Cisplatin-modification of DNA repair and ionizing radiation lethality in yeast, Saccharomyces cerevisiae

Cis-diamminedichloroplatinum II (cisplatin) is a DNA inter- and intrastrand crosslinking agent which can sensitize prokaryotic and eukaryotic cells to killing by ionizing radiation. The mechanism of radiosensitization is unknown but may involve cisplatin inhibition of repair of DNA damage caused by radiation. Repair proficient wild type and repair deficient (rad52, recombinational repair or rad3, excision repair) strains of the yeast Saccharomyces cerevisiae were used to determine whether defects in DNA repair mechanisms would modify the radiosensitizing effect of cisplatin. We report that cisplatin exposure could sensitize yeast cells with a competent recombinational repair mechanism (wild type or rad3), but could not sensitize cells defective in recombinational repair (rad52), indicating that the radiosensitizing effect of cisplatin was due to inhibition of DNA repair processes involving error free RAD52-dependent recombinational repair. The presence or absence of oxygen during irradiation did not alter this radiosensitization. Consistent with this result, cisplatin did not sensitize cells to mutation that results from lesion processing by an error prone DNA repair system. However, under certain circumstances, cisplatin exposure did not cause radiosensitization to killing by radiation in repair competent wild type cells. Within 2 h after a sublethal cisplatin treatment, wild type yeast cells became both thermally tolerant and radiation resistant. Cisplatin pretreatment also suppressed mutations caused by exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a response previously shown in wild type yeast cells following radiation pretreatment. Like radiation, the cisplatin-induced stress response did not confer radiation resistance or suppress MNNG mutations in a recombinational repair deficient mutant (rad52), although thermal tolerance was still induced. These results support the idea that cisplatin adducts in DNA interfere with RAD52-dependent recombinational repair and thereby sensitize cells to killing by radiation. However, the lesions can subsequently induce a general stress response, part of which is induction of RAD52-dependent error free recombinational repair. This stress response confers radiation resistance, thermal tolerance, and mutation resistance in yeast.     

Teratogenic effects of mild heat stress during mouse embryogenesis: effect of Trp53

Hyperthermia can be teratogenic in fetal mice exposed during organogenesis, an effect considered to be due to heat-induced apoptosis of cells in the developing organs. We exposed pregnant mice carrying Trp53(+/+), Trp53(+/-) and Trp53(-/-) fetuses to mild whole-body hyperthermia that raised their core temperature to 40.5 degrees C for 60 min on either day 10 or 11 of gestation. On day 18 of gestation, the fetuses were removed from control and hyperthermia-treated mice and genotyped, and tail length was measured. Limb digits were examined for abnormalities. Tail length in unheated control fetuses was influenced by Trp53 status. A complete lack of functional Trp53 (Trp53(-/-)) but not partial lack of function (Trp53(+/-)) resulted in shorter tails compared to Trp53(+/+) fetuses, indicating a role for Trp53 in the regulation of tail lengthening in mouse fetuses. In all three genotypes, hyperthermia on gestation day 10 resulted in tails shorter than unheated controls, and hyperthermia on day 11 resulted in tails longer than controls. There was no effect on limb digit abnormalities. The data suggest that Trp53-dependent or independent apoptosis may not be directly involved in heat-induced teratogenesis, but that the primary teratogenic effect of heat results from the disruption of another tail length-regulating process that is independent of Trp53. However, the nature of the teratogenic outcome of that disruption depends on the gestation time. The ability of Trp53 to additionally regulate the tail lengthening process was also sensitive to the effects of heat, but that sensitivity again depended on the time of the heat stress during gestation.     

Radiation-induced teratogenic effects in fetal mice with varying Trp53 function: influence of prior heat stress

Teratogenesis induced by radiation in fetal mice has been closely linked to Trp53-dependent apoptosis. This study examined teratogenesis in tails and limb digits of fetal mice with varying Trp53 status after a 4-Gy radiation exposure, with and without a prior 40.5 degrees C, 60-min heat stress. Irradiation earlier in gestation (day 11) produced greater effects than later (day 12) exposure, but in both cases the maximum teratogenic effect of radiation occurred in Trp53 normal fetuses, the minimum in Trp53 null fetuses, and intermediate effects in Trp53 heterozygotes, indicating dominance of Trp53-dependent apoptosis. Heat stress 24 h prior to irradiation on day 11 did not alter the teratogenic effects in Trp53 normal or heterozygous fetuses, but it reduced effects in the Trp53 null fetuses. Conversely, heat stress immediately before irradiation on day 11 amplified teratogenesis in Trp53 null fetuses, still with only a small or no effect on fetuses with full or partial Trp53 function, respectively. These results indicate little effect of mild heat on Trp53-dependent apoptosis after irradiation, but they also suggest heat-induced amplification of Trp53-independent processes that led to apoptosis when heat was delivered near the time of radiation exposure, and heat-induced protection of that process when sufficient expression time was allowed. However, Trp53-dependent apoptosis, when functional, acted as the ultimate determinant of radiation-induced teratogenic effects during early organogenesis. On gestation day 12, radiation effects were diminished, but heat stress 24 h prior to radiation exposure had a large amplifying effect in Trp53 normal or heterozygous fetuses. In the absence of functional Trp53, the sensitizing effect of the heat was diminished. The results may suggest that at later times in organ development, DNA repair is more active, allowing some cells to escape radiation-induced Trp53-dependent apoptosis. However, heat may be able to significantly inhibit this active repair and increase the teratogenic effect of radiation. A diminished effect in the absence of functional Trp53 is consistent with an influence of heat on inhibiting DNA repair, but with a diminished probability of apoptosis.     

Influence of prior exposure to low-dose adapting radiation on radiation-induced teratogenic effects in fetal mice with varying Trp53 function

Teratogenesis in tails and limb digits of fetal mice with varying Trp53 status was examined after exposure of pregnant females to 4 Gy gamma radiation with and without a prior 30-cGy exposure. Prior low-dose exposure modified the teratogenic effects of radiation in a manner dependent upon Trp53 status and gestation time. A 4-Gy exposure on gestation day 11 resulted in tail shortening and digit abnormalities. A 30-cGy exposure 24 h prior to a 4-Gy radiation exposure on day 11 reduced the extent of both digit abnormalities and the tail-shortening effects in Trp53(+/+) fetuses and also reduced tail shortening in Trp53(+/-) fetuses, but to a lesser extent. However, the pre-exposure enhanced the tail-shortening effects of 4 Gy in Trp53(-/-) fetuses. In contrast, a 30-cGy exposure given 24 h prior to a 4-Gy exposure on gestation day 12 had no effect on the reduced tail length resulting from the 4-Gy exposure of Trp53(+/+) or Trp53(+/-) fetuses, but it partly protected Trp53(-/-) fetuses against reduced tail length. A 4-Gy exposure alone on day 12 did not result in any increase in the frequency of digit abnormalities in Trp53(-/-) fetuses so any protective effect of the preirradiation could not be detected. However, the preirradiation did result in protection against in digit abnormalities in Trp53(+/-) fetuses. We conclude that radiation-induced teratogenesis reflects both Trp53-dependent and independent processes that lead to apoptosis, and these respond differently to prior adapting doses.     

BRD4 Promotes DNA Repair and Mediates the Formation of TMPRSS2-ERG Gene Rearrangements in Prostate Cancer

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