Induce Interstrand Cross-links Research Articles

Double-Stranded DNA Fragments Bearing Unrepairable Lesions and Their Internalization into Mouse Krebs-2 Carcinoma Cells

Murine Krebs-2 tumor-initiating stem cells are known to natively internalize extracellular double-stranded DNA fragments. Being internalized, these fragments interfere in the repair of chemically induced interstrand cross-links. In the current investigation, 756 bp polymerase chain reaction (PCR) product containing bulky photoreactive dC adduct was used as extracellular DNA. This adduct was shown to inhibit the cellular system of nucleotide excision repair while being resistant to excision by this DNA repair system. The basic parameters for this DNA probe internalization by the murine Krebs-2 tumor cells were characterized. Being incubated under regular conditions (60 min, 24°C, 500 μL of the incubation medium, in the dark), 0.35% ± 0.18% of the Krebs-2 ascites cells were shown to natively internalize modified DNA. The saturating amount of the modified DNA was detected to be 0.37 μg per 106 cells. For the similar unmodified DNA fragments, this ratio is 0.73 μg per 106 cells. Krebs-2 tumor cells were shown to be saturated internalizing either (190 ± 40) × 103 molecules of modified DNA or (1,000 ± 100) × 103 molecules of native DNA. On internalization, the fragments of DNA undergo partial and nonuniform hydrolysis of 3' ends followed by circularization. The degree of hydrolysis, assessed by sequencing of several clones with the insertion of specific PCR product, was 30-60 nucleotides.

Abstract C51: Chemosensitisation to cisplatin by STAT3 inhibitors is mediated through the inhibition of DNA interstrand crosslink unhooking

Abstract Signal Transducer and Activator of Transcription 3 (STAT3) is constitutively activated in many cancer cell lines, leading to survival, proliferation, angiogenesis and metastasis. The inhibition of STAT3 by natural or synthetic compounds has been shown to sensitise cancer cell lines to the chemotherapy agent cisplatin, however the molecular mechanisms contributing to this chemosensitisation have not yet been fully elucidated. Therefore we investigated the effect of STAT3 inhibition on cisplatin-induced DNA damage and key DNA repair factors. STAT3 inhibitors stattic and curcumin were investigated in combination with cisplatin using the Suphorhodamine B cell growth inhibition assay in the DU145 prostate cancer and A549 non-small cell lung cancer cell lines. Combination treatments result in a significant (P<0.0001) reduction in cisplatin GI50 values. These combinations were synergistic as determined by combination index value analysis using the software Calcusyn, confirming that inhibition of STAT3 enhances the cytotoxic effect of cisplatin. Using a modification of the alkaline comet assay we observed that STAT3 inhibitors significantly prevent the unhooking step of repair of cisplatin induced interstrand crosslinks (ICLs). Treatment with cisplatin alone induces formation of ICLs which peak at 9 hours and unhook by 24 hours post cisplatin exposure in both cell lines. Pre-treating cells with 12μM stattic or 48μM curcumin followed by cisplatin results in an equivalent peak formation of ICLs at 9 hours, however 24 and 48 hours post cisplatin no unhooking is observed. Therefore both STAT3 inhibitors used in this study block the unhooking stage of ICL repair. Using RT-PCR and immunoblotting we show that treatment with STAT3 inhibitors dose-dependently down-regulates EME1, BRCA1 and FANCD2 mRNA and protein levels. These three DNA repair proteins have been shown to be involved in the early stages of ICL repair and therefore down-regulation of these genes may be responsible for the impairment of ICL unhooking seen with STAT3 inhibition. As the effects observed here have been replicated with two STAT3 inhibitors of differing chemical structure, this suggests that STAT3 is directly involved in the regulation of cisplatin-induced ICL repair through modulation of key ICL repair genes. We propose this as a contributing mechanism to the synergy reported between STAT3 inhibitors and cisplatin. The data presented here will inform the design of future drug combination strategies. Citation Format: Helen Valentine, Emily Dixon, John A. Hartley, Konstantinos Kiakos. Chemosensitisation to cisplatin by STAT3 inhibitors is mediated through the inhibition of DNA interstrand crosslink unhooking. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C51.

Characterization of a novel DNA glycosylase from S. sahachiroi involved in the reduction and repair of azinomycin B induced DNA damage.

Azinomycin B is a hybrid polyketide/nonribosomal peptide natural product and possesses antitumor activity by interacting covalently with duplex DNA and inducing interstrand crosslinks. In the biosynthetic study of azinomycin B, a gene (orf1) adjacent to the azinomycin B gene cluster was found to be essential for the survival of the producer, Streptomyces sahachiroi ATCC33158. Sequence analyses revealed that Orf1 belongs to the HTH_42 superfamily of conserved bacterial proteins which are widely distributed in pathogenic and antibiotic-producing bacteria with unknown functions. The protein exhibits a protective effect against azinomycin B when heterologously expressed in azinomycin-sensitive strains. EMSA assays showed its sequence nonspecific binding to DNA and structure-specific binding to azinomycin B-adducted sites, and ChIP assays revealed extensive association of Orf1 with chromatin in vivo. Interestingly, Orf1 not only protects target sites by protein–DNA interaction but is also capable of repairing azinomycin B-mediated DNA cross-linking. It possesses the DNA glycosylase-like activity and specifically repairs DNA damage induced by azinomycin B through removal of both adducted nitrogenous bases in the cross-link. This bifunctional protein massively binds to genomic DNA to reduce drug attack risk as a novel DNA binding protein and triggers the base excision repair system as a novel DNA glycosylase.

Quantification and repair of psoralen-induced interstrand crosslinks in human cells

Bi-functional alkylating agents that cause crosslinks are commonly used in chemotherapy. However, there is no conclusive knowledge for human cells regarding the number of induced interstrand crosslinks (ICLs) and the unhooking rate when the lesion is removed from one of the DNA strand. Using a newly developed method, we quantified the number of induced ICLs for the five furocoumarins; psoralen, 5-methoxypsoralen, 8-methoxypsoralen, tri-methoxypsoralen and angelicin. In quantitative terms, the results were in agreement with the values found by others. In kinetic studies using mammalian cells, we found that half of the psoralen-induced ICLs were unhooked within 2.5h. The rate in normal human diploid fibroblasts was found to be 20,000 ICLs/h/cell. In comparison to survival, 2500 ICLs per cell led to 50% toxicity, indicating that the unhooking of the ICLs is not the crucial step for ICL tolerance. Surprisingly, only 3500 ICLs per cell corresponded to a significant delay in the replication fork elongation. The results indicate involvements of additional pathway(s) for the delay since the effect on replication elongation could be monitored when only 10% of the replication forks encounter an ICL.

Near-UV Induced Interstrand Cross-Links in Anthraquinone−DNA Duplexes

Anthraquinone (AQ) has been extensively used as a photosensitizer to study charge transfer in DNA. Near-UV photolysis of AQ induces electron abstraction in oligonucleotides leading to AQ radical anions and base radical cations. In general, this reaction is followed by the transport of base radical cations to sites of low oxidation potential, that is, GG, and conversion of G radical cations to DNA breaks. Here, we show that AQ also produces interstrand cross-links in DNA duplexes. About half of the cross-links collapse to single strands in hot piperidine treatment. The structure of stable interstrand cross-links was deduced by MS, NMR, and sequence substitution. The cross-links consist of a covalent link between the methyl group of T on one strand with either C6 or C7 of AQ on the other strand. The formation of interstrand cross-links decreased in O2 compared to deoxygenated solutions. In the presence of O2, the yield of breaks at GG doublets was 10-fold greater than that of cross-links for end tethered AQ, while cross-links exceeded breaks for centrally located AQ. The formation of stable cross-links can be explained by initial charge transfer from T to excited AQ, deprotonation of T radical cations, and condensation of the latter species with AQ radicals. These studies reveal a novel pathway of damage in the photolysis of AQ-DNA duplexes.

Can a mixed damage interfere with DNA-protein cross-links repair?

Some photochemical and photobiological properties of 4,5',8-trimethylpsoralen (TMP) have been studied in comparison with 1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2 one (FQ) and 8-methoxypsoralen (8-MOP). TMP and FQ can photobind to mammalian cell DNA in vivo, by UVA irradiation, forming DNA-protein cross-links (DPC), but only TMP shows a strong capacity of inducing interstrand cross-links (ISC). The mechanism of DPC formation was studied using the double irradiation method in Chinese hamster ovary (CHO) cells, and DPC were detected by alkaline elution. Both TMP and FQ induce covalent diadducts linking together DNA and proteins. Studying the formation of double strand breaks (DSB) in CHO cells we observed that TMP induced a low amount of DSB, similar to 8-MOP. TMP and 8-MOP induced chromosomal aberrations in CHO cells to the same extent, while FQ appeared to be more active. Our data suggest that the ISC induced by TMP could trap enzymes involved in DPC repair.

Induction of AT-specific DNA-interstrand crosslinks by bizelesin in genomic and simian virus 40 DNA

Bizelesin is a bifunctional AT-specific DNA alkylating drug. Our study characterized the ability of bizelesin to induce interstrand crosslinks, a potential lethal lesion. In genomic DNA of BSC-1 cells, bizelesin formed from approx. 0.3 to 6.03±0.85 interstrand crosslinks per 10 6 base pairs, at 5–100 nM drug concentration, respectively, comparable to the number of total adducts previously determined in the same system (J.M. Woynarowski, M.M. McHugh, L.S. Gawron, T.A. Beerman, Biochemistry 34 (1995) 13042–13050). Bizelesin did not induce DNA-protein crosslinks or strand breaks. A model defined target, intracellular simian virus 40 (SV40) DNA, was employed to map at the nucleotide level sites of bizelesin adducts, including potential interstrand crosslinks. Preferential adduct formation was observed at AT tracts which are abundant in the SV40 matrix associated region and the origin of replication. Many sites, including each occurrence of 5′-T(A/T) 4A-3′, co-mapped on both DNA strands suggesting interstrand crosslinks, although monoadducts were also formed. Bizelesin adducts in naked SV40 DNA were found at similar sites. The localization of bizelesin-induced crosslinks in AT-rich tracts of replication-related regions is consistent with the potent anti-replicative properties of bizelesin. Given the apparent lack of other types of lesions in genomic DNA, interstrand crosslinks localized in AT-rich tracts, and to some extent perhaps also monoadducts, are likely to be lethal effects of bizelesin.

Roberts syndrome fibroblasts showing cisplatin hypersensitivity have normal host cell reactivation of cisplatin-treated adenovirus and normal capacity of cisplatin-treated cells for adenovirus DNA synthesis.

Roberts syndrome (RS) is a rare, recessively inherited disorder characterized by growth retardation, limb reductions and craniofacial deformities. Cells from a subset of afflicted individuals, termed RS+, display unusual separation or puffing of the heterochromatic regions of their chromosomes and are hypersensitive to several DNA-damaging agents including mitomycin C (MMC) and cisplatin, both of which can induce interstrand crosslinks in DNA. For this reason, we have investigated the ability of RS+ fibroblasts to repair cisplatin-induced DNA lesions using adenoviris as a probe. Host cell reactivation of cisplatin-treated adenovirus (Ad) was significantly reduced in nucleotide excision repair (NER)-deficient xeroderma pigmentosum (XP) cells but was normal in the two RS+ fibroblast strains and the Fanconi's anemia (FA)fibroblast strain tested. The capacity of cisplatin-treated cells for Ad DNA synthesis was reduced in XP and FA cells compared to normal human cells, but was not reduced in RS+ cells. These results indicate that the hypersensitivity of RS+ cells to cisplatin is not due to a deficiency in NER nor due to a deficiency in the pathway which leads to cisplatin hypersensitivity in FA cells. It is possible that the abnormal heterochromatin organisation of RS+ cells selectively renders the heterochromatic regions of the genome more susceptible to mutagen damage and/or less available for repair.

Increased gene specific repair of cisplatin induced interstrand crosslinks in cisplatin resistant cell lines, and studies on carrier ligand specificity.

Development of resistance to cisplatin in previously treatment-responsive malignancies is a major obstacle to successful treatment. Enhanced DNA repair as well as enhanced replicative bypass of DNA adducts have been suggested to play a role in the development of resistance to cisplatin. However, the relative contribution of these mechanisms is unknown. Second generation platinum compounds containing the 1,2-diaminocyclohexane (dach) carrier ligand have been of particular interest in the studies of resistance mechanisms since they have been effective in treatment of cells resistant to cisplatin. We have investigated the formation and repair of interstrand crosslinks (ICL) in the mouse leukemia cell line L1210/0 and its carrier ligand specific resistant derivatives L1210/DDP and L1210/DACH after treatment with ethylenediamine (en)-Pt and diaminocyclohexane (dach)-Pt compounds. ICL in the overall genome were examined using a modification of the alkaline elution assay. A Southern blot technique was employed for the study of ICL in specific regions of the genome. In the overall genome we found decreased formation of ICL with either -en or -dach carrier ligands in the two resistant cell lines without carrier ligand specificity. Some carrier ligand specificity of ICL formation was observed in the dihydrofolate reductase (DHFR) gene, but it did not correlate with the carrier ligand specificity of resistance. At the level of the overall genome there was no difference in repair of ICL between the sensitive and the two resistant cell lines. When measured in the DHFR gene, however, there was enhanced repair of ICL in the two resistant cell lines compared with the sensitive cell line. The enhanced repair at the level of the gene did not display any carrier ligand specificity.

Different effects of RAD genes of Saccharomyces cerevisiae on incisions of interstrand crosslinks and monoadducts in DNA induced by psoralen plus near UV light treatment.

Abstract— The RAD1, RAD2, RAD3 and RAD4 genes of Saccharomyces cerevisiae are required for incising DNA containing UV induced pyrimidine dimers or psoralen plus 360 nm light induced interstrand crosslinks. We have now determined if these genes are also required for incising DNA at psoralen plus 360 nm light induced monoadducts. For distinguishing between incision breaks occurring at crosslinks and at monoadducts. we have used the cdc9‐2 mutant, defective in DNA ligase activity at the restrictive temperature, and the radl‐2 cdc9‐2, rad2‐5 cdc9‐2, rad3‐2 cdc9‐2 and rad4‐4 cdc9‐2 double mutant combinations. We conclude that the radl, rad2, and rad4 mutants are defective in incising DNA both at crosslinks and monoadducts, whereas the rad3 mutant is proficient in incising DNA at monoadducts but not at crosslinks.

DNA-DNA interstrand crosslinking by dimethanesulphonic acid esters: Correlation with cytotoxicity and antitumour activity in the yoshida lymphosarcoma model and relationship to chain length

Members of the homologous series of nine antitumour dimethanesulphonic acid esters, with the exception of ethylene dimethanesulphonate (EDMS), were found to cause DNA-DNA interstrand crosslinking in cells derived from the transplantable rodent Yoshida lymphosarcoma. The ability of the series to induce interstrand crosslinks was compared with in vitro cytotoxicity and in vivo antitumour activity and is discussed in relation to their chain length and ability to span critical target distances in DNA.

Repair by genetic recombination in bacteria: overview.

DNA molecules that have been damaged in both strands at the same level are not subject to repair by excision but instead can be repaired through recombination with homologous molecules. Examples of two-strand damage include postreplication gaps opposite pyrimidine dimers, two-strand breaks produced by X-rays, and chemically induced interstrand cross-links. In ultraviolet-irradiated bacteria, the newly synthesized DNA is of length equal to the interdimer spacing. With continued incubation, this low-molecular-weight DNA is joined into high-molecular-weight chains (postreplication repair), a process associated with sister exchanges in bacteria. Recombination is initiated by pyrimidine dimers opposite postreplication gaps and by interstrand cross-links that have been cut by excision enzymes. The free ends at the resulting gaps presumably initiate the exchanges. Postreplication repair in Escherichia coli occurs in recB- AND RECC but is greatly slowed in recF- mutants. RecB and recC are the structural genes for exonuclease V, which digests two-stranded DNA by releasing oligonucleotides first from one strand and then from the other. The postreplication sister exchanges in ultra-violet-irradiated bacteria result in the distribution of pyrimidine dimers between parental and daughter strands, indicating that long exchanges involving both strands of each duplex occur. The R1 restriction endonuclease from E. COli has been used to cut the DNA of a bacterial drug-resistance transfer factor with one nuclease-sensitive site, and also DNA from the frog Xenopus enriched for ribosomal 18S and 28S genes. The fragments were annealed with the cut plasmid DNA and ligated, producing a new larger plasmid carrying the eukaryotic rDNA and able to infect and replicate in E. coli.