Mutagenesis vol. 19 no. 6 © UK Environmental Mutagen Society 2004; all rights reserved.
The use of IL-2 cultures to measure chromosomal radiosensitivity in breast cancer patients
Department of Anatomy, Embryology, Histology and Medical Physics and 1Department of Radiopharmacy, Ghent University, L. Pasteurlaan 2, 9000 Gent, Belgium and 2Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Gent, Belgium
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Enhanced chromosomal radiosensitivity in breast cancer patients has been demonstrated in several studies. To investigate the chromosomal radiosensitivity of lymphocytes in breast cancer patients the G2 and micronucleus (MN) assays are often used. In these assays blood samples are exposed to ionizing radiation and the number of radiation-induced micronuclei or chromatid breaks are scored. In most studies investigating the in vitro chromosomal radiosensitivity of breast cancer patients the G2 and MN assays were performed on freshly drawn blood. The disadvantage of working with fresh blood samples is that in most cases only one blood sample can be obtained and that the assay cannot be easily repeated without further blood sampling. To allow repeated testing we propose the use of long-term cultures of T lymphocytes (IL-2 cultures). In this study we therefore investigated whether the radiation-induced MN response in IL-2 cultures was the same as in concordant whole blood cultures. For this study the MN assay (2 Gy) was performed on IL-2 cultures of 11 sensitive breast cancer patients and 20 healthy women. The results demonstrate that the enhanced chromosomal radiosensitivity observed in whole blood cultures of breast cancer patients is not present in IL-2 cultures derived from the same blood samples. Therefore, care has to be taken when IL-2 cultures are used to assess chromosomal radiosensitivity in breast cancer patients.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Ionizing radiation induces chromosomal damage. Enhanced chromosomal radiosensitivity has not only been demonstrated in a large number of patients with cancer-prone genetic diseases, e.g. ataxia telangiectasia (reviewed in Scott et al., 1999
), but also in significant proportions of breast, colorectal and head and neck cancer patients (Scott et al., 1994; Baria et al., 2001; Papworth et al., 2001). The enhanced chromosomal radiosensitivity in breast cancer patients has been confirmed in several independent studies (Scott et al., 1994, 1998, 1999; Jones et al., 1995; Parshad et al., 1996; Patel et al., 1997; Terzoudi et al., 2000; Baria et al., 2001; Riches et al., 2001; Baeyens et al., 2002). To investigate the chromosomal radiosensitivity of lymphocytes in cancer patients the G2 and micronucleus (MN) assays are often used. The MN assay has the advantage that it is less time consuming and requires fewer cytogenetic skills than the analysis of metaphase chromosomes. In the MN assay blood samples are exposed to ionizing radiation and the number of radiation-induced micronuclei is scored in 1000 binucleate lymphocytes. In most studies investigating the in vitro chromosomal radiosensitivity of breast cancer patients the MN assay has been performed on freshly drawn blood. The disadvantage of using fresh blood samples is that the assay cannot be repeated without further blood sampling. To allow repeated testing the use of Epstein–Barr virus (EBV)-transformed cell lines derived from the original blood samples could offer a solution. In a previous study we investigated the use of EBV-transformed cell lines to measure chromosomal radiosensitivity, by means of the MN assay, in breast cancer patients. Our results, however demonstrate, that the enhanced chromosomal radiosensitivity observed in fresh blood cultures of breast cancer patients is not present in EBV-transformed cell lines derived from the same blood samples (Baeyens et al., 2004). Therefore, EBV-transformed cell lines are not a suitable MN culture system for evaluating chromosomal radiosensitivity in breast cancer patients. Another alternative to obtain a large pool of cells to allow repeated testing is the use of long-term cultures of T lymphocytes. A simplified method, using recombinant interleukin-2 (IL-2), fetal bovine serum and freeze-killed feeder cells has been described by Donovan et al. (1995) for the mass culture of T lymphocytes derived from human peripheral blood. Donovan et al. (1995) further showed that under these culture conditions the lymphocytes maintained their normal karyotype and were potentially valuable in genotoxicity testing. These lymphocyte cultures have the further advantage that they represent primary cell cultures of T lymphocytes, while EBV cell cultures represent virus-transformed B lymphocytes.
In this study we have investigated the response to ionizing radiation in long-term T lymphocyte cultures as well as in concordant samples of resting peripheral blood lymphocytes. First, the methodology for long-term T lymphocyte culture was optimized. The MN assay was then performed on long-term T lymphocyte cultures of breast cancer patients and a group of healthy women. We selected those breast cancer patients which showed an elevated chromosomal radiosensitivity in fresh blood samples in a previous study with the 3.5 Gy MN assay (Baeyens et al., 2002).
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Blood samples
Blood samples were obtained from 20 healthy women showing a normal response and 11 familial breast cancer patients showing a high MN response, as described in our previous study (Baeyens et al., 2002
). The breast cancer patients were not carriers of BRCA1/2 mutations. All patients and healthy women signed informed consent forms.
Optimization of long-term T lymphocyte cultures
Preliminary work (unpublished data) on long-term T lymphocyte cultures showed that the use of freeze-killed or radiation-killed feeder cells was not necessary to obtain a large pool of T lymphocytes. Good proliferation was obtained just by adding IL-2 (human, recombinant IL-2; Roche, Brussels, Belgium) to the culture each time the culture medium was changed. By this procedure we obtained, after 7 days culture, 10–20 x 106 cells starting from 2 x 106 cells (Trypan blue staining). This expansion of a lymphocyte population can be repeated three times when IL-2 cells are frozen every 7 days (experimental set-up B).
Flow cytometric analysis of lymphocyte subsets was done on fresh blood samples and long-term T lymphocyte cultures from four healthy donors. Two experimental set-ups were used for the long-term cultures (Table I). In set-up A the IL-2-stimulated lymphocytes were cultured continuously for 21 days. Flow cytometric analysis was performed on days 7, 14 and 21. In set-up B the IL-2-stimulated lymphocytes were also cultured for 21 days in total, but every 7 days they were frozen (see Optimized IL-2 culture protocol), so as to investigate the influence of cryopreservation on lymphocytes subsets. Here flow cytometric analysis was performed every seventh day of culture. Lymphocyte surface antigen markers were analysed using a FACScan (Becton Dickinson) flow cytometer detecting fluorescein isothiocyanate (FITC), R-phycoerythrin (PE) and peridinin–chlorophyll (PerCP) fluorescence. The antibodies used to identify the subsets of T lymphocytes were as follows: CD4/FITC (Beckman Coulter, Mijdrecht, The Netherlands); CD8/PE, CD25/PE, CD56/PE and CD3/PerCP (Becton Dickinson, Erembodegem, Belgium). The following combinations were performed: CD3/CD4; CD3/CD8; CD4/CD8; CD3/CD25, CD3/CD56.
|
For the fresh blood samples 50 µl of blood was stained with the appropriate antibodies for 15 min at room temperature in darkness. After the incubation period the erythrocytes were lysed as indicated in the instructions for the DakoCytomation, Utilyse and Erythrocytes lysing reagents (Heverlee, Belgium). For the IL-2-stimulated lymphocytes 105 cells were stained as described before. Thereafter, the cell suspensions were washed with phosphate-buffered saline and subsequently analysed with a flow cytometer. The results of the analysis with the flow cytometer are presented in Table I. The table shows that experimental set-up B, in which the IL-2-stimulated lymphocytes were frozen every 7 days, gave the best results. Almost all cells were CD3+/CD4+ or CD3+/CD8+. Also, the percentage of activated CD3+/CD25+ cells, expressing IL-2 receptors, remained high. The culture conditions of experimental set-up A resulted in a significant increase in the number of natural killer cells (CD3–/CD56+) and a significant decrease in the number of activated T lymphocytes with increasing culture time. The increase in CD3–/CD56+ cells can be explained by the ‘LAK’ (lymphokine-activated killer) effect, which is induced by IL-2 (Trinchieri et al., 1984
; Phillips et al., 1986). This effect is disturbed when cells are frozen. As the lymphocyte subsets present in set-up B more closely resemble the lymphocyte subsets in phytohaemaglutinin-stimulated peripheral blood cultures, these culture conditions were further used for application of the MN assay.
Optimized IL-2 culture protocol
Lymphocytes were isolated and stimulated with phytohaemaglutinin-P solution (PHA-P solution; Difco Biotrading) and IL-2 in 12-well plates. The IL-2-treated lymphocytes were cultured at 37°C in 5% CO2 in RPMI 1640 medium (Gibco Invitrogen, Merelbeke, Belgium) supplemented with 10% foetal calf serum (Gibco Invitrogen), 0.05% L-glutamine (Gibco Invitrogen), penicillin/streptomycin (50 U/ml penicillin, 50 µg/ml streptomycin; Gibco Invitrogen), sodium pyruvate (Gibco Invitrogen), ß-mercaptoethanol (Gibco Invitrogen) and 100 µl/2 ml IL-2. The cells formed clusters and were not disturbed before day 3. From then on the cells were split every 2 days, extra medium was added and on day 7 the IL-2 cultures were frozen (5 x 106 cells). After 3 days the cryopreserved cells were quickly thawed and seeded at 106 cells/ml culture medium in 12-well plates. The same procedure as in week 1 was repeated. After 2 weeks culture and two rounds of freezing of the cells, the IL-2 cell lines were thawed, seeded at 106 cells/ml in 12-well plates and again stimulated with phytohaemaglutinin and IL-2. On day 2 the cells were transferred to tissue culture flasks (25 cm2) (Nunc, Leuven, Belgium) and the MN assay was performed.
IL-2 cultures were prepared from 31 peripheral blood samples (20 healthy women and 11 breast cancer patients).
The G0 MN assay for whole blood cultures
For the blood samples the MN protocol described by Baeyens et al. (2002) was applied with some minor changes. Briefly, 0.5 ml of heparinized blood was diluted in 1.5 ml of preheated (37°C) complete culture medium consisting of RPMI 1640 medium supplemented with 10% foetal calf serum and 0.05% L-glutamine. The cultures were irradiated with 2 Gy 60Co 
-rays at a high dose rate (1 Gy/min) or sham-irradiated at 37°C (Baeyens et al., 2002; Vral et al., 2002). Immediately after irradiation the cultures were further diluted with complete medium to a final volume of 5 ml and 20 µl of 1% PHA-P solution was added to stimulate the T lymphocytes. Twenty-four hours after irradiation 6 µg/ml cytochalasin B (Sigma-Aldrich) was added to block cytokinesis. Cells were harvested 70 h after stimulation. Fixed cell suspensions of fresh blood samples were dropped onto clean slides and stained with 6% Romanowsky–Giemsa (80 ml Azur B + 20 ml Eosin E; Serva, Zandhoven, Belgium) in HEPES buffer for 20 min. All slides were made in duplicate, coded and MN were scored in binucleate cells (BN) (light microscopy, 400x) according to the criteria of Fenech (1993). All the analyses were performed by two scorers. For all samples scorer 1 analysed 500 BN on slide 1 while scorer 2 analysed 500 BN on slide two. No significant differences between the scorers were observed using a paired t-test (P > 0.05).
The G1/S/G2 MN assay for IL-2 cultures
The MN assay protocol for EBV-transformed cell lines was used (Baeyens et al., 2004). In brief, 1 ml of cell suspension (1 x 106 cells/ml containing cells in different stages of the cell cycle) was diluted in 1 ml of complete medium in tissue culture flasks 24 h before irradiation and kept at 37°C in 5% CO2. The cultures were irradiated at 37°C with 2 Gy 60Co -rays at high dose rate (1 Gy/min). A dose of 2 Gy also resulted in a good BN index for IL-2 cultures. From each culture one tube was also sham-irradiated. Immediately after irradiation 6 µg/ml cytochalasin B was added and the culture flasks returned to the incubator. The cells were harvested 48 h after irradiation by a cold (4°C) hypotonic shock with 3 ml of 0.075 M KCl, followed by fixation in methanol:acetic acid:Ringer (0.9% NaCl) solution (12:1:13). A culture time of 48 h post-irradiation (instead of 24 h) was chosen because this resulted in a better BN index. The cells were stored overnight in a refrigerator (4°C) and then fixed another three times with methanol:acetic acid (12:1). Fixed cell suspensions were dropped onto clean slides and stained with 6% Romanowsky–Giemsa in HEPES buffer for 20 min. All slides were made in duplicate, coded and MN were scored in BN (light microscopy, 400x) as described earlier. All the analyses were performed by two scorers. No significant differences between the scorers were observed using a paired t-test (P > 0.05).
Cell cycle analysis
For three IL-2 cultures derived from three healthy individuals two cultures (2 x 106 cells/ml) were set up. The cells of the first culture were harvested just before irradiation while the cells of the second culture were harvested 48 h post-irradiation (2 Gy). For every culture, 300 µl of cell suspension was stained with 3 µl of propidium iodide (Sigma) and immediately frozen in liquid nitrogen. Immediately after thawing the samples were analysed with a FACScan flow cytometer (Becton Dickinson); the percentages of cells in the G0/G1, S and G2/M phases were calculated.
Comparison between whole blood cultures, isolated lymphocyte cultures and IL-2 cultures
In our group of healthy donors we observed one donor with high MN scores and identified this donor as ‘radiosensitive’. As we had the possibility of obtaining more blood samples from the healthy donors, we repeated the G0 MN assay five times for this donor and twice for the other healthy donors, with normal MN responses, to investigate the reproducibility of the higher MN scores. As each time we were able to obtain enough blood, lymphocytes were isolated and the MN assay was also performed on isolated peripheral blood lymphocytes and IL-2 cultures derived from the blood samples of the ‘radiosensitive’ and the two ‘non-sensitive’ healthy donors. The MN protocol for isolated lymphocytes was comparable with the MN protocol for IL-2 cultures. The only difference was that after isolation (lymphoprep) lymphocytes were cultured (2 x 106 cells/ml) in 14 ml of culture tubes.
Binucleation yield (BN) and nuclear division index (NDI)
Five hundred cells per slide were scored to evaluate the percentages of mononucleate, binucleate and polynucleate cells in whole blood cultures and IL-2 cultures of eight healthy women and five breast cancer patients. The BN yield was also scored in isolated lymphocyte cultures of three healthy donors. The NDI is calculated according to the formula: NDI = [(M1) + (2 x M2) + (3 x M3) + (4 x M4)]/n, where M1–M4 indicate the number of cells with 1–4 nuclei and n is the total number of cells scored.
Statistical analysis
For comparison of cell numbers in the different phases of the cell cycle the Wilcoxon test was applied. To compare the MN responses, percentage BN and NDI, the Mann–Whitney test was used. This statistical test was used as it is a non-parametric, distribution-free test that is suitable to compare groups with small sample sizes where no underlying distribution can be assumed.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Comparison of the MN response in whole blood cultures and IL-2 cultures
The results of the MN assay for the whole blood and IL-2 cultures from breast cancer patients and the healthy donors are summarized in Table II and presented in Figure 1. The mean spontaneous frequency of MN in whole blood cultures of the patients (24 MN/1000 BN) was not significantly different from that of the healthy donors (23 MN/1000 BN) (Mann–Whitney test, P > 0.05). The difference in mean spontaneous MN in the IL-2 cultures of the patients (17 MN/1000 BN) and the healthy donors (14 MN/1000 BN) was also not significant (Mann–Whitney test, P > 0.05). When we compared the number of spontaneous micronuclei between the two culture systems, a significant difference was observed with spontaneous MN frequencies being lower in the IL-2 cultures compared with whole blood cultures (Mann–Whitney test, P = 0.012).
|
|
For each sample the spontaneous yield was subtracted from the yield in irradiated cells to give the radiation-induced yield. For whole blood cultures the results for patients were significantly higher compared with healthy donors at a dose of 2 Gy (431 ± 41 versus 329 ± 51 MN/1000 BN, P=0.001). However, for the IL-2 cultures there was no significant difference between the cultures from patients and healthy donors after 2 Gy in vitro irradiation (411 ± 85 and 408 ± 65 MN/1000 BN, Mann–Whitney test, P = 0.971). Comparison of both culture systems shows that the MN response obtained in IL-2 cultures from healthy donors was higher than that obtained in whole blood cultures from healthy donors. For patients there was no difference in MN response between the two culture systems.
Investigation of the inter-individual variability in radiation-induced MN yields (2 Gy) showed that the coefficients of variation (CV) (CV% = SD/mean x 100) obtained for the different IL-2 cultures of healthy donors (16%) and patients (21%) were in the same range as that obtained for whole blood cultures from the healthy individuals (15%) and patients (9%) (Table II).
Cell cycle analysis of IL-2 cultures
The mean percentages of cells in the different phases of the cell cycle are presented in Figure 2. We could not observe a difference between the pre- and post-irradiation conditions for cells in the G0/G1 phases. The number of cells in S phase was clearly, although not significantly (Wilcoxon test, P = 0.1), decreased after irradiation. In contrast, the number of G2/M phase cells was increased post-irradiation, due to G2/M arrest after irradiation (Hwang et al., 1998). This increase was not significant (Wilcoxon test, P = 0.1).
|
Comparison of the MN response in whole blood cultures, isolated lymphocyte cultures and IL-2 cultures of one ‘radiosensitive’ and two ‘normal responding’ donors
Figure 3 shows a comparison of the mean MN scores obtained for three healthy donors using three different culture systems: whole blood cultures, isolated lymphocyte cultures and IL-2 cultures. The differences in mean MN response between the ‘radiosensitive’ and ‘non-radiosensitive’ donors were significant in whole blood cultures and in isolated lymphocyte cultures (Mann–Whitney test, P = 0.008 and P = 0.015). The mean MN responses in IL-2 cultures of the three donors were clearly not significantly different (Mann–Whitney, P > 0.05).
|
Binucleation yield and nuclear division index (NDI)
The percentages of mononucleate, binucleate and polynucleate cells together with the NDI obtained for whole blood samples, IL-2 cultures and isolated lymphocyte cultures are shown in Table III. The mean values obtained for BN cell yield and NDI (Table III) were significantly higher in the whole blood samples compared with the IL-2 and isolated lymphocyte cultures (Mann–WhitneyBNyield test, P < 0.001; Mann–WhitneyNDI test, P = 0.002). Variation analysis further showed that the CV of the mean values for BN yield and NDI (Table III) lie in the same range for the whole blood samples, the IL-2 cultures and the isolated lymphocyte cultures.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Literature data reporting on the use of long-term T lymphocyte cultures to measure chromosomal radiosensitivity are not available. Only in a study by Donovan et al. (1995)
has the use of long-term T lymphocyte cultures been described as an alternative to primary lymphocytes for use in genotoxicity testing. They demonstrated that MN induction by the chemical agent hycanthone in long-term lymphocyte cultures of five donors showed a similar dose–response relationship. The advantage of using long-term cultures of T lymphocytes are multiple. Compared with whole blood or isolated lymphocyte cultures: (i) they present a much simpler system comprising only T lymphocytes; (ii) a large bulk of cells can be obtained and frozen until use. In comparison with EBV-transformed cell lines they have the advantage that: (i) they are not virus transformed and possess the normal human karyotype; (2) they are easy and safer to handle.
In our study the methodology for long-term lymphocyte cultures of Donovan et al. (1995) was adapted and optimized. A bulk of proliferating T lymphocytes was obtained by phytohaemaglutinin and IL-2 stimulation. Freeze-killed feeder cells were not needed. By cryopreservation of IL-2 cultures every 7 days of culture we obtained a very homogeneous population of T lymphocytes that were CD3+/CD4+, CD3+/CD8+ and CD3+/CD25+.
Although the use of IL-2 cultures looks suitable and may have practical advantages compared with whole blood cultures, they should fulfil our requirements with respect to sensitivity and reproducibility before they can be used in chromosomal radiosensitivity testing. To this end we investigated chromosomal radiosensitivity by the MN assay in IL-2 cultures derived from blood samples of healthy women and breast cancer patients showing an enhanced radiosensitivity after in vitro irradiation of the blood samples with 3.5 as well as 2 Gy irradiation doses.
The number of spontaneous MN was significantly lower in the IL-2 cultures than the whole blood cultures. This may indicate that the IL-2 cultures do not display increased chromosomal instability. In contrast, the mean number of radiation-induced (2 Gy) MN was higher in the IL-2 cultures of healthy donors than in the concordant whole blood cultures (Figure 2). A higher MN yield could be explained by the fact that in our IL-2 cultures the CD4+:CD8+ cells ratio at the moment of irradiation was different from that in G0 irradiated lymphocytes. From the literature it is known that different lymphocyte subsets may vary in their sensitivity to ionizing radiation (Philippé et al., 1997).
Our results further demonstrate that the enhanced chromosomal radiosensitivity observed in whole blood cultures of breast cancer patients is not present in IL-2 cultures derived from the same blood samples (Figure 2). The significant difference in radiosensitivity observed between whole blood cultures of breast cancer patients and healthy women completely disappears in concordant IL-2 cultures. Disappearance of chromosomal radiosensitivity was also observed in a previous study in which we investigated the use of EBV-transformed cell lines to measure chromosomal radiosensitivity in breast cancer patients (Baeyens et al., 2004).
Variance analysis revealed that there is no significant difference in the inter-individual variability in MN formation between IL-2 cultures and whole blood cultures. This is not in agreement with our findings for EBV-transformed cell lines, which were characterized by very high variability in MN formation between cell lines from different donors. This high variability, which was proposed as a possible explanation for the disappearance of a radiosensitive response in EBV-transformed cell lines of breast cancer patients, can, however, not be responsible for disappearance of a radiosensitive response in IL-2 cultures.
A possible explanation for the different radiation responses could be related to the fact that in our whole blood culture system the cells were irradiated in G0 phase of the cell cycle, while in the IL-2 culture system cycling cells were irradiated. Dependent on the cell cycle phase, cells have different sensitivities to ionizing radiation. This cell cycle phase-dependent difference in chromosomal radiosensitivity could be partly due to differential use of two DNA double-strand break repair pathways: non-homologous end joining (NHEJ), which is preferentially used in G0-G1 cells and homologous recombination (HR) which is preferentially used in the S and G2 phases of the cell cycle (Kanaar et al., 1998; Takata et al., 1998; Rothkamm et al., 2001). If the sensitive breast cancer patients included in our study had a defect in NHEJ, which is the main repair system used in resting lymphocytes, the outcome of this defect may be less pronounced when cycling cells, which contain G1, S and G2 phase cells, are exposed to ionizing radiation.
Besides cell cycle differences in the G0 and G1/S/G2 MN assays, the differential response observed between G0 lymphocytes and IL-2 cultures may also be related to the fact that G0 lymphocytes are exposed as whole blood cultures while IL-2 cultures are exposed only in medium. The composition and antioxidant status of the blood of patients and controls may affect the radiation response observed in whole blood cultures but not in IL-2 cultures.
As we had the opportunity to obtain repeated blood samples from our healthy donor population, we investigated the radiation response of G0 lymphocytes in whole blood cultures, isolated lymphocyte cultures and G1/S/G2 cells in IL-2 cultures. Three donors were selected based on their MN score in the first experimental set-up, one with a ‘high’ MN score and two with ‘normal’ MN scores. Figure 3 shows that the chromosomal radiosensitive response observed in one donor, based on results obtained for five repeated blood samples, is also present in the concordant isolated lymphocyte cultures. However, in IL-2 cultures this radiosensitive response disappeared. These results support the fact that chromosomal radiosensitivity is a characteristic of G0 lymphocytes. The observation that the radiosensitive response is more pronounced in whole blood cultures compared with isolated lymphocyte cultures may indicate that components in the blood may also partly affect the response of G0 lymphocytes in whole blood cultures.
In conclusion, the results of this study demonstrate that the enhanced chromosomal radiosensitivity observed in whole blood cultures of breast cancer patients is not present in IL-2 cultures derived from the same blood samples. Therefore, care must be taken when IL-2 cultures are used to investigate chromosomal radiosensitivity in breast cancer patients.
|
Acknowledgments |
|---|
We wish to thank all the patients and control donors who participated in this study. The authors thank V.de Gelder, N.De Temmerman and D.Van Raemdonck for technical assistance. This work was supported by the Program of Scientific Support to the Protection of Workers in the Area of Health of the Prime Minister's Office of the Belgian Government–Federal Office for Scientific, Technical and Cultural Affaires (DWTC), by a grant from the Bijzonder Onderzoeksfonds (Gent University, no. 01109501) and by a grant from the Fonds voor Wetenschappelijk Onderzoek Vlaanderen Grant (G.0050.01).
|
Notes |
|---|
3 To whom correspondence should be addressed. Tel: +32 9 264 92 48; Fax: +32 9 264 94 98; Email: anne.vral{at}ugent.be
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Baeyens,A., Thierens,H., Claes,K., Poppe,B., Messiaen,L., De Ridder,L. and Vral,A. (2002) Chromosomal radiosensitivity in breast cancer patients with a known or putative genetic predisposition. Br. J. Cancer, 87, 1379–1385.[CrossRef][Web of Science][Medline]
Baeyens,A., Thierens,H., Vandenbulcke,K., De Ridder,L. and Vral,A. (2004) The use of EBV-transformed cell lines of breast cancer patients to measure chromosomal radiosensitivity. Mutagenesis, 19, 285–290.
Baria,K., Warren,C., Roberts,S.A., West,C.M. and Scott,D. (2001) Chromosomal radiosensitivity as a marker of predisposition to common cancers? Br. J. Cancer, 84, 892–896.[CrossRef][Web of Science][Medline]
Donovan,M.R.O., Freemantle,M.R., Hull,G., Bell,D.A., Arlett,C.F. and Cole,J. (1995) Extended-term cultures of human T-lymphocytes: a practical alternative to primary human lymphocytes for use in genotoxicity testing. Mutagenesis, 10, 189–201.
Fenech,M. (1993) The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations. Mutat. Res., 285, 35–44.[Web of Science][Medline]
Hwang,A. and Muschel,R.J. (1998) Radiation and the G2 phase of the cell cycle. Radiat. Res., 150, 52–59.[Web of Science][Medline]
Jones,L.A., Scott,D., Cowan,R. and Roberts,S.A. (1995) Abnormal radiosensitivity of lymphocytes from breast cancer patients with excessive normal tissue damage after radiotherapy: chromosome aberrations after low dose-rate irradiation. Int. J. Radiat. Biol., 67, 519–528.[Web of Science][Medline]
Kanaar,R., Hoeijmakers,J.H.J. and Van Gent,D.C. (1998) Molecular mechanisms of DNA double-strand break repair. Cell Biol., 8, 483–489.
Papworth,R., Slevin,N., Roberts,S.A. and Scott,D. (2001) Sensitivity to radiation-induced chromosome damage may be a marker of genetic predisposition in young head and neck cancer patients. Br. J. Cancer, 84, 776–782.[CrossRef][Web of Science][Medline]
Parshad,R., Price,F.M., Bohr,V.A., Cowans,K.H., Zujewski,J.A. and Sanford,K.K. (1996) Deficient DNA repair capacity, a predisposing factor in breast cancer. Br. J. Cancer, 74, 1–5.[Web of Science][Medline]
Patel,R.K., Trivedi,A.H., Arora,D.C., Bhatavdekar,J.M. and Patel,D.D. (1997) DNA repair proficiency in breast cancer patients and their first-degree relatives. Int. J. Cancer, 73, 20–24.[CrossRef][Web of Science][Medline]
Philippé,J., Louagie,H., Thierens,H., Vral,A., Cornelissen,M. and De Ridder,L. (1997) Quantification of apoptosis in lymphocyte subsets and effect of apoptosis on apparent expression of membrane antigens. Cytometry, 29, 242–249.[CrossRef][Web of Science][Medline]
Phillips,J.H. and Lanier,L.L. (1986) Dissection of the lymphokine-activated killer phenomenon. J. Exp. Med., 164, 814–825.
Riches,A.C., Bryant,P.E., Steel,C.M., Gleig,A., Robertson,A.J., Preece,P.E. and Thompson,A.M. (2001) Chromosomal radiosensitivity in G2-phase lymphocytes identifies breast cancer patients with distinctive tumour characteristics. Br. J. Cancer, 85, 1157–1161.[CrossRef][Web of Science][Medline]
Rothkamm,K., Kühne,M.O., Jeggo,P.A. and Löbrich,M. (2001) Radiation-induced genomic rearrangements formed by nonhomologous end-joining of DNA double-strand breaks. Cancer Res., 61, 3886–3893.
Scott,D., Spreadborough,A., Levine,E.L. and Roberts,S.A. (1994) Genetic predisposition in breast cancer. Lancet, 344, 1444.[Web of Science][Medline]
Scott,D., Barber,J.B.P., Levine,E.L., Burrill,W. and Roberts,S.A. (1998) Radiation-induced micronucleus induction in lymphocytes identifies a high frequency of radiosensitive cases among breast cancer patients: a test for predisposition? Br. J. Cancer, 77, 614–620.[Web of Science][Medline]
Scott,D., Barber,J.B.P., Spreadborough,A.R., Burrill,W. and Roberts,S.A. (1999) Increased chromosomal radiosensitivity in breast cancer patients: a comparison of two assays. Int. J. Radiat. Biol., 75, 1–10.[CrossRef][Web of Science][Medline]
Takata,M., Sasaki,M.S., Sonoda,E., Morrison,C., Hashimoto,M., Utsumi,H., Yamaguchi-Iwai,Y., Shinohara,A. and Takeda,S. (1998) Homologous recombination and nonhomologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J., 17, 5497–5508.[CrossRef][Web of Science][Medline]
Terzoudi,G.I., Jung,T., Hain,J., Vrouvas,J., Margaritis,K., Donta-Bakoyiannis,C., Makropoulos,V., Angelakis,P.H. and Pantelias,G.E. (2000) Increased chromosomal radiosensitivity in cancer patients: the role of cdk1/cyclin-B activity level in the mechanisms involved. Int. J. Radiat. Biol., 76, 607–615.[CrossRef][Web of Science][Medline]
Trinchieri,G., Matsumoto-Kobayashi,M., Clark,S.C., Seehra,J., London,L. and Perussia,B. (1984) Response of resting human peripheral blood natural killer cells to interleukin 2. J. Exp. Med., 160, 1147–1169.
Vral,A., Thierens,H., Baeyens,A. and De Ridder,L. (2002) The micronucleus and G2-phase assays for human blood lymphocytes as biomarkers of individual sensitivity to ionising radiation: limitations imposed by intraindividual variability. Radiat. Res., 157, 472–477.[CrossRef][Web of Science][Medline]
Received on August 5, 2004; accepted on October 18, 2004.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Beetstra, C. Salisbury, J. Turner, M. Altree, R. McKinnon, G. Suthers, and M. Fenech Lymphocytes of BRCA1 and BRCA2 germ-line mutation carriers, with or without breast cancer, are not abnormally sensitive to the chromosome damaging effect of moderate folate deficiency Carcinogenesis, March 1, 2006; 27(3): 517 - 524. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||