Mitochondrial modulation of oxygen-dependent radiosensitivity in some human tumour cell lines.

Oxygen-dependent radiosensitivity of tumour cells reflects direct oxidative damage to DNA, but non-nuclear mechanisms including signalling pathways may also contribute. Mitochondria are likely candidates because not only do they integrate signals from each of the main kinase pathways but mitochondrial kinases responsive to oxidative stress communicate to the rest of the cell. Using pharmacological and immunochemical methods, we tested the role of mitochondrial permeability transition (MPT) and the Bcl-2 proteins in oxygen-dependent radiosensitivity. Drug-treated or untreated cervical cancer HeLa, breast cancer MCF-7 and melanoma MeWo cell lines were irradiated at 6.2 Gy under normoxic and hypoxic conditions then allowed to proliferate for 7 days. The MPT blocker cyclosporin A (2 microM) strongly protected HeLa but not the other two lines against oxygen-dependent radiosensitivity. By contrast, bongkrekic acid (50 microM), which blocks MPT by targeting the adenine nucleotide transporter, had only marginal effect and calcineurin inhibitor FK-506 (0.1 microM) had none. Nor was evidence found for the modulation of oxygen-dependent radiosensitivity by Bax/Bcl-2 signalling, mitochondrial ATP-dependent potassium (mitoK(ATP)) channels or mitochondrial Ca(2+) uptake. In conclusion, calcineurin-independent protection by cyclosporin A suggests that MPT but not mitoK(ATP) or the mitochondrial apoptosis pathway plays a causal role in oxygen-dependent radiosensitivity of HeLa cells. Targeting MPT may therefore improve the effectiveness of radiotherapy in some solid tumours.

Resazurin assay of radiation response in cultured cells.

We describe use of resazurin reduction for measurement of cell response to irradiation as a simple and non-destructive assay that complements the conventional colony forming assay and can readily be applied to both adherent and non-adherent cell cultures. The resazurin method yields data comparable with the colony forming assay as well as to assay of DNA synthesis (BrdU incorporation), giving an OER (oxygen enhancement ratio) of 2.5 at 60% isoeffect level versus 3.1 for the colony forming assay. Intraday and interday precisions for the resazurin assay were 4.1% and 5.2%, respectively.

Further evidence for biological effects resulting from ionizing radiation doses in the diagnostic X-ray range.

In this study we assessed cell response to ionizing radiation doses in the diagnostic X-ray range, using metabolic performance as the biological end point. HeLa cells were irradiated between 3.5 mGy (standard deviation (SD=2.5%) and 55.1 mGy (SD=0.9%) with 110 kV X-rays, and after 7 days metabolic capacity of cultures was measured spectrophotometrically with resazurin. Cells receiving 7.5 mGy or greater showed impaired proliferation. At 7.5 mGy this effect was prevented by nitric oxide synthase inhibitor N G-monomethyl-L-arginine but not by ascorbic acid or p38 MAPK inhibitor SB203580. We conclude that HeLa cell proliferation is adversely affected by doses within the range of some radiological examinations, through a mechanism involving nitric oxide.

The collection efficiency of an ionization chamber in a pulsed and magnetically swept electron beam: limits of validity of the two-voltage technique.

The two-voltage method of determining the collection efficiency of an ionization chamber exposed to a pulsed and magnetically swept electron beam depends upon the form of the saturation curve agreeing closely with the formula based on ionic conduction and negligible space charge. The possible disturbing effects of charge transport by free electrons and of overlapping pulses are discussed and the conditions to be satisfied for the reliable use of the method are defined and demonstrated experimentally.