Radiation-induced DNA Strand Breaks Research Articles

Strand break repair, DNA polymerase activity and heat radiosensitization in thermotolerant cells

HeLa S3 cells were made thermotolerant by 'chronic' (5 h at 42 degrees C) or 'acute' (15 min at 44 degrees C followed by 5 h at 37 degrees C) heat treatments. Cell survival, repair of radiation-induced DNA strand breaks, alpha and beta DNA polymerase activity and radiation sensitivity following hyperthermia were all measured in both control and thermotolerant cells. The ability to repair DNA strand breaks correlated well with cell survival following hyperthermia. Hyperthermic inhibition of strand break repair was reduced in thermotolerant relative to control cells, although the thermal tolerance ratios for repair were less than for hyperthermic cell killing. Both radiosensitization and DNA polymerase inactivation by hyperthermia were only slightly reduced in thermotolerant relative to control cells. Hence a poor correlation was found between these two parameters and hyperthermic cell survival. For all heat treatments applied, alpha and beta DNA polymerase activity correlated well with the extent of hyperthermic radiosensitization.

Effect of ionising radiation on the axon reaction of mouse anterior horn motor neurons: A histological and immunocytochemical study using a monoclonal antibody to neurofilament protein

The changes taking place in irradiated central nervous tissue prior to the onset of delayed radionecrosis are poorly understood, but functional abnormalities occurring during the latent interval after irradiation are likely to be of importance. In order to investigate functional disturbances in neurones during this period, unilateral sciatic nerve crush was performed in mice following sub-lethal X-irradiation of the lumbar spinal cord. Alterations in the axon reaction of anterior horn cells were studied using a monoclonal antibody to neurofilament protein. With irradiation immediately prior to crush, the normal, well-defined increase in perikaryal neurofilament protein was significantly diminished, although there was no concurrent radiation necrosis and no alterations were seen in contralateral neurones with intact distal axon processes. The effect was more marked in neurones irradiated one month prior to nerve crush, and in the non-irradiated nerve crush region regeneration was delayed, with diminished neurofilament protein in the regenerating axons. These observations indicate that ionising radiation can progressively impair the ability of neurones to synthesise neurofilament protein during distal axon regeneration. This may result from inadequate repair of radiation induced DNA strand-breaks, but may also follow more generalised damage to protein transcription enzymes and RNA metabolism.

Enzyme action at 3' termini of ionizing radiation-induced DNA strand breaks.

gamma-Irradiation of DNA in vitro produces two types of single strand breaks. Both types of strand breaks contain 5'-phosphate DNA termini. Some strand breaks contain 3'-phosphate termini, some contain 3'-phosphoglycolate termini (Henner, W.D., Rodriguez, L.O., Hecht, S. M., and Haseltine, W. A. (1983) J. Biol. Chem. 258, 711-713). We have studied the ability of prokaryotic enzymes of DNA metabolism to act at each of these types of gamma-ray-induced 3' termini in DNA. Neither strand breaks that terminate with 3'-phosphate nor 3'-phosphoglycolate are substrates for direct ligation by T4 DNA ligase. Neither type of gamma-ray-induced 3' terminus can be used as a primer for DNA synthesis by either Escherichia coli DNA polymerase or T4 DNA polymerase. The 3'-phosphatase activity of T4 polynucleotide kinase can convert gamma-ray-induced 3'-phosphate but not 3'-phosphoglycolate termini to 3'-hydroxyl termini that can then serve as primers for DNA polymerase. E. coli alkaline phosphatase is also unable to hydrolyze 3'-phosphoglycolate groups. The 3'-5' exonuclease actions of E. coli DNA polymerase I and T4 DNA polymerase do not degrade DNA strands that have either type of gamma-ray-induced 3' terminus. E. coli exonuclease III can hydrolyze DNA with gamma-ray-induced 3'-phosphate or 3'-phosphoglycolate termini or with DNase I-induced 3'-hydroxyl termini. The initial action of exonuclease III at 3' termini of ionizing radiation-induced DNA fragments is to remove the 3' terminal phosphate or phosphoglycolate to yield a fragment of the same nucleotide length that has a 3'-hydroxyl terminus. These results suggest that repair of ionizing radiation-induced strand breaks may proceed via the sequential action of exonuclease, DNA polymerase, and DNA ligase. The possible role of exonuclease III in repair of gamma-radiation-induced strand breaks is discussed.

Gamma radiation-induced single strand breaks in DNA and their repair in spheroplasts and nuclei of light-grown and dark-grown Euglena cells.

Exposure of light-grown and dark-grown Euglena cells to gamma radiation causes single strand breaks in nuclear DNA as assessed by sedimentation analysis in alkaline sucrose density gradients. The number of radiation-induced single strand breaks in nuclear DNA of light-grown cells is found to be less than that in dark-grown cells. Post-irradiation incubation of both types of cells in 0 . 1 M phosphate buffer, pH 7 . 0 at 25 degrees C for 1 hour results in restitution of the strand breaks in DNA. Light-grown cells (cells with chloroplasts) are able to rejoin all the single strand breaks in DNA produced by gamma irradiation at D50 and D5 doses. On the other hand, dark-grown cells (cells devoid of chloroplasts) are unable to rejoin all the strand breaks caused by irradiation at either of the doses. The rate of DNA repair in dark-grown cells is also much slower than that in light-grown cells. Radiation-induced single strand breaks in DNA and their repair in nuclei from both types of cells is found to be similar to that observed in the spheroplasts. It is suggested that some factor(s) elaborated by chloroplasts may contribute towards the efficiency of nuclear DNA repair in Euglena cells.

Radiation-induced DNA strand breaks and cell pycnosis in the developing rat cerebellum.

DNA strand breaks and cell pycnosis induced by 60Co gamma radiation were studied in the developing rat cerebellum. Rats 2, 5 or 12 days old were irradiated and the repair of DNA strand breaks was studied at various intervals up to seven hours. DNA strand breaks were repaired in 30 min and post-repair DNA degradation was observed about 90 min after irradiation in 12 day old rats and some time later in the younger ones. The pycnotic activity in the external granular layer of the cerebellar cortex seen in histological preparations developed in a similar way, the older rats showing earlier signs of cell pycnosis and a faster degradation of DNA. A correspondence was found between DNA degradation and cell pycnosis.

Sites and structure of gamma radiation-induced DNA strand breaks.

DNA fragments of defined sequence were used to study DNA strand breakage by gamma radiation in doses ranging from 2,500 to 20,000 rads. The amount of radiation-induced DNA breakage is linearly dose-dependent, and strand scission events occur uniformly at all nucleotide sites, regardless of sequence. The 5'-terminal group at points of breakage is a simple phosphoryl moiety. Two types of radiation-induced 3' termini were identified. One species of 3' terminus is a simple phosphoryl group. The other species of 3' terminus is neither a hydroxyl nor a phosphoryl group. A model for radiation-induced DNA strand scission that involves destruction of the deoxyribose moiety is presented. Similarities among DNA strand scission events created by gamma radiation, ferrous ion, doxorubicin, and bleomycin are discussed.

In vitro studies on the absorption of WR-2721 by tumors and normal tissues

An attempt was made to develop single cell systems which would allow the study of absorption of WR-2721 by individual normal and malignant cells. Simple mechanical dissociation procedures provided adequate single cell suspensions for most normal tissues and these cells concentrated WR-2721 against a gradient. One apparent exception was bone marrow—while the tissue is very responsive to WR-2721 in vivo, our in vitro preparations absorbed the drug only very poorly. It was found, however, that lipid and red blood cell contaminants in the marrow cell preparation masked and interfered with the absorption of WR-2721, causing the anomolous results. In contrast with normal tissue, mechanical or enzymatic dissociation procedures failed to produce adequate single cell preparations from tumors. Problems encountered include low cell yield, contamination by non-tumor cells of host origin, and tumor cell membrane damage. Since published studies of WR-2721 protection of monolayer cultures have probably all been carried out using malignant cells, we repeated these experiments using normal human fibroblasts. As with malignant cells, however, we found only marginal radiation protection of these cultured normal cells by WR-2721. In addition, the drug produced no detectable protection against radiation-induced DNA strand breaks, either in normal fibroblasts or in HeLa cells. Thus, cultured fibroblasts do not appear to provide a useful model for studying the absorption of WR-2721 by normal tissue.

Effect of hyperthermia at 42 and 45 degrees C on repair of radiation-induced DNA strand breaks in CHO cells.

The effect of hyperthermia on DNA strand break repair was studied in CHO cells. DNA strand breaks were analysed by the alkaline DNA-unwinding technique followed by chromatography on hydroxyapatite. Immediately after irradiation with doses ranging from 2 to 7 Gy, cells were exposed to 42 or 45 degrees C. Heat alone was found to induce DNA strand breaks only at temperatures exceeding 45 degrees C. In comparison to 37 degrees, C, the rate of single-strand break repair was increased by hyperthermia at 42 degrees C, but decreased at 45 degrees C. In contrast hyperthermia at either temperature resulted in a higher number of remaining double-strand breaks 1 hour after irradiation. For the three treatments applied, i.e. X-rays alone or combined with hyperthermia at 42 or 45 degrees C, the relation between cell survival and the number of double-strand breaks measured 1 hour after irradiation could be described by the same function.

The sensitivity of active and inactive chromatin to ionizing radiation-induced DNA strand breakage.

The sensitivity of active and inactive chromatin to ionizing radiation-induced DNA strand breakage.

Effects of Hyperthermia on Repair of Radiation-Induced DNA Strand Breaks

Previous reports have suggested a relationship between the heat-induced changes in nucleoprotein and the hyperthermic enhancement of radiation sensitivity. In an effort to further understand these relationships, we measured the level of initial DNA strand break damage and the DNA strand break rejoining kinetics in Chinese hamster ovary cells following combined hyperthermia and ionizing radiation treatments. The amount of protein associated with DNA measured as the ratio of [3H]leucine to ['4C]thymidine was also compared in chromatin isolated from both heated and unheated cells. The results of these experiments show that the initial level of radiation-induced DNA strand breaks (as measured by the sensitive technique of alkaline elution) is significantly enhanced by a prior hyperthermia treatment of 43?C for 30 min. Treatments at higher temperatures and longer treatments at the same temperature magnified this effect. Hyperthermia was also shown to cause a substantial inhibition of the DNA strand break rejoining after irradiation. Both the initial level of DNA damage and the rejoining kinetics recovered to normal levels with incubation at 37?C between the hyperthermia and radiation treatments. Recovery of these parameters coincided with the return of the amount of protein associated with DNA to normal values, further suggesting a relationship between the changes in nucleoprotein and the hyperthermic enhancement of radiation sensitivity.

Hyperthermic Enhancement of Radiation Cell Killing in HeLa S3 Cells and Its Effect on the Production and Repair of DNA Strand Breaks

Cell killing and the induction and repair of DNA strand breaks have been examined after hyperthermia and electron irradiation treatments of HeLa S3 cells. Heat alone did not produce any detectable DNA strand breaks. Preirradiation heat treatment did not alter the initial levels of radiation-induced DNA strand breaks. However, the subsequent rate and extent of DNA strand break repair were significantly reduced by preirradiation heat treatments of 44/sup 0/C for 30 or 60 min. When a time gap of up to 1 h was introduced between heating and irradiation there was no significant reduction of the thermal enhancement effect for cell killing or recovery from the reduced ability to repair DNA strand breaks.

Sensitivity of cells in exponential and stationary growth phase to combined treatment with radiation and quinacrine.

The effect of Quinacrine on the recovery of cells from sublethal radiation injury was analysed by testing cellular survival after exposure to low and high radiation doses, and after irradiation with split doses. Quinacrine was found to inhibit recovery in cell cultures in exponential growth phase but not, or only slightly, in cells in stationary growth. This finding is related to the inhibitory effect of Quinacrine on the repair of radiation induced DNA strand breaks.

Radiation-induced DNA Strand Breaks in Deoxygenated Aqueous Solutions. The Formation of Altered Sugars as End Groups

Gamma irradiation of DNA in deoxygenated, N2O-saturated aqueous solution leads to three bound altered sugars present as end groups in broken DNA strands. These sugars are linked to the DNA by phosphoric acid ester bonds. Two of the end groups have the structures (4) and (5). (Formula: see text) The third end group after dephosphorylation has structure (3). The formation of the bound sugars (4) and (5) is explained by a mechanism postulated earlier for the formation of free altered sugars. Except for the phosphoric acid ester linkage, the free altered sugars have the same chemical structures as the bound altered sugars.

Radiation-induced DNA Strand Breaks and Their Repair in the Developing Rat Brain

Rats, 5, 10 or 25 days old, were 60 Co gamma irradiated. The induction of DNA strand breaks was studied after killing the rats within 1 min after irradiation, and the repair of the induced breaks after various intervals up to 180 min. Cell suspensions were prepared from the brain and samples were transferred into alkaline solutions. The fraction of DNA remaining double-stranded after 30 min alkali treatment was estimated after separation of single- and double-stranded DNA on hydroxylapatite. The amount of DNA strand breaks induced per Gray (1--8 Gray) was found to be in accordance with earlier in vivo studies of the mouse small intestine and mouse spleen. The DNA strand breaks in the rat brain induced by 4 Gray 60Co gamma irradiation were repaired 30 min after irradiation in all age groups studied.

Mammalian cell studies with diamide

Diamide, diazenedicarboxylic acid bis (N,N′-dimethylamide), is a glutathione oxidant that has several advantages over classical sulfhydryl reagents, including reversibility. It is used widely to study glutathione's role in cellular function and radioresistance. It is also an effective radiosensitizer of hypoxic cells and acts by a unique mechanismm increasing the slope of the survival curve of irradiated hypoxic mammalian cells at high concentrations and decreasing the shoulder at low concentrations. Increased yields of radiation-induced chromosome aberrations and DNA strand breaks suggest that diamide sensitizes by mechanisms involving DNA. These mechanisms may include direct interactions with DNA and removal of cellular reducing species that normally repair radiation damage. Diamide sensitizes hypoxic cells synergistically with nitrofuran or nitroimidazole sensitizers, is effective against ascites tumors in vitro, and is useful for elucidating the mechanisms of aminothiol radioprotection. If used carefully, and with due regard for its limitations, it provides a useful tool with which to expand our knowledge of the mechanisms that govern cellular radiosensitivity and to hasten development of effective radiosensitizers for clinical use.

Damages Caused by Irradiation in the Presence of NaCl in Macromolecular Components ofE. coli

Damages in macromolecular components and in their functions of E. coli produced by gamma irradiation in the presence of higher concentration of NaCl were determined with reference to the basic mechanism for NaCl radiosensitization. As the results, inhibitions of DNA repair, of DNA degradation, of macromolecular syntheses and of RNA species formation were found in DNA repair proficient strains. The number of radiation-induced DNA-strand breaks was not increased by the presence of 1 m NaCl during irradiation. It was suggested that inhibition of DNA repair may be of primary importance among these lesions for NaCl radiosensitization.

Damages caused by irradiation in the presence of NaCl in macromolecular components of E. coli.

Damages in macromolecular components and in their functions of E. coli produced by gamma irradiation in the presence of higher concentration of NaCl were determined with reference to the basic mechanism for NaCl radiosensitization. As the results, inhibitions of DNA repair, of DNA degradation, of macromolecular syntheses and of RNA species formation were found in DNA repair proficient strains. The number of radiation-induced DNA-strand breaks was not increased by the presence of 1M NaCl during irradiation. It was suggested that inhibition of DNA repair may be of primary importance among these lesions for NaCl radiosensitization.

Repair of Radiation-induced DNA Strand Breaks in Thymocytes

Repair of Radiation-induced DNA Strand Breaks in Thymocytes

In vitro repair of radiation-induced strand breaks in DNA

DNA, γ-irradiated in vitro or in isolated thymocytes was treated with several enzymes to achieve repair of the radiation-induced single strand braks. Whereas an incubation with polynucleotide ligase can join only 25% of the single strand breaks, a combined treatment with exonuclease III (EC 3.1.4.1), DNA polymerase I (EC 2.7.7.7), and polynucleotide ligase leads to repair of 80% of the breaks. For this in vitro repair the exonuclease III has to remove several, probably damaged, nucleotides from the 3′-terminal producing a single-stranded gap, which will be filled in by DNA polymerase I and joined by ligase. Tests for successful rejoining of the strand breaks were performed by showing the loss of primer 3′-OH sites for DNA polymerase I, by the resistance of incorporated nucleotides in the gap to removal by a second exonuclease III treatment, and by strand break determination in the analytical ultracentrifuge. 20% of the radiation-induced strand breaks will not be repaired by this combined treatment possibly due to an incomplete binding of the ligase on the 5′-terminals and/or an incomplete removal of the damaged 3′-terminals by exonuclease III.

The correlation between mutation frequency and cell survival following different mutagenic treatments

A direct mathematical relationship between mutation frequency per survivor and cell survival is derived from theoretical considerations of the molecular effects of radiation in a cell. It is shown that the relationship is satisfied by analysis of the various data on radiation induced mutations available in the literature. The analysis implies that a common type of lesion may lead to mutation and cell death and is derived on the assumption that radiation-induced double strand breaks in DNA are the critical lesions. The mathematical relationship is independent of the way in which the lesion which leads to mutations and cell death is induced, so the analysis has consequently been applied to other mutagenic treatments such as UV light and chemicals. It is concluded that, although the lesions induced by chemicals may not be the same as those induced by radiation, it is probable that for the chemicals considered common basic damage to the DNA molecule is implicated as the critical lesion.