The two principal rationales for applying hyperthermia in cancer therapy are that: (a) the S phase, which is relatively radioresistant, is the most sensitive phase to hyperthermia, and can be selectively radiosensitized by combining hyperthermia with x-irradiation; the cycling tumor cells in S phase which would normally survive an x-ray dose could thus be killed by subjecting these cells to hyperthermia; and (b) the relatively radioresistant hypoxic cells in the tumor may be selectively destroyed by combinations of hyperthermia and x-irradiation. Both of these rationales have been mentioned as reasons for using high LET irradiation in cancer therapy; therefore where such irradiation may be of use, hyperthermia may also be advantageous.

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Magnetic interactions between dissolved oxygen and nitroxide radical spin probes lead to broadening of the ESR lines. We have used a closed-chamber method based on this property to determine the maximum rate of 02 uptake per cell (Vm. per cell) in cultured mammalian cells. A suitable spin probe and a cell suspension are mixed in an aerated medium, and the rate of disappearance of dissolved 02 is measured. The effects of temperature, pH, and microwave power on the determination of dissolved oxygen in solution were studied. For Most mammalian cells are aerobic, and dissolved oxygen is vital for their growth. Elucidation of the mechanism of 02 uptake during the cell cycle is probably fundamental to the complete understanding of cell growth and cell division.There have been several reports on the measurement of 02 uptake during the cell cycle (1-4). Most previous research has been done on dividing egg cells by using the Cartesian diver technique (1) and on yeast cells by using the Clark electrode (2). To our knowledge, only two reports have appeared concerning 02 uptake by cultured mammalian cells during the cell cycle (3,4). One of the obstacles is that the existing techniques for measurement of 02 uptake by cultured mammalian cells require large amounts of.cells for each measurement, making it difficult to study mitotically synchronized cells. Most chemical means of synchronization induce multiple biochemical perturbations that obscure the interpretations of any 02 uptake measurements.It is well known that the interaction ofdissolved oxygen molecules and nitroxide free radicals through Heisenberg spin-exchange causes broadening of the ESR lines (5-7). With a suitable spin probe, this property can be used to quantitate 02 concentrations in solution (8)(9)(10).In this report, we describe an ESR closed-chamber method for determining the 02 uptake by cultured mammalian cells.Basically, a spin probe and a cell suspension are mixed in an aerated medium and the rate of disappearance of 02 is measured. Because a relatively small number of cells is required for each measurement, it has been possible in the present work to study oxygen uptake of mitotically synchronized populations of Chinese hamster ovary (CHO) cells. MATERIALS AND METHODSCell Lines. CHO cells maintained in monolayer culture from frozen stocks for several years in this laboratory were used because they are well characterized. The procedures for culturing CHO cells, either in monolayer or in suspension, and for synchronization have been described (11). The mitotic index ofcells in mitosis was about 97% and did not change appreciably during the ESR measurement.ESR Closed-Chamber Method. A mixture of 10 ml of phosphate-buffered saline (Oxoid; Dulbecco's solution A without Mg2+ and Ca2+, pH 7.4) containing 0.114 mM spin probe [3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-yloxy (CTPO); Aldrich] and 0.2% methyl cellulose E4M (Dow) or 0.1% agar (buffer A) was equilibrated with air at 370C with stirring for at least 10 min prior to use. About 5-10 X...

Oxygen transport in the Chinese hamster ovary (CHO) plasma membrane has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed in the lipid bilayer portion of the membrane at various distances from the membrane surface using the long-pulse saturationrecovery electron spin resonance (ESR) technique. The collision rate was estimated for 5-, 12-, and 16-doxylstearic acids from spin-lattice relaxation times (TI) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameters across the membrane were obtained showing that the oxygen diffusion-concentration product is lower than in water for all locations at 37°C. From oxygen transport parameter profiles, the membrane oxygen permeability coefficients were estimated according to the procedure developed earlier by Subczynski et al. (Subczynski, W. K., J. S. Hyde, and A. Kusumi. 1989. Proceedings of the National Academy of Sciences, . At 37°C, the oxygen permeability coefficient for the plasma membrane was found to be 42 cm/s, about two times lower than for a water layer of the same thickness as the membrane. The oxygen concentration difference across the CHO plasma membrane at physiological conditions is in the nanomolar range. It is concluded that oxygen permeation across the cell plasma membrane cannot be a rate-limiting step for cellular respiration.Correlations of the form PM = cK~ between membrane permeabilities PM of small nonelectrolyte solutes of mol wt <50, including oxygen, and their partition coefficients K into hexadecane and olive oil are reported. Hexadecane: c = 26 cm/s, s = 0.95; olive oil: c --23 cm/s, s = 1.56. These values ofc and s differ from those reported in the literature for solutes of 50 < mol wt <300 (Walter, A., and J. Gutknecht. 1986. Journal of Membrane Biology. 90:207-217). It is concluded that oxygen permeability through membranes can be reliably predicted from measurement of partition coefficients.

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