Takashi Aoyama

Cultured Cells from a Severe Combined Immunodeficient Mouse Have a Slower than Normal Rate of Repair of Potentially Lethal Damage Sensitive to Hypertonic Treatment

The effects of hypertonic 0.5 M NaCl treatment after irradiation on the repair of DNA damage were examined in fibroblasts of the severe combined immunodeficient (scid) mouse. These cells are hypersensitive to ionizing radiation because of a deficiency in the repair of double-strand breaks. Hypertonic treatment caused radiosensitization due to a fixation of potentially lethal damage (PLD) in scid cells, demonstrating that scid cells normally repair PLD. To assess the kinetics of the repair of PLD, hypertonic treatment was delayed for various times after irradiation. Potentially lethal damage was repaired during these times in isotonic medium at 37 degrees C. It was found that the rate of repair of PLD was much slower in scid cells than in BALB/c 3T3 cells, which have a wild-type level of radiosensitivity. This fact indicates that the scid mutation affects the type of repair of PLD that is sensitive to 0.5 M NaCl treatment. In scid hybrid cells containing fragments of human chromosome 8, which complements the radiosensitivity of the scid cells, the rate of repair was restored to a normal level. An enzyme encoded by a gene on chromosome 8 may also be connected with PLD which is sensitive to hypertonic treatment.

Neurotropin increases in vitro life span of human fibroblasts

Fibroblasts isolated from human fetal tissues (skin, lung and heart) exhibited the following population doubling levels (PDL): about 40 PDL for skin; 60 PDL for lung; 10 PDL for heart. Neurotropin extract from vaccinia-virus infected skin tissues of rabbits increases the growth rate of the fibroblast when added to the old lung cell culture (PDL 40, 70% of the maximum life span) but not when added to the young lung (PDL 5, 8% of the maximum life span). Neurotropin (40 or 80 micrograms/ml) increased the replicative life span of the three lines of fibroblasts (skin, lung and heart) when added to the young cell cultures. The increase of the maximum PDL of skin, lung and heart fibroblasts with the Neurotropin treatment was by 19%, 5% and 17%, respectively. When Neurotropin was added to the old lung cell culture (PDL 44, 73% of the maximum life span), the maximum PDL was increased by 9%. Since the existence of hydrocortisone, known to extend the in vitro life span, was negligible in the fluorimetric test of the medium containing Neurotropin, this agent may belong to the class other than hydrocortisone, for increasing in vitro life span.

Are three forms of potentially lethal damage expressed after X irradiation by treatment with hypertonic solutions in Chinese hamster V79 cells

X rays have been shown to induce two forms of potentially lethal damage (PLD), fast-repairing PLD and slowly repairing PLD, whose repair is completed in 1 h and 4-6 h, respectively. In this study three modes of treatment with hypertonic solutions containing different NaCl concentrations for different durations (0.5 M for 30 min, 0.225 M for 4 h, 0.16 M for 16 h) were examined to determine which form of PLD is expressed under each condition. These three modes of treatment enhanced the cell-killing action of X rays on actively growing V79 cells due to fixation of PLD. The kinetics of recovery from PLD was assessed by delayed treatments with hypertonic solutions. Cells exposed to one of the three treatments (see above) had completed recovery times of 1, 4 and 8 h, respectively, suggesting the possibility that these three modes of treatment cause the expression of different forms of PLD. As has been reported, treatment with 0.5 M NaCl for 30 min expressed fast-repairing PLD. The independence of the PLD expressed by 0.225 M NaCl for 4 h from fast-repairing PLD expressed after 0.5 M NaCl for 30 min was shown by combined treatment with the two modes, which reduced survival to the level that would be reached if the two modes acted independently. The data on the recovery time and on the inhibition by 0.225 M NaCl of recovery from slowly repairing PLD in plateau-phase cells indicated that the PLD expressed after 0.225 M NaCl for 4 h may be related to slowly repairing PLD. The combined treatment of 0.16 M NaCl for 16 h with 0.225 M NaCl for 4 h indicated independent action, albeit incomplete, of the PLD expressed after 0.16 M NaCl for 16 h from slowly repairing PLD. We propose for the first time that very slowly repairing PLD is expressed after 0.16 M NaCl for 16 h in exponentially growing cells and that therefore three forms of PLD are expressed by hypertonic treatments after X irradiation.

Changes with age in swimming performance of X-irradiated mice

Abstract The time required to swim 250 cm was determined once weekly for the entire life of fifteen pairs of male dd/K mice. The irradiated group was exposed to a single 224 rad of X-rays at 20 weeks of age. Median survival time (ST50) for the control was 88.9 weeks and that for the irradiated group was 77.4 weeks, and both regression lines relating to death rate and age were parallel. The swimming ability of control mice began to decrease when the mice were 40 weeks of age, after which there was a gradual reduction with age, at 0.00646/day. In the irradiated group, the swimming ability decreased from seven weeks after the irradiation. The time of 50% reduction of swimming speed (TRS50) for the control was 78.9 weeks and that for the irradiated group was 66.3 weeks, and the slopes of the regression lines relating reduction rate and age were similar. Differences between ST50 and TRS50 were 10 weeks in the control and 11 weeks in the irradiated group, respectively. These results indicate that there is no basic difference in the reduction in swimming ability between control and irradiated mice. The X-irradiation may simply mean that the reduction in the swimming ability is displaced to an earlier time, with no alteration in the rate of reduction, and that the earlier appearance in the irradiated group is related to premature aging as induced by irradiation.

Methylisobutylxanthine, a potent inhibitor of cAMP phosphodiesterase, enhances the sensitivity of Chinese hamster ovary cells to mitomycin C.

The effect of 1-methyl-3-isobutylxanthine (MIX) on the sensitivity of CHO cells to mitomycin C (MC) was examined. Treatment of cells with MIX before MC-exposure greatly enhanced the cellular sensitivity to MC, with no effect on plating efficiency. Treatment with MIX after MC-exposure had only a slight effect. The sensitivity to MC was enhanced, biphasically, with increases in the period of pretreatment with MIX. The sensitivity of cells to MC fluctuated through the cell cycle, in that they were most sensitive to MC in G1 and resistant in the G2/M phase. Flow cytometric studies revealed a partial accumulation of cells in G1 (12% increase) at 16 h after MIX treatment. This means that the enhanced sensitivity with MIX pretreatment may partly depend on the accumulation of the cells in G1 and which are sensitive to MC. However, other cellular processes may also be involved, since the accumulation in G1 cannot explain all of the sensitization.

Modification of the sensitivity of CHO cells to mitomycin C by dibutyryl cyclic AMP.

The survival of CHO cells exposed to mitomycin C was decreased three times that of the cells treated with 1 mM dibutyryl cyclic AMP before mitomycin C treatment, as compared to the absence of treatment with this cyclic nucleotide. The sensitization effect began at 3-4 hours after the start of pre-treatment, reached a maximum at around 10 hours and continued to be effective. Post-treatment with the cyclic nucleotide for more than 12 hours increased the survival of CHO cells exposed to mitomycin C.