Radiation-induced micronuclei in human fibroblasts in relation to clonogenic radiosensitivity.

As part of our programme for developing predictive tests for normal tissue response to radiotherapy, we have investigated the efficacy of the cytokinesis-block micronucleus (MN) assay as a means of detecting interindividual differences in cellular radiosensitivity. A study was made of nine fibroblast strains established from vaginal biopsies of pretreatment cervical cancer patients and an ataxia telangiectasia (A-T) cell strain. Cells were irradiated in plateau phase, replated and treated with cytochalasin B 24 h later. MN formation was examined 72 h after irradiation as the number of MN in 100 binucleate cells. The method yielded low spontaneous MN yields (<7 per 100 cells), and mean induced MN frequencies after 3.5 Gy varied between cell strains from 18 to 144 per 100 cells. However, in repeat experiments, considerable intrastrain variability was observed (CV = 32%), with up to twofold differences in MN yields, although this was less than interstrain variability (CV = 62%). An analysis was made of the relationship between MN results and previously obtained clonogenic survival data. There was a significant correlation between MN yields and clonogenic survival. However, when the A-T strain was excluded from the analysis, the correlation lost significance, mainly because of one slow-growing strain which was the most sensitive to cell killing but had almost the lowest MN frequency. With current methodology, the MN assay on human fibroblasts does not appear to have a role in predictive testing of normal tissue radiosensitivity.     

DNA damage-related RNA expression to assess individual sensitivity to ionizing radiation

Predictive markers of intrinsic radiosensitivity in healthy individuals are needed in monitoring their occupational or environmental radiation exposure and may predict a patient's response to radiotherapy. Ionizing radiation can induce a large spectrum of DNA lesions, but under optimal DNA repair conditions, the principal residual lesions of importance are misrepaired double-strand breaks. The micronucleus (MN) assay represents a useful test in measuring radiosensitivity since it reflects non-repaired DNA breaks at the time of cell division. Spontaneous and radiation-induced MN vary greatly between individuals, and little is known about the molecular mechanisms of this variability. DNA repair and apoptosis processes are involved in the cellular response to radiation-induced DNA damage, and variation in gene expression related to these cellular pathways could be linked to individual radiosensitivity. In this study we analysed by real-time quantitative RT-PCR the basal expression of 12 genes involved both in DNA repair and apoptosis in a series of blood samples obtained from 32 healthy male donors. Relationships between basal RNA expressions and MN frequency and distribution per bi-nucleated cell were studied after ex vivo irradiation of total blood samples. Our results indicate that the variability of mRNA gene expression among the 32 subjects appears to be of the same magnitude or higher than that found for spontaneous or radiation-induced MN frequency and that RAD51 gene expression is negatively correlated with radiation-induced MN frequency.     

Radiosensitivity in breast cancer assessed by the Comet and micronucleus assays

Spontaneous and radiation-induced genetic instability of peripheral blood mononuclear cells derived from unselected breast cancer (BC) patients (n=50) was examined using the single-cell gel electrophoresis (Comet) assay and a modified G2 micronucleus (MN) test. Cells from apparently healthy donors (n=16) and from cancer patients (n=9) with an adverse early skin reaction to radiotherapy (RT) served as references. Nonirradiated cells from the three tested groups exhibited similar baseline levels of DNA fragmentation assessed by the Comet assay. Likewise, the Comet analysis of in vitro irradiated (5 Gy) cells did not reveal any significant differences among the three groups with respect to the initial and residual DNA fragmentation, as well as the DNA repair kinetics. The G2 MN test showed that cells from cancer patients with an adverse skin reaction to RT displayed increased frequencies of both spontaneous and radiation-induced MN compared to healthy control or the group of unselected BC patients. Two patients from the latter group developed an increased early skin reaction to RT, which was associated with an increased initial DNA fragmentation in vitro only in one of them. Cells from the other BC patient exhibited a striking slope in the dose-response curve detected by the G2 MN test. We also found that previous RT strongly increased both spontaneous and in vitro radiation-induced MN levels, and to a lesser extent, the radiation-induced DNA damage assessed by the Comet assay. These data suggest that clinical radiation may provoke genetic instability and/or induce persistent DNA damage in normal cells of cancer patients, thus leading to increased levels of MN induction and DNA fragmentation after irradiation in vitro. Therefore, care has to be taken when blood samples collected postradiotherapeutically are used to assess the radiosensitivity of cancer patients.     

Assessment of the proliferative activity and radiosensitivity of human tumours using the cytokinesis-block micronucleus assay.

We established an in vitro cytokinesis-block micronucleus assay of human tumours for estimation of the proportion of cells undergoing mitosis (the dividing fraction, DF), the time for the number of nuclei to double and the radiosensitivity in terms of the micronucleus frequency, based on a concept described previously. Under certain conditions, the nuclear number doubling time (NNDT) was considered to represent the potential doubling time. Tumour specimens obtained at surgery were disaggregated into single-cell suspensions and were directly cultured in the presence of cytochalasin B with or without irradiation. At various intervals, the percentage of multinucleate cells (the plateau value represented the DF), the average number of nuclei per cell and the number of micronuclei in binucleate cells were determined. DF and NNDT values were obtained in 58 of the 73 tumours investigated, and the micronucleus frequency was obtained in 54 of these 58 tumours. The DF ranged from 4.1% to 71% and the NNDT ranged from 3.1 to 83 days. A DF > or = 20% was associated with a higher recurrence rate in patients undergoing curative operation. A correlation was found between the NNDT and the time to relapse in patients with recurrent disease. The average number of micronuclei per binucleate cell at 2 Gy of irradiation (after subtraction of the value at 0 Gy) ranged from 0.052 to 0.35. Tumours which produced more micronuclei after irradiation showed a better response to radiotherapy. This assay can be readily performed on human tumours and appears to have promise as a predictive assay for radiation therapy.     

Micronucleus formation in human tumour cells: lack of correlation with radiosensitivity.

The micronucleus (MN) test has been carefully characterized in four human tumour cell lines of widely differing radiosensitivity. Two radioresistant bladder carcinoma cell lines (MGH-U1 and RT112), one sensitive medulloblastoma cell line (D283MED) and a sensitive neuroblastoma cell line (HX142) were used. The number of MN per Gy of ionising radiation was 0.13 for HX142, 0.17 for D283MED, 0.21 for RT112 and 0.26 for MGH-U1. This does not rank the cell lines in the same order of radiosensitivity as clonogenic cell survival where the surviving fraction at 2 Gy (SF2) was 0.11 for HX142, 0.2 for D283MED, 0.62 for RT112 and 0.53 for MGH-U1. This discrepancy between MN formation and cell death leaves doubt as to the potential usefulness of the MN test as a rapid assay of radiosensitivity but it has potential implications for the mechanistic basis of radiosensitivity in these cells.     

Studies of the in vivo radiosensitivity of human skin fibroblasts.

BACKGROUND AND PURPOSE: To examine the radiosensitivity of skin cells obtained directly from the irradiated skin of patients undergoing fractionated radiation treatment prior to surgery for treatment of soft tissue sarcoma (STS) and to determine if there was a relationship with the development of wound healing complications associated with the surgery post-radiotherapy. METHODS: Micronucleus (MN) formation was measured in cells (primarily dermal fibroblasts) obtained from human skin at their first division after being removed from STS patients during post-radiotherapy surgery (2-9 weeks after the end of the radiotherapy). At the time of radiotherapy (planned tumor dose - 50Gy in 25 daily fractions) measurements were made of surface skin dose at predetermined marked sites. Skin from these sites was obtained at surgery and cell suspensions were prepared directly for the cytokinesis-blocked MN assay. Cultured strains of the fibroblasts were also established from skin nominally outside the edge of the radiation beam and DNA damage (MN formation) was examined following irradiation in vitro for comparison with the results from the in situ irradiations. RESULTS: Extensive DNA damage (MN) was detectable in fibroblasts from human skin at extended periods after irradiation (2-9 weeks after the end of the 5-week fractionated radiotherapy). Analysis of skin receiving a range of doses demonstrated that the level of damage observed was dose dependent. There was no clear correlation between the level of damage observed after irradiation in situ and irradiation of cell strains in culture. Similarly, there was no correlation between the extent of MN formation following in situ irradiation and the propensity for the patient to develop wound healing complications post-surgery. CONCLUSIONS: Despite the presence of DNA damage in dermal fibroblasts weeks after the end of the radiation treatment, there was no relationship between this damage and wound healing complications following surgery post-irradiation. These results suggest that factors other than the radiosensitivity of the skin fibroblasts likely also play a role in wound healing in deep wound sites associated with surgery for STS following radiation therapy.     

外周血淋巴细胞松胞素阻滞微核法预测鼻咽癌放射敏感性的临床和实验研究

目的探讨用外周血松胞素阻滞微核法来预测鼻咽癌放射敏感性的可行性。方法前瞻性研究42例鼻咽癌患者放疗前照射0,0.5,1,2,4Gy的外周血,放疗20Gy、60Gy时的外周血行松胞素阻滞微核法检测微核率、微核细胞率,统计分析正常组织敏感组与不敏感组间、鼻咽肿瘤组织局控组与残留组间微核率、微核细胞率、50Gy鼻咽肿瘤消退率等指标有无差异,并分析这些指标间的相互关系。结果在体外照射1Gy及以上剂量、20Gy、60Gy时外周血微核率、微核细胞率在放射敏感组明显比不敏感组增加,在体外照射2Gy及以上剂量、20Gy、60Gy时外周血微核率、微核细胞率在肿瘤局控组比未局控组明显增加。结论外周血淋巴细胞松胞素阻滞微核法有可能作为预测鼻咽癌皮肤、黏膜急性反应敏感性的指标。外周血淋巴细胞微核率、微核细胞率有可能作为预测鼻咽癌根治性放疗近期疗效的指标。     

Radiosensitivity of human tumour cells is correlated with the induction but not with the repair of DNA double-strand breaks

Nine human tumour cell lines (four mammary, one bladder, two prostate, one cervical, and one squamous cell carcinoma) were studied as to whether cellular radiosensitivity is related to the number of initial or residual double-strand breaks (dsb). Cellular sensitivity was measured by colony assay and dsb by means of constant- and graded-field gel electrophoresis (CFGE and GFGE, respectively). The nine tumour cell lines showed a broad variation in cellular sensitivity (SF2 0.17-0.63). The number of initial dsb as measured by GFGE ranged between 14 and 27 dsb/Gy/diploid DNA content. In contrast, normal fibroblasts raised from skin biopsies of seven individuals showed only a marginal variation with 18-20 dsb/Gy/diploid DNA content. For eight of the nine tumour cell lines, there was a significant correlation between the number of initial dsb and the cellular radiosensitivity. The tumour cells showed a broad variation in the amount of dsb measured 24 h after irradiation by CFGE, which, however, was not correlated with the cellular sensitivity. This residual damage was found to be influenced not only by the actual number of residual dsb, but also by apoptosis and cell cycle progression which had impact on CFGE measurements. Some cell line strains were able to proliferate even after exposure to 150 Gy while others were found to degrade their DNA. Our results suggest that for tumour cells, in contrast to normal cells, the variation in sensitivity is mainly determined by differences in the initial number of dsb induced.     

Lack of a correlation between micronucleus formation and radiosensitivity in established and primary cultures of human tumours.

The radiation-induced genotoxic damage in three established cell lines and 15 primary cultures of human malignant melanoma and ovarian carcinoma showing different radiosensitivity was tested by the cytokinesis-block micronucleus assay. A dose-related increase in micronucleus frequency was observed in all the cell systems. The mean number of micronuclei per Gy of ionising radiation per binucleated cell was respectively 0.44 +/- 0.0075 and 0.43 +/- 0.04 for M14 and JR8 malignant melanoma cell lines and 0.19 +/- 0.013 for the A2780 ovarian cancer cell line. The number of micronuclei did not rank the cell lines in the same order of radiosensitivity as clonogenic cell survival, which showed a surviving fraction at 2 Gy of 0.38 +/- 0.02 for JR8, 0.34 +/- 0.05 for M14 and 0.22 +/- 0.007 for A2780. As regards primary tumour cultures, no correlation was observed between micronucleus induction and surviving fraction at 2 Gy. In conclusion, the discrepancy we observed between micronucleus formation and cell death raises doubts about the potential of the micronucleus assay as a preclinical means to predict radiosensitivity.     

Anti-Genotoxicity Evaluation of Cratoxylum Formosum Dyer Leaves by Comet Assay and Micronucleus Test

Cratoxylum formosum Dyer is the Thai vegetable which commonly consumed a fresh leaves. In this study, we extracted Cratoxylum formosum with water and tested the extract for genotoxicity and anti-genotoxicity effects. We carried out the experiment using micronucleus test and comet assay in TK6 cells. In micronucleus experiment, we used cytokinesis-block proliferation technique to stop cell division which produced a cell at binucleated (BNC) stage. The comet assay was carried out after pre-treatment the cell with C. formosum for 18 h. The results revealed not increased the micronucleus frequency of C. formosum at concentration ranging from 50-150 μg/ml. In contract, it showed that the combination between C. formosum at various concentrations (25, 50, 75, 100, 150 and 200 μg/ml) and mitomycin C could decrease significantly in frequency of micronuclei. The mean of micronucleus frequency in the sample were 23.17 ±3.33, 23.33 ±4.72, 21.00 ±3.61, 11.33 ±3.21, 16.67 ±2.08, and 23.33±1.53 MN/ 1,000 BNC, respectively whereas the MMC-treated group was 33.67 ± 8.96 MN/ 1,000 BNC. The comet assay result showed that pre-treatment with Cratoxylum formosum (25, 50, 100, 200 μg/ml) could inhibit the hydrogen peroxide induced DNA damage by 6.95, 12.99, 17.61, and 26.39 respectively.     

Chromosomal instability and radiosensitivity in myelodysplastic syndrome cells.

Myelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells. To investigate whether chromosomal instability and/or DNA repair defects are involved in the development of MDS, we measured the micronucleus (MN) frequency in peripheral blood lymphocytes exposed to various doses of X-rays, using a cytokinesis-block micronucleus assay. The spontaneous MN frequencies in RAEB and RAEB-T patients were significantly higher than those in normal individuals (P = 0.0224, P = 0.008, respectively). Also, the X-ray-induced MN frequencies in RA/RARS, RAEB, and RAEB-T patients were significantly higher than those in normal individuals (P = 0.007, P = 0.003, P = 0.003, respectively, at 2 Gy). In order to elucidate the cause of unusual radiosensitivity, we measured the expression levels of nucleotide excision repair (NER) genes in peripheral blood mononuclear cells using a RT-PCR method. Reduction of NER gene expression was found in only one of 10 patients with low risk MDS, but in four of 11 patients with high risk MDS. Our data suggest that chromosomal instability and DNA repair defects may be involved in the pathophysiology of disease progression of MDS.     

Fractionated irradiation of five human lung cancer cell lines and prediction of survival according to a radiobiology model

BACKGROUND: This study evaluates a predictive radiobiology model by measurements of surviving fraction (SF) by the clonogenic assay or the extrapolation method and the proliferation rate in vitro. It is hypothesized that incorporating proliferation to intrinsic radiosensitivity, measured by SF, to predict radiation responsiveness after fractionated irradiation adds to the model's accuracy. Materials and Methods. Five lung cancer cell lines with known SF after 1 Gy (SF1), and also SF2 and SF5, were irradiated with three different fractionation regimes; 10 × 1 Gy, 5 × 2 Gy or 2 × 5 Gy during the same total time to achieve empirical SF. In addition, the SF1, SF2 and SF5 after fractionated irradiation was calculated for each cell line based on the already known single fraction SF and with or without a proliferation factor. The results were compared to the empirical data. Results and Discussion: By using the clonogenic assay to measure radiosensitivity, prediction of radiosensitivity was improved after fractionated radiotherapy when proliferation was used in the radiobiology model. However, this was not the case in the cell lines where the extrapolation method was used to calculate SF. Thus, a radiobiology model including intrinsic radiosensitivity, measured by the clonogenic assay, as well as proliferation, is better at predicting survival after fractionated radiotherapy, compared to the use of intrinsic radiosensitivity alone.     

Relationship between in vitro chromosomal radiosensitivity of peripheral blood lymphocytes and the expression of normal tissue damage following radiotherapy for breast cancer.

BACKGROUND AND PURPOSE: There is a need for rapid and reliable tests for the prediction of normal tissue responses to radiotherapy, as this could lead to individualization of patient radiotherapy schedules and thus improvements in the therapeutic ratio. Because the use of cultured fibroblasts is too slow to be practicable in a clinical setting, we evaluated the predictive role of assays of lymphocyte chromosomal radiosensitivity in patients having radiotherapy for breast cancer. MATERIALS AND METHODS: Radiosensitivity was assessed using a micronucleus (MN) assay at high dose rate (HDR) and low dose rate (LDR) on lymphocytes irradiated in the G(0) phase of the cell cycle (Scott D, Barber JB, Levine EL, Burril W, Roberts SA. Radiation-induced micronucleus induction in lymphocytes identifies a frequency of radiosensitive cases among breast cancer patients: a test for predispostion? Br. J. Cancer 1998;77;614-620) and an assay of G(2) phase chromatid radiosensitivity ('G(2) assay') (Scott D, Spreadborough A, Levine E, Roberts SA. Genetic predisposition in breast cancer. Lancet 1994; 344: 1444). In a study of acute reactions, blood samples were taken from breast cancer patients before the start of radiotherapy, and the skin reaction documented. 116 patients were tested with the HDR MN assay, 73 with the LDR MN assay and 123 with the G(2) assay. In a study of late reactions, samples were taken from a series of breast cancer patients 8-14 years after radiotherapy and the patients assessed for the severity of late effects according to the'LENT SOMA' scales. 47 were tested with the HDR assay, 26 with the LDR assay and 19 with the G(2) assay. For each clinical endpoint, patients were classified as being normal reactors or 'highly radiosensitive patients' (HR patients (Burnet NG. Johansen J, Turesson I, Nyman J. Describing patients' normal tissue reactions: Concerning the possiblity of individualising radiotherapy dose presciptions based on potential predictive assays of normal tissue radiosensitivity. Int. J. Cancer 1998;79:606-613)). RESULTS: The HR patients could be identified in some of the assays. For example, for acute skin reactions, 9/123 patients were judged as HR; they had significantly higher G(2) scores than normal reactors (P=0.004). For the late reactions, the mean HDR MN scores were higher for the 4/47 patients who had severe telangiectasia (P=0.042) and the 8/47 patients had severe fibrosis (P=0.055). However, there were no trends towards increased chromosomal radiosensitivity with the micronucleus scores at HDR or LDR, or with G(2) chromosomal radiosensitivity. CONCLUSIONS: While these results support the concept of using lymphocytes to detect elevated sensitivity to radiotherapy (as an alternative to fibroblasts), these assays are unlikely to be of assistance for the prediction of normal tissue effects in the clinic in their present form.     

TECIA法体外放射敏感性实验对原发性肺鳞癌放疗个体化方案选择的意义

目的：建立人原发性肺鳞癌的组织培养放疗敏感性检测法,快速确定人原发性肺鳞癌组织的辐射敏感性的个体差异,用于筛选放疗方案及指导临床肿瘤个体化放疗方案。方法：采用TECIA法检测不同个体原发性肺鳞癌组织在不同剂量、不同分次放疗方案照射后不同时间的细胞凋亡水平。比较不同剂量、不同分次放疗方案照射后不同时间的细胞凋亡水平的差异。结果：TECIA法发现,照射前人原发性肺鳞癌组织细胞凋亡指数较低,放疗后出现较高的细胞凋亡指数,与放疗剂量呈正相关。结论：肿瘤放射敏感性实验对指导患者的个体化治疗具有重要的价值。     

Chromosomal radiosensitivity in breast cancer patients: influence of age of onset of the disease

The age dependency of onset of the disease on chromosomal radiosensitivity of an unselected group of breast cancer patients (n=100) was investigated and compared to a group of healthy women (n=100). The chromosomal radiosensitivity was assessed with the G2 and the G0 micro-nucleus (MN) assay. For the G2 assay lymphocytes were irradiated in vitro with a dose of 0.4 Gy 60Co gamma-rays after 70 h incubation and chromatid breaks were scored in 50 metaphases. For the G0 MN assay lymphocytes were exposed in vitro to 3.5 Gy 60Co gamma-rays at low dose rate (LDR). 72 h post-irradiation cultures were arrested and micronuclei were scored in 1000 binucleate cells. The results demonstrated that the group of breast cancer patients was more radiosensitive than a population of healthy women and this with both the G2 and the G0 MN assay. Analyses of the G2 and MN response in different age groups of the breast cancer patients revealed no significant differences in mean G2 and MN scores and suggest that the age of onset of the disease has no effect on chromosomal radiosensitivity in unselected breast cancer patients. Correlations with different clinical parameters were also investigated.     

Wound healing morbidity in STS patients treated with preoperative radiotherapy in relation to in vitro skin fibroblast radiosensitivity, proliferative capacity and TGF-beta activity.

BACKGROUND AND PURPOSE: In a recent study, we demonstrated that the ability of dermal fibroblasts, obtained from soft tissue sarcoma (STS) patients, to undergo initial division in vitro following radiation exposure correlated with the development of wound healing morbidity in the patients following their treatment with preoperative radiotherapy. Transforming growth factor beta (TGF-beta) is thought to play an important role in fibroblast proliferation and radiosensitivity both of which may impact on wound healing. Thus, in this study we examined the interrelationship between TGF-beta activity, radiosensitivity and proliferation of cultured fibroblasts and the wound healing response of STS patients after preoperative radiotherapy to provide a validation cohort for our previous study and to investigate mechanisms. PATIENTS AND METHODS: Skin fibroblasts were established from skin biopsies of 46 STS patients. The treatment group consisted of 28 patients who received preoperative radiotherapy. Eighteen patients constituted a control group who were either irradiated postoperatively or did not receive radiation treatment. Fibroblast cultures were subjected to the colony forming and cytokinesis-blocked binucleation assays (low dose rate: approximately 0.02 Gy/min) and TGF-beta assays (high dose-rate: approximately 1.06 Gy/min) following gamma-irradiation. Fibroblast radiosensitivity and initial proliferative ability were represented by the surviving fraction at 2.4 Gy (SF(2.4)) and binucleation index (BNI), respectively. Active and total TGF-beta levels in fibroblast cultures were determined using a biological assay. Wound healing complication (WHC), defined as the requirement for further surgery or prolonged deep wound packing, was the clinical endpoint examined. RESULTS: Of the 28 patients treated with preoperative radiotherapy, 8 (29%) had wound healing difficulties. Fibroblasts from patients who developed WHC showed a trend to retain a significantly higher initial proliferative ability after irradiation compared with those from individuals in the treatment group with normal wound healing, consistent with the results of our previous study. No link was observed between fibroblast radiosensitivity and WHC. Neither active nor total TGF-beta levels in cultures were significantly affected by irradiation. Fibroblast proliferation in unirradiated and irradiated cultures, as well as radiosensitivity, was not influenced by TGF-beta content. TGF-beta expression in fibroblast cultures did not reflect wound healing morbidity. CONCLUSIONS: These data are consistent with our previous study and combined the results suggest that in vitro fibroblast proliferation after irradiation may be a useful predictor of wound healing morbidity in STS patients treated with preoperative radiotherapy. TGF-beta levels in culture do not predict WHC, suggesting that the role of TGF-beta in wound healing is likely controlled by other in vivo factors.     

Chromosomal radiosensitivity in breast cancer patients with a known or putative genetic predisposition

The chromosomal radiosensitivity of breast cancer patients with a known or putative genetic predisposition was investigated and compared to a group of healthy women. The chromosomal radiosensitivity was assessed with the G2 and the G0-micronucleus assay. For the G2 assay lymphocytes were irradiated in vitro with a dose of 0.4 Gy (60)Co gamma-rays after 71 h incubation, and chromatid breaks were scored in 50 metaphases. For the micronucleus assay lymphocytes were exposed in vitro to 3.5 Gy (60)Co gamma-rays at a high dose rate or low dose rate. 70 h post-irradiation cultures were arrested and micronuclei were scored in 1000 binucleate cells. The results demonstrated that the group of breast cancer patients with a known or putative genetic predisposition was on the average more radiosensitive than a population of healthy women, and this with the G2 as well as with the high dose rate and low dose rate micronucleus assay. With the G2 assay 43% of the patients were found to be radiosensitive. A higher proportion of the patients were radiosensitive with the micronucleus assay (45% with high dose rate and 61% with low dose rate). No correlation was found between the G2 and the G0-micronucleus chromosomal radiosensitivity. Out of the different subgroups considered, the group of the young breast cancer patients without family history showed the highest percentage of radiosensitive cases in the G2 (50%) as well as in the micronucleus assay (75-78%).     

Radiobiological characteristics of solid tumours depending on the p53 status of the tumour cells, with emphasis on the response of intratumour quiescent cells

Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mTP53) or with a neo vector as a control (SAS/neo) were inoculated subcutaneously (s.c.) into both hind legs of Balb/cA nude mice. Mice bearing tumours received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumours. The mice then received gamma-ray irradiation. Another group of mice received a series of test doses of gamma-rays while alive or after tumour clamping to obtain hypoxic fractions (HFs) in the tumours. Right after irradiation, the tumour cells were isolated and incubated with a cytokinesis blocker. The micronucleus (MN) frequency in the cells without BrdU labelling (=quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, tumour cell suspensions obtained in the same manner were used for determining the frequency of apoptosis in the Q cells. The MN frequency and apoptosis frequency in total (P+Q) tumour cells were determined from the tumours that were not pretreated with BrdU. In total cell populations, SAS/mTP53 cells were more radioresistant than SAS/neo cells in clonogenic survival. Q tumour cells exhibited a significantly lower apoptosis and MN frequency, probably due to their much larger HF, than total cells. In both total and Q cell fractions, SAS/mTP53 cells were less susceptible to apoptosis and more susceptible to micronucleation than SAS/neo cells. Obviously, TP53 status had the potential to influence the radiosensitivity of not only the total cells, but also the Q cells. However, irrespective of the TP53 status, significant differences in radiosensitivity between total and Q tumour cells were consistently observed. From the viewpoint of tumour control as a whole, including intratumour Q tumour cell control, a treatment modality for enhancing the Q cell response has to be considered.     

Alteration of Radiosensitivity of Quiescent Cell Populations in Solid Tumors Irradiated with X-Rays Twice at Various Intervals

5-Bromo-2'-deoxyuridine (BUdR) was injected into SCC VII or EMT6/KU tumor-bearing mice intraperitoneally to label all the proliferating tumor cells. First, the mice were irradiated with X-rays at a dose of 10 Gy, followed by a dose of 0–20 Gy at 0, 12, 24 or 48 h later. During the interval, no BUdR was injected. Immediately after the second irradiation, the tumors were excised and trypsinized. The micronucleus (MN) frequency in cells without BUdR labeling was determined by means of incubation with cytochalasin-B (a cytokinesis-blocker) and immunoftuorescence staining for BUdR. When the tumors were not pretreated with BUdR before the first irradiation, the MN frequency in all tumor cells was determined. To determine the labeling indices of SCC VII and EMT6/KU tumors at the time of the second irradiation, each group also included mice that were continuously administered BUdR until just before the second irradiation using mini-osmotic pumps which had been implanted subcutaneously 5 days before the first irradiation. The MN frequency of all tumor cell populations obtained immediately after the second irradiation decreased in proportion to the increase in interval time. However, in both tumor systems, the MN frequency of unlabeled cell populations, which could be regarded as quiescent cells in the tumors at the time of the first irradiation, was raised with increase in the interval time. In addition, the labeling index at the second irradiation was higher than that at the first irradiation. These findings support the occurrence of recruitment from quiescent to proliferating state during fractionated irradiation.     

Partial volume rat lung irradiation; assessment of early DNA damage in different lung regions and effect of radical scavengers.

PURPOSE: These studies were designed to examine radiation-induced in-field and out-of-field DNA damage in rat lung as a function of dose and various volumes of irradiation. They also determined whether superoxide dismutase (SOD) and nitro-L-arginine methyl ester (L-NAME) protected against this damage. METHODS AND MATERIALS: The whole lung, or various volumes of the lower or upper lungs of Sprague-Dawley rats were exposed to doses up to 20 Gy of 60Co gamma rays. Radiation-induced DNA damage was quantified in fibroblasts obtained at 18 h after irradiation from both irradiated and shielded lung regions using a micronucleus assay. The radioprotective role of SOD (CuZnSOD: 10 mg/kg body weight; MnSOD: 50-100mg/kg body weight) and L-NAME (0.2 mg/kg body weight.) in vivo was determined by injecting them into rats 30 min before or immediately after a dose of 10 Gy. RESULTS: Micronucleus formation was approximately linear with dose up to 15 Gy. When 70% of the lung volume was irradiated with 10 Gy, irradiated lower lung gave similar numbers of micronuclei (MN)/binucleate cell (BN) to that observed following whole lung irradiation (0.91 MN/BN), whereas the irradiated upper lung gave only 0.66 MN/BN. Following lower lung irradiation, the shielded upper lung (30% of lung volume) showed substantial (out-of-field) damage (0.43 MN/BN). When 30% of the lung was given 10 Gy, irradiated upper or lower lung showed similar amounts of in-field damage (0.43 MN/BN) but this was smaller than that seen following irradiation of 70% of the lung volume. For 30% lower lung irradiation, the shielded upper lung showed only a small out-of-field effect (0.1 MN/BN). For both volumes of irradiation there was a similar or smaller effect in the shielded lower lung after upper lung irradiation. Injection of SOD before or L-NAME after 10 Gy to the lower 70% lung volume resulted in a reduction in DNA damage both in-field and out-of-field but the percentage was much greater for out-of-field damage (50-60%) than for in-field damage (10-30%). Following whole lung irradiation (10 Gy) significantly greater DNA damage was observed in fibroblasts from the left lung than from the right lung (0.93 MN/BN vs. 0.82 MN/BN). Following whole lung irradiation there was no significant difference in DNA damage observed in fibroblasts from the lower lung and the upper lung. CONCLUSIONS: With partial lung irradiation the lower lung sustains more in-field DNA damage following irradiation than the upper lung, whereas out-of-field effects are observed primarily in the upper lung (i.e. following lower lung irradiation). Following whole lung irradiation the left lung sustains more damage than the right lung but there is no difference between the upper and lower lung. The protective effects of SOD and L-NAME suggest that inflammatory cytokines induced by the irradiation may be involved in the initiation of a reaction resulting in the production of reactive oxyradicals and nitric oxide that cause indirect DNA damage both in and out of the radiation field.