Analysis of proteins cross-linked to DNA after treatment of cells with formaldehyde, chromate, and cis-diamminedichloroplatinum(II)

Mol Toxicol. Winter 1989;2(1):11-26.

Abstract

The proteins cross-linked to the DNA of cultured Chinese hamster ovary cells after exposure to cis-diamminedichloroplatinum(II) (cis-Pt), chromate, and formaldehyde were compared by two-dimensional (2D) gel electrophoresis, immunoblotting, and centrifugal assays that measured cross-link stability. Chromate and cis-Pt cross-linked seven of the same nonhistone proteins, such as actin, to DNA. In contrast, formaldehyde selectively formed histone-DNA cross-links. Immunoblotting experiments showed that all three chemicals cross-linked a 97-kDa nuclear protein to the DNA despite their different chemical reactivity with DNA and proteins. The chromate- and cis-Pt-induced cross-links were disrupted by thiourea, 2-mercaptoethanol, and EDTA, indicating that the metal could be chemically displaced from the cross-links. The formaldehyde-induced complexes required degradation with DNase 1 for the resolution of histones on 2D gels and were not chemically labile like the metal-induced cross-links. The agents and methodology used here could be applied to the study of additional nuclear proteins that bind or reside near the DNA.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cell Line
  • Cell Survival / drug effects
  • Chromates / pharmacology*
  • Cisplatin / pharmacology*
  • Cross-Linking Reagents*
  • DNA / drug effects*
  • Electrophoresis, Gel, Two-Dimensional
  • Formaldehyde / pharmacology*
  • Immunoblotting
  • Nuclear Proteins / drug effects
  • Potassium Compounds*
  • Proteins / drug effects*
  • Ultracentrifugation

Substances

  • Chromates
  • Cross-Linking Reagents
  • Nuclear Proteins
  • Potassium Compounds
  • Proteins
  • Formaldehyde
  • potassium chromate(VI)
  • DNA
  • Cisplatin