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PubMed1976

The effect of hyperthermia on DNA repair.

In the past there were many individual observations on the value of hyperthermia in the treatment of human neoplasia but most of the information about the value of hyperthermia as a single agent or in the combined modality approach has come from laboratory investigations. Dose response curves for cell survival after exposure to heat are similar in shape to cell survival curves obtained after irradiation or treatment with some cytostatic agents. The shoulder in such curves suggests that repair of sublethal or potentially lethal damage takes place after hyperthermic treatment. On the level of molecular biology the process of cellular repair should correspond to repair of damage inflicted on deoxyribonucleic acid (DNA). We have shown by means of the BUdR assay that such DNA-repair synthesis does take place upon exposure to heat. Many investigations have provided evidence of a synergism between hyperthermia and ionizing irradiation or some cytostatic agents. It was suggested that such synergism might be caused by the inhibition of repair of sublethal damage by heat. After inflicting DNA damage by a strong alkylating agent (NA-AAF) we could demonstrate DNA-repair synthesis by means of the BUdR-assay during exposure to heat. At the present time results obtained by assaying DNA repair on the basis of cell survival and by means of the BUdR-assay are difficult to reconcile.

Acetoxyacetylaminofluorene↗
PubMed1979

Inhibition of DNA repair in ultraviolet-irradiated human cells by hydroxyurea.

The effect on DNA repair in ultraviolet-irradiated human skin fibroblasts by hydroxyurea has been examined in this study using three independent methods for measuring DNA repair:the 5-bromodeoxyuridine photolysis assay which measures DNA repair replication, chromatographic measurement of thymine-containing dimers, and measurement of specific ultraviolet-endonuclease-sensitive sites in irradiated DNA. Little effect of hydroxyurea was observed at the concentration of 2 mM, which is often used to inhibit semiconservative DNA synthesis; however, 10 mM hydroxyurea resulted in marked inhibition (65--70%) of excision repair. This inhibition was accompanied by a possible doubling in the size of the repaired region. The accumulation of large numbers of single-strand breaks following ultraviolet irradiation and hydroxyurea incubation seen by other investigators was not observed with the normal skin fibroblasts used in this study. A comparison of hydroxyurea effects on the different DNA repair assays indicates inhibition of one step in DNA repair also results in varying degrees of inhibition of other steps as well.

Bromodeoxyuridine↗
PubMed1977

Role of DNA repair in natural resistance of rat ascites hepatomas to nitrogen mustard.

Mechanism of natural resistance of rat ascites hepatomas to nitrogen mustard was examined with tumors, especially AH-13 and AH-44, sensitive and resistant to the chemical, respectively. There was found little difference in the uptake and binding of the chemical among the two lines. In contrast, DNA repair activity of the resistant line was higher than that of the sensitive line. This result suggested that natural resistance of AH-44 to the drug may be, at least partially, due to its higher activity of repairing DNA damage.

Animals↗
PubMed1976

DNA repair synthesis in guineapig pancreas following exposure to nitrosomethylurethane.

NMUT, a known pancreatic carcinogen in guineapigs, alkylates pancreatic DNA and RNA, both in vivo and in vitro. Following the in vivo administration of a single maximum tolerated dose of NMUT (30 mg/kg), a significant increase in 3H-Tdr incorporation into DNA was observed in the duodenal segment of the pancreas after four days; this increase in thymidine incorporation probably represents in vivo DNA repair synthesis. The level of normal DNA synthesis was greater in the duodenal segment than elsewhere in the pancreas. In vitro exposure of pancreatic slices from the duodenal segment to 20 mM NMUT for 30 minutes resulted in a significant increase in 3H-TdR incorporation into DNA in the presence of HU, reflecting DNA repair synthesis following NMUT-induced DNA damage; normal DNA synthesis in the pancreatic slices in vitro was markedly suppressed by 10 mM HU. Studies on the kinetics of DNA repair synthesis in pancreatic slices indicated an initial increase of 3H-TdR incorporation, followed by a steady time-dependent decline. It appears that most of the DNA repair synthesis occurs within two hours after exposure to NMUT.

Animals↗
PubMed1975

DNA repair in Proteus mirabilis. III.Survival, dimer excision, and UV reactivation in comparison with Escherichia coli K12.

Measurements of UV sensitivity of wildtype cells (wt) and UV senistive mutants of E. coli and P. mirabilis suggest that the increased sensitivity of P. mirabilis (wt) is due either to incomplete repair of DNA lesions or to additive lethality probably as a result of UV induction of defective phage(s) present in P. mitabilis (Taubeneck, 1967). Direct estimates of the rate of pyrimidine dimer excision and a comparison of the UV reactivation capacity of E. coli and P. mirabilis for the temperate phages lambda and pi 1, respectively, support this conclusion.

Cell Survival↗
PubMed1977

Nonuniform distribution of DNA repair in chromatin after treatment with methyl methanesulfonate.

The distribution of methyl methanesulfonate induced DNA repair was measured in mouse mammary cell chromatin by digestion of "repair labeled" nuclei with micrococcal nuclease. The results indicate that there is a nonuniform distribution of DNA repair in chromatin. The chromatin fraction digested during the first 5 minutes of incubation with micrococcal nuclease appears to be a primary site of DNA repair after methyl methanesulfoante treatment. The observed nonuniform distribution of DNA repair in chromatin may be due to 1)a nonrandom alkylation of DNA in chromatin by methyl methanesulfonate or 2)areas in chromatin of increased accessibility for the repair enzymes to the DNA lesions.

Animals↗
PubMed1978

The nucleotide-permeable Escherichia coli cell, a sensitive DNA repair indicator for carcinogens, mutagens, and antitumor agents binding covalently to DNA.

Ether-permeabilized (nucleotide-permeable) Escherichia coli cells respond to alkylating and arylalkylating carcinogens with DNA excision repair, as assessed by their stimulation of DNA repair synthesis. In the present work, we have investigated whether DNA repair synthesis in ether-treated E. coli cells can serve as a general indicator to monitor the DNA-binding of carcinogens, mutagens and antitumor agents. Therefore, a standard assay was developed and comparative analyses were performed on 11 ultimate carcinogens, 10 proximate carcinogens, 2 tumor promoters, 6 mutagens, and 12 antitumor agents. All ultimate carcinogens (alkylating, acylating, arylalkylating agents) and mutagens (e.g., hydrogeen peroxide, acridine derivatives) caused DNA excision repair in wild type cells as measured by [3H] dTMP incorporation and simultaneously inhibited replicative DNA synthesis to various extents. Control experiments with the mutant cells uvrA and uvrB were performed to determine whether the pyrimidine-dimer-specific UV-endonuclease was involved in the removal of DNA damage. This was found to be true for the ultimate carcinogens (Ac)2 ONFln, mitomycin C, and for very reactive alkylating carcinogens. None of the ultimate carcinogens induced repair polymerization in mutant cells lacking the 5'-3' exonucleolytic activity of DNA polymerase I. Proximate carcinogens, such as Me2NNO, 4-nitroquinoline-1-oxide and aflatoxins, did not induce excision repair in the standard assay, probably because of the inability of E. coli to perform the activation steps necessary for covalent DNA-binding. However, Me2NNO, when pretreated with Udenfriend's hydroxylating mixture, gave rise to a low level of repair polymerization in ether-treated cells. Intercalating mutagens, such as quinacrine and ethidum bromide, inhibited replicative DNA synthesis. However, they were not found to be repair-inducers. THE TUMOR PROMOters TPA and phorbol-12,13-didecanoate did not cause excision repair, even when applied at high concentrations, nor did they inhibit repair synthesis stimulated by MeNOUr or (Ac)2 ONFln. The antitumor agents may be classified into two groups on the basis of the influence they exert on DNA synthesis: members of the first group (involving BCNU and bleomycin) stimulate repair polymerization and, in addition, inhibit DNA replication. These compounds are known to bind covalently to DNA. The second group of drugs (including adriamycin and cis-Pt(II)diammine complexes) inhibits DNA replication without stimulating repair synthesis. The predominant DNA-interaction of these compounds is known to be a non-covalent (i.e., intercalative, electrostatic) binding. Our experiments show that the ether-permeabilized E. coli cell can be successfully used to test ultimate carcinogens, mutagens and antitumor agents for repair-inducing and replication-inhibiting activity. The standard test might be extended to pre- and proximate carcinogens, provided these can be suitably activated.

Antineoplastic Agents↗
PubMed1976

Studies on DNA repair in early spermatid stages of male mice after in vivo treatment with methyl-, ethyl-, propyl-, and isopropyl methanesulfonate.

In vivo DNA repair occurring in early spermatid stages of the mouse has been studied with four mutagens that are chemical homologs: MMS, EMS, PMS and IMS. Using the well-studied sequence of events that occurs during spermatogenesis and spermiogenesis in the mouse, aatids was measured by the unscheduled incorporation of [3H]dT into these germ cells which were recovered from the caudal epididymides 16 days after chemical treatment. Purification of the caudal sperm DNA at this time verified that the [3H]dT was incorporated into the DNA. For each chemical mutagen a study was made on the level of DNA repair occurring in early spermatids as a function of the administered, in vivo dose. Within experimental errors, all four chemicals produced a linear increase in DNA repair in early spermatids with increasing dose. Only the highest dose of MMS (100 mg/kg) produced a greater repair response than expected for a linear curve. At equimolar doses the most effective chemical in inducing DNA repair was MMS, followed by EMS, IMS and PMS. When testicular injections of [3H]dT were given at the same time as the intraperitoneal injections of the mutagens, the amount of unscheduled incorporation of [3H]dT into the DNA of early spermatids was maximized. Since [3H]dT has been shown to be available for incorporation into germ-cell DNA for only approximately 1 h after injection, all four mutagens must reach the DNA of early spermatids and begin producing "repairable" lesions within 1 h after treatment. The amount of DNA repair occurring at later times after chemical treatment of early spermatids was studied by testicular injections of [3H]dT 1/2, 1, 2 and 3 days after chemical treatment. Repair was still occurring in the early spermatids at 3 days post-treatment; this repair is most likely a manifestation of the finite rate of the repair process rather than resulting from newly alkylated DNA. For MMS and EMS there was a rapid decrease in the level of DNA repair in the first 1/2 day following treatment. This was followed by a much slower, exponential decrease in the level of repair out to 3 days post-treatment. The curves suggest that the amount of repair is proportional to the number of repairable lesions still present in the DNA. For PMS and IMS the level of repair decreases rapidly in the first 1/2 day after treatment and thereafter remains relatively constant through 3 days post-treatment. With all four mutagens, DNA repair in early spermatids was detectable at doses 5 to 10 times lower than those required to observe other genetic end points such as dominant lethals, translocations and specific-locus mutations in any germ-cell stage. The sensitivity of detection of in vivo DNA repair in the germ cells of male mice makes such a system a useful adjunct to other genetic tests for studying chemical mutagenesis in mammals.

1-Propanol↗
PubMed1992

[The regulation of the DNA repair process in mammalian cells. IV. The role of DNA polymerases in the epidermal growth factor regulation of the repair of single-stranded DNA breaks induced by ionizing radiation in Swiss 3T6 mouse cells].

A study was made of the repair of ionizing radiation-induced DNA single-strand breaks (SSB) in proliferating and quiescent mouse Swiss 3T6 cells and in those stimulated from the quiet status by epidermal growth factor in combination with insulin, in the presence of specific inhibitors of DNA polymerase alpha and delta (aphidicolin) and DNA polymerase beta (2', 3'-dideoxythymidine-5'-triphosphate). The repair of DNA SSB induced by X-ray-irradiation (10 Gr) or by gamma-ray irradiation (150 Gr) is more sensitive to aphidicolin independently of cell proliferating status. Aphidicolin inhibits the recovery of single-strand DNA in quiescent and mitogen-stimulated cells three times stronger than in proliferating cells. The influence of 2', 3'-dideoxythymidine-5'-triphosphate on the rate of DNA SSB repair in cells of all the three types does not differ. Thus, the decrease in DNA repair efficiency in quiescent cells is connected with a decrease in the activity of aphidicolin-sensitive DNA polymerase, apparently DNA polymerase alpha. It is suggested that the regulation action of mitogens on the DNA SSB repair may be determined by qualitative changes of this enzyme or of some conditions in which it functions. The involvement of DNA polymerase delta in this process is not excluded.

Animals↗
PubMed2022

Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination.

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of chromatin, show most DNA repair enzymes remove DNA damage at reduced rates as compared to free DNA. The molecular details on how base excision repair (BER) enzymes recognize and remove DNA damage in nucleosomes have not been elucidated. However, biochemical BER data of nucleosomal substrates suggest the nucleosome presents different structural barriers dependent on the location of the DNA lesion and the enzyme. This indicates the mechanisms employed by these enzymes to remove DNA damage in free DNA may be different than those employed in nucleosomes. Given that the majority of genomic DNA is assembled into nucleosomes, structural information of these complexes is needed. To date, the scientific community lacks detailed protocols to perform technically feasible structural studies of these complexes. Here, we provide two methods to prepare a complex of two genetically fused BER enzymes (Polymerase β and AP Endonuclease1) bound to a single-nucleotide gap near the entry-exit of the nucleosome for cryo-electron microscopy (cryo-EM) structural determination. Both methods of sample preparation are compatible for vitrifying quality grids via plunge freezing. This protocol can be used as a starting point to prepare other nucleosomal complexes with different BER factors, pioneer transcription factors, and chromatin-modifying enzymes.

Chromatin↗
PubMed1976

Control of DNA repair linked to neuroblastoma differentiation.

Mouse neuroblastoma cells, which can be induced to undergo reversible differentiation in culture, have been used as a model to investigate the effects of ultra-violet (U.V.) radiation on terminally-differentiated nerve cells. Differentiated neuroblastoma cells were found to be extremely sensitive to U.V.-radiation when compared with proliferating cells from the same clone. However, normal resistance was regained if the differentiated cells were allowed to proceed to the next G1 phase of the cell-cycle before irradiation. Neuroblastoma cells in the differentiated mode are capable of carrying out soem excision repair of DNA damage, but they appear to lack a repair mechanism present in proliferating cells.

Cell Division↗
PubMed1979

Effects of opiates and demographic factors on DNA repair synthesis in human leukocytes.

DNA repair synthesis in leukocytes stressed by far UV irradiation was studied in 90 normal individuals, 38 street-heroin addicts, and 18 methadone maintenance patients. Age, sex, coffee use, and alcohol use had no significant effect on the maximal repair synthesis response of the control subjects, but smoking tobacco significantly decreased the mean response and variance when compared with nonsmoking controls. Heroin addiction had an even more pronounced negative effect, and this may be related to the high rate of chromosome aberrations found in this population. Half of the addicts tested were incapable of repairing UV fluences one-quarter as large as those repaired by the control subjects (5 J/m2 and 20 J/m2, respectively) in the 2-hr assay period. Long-term methadone treatment ameliorated the effects of the street heroin, just as it resulted in a decrease of the chromosome aberration frequency.

Adolescent↗
PubMed1975

Nonspecific inhibition of DNA repair by promoting and nonpromoting phorbol esters.

The effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and its nonpromoting structural analogue, 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (Me-TPA), on N-acetoxy-2-acetylaminofluorene-elicited DNA repair and replicative DNA synthesis was measured in normal human fibroblasts. Both esters inhibited DNA repair synthesis, and Me-TPA was nearly as effective as TPA. In addition, TPA inhibited replicative DNA synthesis. These findings showed that inhibition of DNA repair synthesis may not be a major factor in the mechanisms of action of tumor promoters.

Acetoxyacetylaminofluorene↗
PubMed1975

Nonspecific inhibition of DNA repair synthesis by tumor promoters in human diploid fibroblasts damaged with N-acetoxy-2-acetylaminofluorene.

The effects of selected tumor-promoting agents and their nonpromoting analogs on DNA repair synthesis were examined in human diploid fibroblasts (WI-38) damaged with N-acetoxy-2-acetylaminofluorene. Over a range of doses, three promoters (croton oil, 12-O-tetradecanoylphorbol-13-acetate, and anthralin) were found to inhibit DNA repair synthesis while their nonpromoting analogs (phorbol and 1,8-dihydroxyanthraquinone) had little effect. Another tumor promoter, phenol, inhibited DNA repair synthesis only at very high concentrations while an analog, 4-nitrophenol, produced inhibition of DNA repair synthesis at molar concentrations at which phenol had no effect. To investigate the specificity of this phenomenon, the effects of these agents on DNA-replicative synthesis, RNA synthesis, protein synthesis, and cell morphology were evaluated. At equimolar concentrations, tumor promoters were found to inhibit DNA-replicative synthesis as effectively as repair synthesis. RNA and protein synthesis were similarly inhibited over the same range of concentrations. Extensive morphological changes, interpreted as evidence of toxicity, were seen at concentrations of promoters that inhibited the macromolecular syntheses studied. The nonpromoting analogs, with the exception of nitrophenol, had little effect on these processes and showed only slight morphological damage. Thus tumor-promoting agents appeared to inhibit a number of macromolecular synthetic events, including DNA repair synthesis. It is suggested that the effect of tumor promoters on DNA repair synthesis is part of a general response to cellular injury rather than a selective response involving a single metabolic pathway. Furthermore, it is unlikely that the inhibition of repair synthesis represents the major mode of action of promoting agents in the carcinogenic process.

Acetoxyacetylaminofluorene↗