FR900482 class of anti-tumor drugs cross-links oncoprotein HMG I/Y to DNA in vivo

Background: Overexpression of the high-mobility group, HMG IN, family of ch romatin oncoproteins has been implicated as a clinical diagnostic marker fo r both neoplastic cellular transformation and increased metastatic potentia l of several human cancers. These minor groove DNA-binding oncoproteins are thus an attractive target for anti-tumor chemotherapy. FR900482 represents a new class of anti-tumor agents that bind to the minor groove of DNA and exhibit greatly reduced host toxicity compared to the structurally related mitomycin C class of anti-tumor drugs. We report covalent cross-linking of DNA to HMG I/Y by FR900482 in vivo which represents the first example of a covalent DNA-drug-protein cross-link with a minor groove-binding oncoprotei n and a potential novel mechanism through which these compounds exert their anti-tumor activity. Results: Using a modified chromatin immunoprecipitation procedure, fragment s of DNA that have been covalently cross-linked by FR900482 to HMG. IN prot eins in vivo were polymerase chain reaction-amplified, isolated and charact erized. The nuclear samples from control cells were devoid of DNA fragments whereas the nuclear samples from cells treated with FR900482 contained DNA fragments which were cross-linked by the drug to the minor groove-binding HMG I/Y proteins in vivo. Additional control experiments established that t he drug also cross-linked other non-oncogenic minor groove-binding proteins (HMG-1 and HMG-2) but did not cross-link major groove-binding proteins (El f-l and NF kappaB) in vivo. Our results are the first demonstration that FR 900482 cross-links a number of minor groove-binding proteins in vivo and su ggests that the cross-linking of the HMG IN oncoproteins may participate in the mode of efficacy as a chemotherapeutic agent. Conclusions: We have illustrated that the FR class of anti-tumor antibiotic s, represented in this study by FR900482, is able to produce covalent cross -links between the HMG IN oncoproteins and DNA in vivo. The ability of this class of compounds to cross-link the HMG IN proteins in the minor groove o f DNA represents the first demonstration of drug-induced cross-linking of a specific cancer-related protein to DNA in living cells. We have also demon strated that FR900482 cross-links other minor groove-binding proteins (HMG- 1 and HMG-2 in the present study) in vivo; however, since HMG IN is the onl y minor groove-binding oncoprotein presently known, it is possible that the se non-histone chromatin proteins are among the important in vivo targets o f this family of drugs. These compounds have already been assessed as repre senting a compelling clinical replacement for mitomycin C due to their grea tly reduced host toxicity and superior DNA interstrand cross-linking effica cy. The capacity of FR900482 to cross-link the HMG IN oncoprotein with nucl ear DNA in vivo potentially represents a significant elucidation of the ant i-tumor efficacy of this family of anticancer agents.