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Purpose : To determine if the efficacy of G 2 checkpoint control (measured as the degree of mitotic inhibition) was reduced in breast cancer patients ( n =129) compared with healthy controls ( n =105) after exposure of lymphocytes to X-rays. We had previously shown that the average level of radiation-induced chromosome damage was higher in G 2 lymphocytes of these patients than in the controls, and it was proposed that this was a marker of low penetrance predisposition to cancer. Materials and methods : Proliferating lymphocytes were X-irradiated (50 cGy) and sampled at 90 min post-irradiation, which was the time of maximum mitotic inhibition of G 2 cells, expressed as the extent of reduction in the mitotic index in irradiated compared with unirradiated cells. Results : Repeated measurements on 28 controls showed that there were reproducible differences in mitotic inhibition between individuals. Inhibition was significantly greater in female than in male controls ( p =0.014), but less in patients than in female controls ( p =0.009). There was a weak inverse correlation between the extent of inhibition and the amount of chromosome damage in all females ( r =-0.15, p =0.043). Conclusions : The lesser mitotic inhibition in patients than in female controls might contribute to their greater mean G 2 chromosomal radiosensitivity. However, this hypothesis is not easily reconciled with other observations that (1) the significant difference in inhibition between the sexes in controls was not accompanied by any gender difference in radiosensitivity and (2) there was an inverse correlation between inhibition and age in controls, yet no age-related increase in radiosensitivity. There might, therefore, be no causal relationship between G 2 mitotic inhibition and chromosomal radiosensitivity. [ABSTRACT FROM AUTHOR]

Purpose : The association between enhanced intrinsic radiosensitivity, measured by the level of chromosome damage induced in either the G 2 or G 0 phases of the cell cycle, and the risk of breast cancer has been well demonstrated. Although not originally designed for this purpose, early studies are now sufficiently mature (median follow-up 4.2 years) to enable an assessment of the impact of radiosensitivity on patient outcome. Materials and methods : Breast cancer patients assessed in previous studies of G 2 and G 0 lymphocyte chromosomal radiosensitivity between 1993 and 1997 were identified. Tumour characteristics, recurrence and survival data were obtained from clinical records and outcomes were compared between those identified as having normal and elevated radiosensitivity in the two assays. Results : There was a significant association between G 0 sensitivity and tumour grade ( p =0.03), but otherwise there were no associations in these series between radiosensitivity in the two assays and tumour characteristics. Overall survival in this cohort of patients was high (94% at 5 years) and there was no association between the G 2 or G 0 assay results and outcome as measured by cancer-specific survival or local recurrence. Conclusions : Radiosensitivity, as measured by these two assays, was not a strong prognostic factor following standard radiotherapy treatment. However, the statistical power of the present study was limited and modest prognostic utility could not be discounted. Further studies designed specially to investigate outcome are needed, possibly in association with trials of altered radiotherapy fractionation. [ABSTRACT FROM AUTHOR]

Purpose: To study the role of p53 and bcl-2 in the response of the small intestine to irradiation delivered at low dose-rate. Materials and methods: Mice homozygous for p53 or bcl-2 deletion (-/-), their respective heterozygotes (+/-), and their wild-type littermates (+/+) including a previously used hybrid strain (B6D2F[sub 1]), were irradiated to the whole-body using [sup 60]Co gamma-rays at 1 Gyh[sup -1]. Crypt survival levels in the small intestine were measured at day 3 after the end of irradiation. Results: Crypt survival levels were higher in p53 -/- mice than in the other p53 genotypes after 25–30 Gy, but not after lower or higher doses. Similar experiments with the three genotypes for bcl-2 status showed lower crypt survival after all doses used in the -/- mice, compared with the +/- and +/+ mice, which were similar in response. The marked degree of curvature in the survival curve observed for the p53 genotypes was also observed in B6D2F[sub 1] hybrid mice, was particularly striking in the p53 -/- mice, but was not seen to the same extent in the bcl-2 genotypes. The heterozygotes for p53 or for bcl-2 were nearer in response to their respective +/+ genotypes rather than the -/- genotypes. Conclusion: The increased crypt survival levels at some radiation dose levels in the p53 nulls contrasts with the lack of change reported previously using irradiation at high dose-rate. The decreased survival in the bcl-2 nulls is consistent with the known ‘survival’ function of bcl-2, although bcl-2 expression has not been detected immunohistochemically in this intestinal site. The marked degree of curvature in the dose–response curve at high dose levels for some genotypes was unexpected at this low dose-rate. [ABSTRACT FROM AUTHOR]

Purpose: The feasibility of using cryopreserved lymphocytes to detect inter-individual differences in chromosomal radiosensitivity was investigated. Typically, such studies are conducted with fresh blood samples but, in a clinical setting, when availability of samples is unpredictable, this is not always convenient. The sensitivity of 23 normal healthy donors, 11 breast cancer patients who had shown severe acute skin reactions to radiotherapy and seven ataxia telangiectasia (A-T) heterozygotes was determined. Materials and methods: Thawed lymphocytes were exposed to high (HDR) or low dose rate (LDR) gamma irradiation (3.5 Gy) in G[sub 0], stimulated with PHA, treated with cytochalasin-B 24h later and then harvested at 90h for the determination of micronucleus (MN) yields in binucleate cells. Results: Each normal donor was tested one to three times. Mean MN yields were 76.1 +/- 9.3/100 cells at HDR and 44.5 +/- 5.3 at LDR, giving an LDR sparing effect of 39.6 +/- 9.3%. A relatively high proportion of tests failed to yield sufficient binucleate cells for analysis. Inter-experimental variability was also high and it was not possible to demonstrate inter-individual differences in sensitivity in spite of the use of an internal control sample from a single normal donor in each experiment. There was a small but significant increase in radiation-induced MN in the breast cancer patients compared with the normals at LDR (but not at HDR), but a complete overlap with the normal range. There was no increase in sensitivity in the A-T heterozygotes at HDR. The LDR samples failed because the LDR protocol reduced proliferation rates, and radiation-induced mitotic inhibition in this group was higher than in normals. Conclusions: In comparison with previous experience with fresh blood samples, the use of frozen lymphocytes is not as satisfactory because: (1) experimental failures are higher; (2) inter-experiment variability is higher: (3) dose-rate sparing is lower, suggesting poorer repair; and (4) the ability to discriminate between breast cancer cases and normals is probably lower. [ABSTRACT FROM AUTHOR]

Purpose: It has been shown previously that sensitivity to the induction of chromosome damage by ionizing radiation is, on average, higher in G2 or G0 lymphocytes of breast cancer patients than of normal healthy controls. The authors suggested that elevated chromosomal radiosensitivity may be a marker for breast cancer predisposition. To investigate whether the G0 micronucleus assay is a true surrogate for the more demanding G2 metaphase assay, both tests have now been performed on the same blood samples from 80 patients. Methods: For the G0 micronucleus assay, cells were exposed to 3.5Gy 137Cs gamma -rays 6 h before mitogenic stimulation, treated with cytochalasin B at 24 h post-stimulation and harvested at 90 h. For the G2 assay, at 72 h after stimulation cells were given 0.5Gy X-rays and harvested 90 min later. Results: Previous observations were confirmed, now with much larger numbers of donors, in that approximately 40% of breast cancer patients showed elevated sensitivity in the G2 assay (135 patients, 105 normals) and 25% in the G0 assay (130 patients, 68 normals). However, there was no correlation between G2 and G0 sensitivity for the 80 patients tested (r=-0.001, p=0.99). Most of the sensitive patients were either G2 or G0 sensitive, with only 4% sensitive in both assays. Conclusions: The results suggest that different mechanisms of chromosomal radiosensitivity operate in G2 and G0 cells and that, in general, each chromosomally radiosensitive patient is defective in only one such mechanism, possibly via mutation (or polymorphism) of a single gene. Such mutations may confer cancer predisposition, of low penetrance, in a substantial proportion of patients. [ABSTRACT FROM AUTHOR]

Radiation survival curves have been obtained for crypts in the small and large intestine of male BDF1 mice using the microcolony assay. Four regions of the small intestine and three regions of the large intestine were studied. One of the major objectives was to determine the optimal conditions for the microcolony assay in the large bowel. Various times post-irradiation were studied using different approaches and threshold criteria for surviving crypts. Small, but statistically significant, differences were observed along the length of the small intestine, but no differences were observed between different regions of the large intestine. There was a marked difference (p<0.001) in the response in the large compared with the small intestine (D0 = 291 14 compared with 151 4 cGy respectively with corresponding extrapolation numbers of 18 4 and 377 65). The use of tritiated thymidine or vincristine helped in the identification of true survivors in the large bowel, but these approaches are not necessary in the small bowel. The longer the time after irradiation that the samples were fixed the easier it was to identify survivors in the colon. However, the longer the time the more animals died. The optimum compromize in these studies was to use partial-body irradiation (abdomen only) and to sample on the fifth day after irradiation. With these = criteria the mid-colon survival curve had a D 274 28cGy 0 and an extrapolation number of 22 10. The results can be considered in relation to published data on the levels of p53 and bcl-2 expression and apoptosis in murine small and large bowel. [ABSTRACT FROM AUTHOR]

Purpose: To examine whether in vitro measurements of normal and tumour cell radiosensitivity can be used as prognostic factors in clinical oncology. Materials and methods: Stage I-III cervix carcinoma patients were treated with radical radiotherapy with a minimum of 3 years' follow-up. Lymphocyte and tumour radiosensitivities were assayed using, respectively, a limiting dilution and soft agar clonogenic assay to obtain surviving fraction at 2 Gy (SF2). The results were related, in an actuarial analysis, to late morbidity assessed using the Franco-Italian glossary. Results: Patients with radiosensitive lymphocytes had a significantly increased risk of developing late complications (n 93, p 0.002). Increasing tumour radiosensitivity was associated with an increased risk of morbidity (n 113, p 0.032). A significant correlation was found between fibroblast and tumour cell radiosensitivity (r 0.57, p 0.03), but a weak inverse association was found between lymphocyte and tumour cell radiosensitivity (r 0.32, p 0.03). Patients with radiosensitive lymphocytes and tumour cells had higher levels of late complications than those whose cells were radioresistant. Conclusion: The work described highlights the importance of cellular radiosensitivity as a parameter determining the clinical response to radiotherapy. [ABSTRACT FROM AUTHOR]

Discusses issues in radiation dose-fractionation studies. Dose-response curves; Fractionation sensitivity; Treatment time; Description of other radiotherapy modifications.

Of breast cancer patients, 30% are sensitive in a lymphocyte assay of radiation-induced chromosome damage (micronucleus induction) compared with 10% of healthy controls. Twenty-two first-degree relatives of 11 sensitive patients had an average micronucleus yield significantly higher than that of 68 controls. This suggests that radiosensitivity in this assay may be an inherited characteristic associated with predisposition to breast cancer. [ABSTRACT FROM AUTHOR]

Purpose: To determine if the efficacy of G(2) checkpoint control (measured as the degree of mitotic inhibition) was reduced in breast cancer patients (n=129) compared with healthy controls (n=105) after exposure of lymphocytes to X-rays. We had previously shown that the average level of radiation-induced chromosome damage was higher in G(2) lymphocytes of these patients than in the controls, and it was proposed that this was a marker of low penetrance predisposition to cancer., Materials and Methods: Proliferating lymphocytes were X-irradiated (50 cGy) and sampled at 90 min post-irradiation, which was the time of maximum mitotic inhibition of G(2) cells, expressed as the extent of reduction in the mitotic index in irradiated compared with unirradiated cells., Results: Repeated measurements on 28 controls showed that there were reproducible differences in mitotic inhibition between individuals. Inhibition was significantly greater in female than in male controls (p=0.014), but less in patients than in female controls (p=0.009). There was a weak inverse correlation between the extent of inhibition and the amount of chromosome damage in all females (r=-0.15, p=0.043)., Conclusions: The lesser mitotic inhibition in patients than in female controls might contribute to their greater mean G(2) chromosomal radiosensitivity. However, this hypothesis is not easily reconciled with other observations that (1) the significant difference in inhibition between the sexes in controls was not accompanied by any gender difference in radiosensitivity and (2) there was an inverse correlation between inhibition and age in controls, yet no age-related increase in radiosensitivity. There might, therefore, be no causal relationship between G(2) mitotic inhibition and chromosomal radiosensitivity.

Purpose: The feasibility of using cryopreserved lymphocytes to detect inter-individual differences in chromosomal radiosensitivity was investigated. Typically, such studies are conducted with fresh blood samples but, in a clinical setting, when availability of samples is unpredictable, this is not always convenient. The sensitivity of 23 normal healthy donors, 11 breast cancer patients who had shown severe acute skin reactions to radiotherapy and seven ataxia telangiectasia (A-T) heterozygotes was determined., Materials and Methods: Thawed lymphocytes were exposed to high (HDR) or low dose rate (LDR) gamma irradiation (3.5 Gy) in Go, stimulated with PHA, treated with cytochalasin-B 24 h later and then harvested at 90 h for the determination of micronucleus (MN) yields in binucleate cells., Results: Each normal donor was tested one to three times. Mean MN yields were 76.1 +/- 9.3/100 cells at HDR and 44.5 +/- 5.3 at LDR, giving an LDR sparing effect of 39.6 +/- 9.3%. A relatively high proportion of tests failed to yield sufficient binucleate cells for analysis. Inter-experimental variability was also high and it was not possible to demonstrate inter-individual differences in sensitivity in spite of the use of an internal control sample from a single normal donor in each experiment. There was a small but significant increase in radiation-induced MN in the breast cancer patients compared with the normals at LDR (but not at HDR), but a complete overlap with the normal range. There was no increase in sensitivity in the A-T heterozygotes at HDR. The LDR samples failed because the LDR protocol reduced proliferation rates, and radiation-induced mitotic inhibition in this group was higher than in normals., Conclusions: In comparison with previous experience with fresh blood samples, the use of frozen lymphocytes is not as satisfactory because: (1) experimental failures are higher; (2) inter-experiment variability is higher: (3) dose-rate sparing is lower, suggesting poorer repair; and (4) the ability to discriminate between breast cancer cases and normals is probably lower.

Data are presented for the kinetics of repair of sub-lethal damage in the large intestine of mice. The results are based on experiments using the crypt microcolony assay with two equal sized doses which were delivered with a variable interval of time between the doses. These show that this split-dose repair was largely complete after 5 h, and that there were no significant differences between three regions of the large intestine. Overall the half-time for the repair was 2.3 +/- 0.8 h, and the maximum split-dose repair ratio (the proportion of damage recovered by splitting the dose into two fractions) was 22 +/- 2% and the mean recovery factor (the ratio of the number of surviving crypts using long interfraction intervals to that at zero time) was 11 +/- 2. The split-dose approach (Hendry 1979) using a 5 h interval has been used to estimate the number of clonogenic cells in large intestinal crypts. A range of single and paired doses between 7 Gy and 10.5 Gy were used. There were significant differences between the three regions of the large intestine, the caecum, mid-colon and rectum. The estimate of the number of clonogens also depended in a significant way on the dose of radiation used to make the estimate. At low doses large intestinal crypts contain between 5 and 10 clonogenic cells while if high doses were used they contain an estimated 16 to 36 clonogenic cells. Considerable similarity exists between the small intestine and the large intestine for; (a) the repair kinetics (b), the clonogenic estimates, and (c) their dependence on dose.

We investigated the reproducibility of the cytochalasin B micronucleus (MN) assay in irradiated human lymphocytes to assess its suitability in predicting cancer predisposition and response to radiotherapy by virtue of defects in the processing of clastogenic lesions. G0 lymphocytes were exposed to 3.0 Gy 60Co gamma-rays at high (HDR) or low dose-rate (LDR). Six healthy donors were assayed three times each in nine experiments and compared with six ataxia-telangiectasia (A-T) heterozygotes. In controls, significant interexperiment variability in MN yields was observed at HDR and LDR, also in dose-rate sparing (i.e. reduction in MN yield at LDR compared with HDR). Significant inter-individual variability was seen at HDR, but not at LDR or for sparing. Average sparing was 66.4 +/- 4.8%. In spite of the experimental variability, a significant difference between controls and A-T heterozygotes was detected at LDR, and 5/6 heterozygotes had sparing values below the control range. This gives encouragement for the use of this assay in predictive testing if sources of experimental variability can be identified so as to improve discrimination between individuals. HDR and to a lesser extent LDR irradiation induced significant mitotic inhibition, seen as a reduction in binucleate cells after cytocholasin treatment. A positive correlation between mitotic inhibition and MN frequency suggests that similar lesions may be involved in these effects.