USING CELL-FRACTIONATION AND PHOTOCHEMICAL CROSS-LINKING METHODS TO DETERMINE THE CELLULAR-BINDING SITE(S) OF THE ANTITUMOR DRUG DMP-840

In order to understand its mechanism of action we have begun an effort to better define the cellular target of action of the experimental an titumor agent DMP 840 (NSC D640430; (R,R)-2,2'-(1,2-ethanediylbis(imin o-(1-methyl-2,1- ethanediyl)))-bis(5-nitro-1H-benz(de)isoquinoline- 1, 3-(2H)-dione) dimethanesulfonate). Using a combination of gentle cell fractionation procedures and a previously unidentified photochemical c rosslinking reaction, we have shown that after the drug is added to cu ltured Clone A cells, more than 80% of the drug that is found associat ed with cells partitions to the chromatin-containing structural framew ork of the cell and that the primary target after crosslinking with 36 0 nm light is DNA. While DMP 840 photoreacts quite efficiently with pu rified RNA in vitro, no photoattachment of the drug to RNA was observe d in cells. In vitro photochemical studies also reveal that while GC-r ich DNA is a preferred target for drug interaction, AT-rich DNA is mor e active in the photochemical crosslinking reaction. These results sug gest that DMP 840 probably kills cells by interfering with DNA-metabol ic processes, and that the drug and its derivatives are likely to be u seful photoactive molecular probes for investigating higher order chro matin structures in cells.