A correlation between residual DNA double-strand breaks and clonogenic measurements of radiosensitivity in fibroblasts from preradiotherapy cervix cancer patients.

PURPOSE: To study the relationship between residual DNA damage and clonogenic measurements of radiosensitivity in fibroblasts from pretreatment cervix cancer patients. METHODS AND MATERIALS: Early passage vaginal fibroblasts from nine preradiotherapy cervix cancer patients and two radiosensitive skin fibroblast cell strains were studied. Cell survival was measured by clonogenic assay following both high and low dose rate irradiation. Residual DNA damage was measured using pulsed-field gel electrophoresis (PFGE) after irradiating radiolabeled, plateau-phase cells at 37 degrees C and allowing 24 h for repair. DNA damage was expressed both in terms of the residual damage slope (fitted to data from 60 to 150 Gy) and the fraction of activity released (FAR) following 150 Gy. RESULTS: The surviving fraction at 2 Gy (SF2) values after high dose rate irradiation for the vaginal fibroblasts ranged from 0.15 to 0.32 (a 2.2-fold difference). When the two radiosensitive cell strains were included, residual damage, expressed as the residual damage slope, correlated with alpha (r = 0.82, p = 0.002), D bar (r = -0.91, p < 0.001) and SF2 (p = -0.79, p = 0.004), and when the vaginal fibroblasts alone were studied, the residual damage slope again correlated with clonogenic survival, although less strongly [alpha (r = 0.66, p = 0.053), D bar (r = -0.83, p = 0.006), and SF2 (r = -0.63, p = 0.07)]. Within the group of vaginal fibroblasts there was a 4.0-fold difference in residual DNA damage slope. When residual damage was expressed as FAR at 150 Gy, then for all cell strains the correlations were alpha: r = 0.78, p = 0.004, D bar: r = -0.86, p = 0.001, and SF2: r = -0.78, p = 0.004, and for the vaginal fibroblast strains alone the correlations were alpha: r = 0.60, p = 0.088, D bar: r = -0.75, p = 0.02, and SF2: r = 0.62, p = 0.077. CONCLUSION: This study confirms previous findings that residual DNA damage correlates with clonogenic survival in fibroblasts. In addition, it demonstrates a correlation for fibroblasts from pretreatment cervix cancer patients demonstrating a relatively small range of SF2 values.

The intrinsic radiosensitivity of normal and tumour cells.

PURPOSE: To examine whether in vitro measurements of normal and tumour cell radiosensitivity can be used as prognostic factors in clinical oncology. MATERIALS AND METHODS: Stage I-III cervix carcinoma patients were treated with radical radiotherapy with a minimum of 3 years' follow-up. Lymphocyte and tumour radiosensitivities were assayed using, respectively, a limiting dilution and soft agar clonogenic assay to obtain surviving fraction at 2 Gy (SF2). The results were related, in an actuarial analysis, to late morbidity assessed using the Franco Italian glossary. RESULTS: Patients with radiosensitive lymphocytes had a significantly increased risk of developing late complications (n = 93, p = 0.002). Increasing tumour radiosensitivity was associated with an increased risk of morbidity (n= 113, p=0.032). A significant correlation was found between fibroblast and tumour cell radiosensitivity (r=0.57, p=0.03), but a weak inverse association was found between lymphocyte and tumour cell radiosensitivity (r= -0.32, p=0.03). Patients with radiosensitive lymphocytes and tumour cells had higher levels of late complications than those whose cells were radioresistant. CONCLUSION: The work described highlights the importance of cellular radiosensitivity as a parameter determining the clinical response to radiotherapy.

Comparative effectiveness of avermectins and deltamethrin in suppressing oviposition in Lucilia cuprina (Diptera: Calliphoridae).

The activities of three avermectins and deltamethrin as oviposition suppressants were investigated with a laboratory bioassay in which gravid females of the blowfly Lucilia cuprina (Wiedemann) were exposed to treated oviposition targets. An easily comparable index of suppression, the oviposition suppression concentration (OSC), was defined. All four compounds were effective oviposition suppressants. The three avermectins had similar OSC50 values (approximately 13 ppm). Deltamethrin, with an OSC50 of 0.4 ppm, was the most potent suppressant. The avermectins all produced significant mortality in adults with suppressed oviposition, while deltamethrin did not cause an increase in deaths at concentrations giving up to 100% suppression of oviposition. The toxicities of all four compounds to adult females were similar when assessed by topical application.

Fibroblast radiosensitivity measured using the comet DNA-damage assay correlates with clonogenic survival parameters.

A study was made of the neutral comet assay as a potential method for measuring normal cell radiosensitivity. Eleven fibroblast strains were studied comprising nine derived from vaginal biopsies from pretreatment cervical cancer patients and two strains from radiosensitive individuals. DNA double strand break (dsbs) dose-response curves for both initial and residual (20-h repair time) damage were obtained over the dose range 0-240 Gy, with slopes varying 3.2 and 8-fold respectively. Clonogenic cell survival parameters were available for all the cell strains following both high- and low-dose rate irradiation. There were no correlations between the dose-response slope of the initial level of DNA dsbs and parameters that mainly describe the initial portion of clonogenic radiation survival curves (SF2, alpha, D). A significant correlation (r = -0.63, P = 0.04) was found between the extent of residual DNA dsbs and clonogenicity for all 11 fibroblast strains. The parameter showing the highest correlation with fibroblast cell killing (D) for the nine normal fibroblasts alone was the ratio of initial/residual DNA dsb dose-response slope (r = 0.80, P = < 0.01). A significant correlation (r = -0.67, P = 0.03) with clonogenic radiosensitivity was also found for all 11 cell strains when using the ratio of initial/residual DNA dsb damage at a single dose of 180 Gy. This study shows that fibroblast radiosensitivity measured using the neutral comet assay correlates with clonogenic radiation survival parameters, and therefore may have potential value in predictive testing of normal tissue radiosensitivity.

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.