Proteomic analysis of DNA-protein cross-linking by antitumor nitrogen mustards

Abstract

Nitrogen mustards are antitumor agents used clinically for the treatment of a variety of neoplastic conditions. The biological activity of these compounds is typically attributed to their ability to induce DNA-DNA cross-links. However, nitrogen mustards are able to produce a variety of other lesions, including DNA-protein cross-links (DPCs). DPCs induced by nitrogen mustards are not well-characterized because of their structural complexity and the insufficient specificity and sensitivity of previously available experimental methodologies. In the present work, affinity capture methodology in combination with mass spectrometry-based proteomics was employed to identify mammalian proteins that form covalent cross-links to DNA in the presence of a simple nitrogen mustard, mechlorethamine. Following incubation of 5'-biotinylated DNA duplexes with nuclear protein extracts, DPCs were isolated by affinity capture on streptavidin beads, and the cross-linked proteins were identified by high-performance liquid chromatography-electrospray tandem mass spectrometry of tryptic peptides. Mechlorethamine treatment resulted in the formation of DPCs with nuclear proteins involved in chromatin regulation, DNA replication and repair, cell cycle control, transcriptional regulation, and cell architecture. Western blot analysis was employed to confirm protein identification and to quantify the extent of drug-mediated cross-linking. Mass spectrometry of amino acid-nucleobase conjugates found in total proteolytic digests revealed that mechlorethamine-induced DPCs are formed via alkylation of the N7 position of guanine in duplex DNA and cysteine thiols within the proteins to give N-[2-[S-cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]methylamine lesions. The results described herein suggest that cellular exposure to nitrogen mustards leads to cross-linking of a large spectrum of nuclear proteins to chromosomal DNA, potentially contributing to the cytotoxic and mutagenic effects of these drugs.

Figure 1

Concentration-dependent formation of DPCs in nuclear protein extracts prepared from CHO (A) and HeLa (B) cells following exposure to mechlorethamine. Nuclear protein extracts (500 μg) and 5′-biotinylated double-stranded oligodeoxynucleotides (3.12 nmol) were incubated with 0-1000 μM mechlorethamine (lanes 3-8), subjected to biotin capture enrichment, and resolved by 10% SDS-PAGE. The gels were stained with SimplyBlue SafeStain to visualize the cross-linked proteins.

Figure 2

HPLC-ESI⁺-MS/MS identification of tryptic peptides resulting from biotin capture of mechlorethamine-induced DNA-protein cross-links involving GAPDH (A), PARP (B), and nucleolin (C).

Figure 3

Cellular functions of CHO and human proteins that form DPCs in the presence of mechlorethamine.

Figure 4

Western blot analysis of mechlorethamine-induced DNA-protein cross-links in nuclear extracts from CHO cells. (A) Nuclear extract proteins from CHO cells were incubated with 0 (lane 1), 500 μM (lane 2), or 1000 μM mechlorethamine (lane 3) in the presence of 5′-biotinylated double-stranded oligodeoxynucleotides. Following biotin capture enrichment, cross-linked proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Western blots were performed using primary antibodies specific for: actin, O⁶-alkylguanine DNA alkyltransferase (AGT), nucleolin, poly (ADP-ribose) polymerase (PARP), and X-ray repair cross-complementary protein 1 (XRCC1). Untreated nuclear extract (∼25 μg) was loaded as a positive control (lane 4). In cases where the samples were not loaded in adjacent gel lanes, protein signals are outlined separately. (B) Densitometric analysis of western blots to estimate the extent of protein cross-linking to DNA in the presence of mechlorethamine.

Figure 5

Western blot analysis of mechlorethamine-induced DNA-protein cross-links in nuclear extracts for HeLa cervical carcinoma cells: (A) Nuclear extract proteins form HeLa cells were incubated with 5′-biotinylated double-stranded oligodeoxynucleotides in the presence of 0 (lane 1), 500 μM (lane 2), or 1000 μM mechlorethamine. Following biotin capture enrichment, proteins participating in DPC formation were resolved by 10 or 12% SDS-PAGE and transferred to nitrocellulose membranes. Western blots were performed using primary antibodies specific for actin, nucleolin, poly (ADP-ribose) polymerase (PARP), DNA-(apurinic or apyrimidinic site) lyase (Ref-1), ATP-dependent DNA helicase subunit 2 (Ku), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1-alpha 1 (EF-1α1), O⁶-alkylguanine DNA alkyltransferase (AGT), and X-ray repair cross-complementary protein 1 (XRCC1). Untreated nuclear extract (∼25 μg) was loaded as a positive control (lane 4). In cases where the samples were not loaded in adjacent gel lanes, protein signals are outlined separately. (B) Densitometric analysis of western blots to estimate the extent of protein cross-linking to DNA in the presence of mechlorethamine.

Figure 6

HPLC-ESI⁺-MS/MS analysis of N-[2-[S-cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA) in proteolytic digests. Nuclear protein extracts from HeLa cells were incubated with mechlorethamine in the presence of double-stranded DNA, and the resulting DPCs were subjected to thermal and enzymatic hydrolysis to release nucleic acid-nucleobase conjugates (synthetic Cys-N7G-EMA (A), enzymatic digests of HeLa nuclear extracts following incubation in the presence of DNA (negative control) (B), enzymatic digests of HeLa nuclear extracts following incubation with 1000 μM mechlorethamine in the presence of DNA (C).

Scheme 1

Nitrogen mustard-mediated formation of DNA-protein cross-links.

Scheme 2

Experimental scheme for biotin capture methodology used to isolate DNA-protein cross-links from nuclear protein extracts incubated with mechlorethamine in the presence of double-stranded DNA.