Radiation-induced variations in urothelial expression of intercellular adhesion molecule 1 (ICAM-1): association with changes in urinary bladder function.

PURPOSE: To assess the effect of single-dose irradiation on intercellular adhesion molecule 1 (ICAM-1) expression in the urothelium of mouse urinary bladder and to correlate ICAM-1 variations with fluctuations in storage capacity during the early and late radiation response. MATERIALS AND METHODS: Groups of female C3H mice were subjected to irradiation with either 20 or 0 Gy. The intensity of immunohistochemical ICAM-1 staining in the urothelium was assessed in a semiquantitative way applying an arbitrary score (0-5). Changes in bladder storage function were assessed by transurethral cystometry. RESULTS: For the early radiation response phase, a reduction in bladder capacity by >50%, i.e. a positive functional radiation response, was seen in 40% of the irradiated animals between days 0 and 15, and in 64% of animals during days 16-30. During the late response phase, 71% of the animals sacrificed after day 180 developed a positive functional response. Urothelial cells were found to express ICAM-1 constitutively. Irradiation resulted in an early rise in staining signal by day 2, with a maximum on day 4 and a return to control values on day 13. A permanent increase in ICAM-1 staining signal was observed in the late phase, from day 90 to 360 after irradiation. The expression of ICAM-1 in animals with a positive late response was 4.2+/-1.2 (mean+/-standard deviation), compared with 2.6+/-1.0 in non-responders (p=0.0009). CONCLUSION: Irradiation induces significant acute and chronic changes in urothelial ICAM-1 expression indicating that the urothelium contributes to the pathogenesis of both acute and late radiation effects in the urinary bladder.

Ultrafractionation in A7 human malignant glioma in nude mice.

PURPOSE: Low-dose hyperradiosensitivity (HRS) has been demonstrated in numerous cell lines in vitro, including a number of radioresistant human malignant glioma cell lines such as A7. The aim of our experiment was to show whether HRS can be exploited by using ultrafractionated irradiation (UF) to improve local control of A7 tumours growing in nude mice. Extrapolation of the in vitro results predict a 3.7-fold difference in the efficacy of UF compared with conventional fractionation (CF). MATERIAL AND METHODS: Subcutaneuously growing A7 tumours were irradiated either with UF (126 fractions in 6 weeks, 0.4 Gy per fraction) or CF (30 fractions in 6 weeks, 1.68 Gy per fraction). The total dose was 50.4 Gy in both experimental arms. Fractionated irradiations were given under ambient conditions and followed by graded top-up doses under clamp hypoxia. Endpoints were tumour growth delay and local tumour control 180 days after the end of treatment. RESULTS: UF resulted in a significant decrease of tumour growth delay and in a significant increase of the top-up TCD(50) compared with CF (40.0 Gy [95% CI 29; 61 Gy] versus 28.3 Gy [24; 35 Gy], p=0.047). CONCLUSIONS: Despite a pronounced HRS phenomenon in vitro, UF was significantly less effective than CF in A7 human malignant glioma in nude mice. These results neither disprove the existence of HRS nor do they exclude a possible clinical value of UF. The findings rather indicate that simplistic extrapolation from results obtained after single-dose exposure or few fractions in vitro is not sufficient to predict outcome of UF in vivo and that comprehensive evaluation of novel treatment options in animal models continues to be an essential requirement for clinical translation.

Effect of keratinocyte growth factor on radiation survival and colony size of human epidermal keratinocytes in vitro.

Keratinocyte growth factor (FGF7, also known as KGF) ameliorates the radiation response of mouse oral mucosa and other epithelial tissues. However, the precise mechanisms remain unclear. The aim of the present study was to investigate the effect of FGF7 on the survival and colony size of normal human epidermal keratinocytes in vitro. Primary neonatal keratinocytes (HEKn) were irradiated with doses of 0 and 2 Gy of 200 kV X rays and incubated in the presence or absence of 100 ng/ml FGF7. The plating efficiency (PE) and surviving fraction (SF2) were determined using a clonogenic assay. In cell cultures without FGF7, the mean PE was 4.6 +/- 0.2%. Irradiation with 2 Gy resulted in an SF2 of 51 +/- 2%. In cell cultures with FGF7, the mean PE was identical, and a similar SF2 of 54 +/- 1% was observed (P = 0.4). However, the individual colony size was significantly increased in all cultures incubated with FGF7 compared to those incubated without FGF7. The number of extremely large colonies (> or =2 mm) was clearly higher (P < 0.0001) in cultures with FGF7. This was accompanied by a significant reduction in the diameter of individual cells from 29 microm in controls to 23 microm with FGF7. In conclusion, FGF7 does not affect the survival of keratinocytes after irradiation, but it does stimulate proliferation of surviving cells.

Repopulation of FaDu human squamous cell carcinoma during fractionated radiotherapy correlates with reoxygenation.

PURPOSE: FaDu human squamous cell carcinoma (FaDu-hSCC) showed a clear-cut time factor during fractionated radiotherapy (RT) under ambient blood flow. It remained unclear whether this is caused solely by proliferation or if radioresistance resulting from increasing hypoxia contributed to this phenomenon. To address this question, repopulation of clonogenic FaDu cells during fractionated RT under clamp hypoxia was determined by local tumor control assays, and compared to the results after irradiation with the same regimen under ambient blood flow. METHODS AND MATERIALS: FaDu-hSCC was transplanted into the right hind leg of NMRI nu/nu mice. In the first set of experiments, irradiation was performed under clamp hypoxia. After increasing numbers of 3 Gy fractions (time intervals 24 h or 48 h), graded top-up doses were given to determine the TCD(50) (dose required to control 50% of the tumors). In the second set of experiments, all 3 Gy fractions were applied under ambient conditions, but as in the previous experiments the graded top-up doses were given under clamp hypoxia. A total of 26 TCD(50) assays were performed and analyzed using maximum likelihood techniques. RESULTS: With increasing numbers of daily fractions, the top-up TCD(50) under clamp hypoxia decreased from 39.4 Gy [95% CI 36, 42] after single dose to 19.8 Gy [15, 24] after 18 fractions in 18 days and to 37.8 Gy [31, 44] after 18 fractions in 36 days. The results were consistent with biphasic repopulation, with a switch to rapid repopulation after about 22 days [13, 30]. The clonogen doubling time (T(clon)) decreased from 9.8 days [0, 21] in the beginning of RT to 3.4 days after 22 days. Under ambient blood flow the top-up TCD(50) decreased from 37.6 Gy [34, 40] after single dose irradiation to 0 Gy [0, 1] after 18 fractions in 18 days and 22.4 Gy [18, 27] after 18 fractions in 36 days. Similar to results from irradiations under clamp hypoxia, the ambient data were consistent with a biphasic course of clonogen inactivation. Comparison of both data sets revealed significant reoxygenation after 12 fractions. CONCLUSIONS: Our data are most consistent with a biphasic course of clonogen repopulation during fractionated RT of FaDu-hSCC under clamp hypoxia with a switch in T(clon) after about 22 days of treatment ("dog-leg"). A similar biphasic course of cell repopulation was observed under ambient conditions. The temporal coincidence between repopulation and reoxygenation suggests that the latter might be the stimulus for proliferation in FaDu tumors.