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

<i>N</i>,<i>N</i>-Bis-(2-chloroethyl)-phosphorodiamidic acid (phosphoramide mustard, PM) and <i>N</i>,<i>N</i>-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 <i>N</i>-[2-[cysteinyl]­ethyl]-<i>N</i>-[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

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-<i>cis</i>-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-<i>cis</i>-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