In vivo 31 P nuclear magnetic resonance (<tex-math>${}^{31}{\rm P}\ {\rm NMR}$</tex-math>) spectroscopy has been used to compare metabolic profiles with tumor radiosensitivity. A radioresistant mammary carcinoma (MCa) and a radiosensitive methylcholanthrene-induced fibrosarcoma (Meth-A) were studied by 31 P NMR spectroscopy in the tumor volume range of approximately <tex-math>$100-1200\ {\rm mm}^{3}$</tex-math>. The MCa showed a constant pH in this volume range; the ratio of phosphocreatine to inorganic phosphate (<tex-math>${\rm PCr}/P_{{\rm i}}$</tex-math>) for <tex-math>$160-300\ {\rm mm}^{3}$</tex-math> tumors was 0.33 ± 0.11 (mean ± standard deviation) and did not change (0.29 ± .09) for tumors in the volume range of <tex-math>$600-1200\ {\rm mm}^{3}$</tex-math>. In comparison, the Meth-A showed a decrease in tumor pH as volume increased from <tex-math>$160-300\ {\rm mm}^{3}$</tex-math> (pH 7.16 ± .04) to <tex-math>$600-1200\ {\rm mm}^{3}$</tex-math> (pH 6.94 ± .07). Tumor <tex-math>${\rm PCr}/P_{{\rm i}}$</tex-math> decreased from 0.70 ± .16 (<tex-math>$160-300\ {\rm mm}^{3}$</tex-math>) to 0.33 ± .16 (<tex-math>$600-1200\ {\rm mm}^{3}$</tex-math>). The radiation doses for control of MCa-induced tumors in 50% of the treated tumors ranged from 65 (<tex-math>$150-250\ {\rm mm}^{3}$</tex-math>) to 71 Gy (<tex-math>$1000-1300\ {\rm mm}^{3}$</tex-math>) and for the Meth-A-induced tumors ranged from 35 (<tex-math>$150-250\ {\rm mm}^{3}$</tex-math>) to 38 Gy (<tex-math>$1000-1300\ {\rm mm}^{3}$</tex-math>). These results suggest that 31 P NMR spectra may be a qualitative predictor of tumor hypoxia, although further studies of human and rodent tumors are necessary to support this hypothesis.

Cell culture studies were carried out to determine whether moderate hyperthermia reduces the oxygen enhancement ratio of cells under well-defined cultural conditions. Using asynchronously growing HeLa cells, the OER of cells with and without glucose was determined following exposure of cells to moderate hyperthermia, 40.5°C for 1 hr, immediately after X irradiation. The OER of cells with 5 mM glucose was 3.2, whereas the OER of glucose-deprived cells was reduced to 2.0. The pH of the cell culture medium was kept at 7.4 throughout the experiments. The present finding may provide a clue toward further enhancing the radiosensitization of hypoxic cells by heat.

Investigations were carried out to determine the importance of glucose as a modifying factor for hyperthermic cellular damage. HeLa S-3 cells were heated under oxic and hypoxic conditions, in the presence and absence of D-glucose. Temperatures as low as 41°C selectively enhanced killing of the glucose deprived hypoxic cell. Glucose deprivation (37°C, 2 hr) or heat alone (41°C for 2 hr in regular medium with glucose) produced minimal cell kill under oxic or hypoxic conditions. No enhancement was seen with hyperthermia under oxic condition in the absence of glucose. The result suggests that the interplay between glucose concentration is important for hyperthermic cell kill.

Mitotically synchronized cultures of HeLa S-3 cells were subjected to the treatment of radiation (400 rad), hyperthermia (43°C), and a combination of both at different phases of the division cycle. The radioresistance was most pronounced in the mid G-1 and late S phases, while thermal resistance was greatest in the early G-1 phase and steadily decreased as cells entered the S phase. Cells in the late S and early G-2 phases were found to be most sensitive to hyperthermia. The sequential treatment of radiation immediately followed by hyperthermia resulted in an enhanced cell killing throughout the cell cycle with a marked synergism occurring in cells in the late S phase. The age-response function of the combined treatment was more similar to that of the thermal age response.

The periodic synthesis of thymidine kinase during the cell cycle has been studied in synchronous populations of HeLa cells. There is a sharp decrease in enzyme activity after cell division followed by a rise in thymidine kinase levels which reaches a maximum several hours after the peak of DNA synthetic activity. Cells treated with inhibitors of protein or RNA synthesis early in the cycle have low levels of thymidine kinase activity. Cells exposed to irradiation, however, have a pattern of prolonged elevated levels of thymidine kinase activity. This altered pattern of enzyme activity resulting from x-irradiation can be mimicked by treatment of the cells with inhibitors of DNA synthesis or of cell division. The data indicate that radiation of a dose of 2000 rads does not interfere with the synthesis of the enzyme and that the prolonged elevation of thymidine kinase activity in x-irradiated cells reflects interference with the progression of the cell population to a phase of the cell cycle that is detrimental to the enzyme.

Alterations in protein and RNA metabolism in asynchronously growing HeLa cell population following 2000 rads of x-irradiation were studied using sucrose density gradients, radioactive precursor labeling, and autoradiographic procedures. It was found that irradiation reduced protein synthesis within a 2-hour period while a depression of RNA synthesis by x-irradiation was evident somewhat later. Rapidly labeled ribosome precursor RNA was delayed in its transformation into smaller RNA molecules as revealed by pulse chase experiments at a time when inhibition of total RNA synthesis becomes noticeable after 2000 rads of x-irradiation. Specific activities of polysomal fractions from irradiated and control cells labeled with radioactive amino acids are almost identical, but the total amount of radioactivity was less in the sedimentation profile of irradiated cells than in controls. Several possible mechanisms that could result in the depression of protein synthesis are discussed.

Synchronized cultures of HeLa cells x-irradiated at different stages of the division cycle were incubated with inhibitors of DNA and protein synthesis. Thymidine at concentrations that inhibit DNA synthesis increases survival of cells irradiated in the G 1 -S transition of S phase when present in the medium during a 3.5-hour postirradiation period. There was no such effect of thymidine on cells irradiated in the early G 1 phase. Inhibitors of DNA synthesis such as hydroxyurea, which at high concentrations are toxic even to unirradiated cells, at nontoxic concentrations will enhance survival of cells irradiated in the S phase, but will decrease survival of cells irradiated in the G 1 phase. At slightly toxic concentrations, hydroxyurea enhances the effect of irradiation even in cells irradiated in the S phase. Acetoxycycloheximide, an inhibitor of both DNA and protein synthesis, increases survival in irradiated cells in a manner similar to that of thymidine, while puromycin, an inhibitor of protein synthesis decreases survival of cells irradiated in the S phase. It was concluded that inhibition of DNA synthesis, but not inhibition of protein synthesis, has a beneficial effect on survival of x-irradiated cells.

HeLa S-3 cells in culture were used to determine the biological effectiveness of a 20-MeV electron beam at the depths of the relative 100% and 25% dose in a polystyrene phantom. It was found that the survival curves at the two different depths were similar and that the extent of the recovery from sublethal irradiation was not significantly different. The clonal size distribution revealed no qualitative differences between the two depths. It was thus concluded that the biological effectiveness of 20-MeV electrons at the two depths was not significantly different. The variability in the biological effectiveness observed in some reports with depth may have resulted from uncertainties in dosimetry or changes in environmental conditions.