Radiosensitivity and Capacity to Recover from Radiation-Induced Damage in Pimonidazole-Unlabeled Intratumor Quiescent Cells Depend on p53 Status

Background Using our method for selectively detecting the response of intratumor quiescent (Q) cells to irradiation, the Q cells was shown to have a much larger hypoxic fraction (HF) than total (= proliferating (P) + Q) tumor cell population irrespective of the p53 status of tumor cells. However, the size of the HF was clearly less than 100%, meaning the Q cell population was never fully hypoxic. Thus, the dependency of the radio-sensitivity and recovery capacity from radiation-induced damage on p53 status was investigated in pimonidazole-unlabeled oxygenated Q tumor cells. Methods Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53), or with neo vector as a control (SAS/neo), were inoculated subcutaneously into left hind legs of Balb/cA nude mice. The tumor-bearing mice received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all intratumor P cells. Tumors were irradiated with γ-rays at a high dose-rate or a reduced dose-rate at 1 h after the administration of pimonidazole. The responses of Q and total cell populations were evaluated with the frequencies of micronucleation and apoptosis using immunofluorescence staining for BrdU. The response of pimonidazole unlabeled tumor cell fractions was assessed with apoptosis frequency using immunofluorescence staining for pimonidazole. Results The pimonidazole-unlabeled tumor cell fraction showed significantly enhanced radio-sensitivity compared with the whole tumor cell fraction more remarkably in Q cells and p53-mutated tumors than total cells and p53-wild type tumors, respectively. However, a significantly greater decrease in radio-sensitivity in the pimonidazole-unlabeled than the whole cell fraction, evaluated using a delayed assay or a decrease in radiation dose rate, was more clearly observed in Q cells and p53-wild type tumors than total cells and p53-mutated type tumors, respectively. Concerning the whole tumor cell fraction, the Q cells showed significantly greater radio-resistance and recovery capacity from radiation-induced damage than the total cells both in p53-wild and p53-mutated type tumors. Conclusions The pimonidazole-unlabeled sub-fraction of the Q tumor cells, probably oxygenated, may be a critical target in the control of solid tumors, although its radio-sensitivity and recovery capacity from radiation-induced damage depend on p53 status of the tumor cell.