G.G. Steel

In vitro studies of cell proliferation in tumours—I: Critical appraisal of methods and theoretical considerations

Resume La proliferation cellulaire dans les tumeurs humaines est etudiee par un marquage in vitro a la thymidine- 3 H, realise sous une forte pression doxygene, de facon a preserver la synthese de lADN dans les cellules situees dans la profondeur des explants. Les ‘indices de marquage’ par la thymidine- 3 H servent a predire la vitesse de la proliferation cellulaire dans la tumeur et de la, un temps potentiel de duplication de la tumeur, Les postulats utilises dans la methode sont discutes, en particulier le role de la perte cellulaire.

Planned and unplanned gaps in radiotherapy: the importance of gap position and gap duration

Local tumour control in 971 patients with squamous carcinoma of the supraglottic larynx has been examined in relation to the occurrence of gaps in radiation therapy. The minimum follow-up time was 3 years. The reasons for a gap in radiotherapy fell into four categories: independent of the patient (national holidays, machine break-down, etc.), planned gaps (split-course therapy), severe normal-tissue reactions, and intercurrent disease. Only 11.7% of patients had no gap at all, 75.5% a single gap, and 2.1% had more than four gaps. The probability of tumour control increased with dose in all patient sub-groups; the average percentage increase for a 1% increase in dose was 4.3. The data were subjected to multivariate analysis, leading to the following conclusions. Patients in whom there was a single gap showed a remarkable trend of local control: if the gap began before day 19 after the start of therapy, the local tumour control was considerably below that in patients who did not suffer a gap in treatment. The local tumour control in patients whose gap began at day 20-29 was indistinguishable from that in patients who had no gap in treatment. A gap further towards the end of treatment was again associated with a severe drop in local control. This trend was independent of the recorded cause of the gap. The mechanism of this phenomenon is not clear. The effect of the timing of a treatment gap appears in this data set to have had a considerable impact on outcome and our observations should stimulate further study of this phenomenon in other clinical settings.

Radio sensitivity, recovery and dose-rate effect in three human glioma cell lines

Abstract 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 radio sensitivities 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.

The Intrinsic α/β 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 α and β (termed αac and βac), low dose-rate and split-dose experiments have been used to derive independent values of α and β (αldr and βRR), respectively. αldr 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. βRR derived from split-dose experiments represents a unique measure of recovery for each cell line. Large differences were found between individual values of βac and βRR, especially in the radiosensitive cell lines. Since βRR 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 α/β ratios. No ...

Differences in radiation response among human cervix carcinoma cell lines.

The radiobiological properties of five newly established carcinoma of the cervix cell lines have been determined. By means of an in vitro clonogenic assay, survival curves were compared at radiation dose rates of 150, 3.2 and 1.6 cGy/min. In terms of both acute radiosensitivity and the extent of low dose-rate sparing, large differences existed among the lines. Two of the lines, HX151c and HX160c, were radiosensitive (surviving fractions at 2 Gy of 0.23 and 0.33 respectively) and showed little radiation recovery capacity during protracted irradiation, whereas the remaining three lines had much higher surviving fraction at 2 Gy (up to 0.6) and showed considerable low dose-rate sparing. The data were well fitted by the Incomplete Repair model of Thames; repair half times ranged from 0.25 to 5.7 h. These findings indicate that large differences in intrinsic radiation recovery capacity exist within this tumour type, differences which emphasise that predictive testing of intrinsic radiosensitivity may be of clinical value in this disease.

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)

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.

Cell-cycle variation in DNA migration in pulsed-field gel electrophoresis

Pulsed-field electrophoresis is being used extensively in the gene mapping studies and in the analysis of DNA strand breakage by ionizing radiation. We have evaluated the relationship between the fraction of S phase DNA in a cell population and its ability to modify the migration of DNA in pulsed-field gel electrophoresis. We have shown that increasing the proportion of S phase DNA reduced the effective rate of migration of MGH-U1 cellular DNA. This effect was observed after treatment with ionizing radiation or the restriction enzyme Not I. However, when radiation-induced damage was studied using intact cells, only the DNA with 70 percent S phase showed apparent differences in damage induction. These studies therefore provide data to indicate the percentage of S phase cells at which overall DNA migration might be affected significantly.

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.

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.