posted on 2017-03-10, 00:00authored byXun Ming, Arnold Groehler, Erin D. Michaelson-Richie, Peter W. Villalta, Colin Campbell, Natalia Y. Tretyakova
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.