G.P. Raaphorst

A comparison of cell killing by heat and/or x rays in Chinese hamster V79 cells, Friend erythroleukemia mouse cells, and human thymocyte MOLT-4 cells

The radiation and/or heat sensitivity of Chinese hamster V79 cells, Friend erythroleukemia (FELC) mouse cells, and MOLT-4 human transformed thymocytes were compared. MOLT-4 cells were more radiosensitive (D0 = 0.50 Gy) than FELC (D0 = 0.65 Gy) and V79 cells (D0 = 1.43 Gy). Arrhenius analysis showed that MOLT-4 cells were more heat sensitive than FELC or V79 cells below 42.0 degrees C, but more heat resistant at higher temperatures. In addition, the MOLT-4 cells showed a single-heat inactivation energy between 41.0 and 45.0 degrees C, while FELC and V79 cells both showed a transition in the inactivation energy at about 43.0 and 43.5 degrees C, respectively. These differences may be related to the fact that the upper temperature limit for the development of thermal tolerance during continuous heating was lower for MOLT-4 cells than for FELC or V79 cells. Killing of FELC and V79 cells was dependent on the sequence in which heat and X rays were applied, but the greatest effect was obtained when both treatments were given simultaneously. Recovery occurred when treatments were separated by incubation at 37.0 degrees C. The MOLT-4 cells did not show a sequence dependence for heating and irradiation. Survival of MOLT-4 cells after heating and/or irradiation was compared using trypan blue dye exclusion or colony formation. Both assays showed similar qualitative responses, but survival levels measured by the trypan blue assay were much higher than those determined from the colony-forming assay.

Thermal sensitivity and radiosensitization in V79 cells after BrdUrd or IdUrd incorporation

Chinese hamster V79 cells were exposed to 10(-5) moles/liter bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd) for 16 or 29 hr and then tested for thermal sensitivity, radiosensitivity, and sensitivity to the combined treatments of heat and radiation. BrdUrd and IdUrd treatment of cells resulted in enhanced radiosensitivity which increased with exposure time but had little or no effect on thermal sensitivity. For 42.0 degrees C heating, no effect was observed, while for 45.0 degrees C heating, a small decrease in thermal sensitivity occurred for both 16- and 29-hr exposure times, in the combined treatment of heat and radiation, the presence of BrdUrd or IdUrd resulted in about the same thermal enhancement in radiosensitivity. BrdUrd and IdUrd uptake into cellular DNA were measured using high-pressure liquid chromatography and, after a 29-hr exposure to 10(-5) moles/liter of BrdUrd or IdUrd, approximately 40% of the thymidine was substituted.

Effect of Anisotonic NaCl Treatment on Cellular Ultrastructure of V79 Chinese Hamster Cells. Part 1 Mitotic Cells

The ultrastructural modifications produced by anisotonic NaCl treatment of Chinese hamster mitotic cells were observed at three NaCl concentrations which have been frequently used in radiosensitization studies: 0.05, 0.5 and 1.5 M. After exposure to 0.05 M NaCl, many well-spread chromosomes are visible. The chromatin fibres are well dispersed and membraneous material is associated with the chromosomes. After hypertonic treatment with 0.5 M NaCl, the chromosomes have a uniform, structureless appearance with some coalescing into larger anaphase-like masses. At 1.5 M NaCl, large scale cellular dehydration is apparent, and filamentous structures such as microfilaments are tightly constricted. The degree of chromosome staining is also reduced below the level of the cytoplasm. After both hypo- and hypertonic NaCl treatment the chromosomes appear swollen relative to untreated cells, but hypertonic treatment causes chromosome clumping and dissociates chromatin. Conformational changes in the chromatin may restrict the capacity for DNA repair and be related to cellular radiosensitivity.

Fixation and repair of radiation damage in normal and ataxia-telangiectasia human cells.

Post-irradiation exposure to anisotonic treatment showed that fixation and repair of potentially lethal damage occurred in both normal and ataxia-telangiectasia human fibroblasts when treated in plateau or exponential growth phase.

Fixation and repair by anisotonic treatment of radiation damage leading to oncogenic transformation

Mouse embryo (C3H 10T1/2) cells were exposed to anisotonic NaCl solutions or combined treatments of radiation and anisotonic solutions. Anisotonic treatment with 0.05 or 0.5 mol/l solutions did not cause transformation and only prolonged exposure at 1.5 mol/l caused significant increases in transformation. When cells were irradiated in the presence of 0.05 mol/l NaCl, increased transformation occurred than when cells were irradiated in medium. Thus, anisotonic treatment after irradiation resulted in fixation of potentially lethal and transformed radiation damage. Fixation of potentially transformed damage was greater for cells irradiated in the presence of 0.05 mol/l NaCl than for cells irradiated in medium. When the NaCl treatment after irradiation was delayed by incubation at 37 degrees C, recovery of potentially lethal and potentially transformed damage was observed.

In vitro transformation by bromodeoxyuridine and X irradiation in C3H 10T1/2 cells

The effect of 10(-5) M bromodeoxyuridine (BrdUrd) substitution in C3H 10T1/2 cells was evaluated. Cellular toxicity increased rapidly for BrdUrd exposure times that were longer than the population doubling time. Radiosensitization by BrdUrd exposure was almost complete after one cell doubling time and was characterized by a decrease in D0 and the survival curve shoulder. Exposure to BrdUrd for one cell doubling time produced only very low transformation levels, but for prolonged BrdUrd exposure times, the transformation frequency per viable cell increased significantly. BrdUrd incorporation also enhanced radiation induction of transformation above the transformation levels resulting from the independent action of X rays or BrdUrd treatment. These results show that BrdUrd is a transforming agent in C3H 10T1/2 cells and thus may be a carcinogen and that BrdUrd can enhance radiation-induced transformation.

Effects of oxygen and misonidazole on cell transformation and cell killing in C3H 10T1/2 cells by X rays in vitro

The effects of oxygen (air) and misonidazole on the transformation and killing of 10T1/2 cells by X rays were examined. The oxygen effect for the cell transformation end point was very similar to that for cell killing. Misonidazole enhanced both cell killing and cell transformation to a similar extent. The enhancement of both end points by misonidazole occurred only in the absence of oxygen during irradiation and was of lesser magnitude than that observed for oxygen. These results demonstrate that the radiation chemical processes leading to cell killing and cell transformation, respectively, are affected similarly by these two enhancers of radiation action.

The Effect of Hypo- and Hypertonic NaCl Solutions on Cellular Damage Resulting from Combined Treatments of Heat and X-rays

The exposure of Chinese hamster V79 cells to 0.05 or 1.5 M NaCl immediately after combined treatment with heat and X-rays resulted in fixation of potentially lethal damage. The synergistic damage resulting from the interaction of heat and X-rays responded to anisotonic treatment like X-ray damage when heating was at 44.0 or 45.0 degrees C and like heat damage when heating was at 41.5 or 42.0 degrees C. When anisotonic treatment was given before heat plus X-ray exposure, sensitization occurred but the differences between heating at 42.0 and 45.0 degrees C were not as large. Incubation at 37 degrees C between heat and X-ray treatments or between X-ray and heat treatments resulted in recovery as manifested by a decrease in the fixation of damage by subsequent anisotonic treatment. Recovery was also observed when cells were incubated at 37 degrees C between the combined exposure to heat plus X-rays and the anisotonic treatment. These data imply that the lesions produced by heat plus X-ray treatments, which are susceptible to salt fixation, can be repaired.

Inhibitors of repair of potentially lethal damage and DNA polymerases also influence the recovery of potentially neoplastic transforming damage in C3H10T-1/2 cells.

The effects of aphidicolin and beta Ara A on radiation sensitivity were evaluated in terms of cell killing, recovery, and neoplastic transformation in the C3H10T-1/2 cell system. When cells were held in plateau phase, recovery of potentially lethal damage (PLD) and potentially transforming damage (PTD) occurred. The addition of beta Ara A resulted in reduced PLD recovery for both the survival and neoplastic transformation end points. The addition of aphidicolin did not affect recovery of PLD or PTD. These data show that the inhibition of polymerase alpha by aphidicolin does not affect recovery of damage leading to cell death or neoplastic transformation. However, the inhibition of both polymerase alpha and beta by beta Ara A resulted in inhibition of recovery of damage leading to both cell death and neoplastic transformation. These data indicated that polymerase beta may be involved in both PLD and PTD recovery.

Reduced pH increases recovery from radiation damage potentially leading to cell death and to in vitro transformation.

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.