Relationship between the recovery from sublethal X-ray damage and the rejoining of chromosome breaks in normal human fibroblasts

Using plateau-phase cultures of AG1522 normal human fibroblasts, we examined relationships between the breakage and rejoining of chromosomes and the induction and repair of sublethal damage (SLD) following fractionated doses of X rays. The rate constant for the rejoining of breaks in prematurely condensed interphase chromosomes, measured previously, accurately predicts both the rate of change in survival due to potentially lethal damage (PLD) repair and the rate of change in survival for dose fractionation due to SLD repair. Further, changes in the frequency of chromosome-type deletions and asymmetrical exchange aberrations measured in the first postirradiation mitosis corresponded closely with changes in cell killing when doses were fractionated, and a dose-fractionation- or dose-rate-independent alpha component of damage was similar for aberration and cell killing end points. These results substantiate the hypothesis that sublethal damage repair results from the rejoining of breaks in interphase chromatin produced by a first dose so they no longer are capable of interacting with those produced by a second dose. The fact that the repair of potentially lethal damage is also readily explained on the basis of chromosome break rejoining (M. N. Cornforth and J. S. Bedford, Radiat. Res. 111, 385-405 (1987)) strongly suggests that PLD and SLD repair are different manifestations of the same basic process operating on the same basic lesions.     

Age-dependent modification by lactate of repair of potentially lethal damage in normal human diploid fibroblasts

We investigated age-related changes in normal human diploid fibroblasts (strain AG 1522) followed throughout their lifespan in vitro. Radiosensitivity and the capacity to repair potentially lethal radiation damage (PLD) remained constant until senescence was reached. However, the effects of incubation with lactate on PLD repair were age dependent and complex. Low millimolar concentrations of lactate increased the capacity to repair PLD, whereas higher concentrations suppressed it in a concentration-dependent manner. With increasing in vitro age, the cells became less sensitive to the modifying effects of lactate; in presenescent cells, the modifying effects of lactate on PLD repair had completely vanished.     

Reduced repair of potentially lethal radiation damage in glutathione synthetase-deficient human fibroblasts after X-irradiation

Using a human fibroblast strain deficient in glutathione synthetase and a related proficient control strain, the role of glutathione (GSH) in repair of potentially lethal damage (PLD) has been investigated in determining survival by plating cells immediately or 24 h after irradiation. After oxic or hypoxic irradiation, both cell strains repair radiation-induced damage. However, under hypoxic conditions, the proficient cells repair PLD as well as under oxic conditions while the deficient cells repair less PLD after irradiation under hypoxic than under oxic conditions. Therefore, the oxygen enhancement ratio (o.e.r.) for proficient cells is similar whether the cells are plated immediately or 24 h later (2.0 and 2.13, respectively). In contrast, the o.e.r. for deficient cells is lower when the cells are plated 24 h after irradiation than when they are plated immediately thereafter (1.16 as compared to 1.55). The results indicate that GSH is involved in PLD repair and, in particular, in the repair of damage induced by radiation delivered under hypoxic conditions.     

Potentially lethal damage repair in cell lines of radioresistant human tumours and normal skin fibroblasts

Radiation cell survival data were obtained in vitro for three cell lines isolated from human tumours traditionally considered to be radioresistant--two melanomas and one osteosarcoma--as well as from a diploid skin fibroblast cell line. One melanoma cell line was much more radioresistant than the other, while the osteosarcoma and fibroblast cell lines were more radiosensitive than either. For cells growing exponentially, little potentially lethal damage repair (PLDR) could be demonstrated by comparing survival data for cells in which subculture was delayed by 6 h with those sub-cultured immediately after treatment. For the malignant cells in plateau phase, which in these cells might be better termed 'slowed growth phase', since an appreciable fraction of the cells are still cycling, a small amount of PLDR was observed, but not as much as reported by other investigators in the literature. The normal fibroblasts, which achieved a truer plateau phase in terms of noncycling cells, showed a significantly larger amount of PLDR than the tumour cells.     

Effect of confluent holding on potentially lethal damage repair, cell cycle progression, and chromosomal aberrations in human normal and ataxia-telangiectasia fibroblasts

The effects of confluent holding recovery on survival, chromosomal aberrations, and progression through the life cycle after subculture of human diploid fibroblasts X-irradiated during density inhibition of growth have been examined. The responses of three normal strains were determined and compared with those of four ataxia-telangiectasia (AT), an AT heterozygote, and two hereditary retinoblastoma strains. The capacity for potentially lethal damage repair (PLDR) was slightly reduced in retinoblastoma cells and almost absent in AT cells, but normal in an AT heterozygote. The decline in chromosomal aberrations seen in normal cells during confluent holding was absent in AT cells, consistent with the lack of PLDR. Following subculture, all irradiated AT fibroblasts progressed through the cell cycle to the first mitosis with no delay. AT heterozygotic and retinoblastoma cells showed both an enhanced delay in the initiation of DNA synthesis and a large fraction of cells irreversibly blocked in G1 as compared with normal cells. Both the delayed entry into S and the G1 block were reduced by confluent holding. These results indicate that AT homozygotic and heterozygotic cells respond quite differently to X irradiation.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on repair of DNA breaks, interphase chromatin breaks, and potentially lethal damage in plateau-phase CHO cells.

There is evidence suggesting that radiosensitization induced in mammalian cells by substitution in the DNA of thymidine with BrdU has a component that relies on inhibition of repair and/or fixation of radiation damage. Here, experiments designed to study the mechanism of this phenomenon are described. The effect of BrdU incorporation into DNA was studied on cellular repair capability, rejoining of interphase chromosome breaks, as well as induction and rejoining of DNA double- and single-stranded breaks (DSBs and SSBs) in plateau-phase CHO cells exposed to X rays. Repair of potentially lethal damage (PLD), as measured by delayed plating of plateau-phase cells, was used to assay cellular repair capacity. Rejoining of interphase chromosome breaks was assayed by means of premature chromosome condensation (PCC); induction and rejoining of DNA DSBs were assayed by pulsed-field gel electrophoresis and induction and rejoining of DNA SSBs by DNA unwinding. A decrease was observed in the rate of repair of PLD in cells grown in the presence of BrdU, the magnitude of which depended upon the degree of thymidine replacement. The relative increase in survival caused by PLD repair was larger in cells substituted with BrdU and led to a partial loss of the radiosensitizing effect compared to cells tested immediately after irradiation. A decrease was also observed in the rate of rejoining of interphase chromosome breaks as well as in the rate of rejoining of the slow component of DNA DSBs in cells substituted with BrdU. The time constants measured for the rejoining of the slow component of DNA DSBs and of interphase chromosome breaks were similar both in the presence and in the absence of BrdU, suggesting a correlation between this subset of DNA lesions and interphase chromosome breaks. It is proposed that a larger proportion of radiation-induced potentially lethal lesions becomes lethal in cells grown in the presence of BrdU. Potentially lethal lesions are fixed via interaction with processes associated with cell cycle progression in cells plated immediately after irradiation, but can be partly repaired in cells kept in the plateau-phase. It is hypothesized that fixation of PLD is caused by alterations in chromatin conformation that occur during normal progression of cells throughout the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Repair of potentially lethal and sublethal damage induced by neocarzinostatin in normal and ataxia-telangiectasia skin fibroblasts

Changes in 32P labeling of phosphatidylinositol-4,5-bisphosphate (PIP2) were examined during ADP-induced aggregation of washed rabbit platelets prelabeled with [32P]phosphate. ADP caused a significant decrease in the amount and 32P labeling of PIP2 at 10 and 60 sec. The decrease in labeling persisted at 2.5 min when the platelets were still aggregated, but disappeared by 10 min. Platelets refractory to ADP showed no further significant change in 32P in PIP2 when exposed to ADP; a decrease in PIP2 labeling could be induced, however, after platelets had recovered their disc shape and sensitivity to ADP. These data indicate that PIP2 may play a role in the response of platelets to ADP.     

Novobiocin inhibits the repair of potentially lethal damage but not the repair of sublethal damage

Because of the critical role of the DNA topoisomerases in the synthesis and conformation of DNA, and the well-known observation that radiation inhibits replicative DNA synthesis, we have examined the possibility that inhibitors of these enzymes might influence radiation lethality. In particular, using protocols involving the administration of either fresh or conditioned medium, we examined the ability of intercalative and nonintercalative inhibitors to affect the expression of potentially lethal damage and/or sublethal damage. The inhibitors examined were amsacrine, teniposide, etoposide, and novobiocin; only the latter compound was clearly effective in a selective way at nontoxic concentrations, and this was observed specifically in reference to the repair of potentially lethal damage effected by incubation in conditioned medium. These results are another example of differences between the repair of sublethal versus potentially lethal damage that further support distinctions between the two. At a mechanistic level, these and other data suggest that the property of novobiocin that is relevant in the foregoing is its metabolic inhibition of replicative DNA synthesis, a process which may be more important in the repair of potentially lethal damage as opposed to sublethal damage.     

Incomplete rejoining of DNA double-strand breaks in unstimulated normal human lymphocytes

We investigated the repair kinetics of DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in unstimulated normal human peripheral blood lymphocytes (HPBL). SSBs and DSBs induced by gamma-irradiation (at 0 degree C) were assayed without radiolabel by alkaline and neutral filter elution, respectively. Incubation of irradiated cells at 37 degrees C for various lengths of time demonstrated that the percent DNA rejoined increased until it reached a plateau at approximately 60 min; this repair plateau underwent no substantial change when incubation continued for 20-24 h. The level of the plateau indicated how closely the elution profile of DNA from cells irradiated and incubated (experimental) resembled the elution profile of DNA from unirradiated cells (control). After 6 Gy and 60 min incubation, the alkaline elution profile of DNA from experimental cells from 5 donors was indistinguishable from that seen in DNA from control cells, suggesting that rejoining of SSBs was complete. In contrast after 100 Gy and 60 min incubation the neutral elution profile of DNA from cells from the same donors demonstrated that, compared to DNA from control cells, rejoining of DSBs was approximately two-thirds complete. In the range of 2-8 Gy, 85-104% of SSBs were rejoined after 60 min incubation; in the range of 30-120 Gy, 46-80% of DSBs were rejoined after 60 min incubation. These unexpected results stand in contrast to our previous studies with confluent normal human diploid fibroblasts (HDF), in which rejoining of both SSBs and DSBs was greater than 90% complete by 60 min repair incubation and 100% complete after 18-24 h.     

A comparison of chromosome repair kinetics in G(0) and G(1) reveals that enhanced repair fidelity under noncycling conditions accounts for increased potentially lethal damage repair

Potentially lethal damage (PLD) and its repair were studied in confluent human fibroblasts by analyzing the kinetics of chromosome break rejoining and misrejoining in irradiated cells that were either held in noncycling G(0) phase or allowed to enter G(1) phase of the cell cycle immediately after 6 Gy irradiation. Virally mediated premature chromosome condensation (PCC) methods were combined with fluorescence in situ hybridization (FISH) to study chromosomal aberrations in interphase. Flow cytometry revealed that the vast majority of cells had not yet entered S phase 15 h after release from G(0). By this time some 95% of initially produced prematurely condensed chromosome breaks had rejoined, indicating that most repair processes occurred during G(1). The rejoining kinetics of prematurely condensed chromosome breaks was similar for each culture condition. However, under noncycling conditions misrepair peaked at 0.55 exchanges per cell, while under cycling conditions (G(1)) it peaked at 1.1 exchanges per cell. At 12 h postirradiation, complex-type exchanges were sevenfold more abundant for cycling cells (G(1)) than for noncycling cells (G(0)). Since most repair in G(0)/G(1) occurs via the non-homologous end-joining (NHEJ) process, increased PLD repair may result from improved cell cycle-specific rejoining fidelity of the NHEJ pathway.     

Evidence for the repair of potentially lethal damage in irradiated bone marrow

Summary The effects on cell survival of maintaining bone marrow cells (CFU-S) in situ following irradiation and before assay by transplantation was investigated. When the CFU-S cells are maintained in situ following irradiation survival drops and plateaus at about 9 h post-irradiation. Evidence is presented that this decrease in survival may be due to potentially lethal damage repair (PLD) inhibition caused by post-irradiation in situ holding. This effect on PLD repair is different than that usually found in cells in vitro and in vivo tumors in that it mainly alters the shoulder rather than the slope of the survival curve of CFU-S cells. It is different than PLDR found in vivo for normal mammary and thyroid gland epithelial cells because in situ holding decreases rather than increases the survival of CFU-S cells. Evidence is also presented that the radiation survival curve for in situ bone marrow cells (CFU-S) may not have a shoulder.Supported in part by NIH, NCI grants P01 CA 19298 and P30 CA 14520Supported in part by an American Cancer Society Clinical Fellowship     

X-ray-related potentially lethal damage expressed by chromosome condensation and the influence of caffeine

Caffeine has been reported to induce premature chromosome condensation (PCC) in S-phase cells in the presence of an inhibitor of DNA synthesis. We found that when S-phase cells are treated with caffeine and hydroxyurea after X irradiation, substantially more potentially lethal damage (PLD) is expressed, but the addition of cycloheximide, which inhibits PCC induction in S-phase cells, in the presence of caffeine and hydroxyurea reduces the expression of PLD to the same level as seen with caffeine alone. This can be interpreted to mean that the expression of PLD seen with caffeine in the absence of an inhibitor of DNA synthesis is not associated with chromosome condensation. Evidence that PCC induction in S-phase cells and the influence of caffeine on PLD expression were suppressed by incubation at 40 degrees C of tsBN75 cells with a ts defect in ubiquitin-activating enzyme indicates the involvement of ubiquitin in these two processes. These observations as well as previous findings on ubiquitin suggest to us that caffeine induces changes in DNA-chromatin conformation, which are caused by induction of PCC or ubiquitination of chromosomal protein. Such changes occurring postirradiation would favor expression of PLD.     

Age-dependent decline in rejoining of X-ray-induced DNA double-strand breaks in normal human lymphocytes

Unstimulated human peripheral blood lymphocytes (HPBL), separated by density centrifugation from anticoagulated whole blood, were X-irradiated (30 Gy) on ice and incubated in medium at 37 degrees C for repair times of 15, 30, and 120 min. Blood donors were 18 normotensive, non-smoking Caucasians aged 23-78, free from overt pathology and not taking any medications. Neutral filter elution was used to assay DNA double-strand break (DSB) induction and completeness of DSB rejoining (plus rejoining of any X-ray-induced alkali-labile sites converted to DSBs in vitro at pH 9.6). After 30 or 120 min repair incubation, the percentage of DSBs rejoined by cells from older donors (aged 66-78 years) was less than half the percentage of DSBs rejoined by cells from younger donors (aged 23-39 and 42-57). When data from the 3 age groups were pooled, the age-related decline in percent DSBs rejoined was significant for repair times 30 min (r = -0.63, p less than 0.005) and 120 min (r = -0.64, p less than 0.005) but not for 15 min (r = -0.04). These age-related declines were observed even though DNA from older donors sustained fewer strand breaks as demonstrated by the negative correlation between donor age and DSB induction (r = -0.65, p less than 0.005). These results suggest that the efficacy of X-ray-induced DSB repair diminishes with in vivo age in unstimulated HPBL.     

Quantitative aspects of repair of potentially lethal damage in mammalian cells.

Stationary cultures of Ehrlich ascites tumour cells have been irradiated with X-rays and then immediately or after a time interval trep plated to measure the survival. The increase in survival observed after delayed plating is interpreted as repair of potentially lethal damage. A cybernetic model is used to analyse these data. Three states of damage are assumed for the cells. In state A the cells can grow to macrocolonies, in state B the cells have suffered potentially lethal damage and can grow to macrocolonies only if they are allowed to repair the damage and in state C the cells are lethally damaged. A method of deriving the values of the parameters of the model from the experimental data is given. The dependence of the reaction rate constant of the repair of potentially lethal damage on the dose D is used to derive a possible mechanism for the production of the shoulder in the dose effect curve. Finally this model is compared with other models of radiation action on living cells.     

Fixation and repair of radiation-induced potentially mutagenic damage sensitive to hypertonic treatment in human diploid fibroblasts

The effect of hypertonic salt treatment on the repair of potentially lethal damage and potentially mutagenic damage in X-irradiated asynchronous and synchronous human diploid fibroblasts (IMR91) have been studied. Resistance to 6-thioguanine was used for the mutagenic end point. When cells in late-S-phase were treated with hypertonic salt solution immediately after X-irradiation, both cell killing and mutation induction were enhanced, as compared to X-irradiation alone. This suggests that X-irradiation of cells in late S phase induces both potentially lethal damage and potentially mutagenic damage and that both are sensitive to hypertonic salt solution. When cells were allowed 2 h for repair after exposure to X-rays, both types of damage were completely repaired. Almost the same results were obtained with asynchronous cells. These results are discussed in terms of the relationship between radiation damage leading to cell lethality and mutagenesis.     

Exogenous lactate modifies the repair of potentially lethal damage in three human tumor cell lines irradiated in vitro

Lactate is one of several pathophysiological factors accumulating in the micromilieu of tumors under both hypoxic and well-oxygenized conditions, and thus may affect the recovery of irradiated tumor cells in vivo. In the present study, we investigated the effects of postirradiation incubation with exogenous lactate during confluent holding recovery on the repair of potentially lethal damage in three human tumor cell lines. Recovery was either unaffected or enhanced by low concentrations of exogenous lactate (2-5 mM), whereas it was suppressed by higher concentrations (10-50 mM). With high concentrations, survival in all three cell lines was lower at the end of the confluent holding period than at the beginning, yielding recovery ratios of less than 1.0. The effects differed quantitatively among the three tumor cell lines, and between the tumor cells and the normal diploid fibroblasts (AG 1522) studied previously.     

Induction of chromosome damage by Neurospora endonuclease in repair-inhibited quiescent normal human fibroblasts

The purpose of these experiments was to determine the role of double-strand breaks in chromosome aberration formations. Quiescent normal human fibroblasts were treated with 3 μM nitrogen mustard and then allowed to repair their DNA damage for 24 h prior to cell fusion and induction of premature chromosome condensation. The extent of chromosome damage was determined in the G1 prematurely condensed chromosomes (G1 PCC). The presence of cytosine arabinoside and hydroxyurea during the repair period in order to accumulate single-strand DNA breaks resulted in an increase in the chromosome-break frequency. Treatment of these repair-inhibited cells with single-strand-specific neurospora endonuclease during fusion to change single-strand lesions into double-strand breajs resulted in a doubling of the aberration frequency. These results support the notion that double-strand breaks are important in chromosome-aberration formation.     

Differential response of human and rodent cell lines to chemical inhibition of the repair of potentially lethal damage

Summary We have examined the effects of several classes of metabolic inhibitors on the repair of potentially lethal damage in density-inhibited cultures of two rodent and two human cell systems which differ in their growth characteristics. Aphidicolin, 1--d-arabinofuranosylcytosine (ara-C) and hydroxyurea showed no effect on PLD repair, whereas the effects of 9--d-arabinofuranosyladenine (ara-A) and 3-aminobenzamide (3-AB) were cell line dependent. For example, 3-AB suppressed PLD repair almost completely in CHO cells, but showed no inhibitory effects in human diploid fibroblasts. These results indicate that inhibitors of DNA replication and poly(ADP-ribose) synthesis are not efficient inhibitors of cellular recovery in irradiated cells and, moreover, that such effects may be cell line dependent.     

Deficiency in fast repair process of potentially lethal damage induced by X-irradiation in fibroblasts derived from LEC strain rats.

The time course for the repair of PLD in LEC and WKAH rat cells irradiated at 5 Gy was examined. In the case of WKAH rat cells, the surviving fraction increased with increasing incubation times after X-irradiation. When hypertonic treatment was performed at each incubation time with 0.5 M NaCl for 20 min, increase in the surviving fractions was not shown. In contrast, no significant recovery of the surviving fraction in LEC rat cells was observed after incubation of irradiated cells with or without 0.5 M NaCl for 20 min. On dose-survival curves, hypertonic treatment with 0.5 M NaCl enhanced radiosensitivity of WKAH rat cells, but not LEC rat cells. Although the surviving fraction of the cells from backcross mice with normal radiosensitivity reduced by treatment with 0.5 M NaCl, the survival fraction was not affected in the cells from backcross mice with higher radiosensitivity by treatment with 0.5 M NaCl. When the cells were X-irradiated and incubated with or without 0.225 M NaCl, the radiosensitivities of LEC and WKAH rat cells treated with 0.225 M NaCl for 4 h were approximately two-fold higher than those of untreated cells. Treatment with caffeine also reduced the surviving fractions of both X-irradiated LEC and WKAH rat cells, compared with those of untreated cells. These results indicated that the slow repair of PLD occurred in LEC rat cells but not the fast repair of PLD.