A small molecule inhibitor of monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA) inhibits repair of interstrand DNA cross-link, enhances DNA double strand break, and sensitizes cancer cells to cisplatin.
Small molecule inhibitors of proliferating cell nuclear antigen (PCNA)/PCNA interacting protein box (PIP-Box) interactions, including T2 amino alcohol (T2AA), inhibit translesion DNA synthesis. The crystal structure of PCNA in complex with T2AA revealed that T2AA bound to the surface adjacent to the subunit interface of the homotrimer of PCNA in addition to the PIP-box binding cavity. Because this site is close to Lys-164, which is monoubiquitinated by RAD18, we postulated that T2AA would affect monoubiquitinated PCNA interactions. Binding of monoubiquitinated PCNA and a purified pol η fragment containing the UBZ and PIP-box was inhibited by T2AA in vitro. T2AA decreased PCNA/pol η and PCNA/REV1 chromatin colocalization but did not inhibit PCNA monoubiquitination, suggesting that T2AA hinders interactions of pol η and REV1 with monoubiquitinated PCNA. Interstrand DNA cross-links (ICLs) are repaired by mechanisms using translesion DNA synthesis that is regulated by monoubiquitinated PCNA. T2AA significantly delayed reactivation of a reporter plasmid containing an ICL. Neutral comet analysis of cells receiving T2AA in addition to cisplatin revealed that T2AA significantly enhanced formation of DNA double strand breaks (DSBs) by cisplatin. T2AA promoted colocalized foci formation of phospho-ATM and 53BP1 and up-regulated phospho-BRCA1 in cisplatin-treated cells, suggesting that T2AA increases DSBs. When cells were treated by cisplatin and T2AA, their clonogenic survival was significantly less than that of those treated by cisplatin only. These findings show that the inhibitors of monoubiquitinated PCNA chemosensitize cells by inhibiting repair of ICLs and DSBs.
Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee 38138.
Akira
A
Inoue
Department of Medical Life Science, Graduate School of Medical Life Science, Yokohama City University, Yokohama 230-0045, Japan.
Sotaro
S
Kikuchi
Department of Medical Life Science, Graduate School of Medical Life Science, Yokohama City University, Yokohama 230-0045, Japan.
Asami
A
Hishiki
Protein Production Facility, St. Jude Children's Research Hospital, Memphis, Tennessee 38138.
Youming
Y
Shao
Protein Production Facility, St. Jude Children's Research Hospital, Memphis, Tennessee 38138.
Richard
R
Heath
Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee 38138.
Benjamin J
BJ
Evison
Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee 38138.
Marcelo
M
Actis
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109.
Christine E
CE
Canman
Department of Medical Life Science, Graduate School of Medical Life Science, Yokohama City University, Yokohama 230-0045, Japan.
Hiroshi
H
Hashimoto
Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee 38138. Electronic address: naoaki.fujii@stjude.org.
Naoaki
N
Fujii
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D019556:COS Cells; D002522:Chlorocebus aethiops; D002945:Cisplatin / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D004260:DNA Repair / Q000187:drug effects*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism*; D010647:Phenyl Ethers / Q000737:chemistry / Q000494:pharmacology*; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000378:metabolism*; D011412:Propanolamines / Q000737:chemistry / Q000494:pharmacology*; D054852:Small Molecule Libraries / Q000494:pharmacology*
D000970:Antineoplastic Agents; D010647:Phenyl Ethers; D018809:Proliferating Cell Nuclear Antigen; D011412:Propanolamines; D054852:Small Molecule Libraries; D002945:Cisplatin
Crystallography; DNA Damage; Interstrand Cross-link; PCNA; Small Molecules; Structural Biology; Ubiquitination