ABSTRACT
Cisplatin (CP) is a common anti-tumor drug used to treat many solid tumors. The activity of CP is attributed to the formation of DNA-DNA cross-links, which consist of 1,2-intra-, 1,3-intra-, and interstrand cross-links. To better understand how each intrastrand cross-link contributes to the activity of CP, we have developed comprehensive ultraperformance liquid chromatography-selective ion monitoring (UPLC-SIM) assays to quantify 1,2-GG, 1,2-AG, 1,3-GCG, and 1,3-GTG-intrastrand cross-links. The limit of quantitation for the developed assays ranged from 5 – 50 fmol, or as low as 6 cross-links per 108 nucleotides. To demonstrate the utility of the UPLC-SIM assays, we first performed in vitro cross-link formation kinetics experiments. We confirmed 1,2-GG-intrastrand cross-links were the most abundant intrastrand cross-link and formed at a faster rate compared to 1,2-AG- and 1,3-intrastrand cross-links. Furthermore, we investigated the repair kinetics of intrastrand cross-links in CP-treated wild type and nucleotide excision repair (NER)-deficient U2OS cells. We observed slow repair of both 1,2- and 1,3-intrastrand cross-links in wild type cells, and no evidence of repair in the NER-deficient cells. Taken together, we have demonstrated that our assay is capable of accurately quantifying intrastrand cross-links in CP-treated samples and can be utilized to better understand the activity of CP.
Competing Interest Statement
The authors have declared no competing interest.
ABBREVIATIONS
- (ACN)
- Acetonitrile
- (AAS)
- Atomic absorption spectroscopy
- (AGC)
- Automatic gain control
- (CTDNA)
- Calf thymus DNA
- (CP)
- Cisplatin
- (CV)
- Column volumes
- (DMEM)
- Dulbeccos modified eagles medium
- (dsDNA)
- Double stranded DNA
- (EtOH)
- Ethanol
- (ESI)
- Electrospray ionization
- (Exo I)
- Exonuclease I
- (Exo III)
- Exonuclease III
- (Exo V)
- Exonuclease V
- (Exo VII)
- Exonuclease VIII
- (FPLC)
- Fast protein liquid chromatography
- (HPLC)
- High performance liquid chromatography
- (HPLC-MS/MS)
- High performance liquid chromatography – tandem mass spectrometry
- (HRAM)
- High resolution accurate mass
- (ICP-MS)
- Inductively coupled plasma mass spectrometry
- (IS)
- Internal standard
- (ICL)
- Interstrand cross-link
- (kDa)
- kilodalton
- (LCMS)
- Liquid chromatography – mass spectrometry
- (MS)
- Mass spectrometry
- (µg)
- microgram
- (mM)
- microliter
- (µL)
- milligram
- (mg)
- milliliter
- (mL)
- millimolar
- (mMol)
- millimole
- (NEB)
- New England Biolabs
- (NucP1)
- Nuclease P1
- (NucS1)
- Nuclease S1
- (NER)
- Nucleotide excision repair
- (Pt)
- Platinum
- (Quick CIP)
- Quick calf intestinal alkaline phosphatase
- (rcf)
- Relative centrifugal force
- (NaCl)
- Sodium chloride
- (NaClO4)
- Sodium perchlorate
- (TBE)
- Tris-borate-EDTA
- (UPLC-PRM)
- Ultra performance liquid chromatography – parallel reaction monitoring
- (UPLC-SIM)
- Ultra performance liquid chromatography – selective ion monitoring
- (UP)
- Ultrapure
- (UV)
- Ultraviolet