UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway that is associated with inactivation of the Wee1Hu kinase and activation of the Cdc25C phosphatase.
We have previously demonstrated that UCN-01, a potent protein kinase inhibitor currently in phase I clinical trials for cancer treatment, abrogates G2 arrest following DNA damage. Here we used murine FT210 cells, which contain temperature-sensitive Cdc2 mutations, to determine if UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway. We report that UCN-01 cannot induce mitosis in DNA-damaged FT210 cells at the non-permissive temperature for Cdc2 function. Failure to abrogate G2 arrest was not due to UCN-01-inactivation at the elevated temperature because parental FM3A cells, which have wild-type Cdc2, were sensitive to UCN-01-induced G2 checkpoint abrogation. Having established that UCN-01 acted through Cdc2, we next assessed UCN-01's effect on the Cdc2-inhibitory kinase, Wee1Hu, and the Cdc2-activating phosphatase, Cdc25C. We found that Wee1Hu was indeed inactivated in UCN-01-treated cells, possibly just prior to Cdc2 activation and entry of DNA-damaged cells into mitosis. This inhibition appeared, however, to be a consequence of a further upstream action since in vitro studies revealed purified Wee1Hu was relatively resistant to UCN-01-inhibition. Consistent with such an upstream action, UCN-01 also promoted the hyperphosphorylation (activation) of Cdc25C in DNA-damaged cells. Our results suggest that UCN-01 abrogates G2 checkpoint function through inhibition of a kinase residing upstream of Cdc2, Wee1Hu, and Cdc25C, and that changes observed in these mitotic regulators are downstream consequences of UCN-01's actions.
Yu
L
L
Laboratory of Molecular Pharmacology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Orlandi
L
L
Wang
P
P
Orr
M S
MS
Senderowicz
A M
AM
Sausville
E A
EA
Silvestrini
R
R
Watanabe
N
N
Piwnica-Worms
H
H
O'Connor
P M
PM
D000470:Alkaloids / Q000494:pharmacology*; D000475:Alkenes / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016195:G2 Phase / Q000187:drug effects*; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011090:Polyenes; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011753:Pyrones; D019311:Staurosporine / Q000494:pharmacology / Q000031:analogs & derivatives; D014407:Tumor Cells, Cultured; D020687:cdc25 Phosphatases
D000470:Alkaloids; D000475:Alkenes; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; D011090:Polyenes; D011753:Pyrones; C054852:7-hydroxystaurosporine; D011505:Protein-Tyrosine Kinases; C499348:Wee1 protein, mouse; D016203:CDC2 Protein Kinase; C517010:Cdc25c protein, mouse; D020687:cdc25 Phosphatases; D019311:Staurosporine; C040313:fostriecin