5 research outputs found
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>-diamminedichloroplatinumÂ(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<sup>+</sup>-MS/MS
was employed to detect 1,1-<i>cis</i>-diammine-2-(5-amino-5-carboxypentyl)Âamino-2-(2′-deoxyguanosine-7-yl)-platinumÂ(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
DNA-Reactive Protein Monoepoxides Induce Cell Death and Mutagenesis in Mammalian Cells
Although
cytotoxic alkylating agents possessing two electrophilic
reactive groups are thought to act by cross-linking cellular biomolecules,
their exact mechanisms of action have not been established. In cells,
these compounds form a mixture of DNA lesions, including nucleobase
monoadducts, interstrand and intrastrand cross-links, and DNA–protein
cross-links (DPCs). Interstrand DNA–DNA cross-links block replication
and transcription by preventing DNA strand separation, contributing
to toxicity and mutagenesis. In contrast, potential contributions
of drug-induced DPCs are poorly understood. To gain insight into the
biological consequences of DPC formation, we generated DNA-reactive
protein reagents and examined their toxicity and mutagenesis in mammalian
cells. Recombinant human <i>O</i><sup>6</sup>-alkylguanine
DNA alkyltransferase (AGT) protein or its variants (C145A and K125L)
were treated with 1,2,3,4-diepoxybutane to yield proteins containing
2-hydroxy-3,4-epoxybutyl groups on cysteine residues. Gel shift and
mass spectrometry experiments confirmed that epoxide-functionalized
AGT proteins formed covalent DPC but no other types of nucleobase
damage when incubated with duplex DNA. Introduction of purified AGT
monoepoxides into mammalian cells via electroporation generated AGT–DNA
cross-links and induced cell death and mutations at the hypoxanthine-guanine
phosphoribosyltransferase gene. Smaller numbers of DPC lesions and
reduced levels of cell death were observed when using protein monoepoxides
generated from an AGT variant that fails to accumulate in the cell
nucleus (K125L), suggesting that nuclear DNA damage is required for
toxicity. Taken together, these results indicate that AGT protein
monoepoxides produce cytotoxic and mutagenic DPC lesions within chromosomal
DNA. More generally, these data suggest that covalent DPC lesions
contribute to the cytotoxic and mutagenic effects of bis-electrophiles