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Detection and quantification of melphalan-DNA adducts at the single cell
level in hematopoietic tumor cells
AJ Frank, SJ Proctor and MJ Tilby
Leukemia Research Fund Unit, University of Newcastle, Upon Tyne, England.
Bifunctional alkylating agents, such as melphalan, are widely used in the
treatment of hematological malignancies. The effects of these drugs on
particular types of hematological cells and the causes of treatment failure
are poorly understood. The aim of this work was to establish an ability to
measure the extent to which melphalan reacts with the DNA of individual
tumor cells, thereby creating new possibilities for molecular
pharmacological studies on clinical samples. A novel approach for staining
drug-DNA adducts is described in which cells were embedded in agarose and
then lysed. The DNA from each cell remained in an ideal state for
quantitative immunofluorescent staining using a previously described
monoclonal antibody. Immunofluorescence and DNA-Hoechst dye fluorescence
were quantified using a cooled slow scan charge coupled device camera and
image analysis procedures. Immunofluorescence of drug- treated cells from a
human leukemia cell line was partially correlated with DNA content. Mean
integrated immunofluorescence of 50 to 100 cells was dependent on drug
concentration and was linearly related to adduct levels. In these cells and
in chronic lymphocytic leukemia cells obtained from patients, there was
considerable intercell heterogeneity in apparent adduct levels. This was
also seen in peripheral blood mononuclear cells isolated from a patient
after melphalan therapy.
Volume 88,
Issue 3,
pp. 977-984,
08/01/1996
Copyright © 1996 by The American Society of Hematology
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