In vitro radiosensitivity of human medulloblastoma cell lines.
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Brain Neoplasms↗
Biomedical subjects
G G Steel
Publications and source records attributed to G G Steel.
Cell-cycle progression during continuous low dose rate irradiation of a human bladder carcinoma cell line.
At very low radiation dose rates, the proliferation of mammalian cells continues unaffected but as the dose rate is increased there comes a point at which it is interrupted. The dose rate at which this happens is often thought to be a significant factor in the effects of brachytherapy: it may determine the range from an implanted source at which cell-cycle redistribution and repopulation effects will occur. By means of mitotic counts and DNA flow cytometry, we have examined the dose rate effect in a human bladder carcinoma cell line (MGH-U1). Irradiation at dose rate 0.1 cGy/min had little or no effect on cell-cycle progression. Suppression of mitosis and arrest of cells in G2 was observed at 0.4 cGy/min and above. Surprisingly, the duration of mitotic arrest showed little dose rate dependence; it was followed by an overshoot of cells in mitosis after 24-39 h of irradiation. An even more pronounced overshoot of cells in G2 occurred and persisted throughout the irradiation period. The cell kinetic data indicate that after the temporary block in cell-cycle progression, cell proliferation continued at all dose rates up to 1.4 cGy/min. We have evaluated these results in the light of previous studies in this department of the dose rate effect for cell survival in the MGH-U1 cell line. After 24 h irradiation at 1.4 cGy/min the surviving fraction was below 10(-2), also after 30 h at 1.0 cGy/min. When cell-cycle blockade is considerable, so is the level of cell killing. Flow-cytometric data therefore are dominated by the properties of cells that are doomed to die.(ABSTRACT TRUNCATED AT 250 WORDS)
Cell Cycle↗
Mouse hemithoracic irradiation and its interaction with cytotoxic drugs.
Irradiation of the mouse right or left hemithorax at 14 or 18 Gy produced a dose-dependent rise in breathing rate 16 weeks after irradiation without significant mortality. The measurement of breathing rate changes following right hemithoracic irradiation combined with the maximally tolerated dose of cytotoxic drugs was assessed as a method for qualitatively detecting drug-irradiation interactions which either exacerbate pneumonitis or alter its time course. Cyclophosphamide at 100 mg/kg accentuated and accelerated the rise in breathing rate, culminating in early mortality. BCNU 30 mg/kg delayed the appearance of the radiation response. Busulphan 30 mg/kg appeared to be radioprotective, but this was shown to be due to the DMSO-containing vehicle. Doxorubicin 6 mg/kg had no effect when combined with right or left hemithoracic irradiation. Carboplatin 100 mg/kg, vindesine 4 mg/kg and vinblastine 4 mg/kg had no substantial effect upon the changes in breathing rate.
Animals↗
Heterogeneity of radiosensitivity in a human glioma cell line.
Sixteen clones were isolated from an early-passage human glioma cell line (IN859) and have been found to show variation in several biological characteristics including DNA content, modal chromosome number, and morphology. In addition, heterogeneity of radiosensitivity was detected: the doses that gave a surviving fraction of 0.01 varied by a factor of approximately 1.5. The most sensitive (clone 6) and the most resistant (clone 9) clones were selected for further study; their surviving fractions at 2Gy (SF2) were 0.37 and 0.64, respectively. When compared at a fixed radiation dose the sensitive clone surprisingly demonstrated greater split-dose recovery than the resistant clone; it also showed greater low dose-rate sparing.
Aged↗
Differences in the level of DNA double-strand breaks in human tumour cell lines following low dose-rate irradiation.
It is now well accepted that differences exist in the intrinsic radiosensitivity of human tumour cells although the molecular basis of this is still unclear. Current evidence suggests that of the lesions induced in DNA by ionising radiation, double-strand breaks (DSB) are the most closely linked to cell death. In this study, levels of DSB were measured by neutral filter elution under conditions of both repair inhibition and maximum recovery and compared with clonogenic survival curves for high (HDR) and low dose-rate (LDR) irradiation in human carcinoma lines of differing radiosensitivity. Four human lung carcinoma lines were used, two small-cell (SCLC; HC12 and HX149) and two non-small cell lines (NSCLC; HX147A7 and HX148G7). Cell survival was measured by soft agar and monolayer colony-forming assays as appropriate and a large variation in sensitivity of the cell lines was seen (alpha values of 0.06 to 0.56 Gy-1). We have previously reported that the damage induced at high dose rate does vary in these cell lines but not in a way which correlates with their cell survival response [5]. Following irradiation to 15 Gy at low dose rate essentially no DSBs were detected in any of the four lines but at 70 Gy the more sensitive SCLC showed more residual damage than in the more radioresistant NSCLC lines. The prime determinant of the difference between the LDR and HDR damage curves is likely to be repair occurring during irradiation.(ABSTRACT TRUNCATED AT 250 WORDS)
Cell Survival↗
Cellular recovery in two sub-lines of the L5178Y murine leukaemic lymphoblast cell line differing in their sensitivity to ionizing radiation.
Cellular recovery was assessed in two sublines of L5178Y murine lymphoma cells of differing radiosensitivity (LY-S and LY-A4) using low dose-rate irradiation and split-dose experiments. No increase in cell survival was observed in the LY-S cell line until the dose-rate was reduced to 2 cGy/min, whereas in the LY-A4 cell line 20 cGy/min was low enough to detect changes in survival. The extent of this change, as assessed by dose reduction factors at 2 logs of cell kill, was greater in the LY-A4 cell line. Fitting these data with the incomplete repair model of Thames led to anomalous values for the half-time of repair. In split-dose experiments the maximum observed recovery ratio increased as a function of dose in a manner that is consistent with the linear-quadratic equation. As was found previously with radiosensitive human tumour cells, the LY-S cell line showed more split-dose recovery at any given dose than the LY-A4 cell line.
Animals↗
The intrinsic alpha/beta ratio for human tumour cells: is it a constant?
The radiation response of 15 mammalian cell lines comprising 11 human tumour, two human fibroblast and two murine lymphoma cell lines, has been analysed using the linear-quadratic equation. As well as using conventional analysis of acute dose-survival curves to derive values for alpha and beta (termed alpha ac and beta ac), low dose-rate and split-dose experiments have been used to derive independent values of alpha and beta (alpha 1dr and beta RR), respectively. alpha 1dr provides a measure of irrecoverable damage, the magnitude of which agreed well with the initial slope of the acute survival curve for most cell lines. beta RR derived from split-dose experiments represents a unique measure of recovery for each cell line. Large differences were found between individual values of beta ac and beta RR, especially in the radiosensitive cell lines. Since beta RR is a functional measure of recovery we suggest that this is the more relevant parameter in studies of dose sparing. The most striking result of this analysis was found in considering the alpha/beta ratios. No relationship was observed between alpha ac and beta ac resulting in values of alpha ac/beta ac ranging from 1 to 175. In contrast a positive correlation was observed between alpha 1dr beta RR in the 11 tumour cell lines, giving an alpha/beta ratio of 9.4 +/- 1.8 Gy. This observation of the relative constancy of the ratio for human tumour cells leads to an hypothesis about the role of initial damage as a determinant of radiosensitivity.
Animals↗
The nature of the initial slope of radiation cell survival curves.
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Animals↗
Evaluation of cell attachment matrix (CAM) coated plates for primary culture of human tumour biopsies.
Baker et al. [Cancer Res. 46: 1263-1274, 1986] developed an adhesive tumour cell culture system (ATCCS) using a culture surface coated with a cell attachment matrix (CAM). The ability of CAM-coated plates to support the growth of cells from human tumour biopsies has been evaluated. Successful growth was obtained in 9/22 samples (41%), but fibroblasts, rather than tumour cells, grew in the majority. Comparison of CAM with other surfaces showed that CAM was no better for establishing tumour cell growth than the presence of feeder cells or an alternative attachment factor vitronectin.
Animals↗
The ESTRO Breur lecture. Cellular sensitivity to low dose-rate irradiation focuses the problem of tumour radioresistance.
This paper emphasises the radiobiology of human tumour cells irradiated at the relatively low dose rate of 1 Gy/h (i.e.1-2 cGy/min), described here as the "Regaud dose rate". Continuous irradiation at this dose rate is approximately isoeffective with fractionated radiotherapy using 2 Gy/fractions. At the Regaud dose rate, cell survival curves are approximately exponential and they appear to extrapolate the initial slope of the high dose rate survival curve. Little recovery occurs after such treatments since it has largely taken place during irradiation. At the Regaud dose rate human tumour cell lines show a wide range of radiosensitivities, differing by a factor of around 7. This may well be the most clinically-relevant way of describing the radiosensitivity of tumour cells. Current models of radiation cell killing envisage a component of damage that increases linearly with dose. It is this component that dominates the slope of the Regaud survival curve. It may be produced by DNA damage due to clusters of ionisation events, or perhaps by damage to hypersensitive parts of the genome. The steepness of this component of damage may be modified by exogenous inhibitors of DNA damage repair.
Cell Survival↗
Cell proliferation in human tumour xenografts: measurement using antibody labelling against bromodeoxyuridine and Ki-67.
Cell proliferation was investigated in human tumour xenografts using bromodeoxyuridine (BrdUrd) labelling, evaluated either by flow cytometry or in tissue sections, and also using the proliferation marker Ki-67. BrdUrd labelling was found to increase when cryostat tumour sections were digested with an enzymic solution. This yielded a labelling index up to four times higher than that obtained using the flow cytometer. Ki-67 indices were found to be higher than those reported for human tumour biopsies, as may be expected due to the enhanced growth rate of the xenografts. Significant heterogeneity was observed in the results for cervix, breast and bladder tumours, and the results of the three methods were poorly correlated. However, three of the four tumour types showed that the tumour with the lowest Ki-67 index also had the longest potential doubling time. Since the measurement of Ki-67 index was found technically easier to perform, and also adequately reflects relative tumour cell proliferation, it is preferred over the other techniques.
Animals↗
Radiosensitivity, recovery and dose-rate effect in three human glioma cell lines.
The results of radiotherapy in the treatment of high-grade gliomas are disappointing. In this study three recently established cell lines from high-grade human gliomas have been found to exhibit a sensitivity that is at the resistant end of the spectrum of radiosensitivities seen in human tumour cells generally. The results support the view that inherent cellular radioresistance may be an important cause of failure in this disease. All three cell lines showed an increase in survival when the radiation dose rate was reduced. In split-dose experiments, recovery was found to increase with dose in a manner consistent with the predictions of the linear-quadratic equation.
Cell Line↗
The inhibition of cellular recovery in human tumour cells by inhibitors of topoisomerase.
A human bladder carcinoma cell line was irradiated at high and low dose rates and exposed to camptothecin and VP16, inhibitors of topoisomerase I and topoisomerase II respectively. Although camptothecin substantially modified the cytotoxic effects of high dose rate irradiation, abolished low dose rate sparing and inhibited the repair of sublethal and potentially lethal damage, VP16 had no effect on the survival curves even at highly cytotoxic doses. Thus, it is argued that there is a role for topoisomerase I but not topoisomerase II in the repair of DNA damage induced by ionising radiation.
Camptothecin↗
The radiation dose-rate effect in two human neuroblastoma cell lines.
The current use of targeted radiotherapy in the treatment of neuroblastoma has generated a requirement for further information on the radiobiology of these cells. Here we report on studies of the dose-rate effect in two human neuroblastoma cell lines (HX138 and HX142) and the recovery that they demonstrate in split-dose experiments. The sensitivity of the two cell lines to high dose-rate irradiation was confirmed. Surviving fractions at 2 Gy were 0.083 for HX138 and 0.11 for HX142. There was little evidence of a dose-rate effect above 2 cGy min-1 but significant sparing was seen at lower dose rates. Substantial recovery was seen in split-dose experiments on both cell lines, to an extent that was consistent with the linear quadratic equation. The data were used to derive values for the beta parameter of the linear-quadratic equation; the values for the neuroblastomas were higher than for any of the other human tumour cell lines that we have investigated to date. Thus, despite their high sensitivity to ionising radiation HX138 and HX142 do exhibit substantial levels of cellular recovery, suggesting that they may have a significant capacity for repair of radiation-induced lesions.
Cell Survival↗
Growth and radiosensitivity testing of human tumour cells using the adhesive tumour cell culture system.
The radiosensitivity of human tumour cell lines and cells cultured from xenografts or biopsy specimens was measured using the adhesive tumour cell culture system (ATCCS). For cell lines the derived surviving fractions at 2 Gy were in good agreement with values obtained by clonogenic assay. However, the assay tended to overestimate survival at higher radiation doses, and thus to give a false impression of radioresistance. When cells taken from xenografts or tumour biopsies were cultured there was no evidence for selective growth of tumour cells: fibroblast-like cells commonly grew. Immunohistochemical staining against the intermediate filament, vimentin, supported the mesenchymal origin of the fibroblast-like cells. In cultures of artificial mixtures of tumour cells and fibroblasts, low proportions of fibroblasts were not excluded by the assay and consequently modified the radiation response. The majority of cultures grown from bladder carcinoma biopsy specimens appeared fibroblast-like, although in some cases clearly distinguishable colonies of tumour cells were also grown. In such tumour types the reliable measurement of radiosensitivity in cells taken from biopsies will require further development of techniques that allow the selective growth of tumour cells.
Animals↗
Override of the radiation-induced mitotic block in human tumour cells by methylxanthines and its relationship to the potentiation of cytotoxicity.
The four methylated xanthine derivatives, caffeine, theophylline, theobromine and paraxanthine, were tested for their ability to override the mitotic block induced by ionizing radiation in the human bladder carcinoma cell line RT112. All four agents were found to partially override the block, ranking in terms of potency at a concentration of 1 mM in the order caffeine greater than theophylline greater than theobromine = paraxanthine. However, the effects of the four agents on the clonal survival of irradiated cells failed to correlate with the extent of override, both in terms of the relative effects of the four agents and the dose-response relationships; at a concentration of 1 mM only caffeine was found to potentiate cell killing as well as causing block override, whilst at higher concentrations all the agents had a significant effect on survival but little or no further influence on the degree of block override. It is therefore concluded that override of a mitotic block is not in itself sufficient to cause the increased killing seen when irradiated cells are incubated in the presence of caffeine, and that caffeine exerts its potentiating effect either by directly inhibiting repair of damage in DNA or by causing override of the radiation-induced inhibition of DNA synthesis.
Caffeine↗
A comparison of the in vivo and in vitro radiation response of three human cervix carcinomas.
The radiation response of three carcinoma of the cervix tumours has been compared in vivo in xenografts using growth delay and in vitro by means of a monolayer-based clonogenic assay. Tumours have been irradiated with 60Co gamma-rays at both high (70-100 cGy/min) and continuous low dose rates (3-5 cGy/min) in order to determine the relative in vivo and in vitro sparing effects associated with lowering radiation dose rate. In vitro, two of the lines (HX155c and HX156c) showed significant low dose rate sparing when compared to the HX160c line (which showed little sparing) (p = 0.012). Despite greater scatter for the in vivo data, there was a general tendency for the in vivo results to follow that predicted from the in vitro experiments. In vivo, HX156 exhibited significant sparing (p = 0.011) whereas HX160 again exhibited no significant sparing (p = 0.15). Although the HX155 line did show some sparing in vivo this did not reach significance (p = 0.111). All three lines showed less actual specific growth delay in vivo than that predicted from in vitro data. These in vitro/in vivo findings lead to the conclusion that rapid predictive testing of radiosensitivity (ideally utilising low dose rate irradiation) would be beneficial in determining the choice of radiotherapy regimens for the treatment of this disease.
Animals↗
Why are some human tumours more radiosensitive than others?
There is now good evidence that the radiosensitivity of human tumour cells varies form one tumour type to another, and that the steepness of the initial part of the cell survival curve correlates with clinical radioresponsiveness. Studies at low dose rate allow differences between tumour cells to be seen more clearly. Current mathematical models of radiation cell killing include two components: a linear (i.e. exponential "alpha-component") and a bending component ("beta-component"). Repair of radiation damage affects only the beta-component. Among the 17 human tumour cell lines that we have studied, the average surviving fraction at 2 Gy due to the alpha-component is 0.44 and that due to the beta-component is 0.88. The beta-effect at 2 Gy appears to be similar in radiosensitive and radioresistant tumours; thus among radiosensitive tumours where the survival due to the alpha-component is below 0.3 the beta-effect makes a very small contribution to overall radiosensitivity in the low-dose region. Steep cell survival curves may appear straight but still be consistent with a modest beta-value. The overall effect of many small fractions will be to amplify the dominance of the alpha-component. The beta-effect is then unimportant because repair will be almost complete. Repair inhibitors may however change this situation, reducing recovery, increasing the beta-effect, and thereby increasing sensitivity. But in the absence of such inhibitors the differences between radiosensitive and radioresistant tumours must be looked for not in repair capacity but in the nature of the alpha-component. The most radiocurable tumours do not have low beta-values; they have higher alpha-values than the less curable tumours.
Cell Line↗