FR900482 class of anti-tumor drugs cross-links oncoprotein HMG I/Y to DNA in vivo.
BACKGROUND
Overexpression of the high-mobility group, HMG I/Y, family of chromatin oncoproteins has been implicated as a clinical diagnostic marker for both neoplastic cellular transformation and increased metastatic potential 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 oncoprotein and a potential novel mechanism through which these compounds exert their anti-tumor activity.


RESULTS
Using a modified chromatin immunoprecipitation procedure, fragments of DNA that have been covalently cross-linked by FR900482 to HMG I/Y proteins in vivo were polymerase chain reaction-amplified, isolated and characterized. 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 the 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 (Elf-1 and NFkappaB) in vivo. Our results are the first demonstration that FR900482 cross-links a number of minor groove-binding proteins in vivo and suggests that the cross-linking of the HMG I/Y oncoproteins may participate in the mode of efficacy as a chemotherapeutic agent.


CONCLUSIONS
We have illustrated that the FR class of anti-tumor antibiotics, represented in this study by FR900482, is able to produce covalent cross-links between the HMG I/Y oncoproteins and DNA in vivo. The ability of this class of compounds to cross-link the HMG I/Y proteins in the minor groove of DNA represents the first demonstration of drug-induced cross-linking of a specific cancer-related protein to DNA in living cells. We have also demonstrated that FR900482 cross-links other minor groove-binding proteins (HMG-1 and HMG-2 in the present study) in vivo; however, since HMG I/Y is the only minor groove-binding oncoprotein presently known, it is possible that these non-histone chromatin proteins are among the important in vivo targets of this family of drugs. These compounds have already been assessed as representing a compelling clinical replacement for mitomycin C due to their greatly reduced host toxicity and superior DNA interstrand cross-linking efficacy. The capacity of FR900482 to cross-link the HMG I/Y oncoprotein with nuclear DNA in vivo potentially represents a significant elucidation of the anti-tumor efficacy of this family of anticancer agents.
Beckerbauer
L
L
School of Molecular Biosciences, Biochemistry/Biophysics, Washington State University, Pullman, WA 99164-4660, USA.
Tepe
J J
JJ
Cullison
J
J
Reeves
R
R
Williams
R M
RM
D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D025741:HMGA1a Protein; D006609:High Mobility Group Proteins / Q000737:chemistry / Q000378:metabolism*; D006651:Histocytochemistry; D006801:Humans; D019169:Jurkat Cells; D016685:Mitomycin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009336:Necrosis; D015513:Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D010078:Oxazines / Q000378:metabolism / Q000494:pharmacology / Q000633:toxicity; D010750:Phosphoproteins / Q000378:metabolism; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D000970:Antineoplastic Agents; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D015513:Oncogene Proteins; D010078:Oxazines; D010750:Phosphoproteins; D014157:Transcription Factors; C053335:FR 900482; D025741:HMGA1a Protein; D016685:Mitomycin; D004247:DNA