Flavopiridol binds to duplex DNA.
Flavopiridol, the first potent cyclin-dependent kinase inhibitor to enter clinical trials, was recently found to be cytotoxic to noncycling cells. The present studies were performed to examine the hypothesis that flavopiridol, like several other antineoplastic agents that kill noncycling cells, might also interact with DNA. Consistent with this possibility, treatment of A549 human lung cancer cells with clinically achievable concentrations of flavopiridol resulted in rapid elevations of the DNA damage-responsive protein p53. In further studies, the binding of flavopiridol to DNA was examined in vitro by four independent techniques. Absorption spectroscopy revealed that addition of DNA to aqueous flavopiridol solutions resulted in a red shift of the flavopiridol lambda(max) from 311 to 344 nm, demonstrating an isosbestic point typical of changes seen with DNA-binding compounds. Reverse-phase high-performance liquid chromatography demonstrated that flavopiridol binds to genomic DNA to a similar extent as ethidium bromide and Hoechst 33258. Nuclear magnetic resonance spectroscopy revealed that DNA caused extreme broadening of flavopiridol 1H nuclear magnetic resonance signals that could be reversed by addition of ethidium bromide or by DNA melting, suggesting that flavopiridol binds to (and likely intercalates into) duplex DNA. Equilibrium dialysis demonstrated that the equilibrium dissociation constant of the flavopiridol-DNA complex (5.4+/-3.4 x 10(-4) M) was in the same range observed for binding of the intercalators doxorubicin and pyrazoloacridine to DNA. Molecular modeling confirmed the feasibility of flavopiridol intercalation into DNA and analysis of the effects of flavopiridol in the National Cancer Institute tumor cell line panel using the COMPARE algorithm demonstrated that flavopiridol most closely resembles cytotoxic antineoplastic intercalators. Collectively, these data suggest that DNA might be a second target of flavopiridol, providing a potential explanation for the ability of this agent to kill noncycling cancer cells.
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KC
Division of Medical Oncology, Mayo Clinic, Rochester, Minnesota 55905, USA.
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R H
RH
Kottke
T J
TJ
Svingen
P A
PA
Xu
K
K
Pang
Y P
YP
Hajdu
E
E
Kaufmann
S H
SH
D000166:Acridines / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism*; D047310:Apigenin; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D004247:DNA / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D004996:Ethidium / Q000494:pharmacology; D005419:Flavonoids / Q000378:metabolism* / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007364:Intercalating Agents / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010880:Piperidines / Q000378:metabolism* / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D012313:RNA / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis
D000166:Acridines; D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D005419:Flavonoids; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D010880:Piperidines; D011720:Pyrazoles; D016159:Tumor Suppressor Protein p53; C077990:alvocidib; D012313:RNA; D047310:Apigenin; D004247:DNA; D018844:Cyclin-Dependent Kinases; D004264:DNA Topoisomerases, Type I; D004996:Ethidium; C064541:NSC 366140