A filter incubation method for the determination of potentially crosslinkable sites in DNA in mammalian cells

Maurizio D'Incalci, Leszek Szmigiero, Leonard C. Erickson, John A. Hartley, Kurt W. Kohn

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

A method for the determination of DNA monoadducts capable of forming interstrand crosslinks in mammalian cells is described. Such monoadducts were produced by brief treatment of cells with cis-diamminedichloro-Pt(II) (cis-DDP), 1-(2-chloroethyl)-1-nitrosourea (ClEtNU), l-phenylalanine mustard (L-PAM), or diaziridinylbenzoquinone (AZQ). The method is an alkaline elution procedure in which the DNA from lysed cells is incubated on polycarbonate filters at pH 10 and 37°C. During this incubation, the progressive formation of interstrand crosslinking was observed in drug-treated cells. In the case of ClEtNU and AZQ, DNA strand breaks also formed, due to the presence of labile lesions in the DNA. This made quantitation of interstrand crosslinks difficult for these drugs. For cis-DDP and l-PAM, however, there was no significant production of strand breaks and the assay for interstrand crosslinks was quantifiable.

Original languageEnglish
Pages (from-to)161-165
Number of pages5
JournalAnalytical Biochemistry
Volume150
Issue number1
DOIs
Publication statusPublished - 1985

Fingerprint

diaziquone
Cells
polycarbonate
DNA
Pulse amplitude modulation
DNA Breaks
Melphalan
Pharmaceutical Preparations
Crosslinking
Assays

Keywords

  • alkaline elution
  • antineoplastic agents
  • cis-platinum
  • DNA interstrand crosslinks

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

A filter incubation method for the determination of potentially crosslinkable sites in DNA in mammalian cells. / D'Incalci, Maurizio; Szmigiero, Leszek; Erickson, Leonard C.; Hartley, John A.; Kohn, Kurt W.

In: Analytical Biochemistry, Vol. 150, No. 1, 1985, p. 161-165.

Research output: Contribution to journalArticle

D'Incalci, Maurizio ; Szmigiero, Leszek ; Erickson, Leonard C. ; Hartley, John A. ; Kohn, Kurt W. / A filter incubation method for the determination of potentially crosslinkable sites in DNA in mammalian cells. In: Analytical Biochemistry. 1985 ; Vol. 150, No. 1. pp. 161-165.
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