of cdk/cyclin-B activity level in the mechanisms involved. Int. J. Radiat. Biol. (2000) 76:607–615.[CrossRef][Web of Science][Medline]

    11. Sanford KK, Parshad R, Gantt R, Tarone RE, Jones GM, Price FM. Factors affecting the significance of G2 chromatin radiosensitivity in predisposition to cancer. Int. J. Radiat. Biol. (1989) 55:963–981.[Web of Science][Medline]

    12. Bender MA, Viola MV, Fiore J, Thompson MH, Leonard R. Normal G2 chromosomal radiosensitivity and cell survival in the cancer family syndrome. Cancer Res. (1988) 48:2579–2584.[Abstract/Free Full Text]

    13. Scott D, Spreadborough AR, Jones LA, Roberts SA, Moore CJ. Chromosomal radiosensitivity in G2-phase lymphocytes as an indicator of cancer predispostion. Radiat. Res. (1996) 145:3–16.[Web of Science][Medline]

    14. Papworth R, Slevin N, Roberts SA, Scott D. Sensitivity to radiation-induced chromosome damage may be a marker of genetic predisposition in young head and neck cancer patients. Br. J. Cancer (2001) 84:776–782.[CrossRef][Web of Science][Medline]

    15. Baria K, Warren C, Eden OB, Roberts SA, West CM, Scott D. Chromosomal radiosensitivity in young cancer patients: possible evidence of genetic predisposition. Int. J. Radiat. Biol. (2002) 78:341–346.[CrossRef][Web of Science][Medline]

    16. Baeyens A, Thierens H, Claes K, Poppe B, Messiaen L, de Ridder L, Vral A. Chromosomal radiosensitivity in breast cancer patients with a known or putative genetic predisposition. Br. J. Cancer (2002) 87:1379–1385.[CrossRef][Web of Science][Medline]

    17. Patel RK, Trivedi AH, Arora DC, Bhatavdekar JM, Patel DD. DNA repair proficiency in breast cancer patients and their first-degree relatives. Int. J. Cancer (1997) 73:20–24.[CrossRef][Web of Science][Medline]

    18. Schwartz JL, Cowan J, Grdina DJ, Weichselbaum RR. Attenuation of G2-phase cell-cycle checkpoint control is associated with frequencies of unrejoined chromosome breaks in human tumor cells. Radiat. Res. (1996) 146:139–143.[Web of Science][Medline]

    19. Scott D, Spreadborough AR, Roberts SA. Less G2 arrest in irradiated cells of breast cancer patients than in female controls: a contribution to their enhanced chromosomal radiosensitivity? Int. J. Radiat. Biol. (2003) 79:405–411.[CrossRef][Web of Science][Medline]

    20. Pretazzoli V, Salone B, Bosi A, Olivieri G. Variability of G2 checkpoint sensitivity to low doses of X-rays (2 cGy): correlation with G2 chromatid aberrations but not with an adaptive response. Mutagenesis (2000) 15:531–535.[Abstract/Free Full Text]

    21. Gotoh E, Asakawa Y, Kosaka H. Inhibition of protein serine/threonine phosphatases directly induces premature chromosome condensation in mammalian cells. Biomed. Res. (1995) 16:63–68.[Web of Science]

    22. Gotoh E, Kawata T, Durante M. Chromatid break rejoining and exchange aberration formation following g-ray exposure: analysis in G2 human fibroblasts by chemical-induced premature chromosome condensation. Int. J. Radiat. Biol. (1999) 75:1129–1135.[CrossRef][Web of Science][Medline]

    23. Durante M, Furusawa Y, Gotoh E. A simple method for simultaneous interphase-metaphase chromosome analysis in biodosimetry. Int. J. Radiat. Biol. (1998) 74:457–462.[CrossRef][Web of Science][Medline]

    24. Durante M, Furusawa Y, Majima H, Kawata T, Gotoh E. Association between G2-phase block and repair of radiation-induced chromosome fragments in human lymphocytes. Radiat. Res. (1999) 151:670–676.[CrossRef][Web of Science][Medline]

    25. Gotoh E, Durante M. Chromosome condensation outside of mitosis: mechanisms and new tools. J. Cell. Physiol. (2006) 209:297–304.[CrossRef][Web of Science][Medline]

    26. Kawata T, Ito H, Uno T, et al. G2 chromatid damage and repair kinetics in normal human fibroblast cells exposed to low- or high-LET radiation. Cytogenet. Genome Res. (2004) 104:211–215.[CrossRef][Web of Science][Medline]

    27. Bryant PE, Mozdarani H. G2-phase chromatid break kinetics in irradiated DNA repair mutant hamster cell lines using calyculin-induced PCC and colcemid-block. Mutagenesis (2007) 22:359–362.[Abstract/Free Full Text]

    28. Terzoudi G, Manola KN, Pantelias GE, Iliakis G. Checkpoint abrogation in G2 compromises repair of chromosomal breaks in ataxia telangiectasia cells. Cancer Res. (2005) 65:11292–11296.[Abstract/Free Full Text]

    29. Bryant PE, Gray L, Riches AC, et al. The G2 assay: a technical report. Int. J. Radiat. Biol. (2002) 78:863–866.[CrossRef][Web of Science][Medline]

Received on December 5, 2007; revised on January 17, 2008; accepted on January 25, 2008.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
23/4/267    most recent
gen009v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Shovman, O.
Right arrow Articles by Bryant, P. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shovman, O.
Right arrow Articles by Bryant, P. E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?