Differential Effects of Procaine and Phenethyl Alcohol on Excision Repair of DNA in u.v.-irradiated Escherichia Coli

Summary

Experiments were performed to investigate the involvement of the cell membrane in the excision DNA repair process in Escherichia coli. Two membrane-binding drugs, procaine and phenethyl alcohol (PEA), inhibited liquid-holding recovery (LHR) in u.v.-irradiated E. coli wild-type and recA strains. In uvrB and polA strains where, after u.v.-irradiation, LHR was absent the two drugs had no effect. Both drugs markedly reduced the removal of u.v.-induced thymine dimers in the DNA of wild-type cells (H/r30). Analysis by alkaline sucrose gradients revealed that PEA inhibited the incision step in excision repair. In contrast, procaine had no effect on incision but apparently inhibited the late steps in excision repair. PEA dissociated DNA from the cell membrane, whereas procaine did not. The results suggest that the two drugs PEA and procaine inhibit LHR and the excision repair process operating on u.v.-induced damage in E. coli by at least two different mechanisms each of which may involve the cell membrane.     

Synergistic effect of aphidicolin and 1-beta-D-arabinofuranosylcytosine on the repair of gamma-ray-induced DNA damage in normal human fibroblasts.

The effects on enzymatic DNA repair of aphidicolin and 1-beta-D-arabinofuranosylcytosine (araC), two potent inhibitors of long-patch excision repair, were investigated in cultured human cells exposed to 60Co gamma-radiation. Using alkaline-sucrose velocity sedimentation analysis, both drugs were shown to inhibit markedly the repair of radioproducts in cultures exposed to greater than or equal to 150 Gy, indicating that a significant component of gamma-ray-induced DNA damage is operated on by a long-patch excision pathway. Moreover, while the extent of repair inhibited by aphidicolin was comparable to that suppressed by araC, combined exposure of irradiated cultures to the two drugs elicited a synergistic response. Specifically, in all three normal fibroblast strains examined, the yield of aphidicolin- or araC-detectable sites (lesions whose repair could be blocked by each drug alone) observed during the first 2 h after irradiation with 150 Gy ranged from 0.8 to 1.2 per 10(8) daltons genomic DNA, whereas the incidence of sites detected by combined exposure to the inhibitors was increased 4-fold (i.e. 3.8 per 10(8) daltons). This difference in site yield leads us to propose that simultaneous administration of aphidicolin and araC serves to block, in addition to long-patch repair, a second mode of excision repair which is refractory to each drug alone.     

突变型DNA聚合酶β碱基切除修复活性的分析

目的分析162位Val→Ala突变型DNA聚合酶β（DNA polymerase β,polβ）碱基切除修复活性的变化,为探讨修复基因DNA聚合酶β突变在肿瘤发生发展中的作用积累实验依据。方法异丙基-β—D-硫代半乳糖苷（IPTG）分别诱导含有野生型和162位Val→Ala突变型DNA聚合酶β表达载体（pQE80L—Wpolβ和pQE80L-Mpolβ）的大肠杆菌DH5α;通过镍柱亲和层析法纯化蛋白,复性后蛋白定量;退火合成含有单碱基缺失的DNA底物,与纯化的野生型和突变型DNA聚合酶β蛋白进行BER修复试验。结果通过诱导、纯化得到38kd野生型和突变型DNA聚合酶β蛋白;在BER实验中。野生型DNA聚合酶β能够在DNA底物的酶切位点处将其完全切开,而突变型DNA聚合酶β仅部分将其切开。结论162位Val→Ala突变型DNA聚合酶β碱基切除修复能力显著减弱,提示肿瘤发生发展与polβ基因修复活性降低密切相关。     

Commentary on the Second Event Theory of Busby

Summary

The responses to u.v. of two cell lines derived from the Indian muntjac are described. The u.v. sensitivity of the diploid cell falls within the range of most normal mammalian cells while the other, a heteroploid cell, transformed by SV40, is much more sensitive to killing. This hypersensitivity cannot be explained by defective excision repair: the two cell types are indistinguishable in this activity as judged by inhibitor-associated DNA break accumulation and unscheduled DNA synthesis. Rather, the SV40 transformed cells have a pronounced inability to recover normal DNA replication after u.v. These cells are, therefore, defective in a post-replication recovery mechanism and in this respect resemble the behaviour of the variant form of xeroderma pigmentosum. Their limited ability to recover normal levels of RNA synthesis after u.v. hints at the complexity of the phenotype.     

Reduction of DNA-polymerase β Activity of CHO Cells by Single and Combined Heat Treatments

Summary

The effect of single and combined heat treatments on the activity of DNA polymerase β was studied in CHO cells. The activity of polymerase β was determined by measuring the amount of [³H]TTP incorporated into activated calf thymus DNA in the presence of aphidicolin, a specific inhibitor of DNA polymerase α. Biphasic response curves were obtained for all temperatures tested (40–46°C) showing the sensitivity to decrease during heating. A constant activation energy of E_a = 120 ± 10 kcal/mole was found for the initial heat sensitivity, whereas the Arrhenius plot for the final sensitivity is characterized by an inflection point at 43°C with E_a = 360 ± 40 kcal/mole or E_a = 130 ± 20 kcal/mole for temperatures below or above 43°C, respectively. The observed decrease of the polymerase activity is not due to a decrease in the number of active enzyme molecules but to a change in its affinity, since the inhibition is reversible when increasing concentrations of TTP are applied. When acute or chronic thermotolerance was induced by a priming heat treatment at 43°C for 45 min followed by a time interval at 37°C for 16 h or by a preincubation at 40°C for 16 h, respectively, the thermal sensitivity of polymerase β was lowered by a factor of up to 5. By contrast, pretreatment at a higher temperature followed by a lower temperature (step-down heating) did not alter the sensitivity of polymerase β to the second treatment. The results indicate that heat-induced cell death cannot be the consequence of the reduction of the polymerase β activity, confirming earlier studies on this subject.     

Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells.

DNA breaks in eukaryotic cells can be detected by alkaline electrophoresis of cells embedded in agarose. DNA containing breaks extends in the direction of the anode forming an image resembling the tail of a comet. We have adapted this procedure of single cell gel electrophoresis (SCGE) for studying DNA damage and repair induced by UV-C-radiation, using HeLa cells. UV-C itself does not induce DNA breakage, and though cellular repair of UV-C damage produces DNA breaks as intermediates, these are too short-lived to be detected by SCGE. Incubation of UV-C-irradiated cells with the DNA synthesis inhibitor aphidicolin causes accumulation of incomplete repair sites to a level readily detected by SCGE even after doses as low as 0.5 J m-2 and incubation for as little as 5 min. We have also used SCGE to study UV-C-dependent incision, repair synthesis and ligation in permeable cells. Finally, we have incubated permeable cells, after UV-C-irradiation, with exogenous UV endonuclease, examining the consequent breaks both by SCGE and by alkaline unwinding in order to express results of the electrophoretic method in terms of DNA break frequencies. The sensitivity of the SCGE technique can thus be estimated; as few as 0.1 DNA breaks per 10(9) daltons are detected.     

Effects of Hypoxic Cell Radiosensitizers on the Activity of ATPases

Summary

The yield and repairability of DNA—protein cross-links have been compared after γ- or U.V.-irradiation of Chinese hamster V79-379 lung fibroblasts. Using a filter-binding assay, cross-linked DNA can be specifically isolated after doses between 10 and 100 Gy of γ-radiation and fluences between 20 and 300 J/m² of U.V.-radiation. After ionizing radiation, the majority of DNA cross-linked to protein is released with biphasic kinetics, requiring 1 h for removal of 50 per cent of the cross-linked DNA and 24 h for 90 per cent release. In these cells, U.V.-induced cross-linked DNA is not removed; on the contrary, the yield of apparent DNA—protein complexes increases during postirradiation incubation. Prior γ-irradiation, to initiate the associated repair system, does not stimulate release of U.V.-induced cross-linked DNA. Inhibition of protein synthesis by cycloheximide affects neither the removal of γ-ray-induced cross-linked DNA nor the increase in U.V.-induced cross-linked DNA. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, slows the second phase of release after γ-irradiation as well as the increase in apparent cross-links after U.V.-irradiation. Thus, even though both types of DNA—protein cross-links can be detected by the same assay, their structures or other factors must be substantially different, since the repair system for one type does not recognize the other.     

Cellular Responses to Ionizing Radiation: Effects of Interrupting DNA Repair with Chemical Agents

Summary

This review is concerned with the influence of different classes of chemical agents on cellular repair of DNA damage induced by ionizing radiation. Single-strand break rejoining is little affected by inhibitors of DNA synthesis; however, such inhibitors do lead to a persistence of double-strand breaks in the DNA, and this correlates with an enhancement of chromosome aberrations and cell killing. Experiments with antagonists of topoisomerase II suggest an intriguing role for this DNA unwinding enzyme in double-strand break repair. Interference with poly(ADP-ribose) synthesis, by means of the inhibitor 3-aminobenzamide, does not have a clear-cut effect on recovery from ionizing radiation damage. Various substances (for example, caffeine and trypsin) affect DNA repair via a modulation of the cell cycle, altering the time available to the cell for repairing potentially lethal DNA damage before such damage is ‘fixed’ by the process of DNA replication. Finally, disturbing cellular energy metabolism, and depressing the level of ATP, can inhibit the repair of radiation damage.     

Effects of aphidicolin on cell recovery and repair of DNA damage in irradiated human fibroblasts]

The effect of aphidicolin, a specific inhibitor of DNA polymerases alpha and delta, on recovery and repair of X-irradiated human fibroblasts was investigated. Aphidicolin concentrations from 0.5 to 5 micrograms/ml reduce DNA synthesis in exponentially growing cells after short incubation periods (1 h), longer incubation times (24 h) enhance this effect. Colony forming ability of unirradiated fibroblasts, treated in confluent growth state with aphidicolin, was not reduced. After incubation with aphidicolin contact-inhibited fibroblasts show reduced PLD recovery. For the short preincubation time (1 microgram/ml) the effect depends on aphidicolin concentration, for the long preincubation period the effect is independent of concentration. DNA double-strand break induction and rejoining was investigated after combined action of aphidicolin and X-rays using the neutral filter elution technique. The yield of initial DNA double-strand breaks was not affected by aphidicolin. After a 90-min recovery time, however, DNA double-strand break rejoining was markedly reduced. This effect was dependent on concentration for the short incubation time (1 h) but not for the long preincubation (24 h).     

PARP inhibitors diminish DNA damage repair for the enhancement of tumor photodynamic therapy

Pancreatic cancer is a lethal malignancy and only around 4% of patients will live 5 years post-diagnosis. Photodynamic therapy (PDT) is a promising strategy for treating malignant tumors because of its high selectivity. Through the colocalization of light, oxygen and photosensitizer, a large number of reactive oxygen species (ROS) are generated under excitation at a specific wavelength of a laser, which can induce DNA damage and destroy cancer cells. However, the repair mechanism of cell will repair part of the damaged DNA, which could reduce the efficiency of PDT. The poly (ADP-Ribose) polymerase (PARP) plays a wide and multifaceted role in the cellular response to DNA damage, with growing evidence for participation in multiple pathways of DNA damage repair and genome maintenance. Cells require PARP to resolve single-strand DNA breaks (SSBs) induced by chemotherapy agents. Its inhibition is thought to result in the accumulation of damage in DNA, which may eventually lead to cell death. The combination therapy of PDT and PARP inhibitors may benefit patients. In this study, we design and synthesize a zeolitic imidazolate framework-8 (ZIF-8) to co-deliver DNA damaging agent Chlorin e6 (Ce6) and PARP inhibitor Olaparib (Ola). Ce6 and Ola demonstrate strong synergistic actions, providing a novel approach for the treatment of pancreatic cancer.     

Effect of aphidicolin on DNA synthesis, PLD-recovery and DNA repair of human diploid fibroblasts.

The effect of aphidicolin, a specific inhibitor of DNA polymerases alpha and delta, was studied on DNA synthesis, PLD-recovery and DNA double-strand break rejoining in X-irradiated human fibroblasts. In unirradiated, exponentially growing cells, aphidicolin (0.5-5 micrograms ml) inhibited DNA synthesis almost completely. This effect depended not only on aphidicolin concentration but also on the duration of pre-incubation. The action of aphidicolin was found to be reversible. When aphidicolin had been removed, colony forming ability was not affected in aphidicolin pretreated cells. Aphidicolin pretreated and irradiated cells showed a reduction in PLD-recovery, dependent on aphidicolin concentration and duration of pretreatment. The initial number of DNA double-strand breaks (calibrated by 125I decay) was not affected by aphidicolin. However, after incubation for 90 min in the presence of aphidicolin there was a large reduction in double-strand break rejoining. With long incubation periods in aphidicolin rejoining was almost completely inhibited.     

Heavy Ion Effects on Cellular DNA: Strand Break Induction and Repair in Cultured Diploid Lens Epithelial Cells

Summary

The relative biological effectiveness (RBE) for the induction of DNA strand breaks and the efficiency of repair of these breaks in cultured diploid bovine lens epithelial cells was measured, using accelerated heavy ions in the linear energy transfer (LET)-range up to 16 200 keV/µm. At LET values above 800 keV/µm, the number of DNA strand breaks induced per particle increases both with the atomic number of the projectile and with its kinetic energy. About 90 per cent or more of the strand breaks induced by ions with an LET of less than 10 000 keV/µm are repaired within 24 h. Repair kinetics show a dependence on the particle fluence (irradiation dose). At higher particle fluences a higher proportion of non-rejoined breaks is found, even after prolonged periods of incubation. At any LET value, repair is much slower after heavy-ion exposure than after X-irradiation. This is especially true for low energetic particles with a very high local density of energy deposition within the particle track. At the highest LET value (16 200 keV/µm), no significant repair is observed.     

Enhancement of Radiosensitivity of Cultured Mammalian Cells by Neocarzinostatin. II. Fixation of Potentially Lethal Damage

Summary

The effects of neocarzinostatin (NCS), an anti-tumour drug, on the repair of potentially lethal damage (PLD) were studied using cultured Chinese hamster V79, malignant human melanoma and mouse lymphoma L5178Y cells in the stationary phase. The repair of PLD was observed in the melanoma and L5178Y cells but no such repair was observed in the V79 cells, when studied by delayed plating. NCS added to the culture medium immediately after X-irradiation evoked fixation of PLD within 10 min of the addition of NCS. The ratios of D₀ values of the survival curves of the cells treated with NCS to those plated immediately after X-irradiation were 0·78, 0·88 and 0·85 for V79, melanoma and L5178Y cells, respectively. The extent of the fixation by NCS was similar to that caused by 0·5m NaCl solution. The results in the present study and the inhibition of sublethal damage (SLD) by NCS reported previously, suggest that NCS might react with the DNA damage induced by radiation and modify it to lethal damage. The study indicates that SLD and PLD appear to be closely related to one another.     

Thermal Radiosensitization in CHO Cells by Prior Heating at 41–46°C

Summary

CHO cells were exposed to heat at temperatures ranging from 41°C to 46°C followed by irradiation on ice with graded doses of X-rays. The dose–response curves obtained were analysed in terms of D_10%, D₀ and Dq and thermal enhancement was expressed by the corresponding values TER_10%, TEF and TEQ, respectively. TER_10% and TEF were shown to increase linearly with heating time, the increase being steeper at higher temperatures. The dose–response curves were also analysed using the equation ? ln(S/S₀) = αD + βD²; the values of α and β obtained from curve-fitting were found to increase with heating time. For temperatures below 43°C the relative increase in α was greater than that in β; the Arrhenius activation energies were E_a = 890 kJ mol^?1 for α and E_a = 1830 kJ mol^?1 for β. At temperatures exceeding 43°C the relative increase of α and β was similar and the corresponding activation energies were about the same (E_a ≈ 700 kJ mol^?1). The increase in the α-term was attributed to a depressed repair of double-strand breaks, whereas the increase of β was assumed to be a consequence of an insufficient repair of base damage.     

Enhancement of radiosensitivity of cultured mammalian cells by neocarzinostatin. II. Fixation of potentially lethal damage

The effects of neocarzinostatin (NCS), an anti-tumour drug, on the repair of potentially lethal damage (PLD) were studied using cultured Chinese hamster V79, malignant human melanoma and mouse lymphoma L5178Y cells in the stationary phase. The repair of PLD was observed in the melanoma and L5178Y cells but no such repair was observed in the V79 cells, when studied by delayed plating. NCS added to the culture medium immediately after X-irradiation evoked fixation of PLD within 10 min of the addition of NCS. The ratios of D0 values of the survival curves of the cells treated with NCS to those plated immediately after X-irradiation were 0.78, 0.88 and 0.85 for V79, melanoma and L5178Y cells, respectively. The extent of the fixation by NCS was similar to that caused by 0.5 M NaCl solution. The results in the present study and the inhibition of sublethal damage (SLD) by NCS reported previously, suggest that NCS might react with the DNA damage induced by radiation and modify it to lethal damage. The study indicates that SLD and PLD appear to be closely related to one another.     

Deoxyribonucleic acid synthesis by rat thymus and spleen cells in vitro following hyperthermia

The inhibition of the semiconservative and restorative DNA synthesis caused by hyperthermia (30 to 60 min, 43 degrees C) was significantly higher in spleen cells than in thymus cells. The DNA repair synthesis of thymus cells measured at 37 degrees C was increased by about two times the initial value after a pre-incubation of 30 to 90 min and 30 to 60 min, respectively, with 37 and 43 degrees C, respectively. Under the same conditions, the 3H-thymidine incorporation into the DNA of spleen cells diminished proportionally to the pre-incubation time after a pre-incubation of 30 and 45 min, respectively, with 43 and 37 degrees C, respectively. When hyperthermia and inhibitors of DNA synthesis or DNA repair (hydroxyurea, 1-beta-D-arabinofuranosylcytosine, 3',5'-didesoxythymidine, and 3-aminobenzamide) were combined, overadditive effects--without cell specific particularities--were seen only in the case of 3-aminobenzamide. Only in thymus cells, the inhibitor of DNA topoisomerase II novobiocin caused an overadditive reinforcement of the inhibition induced by hyperthermia of the semiconservative DNA synthesis. The stimulation of DNA repair synthesis in thymus cells caused by novobiocin with the aid of DNA polymerase beta could be compensated by hyperthermia. The sedimentation of thymus and spleen cell nucleoids was increased after hyperthermia. The results suggest a special importance of DNA topology and of the DNA polymerase beta activity for the cellular effect of hyperthermia.     

Enhancement of Radiosensitivity of Cultured Mammalian Cells by Neocarzinostatin. I. Inhibition of the Repair of Sublethal Damage

Summary

The enhancement of radiosensitivity by neocarzinostatin (NCS), an antitumour drug, was studied using three strains of cultured mammalian cells with different repair capabilities for sublethal damage. NCS enhanced the radiosensitivity of the cells when applied both during and after X-irradiation under aerobic conditions. The enhancement ratios of NCS during X-irradiation were 1·25, 1·27 and 1·38 for mouse lymphoma L5178Y, Chinese hamster V79 and mouse mammary tumour FM 3A cells, respectively. The corresponding ratios after X-irradiation were 1·18, 1·27 and 1·38, respectively. These ratios were proportional to the repair capabilities of the cells for sublethal damage. NCS completely inhibited the repair of sublethal damage regardless of the repair capabilities of the cells for sublethal damage. NCS was equally effective for hypoxic cells. These results suggested that NCS enhanced the radiosensitivity of the cells probably by interacting with the residual damage after X-irradiation, thereby converting the sublethal damage or potentially lethal damage into lethal damage.     

Sensitization of Cultured Chinese Hamster Cells to 42°C Hyperthermia by Pentalenolactone,an Inhibitor of Glycolytic ATP Synthesis

Summary

The antibiotic pentalenolactone, a specific inhibitor of glyceraldehydephosphate dehydrogenase, was used to investigate the effect of glycolytic adenosine triphosphate (ATP) synthesis on the survival response of aerobic and hypoxic Chinese hamster cells treated with 42°C hyperthermia. Data obtained with aerobic cells, incubated in balanced salt solutions supplemented with different substrates for ATP production, showed that 50 μm pentalenolactone blocked ATP synthesis via glycolysis but not by oxidative phosphorylation. The glycolytic inhibition was reversed upon transfer of the cells to antibiotic-free medium, and minimal cytotoxicity (< 20 per cent) was observed.

Hypoxic cultures were obtained by incubating dense cell suspensions (2 × 10⁶/ml) to produce metabolic oxygen depletion. Concomitant with the development of hypoxia, pentalenolactone-treated cells became ATP-depleted; cellular ATP levels were reduced by about 70-fold as compared to hypoxic cells in the antibiotic-free medium. The ATP-depleted cells were more sensitive to killing by hyperthermia. Comparison of the 42°C survival curves for control and the antibiotic-treated hypoxic cells yielded a dose-modifying factor of 4 (5 per cent survival level). The results indicate that inhibition of glycolytic ATP synthesis, for example by pentalenolactone, can selectively sensitize hypoxic cells to the lethal effects of mild hyperthermia.     

Reply to Letter by C. B. Seymour and C. Mothersill

Summary

V79 379A cells were irradiated and then exposed to anisotonic PBS for 20 min. This enhanced the radiation effect resulting from the fixation of potentially lethal damage. The induction of DNA single- and double-strand breaks is not increased by this treatment. Anisotonic treatment delayed the onset of repair of DNA damage. However when cells were returned to normal medium, they repaired the damage to a similar extent as cells not exposed to the anisotonic treatment. We suggest that the fixation of damage by post-irradiation anisotonic treatment is mediated through an increased probability of misrepair of DNA damage due to the delay in the onset of repair. This is supported by the observation that there is a reduced effect of post-irradiation anisotonic treatment on cells that have a markedly reduced ability to repair double-strand breaks.     

Time-course of the hazard of murine nephropathy induced by total-body irradiation

Purpose : We investigated the form of the hazard function for total-body irradiation (TBI) induced nephropathy in mice during 1.5 years of follow-up. Material and methods : The material was collected from our previously published data. Following single-dose or fractionated irradiation and bone marrow transplantation (BMT) the mice were tested regularly for kidney damage using the CrEDTA residual activity, percentage haematocrit and blood urea nitrogen endpoints. The hazard rate was studied in the pooled data of 172 mice for all three endpoints, while fractionation sensitivity was estimated from the direct analysis, which was performed using the CrEDTA residual activity endpoint and the actual follow-up data in individual mice. Results : The hazard rate of kidney damage following TBI and BMT showed a biphasic pattern that is most evident with the CrEDTA residual activity endpoint, with a reduced risk of renal failure around week 36 after TBI. Assessment of kidney function in individual animals showed evidence of recovery from radiation damage around week 36 after TBI. An analysis of fractionation sensitivity showed that the first wave was characterized by an α / β ratio of 8.4 Gy (95% CI: 4.0-14.3 Gy), while the α / β ratio for the second wave was estimated at 6.1 Gy (95% CI: 3.3-9.8 Gy). Conclusions : The biphasic nature of the hazard function reported here may be a unique feature of TBI-induced renal damage. Differentiation between the two phases in terms of their α / β ratio was not possible. The biological basis of this observation remains to be clarified. The reported high α / β ratio of kidney damage in the TBI situation may have important clinical implications.