Changes in Cell Proliferative Parameters of SCCVII and EMT6 Murine Tumors after Single-dose Irradiation

To understand better the repopulation kinetics of tumor cells after radiotherapy, we Investigated changes in cell proliferative parameters after single-dose irradiation of SCCVII tumors in C3H/He mice and EMT6 tumors in Balb/c mice. The following parameters were determined 0–15 days after single irradiation at 20 or 30 Gy; dividing fraction (DP), potential doubling time (Tpot), number of clonogenic cells per tumor (Ncln), and volume doubling time (Tvol). DF and Tpot were determined by in vivo - in vitro cytokinesis-block assay with cytochalasin B, Ncln was measured by in vivo - in vitro colony-forming assay, and Tvol was determined by growth delay assay. In both tumors, longer Tpot and lower DF and Ncln were obtained for 3–4 days after irradiation, but in SCCVII tumors these values returned to the pretreatment levels 9 days after irradiation. In EMT6 tumors, Tpot, DF, and Ncln did not return to the pretreatment levels even 12 days after irradiation. In the regrowth phase of both tumors following irradiation at 20 Gy, Tvol was longer than the pretreatment level, although Tpot was similar in SCCVII and only slightly longer in EMT6. Therefore, the cell loss factor in the regrowth phase was considered to be higher than the pretreatment level in both tumors. From the results, recruitment of previously quiescent cells into the proliferative pool in these tumors was suggested to contribute to repopulation after radiation.     

Determination of radiation-induced damage in lymphocytes using the micronucleus and microgel electrophoresis ‘Comet’ assays

DNA damage assays may be useful as rapid predictors of normal tissue radiosensitivity in clinical samples. We measured in vitro radiation-induced (2 Gy) damage to lymphocytes from cancer patients and normal healthy donors using both the micronucleus and microgel electrophoresis (Comet) assays simultaneously. For damage assessment, there was a good correlation (P < 0.001) between the mean comet lengths and the fraction of cells with comets. There was no correlation with initial damage, determined as the proportion of cells within a sample that formed comets, in comparison with the mean frequency of micronuclei per binucleate cell. However, there appeared to be an association between the determination of repair proficiency in the Comet assay and the mean frequency of micronuclei per binucleate cell in lymphocytes from cancer patients.     

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.     

Influence of Treatment Time on the Response of Rat Rhabdomyosarcoma Rlh to Fractionated Irradiation

The aim of this work was to study influence of overall treatment time on response of an experimental tumour to fractionated radiotherapy. Rhabdomyosarcoma RlH of the rat was treated by fractionated irradiation applying 30 fractions in different overall treatment times of 10, 18.5, 29, 39, and 67 days. The doses per fraction ranged from 1.50 to 2.67 Gy. Tumour response was assessed by net growth delay and by an in vitro colony assay. The results indicate that repopulation of the tumour with clonogenic tumour cells slowed down during treatment leading to an average doubling time of clonogenic tumour cells of 8 ± 1 days, which is considerably longer than that of unirradiated tumours (3 days). The repopulation rate seemed to increase after about 3 weeks of treatment with a doubling time of tumour clonogens of 5 ± I days, which was still lower than in control tumours.     

The increment of micronucleus frequency in cervical carcinoma during irradiation in vivo and its prognostic value for tumour radiocurability.

A potential usefulness of micronucleus assay for prediction of tumour radiosensitivity has been tested in 64 patients with advanced stage (II B-IV B) cervical carcinoma treated by radiotherapy. The study of cellular radiosensitivity in vitro was conducted in parallel with the study of cellular damage after tumour irradiation in vivo. Radiosensitivity of in vitro cultured primary cells isolated from tumour biopsies taken before radiotherapy was evaluated using cytokinesis-block micronucleus assay. Frequency of micronuclei per binucleated cell (MN/BNC) at 2 Gy was used as a measure of radiosensitivity. Radiation sensitivity in vivo was expressed as per cent increment of micronucleus frequency in cells isolated from biopsy taken after 20 Gy (external irradiation, 10 x 2 Gy) over the pre-treatment spontaneous micronucleus level and was called MN20. Very low correlation (r = 0.324) was observed between micronucleus frequency in vitro and in vivo. Although micronucleus frequency at 2 Gy differed widely between tumours evaluated (mean MN/BNC was 0.224; range 0.08-0.416), no significant correlation was observed between this parameter and clinical outcome. The average increment of micronucleus frequency after 20 Gy amounted to 193% of spontaneous level (range 60-610%) and was independent of spontaneous micronucleation before radiotherapy. In contrast to in vitro results, these from in vivo assay seem to have a predictive value for radiotherapy of cervix cancer. The micronucleus increment in vivo that reached at least 117.5% of pretreatment value (first quartile for MN20 data set) correlated significantly with better tumour local control (P < 0.008) and overall survival (P < 0.045). Our results suggest that evaluation of increment of micronucleus frequency during radiotherapy (after fixed tested dose of 20 Gy) offers a potentially valuable approach to predicting individual radioresponsiveness and may be helpful for individualization of treatment strategy in advanced stage cervical cancer.     

Chemical Modification of Adrenal Response to External Whole Body Irradiation in Mice

Adult Swiss albino mice were exposed to whole body gamma irradiation with 1.5, 3.0, 6.0 and 9.0 Gy in the presence or absence of the protective drugs MPG and WR-2721. The changes in the total cell population, pyk-notic nuclei and necrotic cells, and binucleate cells were observed at various post-irradiation times. The degree of damage increased with the radiation dose. The number of total cells decreased with a corresponding increase in pyknotic nuclei, necrotic cells and binucleate cells. Both drugs provided protection against radiation injury, and reduced early post-irradiation cell damage and enhanced recovery after sublethal exposures. After higher doses, especially after lethal doses, the drugs became less effective in protecting the tissue.     

Repopulation of gamma-irradiated Lewis lung carcinoma by malignant cells and host macrophage progenitors.

Cellular repopulation in Lewis carcinoma irradiated with 60Co gamma-rays was examined by performing sequential cell-survival estimations using an in vitro soft-agar-colony assay. Following local irradiation (15--35 Gy) two distinct types of colony were seen: compact colonies with tightly packed cells and diffuse colonies with widely dispersed cells. Maximal diffuse colony formation in vitro was only obtained in the simultaneous presence of adequate numbers of compact colonies. After whole-body irradiation only compact colonies were observed. Only-cell survival data from compact colony counts correlated with cell survival estimated by the lung colony assay and we conclude that compact colonies are produced by clonogenic tumour cells. Cytochemical and immunological evidence showed that diffuse colonies were composed of macrophages. After local irradiation the initial kill of clonogenic tumour cells was dose dependent. At each dose level, repopulation began immediately and proceeded with a doubling time of about 1 day. Macrophage colony-forming cells (macrophage progenitors) per tumour were initially reduced by about 3 decades, but recovered very rapidly to reach pretreatment levels within 2 days. We conclude that at least two populations of clonogenic cells are present in Lewis lung carcinoma, tumour cells that repopulate irradiated tumours by in situ proliferation and host-macrophage progenitors that repopulate locally irradiated tumours by infiltration. The hazards of confusing host and tumour cell colonies in in vitro assay systems are stressed.     

Relationship between radiation induced dicentric chromosome aberrations and micronucleus formation in human lymphocytes

Chromosome damage measured by the chromosome aberration technique is a reliable method to assess the radiation dose absorbed by cells. However, this technique has some disadvantages. Scoring is difficult and requires skill and experience which of these lead low number of cell counts. The micronucleus (MN) technique which also measures chromosome losses has easy scoring criteria leading high numbers of cell counts and therefore holds more statistical power. In this study, the relationship between the results of the micronucleus technique and those obtained by the chromosome aberration technique was investigated after radiation doses of 1Gy, 2Gy, 3Gy and 4Gy to peripheral blood lymphocytes of 3 healthy individuals. Increases in the chromosome damage after radiation were observed in both techniques. When the dicentric aberration frequencies that were measured in the chromosome aberration technique and the micronucleus frequencies were compared, no difference (p > 0.05) between these two independent measures of radiation damage was reported. The relationship between the micronuclei and the free acentric chromosome aberrations measured in the chromosome aberration technique was not significant as well as that between the dicentrics and micronuclei. On the basis of the relationship between the dicentric aberrations and the micronucleus frequencies, the micronucleus technique with an easy and short-term application and with an easy scoring can be used as an alternative to the chromosome aberration technique.     

Inhibition of radiation-induced DNA damage by jamun, Syzygium cumini, in the cultured splenocytes of mice exposed to different doses of γ-radiation

The radioprotective property of 50 mg/kg body weight jamun (Syzygium cumini) extract was studied in the cultured splenocytes of mice exposed to 0, 0.5, 1, 2, 3, or 4 Gy of γ-radiation. The spleens of irradiated mice were removed aseptically and the splenocytes were extracted from the individual spleens and cultured. The micronuclei were prepared 72 hours after irradiation in binucleate splenocytes by blocking cytokinesis with cytochalasin-B. Irradiation of mice resulted in a dose-dependent elevation in the micronucleated splenocytes. The exposure of mice not only elevated splenocytes bearing one micronucleus but also cells bearing 2 and multiple (>2) micronuclei indicating induction of complex DNA damage after irradiation. Oral treatment of mice with 50 mg/kg body weight of jamun leaf extract protected against the radiation-induced micronuclei formation. Jamun extract also protected against the formation of 2 and multiple micronuclei indicating repair or inhibition of complex DNA damage. The assessment of lipid peroxidation in mice brain homogenate has indicated a concentration dependent inhibition of lipid peroxidation by jamun extract. Studies in a cell free system revealed that jamun extract inhibited the formation of OH, O(2)-, DPPH, and ABTS(+) free radicals in a concentration dependent manner. Our study demonstrates that jamun extract protected mice against the radiation-induced DNA damage and inhibition of radiation-induced free radical formation may be one of the mechanisms of radioprotection.     

Peripheral Blood Lymphocytes as In Vitro Model to Evaluate Genomic Instability Caused by Low Dose Radiation

Diagnostic and therapeutic radiation fields are planned so as to reduce side-effects while maximising the dose to site but effects on healthy tissues are inevitable. Radiation causes strand breaks in DNA of exposed cells which can lead to chromosomal aberrations and cause malfunction and cell death. Several researchers have highlighted the damaging effects of high dose radiation but still there is a lacuna in identifying damage due to low dose radiation used for diagnostic purposes. Blood is an easy resource to study genotoxicity and to estimate the effects of radiation. The micronucleus assay and chromosomal aberration can indicate genetic damage and our present aim was to establish these with lymphocytes in an in vitro model to predict the immediate effects low dose radiation. Blood was collected from healthy individuals and divided into 6 groups with increasing radiation dose i.e., 0Gy, 0.10Gy, 0.25Gy, 0.50Gy, 1Gy and 2Gy. The samples were irradiated in duplicates using a LINAC in the radiation oncology department. Standard protocols were applied for chromosomal aberration and micronucleus assays. Metaphases were stained in Giemsa and 200 were scored per sample for the detection of dicentric or acentric forms. For micronuclei detection, 200 metaphases. Giemsa stained binucleate cells per sample were analysed for any abnormality. The micronuclei (MN) frequency was increased in cells exposed to the entire range of doses (0.1- 2Gy) delivered. Controls showed minimal MN formation (2.0%0.05) with triple MN (5.6%2.0) frequency at the lowest dose. MN formation increased exponentially with the radiation dose thereafter with a maximum at 2Gy. Significantly elevated numbers of dicentric chromosomes were also observed, even at doses of 0.1- 0.5Gy, compared to controls, and acentric chromosomes were apparent at 2Gy. In conclusion we can state that lymphocytes can be effectively used to study direct effect of low dose radiation.     

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%).     

人结肠癌放射抗拒性细胞株的建立

目的应用连续照射的方法建立人结肠癌放射抗拒细胞株SW480-R,为研究人结肠癌放射抗拒的机制提供模型。方法用2Gv剂量的x射线照射人结肠癌SW480细胞株,每天1次,每周5d,分别照射不同周期后,按照细胞存活率筛选出放射抗拒细胞株SW480．R,并检测细胞的倍增时间。以细胞克隆形成实验检测细胞的放射敏感性,MTT法检测照射后不同时间点的细胞存活分数,流式细胞仪检测细胞的细胞周期分布,并分别与亲代结肠癌细胞株进行比较。结果经2Gy照射3周后细胞仍有存活,可作为放射抗拒细胞株的模型。SW480．R细胞株的倍增时间明显长于亲代SW480细胞株（脚．05）,SW480-R细胞株的放射抗拒l生增加,照射后12h的细胞存活率较SW480细胞株显著增高（98．40％US92．81％,P〈0．001）。流式细胞仪检测细胞周期显示,SW480一R细胞株的细胞周期分布与SW480细胞株不同,G2／M期明显增高。结论人结肠癌细胞株SW480每天经x射线照射2Gy,连续照射3周后可以得到具有放射抗拒细胞株SW480．R。此细胞株具有稳定的放射抗拒性,且与亲代细胞株有不同的细胞周期分布。     

Estimation of the dividing fraction and potential doubling time of tumors using cytochalasin B

We devised a new in vitro method to estimate the proportion of dividing cells and the potential doubling time (Tpot) of tumors using the same technique as with the cytokinesis-block micronucleus assay. The usefulness of this methodology was confirmed by comparing the data with those obtained by flow cytometry after bromodeoxyuridine (BrdUrd) incorporation. Xenografted human and murine tumors were excised 0.5-8 h after BrdUrd injection and disaggregated to single cells. A portion of these cells was then plated in dishes to which 1 or 2 micrograms/ml cytochalasin B were added. These concentrations of cytochalasin B blocked cytokinesis but not karyokinesis with the result that cells became multinucleate after mitoses. At every 12 or 24 h of culture, the proportion of multinucleate cells and the total number of nuclei and cells were scored. The remaining cells were analyzed with a flow cytometer and the BrdUrd-labeling index and Tpot were determined. In all 8 tumor lines studied, the proportion of multinucleate cells reached a plateau within 3-7 days of culture, and we therefore defined the dividing fraction as the plateau value. The dividing fraction ranged between 33 and 98% and clearly tended to be high in rapidly growing tumors. A significant correlation was seen between the dividing fraction and BrdUrd-labeling index (r = 0.74, P less than 0.001). The increase in the average number of nuclei per cell also tended to be higher in rapidly growing tumors. The Tpot was estimated as the time for this nucleus/cell ratio to reach 2.0. In 7 of 8 tumor lines, Tpot values estimated by this method compared reasonably with those estimated by the BrdUrd method. Therefore, this simple technique, originally developed for radiosensitivity prediction, would also seem to be useful in estimating tumor proliferative activity.     

Microscopic and flow cytometric study of micronuclei in iododeoxyuridine labelled cells irradiated with soft x-rays

Iododeoxyuridine labelled (IUdR(+)) and unlabelled (IUdR(-)) CHO cells irradiated with 2 Gy of soft x-rays showed only minor differences in the kinetics of micronuclei formation during the first 20 hours postirradiation period. Between 20 to 40 hours, the IUdR(-) cells showed approximately a constant number # of micronuclei while the number of micronuclei in IUdR(+) cells was still increasing. The frequency of micronuclei was higher in IUdR(+) cells compared to IUdR(-) cells at 24 hours after irradiation with various doses up to 4.0 Gy. Dose modifying factors were found to be 1.3 (microscopic evaluation) and 1.8 (flow cytometric evaluation). Flow cytometry with use of two parameters, fluorescence from propidium iodide and light scattering, seems to be a good tool to estimate the frequency of micronuclei in CHO cells in the dose range up to about 4 Gy. At higher doses perturbation of the cell cycle and the appearance of dying cells will influence the results.     

Chromosomal damage in two X-ray irradiated cell lines: influence of cell cycle stage and irradiation temperature

The aim of this study was to investigate if irradiation with X-rays in different cell cycle phases resulted in a different response as measured with the micronucleus technique. In addition, the influence of irradiation temperature was investigated. MATERIALS AND METHODS: Cells from a non-transformed human fibroblast cell line, HS2429, and a human breast cancer cell line, MCF-7, were synchronized by thymidine block and irradiated at either 2 degrees C or 37 degrees C in the G1-, S- and G2/M-phases. After cytokinesis-block by cytochalasin B, the frequency of micronuclei was determined. RESULTS: Clear dose-response relationships were found. More micronuclei were detected in fibroblast cells irradiated in G1 and S than in G2/M, while the differences were not as prominent in MCF-7 cells. The irradiation temperature had no significant influence on the formation of micronuclei in either of the cell lines. CONCLUSION: The formation of micronuclei varies with the cell cycle stage at the time of irradiation.     

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.     

Radiation-induced DNA damage and its repair in lymphocytes of patients with head and neck cancer and healthy donors

BACKGROUND: DNA repair capacity may be an important factor in determining both individual susceptibility to cancer and the response to cancer therapy. The aim of this work was to compare DNA damage and the repair process in cells originating from healthy donors and cancer patients. MATERIALS AND METHODS: Using the micronucleus and comet assays, we compared the induction of DNA damage and its repair in lymphocytes isolated from blood samples of 14 healthy donors and 24 patients with head and neck tumours. Gamma-rays at the dose of 2 or 4 Gy were used as the damaging factor. The micronucleus test was performed according to Fenech (1) and the comet assay according to Green et al. (2). RESULTS AND CONCLUSION: Lymphocytes of both healthy donors and tumour patients showed great diversification in reaction to the same dose of gamma irradiation as well as differences in the kinetics of DNA repair. The patient group contained significantly more individuals whose lymphocytes were characterized by higher background DNA damage and higher damage inducibility. Blood cells of donors showing high damage inducibility also showed increased levels of micronuclei induced by ionizing radiation. Micronuclei induction did not correlate with a high level of unrepaired DNA damage.     

脑胶质瘤不同照射方案生物效应的实验研究

目的 通过对人脑多形性胶质母细胞瘤(BT325细胞系)裸小鼠移植瘤不同分割模式照射后生物效应的实验研究,加深对人脑胶质瘤放射反应生物学特性认识,为临床设计照射计划、制定合理分次治疗方案提供实验依据.方法 对BT325细胞系裸小鼠移植瘤进行单次10、20、30、40、60 Gy照射和2 Gy每周5次每天照射、3 Gy每周3次隔天照射、3 Gy每周5次每天照射、4 Gy每周3次隔天照射,总剂量分别为125、114、126、112 Gy.用肿瘤生长曲线评价剂量效应关系并测定肿瘤体积倍增时间.取贴壁生长的指数生长期BT325细胞,用生长曲线测定细胞倍增时间,细胞克隆形成分析法绘制细胞存活曲线(照射剂量为0、1、2、4、6、8、10 Gy)计算曲线参数值,彗星分析法测定DNA单链断裂半修复时间(T1/2).结果 单次照射各剂量点均不能有效控制肿瘤.2 Gy5次/周和3 Gy3次/周治疗方案对人脑胶质瘤实体瘤也无明显治疗效果.增加分次剂量可使脑胶质瘤实体肿瘤有较明显的消退,其中4 Gy3次/周方案好于其他方案但仍未能达到治愈肿瘤的效果.表明对肿瘤干细胞的控制不理想.各项生物学参数的测定结果:BT325细胞倍增时间为30.16 h,裸小鼠移植瘤肿瘤倍增时间为43 d;细胞存活曲线LQ模型拟合的α=0.360 Gy-1、β=0.057 Gy-2,多靶单击模型拟合的D0=1.394 Gy、Dq=2.127 Gy、SF2=0.714;5 Gy照射DNA单链断裂的T1/2=9.999 min.结论 本实验从实证实验角度印证了临床上脑胶质瘤是一种放射耐受性较高的肿瘤的看法.研究结果提示增高分割剂量有可能提高肿瘤的近期疗效(使肿瘤消退率增加),但如何提高对肿瘤干细胞的控制还需进一步深入研究.各项生物学参数测定结果提示脑胶质瘤细胞内在放射敏感性较差.     

Increase in the fraction of necrotic, not apoptotic, cells in SiHa xenograft tumours shortly after irradiation.

BACKGROUND AND PURPOSE: Approximately 18% of the cells recovered by rapid mechanical dissociation of SiHa xenograft tumours contain large numbers of DNA strand breaks. The number of damaged cells increases to 30-40% 4-6 h after exposure to 5 or 15 Gy, returning to normal levels by 12 h. This observation is reminiscent of the rate of production of apoptotic cells in other murine and human xenograft tumours. The nature of this damage, rate of development and relation to cell proliferation rate were therefore examined in detail. MATERIALS AND METHODS: SiHa human cervical carcinoma cells were grown as xenograft tumours in SCID mice. Single-cell suspensions were prepared as a function of time after irradiation of the mouse and examined for DNA damage using the alkaline comet assay. Cell cycle progression was measured by flow cytometry evaluation of anti-bromodeoxyuridine-labelled tumour cells. RESULTS: Significant numbers of apoptotic cells could not be detected in irradiated SiHa tumours using an end-labelling assay, electron microscopy, or histological examination of thin sections. Instead, xenograft cells exhibiting extensive DNA damage in the comet assay were predominantly necrotic cells. The increase in the proportion of heavily damaged cells 4-6 h after irradiation could be the result of an interplay between several factors including loss of viable cells and change in production or loss of necrotic cells. Analysis of the progression of BrdUrd-labelled cells confirmed that while 35% of cells from untreated SiHa tumours had divided and entered G1 phase by 6 h after BrdUrd injection, none of the labelled cells from tumours exposed to 5 or 15 Gy had progressed to G1. CONCLUSIONS: The increase in the percentage of SiHa tumour cells with extensive DNA damage 4-6 h after irradiation is attributable to necrosis, not apoptosis. Cell cycle progression and cell loss are likely to influence the kinetics of appearance of both apoptotic and necrotic cells in irradiated tumours.     

PLD-repair in human melanoma xenografts following single dose and fractionated irradiation

PLD-repair following single dose and fractionated irradiation was studied in vivo using five human melanoma xenograft lines. Tumours given single graded radiation doses were excised immediately after or 24 h after the radiation exposure for assay of cell survival in vitro. All melanoma lines showed PLD-repair after single dose irradiation: the PLD-repair factors, i.e. the ratio of the Do values for tumours excised 24 h after and immediately after irradiation, ranged from 1.2 +/- 0.1 to 1.4 +/- 0.1. PLD-repair following fractionated irradiation was studied by giving tumours seven fractions of 2.0 Gy over 7 days and then, after an interval of 24 h, single graded radiation doses in the range 6-21 Gy. Cell survival was assayed in vitro immediately after or 24 h after the last radiation exposure. The Do values as well as the surviving fractions were approximately equal after immediate and delayed cell seeding, i.e. none of the melanoma lines showed significant PLD-repair after fractionated irradiation. The lack of PLD-repair after fractionated irradiation was possibly a consequence of radiation-induced recruitment of quiescent tumour cells into the cell cycle. Consequently, PLD-repair is probably not a major cause of failure in the radiation therapy of malignant melanoma when treated with 2.0 Gy fractions.