SUNY at Albany
June 19-23, 2001
Kinetics and Specificity of Cisplatin Binding to Genomic DNA in Peripheral Blood Mononuclear Cells.
The amount of platination of DNA by cisplatin, CDDP, in peripheral blood mononuclear cells (PBMC) was investigated using atomic absorption spectroscopy (AAS). Plots showing the amount of platinum bound to DNA versus the concentration of cisplatin, [CDDP], in the incubation medium were non-linear. For [CDDP] less than ~5 mM, the amount of platinum bound to DNA was proportional to the concentration of cisplatin in the incubation medium. However, when the drug concentration is above ~5 mM, the slope of the plot of DNA-bound Pt vs. [CDDP] significantly increases. To study the effect of thiol groups on the binding of platinum to DNA, cells were treated with N-ethylmaleimide (NEM), which alkylates thiol functional groups, preventing their binding platinum. Analysis using HPLC showed that ~99% of all cellular glutathione (GSH) was modified by NEM. For sulfhydryl-blocked cells, a plot of the amount of platinum bound to DNA vs.[CDDP] is linear with a slope similar to that for thiol-competent cells at high platinum concentrations. This demonstrates that sulfhydryl groups act to block platination of DNA by cisplatin in peripheral blood mononuclear cells.
The sites of platination within a 200 nt segment of the c-jun gene were identified using the method developed by Hartley and coworkers . A biotinylated primer is hybridized to platinated DNA and extended to a platinum site on the opposite strand. After attachment of about three guanine ribonucleotides to the extended products using terminal deoxynucletidyl transferase, a double stranded linker containing the 3'-overhang sequence, CCC, is ligated to the ribo-G tailed products and 30 rounds of gene-specific PCR carried out. These lengths of the PCR-generated products indicate the locations of the platinum lesions on the original DNA. The lengths are obtained by extending a radioactive gene-specific primer to the end of each fragment and carrying out PAGE analysis and microdensitometry. Our efforts to make this multi-step analysis quantitative will be reported and discussed.
A simple kinetic model for the binding of cisplatin to cellular DNA is proposed. It takes into account passage of Pt through the cell membrane, binding of Pt to sulfhydryl sites, passage of Pt through the nuclear envelope, and binding of Pt to genomic DNA. The model shows how the amount of Pt bound to DNA depends on the cisplatin concentration in the extracellular solution, the concentration of unblocked sulfhydryl sites, and the incubation time.References and Footnotes
Bradley A. Hubbard (1), Kirk Tacka, James C. Dabrowiak, Jerry Goodisman (2), Mehmet K. Aktas, Peter D. Sadowitz, Ronald L. Dubowy and Abdul-Kader Souid (1)
State University of New York, Upstate Medical University, Department of Pediatrics, 750 East Adams Street, Syracuse, NY 13210 (1) and
Syracuse University, Department of Chemistry, 111 College PL, CST 1-014, Syracuse, NY 13244 (2)