UCN-01 inhibits p53 up-regulation and abrogates gamma-radiation-induced G(2)-M checkpoint independently of p53 by targeting both of the checkpoint kinases, Chk2 and Chk1.
UCN-01 (7-hydroxystaurosporine) is a cell-cycle checkpoint abrogator that sensitizes cells to ionizing radiation (IR) and chemotherapeutic agents. It has been shown previously that UCN-01 abrogates DNA-damage-induced G(2) checkpoint most selectively in p53-defective cells, by primarily targeting Chk1. Here we show that UCN-01 prevented IR-induced p53 up-regulation and p53 phosphorylation on serine 20, a site previously identified for Chk2 (or/and Chk1) kinase. We found that in human colon carcinoma HCT116 cells, IR treatment enhanced Chk2 kinase activity, whereas Chk1 activity remained unchanged, which suggested that UCN-01 may interrupt IR-induced p53 response by inhibiting Chk2 kinase. This conclusion is supported by in vitro kinase assays, showing that UCN-01 inhibits Chk2 immunoprecipitated from HCT116 cells (IC(50), approximately 10 nM). In addition, UCN-01 efficiently abrogated both the initiation and maintenance of IR-induced G(2) arrest in HCT116 cells and their isogenic p53 (-/-) derivative, indicating that G(2) checkpoint abrogation by UCN-01 is p53 independent. In the p53 (-/-) cells, there was no p21(Waf1/Cip1) induction nor UCN-01-induced apoptosis. Taken together, these observations indicate that UCN-01 can modulate both Chk1 and Chk2 in intact cells and enhance IR-induced apoptosis in p53-deficient, and consequently p21-deficient, cells.
Yu
Qiang
Q
Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
La Rose
JiHyun
J
Zhang
Hongliang
H
Takemura
Haruyuki
H
Kohn
Kurt W
KW
Pommier
Yves
Y
D000470:Alkaloids / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D003110:Colonic Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016195:G2 Phase / Q000187:drug effects / Q000528:radiation effects*; D005720:Gamma Rays; D006801:Humans; D008938:Mitosis / Q000187:drug effects / Q000528:radiation effects; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D011233:Precipitin Tests; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine / Q000031:analogs & derivatives; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000528:radiation effects; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
D000470:Alkaloids; D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D016159:Tumor Suppressor Protein p53; C054852:7-hydroxystaurosporine; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine