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

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.

Lactic acidosis in a newborn with adrenal calcifications.

A patient is reported who presented in the newborn period with an unusual combination of congenital lactic acidosis and bilateral calcifications in the adrenal medulla, visible on standard abdominal x-ray and ultrasound examination. At birth, the proband was hypotonic and dystrophic. She developed respiratory insufficiency, cardiomegaly, and hepatomegaly and died at the age of 38 d. Examination of postmortem heart muscle revealed multiple areas of myocardial infarction with dystrophic calcifications. In the medulla of the adrenal glands, foci of necrosis and calcifications, and in the liver, multiple zones of necrosis and iron deposition were detected. Biochemical analysis in heart muscle revealed a decreased activity of complex IV of the oxidative phosphorylation (OXPHOS) and in liver a combined deficiency involving the complexes I, III, IV, and V. The findings were suggestive of a defect in biosynthesis of the mitochondrially encoded subunits of the OXPHOS complexes. Extensive analysis of the proband's mitochondrial DNA revealed neither pathogenic deletions and point mutations nor copy number alterations. Relative amounts of mitochondrial transcripts for the ribosomal mitochondrial 12S rRNA (12S) and mitochondrial 16S rRNA (16S) were significantly increased suggesting a compensatory mechanism involving the transcription machinery to low levels of translation. The underlying molecular defect has not been identified yet.