E.P. Malaise

Mean Inactivation Dose: A Useful Concept for Intercomparison of Human Cell Survival Curves

The mean inactivation dose (D) is calculated for published in vitro survival curves obtained from cell lines of both normal and neoplastic human tissues. Cells belonging to different histological categories (melanomas, carcinomas, etc.) are shown to be characterized by distinct values of D which are related to the clinical radiosensitivity of tumors from these categories. Compared to other ways of representing in vitro radiosensitivity, e.g., by the multitarget parameters D0 and n, the parameter D has several specific advantages: (i) D is representative for the whole cell population rather than for a fraction of it; (ii) it minimizes the fluctuations of the survival curves of a given cell line investigated by different authors; (iii) there is low variability of D within each histological category; (iv) significant differences in radiosensitivity between the categories emerge when using D. D appears to be a useful concept for specifying intrinsic radiosensitivity of human cell lines.

Hypersensitivity of ataxia telangiectasia fibroblasts to ionizing radiation is associated with a repair deficiency of DNA double-strand breaks.

We have studied the intrinsic radiosensitivity, repair of potentially lethal damage (PLD) and the repair rate of radiation-induced DNA double-strand breaks (DSB) in 11 non-transformed human fibroblast cell lines, four of which were homozygous for the A-T mutation and two that were heterozygous (A-TH). All the experiments were done on cells in plateau phase of growth (97-99% of cells in G0/G1). With a dose of 30 Gy delivered at 4 degrees C, the A-T cell lines had faster repair rates of up to 6 h, after which the repair curve crossed that of the control so that the residual damage at 24 h was higher in the A-T cells. Irradiation at 37 degrees C at low dose rate 1 cGy.min-1) produced even more marked differences between the A-T cells and controls: the residual DSB level was always higher in A-T cells than controls at doses of 5-40 Gy, due to defective repair of a small fraction of DSB in A-T cells. The two protocols showed DSB repair rates for the A-TH cell lines that were intermediate between those of the A-T and control cells. There was a quantitative relationship between the residual DSB after irradiation at 37 degrees C and the intrinsic radiosensitivity, and with the extent of PLD repair. There were very few apoptotic cells in the non-transformed control and A-T cell line, both before and after irradiation. In combination, these result support the contention that the defective repair of DSB is a mechanism of the hypersensitivity linked to the A-T mutation.

Initial slope of radiation survival curves is characteristic of the origin of primary and established cultures of human tumor cells and fibroblasts.

The published survival curves of 110 human tumor cell lines and 147 nontransformed human fibroblast strains have been reanalyzed using three different statistical methods: the single hit multitarget model, the linear-quadratic model, and the mean inactivation dose. The 110 tumor cell lines were classified in two ways: (a) into three categories defined by clinical radiocurability criteria, and (b) into seven categories based on histopathology. The 147 fibroblast strains were divided into eight genetic groups. Differences in the radiosensitivities of both the tumor cell and fibroblast groups could be demonstrated only by parameters that describe the slopes of the initial part of the survival curves. The capacity of the survival level to identify significant differences between groups was dose dependent over the range 1 to 6 Gy. This relationship showed a bell-shaped curve with a maximum at 1.5 Gy for the tumor cell lines and 3 Gy for the fibroblasts. Values for intrinsic radiosensitivity for a number of groups of tumors have also been obtained by primary culture of tumor cells. These values are strictly comparable to those obtained by clonogenic methods. This confirms that intrinsic radiosensitivity is a determinant of the response of tumor cells to radiotherapy and suggests that tissue culture methods may be used as a predictive assay.

Induction and Rejoining of DNA Double-Strand Breaks and Interphase Chromosome Breaks after Exposure to X Rays in One Normal and Two Hypersensitive Human Fibroblast Cell Lines

The aim of this work was to measure simultaneously and in a quantitative manner double-strand breaks (DSBs), interphase chromosome breaks and cell lethality either immediately after irradiation, or at various times thereafter (up to 24 h), in cells of three nontransformed human fibroblast cell lines of widely different intrinsic radiosensitivity. We wished to assess initial damage, repair kinetics and residual damage at the DNA and the chromosome level, and to correlate these parameters with cell killing. We employed HF19 cells, a normal fibroblast cell line, AT2 cells, a radiosensitive cell line from a patient suffering from ataxia telangiectasia (AT), and 180BR cells, a radiosensitive cell line from a patient with no clinical symptoms of AT. AT2 and 180BR cells, in addition to being radiosensitive, also display a reduced ability to repair potentially lethal damage compared to HF19 cells. The yield of DSBs, as measured by pulsed-field gel electrophoresis, is similar in all three cell lines (slopes correspond to 1.6-1.7% Gy-1 of DNA-associated radioactivity released from the gel well into the lane). In contrast, residual DSBs measured 24 h after irradiation are almost zero for HF19 cells (0.1% confidence interval = 0-1.4%), but are 12.5% (+/- 2.3%) and 43.8% (+/- 1.2%) of those measured immediately after irradiation in AT2 and 180BR cells, respectively. Residual interphase chromosome breaks are 11.6% (+/- 1.6%), 29.7% (+/- 5.7%) and 41.4% (+/- 2.2%) of those measured immediately after irradiation in HF19, AT2 and 180BR cells, respectively. Neither the initial yield of DSBs nor that of excess interphase chromosome breaks can explain the differences in radiosensitivity between the three cell lines; however, there is a correlation between residual DSBs, rate of DSB rejoining at 24 h, residual interphase chromosome breaks on the one hand and cell survival on the other hand.

Might intrinsic radioresistance of human tumour cells be induced by radiation

Survival measurements were made on six human tumour cell lines in vitro after irradiation with single doses of X rays. Doses up to 5 Gy were used giving surviving fractions down to 20%, but the majority of the measurements were made at doses < 1 Gy. These six cell lines have very different intrinsic radiosensitivities: HT29, Be11, and RT112 are radioresistant with surviving fractions at 2 Gy (SF2) between 60 and 74%, while MeWo, SW48, and HX142 are radiosensitive (SF2 = 3-29%). For all the cell lines, response over the dose range 2-5 Gy showed a good fit to a Linear-Quadratic (LQ) model. However, HT29, Be11, and RT112 cells showed a significant increase in X-ray radiosensitivity at doses below < 1 Gy compared with the prediction extrapolated from a LQ model fitted to the data at higher doses. The LQ model also slightly underpredicted the effect of low-dose X rays in MeWo cells, but the response of SW48 and HX142 cells was well described by the LQ model at all doses, with no evidence of increased low-dose effectiveness. The most plausible explanation for this phenomenon is that it reflects an induced radioresistance so that low doses of X-rays in vitro are more effective per Gy than higher doses, because only at higher doses is there sufficient damage to trigger repair systems or other radioprotective mechanisms. It follows that variation in the amount of inducible radioresistance might explain, in part, differences in intrinsic radiosensitivity above > 1 Gy between cell lines: cells would be intrinsically radiosensitive because they have a diminished inducible response.

Rapid tumor cell proliferation after induction chemotherapy in oropharyngeal cancer

Tumor cell kinetics were studied in vivo in a series of 97 patients with oropharyngeal cancer. The duration of S phase (tS), the labeling index (LI), and the potential doubling time (Tpot) were obtained by flow cytometry measurements of a tumor biopsy obtained after intravenous injection of 200 mg 5‐bromodeoxyuridine to the patient. The mean LI was 9.7% (standard deviation [SD], 5.4), the mean tS was 10.1 hours (SD, 3.6), and the mean Tpot was 4.6 days (SD, 3.5). No significant relationship was found between the Tpot or LI and the size of the tumor, nodal status, histological grade, or the site of the primary within the oropharynx. Conversely, aneuploid tumors had longer tS (P<.001), higher LI (P<.001), and shorter Tpot (P<.05) than the diploid tumors. The mean LI and Tpot of the tumors obtained after induction chemotherapy were significantly higher and shorter, respectively, than those measured before any treatment. The data strongly suggest that rapid tumor cell proliferation frequently occurs in oropharyngeal cancer which had responded poorly to chemotherapy.

The effect of very low radiation doses on the human bladder carcinoma cell line RT112

Human RT112 cells were exposed to single doses of X-rays (0.05-5 Gy), and cell survival was measured using a Dynamic Microscopic Imaging Processing Scanner (DMIPS) with which individual cells can be located in tissue-culture flasks, their positions recorded, and after an appropriate incubation time the recorded positions revisited to allow the accurate scoring of survivors. The response over the X-ray dose range 1-5 Gy showed a good fit to a Linear-Quadratic (LQ) model. For X-ray doses < 1 Gy, an increased effect of X-rays was observed with cell survival below the prediction from the LQ model extrapolated from higher doses. Several arguments suggest that this phenomenon could reflect an induced radioresistance so that in this cell line, low single doses of X-rays are more effective per Gray than higher doses in reducing cell survival because only at higher doses, above a threshold, is there sufficient damage to trigger radioprotective mechanisms.

In vivo measurement of the potential doubling time by flowcytometry in oropharyngeal cancer treated by conventional radiotherapy

PURPOSE Experimental and clinical studies suggest that the pre-treatment potential doubling time could be predictive of tumor control in patients treated by conventional radiotherapy and could help to identify the rapidly growing tumors for which accelerated radiotherapy is required. METHODS AND MATERIALS To test this hypothesis, we studied prospectively 48 patients with a squamous cell carcinoma of the oropharynx and treated by conventional radiotherapy (70 Gy/7 weeks). The duration of S phase, the labeling index and the potential doubling time were obtained by flowcytometry measurements of a tumor biopsy obtained after injection of 200 mg bromodeoxyuridine to the patient. RESULTS Three parameters were significantly associated with an increased risk of relapse namely the tumors size (T4; p < 0.01), the nodal status (> or = N2; p < 0.05) and the site of the primary within the oropharynx (p = 0.08). The S phase, labeling index, DNA index and potential doubling time were not significantly associated with an increased risk of relapse. However when considering only the T2 subgroup of patients, high labeling indexes and short potential doubling time were associated with an increased risk of relapse: the mean pre-treatment potential doubling time of the tumors which relapsed was 3.21 versus 5.5 days when there was no evidence of local relapse (p < 0.05). The mean labeling index for the group of tumors associated with a tumor recurrence was 11.7% compared to 7.3% when there was no evidence of relapse (p = 0.02). CONCLUSION Factors other than proliferation play a role in determining the outcome of oropharyngeal cancers treated by conventional radiotherapy. However there was a significant correlation between short potential doubling time, high labeling index and tumor recurrence in the T2 subgroup of patients. The finding of significance for potential doubling time and labeling index in the T2 subset of tumors may be a reflexion of the more homogeneous nature of these tumors with regard to prognostic variables.

Underestimation of the small residual damage when measuring DNA double-strand breaks (DSB): is the repair of radiation-induced DSB complete?

PURPOSE To overcome the underestimation of the small residual damage when measuring DNA double-strand breaks (DSB) as fraction of activity released (FAR) by pulsed-field gel electrophoresis. MATERIALS AND METHODS The techniques used to assess DNA damage (e.g. pulsed-field gel electrophoresis, neutral elution, comet assay) do not directly measure the number of DSB. The Blöcher model can be used to express data as DSB after irradiation at 4 degrees C by calculating the distribution of all radiation-induced DNA fragments as a function of their size. We have used this model to measure the residual DSB (irradiation at 4 degrees C followed by incubation at 37 degrees C) in untransformed human fibroblasts. RESULTS The DSB induction rate after irradiation at 4 degrees C was 39.1+/-2.0 Gy(-1). The DSB repair rate obtained after doses of 10 to 80 Gy followed by repair times of 0 to 24 h was expressed as unrepaired DSB calculated from the Blöcher formula. All the damage appeared to be repaired at 24h when the data were expressed as FAR, whereas 15% of DSB remained unrepaired. The DSB repair rate and the chromosome break repair rate assessed by premature condensation chromosome (PCC) techniques were similar. CONCLUSION The expression of repair data in terms of FAR dramatically underestimates the amount of unrepaired DNA damage. The Blöcher model that takes into account the size distribution of radiation-induced DNA fragments should therefore be used to avoid this bias. Applied to a normal human fibroblast cell line, this model shows that DSB repair is never complete.

The Relationship between Potentially Lethal Damage Repair and Intrinsic Radiosensitivity of Human Cells

The intrinsic radiosensitivity of exponentially growing cells (exp) was compared to that of immediately plated plateau phase cells (ip) using published data on 60 human cell lines (27 fibroblast lines and 33 tumour cell lines). The values for alpha, D and S2 are not significantly different for the two groups; beta is significantly higher in ip cells. This produces a smaller alpha/beta ratio in ip cells than in exp cells. The influence of potentially lethal damage (PLD) repair was assessed by comparing the radiosensitivities of ip cells and plateau phase cells with delayed plating (dp). The published data for 81 human cell lines (48 fibroblasts and 33 tumour lines) were used. PLD repair was found to lead to a decrease in alpha and an increase in D and S2, whereas neither beta nor the alpha/beta ratio changed significantly. The relationship between PLD repair and intrinsic radiosensitivity was assessed by repair capacity and the repair ratio. The fitted relationship is a bell-shaped curve with a maximum at 2.2 Gy for repair capacity. The fitted curve predicts that repair capacity is zero at a D up of 0.28 Gy and at 4 Gy. Thus, PLD repair is a reasonable reflection of intrinsic radiosensitivity up to 2.2 Gy. Above 2.2 Gy, the relationship is reversed: the greater the radioresistance, the lower the PLD repair.