Detection and quantification of melphalan-DNA adducts at the single cell level in hematopoietic tumor cells

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.