C3H-10T1/2 cells were grown to plateau phase and assessed for recovery from radiation damage under acidic and normal pH conditions. Repair of potentially lethal damage (PLD) was observed after X-ray doses of 5.0, 6.0 and 12.0 Gy and repair increased with dose. The repair at an acidic pH of 6.8 was greater than at the normal pH of 7.4. Repair of potentially transforming damage (PTD) was also observed when cells were held in plateau phase after irradiation. Repair of PTD was greater at the acidic pH than at the normal pH. When PTD recovery was plotted vs PLD recovery the results showed a good linear correlation with approximately twice as much PTDR. The experimental conditions allowing PLDR and PTDR indicate that the repair process may be error-free, since transformation was reduced in all experiments which showed recovery from PLD.
Since hyperthermia is being used extensively in cell biology studies and as a tool for cancer therapy with or without irradiation (Proc. Int. Cancer Symposium on Cancer Therapy by Hyperthermia and Radiation, 1975; Streffer et al., 1978; Dewey et al., 1979), it is important to determine whether heat treatment can cause oncogenic transformation. In a recent article by Harisiades et al. (1980), it was shown that heat treatment alone in the temperature range 40.0–45.0°C did not cause oncogenic transformation in cultured C3H–10T½ cells, while heat treatments in the temperature range 40.0–42.2°C reduced the incogenic transformation frequency resulting from X rays when heating was performed immediately after X irradiation. We have also investigated the effect of heat alone or combined with X rays on induction of oncogenic transformation in the C3H–10T½ cell system developed by Reznikoff et al. (1973a; b). These data were first presented at the 28th Annual Meeting of the Radiation Research Society (Raaphorst et al., 1980). We have used experimental protocols which include heating either before or after X irradiation such that the two treatments were done one immediately after the other or separated by an incubation interval at 37°C.
SummaryIonizing radiation markedly alters the response of the reovirus transcriptase unblocking mechanism to stimulation by K+ ions, which normally trigger the switch-on of transcription in this system. In irradiated subviral particles the concentration of K+ ions needed to trigger switch-on is reduced in a dose-dependent way. The observed alteration of switch-on characteristics appears to correlate with alteration of the electrophoretic behaviour of a single major polypeptide species. These observations have important implications for understanding some of the effects of ionizing radiation on cells, most notably the induction of both latent virus and cell differentiation.
Activation of the transcriptase in purified reovirus by chymotrypsin requires facilitating conditions in the digestion mix. In the presence of facilitation the virions are uncoated to cores. Without facilitation, uncoating is blocked at an intermediate stage, with transcriptase latent. Two independent types of facilitation have been discovered. The first type is mediated by K+, Rb+ or Cs+ ions. A second type, described here, is mediated by suitably high concentrations of virus in the digestion mix and requires collisions between intermediate subviral particles for its occurrence. The virus-concentration-mediated process is reversibly inhibited by high levels of active chymotrypsin.
Effects of Ca 2+ and Mg 2+ on the "switch-on" of transcriptase function in reovirus intermediate subviral particles were examined. Switch-on in this system is triggered by K + ions, whereas Na + ions antagonize the effects of K + ions. Once effected, switch-on is irreversible. In the absence of Ca 2+ and Mg 2+ ions, switch-on of transcriptase is effected by incubation at 37 °C in the presence of a particular ratio of K + /Na + (total K + plus Na + being kept constant). Inclusion of Ca 2+ in the activation step lowers the ratio that triggers the switch-on event. This indicates that Ca 2+ destabilizes the switched-off state of the transcriptase. Inclusions of Mg 2+ ions in the activation step raises the K + /Na + ratio necessary to trigger switch-on. Thus Mg 2+ tends to stabilize the switched-off state of the transcriptase to the triggering action of K + ions. Inclusion of both Ca 2+ and Mg 2+ together during the activation step results in mutual inhibition of the effects expected from each divalent cation alone. These results indicate that switch-on of transcriptase function, which is triggered by K + ions, can be modulated by interactions involving the major physiological cations (Na + , K + , Mg 2+ , Ca 2+ ).
Specific monovalent cations control the modification of reovirus infectivity by chymotrypsin. Digestion in K(+), Rb(+), or Cs(+) reduces infectivity several logs, whereas in Na(+) or Li(+) digestion markedly enhances infectivity.
In 25 out of 33 cases the survival response of myeloid progenitor cells from fresh human bone marrows, X-irradiated in vitro in phosphate-buffered saline at 23 degrees C, was characterized by a two-term exponential relationship, with D0 values of 0.42 +/- 0.19 Gy and 1.38 +/- 0.37 Gy, respectively. In the remaining eight cases for which the colony-forming efficiency was 10 times lower, survival followed a single exponential function with D0 = 1.18 +/- 0.15 Gy. The biphasic response at 23 degrees C became a single exponential response when the temperature at irradiation was 30 or 37 degrees C.
Effects of two anticalmodulin drugs, trifluoperazine and calmidazolium, on normal and transformed C3H10T1/2 cells were examined in vitro. As indicated by reduction of plating efficiencies in the presence of these drugs, the intrinsic sensitivities of normal and transformed cells were similar and showed no consistent differences. Comparison of cell killing kinetics in cycling and noncycling cell populations revealed that both drugs were preferentially cytotoxic for cycling cells. This differential cytotoxicity for cycling versus noncycling cells could provide a basis for exploitation of anticalmodulin drugs in cancer chemotherapy.
