title: Wilms' tumor gene 1 enhances nutlin-3-induced apoptosis.
abstract: Nutlin-3, a human double minute 2 (HDM2) antagonist, induces cell cycle arrest or apoptosis by upregulating p53 in cancer cells. WT1, the product of Wilms' tumor gene 1, has been shown to interact with p53, but the effect of WT1 on nutlin-3-induced apoptosis has yet to be examined. To address this issue, we analyzed the inhibitory effect of nutlin-3 on cell growth as a function of Wt1 expression status using a Wt1-inducible U2OS cell line. In the absence of Wt1 expression, nutlin-3 induced cell cycle arrest with marginal cytotoxicity. Furthermore, upon Wt1 expression, nutlin-3 exerted a marked degree of cell death, as evidenced by the accumulation of hypo-diploid cells and LDH release. During cell death induction, cytochrome c was released into the cytosol, and caspase-9 and -3 were activated, suggesting that an intrinsic apoptotic pathway may be involved in this cell death. Consistent with this, z-VAD-Fmk, a pan-caspase inhibitor and the overexpression of BCL-XL attenuated the cell death. Nutlin-3 caused an increase in the mRNA levels of both BCL-XL and BAK, as well as their corresponding protein levels in mitochondria. In the presence of Wt1, nutlin-3-induced BCL-XL expression was attenuated while the expression of nutlin-3-induced BAK was potentiated. Collectively, these results suggest that WT1 potentiates nutlin-3-induced apoptosis by downregulating the expression of BCL-XL while upregulating that of BAK, which leads to the activation of an intrinsic apoptotic pathway.
authors.affiliation: Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea.
authors.forename: Sun-Young
authors.initials: SY
authors.lastname: Lee
authors.forename: Yun-Jeong
authors.initials: YJ
authors.lastname: Choe
authors.forename: Jik Young
authors.initials: JY
authors.lastname: Park
authors.forename: Sang Sun
authors.initials: SS
authors.lastname: Lee
authors.forename: Yoon-Hyoung
authors.initials: YH
authors.lastname: Kim
authors.forename: Seok Joon
authors.initials: SJ
authors.lastname: Shin
authors.forename: Yeun Jun
authors.initials: YJ
authors.lastname: Chung
authors.forename: Ho-Shik
authors.initials: HS
authors.lastname: Kim
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D061945:Caspase Inhibitors / Q000494:pharmacology; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D015536:Down-Regulation; D004789:Enzyme Activation; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D008928:Mitochondria; D010879:Piperazines / Q000494:pharmacology*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000096:biosynthesis; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis; D015854:Up-Regulation; D025721:WT1 Proteins / Q000096:biosynthesis* / Q000235:genetics; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000096:biosynthesis / Q000235:genetics; D051020:bcl-X Protein / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000590:Amino Acid Chloromethyl Ketones; C494772:BAK1 protein, human; C494811:BCL2L1 protein, human; D061945:Caspase Inhibitors; D007093:Imidazoles; D010879:Piperazines; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D025721:WT1 Proteins; C583751:WT1 protein, human; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051020:bcl-X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C482205:nutlin 3; D045304:Cytochromes c; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D053148:Caspase 3; D053453:Caspase 9