Abstract
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.
Highlights
§§ Investigator of the Howard Hughes Medical Institute. ¶¶ To whom correspondence should be addressed
We show that UCN-01, an agent currently undergoing clinical trials for the treatment of cancer, requires the function of the Cdc2 gene product to abrogate G2 arrest induced by DNA damage
We report that UCN-01-induced G2 checkpoint abrogation is associated with the inactivation of the Wee1Hu kinase just prior to, or at least at the same time as, Cdc2 is activated and the DNA-damaged cells enter mitosis
Summary
Chemicals and Reagents—UCN-01 (NSC 638850) and fostriecin (NSC 339638) were obtained from Jill Johnson (Drug Synthesis and Chemistry Branch, National Cancer Institute, Bethesda, MD). Cell Culture—The murine mammary carcinoma FT210 cell line, which contains temperature-sensitive mutant Cdc genes, and the parental FM3A cell line, which is wild type for Cdc, were kindly provided by Dr Fuumio Hanaoka (Osaka University, Osaka, Japan). These cells were maintained at 32 °C in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Sigma). Cells were washed once with ice-cold PBS (5 ml) and treated with RNase (30 min at 37 °C, 500 units/ml; Sigma), and DNA was stained with propidium iodide (50 g/ml).
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