Hydrated electrons induce the formation of interstrand cross-links in DNA modified by cisplatin adducts.

Double-stranded oligonucleotides containing cisplatin adducts, with and without a mismatched region, were exposed to hydrated electrons generated by gamma-rays. Gel electrophoresis analysis demonstrates the formation of cisplatin-interstrand crosslinks from the cisplatin-intrastrand species. The rate constant per base for the reaction between hydrated electrons and the double-stranded oligonucleotides with and without cisplatin containing a mismatched region was determined by pulse radiolysis to be 7 × 109 and 2 × 109 M-1 s-1, respectively. These results provide a better understanding of the radiosensitizing effect of cisplatin adducts in hypoxic tumors and of the formation of interstrand crosslinks, which are difficult for cells to repair.

Absolute cross section for DNA damage induced by low-energy (10 eV) electrons: Experimental refinements and sample characterization by AFM.

This work describes multiple experimental improvements for measuring absolute cross sections of DNA damage induced by low-energy electrons in nanometer-thick films in vacuum. Measurements of such cross sections are particularly sensitive to film thickness and uniformity. Using atomic force microscopy in 70% ethanol, we present a novel and effective method to determine plasmid DNA film thickness and uniformity that combines height histograms and force-distance curves. We also investigate film deposition with DNA intercalated with 1,3-diaminopropane (Dap) on tantalum-coated substrates as a convenient and cost-effective alternative to the previously-used graphite substrate. The tantalum substrate permits deposition of films very similar to those formed on graphite. Using these refinements and further optimizations of the experimental procedure, we measure an absolute cross section of (7.4 ± 2.3) × 10-18 cm2 per nucleotide for conformational damage to a 3197 base-pair plasmid, induced by 10 eV electrons, which we believe should be considered as a reference value.

Radiosensitization of DNA by Cisplatin Adducts Results from an Increase in the Rate Constant for the Reaction with Hydrated Electrons and Formation of Pt(I).

Pulse radiolysis measurements of the decay of hydrated electrons in solutions containing different concentrations of the oligonucleotide GTG with and without a cisplatin adduct show that the presence of a cisplatin moiety accelerates the reaction between hydrated electrons and the oligonucleotide. The rate constant of the reaction is found to be 2.23 × 10(10) mol(-1) L s(-1), which indicates that it is diffusion controlled. In addition, we show for the first time the formation of a Pt(I) intermediate as a result of the reaction of hydrated electrons with GTG-cisplatin. A putative reaction mechanism is proposed, which may form the basis of the radiosensitization of cancer cells in concomitant chemoradiation therapy with cisplatin.

Electron Resonance Decay into a Biological Function: Decrease in Viability of E. coli Transformed by Plasmid DNA Irradiated with 0.5-18 eV Electrons.

Transient negative ions (TNIs) are ubiquitous in electron-molecule scattering at low electron impact energies (0-20 eV) and are particularly effective in damaging large biomolecules. Because ionizing radiation generates mostly 0-20 eV electrons, TNIs are expected to play important roles in cell mutagenesis and death during radiotherapeutic cancer treatment, although this hypothesis has never been directly verified. Here, we measure the efficiency of transforming E. coli bacteria by inserting into the cells, pGEM-3ZfL(-) plasmid DNA that confers resistance to the antibiotic ampicillin. Before transformation, plasmids are irradiated with electrons of specific energies between 0.5 and 18 eV. The loss of transformation efficiency plotted as a function of irradiation energy reveals TNIs at 5.5 and 9.5 eV, corresponding to similar states observed in the yields of DNA double strand breaks. We show that TNIs are detectable in the electron-energy dependence of a biological process and can decrease cell viability.

Cisplatin intrastrand adducts sensitize DNA to base damage by hydrated electrons.

The oligonucleotide TTTTTGTGTTT with or without a cisplatin adduct was reacted with hydrated electrons generated by ionizing radiation. Hydroxyl radicals were quenched with ethylenediaminetetraacetic acid (EDTA), and the solutions were bubbled with wet nitrogen to eliminate oxygen, a scavenger of hydrated electrons. Prior to irradiation, the structure of the initial cisplatin adduct was identified by mass spectrometry as G-cisplatin-G. Radiation damage to DNA bases was quantified by high-performance liquid chromatography (HPLC), after enzymatic digestion of the TTTTTGTGTTT-cisplatin complex to deoxyribonucleosides. The masses of the platinum adducts following digestion and separation by HPLC were measured by mass spectrometry. Our results demonstrate that hydrated electrons induce damage to thymines as well as detachment of the cisplatin moiety from both guanines in the oligonucleotide. This detachment regenerates both unmodified guanine and damaged guanine, in equimolar amounts. At 1000 Gy, a net average of 2.5 thymines and 1 guanine are damaged for each platinum lost from the oligonucleotide. Given the extensive base damage that occurs for each cisplatin adduct lost, it is clear that, prior to undergoing detachment, these adducts must catalyze several cycles of reactions of hydrated electrons with DNA bases. It is likely that a single reaction leads to the loss of the cisplatin adduct and the damage observed on the guanine base; however, the damage to the thymine bases must require the continued presence of the cisplatin adduct, acting as a catalyst. To our knowledge, this is the first time that platinum-DNA adducts have been shown to have catalytic activity. We propose two pathways for the interaction of hydrated electrons with TTTTTGTGTTT-cisplatin: (1) the hydrated electron is initially captured by a thymine base and transferred by base to base electron hopping to the guanine site, where the cisplatin moiety detaches from the oligonucleotide via dissociative electron attachment, and (2) the hydrated electron interacts directly with the platinum-guanine adduct and induces detachment of the cisplatin moiety via dissociative electron attachment. Although the precise mechanism remains to be elucidated, our results provide important insights into the radiosensitization of DNA by cisplatin.

Non-DSB clustered DNA lesions induced by ionizing radiation are largely responsible for the loss of plasmid DNA functionality in the presence of cisplatin.

The combination of cisplatin and ionizing radiation (IR) increases cell toxicity by both enhancing DNA damage and inhibiting repair mechanisms. Although the formation of cluster DNA lesions, particularly double-strand breaks (DSB) at the site of cisplatin-DNA-adducts has been reported to induce cell death, the contribution of DSB and non-DSB cluster lesions to the cellular toxicity is still unknown. Although both lesions are toxic, it is not always possible to measure their frequency and cell survival in the same model system. To overcome this problem, here, we investigate the effect of cisplatin-adducts on the induction of DSB and non-DSB cluster DNA lesions by IR and determine the impact of such lesions on plasmid functionality. Cluster lesions are two or more lesions on opposite DNA strands with a short distance such that error free repair is difficult or impossible. At a ratio of two cisplatin per plasmid, irradiation of platinated DNA in solution with (137)Cs γ-rays shows enhancements in the formation of DNA DSB and non-DSB cluster lesions by factors of 2.6 and 2.1, respectively, compared to unmodified DNA. However, in absolute terms, the yield for non-DSB cluster lesions is far larger than that for DSB, by a factor of 26. Unmodified and cisplatin-modified DNA were irradiated and subsequently transformed into Escherichia coli to give survival curves representing the functionality of the plasmid DNA as a function of radiation dose. Our results demonstrate that non-DSB cluster lesions are the only toxic lesions present at a sufficient frequency to account for the loss of DNA functionality. Our data also show that Frank-DSB lesions are simply too infrequent to account for the loss of DNA functionality. In conclusion, non-DSB cluster DNA damage is known to be difficult to repair and is probably the lesion responsible for the loss of functionality of DNA modified by cisplatin.

Hydrated electrons react with high specificity with cisplatin bound to single-stranded DNA.

Short oligonucleotides TTTTTGTGTTT and TTTTTTTGTTT in solution with and without cisplatin (cisPt) bound to the guanine bases were irradiated with γ-rays at doses varying from 0 to 2500 Gy. To determine the effect of hydrated electrons from water radiolysis on the oligonucleotides, we quenched (•)OH radicals with ethylenediaminetetraacetic acid (EDTA) and displaced oxygen, which reacts with hydrated electrons, by bubbling the solution with wet nitrogen. DNA strand breaks and platinum detachment were quantified by gel electrophoresis. Our results demonstrate that hydrated electrons react almost exclusively at the position of the cisPt adduct, where they induce cisPt detachment from one or both guanines in the oligonucleotide. Given the high yield of hydrated electrons in irradiated tissues, this reaction may be an important step in the mechanism of radiosensitization of DNA by cisPt.

Single-strand-specific radiosensitization of DNA by bromodeoxyuridine.

The effects of bromodeoxyuridine (BrdUrd) substitution for thymidine on gamma-ray-induced strand breakage were determined in single- and double-stranded oligonucleotides and double-stranded oligonucleotides containing a mismatched bubble region. BrdUrd does not sensitize complementary double-stranded DNA to gamma-ray-induced strand breakage, but it greatly sensitizes single-stranded DNA. However, when the BrdUrd is present in a single-stranded bubble of a double-stranded oligonucleotide, the non-base-paired nucleotides adjacent to the BrdUrd as well as several unpaired sites on the opposite unsubstituted strand are strongly sensitized. The radiosensitization properties of BrdUrd result primarily from the electrophilic nature of the bromine, making it a good leaving group and leading to the irreversible formation of the uridine-yl radical (dUrd(.)) or the uridine-yl anion (dUrd(-)) upon addition of an electron. The radiolytic loss of the bromine atom is greatly suppressed in double-stranded compared to single-stranded DNA. Thus we propose that the radiosensitization effects of bromouracil in vivo will likely be limited to single-strand regions such as found in transcription bubbles, replication forks, DNA bulges and the loop region of telomeres. Our results may have profound implications for the clinical use of bromodeoxyuridine (BrdUrd) as a radiosensitizer as well as for the development of targeted radiosensitizers.

Inhibition of ultraviolet B (UVB) induced apoptosis in A431 cells by mimosine is not dependent on cell cycle arrest.

Ultraviolet (UV) radiation is a strong apoptotic trigger in many cell types. We have previously reported that a plant amino acid, mimosine (beta [N-(3-hydroxy-4-pyridone)]-alpha-aminopropionic acid), with a well-known reversible G1 cell cycle arrest activity can inhibit apoptosis induced by UV irradiation and RNA polymerase II blockage in human A431 cells. Here, apoptosis was measured with a fluorimetric caspase activation assay. Interestingly, the protective state was effective up to 24 h following removal of mimosine from the culture medium while cells were progressing in the cell cycle. Our results demonstrate that the protective effect of mimosine against UV-induced apoptosis can be dissociated from its G1 cell-cycle arrest activity.

p21-induced cycle arrest in G1 protects cells from apoptosis induced by UV-irradiation or RNA polymerase II blockage.

Cells expressing the R273H mutant of p53, which lacks sequence specific DNA binding capacity, do not undergo cell cycle arrest in G1 following exposure to ionizing or UV radiation because of their inability to induce p21Waf1/Cip1, a cyclin-dependent kinase inhibitor and downstream mediator of p53-dependent DNA damage-induced growth arrest. Following UV-irradiation or treatment with an inhibitor of RNA pol II, we observed a rapid induction of the apoptotic process, as evidenced by DNA fragmentation and the proteolytic cleavage of poly(ADP-ribose) polymerase. Using mimosine, a p21Waf1/Cip1 inducer that bypasses the requirement for transcriptional transactivation by p53, we demonstrated that a G1 cell cycle arrest can prevent apoptosis following UV-irradiation or treatment with an RNA polymerase 11 inhibitor. Serum starvation, which also synchronized cells in G1 but did not induce p21Waf1/Cip1, did not protect cells from apoptosis. These results demonstrate that restoring a late G1 checkpoint by inducing p21Waf1/Cip1 expression can protect cells from DNA damage induced apoptosis. Our results suggest that p21Waf1/Cip1 can interrupt the apoptotic process at a point downstream from p53 accumulation but upstream from caspase-3 activation.

The ortho-quinone metabolite of the anticancer drug etoposide (VP-16) is a potent inhibitor of the topoisomerase II/DNA cleavable complex.

Epipodophyllotoxin derivatives, such as etoposide (VP-16), constitute an important class of anticancer agents, the major cytotoxic effects of which are associated with trapping of the topoisomerase II/DNA cleavable complex and formation of protein-DNA cross-links and nicked DNA. VP-16, however, can be metabolized to several highly reactive products, including an ortho-quinone (VPQ). The inhibitory activity of VPQ against purified human topoisomerase II processing of supercoiled DNA was studied and compared with that of the parent compound, VP-16. Our results show that VPQ is a powerful inhibitor of topoisomerase II, which prevents DNA strand passage in the presence of ATP. As with VP-16, trapping of the cleavable complex is highly reversible upon removal of divalent ions, which indicating that VPQ alters the cleavage-reunion equilibrium of topoisomerase II and DNA mainly by noncovalent interactions, as does the parent compound. However, we observed several differences between the effects induced by VP-16 and VPQ, including a strong inhibition of the second DNA strand religation, which implies the involvement of additional (asymmetric) mode(s) of interactions of the VPQ, possibly by interference with ATP binding by the homodimeric enzyme, and/or involving covalent interactions. Reduced or oxidized glutathione prevented trapping of the topoisomerase/DNA cleavable complex by VPQ, but not by VP-16, probably by forming covalent adducts with the former.

Inhibition of the topoisomerase II-DNA cleavable complex by the ortho-quinone derivative of the antitumor drug etoposide (VP-16).

Etoposide (VP-16) is a widely used anticancer drug whose toxicity involves poisoning of topoisomerase II. VP-16 undergoes enzymatic oxido-reductive transformations in cells, resulting in the formation of the ortho-quinone derivative (VPQ) as a major product. The actions of VP-16 and VPQ on purified human topoisomerase II have been compared. Both the parent drug and VPQ are very efficient at trapping the topoisomerase II-DNA cleavable complex, suggesting that methoxy groups on the E-ring are not a prerequisite for activity. Our data also imply that VPQ has more effect than VP-16 on the breakage-reunion equilibrium of topoisomerase II and DNA. The stronger inhibition of the religation of the second strand observed with VPQ suggests it interacts asymmetrically with the two homodimers of topoisomerase II bound to DNA.

Enhancement of etoposide (VP-16) cytotoxicity by enzymatic and photodynamically induced oxidative stress.

The effects of glutathione (GSH) depletion by buthionine sulfoximane (BSO) or by photosensitization-induced oxidative stress using metallo-phthalocyanines (MePcS4) on etoposide (VP-16) cytotoxicity against K562 human leukemic cells were investigated. Both treatments enhanced VP-16 toxicity in a markedly synergistic way, as revealed by combination index analysis procedure. Synergistic drug interactions were accompanied by a supra-additive induction of DNA strand breaks. The proposed role of intracellular GSH in preventing metabolic transformations of VP-16 and thus decreasing its toxicity was confirmed by electron spin resonance (ESR) monitoring of the accumulation of the VP-16 phenoxyl radical in cell cytoplasm subjected to GSH depletion. Taken together the results emphasize the beneficial effect of GSH-related oxidative stress in enhancement of etoposide toxicity and possibly in its anticancer applications.

The apoptotic and transcriptional transactivation activities of p53 can be dissociated.

Previous studies have shown that the apoptotic response of cells following DNA damage requires p53 expression. Wild-type p53 protein levels increase in response to DNA damage and its growth-suppressive action is thought to be mediated by transcriptional activation of the p21/WAF1/CIP1 gene, the product of which is a potent inhibitor of cyclin-dependent kinases. The mechanism by which elevated p53 levels lead to apoptosis is not known, but is believed to involve transcriptional activation of apoptotic genes, such as BAX. We have studied transformed human cells that constitutively express high levels of the R273H mutant p53, which has been reported to lack transcriptional activation activity. We used the inability to induce the p21/Waf1/Cip1 protein as a marker to verify the lack of transcriptional activation activity. Cells expressing the R273H mutant of p53 do not show an increase in p21/Waf1/Cip1 following irradiation with ionizing or UVB radiation. Surprisingly, these cells are very susceptible to induction of apoptosis by UVB radiation, as seen by the formation of a nucleosomal ladder and the proteolytic cleavage of poly(ADP-ribose) polymerase. This suggests that the R273 mutant p53 can function normally in apoptosis but not in transcriptional activation following DNA damage. Furthermore, an inhibitor of RNA polymerase II is a potent inducer of apoptosis in these cells, demonstrating that transcription is not required for apoptosis and suggesting that stalled RNA polymerase II complexes can initiate apoptosis. Interestingly, proteolytic cleavage of p53 occurs during apoptosis in these cells, generating a 45-kDa fragment and liberating the DNA repair helicase binding domain of p53. We propose that the peptide liberated from the carboxy terminus of p53 may contribute to its apoptotic activity, possibly through interaction with the XPB and XPD DNA helicases.

Incorporation of two deoxycytidine oxidation products into cellular DNA.

The oxidation of cytosine in DNA by free radicals and other oxidants leads to an assortment of products including pyrimidine ring 5,6-saturated, 5,6-unsaturated, contraction, and fragmentation products. The formation of these products in cellular DNA may explain in part the preponderance of C to T transitions induced spontaneously and by H2O2 or ionizing radiation. Our studies have focused on the biological effects of two major 5,6-unsaturated oxidation products of cytosine: 5- hydroxycytosine and 5-hydroxyuracil. In the present work, we have attempted to study the repair of these two lesions by specifically incorporating them into cellular DNA upon incubation of cells with 5-hydroxy-2'deoxycytidine and 5-hydroxy-2'-deoxyuridine. Incubation of mouse L1210 cells with 250 M 5-hydroxy-2'-deoxycytidine led to the incorporation of this lesion to a level 20 times higher (43 lesions/10(5) cytosines) than base-line levels; however, there was no evidence for its repair following a 15-h chase. In contrast, we did not observe any significant incorporation of 5-hydroxy-2'-deoxyuridine into the DNA of L1210 cells but did observe an unidentified product, presumably an oxidation product. This unidentified pyrimidine was incorporated at a very high level (about 2000 lesions/10(5) cytosine residues) and then partially repaired in chase experiments.

DNA strand scission and base release photosensitized by metallo-phthalocyanines.

Photoirradiation of aqueous solutions of DNA in the presence of Al- or Zn-tetrasulphonated phthalocyanines (AIPcS4 and ZnPcS4) causes formation of strand breaks and liberation of nucleobases. The effect of added D2O, which enhances singlet oxygen (1O2) lifetime, radical scavengers including alcohols and the spin-trap DMPO, as well as superoxide dismutase, indicates that both singlet oxygen (1O2) and free radicals contribute to the production of strand breaks. However, in the case of base release, only free radicals, such as the hydroxyl radical (.OH), appear to be involved in the degradation process. Detection of the characteristic free-radical oxidation products of deoxyribose provides evidence that .OH are involved in the photosensitized DNA damage. EPR and spin trapping data suggest that superoxide (O2.-) is the most likely precursor of .OH and a Fenton-type mechanism is proposed for their formation.

Photocytotoxicity and intracellular generation of free radicals by tetrasulphonated Al- and Zn-phthalocyanines.

The photosensitizing properties of tetrasulphonated Al- and Zn-phthalocyanines (AlPcS4 and ZnPcS4) in lymphoma cells were studied as a function of the pre/post-illumination incubation time. Photocytotoxicity increased with incubation time, ranging from a transient cell-cycle arrest to cell killing. Under all experimental conditions, the phototoxicity of ZnPcS4 was markedly higher than that of AlPcS4. The primary photoprocesses initiated by metallo-phthalocyanines (MePcS4) in the cells were probed with DMPO/esr spin-trapping techniques. Under all incubation conditions the intracellularly bound MePcS4 sensitized formation of three different types of DMPO spin-adducts: DMPO/OH (hydroxyl radical), DMPO/R (organic carbon-centred radical(s)) and an unidentified simple nitroxyl, referred to as DMPO/ox. The yields of trapped radicals depended on the length of the incubation with the dyes prior to illumination and the formation of spin-adducts was shown to be intracellular. The ability of DMPO to protect cells from the photocytotoxic effects of Al- and ZnPcS4, combined with the generation of carbon-centred spin-adducts is direct evidence for the involvement of free-radical-mediated damage of cellular constituents.

Purification of DNA fragments containing excision-repair patches from human cells using streptavidin-biotin.

We have developed a method for purifying DNA fragments containing excision-repair patches which involves incorporation of biotinated deoxyuridine monophosphate into repair patches followed by isolation of biotin-containing DNA fragments using streptavidin and either isopycnic density gradient centrifugation or gel electrophoresis. Normal human fibroblasts were damaged with UV radiation, rendered permeable and allowed to perform repair synthesis in the presence of ATP, dATP, dGTP, [3H]dCTP and biotinated deoxyuridine triphosphate. The DNA was purified, sonicated to a number-average molecular weight of 150 bp, then incubated with streptavidin, a protein with a high affinity for biotin and with a density of 1.3 g/ml in cesium trifluoroacetate compared to 1.6 g/ml for DNA. Isopycnic centrifugation in cesium trifluoroacetate resulted in the separation of the streptavidin-DNA complex with little or no dissociation. The streptavidin-DNA complex was also separated from free DNA by electrophoresis in 2% agarose. This method is applicable to any type DNA damage repaired by the excision repair pathways in which thymine is present in the repair patches, including damage from chemical carcinogens and ionizing radiation.

DNA polymerase delta mediates excision repair in growing cells damaged with ultraviolet radiation.

In confluent, stationary phase cells, an aphidicolin-sensitive DNA polymerase mediates UV-induced excision repair, but the situation in growing cells is still controversial. The sensitivity of repair synthesis to aphidicolin, an inhibitor of DNA polymerases alpha and delta, was determined in growth phase and confluent normal human fibroblasts (AG1518) using several techniques. Repair synthesis in confluent cells was always inhibited by aphidicolin, no matter which measurement technique was used. However, the inhibition of repair synthesis in growth-phase cells by aphidicolin was only detectable when techniques unaffected by changes in nucleotide metabolism were used. We conclude that UV-induced repair synthesis in growing cells is actually aphidicolin sensitive, but that this inhibition can be obscured by changes in nucleotide metabolism. Employing butylphenyl-deoxyguanosine triphosphate, a potent inhibitor of polymerase alpha and a weak inhibitor of delta, we have obtained evidence that polymerase delta is responsible for repair synthesis in growth-phase cells following UV irradiation.

Reactive oxygen production against malaria--a potential cancer risk factor.

In response to malaria infection, phagocytes, such as macro-phages and neutrophils, produce superoxide and thence the other reactive oxygen species (ROS) with which to kill the parasites. Excess ROS is normally eliminated by the body's natural scavenger molecules; however, in the event of a vast excess of ROS, as may be the case in acute as well as chronic malaria patients, the natural scavengers may be overwhelmed. We hypothesize that unscavenged ROS in malaria patients causes DNA damage in normal host cells which, if unrepaired or incorrectly repaired, could result in oncogene activation and eventually lead to cancer. An epidemiologic study may be warranted in malaria-endemic regions to investigate the possible relationship between malaria infection and cancer risk.