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

Abstract

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.

Figure 1

Cytoxicity of phosphoramide mustard in human fibrosarcoma (HT1080) cells. Cells (in triplicate) were treated with 0 – 2000 μM PM for 3 h. Following exposure, the cells were allowed to recover in fresh media for 48 h. Cell viability was measured by the Alamar Blue assay using a Synergi H1 Microplate reader.

Figure 2

(A) Concentration-dependent formation of DPCs in HT1080 cells treated with 0 – 500 μM PM for 3 h. Chromosomal DNA containing drug-induced DPCs was extracted using a modified phenol/chloroform extraction method described in the Experimental section. The proteins were released from the DNA backbone by neutral thermal hydrolysis to yield guanine-protein conjugates (Scheme 2), which were resolved by 12% SDS-PAGE and visualized by staining with SimplyBlue SafeStain. Lane 1: molecular weight marker. Lanes 2, 4, 6, 8, 10 are blank. Lane 3: 50 μM PM. Lane 5: 100 μM PM. Lane 7: 250 μM PM. Lane 9: 500 μM PM. (B) Densitometry measurements of the band intensity form Figure 2A, normalized to the untreated control.

Figure 3

DNA-protein cross-linking in human fibroblasts used for proteomics experiments. HT1080 cells (~5.0 × 10⁶, in triplicate) were treated with 100 μM PM (lanes 9 – 11) or buffer only (lanes 3 – 5) for 3 h. Following DPC extraction and thermal hydrolysis to release guanine-protein conjugates, the cross-linked proteins were resolved by 12% SDS-PAGE and visualized by staining with SimplyBlue SafeStain. Proteins within the molecular weight ranges of 30 – 5 kDa (A), 50 – 30 kDa (B), 80 – 50 kDa (C), 110 – 80 kDa (D), and 260 – 110 kD (E) were excised from the gels, subjected to in-gel trypsin digestion, and analyzed by HPLC-ESI⁺-MS/MS.

Figure 4

GO annotations for the proteins involved in PM-induced DPC formation in human HT1080 cells according to cellular distribution, molecular function, and biological processes. The numbers of proteins falling into each category are included.

Figure 5

Representative HPLC-ESI⁺-MS/MS trace (A) and quantitative results for Cys-NOR-N7G conjugates in HT1080 cells treated with 0–500 μM NOR for 3 h. Following treatment, genomic DNA containing DPCs was extracted, hydrolyzed to release DNA-protein conjugates, and digested by proteinase K to amino acids. NOR-induced Cys-NOR-N7G conjugates were analyzed by isotope dilution HPLC-ESI⁺-MS/MS using the corresponding ¹⁵N₅-labeled internal standard.

Figure 6

Cellular viability (A) and DPC formation (B) in HT1080, XPA, PD20, and PD20- corrected cells treated with 250 μM NOR for 3 h. Following treatment, NOR-containing media was removed, and the cells were allowed to recover in fresh media for 4 h. Cys-NOR-N7G conjugates were quantified by isotope dilution HPLC-ESI⁺-MS/MS.

Figure 7

Venn diagrams showing the overlaps between the lists of proteins that form DPCs in human cells following exposure to PM, DEB, and cisplatin.

Scheme 1

Metabolism of cyclophosphamide to phosphoramide mustard and nornitrogen mustard and the formation of DNA-protein crosslinks.

Scheme 2

Experimental strategy for isolation of DNA-protein cross-links from phosphoramide mustard-treated cells and their characterization by mass spectrometry.