Development of Comprehensive Ultraperformance Liquid Chromatography-High-Resolution Mass Spectrometry Assays to Quantitate Cisplatin-Induced DNA-DNA Cross-Links.

Cisplatin (CP) is a common antitumor drug that is used to treat many solid tumors. The activity of CP is attributed to the formation of DNA-DNA cross-links, which consist of 1,2-intra-, 1,3-intra-, and interstrand cross-links. To better understand how each intrastrand cross-link contributes to the activity of CP, we have developed comprehensive ultraperformance liquid chromatography-selective ion monitoring (UPLC-SIM) assays to quantify 1,2-GG-, 1,2-AG-, 1,3-GCG-, and 1,3-GTG-intrastrand cross-links. The limit of quantitation for the developed assays ranged from 5 to 50 fmol or as low as 6 cross-links per 108 nucleotides. To demonstrate the utility of the UPLC-SIM assays, we first performed in vitro cross-link formation kinetics experiments. We confirmed that the 1,2-GG-intrastrand cross-links were the most abundant intrastrand cross-link and formed at a faster rate compared to 1,2-AG- and 1,3-intrastrand cross-links. Furthermore, we investigated the repair kinetics of intrastrand cross-links in CP-treated wild-type and nucleotide excision repair (NER)-deficient U2OS cells. We observed a slow decrease of both 1,2- and 1,3-intrastrand cross-links in wild-type cells and no evidence of direct repair in the NER-deficient cells. Taken together, we have demonstrated that our assays are capable of accurately quantifying intrastrand cross-links in CP-treated samples and can be utilized to better understand the activity of CP.

Recent Advances in DNA Lesion Mapping and Repair Mechanisms.

Recognition and repair of DNA lesions are critical for cell survival. Herein, we highlight recent advances in the sequencing, repair mechanisms, and biological consequences of DNA lesions presented at the 2022 Fall American Chemical Society meeting.

New Synthetic Analogs of Nitrogen Mustard DNA Interstrand Cross-Links and Their Use to Study Lesion Bypass by DNA Polymerases.

Nitrogen mustards are a widely used class of antitumor agents that exert their cytotoxic effects through the formation of DNA interstrand cross-links (ICLs). Despite being among the first antitumor agents used, the biological responses to NM ICLs remain only partially understood. We have previously reported the generation of NM ICL mimics by incorporation of ICL precursors into DNA using solid-phase synthesis at defined positions, followed by a double reductive amination reaction. However, the structure of these mimics deviated from the native NM ICLs. Using further development of our approach, we report a new class of NM ICL mimics that only differ from their native counterpart by substitution of dG with 7-deaza-dG at the ICL. Importantly, this approach allows for the synthesis of diverse NM ICLs, illustrated here with a mimic of the adduct formed by chlorambucil. We used the newly generated ICLs in reactions with replicative and translesion synthesis DNA polymerase to demonstrate their stability and utility for functional studies. These new NM ICLs will allow for the further characterization of the biological responses to this important class of antitumor agents.

Cross-linking of the DNA repair protein O6-alkylguanine DNA alkyltransferase to DNA in the presence of cisplatin.

1,1,2,2-cis-diamminedichloroplatinum (II) (cisplatin) is a chemotherapeutic agent widely used in the clinic to treat various cancers. The antitumor activity of cisplatin is generally attributed to its ability to form intrastrand and interstrand DNA-DNA cross-links via sequential platination of two nucleophilic sites within the DNA duplex. However, cisplatin also induces DNA- protein lesions (DPCs) that may contribute to its biological effects due to their ability to block DNA replication and transcription. We previously reported that over 250 nuclear proteins including high mobility group proteins, histone proteins, and elongation factors formed DPCs in human HT1080 cells treated with cisplatin (Ming et al. Chem. Res. Toxicol. 2017, 30, 980-995). Interestingly, cisplatin-induced DNA-protein conjugates were reversed upon heating, by an unknown mechanism. In the present work, DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) was used as a model to investigate the molecular details of cisplatin-mediated DNA-protein cross-linking and to establish the mechanism of their reversal. We found that AGT is readily cross-linked to DNA in the presence of cisplatin. HPLC-ESI+-MS/MS sequencing of tryptic peptides originating from dG-Pt-AGT complexes revealed that the cross-linking occurred at six sites within this protein including Glu110, Lys125, Cys145, His146, Arg147, and Cys150. Cisplatin-induced Lys-Gua cross-links (1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2'-deoxyguanosine-7-yl)-platinum(II) (dG-Pt-Lys) were detected by HPLC-ESI+-MS/MS of total digests of modified protein in comparison with the corresponding authentic standard. Upon heating, dG-Pt-AGT complexes were subject to platination migration from protein to DNA, forming cis-[Pt(NH3)2{d(GpG)}] cross-links which were detected by HPLC-ESI+-MS/MS. Our results provide a new insight into the mechanism of cisplatin-mediated DNA-protein cross-linking and their dynamic equilibrium with the corresponding DNA-DNA lesions.

N6-(2-Deoxy-d- erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5- N-(2-hydroxy-3-buten-1-yl)-formamidopyrimidine Adducts of 1,3-Butadiene: Synthesis, Structural Identification, and Detection in Human Cells.

1,3-Butadiene (BD) is an environmental and occupational toxicant classified as a human carcinogen. BD is metabolically activated by cytochrome P450 monooxygenases to 3,4-epoxy-1-butene (EB), which alkylates DNA to form a range of nucleobase adducts. Among these, the most abundant are the hydrolytically labile N7-guanine adducts such as N7-(2-hydroxy-3-buten-1-yl)-guanine (N7-EB-dG). We now report that N7-EB-dG can be converted to the corresponding ring open N6-(2-deoxy-d- erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5- N-(2-hydroxy-3-buten-1-yl)-formamidopyrimidine (EB-Fapy-dG) adducts. EB-Fapy-dG lesions were detected in EB-treated calf thymus DNA and in EB-treated mammalian cells using quantitative isotope dilution nanoLC-ESI+-MS/MS. EB-Fapy-dG adduct formation in EB-treated calf thymus DNA was concentration dependent and was greatly accelerated at an increased pH. EB-FAPy-dG adduct amounts were 2-fold higher in base excision repair-deficient NEIL1-/- mouse embryonic fibroblasts (MEF) as compared to isogenic controls (NEIL1+/+), suggesting that this lesion may be a substrate for NEIL1. Furthermore, NEIL1-/- cells were sensitized to EB treatment as compared to NEIL1+/+ fibroblasts. Overall, our results indicate that ring-opened EB-FAPy-dG adducts form under physiological conditions, prompting future studies to determine their contributions to genotoxicity and mutagenicity of BD.

Oxidative cross-linking of proteins to DNA following ischemia-reperfusion injury.

Myocardial infarction (MI) is a life-threatening condition that can occur when blood flow to the heart is interrupted due to a blockage in one or more of the coronary vessels. Current treatments of MI rapidly restore blood flow to the affected myocardium using thrombolytic agents or angioplasty. Adverse effects including inflammation, tissue necrosis, and ventricular dysfunction are, however, not uncommon following reperfusion therapy. These conditions are thought to be caused by a sudden influx of reactive oxygen species (ROS) to the affected myocardium. We employed the model of left anterior descending artery ligation/reperfusion surgery in a rat model to show that ischemia/reperfusion injury is associated with the formation of toxic DNA-protein cross-links (DPCs) in cardiomyocytes. Mass spectrometry based experiments have revealed that these conjugates were formed by a free radical mechanism and involved thymidine residues of DNA and tyrosine side chains of proteins (dT-Tyr). Quantitative proteomics experiments have identified nearly 90 proteins participating in hydroxyl radical-induced DPC formation, including ROS scavengers, contractile proteins, and regulators of apoptosis. Global proteome changes were less pronounced and included increased expression of mitochondrial proteins required for aerobic respiration and biomarkers of sarcomere breakdown following ischemia/reperfusion injury. Overall, our results are consistent with a model where sudden return of oxygen to ischemic tissues induces oxidative stress, inflammation, and the formation of DNA-protein cross-links that may contribute to reperfusion injury by desregulating gene expression and inducing cardiomyocyte death.

Mass Spectrometry Based Proteomics Study of Cisplatin-Induced DNA-Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells.

Platinum-based antitumor drugs such as 1,1,2,2-cis-diamminedichloroplatinum(II) (cisplatin), carboplatin, and oxaliplatin are currently used to treat nearly 50% of all cancer cases, and novel platinum based agents are under development. The antitumor effects of cisplatin and other platinum compounds are attributed to their ability to induce interstrand DNA-DNA cross-links, which are thought to inhibit tumor cell growth by blocking DNA replication and/or preventing transcription. However, platinum agents also induce significant numbers of unusually bulky and helix-distorting DNA-protein cross-links (DPCs), which are poorly characterized because of their unusual complexity. We and others have previously shown that DPCs block DNA replication and transcription and causes toxicity in human cells, potentially contributing to the biological effects of platinum agents. In the present work, we have undertaken a system-wide investigation of cisplatin-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells using mass spectrometry-based proteomics. DPCs were isolated from cisplatin-treated cells using a modified phenol/chloroform DNA extraction in the presence of protease inhibitors. Proteins were released from DNA strands and identified by mass spectrometry-based proteomics and immunological detection. Over 250 nuclear proteins captured on chromosomal DNA following treatment with cisplatin were identified, including high mobility group (HMG) proteins, histone proteins, and elongation factors. To reveal the exact molecular structures of cisplatin-mediated DPCs, isotope dilution HPLC-ESI+-MS/MS was employed to detect 1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2'-deoxyguanosine-7-yl)-platinum(II) (dG-Pt-Lys) conjugates between the N7 guanine of DNA and the ε-amino group of lysine. Our results demonstrate that therapeutic levels of cisplatin induce a wide range of DPC lesions, which likely contribute to both target and off target effects of this clinically important drug.

Mass Spectrometry-Based Tools to Characterize DNA-Protein Cross-Linking by Bis-Electrophiles.

DNA-protein cross-links (DPCs) are unusually bulky DNA adducts that form in cells as a result of exposure to endogenous and exogenous agents including reactive oxygen species, ultraviolet light, ionizing radiation, environmental agents (e.g. transition metals, formaldehyde, 1,2-dibromoethane, 1,3-butadiene) and common chemotherapeutic agents. Covalent DPCs are cytotoxic and mutagenic due to their ability to interfere with faithful DNA replication and to prevent accurate gene expression. Key to our understanding of the biological significance of DPC formation is identifying the proteins most susceptible to forming these unusually bulky and complex lesions and quantifying the extent of DNA-protein cross-linking in cells and tissues. Recent advances in bottom-up mass spectrometry-based proteomics have allowed for an unbiased assessment of the whole protein DPC adductome after in vitro and in vivo exposures to cross-linking agents. This MiniReview summarizes current and emerging methods for DPC isolation and analysis by mass spectrometry-based proteomics. We also highlight several examples of successful applications of these novel methodologies to studies of DPC lesions induced by bis-electrophiles such as formaldehyde, 1,2,3,4-diepoxybutane, nitrogen mustards and cisplatin.

Covalent DNA-Protein Cross-Linking by Phosphoramide Mustard and Nornitrogen Mustard in Human Cells.

N,N-Bis-(2-chloroethyl)-phosphorodiamidic acid (phosphoramide mustard, PM) and N,N-bis-(2-chloroethyl)-amine (nornitrogen mustard, NOR) are the two biologically active metabolites of cyclophosphamide, a DNA alkylating drug commonly used to treat lymphomas, breast cancer, certain brain cancers, and autoimmune diseases. PM and NOR are reactive bis-electrophiles capable of cross-linking cellular biomolecules to form covalent DNA-DNA and DNA-protein cross-links (DPCs). In the present work, a mass spectrometry-based proteomics approach was employed to characterize PM- and NOR-mediated DNA-protein cross-linking in human cells. Following treatment of human fibrosarcoma cells (HT1080) with cytotoxic concentrations of PM, over 130 proteins were found to be covalently trapped to DNA, including those involved in transcriptional regulation, RNA splicing/processing, chromatin organization, and protein transport. HPLC-ESI(+)-MS/MS analysis of proteolytic digests of DPC-containing DNA from NOR-treated cells revealed a concentration-dependent formation of N-[2-[cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]amine (Cys-NOR-N7G) conjugates, confirming that it cross-links cysteine thiols of proteins to the N7 position of guanines in DNA. Cys-NOR-N7G adduct numbers were higher in NER-deficient xeroderma pigmentosum cells (XPA) as compared with repair proficient cells. Furthermore, both XPA and FANCD2 deficient cells were sensitized to PM treatment as compared to that of wild type cells, suggesting that Fanconi anemia and nucleotide excision repair pathways are involved in the removal of cyclophosphamide-induced DNA damage.

DNA-Protein Cross-Links: Formation, Structural Identities, and Biological Outcomes.

Noncovalent DNA-protein interactions are at the heart of normal cell function. In eukaryotic cells, genomic DNA is wrapped around histone octamers to allow for chromosomal packaging in the nucleus. Binding of regulatory protein factors to DNA directs replication, controls transcription, and mediates cellular responses to DNA damage. Because of their fundamental significance in all cellular processes involving DNA, dynamic DNA-protein interactions are required for cell survival, and their disruption is likely to have serious biological consequences. DNA-protein cross-links (DPCs) form when cellular proteins become covalently trapped on DNA strands upon exposure to various endogenous, environmental and chemotherapeutic agents. DPCs progressively accumulate in the brain and heart tissues as a result of endogenous exposure to reactive oxygen species and lipid peroxidation products, as well as normal cellular metabolism. A range of structurally diverse DPCs are found following treatment with chemotherapeutic drugs, transition metal ions, and metabolically activated carcinogens. Because of their considerable size and their helix-distorting nature, DPCs interfere with the progression of replication and transcription machineries and hence hamper the faithful expression of genetic information, potentially contributing to mutagenesis and carcinogenesis. Mass spectrometry-based studies have identified hundreds of proteins that can become cross-linked to nuclear DNA in the presence of reactive oxygen species, carcinogen metabolites, and antitumor drugs. While many of these proteins including histones, transcription factors, and repair proteins are known DNA binding partners, other gene products with no documented affinity for DNA also participate in DPC formation. Furthermore, multiple sites within DNA can be targeted for cross-linking including the N7 of guanine, the C-5 methyl group of thymine, and the exocyclic amino groups of guanine, cytosine, and adenine. This structural complexity complicates structural and biological studies of DPC lesions. Two general strategies have been developed for creating DNA strands containing structurally defined, site-specific DPCs. Enzymatic methodologies that trap DNA modifying proteins on their DNA substrate are site specific and efficient, but do not allow for systematic studies of DPC lesion structure on their biological outcomes. Synthetic methodologies for DPC formation are based on solid phase synthesis of oligonucleotide strands containing protein-reactive unnatural DNA bases. The latter approach allows for a wider range of protein substrates to be conjugated to DNA and affords a greater flexibility for the attachment sites within DNA. In this Account, we outline the chemistry of DPC formation in cells, describe our recent efforts to identify the cross-linked proteins by mass spectrometry, and discuss various methodologies for preparing DNA strands containing structurally defined, site specific DPC lesions. Polymerase bypass experiments conducted with model DPCs indicate that the biological outcomes of these bulky lesions are strongly dependent on the peptide/protein size and the exact cross-linking site within DNA. Future studies are needed to elucidate the mechanisms of DPC repair and their biological outcomes in living cells.