Biomedical subjects
Publications and source records attributed to D Hunting.
Electron-stimulated desorption of anions from thin films of linear and supercoiled DNA is investigated in the range 3-20 eV. Resonant structures are observed with maxima at 9.4+/-0.3, 9.2+/-0.3, and 9.2+/-0.3 eV, respectively, in the yield dependence of H-, O-, and OH- on the incident electron energy. Their formation is attributed to dissociative electron attachment.
We report direct measurements of the formation of single-, double- and multiple strand breaks in pure plasmid DNA as a function of exposure to 10-50 eV electrons. The effective cross sections to produce these different types of DNA strand breaks were determined and were found to range from approximately 10(-17) to 3 x 10(-15) cm(2). The total effective cross section and the effective range for destruction of supercoiled DNA extend from 3.4 to 4.4 x 10(-15) cm(2) and 12 to 14 nm, respectively, over the range 10-50 eV. The variation of the effective cross sections with electron energy is discussed in terms of the electron's inelastic mean free path, penetration depth, and dissociation mechanisms, including resonant electron capture; the latter is found to dominate the effective cross sections for single- and double-strand breaks at 10 eV. The most striking observations are that (1) supercoiled DNA is approximately one order of magnitude more sensitive to the formation of double-strand breaks by low-energy electrons than is relaxed circular DNA, and (2) the dependence of the effective cross sections on the incident electron energy is unrelated to the corresponding ionization cross sections. This finding suggests that the traditional notion that radiobiological damage is related to the number of ionization events would not apply at very low energies.
Most of the energy deposited in cells by ionizing radiation is channeled into the production of abundant free secondary electrons with ballistic energies between 1 and 20 electron volts. Here it is shown that reactions of such electrons, even at energies well below ionization thresholds, induce substantial yields of single- and double-strand breaks in DNA, which are caused by rapid decays of transient molecular resonances localized on the DNA's basic components. This finding presents a fundamental challenge to the traditional notion that genotoxic damage by secondary electrons can only occur at energies above the onset of ionization, or upon solvation when they become a slowly reacting chemical species.
PURPOSE: To investigate the induction of DNA strand breaks by electrons with energies ranging from 0.1 to 1.5 keV. MATERIALS AND METHODS: Dry supercoiled plasmid DNA was irradiated with electrons of energies ranging from 0.1 to 1.5 keV and the results were compared with those obtained by gamma-irradiation of the same plasmid in solution. For electron irradiation, the plasmid was deposited on a gold substrate under a controlled atmosphere to minimize contamination of the DNA film. Electron bombardments were performed under ultra-high vacuum conditions (UHV 10(-9) torr). DNA damage was detected by gel electrophoresis followed by quantitation of the DNA bands by fluorescence or by hybridization with a radioactive probe. RESULTS: Electrons with energies from 0.1 to 1.5 keV induced single, double and multiple double-strand breaks in supercoiled plasmid DNA. For equal doses, we observed a marked increase in the efficiency of induction of double- and multiple-strand breaks in supercoiled DNA as a function of electron energy. In contrast to gamma-irradiation, the formation of small DNA fragments by electrons did not seem to be related to the production of the linear form of the plasmid. CONCLUSIONS: Electrons within the energy; range of the secondary electrons generated by high-energy ionizing radiation induce single, double and multiple double-strand breaks in DNA. Problems associated with low-energy electron irradiation experiments and dose calculations in thin films are also discussed.
UV irradiation induces the dimerization of synthetic single-stranded, 80-mer oligonucleotides with self-complementary, alternating purine-pyrimidine sequences, and terminal 5'- and 3'-thymines; this process can be reversed by photoreactivation. The UV-induced 160-mers are sensitive to digestion by the restriction enzyme SnaBI, but monomers are insensitive to digestion, indicating that UV irradiation stabilizes the formation of double-stranded DNA. These results suggest that UV irradiation of these 80-mer oligonucleotide substrates induces the formation of a novel cyclobutane thymine dimer which lacks an intradimer phosphodiester bond (CPD*). This CPD*, linking the terminal thymines of two separate 80-mer molecules, is formed in a double-stranded DNA region created by self-annealing and intermolecular hybridization of the two 80-mer strands. We have found that these UV-induced CPD* in 160-mers are sensitive to cleavage by the nucleotide excision enzyme complex UvrABC nuclease, but resistant to cleavage by the cyclobutane pyrimidine dimer-specific enzyme T4 endonuclease V. However, pretreatment of the 160-mers with ligase reverses their sensitivity to these two enzymes, significantly reducing their susceptibility to cleavage by UvrABC nuclease but dramatically increasing their susceptibility to cleavage by T4 endonuclease. The biological significance of these findings is discussed.
Development of drug resistance is a major factor that limits the effectiveness of chemotherapy treatments. In this study, we determined whether estradiol or its metabolites 2-, 4- and 16alpha-hydroxyestrone could enhance the development of methotrexate resistance in the breast carcinoma cell line, MCF-7. Cells were incubated with the estrogens at a concentration of 10(-8) M for 12 cell doublings and enhancement of methotrexate resistance was measured with the Luria-Delbrück assay. The most efficient estrogens were the 4-hydroxyestrone and 16alpha-hydroxyestrone, which both stimulated methotrexate resistance by 88-fold as compared with the control without estrogen. 2-Hydroxyestrone had an enhancement factor of 33-fold, whereas estradiol showed a slight effect with an enhancement factor of 3.2-fold. To determine whether the estrogen receptor was involved in the development of resistance, expression of the pS2 gene, which contains an estrogen-responsive element, was measured. Both estradiol and 16alpha-hydroxyestrone stimulated expression of the pS2 gene. In contrast, 2- and 4-hydroxyestrone did not increase the level of pS2 mRNA. This suggests that tumors classified as estrogen receptor negative could also develop methotrexate resistance as the result of exposure to estrogens. The status of the tumor suppressor gene p53 was analyzed in methotrexate sensitive and resistant clones. In all the methotrexate resistant clones analyzed, the western blots indicated that the p53 protein was still present and transcriptionally competent, as measured by its capacity to stimulate transcription of the p21waf1/cip1 gene following UVB irradiation. However, the basal level of p53 was higher in resistant clones and addition of 2- or 4-hydroxyestrone increased p53 to levels equivalent to those observed following UVB irradiation. However, this induction of p53 accumulation by estrogens failed to stimulate the transcription of p21waf1/cip1, which indicates that a transcriptionally inactive form of p53 accumulated in methotrexate resistant cells.
Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-dependent eukaryotic DNA-binding protein that specifically recognizes DNA strand breaks produced by various genotoxic agents. To study the biological function of this enzyme, we have established stable HeLa cell lines that constitutively produce the 46-kDa DNA-binding domain of human PARP (PARP-DBD), leading to the trans-dominant inhibition of resident PARP activity. As a control, a cell line was constructed, producing a point-mutated version of the DBD, which has no affinity for DNA in vitro. Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for cells treated with genotoxic agents. Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their doubling time, a G2 + M accumulation, and a marked reduction in cell survival. However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD. These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges. Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves more than its DNA-binding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.
alpha-Amanitin, an inhibitor of RNA polymerase II, has little effect on either UV-induced incision or repair synthesis in cultured normal human fibroblasts but almost completely inhibits both processes in xeroderma pigmentosum group C fibroblasts. Cycloheximide, at a concentration which inhibits protein synthesis by 75-80%, has no effect on incision or repair synthesis in either cell type, which argues that the effects of alpha-amanitin on repair occur at the level of transcription. Cot analysis demonstrates that UV-induced repair synthesis occurs at similar levels in highly repetitive, middle repetitive and single copy sequence in both normal and xeroderma group C cells. We conclude that normal cells must have at least two excision repair pathways for repair of UV-induced damage, one dependent on transcription and the other independent.
The purpose of this study was to determine if the cytotoxic effects of 6-aminonicotinamide are solely the result of an inhibition of poly(ADP-ribose) synthesis. The effects of 6-aminonicotinamide on cell growth, poly(ADP-ribose) synthesis and nucleotide concentrations were compared with the effect of 3-aminobenzamide, a more potent inhibitor of poly(ADP-ribose) synthesis. The growth of L1210 cells was not inhibited by 1 mM 3-aminobenzamide and was only slightly inhibited by 5 mM 3-aminobenzamide even though poly(ADP-ribose) synthesis, as measured by the N-methyl N-nitrosourea induced depletion of NAD+, was inhibited substantially. In contrast, 6-aminonicotinamide was found to be a potent inhibitor of L1210 and CHO cell growth. A 5 mM concentration of 3-aminobenzamide had no effect on purine and pyrimidine ribonucleotide concentrations or on the ATP to ADP ratio, but it did cause a slight elevation of NAD+. 6-Aminonicotinamide at 0.01 mM caused a depletion of purine and pyrimidine nucleotides and NAD+ as well as a reduction in the ATP to ADP ratio. 6-Aminonicotinamide at 1 mM caused a substantial inhibition of purine nucleotide synthesis from [14C] glycine but did not stimulate ATP breakdown. We conclude that inhibition of poly(ADP-ribose) synthesis caused little growth inhibition in itself and that the effects of 6-amininicotinamide on nucleotide metabolism were sufficient to produce an inhibition of both cell growth and DNA repair.
Human lymphocytes lose viability when incubated in vitro with either aphidicolin, an inhibitor of DNA polymerase alpha, or with the combination of aphidicolin and deoxycoformycin (an adenosine deaminase inhibitor). Loss of viability was assayed by vital staining with fluorescein diacetate as well as examination of Wright stained preparations and the appearance of cellular debris observed using an electronic cell counter. The loss of viability was rapid with the combination of aphidicolin (2 micrograms/ml) and deoxycoformycin (1 microgram/ml) with essentially complete loss of viability after 72 hours of incubation. This drug combination produces DNA single strand breaks after 24 and 48 hours of incubation at a level equivalent to that produced by 200 or 400R of X-irradiation, respectively.
The relationship between ribo- and deoxyribonucleotide concentrations, growth rate and cell visibility has been studied. Nucleotide pools were manipulated using mycophenolic acid, pyrazofurin, phosphonoacetyl-L-aspartate (PALA) or thymidine. Growth rate, cell viability, nigrosin exclusion, progression through the cell cycle, and the rates of nucleic acid synthesis were measured. The observed qualitative relationships between nucleotide concentrations and growth rate can be rationalized in terms of the effects of changes in substrate availability on nucleic acid synthesis. However, there is no discernible relationship between changes in nucleotide concentrations and the loss of cell viability. More detailed studies will be required to elucidate each step in the complex process leading from the initial changes in nucleotide concentrations to cell death.
Enzymological studies have shown that ribonucleotide reductase is allosterically inhibited or stimulated by endproduct deoxyribonucleoside triphosphates, and various investigators have put forward schematized models of the regulation of this enzyme; the various models have in practice been considered to be equivalent or at least very similar. Here each of eleven published models is critically examined, and it is shown that there are important differences among them that have not previously been appreciated. In addition, studies using intact mammalian cells have generally been taken as being consistent with the enzymological data, but this point has not received much systematic attention. The available data are examined critically, and the implications of multiple, partially different models are considered.
There are more than twenty appreciably different deoxyribonucleoside triphosphate assays using DNA polymerase in the literature. Therefore, each aspect of this method has been critically evaluated, including the purity of the substrates and of DNA polymerase, the reaction conditions, product isolation, and the effect of cell extracts on the assay. The final method uses a phosphatase-free DNA polymerase preparation, 2'-dAMP to inhibit the 3' leads to 5' exonuclease of DNA polymerase I, and includes a correction of both the background incorporation and the sample incorporation for dilution of the specific activity of the radioactive precursor by the sample. The sensitivity, range, accuracy, and reproducibility are reported as well as values for the deoxyribonucleoside triphosphate content of Chinese hamster ovary K-1 cells.
The effect of changes in ribonucleoside triphosphate concentrations on those of deoxyribonucleotides has been determined in Chinese hamster ovary K-1 cells. Ribonucleoside triphosphate concentrations were manipulated by the use of drugs or naturally occurring purines or pyrimidines. Changes in the concentration of each ribonucleoside triphosphate led to changes in the concentration of each corresponding deoxyribonucleoside triphosphate. The relationships between the concentrations of CTP and dCTP and between those of GTP and dGTP were linear over a wide range. The relationship between the concentrations of ATP and dATP was linear over the narrow range studied, while the relationship between UTP and dTTP concentrations was curvilinear. Overall, these results demonstrate that ribonucleotides play an important role in controlling deoxyribonucleotide concentrations but the exact mechanism of this control remains to be determined.
The ribonucleotide reductase model of Thelander and Reichard, which is based on studies of the purified enzyme, has been tested in cultured Chinese hamster ovary K-1 cells. Deoxyribonucleoside triphosphate concentrations were manipulated by the use of drugs or naturally occurring deoxyribonucleosides, and ribo- and deoxyribo-nucleoside triphosphate concentrations were measured. The results are consistent with the hypothesis that dTTP inhibits CDP reduction and stimulates GDP reduction, and that dGTP inhibits CDP reduction. However, the results are contrary to the hypothesis that dGTP inhibits UDP reduction and stimulates ADP reduction, and that dATP inhibits CDP reduction. It is concluded that the model of Thelander and Reichard applies only in part to intact CHO cells.
A detailed quantitative study of purine and pyrimidine metabolism in logarithmically growing Chinese hamster ovary cells has been performed. Direct measurements of the ribonucleoside and deoxyribonucleoside triphosphate pool sizes have been made, and the pathways and rates of metabolism of adenine, hypoxanthine, guanine, guanosine, deoxyadenosine, and deoxyguanosine and of uridine, cytidine, deoxycytidine, deoxyuridine, and thymidine have been determined.
Explore the source record for details and available documents.
Explore the source record for details and available documents.