Photodynamic mutagenic action of acridine compounds on yeast Saccharomyces cerevisiae

The photodynamically produced mutagenicity and toxicity of 8 acridine compounds were compared in Saccharomyces cerevisiae under resting and growing conditions. Without irradiation none of the acridines induced respiratory-deficient ('petite') colonies, indicative of mitochondrial DNA damage, in resting cells; and only acriflavine and proflavine induced 'petites' in growing cells. Also, without irradiation none of the acridines were significantly toxic or mutagenic for nuclear DNA under resting or growing conditions. However, with irradiation, acriflavine, proflavine, acridine yellow and rivanol became effective 'petite'-inducing mutagens and highly toxic for resting cells, while acriflavine, proflavine, and acridine orange became effective nuclear mutagens for resting cells. Acridine, quinacrine and 9-aminoacridine were not at all biologically effective with irradiation for resting cells. The results presented here indicate that singlet oxygen is generated by a photodynamic mechanism when acriflavine is irradiated, and further, that acridine, quinacrine and 9-aminoacridine are ineffective photosensitizers, because they are incapable of generating singlet oxygen with irradiation.     

Some mechanisms involved in the radiosensitization of E. coli B/r by paracetamol.

Paracetamol, a widely-used analgestic and antipyretic drug, sensitized E. coli B/r to 60Co gamma-rays under hypoxic conditions. Part of the sensitizing effect has been shown to be due to an electron adduct of the drug. Paracetamol inhibited both post-irradiation DNA and protein syntheses. The targets involved in the inhibition of post-irradiation DNA synthesis have been shown to be different in the presence of the sensitizer. Increased DNA degradation after irradiation was also observed when E. coli B/r were irradiated in the presence of the drug. The presence of paracetamol during hypoxic irradiation of E. coli B/r resulted in the enhancement of DNA single-strand scissions with no apparent effect on their rejoining.     

The inhibition of ribonucleic acid polymerase by acridines

1. The aminoacridines, proflavine (3,6-diaminoacridine) and 9-aminoacridine, and a hydrogenated derivative, 9-amino-1,2,3,4-tetrahydroacridine, were shown to inhibit in vitro the DNA-primed RNA polymerase of Escherichia coli. The inhibition is strong with both proflavine and 9-aminoacridine, but weak with 9-amino-1,2,3,4-tetrahydroacridine. 2. The extent to which the three acridines bind to calf-thymus DNA in the enzyme medium was studied spectrophotometrically. The extent of binding decreases in the order: proflavine, 9-aminoacridine, 9-amino-1,2,3,4-tetrahydroacridine. Some evidence was also obtained for interaction between the nucleoside triphosphate substrates and proflavine or 9-aminoacridine; no such interaction was detectable with 9-amino-1,2,3,4-tetrahydroacridine. 3. Although the amount of acridine bound to DNA increases with increasing inhibition, a stage is reached where an increase in acridine concentration still causes an increase in inhibition, with practically no increase in the amount bound to DNA. 4. Plots of reciprocal rates against the reciprocal of DNA concentration were linear and had a common intercept when proflavine or 9-aminoacridine was present. Similar relations were obtained when the reciprocal concentration of nucleoside triphosphates was plotted. The observations are interpreted kinetically in terms of a competitive inhibition of the enzyme by proflavine or 9-aminoacridine and of a kinetic role for the DNA analogous to ;activation'. 5. This suggests that inhibitory acridine molecules can occupy the sites on the RNA polymerase that are specific for binding the nucleoside triphosphate substrate or the bases of the DNA, when these become accessible during the copying process.     

Features of the damage produced by proflavine on transforming deoxyribonucleic acid.

Proflavine formed a complex with transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae, with optimal formation at a ratio of proflavine to DNA of 0.06. The rate of dissociation of the complex by dialysis increased in the order: native, denatured, renatured DNA. The transforming activity of the DNA was reduced by its interaction with proflavine. This inactivation was dependent on the physical state of the DNA, the proflavine concentration, and the temperature. DNA that had been denatured and renatured was most sensitive; native DNA was much less sensitive. The inactivation remained after dialysis and was stable to prolonged storage. It is concluded that the inactivation of transforming DNA by proflavine takes place by a mechanism different from that of DNA-proflavine complex formation.     

Studies of electron migration in DNA in aqueous solution using intercalating dyes.

The reactions of the hydrated electron (e-aq) and of the hydroxyl radical (OH) with double-stranded DNA in aqueous solution at room temperature have been studied through the use of the intercalating dyes, proflavine and ethidium. These dyes react with e-aq with rate constants of (2.5 +/- 0.2) - 10(10) M-1 - s-1 and (3.0 +/- 0.3) - 10(10) M-1 - s-1, respectively; the rate constant for the reaction of OH with proflavine is (1.0 +/- 0.2) - 10(10) M-1 - s-1. When these molecules are bound within the DNA structure both the yields and the rate constants of reaction with e-aq are reduced in a manner entirely consistent with a simple competition between the DNA bases and restricted dye molecules reacting with a bimolecular rate constant of about 2 - 10(9) M-1 - s-1. No evidence of free electron migration in the DNA was obtained, and an upper limit of five base pairs for the range of such migration was derived. Reactions of the hydroxyl radical with DNA-bound proflavine also lead to a rate constant of about 2 - 10(9) M-1 - s-1. These rate constants are in good agreement with rate predictions (per base unit) for a diffusion-controlled reaction with the DNA structure.     

Terminations of DNA synthesis on 'proflavine and light'-treated phi X174 single-stranded DNA

Bacteriophage phi X174 single-stranded DNA molecules were primed with five different restriction fragments and irradiated with visible light in the presence of proflavine. This photodamaged DNA was used as template for the in vitro complementary chain synthesis by E. coli DNA polymerase I (Klenow fragment). Chain terminations were observed by polyacrylamide gel electrophoresis of the synthesized products and localized by comparison with standard sequencing performed simultaneously on the untreated template. 90% of the chain terminations occurred one nucleotide before a guanine residue in the template strand. More than 80% of the sequenced guanine residues were blocking lesions demonstrating the absence of 'hot-spots' for the photodamaging effect of proflavine. At a defined position, the chain termination frequency increased linearly with the irradiation time and was directly influenced by the proflavine concentration present. An important part of lesions resulted from the action of singlet oxygen produced by excited proflavine as shown by the effect that both NaN3 and 2H2O exerted on the reaction. The induced blocking lesions must be important in vivo since no complete replicative forms could be extracted from cell infected with bacteriophages inactivated by 'proflavine and light' treatment.     

E.s.r. study of the effect of oxygen on the irradiation of polyglycine

Radiation damage in polycrystalline polyglycine irradiated under different partial pressures of oxygen has been investigated by e.s.r. spectroscopy. Although the general form of the e.s.r. spectrum remained unaltered as compared to irradiation under vacuum, a decrease in the intensity of the e.s.r. signal was observed. The results were analysed in correlation with Alper's formula. A correspondence between free radical transformation into nonradical species in the presence of oxygen and the biological centres of damage is suggested.     

Extrinsic cotton effects of aminoacridines bound to DNA

The optical rotatory dispersion curves of the proflavine cation were measured in the spectral range 400–500 mμ. No optical activity was observed for the free cation but a large positive Cotton effect appeared in the presence of DNA. The effect of ionic strength, denaturation of the DNA, and the DNA/proflavine ratio were studied. The dependence of the magnitude of the Cotton effect on the DNA/proflavine ratio suggests that a nearest-neighbor interaction between bound proflavine molecules is necessary for optical activity. A simple statistical treatment was made which indicated that only a small number of proflavine molecules are required in close proximity for optical activity to occur. Denaturation of the DNA did not destroy the optical activity, which shows that long runs of DNA double helix are not necessary for optical activity of the ligand molecules. The optical rotatory dispersion curves of acridine orange which was bound to DNA were also measured. Two Cotton effects of opposite sense could be distinguished, the relative magnitudes of which depended on the DNA/acridine orange ratio and the state of denaturation of the DNA. The apparent differences from the proflavine-DNA system can to a large extent be explained in terms of the tendency of acridine orange to form aggregates.     

Identification of the Block in the Intracellular Replication of Single-Stranded DNA of Photodynamically Inactivated Bacteriophage φX174

(32)P-labeled single-stranded DNA phage phiX174 was photodynamically inactivated by irradiation in air with visible light in the presence of the acridine dye, proflavine sulfate. The inactivated phages could adsorb to the host cells but failed to lyse them. Formation of intracellular mature phages was almost completely inhibited. Photodynamic lesions in phiX174 DNA caused intracellular formation of defective double-stranded replicative form molecules which ultimately reverted to the single-stranded configuration.     

Dielectric behavior of DNA-proflavine complex

The dielectric relaxation of namtive DNA and DNA–proflavine complexes at different DNA phosphate (P) to dye (D) ratios, were investigated in the frequency range 100 c/sec to 100 Kc/sec. The proflavine molecules were found to have a profound effect on the static dielectric constant and the relaxation time of the polymers. The static dielectric constant was oberserved to decrese with increasing level of added proflavine. At P/D = 1, the variation of dielectric constant with frequency was small. Relaxation time (τ) was greater for the DNA–proflavine complexes compared to that for free DNA, Maximum value of the relaxation time was obtained at P/D = 10. The increase in the relaxation time and decrease in the static dielectric constant were attributed to the increase in length and meutralization of surface charges of the DNA molecules, respectively, as aresult of proflavine binding.     

Mutation induction by heavy ion irradiation of gpt delta transgenic mice.

Using the new transgenic mice produced by mating gpt delta with p53 knockout, mutation induction by heavy-ion irradiation and the effect of p53 background on such induction were studied. After the whole body irradiation with 10 Gy of 135 MeV/u carbon-ion beam, the genomic DNA was isolated from the different organs and the lambda DNA was rescued as a lambda phage. Mutations in the transgene on the lambda DNA were determined by the spi(-) selection (deletion assay). The spi(-) mutation was induced by the above irradiation, but enhancement of the mutant frequency by the knockout of p53 gene was found not in the phages recovered from liver but in those from kidney. We are now making an effort to determine the nature of spi(-) mutation to confirm such p53 effect.     

Interaction of the C-terminal region of p105 with the nuclear localisation signal of p50 is required for inhibition of NF-kappa B DNA binding activity.

DNA binding of the homodimeric p50 subunit of NF-kappa B was inhibited by a bacterially expressed protein containing the ankyrin repeats present in the C-terminus of the p105 precursor but not by the I kappa B protein MAD-3. However p50 was retained on protein affinity matrices containing either the C-terminal ankyrin repeats of p105 or MAD-3. To investigate the interaction between p50 and proteins containing ankyrin repeats we have used a number of approaches to probe the accessibility of the p50 nuclear localisation signal in the protein complex. A monoclonal antibody recognising a linear epitope either very close to, or including, the nuclear localisation signal of the p50 protein could immunoprecipitate p50 homodimers but was unable to precipitate the protein when it was bound to the C-terminal region of p105. A close association between the nuclear localisation signal of p50 and the C-terminal region of p105 was also suggested by protease accessibility experiments. While the nuclear localisation signal of free p50 is extremely susceptible to cleavage with trypsin the same site is masked in the presence of the C-terminal ankyrin repeats of p105 and, to a lesser extent MAD-3. Removal of the nuclear localisation signal by trypsin digestion generates a protein that is fully competent for DNA binding but is refractile to inhibition by the C-terminal ankyrin repeats of p105. Addition of DNA destabilises complexes between p50 and ankyrin repeat containing proteins, increasing the susceptibility of the nuclear localisation signal to trypsin cleavage. The data suggest that there is a rapid exchange of p50 between complexes containing DNA or I kappa B proteins via a metastable complex containing DNA, p50 and I kappa B.     

Stimulatory and inhibitory effects of 1,25-dihydroxyvitamin D3 on thymocyte mitogenesis induced by phorbol ester and calcium ionophore.

Mouse medullary thymocytes have specific receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The mitogenic stimulation of these cells by phytohemagglutinin in the presence or absence of the phorbol ester TPA is inhibited by 1,25(OH)2D3. The calcium ionophore A23187 did not reverse the inhibition by 1,25(OH)2D3 of phytohemagglutinin. Stimulation of thymocytes with either TPA or A23187 alone did not result in proliferation. Co-stimulation of the thymocytes with TPA and A23187 induces cell proliferation. 1,25(OH)2D3 markedly enhanced the TPA and A23187-induced cell proliferation even when added 4 h after the initiation of the culture. In contrast, DNA synthesis by thymocytes incubated for 4 h in the presence of TPA and A23187 and then cultured in medium containing 1,25(OH)2D3 but in the absence of both TPA and A23187, was inhibited by 1,25(OH)2D3. The extent of inhibition was comparable to the inhibition of lectin-induced stimulation by the hormone. Using monoclonal antibodies to neutralize IL-2 and block IL-2 receptors we showed that 1,25(OH)2D3 enhanced the IL-2-independent component of the A23187- and TPA-induced mitogenesis. In conclusion: (1) The nature and presence of the mitogenic signal determines whether 1,25(OH)2D3 enhances or inhibits thymocyte stimulation. (2) Both stimulatory and inhibitory actions of 1,25(OH)2D3 seem to take place at points distal to the initial increase in intracellular calcium or activation of protein kinase C.     

Radiation-induced DNA double-stranded breaks and the dynamics of apoptotic death of human peripheral lymphocytes

DNA double-stranded breaks and their association with the development of radiation-induced peripheral lymphocyte apoptosis were studied in healthy donors exposed to in vitro gamma-irradiation in a dose of 1 Gy. It was shown that irradiation in 1-Gy dose caused a significant (p < 0.05) increase in the frequency of cells in late apoptosis 4 hours after irradiation and a rise in their frequency in early apoptosis 24 hours following this procedure. A significant correlation (r = 0.52, p < 0.05) was recorded between the primary level of radiation-induced DNA double-stranded breaks and the frequency of cells in late apoptosis following 4 hours, which suggests that DNA double-stranded breaks as a signal to trigger cell apoptotic death are of great importance.     

Photodynamic activity of dyes with different DNA binding properties II. T4 phage inactivation.

The photosensitizing efficiency of six dyes--proflavine, 9-aminoacridine, ethidium bromide, thiopyronine, pyronine and acridine red--have been compared on the basis of the inactivation of sensitized T4 phage caused by light irradiation. This reaction was only measurable after diffusion of the dye through the phage capsid and was not observed in the presence of either chloroquine or quinacrine; it followed a single-hit kinetics as a function of the irradiation time. With each dye, a double reciprocal plot of the inactivation constant versus the dye concentration present gave rise to a linear relationship. From this relation, parameters were deduced which expressed the relative photosensitizing efficiencies. Dye-binding to the phages was measured and the proflavine-mediated inactivation appeared to be related to the amount of strongly bound molecules. Such a conclusion could not be reached in the case of 9-aminoacridine and ethidium bromide, which were much less efficient photosensitizers than proflavine, but which were also strongly bound to the phages. Thiopyronine was weakly bound to the phages; it had, however, the highest photosensitizing activity observed. These results indicate that various mechanisms are involved when the phage photosensitization is due to one dye or another.     

Mechanism of radiosensitizing effect of chloride ion on E. coli

Cells of E. coli capable of repairing DNA damage are sensitized to radiation in the presence of NaCl. However, the enhanced radiolethality was suppressed by the addition of compounds such as an amino acid to the irradiation buffer. The protective efficiencies of these compounds depend on their reactivities with Cl-.2 or OH.. ATP synthesis in the cells irradiated in the presence of NaCl was severely inhibited depending on the dose of irradiation. This reduced rate of ATP synthesis can account for the inhibition of protein, RNA and DNA synthesis in the irradiated cells with NaCl.     

A comparison between rates of cell death and DNA damage during irradiation of Saccharomyces cerevisiae in N2 and N2O

Yeast and several other organisms are more sensitive to the lethal effects of ionizing irradiation if exposed in the presence of N2O as compared to N2. It has been suggested that this increased sensitivity is due to the cooperative effects of OH and H2O2 generated external to the cell wall. Using diploid yeast, wild type for radiation resistance, we have compared the rates of cell death due to gamma irradiation in N2 and N2O with the rates of DNA damage measured by gene conversion of trp- to trp+ (a recombinational repair event). We find that DNA damage as measured by gene conversion increases at a faster rate, per unit dose, during irradiation in N2O as compared to N2, just as lethality was higher in N2O. When DNA damage was compared in N2 and N2O at equal levels of survival, however, there was no significant difference between the two irradiation conditions. Therefore, increased lethality during irradiation in N2O seems to be directly due to increased DNA damage. If the observed increased lethality results from external OH and H2O2, the effect of these highly reactive species is expressed by increased internal damage at the level of DNA.     

Hyperactivation of ATM upon DNA-PKcs inhibition modulates p53 dynamics and cell fate in response to DNA damage

A functional DNA damage response is essential for maintaining genome integrity in the presence of DNA double-strand breaks. It is mainly coordinated by the kinases ATM, ATR, and DNA-PKcs, which control the repair of broken DNA strands and relay the damage signal to the tumor suppressor p53 to induce cell cycle arrest, apoptosis, or senescence. Although many functions of the individual kinases have been identified, it remains unclear how they act in concert to ensure faithful processing of the damage signal. Using specific inhibitors and quantitative analysis at the single-cell level, we systematically characterize the contribution of each kinase for regulating p53 activity. Our results reveal a new regulatory interplay in which loss of DNA-PKcs function leads to hyperactivation of ATM and amplification of the p53 response, sensitizing cells for damage-induced senescence. This interplay determines the outcome of treatment regimens combining irradiation with DNA-PKcs inhibitors in a p53-dependent manner.     

Extrinsic Cotton effects of proflavine bound to polynucleotides

The magnitude of the Cotton effect of proflavine which is bound to RNA or to denatured DNA depends on the ratio of bound proflavine to nucleic acid base. A statistical treatment which explains this behavior has been fitted to the experimental curves and indicates that optical activity arises through interaction between two or more bound proflavine molecules. The corresponding requirement with double helical DNA is for interaction between 3–4 proflavine molecules. Although proflavine binds to denatured DNA at pH 2.8, as shown by the shift of the proflavine spectrum, the strong binding process is absent, and to this is attributed the absence of the Cotton effect at low pH. Studies on the Cotton effects of proflavine bound to poly A and poly U at neutral pH, to poly A at acid pH and to poly (A + U) allow the generalization that a relatively rigid configuration of the binding macromolecule is required for the induction of these extrinsic Cotton effects.     

Effect of gamma-irradiation on dye-DNA binding.

The effect of gamma-rays on the binding of proflavine and acridine orange to DNA was investigated by spectrophotometry. The effect of irradiation was observed on the buffered solutions of the free dye and free DNA. A dose of about 35 krad caused a hyperchromicity of 30-40 per cent to the DNA peak at 258 nm, while the same dose introduced a hypochromic effect to the monomer peaks of the dyes by 30 per cent. This implied that gamma-rays have an effect of decreasing the monomer concentration of free-day molecules in solution. From the results, we conclude that more dye is bound to the changed conformation of dye-bound DNA on irradiation. Scratchard-binding isotherms drawn for the unirradiated and irradiated complexes of Pf-DNA showed interesting differences. Similar isotherms could not be obtained for the acridine orange-DNA system.