The majority of studies on lethal radiobiological damage have focused on double-strand breaks (DSBs), a type of clustered DNA damage and the evaluation of their toxicity, while other types of ...clustered DNA damage have received much less attention. The main purpose of this study is to evaluate the contribution of different lesions induced by ionizing radiation to the loss of plasmid DNA functionality. We employed a simple model system comprising E. coli transformed with an irradiated plasmid pGEM-3Zf (–) to determine the effect of DSBs and other lesions including base damage and clustered lesions on the functionality (“viability”) of the plasmid. The yields of γ-radiation-induced single-strand breaks (SSBs) and DSBs were measured by gel electrophoresis. We found that the transformation efficiency decreases with radiation dose, but this decrease cannot be explained by the formation of DSBs. For example, at doses of 500 and 700 Gy, the relative transformation efficiency falls from 100% to 53% and 26%, respectively, while only 5.7% and 9.1% of the plasmids contain a DSB. In addition, it is also unlikely that randomly distributed base lesions could explain the loss of functionality of the plasmid, since cells can repair them efficiently. However, clustered lesions other than DSBs, which are difficult to repair and result in the loss of information on both DNA strands, have the potential to induce the loss of plasmid functionality. We therefore measured the yields of γ-radiation-induced base lesions and cluster damage, which are respectively converted into SSBs and DSBs by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). Our data demonstrate that the yield of cluster damage (i.e., lesions that yield DSBs following digestion) is 31 times higher than that of frank DSBs. This finding suggests that frank DSBs make a relatively minor contribution to the loss of DNA functionality induced by ionizing radiation, while other toxic lesions formed at a much higher frequencies than DSBs must be responsible for the loss of plasmid functionality. These lesions may be clustered lesions/locally multiply damaged sites (LMDS), including base damage, SSBs and/or intrastrand and interstrand crosslinks, leading to the loss of vital information in the DNA. Using a mathematical model, we estimate that at least three toxic lesions are required for the inactivation of plasmid functionality, in part because even these complex lesions can be repaired.
The induction of DNA interstrand cross-links by ionizing radiation has been largely ignored in favour of studies on double-strand break formation and repair. At least part of the problem is ...technical; it is difficult to detect and quantify interstrand cross-links when the same agent forms both cross-links and single strand breaks because the detection of interstrand cross-links generally involves a denaturation step. Our group has studied the induction of interstrand cross-links following irradiation of DNA containing bromouracil at specific sites. We found that the formation of interstrand cross-links requires the presence of a few (3–5) mismatched bases, comprising the bromouracil. In the absence of mismatched bases, no radiation-induced cross-linking was observed; however, even in the absence of bromouracil, cross-linking still occurred, albeit at a lower efficiency. Our molecular modelling studies demonstrate that the mobility of the bases in the mismatched region is essential for the cross-linking process. Thus, our hypothesis is that ionizing radiation induces DNA interstrand cross-links in non-hybridized regions of DNA. Some obvious examples of such DNA regions are replication forks, transcription bubbles and the D-loop of telomeres. However, an abundance of studies have made it clear that there must be many single-stranded regions in the genome, such as hairpins and cruciforms. For example, alpha satellite DNA, in centromere regions of human chromosomes, forms hairpins. Thus, a variety of non-B DNA structures (hairpins, slipped DNA and tetrahelical structures) exist in the genome and should be susceptible to the formation of radiation-induced interstrand cross-links. Although interstrand cross-links have thus far been virtually ignored in radiation biology, it will be worthwhile to develop methods to detect their presence following exposure of cells to biologically relevant levels of ionizing radiation, since, on a per lesions basis, they are probably more toxic than double-strand breaks.
Interstrand cross-links (ICL) represent one of the most toxic types of DNA damage for dividing cells. They are induced both by natural products (e.g., psoralens + UVA) and by several chemical agents, ...some of which are used in chemotherapy (e.g., carboplatin and mitomycin C). Although repair mechanisms exist for interstrand cross-links, these lesions can induce mutations, chromosomal rearrangements, and cell death. Here, we report, for the first time, the formation of ICL by γ-rays in brominated DNA. It is well established that the radiosensitization properties of bromodeoxyuridine (BrdUrd) result primarily from the electrophilic nature of the bromine, making it a good leaving group and leading to the irreversible formation of a uridinyl radical (dUrd•) or uridinyl anion (dUrd−) upon addition of an electron. We observe that the radiolytic loss of the bromine atom is greatly suppressed in double-stranded compared to single-stranded DNA. We have used a model DNA containing a bulge, formed by five mismatched bases, and have observed a linear dose−response for the formation of strand breaks on the single-stranded regions of both the brominated strand and the opposite nonbrominated strand. Surprisingly, we have observed the formation of interstrand cross-links exclusively in the mismatched region. Thus, we propose that the radiosensitization effects of bromodeoxyuridine in vivo will almost certainly be limited to single strand regions such as found in transcription bubbles, replication forks, mismatched DNA, and possibly the loop region of telomeres. Our results suggest that interstrand cross-links may contribute to the radiosensitization effects of BrdUrd. These findings may have profound implications for the clinical use of bromodeoxyuridine as a radiosensitizer, as well as for the development of targeted radiosensitizers.
Interstrand cross-links impede critical cellular processes such as transcription and replication and are thus considered to be one of the most toxic types of DNA damage. Although several studies now ...point to the existence of γ-radiation-induced cross-links in cellular DNA, little is known about the characteristics required for their creation. Recently, we reported the formation of interstrand cross-links that were specific for mismatched nucleotides within 5-bromo-2′-deoxyuridine-substituted DNA. Given the structural specificity for interstrand cross-link formation, it is likely that open or mismatched regions of DNA in cells may be particularly favorable for cross-link production. Herein, we investigated the effect of the local DNA sequence on the formation of interstrand cross-links, using 5-bromo-2′-deoxyuridine to generate radicals in a mismatched region of DNA. We investigated a total of 12 variations of bases in the mismatched region. The oligonucleotides were irradiated with γ-rays, and interstrand cross-link formation was analyzed by denaturing gel electrophoresis. We found that the efficiency of cross-link formation was highly dependent on the nature of mismatched bases and, on the basis of electrophoretic mobility, observed several distinctive cross-link structures with specific DNA sequences. This study provides new insights into the reactivity of mismatched DNA and the mechanisms leading to interstrand cross-link formation. The potential application of 5-bromo-2′-deoxyuridine-induced interstrand cross-links to the field of DNA repair is discussed.
Peptide nucleic acids (PNAs) efficiently hybridize with DNA and are promoted as versatile gene-targeting analytical tools and pharmaceuticals. However, PNAs have never been exploited as ...radiopharmaceuticals, and radiation-induced physicochemical modifications of PNA:DNA heteroduplexes have not been studied. Drug- and radiation-induced creation of covalent cross-links in DNA obstruct crucial cell survival processes such as transcription and replication and are thus considered genotoxic events with a high impact in anticancer therapies. Here we report that γ-irradiation of complementary PNA:DNA heteroduplexes, wherein the PNA contains l-lysine, free amino, or N-methylmorpholinium N- and C-capping groups, results in the formation of irreversible interstrand cross-links (ICL). The number of detected ICL corresponds to the number of available amino functional groups on the PNA. The effect of DNA sequence on the formation of ICL was studied by modifying the terminal nucleotides of the DNA oligonucleotide to create deletions and overhangs. The involvement of abasic sites (ABS) on the DNA strand in the cross-linking reaction was confirmed by independent experiments with synthetic ABS-containing oligonucleotides. Molecular modeling and molecular dynamics (MD) simulations were applied to elucidate the conformation of the N- and C-capping groups of the PNA oligomer and their interactions with the proximal terminus of the DNA. Good agreement between experimental and modeling results was achieved. Modeling indicated that the presence of positively charged capping groups on the PNA increases the conformational flexibility of the PNA:DNA terminal base pairs and often leads to their melting. This disordered orientation of the duplex ends provides conditions for multiple encounters of the short (amino) and bulky (Lys) side chains with nucleobases and the DNA backbone up to the third base pair along the duplex stem. Dangling duplex ends offer favorable conditions for increased accessibility of the radiation-induced free radicals to terminal nucleotides and their damage. It is suggested that the ICL are produced by initial formation of Schiff base adducts between the PNA amino functions and the opposed DNA oxidation-damaged bases or abasic 2′-deoxyribose-derived aldehydic groups. The subsequent reduction by solvated electrons (e− aq) or other radiation-produced reducing species results in irreversible covalent interstrand cross-links. The simultaneous involvement of oxidizing, •OH, and reducing, e− aq, radicals presents a case in which multiple ionization events along a γ-particle path lead to DNA injuries that also encompass ICL as part of the multiply damaged sites (MDS). The obtained results may find applications in the development of a new generation of gene-targeted radiosensitizers based on PNA vectors.
Peptide nucleic acids (PNAs) efficiently hybridize with DNA and are promoted as versatile gene-targeting analytical tools and pharmaceuticals. However, PNAs have never been exploited as ...radiopharmaceuticals, and radiation-induced physicochemical modifications of PNA:DNA heteroduplexes have not been studied. Drug- and radiation-induced creation of covalent cross-links in DNA obstruct crucial cell survival processes such as transcription and replication and are thus considered genotoxic events with a high impact in anticancer therapies. Here we report that *g-irradiation of complementary PNA:DNA heteroduplexes, wherein the PNA contains l-lysine, free amino, or N-methylmorpholinium N- and C-capping groups, results in the formation of irreversible interstrand cross-links (ICL). The number of detected ICL corresponds to the number of available amino functional groups on the PNA. The effect of DNA sequence on the formation of ICL was studied by modifying the terminal nucleotides of the DNA oligonucleotide to create deletions and overhangs. The involvement of abasic sites (ABS) on the DNA strand in the cross-linking reaction was confirmed by independent experiments with synthetic ABS-containing oligonucleotides. Molecular modeling and molecular dynamics (MD) simulations were applied to elucidate the conformation of the N- and C-capping groups of the PNA oligomer and their interactions with the proximal terminus of the DNA. Good agreement between experimental and modeling results was achieved. Modeling indicated that the presence of positively charged capping groups on the PNA increases the conformational flexibility of the PNA:DNA terminal base pairs and often leads to their melting. This disordered orientation of the duplex ends provides conditions for multiple encounters of the short (amino) and bulky (Lys) side chains with nucleobases and the DNA backbone up to the third base pair along the duplex stem. Dangling duplex ends offer favorable conditions for increased accessibility of the radiation-induced free radicals to terminal nucleotides and their damage. It is suggested that the ICL are produced by initial formation of Schiff base adducts between the PNA amino functions and the opposed DNA oxidation-damaged bases or abasic 2'-deoxyribose-derived aldehydic groups. The subsequent reduction by solvated electrons (e-aq) or other radiation-produced reducing species results in irreversible covalent interstrand cross-links. The simultaneous involvement of oxidizing, -OH, and reducing, e-aq, radicals presents a case in which multiple ionization events along a *g-particle path lead to DNA injuries that also encompass ICL as part of the multiply damaged sites (MDS). The obtained results may find applications in the development of a new generation of gene-targeted radiosensitizers based on PNA vectors.
Genomic instability is thought to underlie tumor progression in solid tumors, such as breast cancer. Although evidence that the hereditary breast cancer genes, BRCA1 and BRCA2, are involved in DNA ...repair suggests that genomic instability plays an important role in hereditary breast tumorigenesis, genomic instability remains poorly characterized in sporadic breast cancers. Using a DNA fingerprinting technique, inter-(simple sequence repeat) PCR (inter-SSR PCR), the degree of genomic instability was quantified in 47 sporadic breast cancers compared with matched adjacent normal breast tissues. Almost all sporadic breast cancers show significant genomic instability by inter-SSR PCR. The distribution of this instability is bimodal; 57% of the tumors show fewer changes, whereas 43% show striking genomic alterations. Further analysis of two inter-SSR PCR tumor-normal differences revealed a genomic amplification and probable deletion. Thus, inter-SSR PCR can detect chromosomal breakage-related genomic alterations in most sporadic breast cancers. Genomic instability as detected by inter-SSR PCR is not correlated with aneuploidy, suggesting that this technique preferentially detects intrachromosomal alterations. Chromosomal instability in breast cancer can therefore be subdivided into at least two groups: (a) intrachromosomal and (b) gross chromosomal. Allelic imbalance at markers at the 13q13 and retinoblastoma loci (13q) and not at 17q loci was significantly associated with high levels of intrachromosomal instability, suggesting genes at 13q13 and retinoblastoma loci are either selectively targeted or involved in the genesis of genomic instability in sporadic breast cancers.
cAMP and dexamethasone are known to modulate Na+ transport in epithelial cells. We investigated whether dibutyryl cAMP (DBcAMP) and dexamethasone modulate the mRNA expression of two key elements of ...the Na+ transport system in isolated rat alveolar epithelial cells: alpha-, beta-, and gamma-subunits of the epithelial Na+ channel (ENaC) and the alpha1- and betal-subunits of Na+-K+-ATPase.
cAMP and dexamethasone are known to modulate Na
+
transport in epithelial cells. We investigated whether dibutyryl cAMP (DBcAMP) and dexamethasone modulate the mRNA expression of two key elements of ...the Na
+
transport system in isolated rat alveolar epithelial cells: α-, β-, and γ-subunits of the epithelial Na
+
channel (ENaC) and the α
1
- and β
1
-subunits of Na
+
-K
+
-ATPase. The cells were treated for up to 48 h with DBcAMP or dexamethasone to assess their long-term impact on the steady-state level of ENaC and Na
+
-K
+
-ATPase mRNA. DBcAMP induced a twofold transient increase of α-ENaC and α
1
-Na
+
-K
+
-ATPase mRNA that peaked after 8 h of treatment. It also upregulated β- and γ-ENaC mRNA but not β
1
-Na
+
-K
+
-ATPase mRNA. Dexamethasone augmented α-ENaC mRNA expression 4.4-fold in cells treated for 24 h and also upregulated β- and γ-ENaC mRNA. There was a 1.6-fold increase at 8 h of β
1
-Na
+
-K
+
-ATPase mRNA but no significant modulation of α
1
-Na
+
-K
+
-ATPase mRNA expression. Because DBcAMP and dexamethasone did not increase the stability of α-ENaC mRNA, we cloned 3.2 kb of the 5′ sequences flanking the mouse α-ENaC gene to study the impact of DBcAMP and dexamethasone on α-ENaC promoter activity. The promoter was able to drive basal expression of the chloramphenicol acetyltransferase (CAT) reporter gene in A549 cells. Dexamethasone increased the activity of the promoter by a factor of 5.9. To complete the study, the physiological effects of DBcAMP and dexamethasone were investigated by measuring transepithelial current in treated and control cells. DBcAMP and dexamethasone modulated transepithelial current with a time course reminiscent of the profile observed for α-ENaC mRNA expression. DBcAMP had a greater impact on transepithelial current (2.5-fold increase at 8 h) than dexamethasone (1.8-fold increase at 24 h). These results suggest that modulation of α-ENaC and Na
+
-K
+
-ATPase gene expression is one of the mechanisms that regulates Na
+
transport in alveolar epithelial cells.
