Faster repair of DNA double-strand breaks in radioresistant human tumor cells

To investigate the molecular basis of radiation resistance in human tumor cells, the induction and repair of radiation-induced DNA single- and double-strand breaks was determined by DNA elution analysis in two normal human cell lines and 12 early-passage human tumor cell lines of varying radiosensitivities. The radiosensitivities (D0) of the cell lines ranged from 1 to 2.9 Gy. Inherent cellular radiosensitivity was found to directly correlate with the rate at which the DNA double-strand breaks were repaired. Radioresistant cell lines repaired approximately 90% of their radiation-induced DNA double-strand breaks within 1 hr of irradiation while more radiosensitive cell lines required 2-4 hr to repair the same fraction of damage. Radioresistant cell lines also had lower initial DNA double-strand break frequencies. DNA single-strand break induction and repair was not found to be an important factor in the radiation response of human tumor and normal cell lines. Therefore, the rate at which DNA double strand breaks are repaired is a critical factor underlying radioresistance in human tumor cell lines.     

DNA Damage in Human Endothelial Cells after Irradiation in Anoxia

Endothelial cells and fibroblasts have been reported to respond differently to oxidative stress. Both the effects of high oxygen tension and radiation involve the action of free radicals. DNA damage (single strand breaks, SSB, and double strand breaks, DSB) was assayed in human umbilical cord vein (HUV) cells and in Chinese hamster fibroblasts (V79) after irradiation under oxic or anoxic conditions. The cells were exposed to single doses in the range of 2-18 Gy of γ-radiation from ⁶⁰Co. Significantly more DNA damage was induced in the V79 cells than in the HUV cells. As a consequence, a higher oxygen enhancement ratio was obtained for the HUV cells (6.3) as compared to the V79 cells (2.8). The repair of SSB was slower in the HUV cells than in the V79 cells, irrespective of oxic state. For the higher doses, the damage remaining at 60 min after anoxic irradiation, i.e. DSB, was only detected in the V79 cells.     

The sizes of cellular deoxynucleoside 5'-triphosphate pools in relation to sensitivity to electron irradiation using sensitive and resistant cell lines

The deoxynucleoside 5'-triphosphate (dNTP) pool sizes have beendetermined before and after electron (e^–) irradiationin sets of radiation sensitive and resistant cell lines. Inthe L5178Y mouse lymphoma radiosensitive line (LS), the dTTPpool fell 50% following irradiation, whilst the three otherdNTP pools remained unaltered. On the other hand, for the radioresistantline (AII) all four dNTP pools increased by 2-to 3-fold. ThedNTP pools of the Chinese hamster radiosen sitive (V79/) lineand radioresistant (V79/79) lines were unaltered by the radiation,but a difference in pool size was present before irradiation,with the pools of the V79 cells being approximately twice thoseof the V79/79 cells. Two out of the three ataxia telangiectasiacell lines studied show reduced dNTP pools when compared withthose of normal human fibroblasts and these pools were alsounaltered by the radia tion. In the L5178Y and Chinese hamstercells the levels of enzymes involved in the biosynthesis ofdNTPs have been determined. In general the higher the levelof ribonucleoside diphosphate reductase (RDR) the larger thecellular pools. The observed levels of RDR could, in part, explainthe observed results. Increasing the dTTP pool by the additionof deoxy thymidine and deoxycytidine to the cell culture withthe V79/79 cells reduced their sensitivity to the radiation.These results indicate a relationship between a cell's sensitivityto e irradiation and the sizes of the cellular dNTP pools. However,the exact nature of any such relationship is unknown.     

Herpes-zoster and procarbazine therapy in patients with malignant glioma

The radiosensitizing effect of AK-2123, a 3-nitrotriazole, and of misonidazole, a 2-nitroimidazole, was studied on hypoxically irradiated Chinese hamster cells (V79-379A) with DNA damage as end-point. The damage was estimated with the alkaline unwinding assay and the DNA precipitation technique, carried out in parallel or in separate experimental series. The sensitizers were used in different concentrations together with standard radiation doses of 30, 60 and 90 Gy. Sensitizer-untreated cell cultures served as controls. The data indicated a radiation dose-dependent sensitization when the effect of AK-2123 was tested with the two assays, the lower radiation dose being associated with larger sensitization. In contrast, sensitization was found to be dose-independent when the effect of misonidazole was tested.     

Effects of 5'-iododeoxyuridine on the repair of radiation induced potentially lethal damage interphase chromatin breaks and DNA double strand breaks in Chinese hamster ovary cells.

The effect of 5'-iododeoxyuridine incorporation into DNA on radiation sensitivity, cellular repair capability, induction and repair of interphase chromatin breaks, as well as induction and repair of DNA double strand breaks was investigated in plateau-phase Chinese hamster ovary cells exposed to X rays. Repair of potentially lethal damage, as measured by delayed plating plateau-phase cells, was used to assay cellular repair capacity. Induction and repair of interphase chromatin breaks were assayed by means of premature chromosome condensation, whereas induction and repair of DNA double strand breaks were assayed by pulsed field gel electrophoresis. Incorporation of 5'-iododeoxyuridine into DNA sensitized cells to radiation. Radiosensitization increased with increasing percent thymidine replacement and was accompanied by an increase in the number of chromatin breaks scored per Gy and a small increase in the number of DNA double strand breaks produced. Cells grown in the presence of 5'-iododeoxyuridine were able to repair potentially lethal damage. When the comparison was made at equal doses, the extent of this repair was higher but its rate slower in 5'-iododeoxyuridine containing cells. At equal survival levels, cells that had incorporated IdU also repaired PLD to a slightly higher extent than control cells grown in IdU free medium. The magnitude of potentially lethal damage repair increased as cells "aged" in the plateau-phase, particularly for high 5'-iododeoxyuridine concentrations (8 microM). Incorporation of 5'-iododeoxyuridine reduced the rate of repair of interphase chromatin breaks and the rate of repair of DNA double strand breaks (both the fast and the slow component). The results suggest that reduction in the efficiency of repair of DNA double strand breaks and chromatin breaks, produced by radiation in 5'-iododeoxyuridine containing cells, is one determinant of the radiosensitization observed.     

Telomere length abnormalities in mammalian radiosensitive cells

Telomere lengths in radiosensitive murine lymphoma cells L5178Y-S and parental radioresistant L5178Y cells were measured by quantitative fluorescence in situ hybridization. Results revealed a 7-fold reduction in telomere length in radiosensitive cells (7 kb) in comparison with radioresistant cells (48 kb). Therefore, it was reasoned that telomere length might be used as a marker for chromosomal radiosensitivity. In agreement with this hypothesis, a significant inverse correlation between telomere length and chromosomal radiosensitivity was observed in lymphocytes from 24 breast cancer patients and 5 normal individuals. In contrast, no chromosomal radiosensitivity was observed in mouse cell lines that showed shortened telomeres, possibly reflecting differences in radiation responses between primary cells and established cell lines. Telomere length abnormalities observed in radiosensitive cells suggest that these two phenotypes may be linked.     

Ataxia-telangiectasia-like Chinese hamster V79 cell mutants with radioresistant DNA synthesis, chromosomal instability, and normal DNA strand break repair

We have isolated three radiosensitive mutants (V-C4, V-E5, and V-G8) of the Chinese hamster V79 cell line which also show increased sensitivities to killing by bleomycin (approximately 2-5-fold) and ethyl methanesulfonate (approximately 2-fold). Genetic complementation analysis indicates that all three mutants belong to one complementation group. The mutants show a radioresistant DNA synthesis following X-ray irradiation when compared to wild-type V79 cells. Both the level and the rate of repair of DNA single- and double-strand breaks measured by DNA elution were similar to those observed in wild-type V79 cells. The level of spontaneously occurring chromosome aberrations in two of these mutants differs severalfold from the level observed in wild-type V-79 cells and in V-G8, to approximately 2- and 6-fold increase in V-E5 and V-C4, respectively. X-irradiation of the mutants resulted in consistently 3-4-fold higher levels of chromatid gaps, breaks, and exchanges than observed in wild-type V79 cells. In addition, G1 irradiation of the mutant cells yielded both chromosome and chromatid types of aberrations. The level and pattern of chromosomal aberrations induced by X-rays in V-C4, V-E5, and V-G8 are similar to those observed in ataxia-telangiectasia cells. These results indicate that our mutants represent the first rodent cell mutants which show phenotypic characteristics strongly resembling those in cells from ataxia-telangiectasia patients.     

Role of the adenylate energy charge in the response of Chinese hamster ovary cells to radiation

Steady-state modification of the adenylate energy charge (EC; ATP + 1/2ADP/ATP + ADP + AMP) in aerobic Chinese hamster ovary (CHO) cells was achieved with a combination of rotenone and 2-deoxy glucose (2dG). Neither agent significantly affected the EC by itself. All incubations were in Eagle's minimum essential medium containing 15% fetal bovine serum. The radiation response of these cells was not significantly affected by this treatment when cells were irradiated either one or three hours after addition of the drugs, and held in the presence of the inhibitors for one hour after irradiation. The ability of cells to repair radiation-induced single-strand breaks was studied by the alkaline elution method. Energy depleted cells repaired single-strand breaks at a slightly slower rate than the controls. However, thymidine incorporation was also inhibited, suggesting that repair may still have preceded events leading to the fixation of that damage (e.g., DNA replication).     

Excessive base excision repair of 5-hydroxymethyluracil from DNA induces apoptosis in Chinese hamster V79 cells containing mutant p53

We have demonstrated previously that the toxicity of 5-hydroxymethyl-2'-deoxyuridine (hmdUrd) to Chinese hamster fibroblasts (V79 cells) results from enzymatic removal of large numbers of hydroxymethyluracil residues from the DNA backbone [Boorstein,R. et al. (1992) Mol. Cell. Biol., 12, 5536-5540]. Here we report that a significant portion of the hmdUrd-induced cell death that is dependent on DNA base excision repair in V79 cells is apoptosis. Incubation of V79 cells with pharmacologically relevant concentrations of hmdUrd resulted in the characteristic changes of apoptosis as measured by gel electrophoresis, flow cytometry and phase contrast microscopy. However, hmdUrd did not induce apoptosis in V79mut1 cells, which are deficient in DNA base excision repair of 5-hydroxymethyluracil (hmUra). Apoptosis was not prevented by addition of 3-aminobenzamide, which inhibits synthesis of poly(ADP-ribose) from NAD, indicating that apoptosis was not the direct consequence of NAD depletion. Pulsed field gel electrophoresis indicated that hmdUrd treatment resulted in high molecular weight (2.2-4.5 Mb) DNA double-strand breaks prior to formation of internucleosomal ladders in V79 cells. Simultaneous measurement of DNA strand breaks with bromodeoxyuridine/terminal deoxynucleotidyl transferase-fluorescein isothiocyanate labeling and of cell cycle distribution indicated that cells with DNA strand breaks accumulated in late S/G(2) and that hmdUrd-treated cells underwent apoptosis after arrest in late S/G(2) phase. Our results indicate that excessive DNA base excision repair results in the generation of high molecular weight DNA double-strand breaks and eventually leads to apoptosis in V79 cells. Thus, delayed apoptosis following DNA damage can be a consequence of excessive DNA repair activity. Immunochemical analysis showed that both V79 and V79mut1 cells contained mutant p53, indicating that apoptosis induced by DNA base excision repair can be independent of p53.     

The sensitivity to DNA topoisomerase inhibitors in L5178Y lymphoma strains is not related to a primary defect of DNA topoisomerases

DNA topoisomerase-targeting antitumor drugs are potent inducersof protein-concealed strand breaks in mammalian cells and actby trapping DNA topoisomerases on chromosomal DNA in the formof drug-enzyme-DNA cleavable complexes. It has been proposedthat the cleavable complex is an unusual form of DNA damagethat elicits cellular responses analogous to those caused byDNA damaging agents. The relationship between topoisomerase-targetingdrug-induced damage and radiation-induced damage has been investigatedby analyzing the properties of DNA topoisomerases in mouse L5178Ylymphoma strains that are cross-sensitive to topoisomerase I-IIinhibitors and to UV light or X-ray irradiation. The strainsare LY-R, isolated from L5178Y cells on the basis of increasedresistance to ionizing radiation, and strains LY-S, isolatedfrom LY-R cells following a spontaneous increase in the sensitivityto ionizing radiation. LY-S cells, deficient in the rejoiningof DNA double-strand breaks, show enhanced sensitivity to topoisomeraseII-targeting inhibitors, whereas LY-R cells have an increasedsensitivity to UV radiation and to the topoisomerase I inhibitor,camptothecin. The cellular availability of DNA topoisomeraseI and II and the sensitivity of the enzymes to their specificinhibitors have been measured in the two related strains. Inthe LY-R strain, we found a 30% decrease in topoisomerase Icontent but no difference in camptothecin sensitivity, whileno quantitative or qualitative differences were observed forthe topoisomerase II. The results indicate that variations insensitivity of the L5178Y strains to topoisomerase inhibitorsare unlikely to be related to primary defects of the targetenzymes, and thus it is possible that common pathways existfor processing of topoisomerase-and radiation-induced damage.     

Detection of hypoxia by measurement of DNA damage in individual cells from spheroids and murine tumours exposed to bioreductive drugs. I. Tirapazamine.

The possibility of using tirapazamine (SR 4233) to identify hypoxic cells in multicell spheroids and murine tumours was examined by measuring tirapazamine-induced DNA damage to individual cells from multicell spheroids and SCCVII murine tumours. Fluorescence microscopy and image analysis were used to measure the extent of migration of DNA from individual cells embedded in agarose and exposed to an electric field. Using both the alkaline and neutral versions of the comet assay, at least 20 times more single-strand breaks were observed in cells from fully anoxic than fully oxic Chinese hamster V79 spheroids exposed to 30 microM tirapazamine, and about 10 times more single- than double-strand breaks were observed. Cells from spheroids containing about 50% radiobiologically hypoxic cells showed a pattern of tirapazamine breaks which translated to approximately 30% well-oxygenated in SCCVII tumors growing in C3H mice was also demonstrated. Cells close to tumour blood vessels showed less DNA damage by 20 mg kg-1 tirapazamine than cells distant from blood vessels. Rejoining of single-strand breaks was exponential, with a half-time of about 1 h under aerobic conditions, but rejoining half-time increased to 2 h for cells allowed to repair under anoxic conditions. While tirapazamine damage to DNA measured using the comet assay cannot provide a direct measure of hypoxic fraction, the degree of heterogeneity in DNA damage can be used to estimate the range and distribution of individual cell oxygen contents within spheroids and tumours.     

Abrogation by novobiocin of cytotoxicity due to the topoisomerase II inhibitor amsacrine in Chinese hamster cells

Using cultured V79 Chinese hamster cells, we found that novobiocin (or 2,4-dinitrophenol) can abrogate, almost completely, the cytotoxicity due to the topoisomerase II inhibitor amsacrine (mAMSA). V79 cells were sensitive to mAMSA killing at all stages in the cell cycle but mainly in S phase followed by late G1 phase; however, novo rescued cells of all ages. The properties of two kinds of radiation-sensitive Chinese hamster cells were also examined, i.e., the line of V79 cells that can be rescued by caffeine, designated S-10 (H. Utsumi and M.M. Elkind, Radiat. Res., 96: 348-358, 1983); and Chinese hamster ovary cells (P.A. Jeggo and L.M. Kemp, Mutat. Res., 112: 313-327, 1983) which are also sensitive to other DNA-damaging agents. As is the case for exposure to radiation, after mAMSA treatment caffeine rescued V79/S-10 cells. Although Jeggo's Chinese hamster ovary cells were more responsive to mAMSA, novo still abrogated mAMSA toxicity in the mutant cells as well as in the parental Chinese hamster ovary cells 2,4-Dinitrophenol acted similarly to novo with respect to mAMSA killing, but neither compound reduced the ATP content of V79 cells. We propose that one reason for the rescue from mAMSA killing of at least S-phase cells by novo or 2,4-dinitrophenol is their ability transiently to inhibit replicative DNA synthesis.     

Radiopotentiation by the oral platinum agent, JM216: role of repair inhibition

PURPOSE: To test for in vitro radiopotentiation by the orally-administered platinum (IV) complex, JM216; to compare these results to cisplatin and carboplatin; and to investigate whether the mechanism of radiopotentiation involves repair inhibition of radiation-induced DNA damage. METHODS AND MATERIALS: H460 human lung carcinoma cells were incubated with the drugs for 1 h at 37 degrees C, irradiated at room temperature, and returned to 37 degrees C for 20 min. Cells were then rinsed and colony forming ability was assessed. Wild-type V79 Chinese hamster cells and radiosensitive, DNA repair-deficient mutant cells (XR-V15B) were also studied along with H460 cells. Ku86 cDNA, which encodes part of a protein involved in DNA repair, was transfected into XR-V15B cells as previously described. The effect of JM216 on sublethal damage repair (SLDR) was also assessed using split-dose recovery. RESULTS: Using equally cytotoxic doses of JM216, cisplatin, and carboplatin, the radiation dose enhancement ratios (DER) were 1.39, 1.31, and 1.20, respectively; the DER with 20 microM JM216 was 1.57. JM216 (20 microM) did not significantly change the final slope of radiation survival curves, but greatly reduced the survival curve shoulder. V79 cells also showed radioenhancement using 20 microM JM216, but no enhancement occurred using XR-V15B cells. Transfection of Ku86 cDNA into XR-V15B cells restored radiopotentiation by JM216 to wild-type V79 levels. In addition, 20 microM JM216 completely inhibited sublethal damage repair in H460 cells. CONCLUSION: Our results show that JM216 can potentiate the effects of radiation in human lung cancer cells, and that the mechanism of this effect is probably inhibition of DNA repair by JM216.     

The formation of radiation-induced DNA breaks: the ratio of double-strand breaks to single-strand breaks

Ionizing radiation causes the formation of strand breaks in cellular DNA, as well as other types of lesions in the chromatin of cells. Some of the earliest investigations of the molecular basis of radiation-induced damage and the implications of enzymatic repair were done by Dr. H. S. Kaplan. The induction frequency of DNA double-strand breaks is of special importance, and it is of interest to know the relative proportions of single-strand and double-strand breaks. This ratio changes noticeably with the radiation quality (ionization density). Because it is difficult to assay for DNA lesions in the large mammalian genome, we have developed a method of assaying for DNA double-strand breaks in the supercoiled nucleosome-complexed Simian virus 40 (SV40) genome, irradiated intracellularly. In this communication we present our measurements of the DNA double-strand breaks (DSBs) to single-strand breaks (SSBs) ratio obtained from the intracellularly irradiated SV40 genome. After cobalt gamma ray and X ray irradiations, this ratio is about 1/10. Our methods and results are compared with pertinent data in the literature. If the DSBs/SSBs ratio of 1/10 for cellular chromatin is correct, a substantial number of DNA double-strand breaks are formed in a mammalian cell after moderate doses (1 Gy) of radiation. The implications of different types of DNA double-strand breaks are discussed.     

Repair of DNA and chromosome breaks in cells exposed to SR 4233 under hypoxia or to ionizing radiation.

3-Amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) is a bioreductive anticancer drug which has a high selective toxicity to hypoxic cells. We have characterized the DNA and chromosome damage in wild-type Chinese hamster ovary (CHO) cells and mutant XR-1 cells after exposure to SR 4233 under hypoxia and compared it to the damage produced by ionizing radiation (gamma-rays). Using the technique of pulsed field gel electrophoresis, we found that the kinetics of rejoining of DNA double-strand breaks in CHO cells after treatment with SR 4233 was concentration dependent, varying from 95% (less than 50 microM) to 10% (200 microM) by 24 h. This contrasted with the dose-independent kinetics exhibited in cells after gamma-ray exposure. XR-1 cells were deficient in rejoining double-strand breaks produced by either SR 4233 or gamma-rays. XR-1 cells were 2-fold more sensitive than wild-type CHO cells to SR 4233 but were 10-fold more sensitive than CHO to gamma-rays. These results suggested that DNA double-strand breaks are involved in hypoxic cell killing by SR 4233, but the specific type of lesion produced is not identical with that causing cell killing by gamma-rays. To further investigate this, we measured chromosome breaks in CHO cells by premature chromosome condensation after equitoxic doses of SR 4233 under hypoxia and gamma-rays. SR 4233 produced lower initial but similar final (after 6 h of repair) numbers of chromosome breaks compared to gamma-rays at equitoxic doses. These results suggest that, at low doses, chromosome breaks can entirely account for hypoxic cell killing by SR 4233 and that chromosome breaks produced by SR 4233 are more damaging and/or more difficult to repair than those produced by gamma-rays.     

Yield of DNA-protein cross-links in gamma-irradiated Chinese hamster cells

gamma-Irradiation of Chinese hamster V79 cells increases the percentage of nuclear DNA cross-linked to proteins. Studies were carried out to ascertain whether the radiation-induced increase in DNA-protein cross-links (DPC) is due to an increase in the number of DNA fragments which are cross-linked to protein or to an increase in the size of bound DNA fragments. Cells were prelabeled with [3H]thymidine and irradiated (10-600 Gy), and DPCs were collected on nitrocellulose filters. Native gel analyses of the DNA recovered from the filters indicate that the number average molecular weight of cross-linked DNA (1.22 X 10(7) Da) is the same in unirradiated cells and in cells given up to 100 Gy. Assuming 5 pg of DNA per V79 cell, it was possible to calculate that there are approximately 6 X 10(3) DPC per unirradiated cell and that 150 DPC are formed per gray of gamma-radiation for doses of 0-100 Gy. Thus, radiation increases the number of new linkages between DNA and protein. At radiation doses greater than 200 Gy the percentage of nuclear DNA cross-linked to protein approaches a plateau value. The number of DPC (greater than 6 X 10(4)) formed at higher doses is within the range of the estimated number of DNA attachment sites on the nuclear matrix.     

Deficiency in BRCA2 leads to increase in non-conservative homologous recombination

The BRCA2 tumor suppressor has been implicated in the maintenance of genomic integrity through a function in cellular responses to DNA damage. The BRCA2 protein directly associates with Rad51, that is essential for repair of double-strand breaks (DSBs) by homologous recombination (HR). In this report, we study the BRCA2-defective Chinese hamster cell mutant V-C8 for its ability to perform homology-directed repair (HDR) between repeated sequences. V-C8 cells were recently shown to be defective in Rad51 foci formation in response to DNA damage. Strikingly, we find that these BRCA2 mutant cells exhibit a strong stimulation of HDR activity compared to the V79 parental cells, which harbor a wild-type BRCA2. Furthermore, molecular characterization of the HDR products shows that loss of BRCA2 in V-C8 cells leads to significant reduction in Rad51-dependent gene conversion but strong enhancement of Rad51-independent single-strand annealing (SSA) events frequency. These data imply that, when HDR by conservative gene conversion is impaired, DSBs usually repaired by this pathway are instead resolved by other non-conservative HDR subpathways. Therefore, high chromosomal instability in BRCA2-deficient cells presumably results from enhancement of error-prone repair mechanisms, such as SSA.     

Neocarzinostatin-mediated DNA damage and repair in wild-type and repair-deficient Chinese hamster ovary cells

The formation and repair of neocarzinostatin (NCS)-mediated DNA damage were examined in two strains of Chinese hamster ovary cells. The response in strain EM9, a mutant line selected for its sensitivity to ethyl methanesulfonate and shown to have a defect in the repair of X-ray-induced DNA breaks, was compared with that observed in the parental strain (AA8). The DNA strand breaks and their subsequent rejoining were measured using the method of elution of DNA from filters under either alkaline (for single-strand breaks), or nondenaturing conditions (for double-strand breaks). Colony survival assays showed that the mutant was more sensitive to the action of NCS than was the parental strain by a factor of approximately 1.5. Elution analyses showed that the DNA from both strains was damaged by NCS; the mutant displayed more damage than the parent under the same treatment conditions. Single-strand breaks were produced with a frequency of about 10 to 15 times the frequency of double-strand breaks. Both strains were able to rejoin both single-strand breaks and double-strand breaks induced by NCS treatment. The strand break data suggest that the difference in NCS-mediated cytotoxicity between EM9 and AA8 cells may be directly related to the enhanced production of DNA strand breaks in EM9. However, the fact that much higher doses of NCS were required in the DNA studies compared to the colony survival assays implies that either a small number of DNA breaks occur in a critical region of the genome, or that lesions other than DNA strand breaks are partly responsible for the observed cytotoxicity.     

Restriction enzyme-induced DNA double-strand breaks as a model system for cellular responses to DNA damage.

To learn more about cellular responses to DNA double-strand breakage, we used three methods to assay cellular damage after treatment with a restriction enzyme that causes DNA double-strand breaks by cleaving at specific recognition sites in the DNA. Chinese hamster ovary cells were treated with increasing doses of Pvu II and studied for double-strand breakage, chromosomal aberration yield, and cell survival. The yield of DNA double-strand breaks, as measured by pulsed-field gel electrophoresis, increased at concentrations up to 500 units and saturated thereafter. The maximum yield of metaphase cells showing aberrant chromosomes was reached at 100 units and stayed constant up to 1,000 units. Although exchange-type aberrations saturated at approximately 4.5 per cell at 100 units, deletion-type aberrations appeared to increase at concentrations up to 500 units. Cell survival, as measured by colony-forming ability after Pvu II treatment, saturated at 100 units. The observed dose-response data are probably due to the saturation of accessible Pvu II cleavage sites within the cell. These data indicate that restriction enzymes induce the same DNA-damaging effects as many of the agents used in cancer treatment. Because the primary DNA lesion induced by restriction enzymes is known, they provide a unique opportunity to understand cellular responses to DNA damage and repair.     

Double-strand break yield following 125I decay--effects of DNA conformation

The decay of iodine-125 (125I) is accompanied by the emission of low-energy electrons that dissipate most of their energy in approximately 10 nm from the decay site. In mammalian cells, the .OH generated by these electrons are also confined to a small volume. Iodine-125 is thus an excellent probe for assessing the radiobiologic effects produced by .OH in close proximity to the site of a decaying atom. We have compared in pUC19 plasmids (naked DNA) and in Chinese hamster V79 lung fibroblasts (chromatin) the modulation by the .OH scavenger dimethyl sulfoxide (DMSO) of 125I-induced DNA double-strand breaks (DSB). The data indicate that DMSO cannot protect plasmid DNA against DSB damage from 125I decaying within a few angstroms from DNA. However, DMSO attenuated DSB production in V79 cells following the decay of DNA-incorporated 125I, thus suggesting that chromatin structure fosters some DSB formation by indirect mechanism(s). DSB production depends on the environment and/or conformation of DNA. Consequently, current biophysical modeling of DNA damage that is based on naked and non-compacted DNA is inadequate for explaining radiobiologic effects at the cellular level.