Mutagenesis vol. 19 no. 4 pp. 285-290,
July 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
The use of EBV-transformed cell lines of breast cancer patients to measure chromosomal radiosensitivity
1Department of Anatomy, Embryology, Histology and Medical Physics, University of Gent, L. Pasteurlaan 2, B-9000 Gent, Belgium and 2Department of Radiopharmacy, University of Gent, Harelbekestraat 72, B-9000 Gent, Belgium
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
To investigate the chromosomal radiosensitivity of lymphocytes in cancer patients the micronucleus (MN) assay is often used and performed on freshly drawn peripheral blood lymphocytes. The use of Epstein–Barr virus (EBV)-transformed lymphoblastoid cell lines may have a lot of advantages (e.g. large pool of cells) compared with fresh blood samples. In this study we have investigated whether the response of EBV-transformed lymphoblastoid cell lines to irradiation in the G1/S/G2 phases of the cell cycle is the same as in concordant whole blood cultures where primary lymphocytes were irradiated in the G0 phase of the cell cycle. For this study the MN assay (2 Gy) was performed on EBV-transformed cell lines of breast cancer patients and a group of healthy women. Those breast cancer patients were selected who showed an elevated chromosomal radiosensitivity in fresh blood samples in a previous study. The results demonstrated 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. Therefore, care must be taken when EBV cell lines are used to assess chromosomal radiosensitivity in breast cancer patients.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Enhanced chromosomal radiosensitivity has been observed in a large number of patients with cancer prone genetic diseases such as ataxia telangiectasia (AT), Nijmegen breakage syndrome and hereditary retinoblastoma (Sanford et al., 1989
; reviewed in Scott et al., 1999). More recently it has also been observed in a significant proportion of patients with breast cancer, colorectal cancer and head and neck cancer (Scott et al., 1994; Baria et al., 2001; Papworth et al., 2001). For breast cancer the enhanced chromosomal radiosensitivity 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), with the G2 assay as well as with the micronucleus (MN) assay for peripheral blood lymphocytes (PBL). In the G2 assay chromatid breaks are analysed in metaphase cells that are irradiated during the G2 phase of the cell cycle. In the MN assay the cells are irradiated in G0 and then stimulated into division and MN are scored in binucleate cells (BN) resulting from cytokinesis block. However, no significant correlation between G2 and G0 radiosensitivity was observed in cancer patients (Scott et al., 1999; Baeyens et al., 2002). The link between breast cancer predisposition and chromosomal radiosensitivity observed with these cytogenetic techniques has led to the suggestion that these assays may be used as a biomarker for increased cancer risk. The enhanced chromosomal sensitivity observed with the MN and G2 assays may be a consequence of differences in DNA repair capacity due to mutations or polymorphisms in genes involved in the processing of DNA damage. Further studies investigating the expression of DNA repair genes in breast cancer patients showing elevated chromosomal radiosensitivity are needed to further unravel the underlying mechanisms of chromosomal radiosensitivity and breast cancer predisposition.
Until now most studies investigating the in vitro chromosomal radiosensitivity of breast cancer patients have been performed on freshly drawn PBL. The use of fresh blood as a biological test system has the disadvantage that in most cases only one blood sample can be obtained, which does not allow repeated testing nor the application of additional molecular techniques such as microarray and real-time PCR on the same sample. A large pool of cells could be obtained by working with Epstein–Barr virus (EBV)-transformed lymphoblastoid cell lines derived from the original blood samples. As EBV-transformed lymphoblastoid cells originate from a different subpopulation of lymphocytes in the peripheral blood, i.e. B lymphocytes, and are exposed in the G1/S/G2 phase of the cell cycle, differences in radiation response could be expected when compared with the response obtained in stimulated T lymphocytes irradiated in G0. So, before using EBV-transformed lymphoblastoid cell lines in these kind of studies it is important to determine whether the chromosomal radiosensitivity observed in PBL is also expressed in EBV-transformed cell lines. EBV-transformed lymphoblastoid cell lines have been widely used to study chromosomal radiosensitivity in AT patients (Antoccia et al., 1994; Liu et al., 1997). More recently they were also used to test chromosomal radiosensitivity in BRCA1/2 heterozygotes (Foray et al., 1999; Trenz et al., 2002). The results obtained were, however, contradictory. Moreover, in none of these studies was a direct comparison made between the response obtained in a fresh blood sample from breast cancer patients and the response obtained in an EBV-transformed cell line derived from that blood sample.
In our study we wanted to investigate whether the response of an EBV-transformed lymphoblastoid cell line is the same as in the concordant sample of resting PBL. For this study the MN assay was performed on EBV-transformed cycling lymphoblastoid cells of breast cancer patients and a group of healthy women. Those breast cancer patients were selected 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 obtained from 12 healthy women showing a normal MN response and 8 familial breast cancer patients showing a high MN response as described in our previous paper (Baeyens et al., 2002
) were included in this study. None of the familial breast cancer patients were carriers of a BRCA1 or BRCA2 mutation.
Cell lines
Lymphocytes were isolated from the 20 peripheral blood samples (12 healthy women and 8 breast cancer patients) and immortalized in our laboratory with the supernatant of the EBV-producing cell line B95-8 using a standard protocol (Neitzel, 1986). None of the cell lines carried a mutation in BRCA1 or BRCA2. Two EBV-transformed lymphoblastoid cell lines (AT-KM and NC) were obtained from P.Bryant (University of St Andrews, UK). The AT-KM cell line, used as a positive control in our study, carried a homozygous mutation of the ATM gene. The NC cell line was derived from a healthy individual. One cell line (P720) was obtained from the Medical Genetics Department, University Hospital Gent, Belgium. This cell line was established from PBL from a breast cancer patient. Neither cell line (NC or P720) had mutations in the BRCA1, BRCA2 and ATM genes.
The lymphoblastoid cells were cultured at 37°C in 5% CO2 in RPMI 1640 medium (Gibco) supplemented with 10% foetal calf serum (Gibco), 0.05% L-glutamine, penicillin, streptomycin, sodium pyruvate and ß-mercaptoethanol. At the time of the MN assays these cell lines had been in culture for less than 2 months.
The G0 MN assay
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 (Life Technologies) supplemented with 10% foetal calf serum (Life Technologies) and 0.05% L-glutamine (Sigma-Aldrich). The cultures were irradiated with 2 Gy 60Co 
-rays at a high dose rate (HDR) (1 Gy/min) or sham-irradiated at 37°C (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% phytohaemaglutinin-P solution (Difco Biotrading) was added to stimulate the T lymphocytes. Twenty-four hours post-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 on 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) 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 2. No significant differences between the scorers were observed using a paired t-test (P > 0.05).
The G1/S/G2 MN assay
The protocol for the MN assay for EBV-transformed cell lines is very similar to the protocol for the G0 MN assay for fresh blood samples. In brief, 1 ml (1 x 106 cells/ml) of cell suspension (lymphoblastoid cells at different stages of the cell cycle) was diluted in 1 ml of complete medium in centrifuge tubes 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 HDR (1 Gy/min). The choice of dose was the result of the optimization procedure for the MN assay for EBV-transformed cell lines. A dose of 3.5 Gy, as used for the irradiation of fresh blood samples, resulted in a very low BN index and MN values reaching a plateau level. A dose of 2 Gy, in contrast, resulted in a good BN index and induction of MN following a linear quadratic dose–response effect. These findings were recently confirmed by the group of Gutiérrez-Enriquez and Hall (2003).
From each cell line one tube was also sham-irradiated. Immediately after irradiation, 6 µg/ml cytochalasin B (Sigma-Aldrich) was added and the tubes were returned to the incubator. The cells were harvested 48 h post-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 (33% BN after 48 h compared with 12% BN after 24 h; n = 5). The cells were stored overnight in the 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) according to the criteria of Fenech (1993). All the analyses were performed by two scorers. No significant differences between the scorers were observed using a paired t-test (P > 0.05).
Binucleation yield and nuclear division index (NDI)
500 cells per slide were scored to evaluate the percentages of mononucleate, binucleate and polynucleate cells in fresh blood cultures and EBV-transformed cell cultures of five healthy women and five breast cancer patients. The NDI is calculated according to the formula: NDI = (M1 + 2M2 + 3M3 + 4M4)/N, where M1–M4 indicate the number of cells with 1–4 nuclei and N the total number of cells scored.
Cell cycle analysis
For nine EBV-transformed cell lines (five of healthy women and four of breast cancer patients) two cultures (1 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 one EBV-transformed cell line 10 cultures were set up simultaneously to study the intraindividual/interculture variance. 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.
Statistical analysis
For the comparison of the MN responses, percentages of BN and NDI, the Mann–Whitney test was applied. To compare the cell number in the different stages of the cell cycle the Wilcoxon test was used. The variance ratio F-test was used to analyse the variabilities obtained in PBL and EBV-transformed cell lines.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The G0 and the G1/S/G2 MN assays
The number of spontaneous micronuclei was not significantly higher in the EBV-transformed cell lines than in the fresh blood samples (Mann–Whitney test, P = 0.51 and 0.60), demonstrating that the EBV-transformed cell lines do not display increased chromosomal instability. The EBV-transformed cell lines derived from breast cancer patients had no mutations in BRCA1/2.
The results obtained with the MN assay for the blood samples and the EBV-transformed cell lines of the breast cancer patients and the healthy women are summarized in Table I and presented graphically in Figure 1. The mean spontaneous frequency of MN in the fresh blood samples of the patients (22 MN/1000 BN) was not significantly different to that of the controls (25 MN/1000 BN) (Mann–Whitney test, P = 0.92). The difference in mean spontaneous MN in the EBV-transformed cell lines of the patients (29 MN/1000 BN) and the healthy women (31 MN/1000 BN) was also not significant (Mann–Whitney test, P = 0.94). For each sample the spontaneous yield was subtracted from the yield in the irradiated cells to give the radiation-induced yield. For whole blood samples the results for the patients were significantly higher compared with healthy individuals for both 2 [429 ± 43 (SD) versus 318 ± 30 MN/1000 BN, P = 0.001] and 3.5 Gy (924 ± 89 versus 754 ± 93 MN/1000 BN, P = 0.007). For the EBV-transformed lymphoblastoid cells there was no significant difference between the cell lines of the patients and the controls after 2 Gy in vitro irradiation (181 ± 50 and 173 ± 57 MN/1000 BN, Mann–Whitney test, P = 0.758). In general, the radiation response of the EBV-transformed cell lines was less than the response of the fresh blood samples, for the patients as well as for the healthy women.
|
|
Investigation of the variability in radiation-induced MN yields (2 Gy) showed that the coefficients of variation (CVs) (CV% = SD/mean x 100) obtained for the different EBV-transformed cell lines of healthy individuals (33%) and patients (27%) were greater than the CVs obtained for the blood samples of the healthy individuals (9%) and patients (10%) (Table I). To investigate whether this large variability observed between the different cell lines (interindividual variance) is an intrinsic characteristic of the cell system used, four EBV-transformed cell lines (AT-KM, NC, C003 and P720) were tested six times in different experiments with the MN assay to study the variability present between different cultures of the same EBV-transformed cell line (intraindividual/interculture variance). The results are presented graphically in Figure 2. The mean CV for intraindividual/interculture differences was 27%, compared with a mean CV of 30% for interindividual variance. There was no significant difference between the intraindividual/interculture and interindividual variability (F-test, P > 0.1).
|
Binucleation yield and nuclear division index (NDI)
The percentages of mononucleate, binucleate and polynucleate cells together with the NDI obtained for blood samples and EBV-transformed cell lines are shown in Table II. The mean values obtained for the BN cell yield and NDI (Table II) were significantly higher in the fresh blood samples compared with the EBV-transformed cell lines (Mann–WhitneyBN yield, P = 0.034; Mann–WhitneyNDI, P = 0.028). Variation analysis further showed that the CV of the mean values for BN yield and NDI (Table II) were significantly higher in the EBV- transformed cell lines compared with fresh blood samples (F-testBN yield, P = 0.001; F-testNDI, P = 0.017).
|
Cell cycle analysis
The mean percentages of cells in the different phases of the cell cycle are represented in Figure 3. When controls and patients are compared significant differences were observed in the distribution of cells in the G0/G1 and G2/M phases of the cell cycle (Mann–Whitney test, P = 0.014). The numbers of cells in S phase were comparable in both groups. The largest proportion of cells were in G0/G1 phase of the cell cycle in the healthy women as well as in the breast cancer patients (range 66–81%).
|
Significant differences were also observed between the pre- and post-irradiation conditions in the group of healthy women for the cells in the G0/G1 and G2/M phases (Wilcoxon test, P = 0.043). The number of cells in S phase was not affected by irradiation. The increase in the number of G2/M phase cells post-irradiation, due to G2/M arrest after irradiation (Hwang et al., 1998
), was also observed in breast cancer patients, but was not significant. Variance analysis on the distribution of cells in the different cell cycle phases showed that the CV values for the different phases of the cell cycle were extensive (range 2–48%). The CV values for intraindividual/interculture differences (n = 10) ranged between 5 and 28%. There was no significant difference between the intraindividual/interculture and interindividual variability (F-test, P > 0.1).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Although EBV-transformed cell lines represent a reliable cell culture system to study radiosensitivity in ATM mutation carriers (Antoccia et al., 1994
; Ramsay and Birrel, 1995; Liu et al., 1997), the potential of EBV-transformed cell lines for radiosensitivity testing in breast cancer patients is not yet clear. The existing literature data are not always consistent. Ramsay and Birrel (1995) have used the tetrazolium-based colorimetric assay (MTT) to assess radiosensitivity in EBV-transformed lymphocytes derived from breast cancer patients and found a considerable percentage (16%) of breast cancer patients with a radiosensitivity lying between AT heterozygotes and AT homozygotes. They suggested that the increased sensitivity of breast cancer patients is retained after EBV transformation. Lavin et al. (1994) screened EBV-transformed cell lines from breast cancer patients for enhanced levels of radiation induced arrest in the G2 phase of the cell cycle, such as is observed in AT patients. They compared the levels of radiation-induced G2 phase arrest as an indicator of radiosensitivity and identified a subgroup of breast cancer patients who exhibited a high level of G2 arrest after irradiation. Concerning chromosomal radiosensitivity, Foray et al. (1999) reported a small study in which EBV-transformed lymphoblastoid cell lines of BRCA1 and BRCA2 heterozygotes had a reduced capacity for DNA double-strand repair and an enhanced sensitivity to micronucleus induction after -ray exposure when compared with lymphoblastoid cells from individuals without mutations in BRCA genes. A recent study of Trenz et al. (2002) indicated that lymphoblastoid cell lines with a BRCA1 mutation do not generally show a high chromosomal radiosensitivity.
In all these studies no direct comparison was made with PBLs. Although the use of EBV-transformed cell lines may have a lot of practical advantages compared with fresh blood lymphocytes 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 in EBV-transformed cell lines derived from blood samples of healthy women and breast cancer patients using the MN assay, showing enhanced radiosensitivity after in vitro irradiation of the blood samples with 3.5 and 2 Gy irradiation dose. The number of spontaneous MN seems to be higher in the EBV-transformed cell lines but this was not significantly different from the fresh blood samples. On the contrary, the number of radiation-induced MN in the EBV-transformed cell lines is always lower than the number of radiation-induced MN in the fresh blood samples when the same irradiation dose of 2 Gy was applied (Figure 1). When the MN assay is performed on fresh blood samples, chromosomal radiosensitivity is studied in T lymphocytes, as this is the only subpopulation stimulated by PHA. EBV-transformed cell lines in contrast are derived from peripheral blood B lymphocytes. Although B lymphocytes are in general more radiosensitive than T lymphocytes, literature data have shown that for doses exceeding 1 Gy B lymphocytes show fewer MN compared with T lymphocytes. This was due to severe damage at the level of the DNA and spindle apparatus which interferes with cell division and by this with the formation of MN in those cells containing the greatest radiation damage (Vral et al., 1998, 2001). Trenz et al. (2002) suggested that the process of EBV transformation or genetic changes during in vitro cultivation of EBV-transformed cell lines might have an influence on the expression of radiosensitivity.
Our results further demonstrate that the enhanced chromosomal radiosensitivity observed in fresh blood samples of breast cancer patients is not present in EBV-transformed cell lines derived from the same blood samples (Figure 1). The significant difference in radiosensitivity observed between fresh blood samples of breast cancer patients and healthy women completely disappears in the concordant EBV-transformed cell lines. In comparison with the control EBV-transformed cell lines, the AT-KM EBV-transformed cell line shows an enhanced yield of radiation-induced MN, in agreement with the literature data (Foray et al., 1999; Gutiérrez-Enriquez and Hall, 2003). The presence of enhanced radiosensitivity in EBV-transformed cell lines of AT patients can be explained by the fact that these cells are homozygous carriers of a mutation in the ATM gene resulting in a highly radiosensitive behaviour.
Variance analysis demonstrates that the variability in MN formation in EBV-transformed cell lines is much higher than in the fresh blood samples (Table I). This high variability is also observed in EBV-transformed cell lines from different individuals (interindividual variance) as well as in different cultures from the same EBV-transformed cell line (intraindividual/interculture variability) (Table I and Figure 2) and may be the reason why the elevated radiation response of the breast cancer patients cannot be observed in the EBV-transformed cell lines. Proliferation and cell cycle analysis (Table II and Figure 3) further demonstrated that the higher variability in MN formation in EBV-transformed cell lines may find its origin in a higher variability in the BN yield and NDI in EBV-transformed cell cultures compared with fresh blood cultures and a high variation in the distribution of cells in G1, S and G2 between different EBV-transformed cell cultures. These results suggest that the large variations in cell cycle activity may be the primary cause of the disappearance of the radiosensitive response in EBV-transformed cell cultures of breast cancer patients. Furthermore, dependent on the cell cycle phase, cells have a different sensitivity to ionizing radiation. The cell cycle phase-dependent difference in chromosomal radiosensitivity could be partly due to a 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 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 the cell cycle differences in the G0 and G1/S/G2 MN assays, the differential response observed between G0 lymphocytes and EBV-transformed lymphoblastoid cells may also be related to the fact that G0 lymphocytes are exposed as whole blood cultures while lymphoblastoid cells 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 lymphoblastoid cultures.
In conclusion, as chromosomal radiosensitivity cannot be demonstrated in EBV-transformed cell lines derived from radiosensitive breast cancer patients (blood samples), care has to be taken when EBV-transformed cell lines are used as a cell culture system to assess chromosomal radiosensitivity and to investigate the molecular mechanisms underlying chromosomal radiosensitivity in breast cancer patients.
|
Acknowledgements |
|---|
The authors thank V.de Gelder and N.De Temmerman 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 Ministers Office of the Belgian Government–Federal Office for Scientific, Technical and Cultural Affairs (DWTC) and by a grant from the Bijzonder Onderzoeksfonds (Gent University, no. 01109501). We wish to thank all the patients and control donors who participated in this study.
|
Notes |
|---|
3To 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 |
|---|
-
Antoccia,A., Palitti,F., Raggi,T., Catena,C. and Tanzarella,C. (1994) Lack of effect of inhibitors of DNA synthesis/repair on the ionising radiation-induced chromosomal damage in G2 stage of ataxia telangiectasia cells. Int. J. Radiat. Biol., 66, 309–317.[Web of Science][Medline]
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]
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]
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]
Foray,N., Randrianarison,V., Marot,D., Perricaudet,M., Lenoir,G. and Feunteun,J. (1999) Gamma-rays-induced death of human cells carrying mutations of BRCA1 or BRCA2. Oncogene, 18, 7334–7342.[CrossRef][Web of Science][Medline]
Gutiérrez-Enriquez,S. and Hall,J. (2003) Use of the cytokinesis-block micronucleus assay to measure radiation-induced chromosome damage in lymphoblastoid cell lines. Mutat. Res., 535, 1–13.[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.
Lavin,M.F., Bennet,I., Ramsay,J., Gardiner,R.A., Seymour,G.J., Farrell,A. and Walsh,M. (1994) Identification of a potentially radiosensitive subgroup among patients with breast cancer. J. Natl Cancer Inst., 86, 1627–1634.
Liu,N. and Bryant,P.E. (1997) Enhancement of frequencies of restriction endonuclease-induced chromatid breaks by arabinoside adenine in normal human and ataxia telangiectasia cells. Int. J. Radiat. Biol., 72, 285–292.[CrossRef][Web of Science][Medline]
Neitzel,H. (1986) A routine method for the establishment of permanent growing lymphoblastoid cell lines. Hum. Genet., 73, 320–326.[CrossRef][Web of Science][Medline]
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 profiency in breast cancer patients and their first-degree relatives. Int. J. Cancer, 73, 20–24.[CrossRef][Web of Science][Medline]
Ramsay,J. and Birrel,G. (1995) Normal tissue radiosensitivity in breast cancer patients. Int. J. Radiat. Oncol. Biol. Phys., 31, 339–344.[CrossRef][Web of Science][Medline]
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., 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.
Sanford,K.K., Parshad,R., Gantt,R., Tarone,R.E., Jones,G.M. and Price,F.M. (1989) Factors affecting and significance of G2 chromatin radiosensitivity in predisposition to cancer. Int. J. Radiat. Biol., 55, 963–981.[Web of Science][Medline]
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]
Trenz,K., Landgraf,J. and Speit,G. (2002) Mutagen sensitivity of human lymphoblastoid cells with a BRCA1 mutation. Breast Cancer Res. Treat., 2463, 1–11.
Vral,A., Louagie,H., Thierens,H., Philippe,J., Cornelissen,M. and De Ridder,L. (1998) Micronucleus frequencies in cytokinesis-blocked human B lymphocytes after low dose -irradiation. Int. J. Radiat. Biol., 73, 549–555.[CrossRef][Web of Science][Medline]
Vral,A., Thierens,H., Bryant,P. and De Ridder,L. (2001) A higher micronucleus yield in B- versus T-cells after low-dose -irradiation is not linked with defective Ku86 protein. Int. J. Radiat. Biol., 77, 329–339. [CrossRef][Web of Science][Medline]
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]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Amoli, D Carthy, H Platt, and W. Ollier EBV Immortalization of human B lymphocytes separated from small volumes of cryo-preserved whole blood Int. J. Epidemiol., April 1, 2008; 37(suppl_1): i41 - i45. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Zijno, F. Saini, and R. Crebelli Suitability of cryopreserved isolated lymphocytes for the analysis of micronuclei with the cytokinesis-block method Mutagenesis, September 1, 2007; 22(5): 311 - 315. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Trenz, P. Schutz, and G. Speit Radiosensitivity of lymphoblastoid cell lines with a heterozygous BRCA1 mutation is not detected by the comet assay and pulsed field gel electrophoresis Mutagenesis, March 1, 2005; 20(2): 131 - 137. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Baeyens, K. Vandenbulcke, J. Philippe, H. Thierens, L. De Ridder, and A. Vral The use of IL-2 cultures to measure chromosomal radiosensitivity in breast cancer patients Mutagenesis, November 1, 2004; 19(6): 493 - 498. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||