The cyclin-dependent kinase inhibitor flavopiridol potentiates the effects of topoisomerase I poisons by suppressing Rad51 expression in a p53-dependent manner.
The results of a phase I clinical trial of the topoisomerase I (Topo I) poison CPT-11 followed by the cyclin-dependent kinase inhibitor flavopiridol in patients with advanced solid tumors indicate that patients whose tumors were wild-type, but not mutant, for p53 obtained the most clinical benefit from this combination therapy. We elected to elucidate the mechanistic basis for this effect in isogenic-paired HCT116 colon cancer cells that were either wild-type (+/+) or null (-/-) for p53. With the combination therapy of SN-38 (the active metabolite of CPT-11) followed by flavopiridol, the induction of apoptosis was 5-fold greater in the p53+/+ cells compared with the p53-/- cells. This sequential treatment induced phosphorylation of p53 at Ser(15), which interacted with Rad51, a DNA repair protein involved in homologous recombination. Rad51 bound to p53-Ser(15) within the first 5 hours of combination therapy, and then was transcriptionally suppressed at 24 hours by flavopiridol only in p53+/+ cells. Microarray analysis also revealed suppression of Rad51 in a p53-dependent manner. Depletion of Rad51 by small interfering RNA (siRNA) sensitized both p53+/+ and p53-/- cells to SN-38-induced apoptosis with increase of gamma H2AX, a marker of DNA damage. Conversely, overexpression of Rad51 rescued p53+/+ cells from SN-->F-induced apoptosis. Because flavopiridol inhibits Cdk9, we found that inhibition of Cdk9 by DRB or by siRNA could recapitulate the flavopiridol effects, with suppression of Rad51 and induction of apoptosis only in p53+/+ cells. In conclusion, after DNA damage by Topo I poisons, flavopiridol targets homologous recombination through a p53-dependent down-regulation of Rad51, resulting in enhancement of apoptosis.
Ambrosini
Grazia
G
Laboratory of New Drug Development, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Seelman
Sharon L
SL
Qin
Li-Xuan
LX
Schwartz
Gary K
GK
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002166:Camptothecin / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D042863:Cyclin-Dependent Kinase 9 / Q000037:antagonists & inhibitors; D015536:Down-Regulation / Q000187:drug effects; D004357:Drug Synergism; D005419:Flavonoids / Q000008:administration & dosage / Q000494:pharmacology*; D045325:HCT116 Cells; D006801:Humans; D000077146:Irinotecan; D010766:Phosphorylation; D010880:Piperidines / Q000008:administration & dosage / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051135:Rad51 Recombinase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D059004:Topoisomerase I Inhibitors; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000378:metabolism*
D005419:Flavonoids; D010880:Piperidines; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; C495901:TP53 protein, human; D059004:Topoisomerase I Inhibitors; D016159:Tumor Suppressor Protein p53; C077990:alvocidib; D000077146:Irinotecan; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D002166:Camptothecin