Radiation-induced DNA Single-strand Breaks Research Articles

LuPARP: Phase 1 trial of 177Lu-PSMA-617 and olaparib in patients with metastatic castration resistant prostate cancer (mCRPC).

5005 Background: 177Lu-PSMA-617, a PSMA-directed, radionuclide therapy improves progression free survival (PFS) and overall survival (OS) in patients (pts) with mCRPC. Although many pts benefit from treatment, approximately a third have primary resistance, median PFS is 5.1 months and all pts inevitably relapse. Poly (ADP-ribose) polymerase (PARP) is implicated in repairing radiation-induced DNA single-strand breaks (SSBs). The potent PARP inhibitor (PARPi) olaparib results in the conversion of DNA SSBs to lethal double-strand breaks. Multiple preclinical studies have shown enhanced anti-tumor activity from combined PARPi and radiotherapy. LuPARP aims to evaluate the safety and efficacy of the combination of 177Lu-PSMA-617 and olaparib. Methods: 48 pts with mCRPC and high PSMA expression (SUVmax 15 at a site of disease, and SUVmax ≥ 10 at other measurable sites) without discordant FDG positive/PSMA negative disease will be enrolled in two stages: dose-escalation (standard 3+3 design) and dose-expansion at the recommended phase 2 dose (RP2D). 177Lu-PSMA-617 is administered at 7.4 GBq IV on day 1, every 6 weeks for up to 6 cycles in conjunction with 9 prespecified dose schedules of olaparib. The dose-limiting toxicity (DLT) period is 6 weeks. The primary objectives are establishing the DLTs and RP2D. Secondary objectives are toxicity, radiological PFS (rPFS), PSA response rate (PSA50-RR), PSA-PFS, objective response rate (ORR) and OS. Responses are assessed by 3-weekly PSA and conventional and PSMA PET/CT imaging every 12 weeks. Results: 29 pts (median age 70 years: range: 52-84; prior docetaxel: 97%; prior androgen receptor pathway inhibitor: 100%) received 177Lu-PSMA-617 on day 1 with escalating doses of olaparib (cohorts 1-6: 50mg - 300 mg BD days 2-15) or alternate olaparib schedules (cohort 7: 200 BD days - 4 to 14; cohort 8: 300 BD days - 4 to 14; cohort 9: 300 mg BD days -4 to 18) for up to 6 cycles. No DLTs were reported. Common treatment related adverse events (TRAE) (≥10%) were Grade (G) 1-2 and included xerostomia (83%), nausea (62%), fatigue (34%), constipation (31%), anorexia (17%), vomiting (14%) and diarrhea (10%). Hematologic TRAE included anemia (G1: 14%; G2: 7%; G3: 7%), thrombocytopenia (G1: 14%; G2: 7%; G3: 3%) and neutropenia (G1: 3%; G3: 7%) that were transient and without clinical sequelae. Across cohorts 1-9, the PSA50-RR was 62% (18/29) and PSA90-RR was 48% (14/29). Five of the 7 pts (71%) with RECIST measurable disease had a partial response. Conclusions: The combination of 177Lu-PSMA-617 and olaparib is well tolerated and has promising activity. Three further patients are being enrolled to cohort 9 to confirm the RP2D ahead of dose-expansion. Clinical trial information: NCT03874884 . [Table: see text]

The effect of gamma radiation on the Common carp (Cyprinus carpio): In vivo genotoxicity assessment with the micronucleus and comet assays

Radioactive wastes may be leached into freshwater, either accidentally or in industrial effluents. We have studied gamma radiation-induced DNA damage in the freshwater fish Cyprinus carpio. Fish were irradiated with 2–10Gy gamma radiation and genotoxic effects in blood cells were studied with the micronucleus (MN) and comet assays. Micronuclei and a dose-dependent increase in comet-tail DNA were seen in dose- and time-dependent studies. The highest % tail DNA was observed at 24h, declining until 72h, which may indicate the repair of radiation-induced DNA single-strand breaks after gamma radiation. However, double-stranded DNA damage may not have been repaired, as indicated by increased micronuclei at later periods. A positive correlation was observed between the comet and micronucleus assay results. This study confirms the mutagenic/genotoxic potential of gamma radiation in the Common carp, as well as the possible combined use of the micronucleus and comet assays for in vivo laboratory studies with fresh-water fish for screening the genotoxic potential of radioactive pollution.

Involvement of the Artemis Protein in the Relative Biological Efficiency Observed With the 76-MeV Proton Beam Used at the Institut Curie Proton Therapy Center in Orsay

Previously we showed that the relative biological efficiency for induced cell killing by the 76-MeV beam used at the Institut Curie Proton Therapy Center in Orsay increased with depth throughout the spread-out Bragg peak (SOBP). To investigate the repair pathways underlying this increase, we used an isogenic human cell model in which individual DNA repair proteins have been depleted, and techniques dedicated to precise measurements of radiation-induced DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). The 3-Gy surviving fractions of HeLa cells individually depleted of Ogg1, XRCC1, and PARP1 (the base excision repair/SSB repair pathway) or of ATM, DNA-PKcs, XRCC4, and Artemis (nonhomologous end-joining pathway) were determined at the 3 positions previously defined in the SOBP. Quantification of incident SSBs and DSBs by the alkaline elution technique and 3-dimensional (3D) immunofluorescence of γ-H2AX foci, respectively, was performed in SQ20 B cells. We showed that the amount of SSBs and DSBs depends directly on the particle fluence and that the increase in relative biological efficiency observed in the distal part of the SOBP is due to a subset of lesions generated under these conditions, leading to cell death via a pathway in which the Artemis protein plays a central role. Because therapies like proton or carbon beams are now being used to treat cancer, it is even more important to dissect the mechanisms implicated in the repair of the lesions generated by these particles. Additionally, alteration of the expression or activity of the Artemis protein could be a novel therapeutic tool before high linear energy transfer irradiation treatment.

Investigations of the contribution of neutrophils to the genotoxicity of respirable quartz particles

Previously we showed that the relative biological efficiency for induced cell killing by the 76-MeV beam used at the Institut Curie Proton Therapy Center in Orsay increased with depth throughout the spread-out Bragg peak (SOBP). To investigate the repair pathways underlying this increase, we used an isogenic human cell model in which individual DNA repair proteins have been depleted, and techniques dedicated to precise measurements of radiation-induced DNA single-strand breaks (SSBs) and double-strand breaks (DSBs).The 3-Gy surviving fractions of HeLa cells individually depleted of Ogg1, XRCC1, and PARP1 (the base excision repair/SSB repair pathway) or of ATM, DNA-PKcs, XRCC4, and Artemis (nonhomologous end-joining pathway) were determined at the 3 positions previously defined in the SOBP. Quantification of incident SSBs and DSBs by the alkaline elution technique and 3-dimensional (3D) immunofluorescence of γ-H2AX foci, respectively, was performed in SQ20 B cells.We showed that the amount of SSBs and DSBs depends directly on the particle fluence and that the increase in relative biological efficiency observed in the distal part of the SOBP is due to a subset of lesions generated under these conditions, leading to cell death via a pathway in which the Artemis protein plays a central role.Because therapies like proton or carbon beams are now being used to treat cancer, it is even more important to dissect the mechanisms implicated in the repair of the lesions generated by these particles. Additionally, alteration of the expression or activity of the Artemis protein could be a novel therapeutic tool before high linear energy transfer irradiation treatment.

TDP1 facilitates repair of ionizing radiation-induced DNA single-strand breaks

Tyrosyl DNA phosphodiesterase-1 (TDP1) is the gene product mutated in spinocerebellar ataxia with axonal neuropathy1 (SCAN1). SCAN1 is a hereditary ataxia that lacks extra-neurological phenotype, pointing to a critical role for TDP1 in the nervous system. Recently, we showed that TDP1 is associated with the DNA single-strand break (SSBR) repair machinery through an interaction with DNA ligase 3α (Lig3α) and that SCAN1 cells are defective in the repair of chromosomal DNA single-strand breaks (SSBs) arising from abortive Topoisomerase 1 (Top1)–DNA intermediates. Here we demonstrate that TDP1 is also required for the repair of SSBs induced by ionizing radiation (IR), though not measurably for IR-induced DNA double-strand breaks (DSBs). In addition, we provide evidence that abortive Top1 cleavage complexes are processed by the proteasome prior to the action of TDP1 in vivo, and we exploit this observation to show that the SSBR defect in SCAN1 following IR reflects, in part at least, the presence of IR-induced protein–DNA cross-links. Finally we show that TDP1 activity at abortive Top1-SSBs is stimulated by XRCC1/Lig3α in vitro. These data expand the type of SSBs processed by TDP1 to include those induced by ionizing radiation, and raise the possibility that TDP1 inhibitors may improve radiotherapy.

DBD-FISH on Neutral Comets: Simultaneous Analysis of DNA Single- and Double-Strand Breaks in Individual Cells

Humanblood leukocytes exposed to X-rays were immersed in an agarose microgel on a slide, extensively deproteinized, and electrophoresed under neutral conditions. Following this single-cell gel electrophoresis assay, characteristics of DNA migration (i.e., area of the comet) are related to the DNA double-strand breaks (dsbs) yield. After electrophoresis, comets were briefly incubated in an alkaline unwinding solution, transforming DNA breaks and alkali-labile sites into restricted single-stranded DNA (ssDNA) motifs. These motifs behave as target sites for hybridization with a whole genome probe, following the DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure. As DNA breakage increases with dose, more ssDNA is produced in the comet by the alkali and more DNA probe hybridizes, resulting in an increase in the mean fluorescence intensity. Since radiation-induced DNA single-strand breaks (ssbs) are far more frequent than dsbs, the mean fluorescence intensity of the DBD-FISH signal from the comet is related to the ssb level, whereas the surface area of the same comet signal is indicative of the dsb yield. Thus, both DNA break types may be simultaneously analyzed in the same cell. This was confirmed in a repair assay performing the DBD-FISH on neutral comets from a human cell line defective in the repair of dsbs. Otherwise, treatment with hydrogen peroxide, a main inducer of ssbs, increased the mean fluorescence intensity, but not the surface, of X-ray-exposed human leukocytes.

Protection from radiation-induced DNA single-strand breaks by induction of nuclear metallothionein

Purpose : To examine the extent to which nuclear metallothionein protects from radiation-induced DNA damage under aerobic and hypoxic conditions. Materials and methods : A semiquantitative fluorescence image analysis method measured the nuclear content of metallothionein (MT) in ME180 and SiHa human squamous cervical carcinoma cell lines under normal growth conditions, and following MT induction by zinc. The extent of initial DNA damage following 60 Co irradiation under aerobic and hypoxic conditions was assessed using the alkaline comet assay. Results : Provided that cells were maintained at 37°C, most of the cellular content of MT was in the nucleus. Incubation at 4°C caused the rapid translocation of MT from the nucleus into the cytoplasm in both cell lines, with no net loss of cellular MT. Baseline nuclear MT levels were about four times greater in ME180 cells, and were much more readily induced by treatment with 100 μ m zinc acetate, compared with SiHa cells. Under aerobic conditions, MT induction by zinc resulted in no protection in either of the cell lines. Under hypoxic conditions, however, the number of DNA single-strand breaks in zinc-treated cells was reduced by ~40% in ME180, but not in SiHa cells, when compared with non-induced controls. Conclusions : Nuclear MT can exert a significant level of protection from radiation by a mechanism that involves competition with oxygen for DNA radical sites and/or scavenging of free radicals. Because increased MT levels have been reported in hypoxic micro-regions of some solid tumours, this protective mechanism might have clinical relevance.

DNA-PK activation by ionizing radiation-induced DNA single-strand breaks

Purpose : To assess the ability of 60Co gamma-radiation-induced plasmid DNA single-strand breaks (gamma-ssb) to activate the DNA-dependent protein kinase (DNA-PK) in vitro. Materials and methods : Plasmid DNA was gamma-irradiated under aerobic conditions to yield 0-6 gamma -ssb and 0.1 double-strand breaks (dsb) per plasmid molecule. The irradiated DNA was used to stimulate DNA-PK in crude HF19 fibroblast nuclear extracts and/or purified HeLa cell DNA-PK protein, and the activation compared with that obtained with a single enzymatically generated plasmid DNA ssb (GpII endonuclease) or dsb (Eco RI endonuclease). Results: gamma-Irradiated plasmid DNA activates DNA-PK in both crude and purified preparations and the kinase activity increases linearly with dose. As significant DNA-PK activation was detectable using irradiated plasmids which contain 0.1dsb/molecule, it was concluded that this activation is due to gamma-ssb. However, using purified DNA-PK, this activation is relatively weak as 3 gamma -ssb is equivalent to one GpII-generated DNA ssb or one end of an Eco RI-generated dsb in DNA-PK assays. Conclusions: As gamma-ssb are in a 20-fold excess of gamma-dsb in vivo for low LET radiation, gamma-ssb may contribute significantly to DNAPK signalling of gamma-radiation-induced DNA damage in vivo.

Thiazolidine Prodrugs of Cysteamine and Cysteine as Radioprotective Agents

The need for protection against the toxic effects of ionizing radiation comes from many different directions: occupational exposure, nuclear accidents, environmental sources and protection of normal tissue during the therapeutic irradiation of cancer. Sulfhydryl-containing compounds, including cysteamine and L-cysteine, have long been known to possess radioprotective properties, but their therapeutic utility is limited by their side effects at radioprotective doses. To avoid this drawback, thiazolidine prodrugs of cysteamine and L-cysteine were prepared by the condensation of each thioalmine with the aldose monosaccharides, D-ribose and D-glucose, producing RibCyst, GlcCyst, RibCys and GlcCys. The prodrugs were designed to liberate the parent thiolamine nonenzymatically, after ring opening and hydrolysis, which is then available to function as a radioprotective agent. Cysteamine's inherent toxicity, measured using Chinese hamster V79 cells growing in culture, was completely eliminated, even at concentrations as high as 25 mM, by providing the thiolamine in the form of a prodrug. Good protection against radiation-induced lethality was demonstrated by the cysteamine prodrugs using a clonogenic assay. Protection against radiation-induced DNA single-strand breaks, as measured by alkaline elution, was also shown by both RibCyst and GlcCyst; this activity was higher than that exhibited by either cysteamine or WR-1065. The L-cysteine prodrugs, RibCys and GlcCys, also possessed radioprotective abilities under most of the conditions studied. Protection against DNA damage was comparable between L-cysteine, WR-1065 and RibCys.

Induction and rejoining of radiation-induced DNA single-strand breaks: “tail moment” as a function of position in the cell cycle

Previous results using a variety of methods have shown no significant difference in induction or rejoining of radiation-induced DNA single-strand breaks (SSBs) as a function of cell-cycle position. However, concurrent of DNA damage and cell-cycle position measured using the alkaline comet assay indicates cycle-dependent differences in “tail momen” which has been shown to be a measure of SSBs. Unirradiated S phase cells showed a significantly higher tail moment presumably as a result of the presence of active replication sites. The time required to repair half of the damage appeared to be consistently shorter for G1 cells than for cells in other phases of the cell cycle (3.7 min versus approximately 5 min). Although early kinetics of rejoining (within 5 min) was identical for CHO, V79 and repair-deficient TK6 cells, TK6 cells subsequently repaired fewer breaks than did CHO or V79 cells. S phase TK6 cells rejoined SSBs more slowly than cells in the other phases; differences in rejoining rates could not be explained by the presence of a subset of slowly repairing TK6 cells. We concluide that the comet assay detects subtle differences in the tail moment when cells in different phases of the cell cycle are irradiated and allowed to repair damage. The biological importance of these observations remains to be determined.

Radiation-Induced DNA Single-Strand Breaks in Freshly Isolated Human Leukocytes

Single-strand breaks are a major form of DNA damage caused by ionizing radiation, and measurement of strand breaks has long been used as an index of overall cellular DNA damage. Most assays for DNA single-strand breaks in cells rely on measuring fractionated DNA samples following alkali denaturation. Quantification is usually achieved by prelabeling cells with radioactive DNA precursors; however, this is not possible in the situation of nondividing cells or freshly isolated tissue. It has previously been demonstrated that the alkali unwinding assay of DNA strand breaks can be quantified by blotting the recovered DNA on nylon membranes and hybridizing with radiolabeled sequence-specific probes. We report here improvements to the technique, which include hot alkali denaturation of DNA samples prior to blotting and the use of carrier DNA that is non-complementary to the radiolabeled probe. Our method allows both single- and double-stranded DNA to be quantified with the same efficiency, thereby improving the sensitivity and reproducibility of the assay, and allows calibration for determination of absolute levels of DNA strand breaks in cells. We also used this method to assay radiation-induced DNA strand breaks in freshly isolated human leukocytes and found them to have a strand break induction rate of 1815 strand breaks/cell/Gy.

Detection of hypoxic cells in a murine tumor with the use of the comet assay.

Hypoxic cells within solid tumors are likely to limit tumor curability by radiation therapy and some chemotherapeutic agents. To quantify a hypoxic fraction in solid tumors, we developed a method which measures radiation-induced DNA single-strand breaks in individual tumor cells and makes use of the fact that three times more strand breaks are produced in aerobic than in hypoxic cells. Immediately after irradiation with doses of 4-20 Gy, SCCVII squamous cell carcinomas growing in C3H mice were removed and cooled, and a single-cell suspension was prepared. These cells were then embedded in agarose, lysed in an alkaline solution, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye. The amount and migration distance of damaged DNA from individual cells were scored by using a fluorescence image processing system, where differentially radio-sensitive aerobic and hypoxic cell populations resulted in bimodal damage distributions. Curve-fitting routines provided quantitative estimates of the fraction of hypoxic cells. After the mice were exposed to 10-20 Gy, the SCCVII tumors (450-600 mg) were shown to have a hypoxic fraction of 18.5% +/- 10.6% (mean +/- SD for 11 tumors), which compares well with the value of 11.6% observed using the paired survival curve method. Our results indicate that this method, which requires only a few thousand cells, is a rapid and sensitive way to detect hypoxic cells in solid animal tumors. Estimating hypoxia in accessible human tumors undergoing radiotherapy may be possible if the sensitivity of the method can be improved to allow detection of hypoxic cells after a dose of 2 Gy.

Radiation sensitivities in various anticancer-drug-resistant human lung cancer cell lines and mechanism of radiation cross-resistance in a cisplatin-resistant cell line.

To determine whether there exists cross-resistance between anticancer drugs and radiation, six drug-resistant human lung cancer cell lines and their parental cell lines were examined for radiosensitivity using a growth-inhibition assay. Only one cisplatin-resistant cell line, PC-9/CDDP, showed cross-resistance to radiation. The other three cisplatin-resistant cell lines (PC-7/CDDP, PC-4/CDDP, and H69/CDDP), an etoposide-resistant cell line (H69/VP) and a camptothecin-resistant cell line (PC-7/CPT) did not show cross-resistance to radiation. To analyze the mechanism of radiation resistance in PC-9/CDDP cells, the formation and repair of radiation-induced DNA single-strand breaks (ssb) and double-strand breaks (dsb) were examined by alkaline elution and neutral elution respectively. Although the formation of DNA ssb and repair of both DNA ssb and DNA dsb were the same for both cell lines, the formation of DNA dsb in PC-9/CDDP cells was significantly less than those in PC-9 cells. Measurement of intracellular glutathione content in all of the cell lines revealed that only PC-9/CDDP cells had a significant increase of glutathione content compared to the parental cells. Buthionine sulfoximine treatment of PC-9/CDDP cells caused an increase of DNA dsb to the same levels as in PC-9 cells after irradiation and caused a complete radiosensitization. These results indicate that cross-resistance to radiation in drug-resistant cells in a rare phenomenon, and increased glutathione content may play a crucial role in the emergence of cross-resistance to radiation in the drug-resistant cells.

Differential protection of radiation-induced DNA single-strand breaks and cell survival by solcoseryl.

V79 Chinese hamster cells were studied in vitro for modification of cobalt-60 gamma radiation effects by solcoseryl. This treatment did not modify cell survival but did protect against DNA single-strand breaks.

Radiation-induced DNA single-strand breaks in the intestinal mucosal cells of mice treated with the radioprotectors WR-2721 or 16-16 dimethyl prostaglandin E2.

Both S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721) and 16-16 dimethyl prostaglandin E2 (dm PGE2) protected the intestinal clonogenic cells to some degree from the effects of 137Cs gamma-irradiation. The D0 was increased from 1.1 +/- 0.12 Gy in controls to 1.55 +/- 0.48 Gy in 16-16 dm PGE2 treated and 2.12 +/- 0.20 Gy in WR-2721 treated mice. Both agents also increased the shoulder of the clonogenic-cell survival curve. Studies were done to measure the effects of these two different radioprotectors on radiation-induction of DNA single-strand breaks in cells comprising the murine intestinal mucosa. The number of DNA single-strand breaks increased with increasing doses of gamma-rays in animals killed immediately following exposure. WR-2721 reduced the number of initial radiation-induced DNA single-strand breaks when given one-half hour before exposure; the time of maximum protection. In contrast, 16-16 dm PGE2 given 1 hour before irradiation (the time required to afford maximum protection from radiation cytotoxicity) did not reduce the number of initial DNA breaks. Both agents impeded the rate of rejoining of DNA breaks with increasing time after irradiation. However, the relationship between these effects on the rate of strand rejoining and cell survival is unknown. These results suggest that either both agents are similarly distributed within the cells but the mechanisms of radioprotection are different, or the mechanisms by which these agents protect are similar, but the two agents affect different subcellular targets, the protection of which contributes to increased cell survival.

Theory of Survival of Bacteria Exposed to Ionizing Radiation: I. X and γ Rays

A new model for the survival of bacteria exposed to ionizing radiation is constructed in the framework of a target theory based on microdosimetric concepts, where single- and double-strand breaks of DNA and their repair in vivo can be described consistently in terms of the microdosimetric quantity j (number of effective primary events per track per target). In this model, the ability of cells to repair DNA damage is taken into consideration in terms of the repair capacities for single- and double-strand breaks of DNA, xi 1 and xi 2 (0 less than or equal to xi 1, xi 2 less than or equal to 1). To apply this model to Escherichia coli K-12 strains with different repair abilities, values of the repair capacity for single-strand breaks, xi 1, were derived from experimental survival curves. The theoretical survival curves for 60Co gamma rays were found to be effectively insensitive to the value of xi 2. Experimental survival curves for the wild-type, uvr, and rec strains of E. coli K-12 were well reproduced in this model. From these results, it is concluded that the theoretical formulation for the survival fraction of bacteria can afford a quantitative method for analysis of the repair process for radiation-induced single-strand breaks in DNA in vivo.

Effects of Nitrous Oxide on the Radiation Sensitivity of Escherichia coli in the Presence and Absence of NPPN

89, 488-502 (1982). N20 had no significant effect on the anoxic radiosensitivity of Escherichia coli B or, at doses below 100 krad, of E. coli K12 strain AB1157. Above 100 krad, a slight sensitization of the latter strain, independent of the presence of 5 mM glycerol, was observed. N20 had no detectable effect on the yield of anoxic radiation-induced DNA single-strand breaks. In the presence of the anoxic radiosensitizer norpseudopelletierine-N-oxyl (NPPN), N20 approximately doubled the radiosensitivity of strain ABl157 (in anoxia) but had little or no effect in the presence of 2,2,6,6-tetramethyl-4-piperidinol-N-oxyl (TMPN) or oxygen. Sensitization by N20 in the presence of NPPN was attributed to the accumulation of toxic product(s) formed from the reaction of extracellular - OH radicals with NPPN. In N2-saturated conditions it appeared that e, reacted with NPPN to give a product (N-hydroxynorpseudopelletierine) which prevented the formation, persistence, or effect of the proposed toxic product(s).

DNA Repair and Replication in Cells of Escherichia coli Made Permeable with Hypotonic Buffers

Hypotonic buffers (20 mM phosphate or 40 mM Tris) were found to make cells of Escherichia coli permeable to low-molecular-weight compounds without significantly affecting cell viability. When cells of E. coli were suspended in hypotonic buffers during irradiation no repair of radiation-induced DNA single-strand breaks occurred. Addition of the four deoxyribonucleoside triphosphates and Mg/sup 2/+ after irradiation brings about a recovery of the rejoining ability. NAD stimulated repair in the presence of the four deoxyribonucleoside triphosphates and Mg/sup 2/+, while the presence of ATP had no significant effect. The initial yield of radiation-induced DNA single-strand breaks was increased in cells treated with hypotonic buffers. This is explained by an efflux of a large fraction of the intracellular compounds containing sulfhydryl groups. Cells of E. coli in 40 mM Tris buffer incorporated exogenous deoxyribonucleoside triphosphates at half the rate observed in cells made permeable by toluene. This DNA synthesis occurred in polAl cells and was ATP dependent and thus probably represents semiconservative replication.

Decreased repair of x-ray induced DNA single-strand breaks in lymphocytes in Down's syndrome.

Gamma-ray irradiation introduces single and/or double strand breaks into the DNA molecule of the cells. In the case of mammalian cells, these breaks are being repaired in general during the first hr following exposure to ionizing radiation. The article reports on the results obtained from testing the ability of cultured lymphocytes from patients with Down's syndrome to repair radiation-induced DNA single-strand breaks. The ability to repair was deduced from the study of the DNA sedimentation profiles in alkaline sucrose gradients. It was found that lymphocytes from Down's syndrome patients are less efficient in repairing single-strand DNA breaks than are lymphocytes from normal individuals. This significantly increased fraction of unrepaired DNA strand breaks might be associated with the unusually high level of radiation-induced chromosome aberrations as compared with normals.

DNA Single-Strand Breaks of Preheated Cultured Mammalian Cells Irradiated under Nitrogen- and Nitrous Oxide-Saturated Conditions

Radiation-induced DNA single-strand breaks were studied using preheated cultured mouse leukemic cells, L5178Y, exposed to60 Co γ rays under ${\rm N}_{2}\text{-}$ and ${\rm N}_{2}{\rm O}\text{-satur...