Mass Spectrometry Based Proteomics Study of Cisplatin-Induced DNA-Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells

Xun Ming, Arnold Groehler, Erin D. Michaelson-Richie, Peter W. Villalta, Colin Campbell, Natalia Y. Tretyakova

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Platinum-based antitumor drugs such as 1,1,2,2-cis-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-cis-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.

Original languageEnglish (US)
Pages (from-to)980-995
Number of pages16
JournalChemical research in toxicology
Volume30
Issue number4
DOIs
StatePublished - Apr 17 2017

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Fibrosarcoma
Proteomics
Cisplatin
Mass spectrometry
Mass Spectrometry
DNA
Platinum
Proteins
oxaliplatin
DNA Replication
Transcription
High Mobility Group Proteins
Platinum Compounds
Peptide Elongation Factors
Cells
Carboplatin
Guanine
Chloroform
Nuclear Proteins
Phenol

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Mass Spectrometry Based Proteomics Study of Cisplatin-Induced DNA-Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells. / Ming, Xun; Groehler, Arnold; Michaelson-Richie, Erin D.; Villalta, Peter W.; Campbell, Colin; Tretyakova, Natalia Y.

In: Chemical research in toxicology, Vol. 30, No. 4, 17.04.2017, p. 980-995.

Research output: Contribution to journalArticle

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