Decreased stability of DNA in cells treated with alkylating agents
- Cedars Medical Center, Miami, FL (United States)
A modified highly sensitive procedure for the evaluation of DNA damage in individual cells treated with alkylating agents is reported. The new methodology is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mg{sup 2+}. Human ovarian carcinoma cells A2780 were treated with nitrogen mustard (HN2), fixed in methanol, and stained with monoclonal antibody (MOAB) F7-26 generated against HN2-treated DNA. Binding of MOAB was measured by flow cytometry with indirect immunofluorescence. Intensive binding of MOAB to control and drug-treated cells was observed after heating in Tris buffer supplemented with MgCl{sub 2}. Thus, the presence of phosphates and MgCl{sub 2} during heating was necessary for the detection of HN2-induced changes in DNA stability. Fluorescence of HN2-treated cells decreased to background levels after treatment with single-strand-specific S{sub 1} nuclease. MOAB F7-26 interacted with single-stranded regions in DNA and did not bind to dsDNA or other cellular antigens. It is suggested that alkylation of guanines decreased the stability of the DNA molecule and increased the access of MOAB F7-26 to deoxycytidines on the opposite DNA strand.
- OSTI ID:
- 5267193
- Journal Information:
- Experimental Cell Research; (United States), Vol. 191:2; ISSN 0014-4827
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ALKYLATING AGENTS
BIOLOGICAL EFFECTS
DNA
CYTOLOGICAL TECHNIQUES
BIOLOGICAL REPAIR
CELL FLOW SYSTEMS
ENZYME IMMUNOASSAY
FLUORESCENCE
GENE AMPLIFICATION
MONOCLONAL ANTIBODIES
NITROGEN MUSTARD
AMINES
ANTIBODIES
BIOASSAY
BIOLOGICAL RECOVERY
IMMUNOASSAY
LUMINESCENCE
NUCLEIC ACIDS
ORGANIC CHLORINE COMPOUNDS
ORGANIC COMPOUNDS
ORGANIC HALOGEN COMPOUNDS
REPAIR
560300* - Chemicals Metabolism & Toxicology