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 (original) (raw)
Related papers
Cancer Research, 2002
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 DNAdamage-induced G 2 checkpoint most selectively in p53-defective cells, by primarily targeting Chk1. Here we show that UCN-01 prevented IRinduced 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 , ϳ10 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.
UCN-01: a Potent Abrogator of G2 Checkpoint Function in Cancer Cells With Disrupted p53
JNCI Journal of the National Cancer Institute, 1996
Background: Arrest of the cell cycle in G2 phase following DNA damage helps protect cell viability by allowing time for DNA repair before entry into mitosis (M phase). Abrogation of G 2 arrest sensitizes cells to the effects of DNA-damaging agents. UCN-01 (7-hydroxystaurosporine), a protein kinase C inhibitor that may block G 2 checkpoint regulation, has been reported to enhance the cytotoxicity of mitomycin C, a known DNA-damaging agent. Purpose: We studied the effect of UCN-01 on G 2 checkpoint control in human lymphoma CA46 cells, whose sensitivity to various DNA-damaging agents and G2 response to DNA damage have been characterized. We also assessed the ability of UCN-01 to enhance the cytotoxicity of y irradiation in CA46 cells and human colon carcinoma HT-29 cells, both of which are mutant for p53 function. The influence of p53 function on UCN-01mediated abrogation of the G 2 checkpoint and enhancement of DNA-damaging agent cytotoxicity was studied in transfected human breast carcinoma MCF-7 cells that either expressed or did not express the human papillomavirus type-16 E6 protein. MCF-7 cells have normal p53 function, and the E6 protein binds p53 protein and promotes its destruction. Methods: The effect of UCN-01 on cell cycle arrest induced by y irradiation was studied in CA46 cells and in transfected MCF-7 cells by use of flow cytometry. A histone HI phosphorylation assay was employed to measure cyclin Bl/Cdc2 kinase activity in extracts derived from irradiated and nonirradiated CA46 cells that had been either treated or not treated with UCN-01; the phosphorylation status of Cdc2 kinase protein in the same extracts was determined by use of western blotting. The effect of UCN-01 on the cytotoxicity of y irradiation in CA46 and HT-29 cells was determined by use of MTT (thiazolyl blue) and clonogenic (colony-forming) assays, respectively; a clonogenic assay was also used to measure the effect of UCN-01 on the cytotoxicity of cisplatin in transfected and nontransfected MCF-7 cells.
Clinical cancer …, 1997
Our goal was to determine the cytotoxicity of 7-OHhydroxystaurosporine (UCN-O1) as a single agent and in combination withcis-diamminedichboroplatinum(II)(CDDP) in a panel of ovarian carcinoma cells. We sought to examine the role of p53 gene function and alterations in cell cycle progression or other mechanisms of action of UCN-01 in. eluding perturbation of the apoptosis pathway mediated by NF-icB and Bcl-2/Bax. Cytotoxicity was determined from dose-response curves established by the Alamar blue vital dye indicator assay. Restoration of wild-type p53 in a p53 null cell line, SKOV 3, was achieved by transfection of a p53 expression vector. Cell cycle distribution was measured by fluorescence-activated cell sorting analysis of ethidium bromide-stained nuclei. Apoptosis was measured by quantitative fluorescence microscopy. NF-KB DNA binding activity was measured by ebectrophoretic mobility shift assay. Bcl-2 and Bax levels were determined by Western immunoblotting. UCN-01 was effective as a cytotoxic agent alone and in combination with CDDP in all cell lines studied, regardless ofp53 status. The degree of sensitization to CDDP conferred by UCN-O1, however, was found to correlate with p53 gene status. p53 wild-type cells seem to be more sensitive to the cytotoxic effects of the combination of UCN-01 + CDDP than the p53 mutant cells. This was confirmed in cells in whichp53 wild-type function was restored by transfection of p53 eDNA, but these cells are also significantly more sensitive to CDDP alone. The effects of UCN-01 on cell cycle progression also appear to bep53 dependent but may not be the primary mechanism of action. The rate of apoptosis is increased 4-fold in UCN-01 + CDDP-treated cells compared
BMC Cancer, 2013
Background: UCN-01 (7-hydroxystaurosporine), a protein kinase inhibitor, has attracted a great deal of attention as a potent antitumour agent. Several clinical trials of UCN-01 alone or in combination with other agents for different tumour types are currently underway, and some of these trials have had positive results. Hepatocellular carcinoma has high incidence rates and is associated with poor prognosis and high mortality rates. Methods: Three different hepatoma cell lines (Huh7, HepG2, and Hep3B) were treated with different concentrations of UCN-01, and the anti-tumour effects of UCN-01 were evaluated. Following UCN-01 treatment, cell growth was measured using an MTT assay, cell cycle arrest was assayed using flow cytometry, and the mechanisms of cell cycle arrest and invasion inhibition were investigated through western blotting and a Matrigel invasion assay. Results: After a 72-h UCN-01 treatment, the growth of different hepatoma cell lines was significantly inhibited in a dose-dependent manner, with IC50 values ranging from 69.76 to 222.74 nM. Flow cytometry results suggested that UCN-01 inhibits proliferation in the hepatoma cells by inducing S and G2/M phase arrest, but not G1/S arrest, which differs from previous reports that used other tumour cell lines. Western blot results illustrated that UCN-01 induces a G2/M phase arrest, regardless of the status of the p53/P21 waf1 pathway, whereas the CHK2/CDC25C pathway and the p53/p21 waf1 pathway were involved in the UCN-01-induced S phase arrest. UCN-01 remarkably inhibited Huh7 cell invasion in a time-dependent manner. Suppression of Huh7 cell invasion may be due to the down-regulation of phosphorylated β-catenin by UCN-01. Conclusions: These findings suggest that UCN-01 induces hepatoma cell growth inhibition by regulating the p53/ p21 waf1 and CHK2/CDC25 pathways. Suppression of Huh7 cell invasion by UCN-01 may be due to the downregulation of phosphorylated β-catenin. These data lend support for further studies on UCN-01 as a promising anti-HCC candidate.
Molecular Cancer Therapeutics, 2008
DNA damage induces cell cycle arrest to provide time for repair and enhance cell survival. The Chk1 inhibitor 7-hydroxystaurosporine (UCN-01) can overcome both S and G2 arrest and drive cells through a lethal mitosis. S-phase arrest induced by the topoisomerase I inhibitor SN38 results from activation of Chk1 and degradation of Cdc25A phosphatase that occurs independent of p53 status. However, p53-mediated induction of p21waf1 and repression of cyclin B prevent abrogation of S and G2 arrest, respectively. Surprisingly, incubation of MCF10A immortalized breast cells with UCN-01 fails to elevate Cdc25A protein due to p53-mediated inhibition of Cdc25A transcription. Suppression of p21waf1 in MCF10A cells overcame this transcriptional inhibition, and the S-phase-arrested cells became sensitive to UCN-01, although they now arrested in G2 as cyclin B expression remained suppressed. We also compared the response of p53 wild-type tumors to the combination of SN38 and UCN-01. In CAKI-1, U87M...
Oncogene
P53 wild-type and p53-null or mutant cells undergo a G 2-phase cell-cycle arrest in response to ionizing radiation (IR). In this study we examined the effect of heat-shock protein 90 (HSP90) inhibitor, geldanamycin (GA), on IRinduced G 2 arrest in human colon adenocarcinoma cells with different p53 status. We show that GA treatment abrogates IR-induced G 2-phase arrest in cells null or mutant for p53. Specifically, GA treatment pushed irradiated p53 signaling-defective cells into a premature mitosis characterized by aberrant mitotic figures, increased cH2AX expression and formation of micronucleated cells. Cells expressing wild-type p53 were resistant to GA-induced G 2 checkpoint abrogation. Notably, GA treatment decreased levels of G 2 regulatory proteins Wee1 and Chk1, and inhibitory phosphorylation of Cdc2, independent of p53 status. Further investigation identified p21 as the potential downstream effector of p53 that mediates resistance to G 2 checkpoint abrogation. Clonogenic survival studies demonstrated higher sensitivity to GA alone or combination IR plus GA treatment in p53 and p21-null cells. Collectively, these data demonstrate potential mechanisms through which HSP90 inhibition can enhance the effects of ionizing radiation in p53compromised cancer cells. Combination IR plus HSP90 inhibitor therapies may be particularly useful in treating cancers that lack wild-type p53.
Cancer Science, 2001
Mechanisms underlying tumor sensitivity to the antitumor agent UCN-01 (7-hydroxystaurosporine) were examined in the nude mouse model using three human tumor xenografts, two pancreatic cancers (PAN-3-JCK and CRL 1420) and a breast cancer (MX-1). UCN-01 antitumor activity was evaluated in terms of relative tumor weights in treated and untreated mice bearing the tumor xenografts. The activity of cyclin-dependent kinase 2 (CDK2), levels of p21 and p27 proteins, pRb status and cell cycle were evaluated. Induction of p21 and apoptosis were also assessed immunohistochemically in CRL 1420. UCN-01 was administered intraperitoneally at a dose of either 5 or 10 mg/kg daily for 5 days followed by a further 5 injections after an interval of 2 days. UCN-01 significantly suppressed the growth of both pancreatic cancers, but was ineffective against MX-1. p21 protein expression was markedly induced in the UCN-01-sensitive pancreatic carcinoma xenografts at both doses, but p21 induction was only evident in the UCN-01-resistant MX-1 at 10 mg/kg. MX-1 exhibited CDK2 activity that was 6-fold higher than that of pancreatic cancer strains, which may explain the resistance of MX-1 to UCN-01 despite the induction of p21 at the dose of 10 mg/kg. The UCN-01-sensitive tumors exhibited G1 arrest and increased levels of apoptosis, changes not observed in resistant MX-1. In conclusion, it appears that a determining factor of in vivo UCN-01 sensitivity involves the balance of CDK2 kinase activity and p21 protein induction, resulting in augmented pRb phosphorylation, G1 cell cycle arrest and apoptosis.
Oncogene, 1999
The p53/p21 Cip1/Waf1-dependent checkpoint control of G1/S and G2/M phases of the cell cycle in response to DNA damage is an important mechanism of genome stability maintenance in normal cells. In many tumor cells, due to frequent point mutations and deletions of p53, the stringent control of the cell cycle and apoptosis is compromised. We have examined the cell cycle control and cell death of the rat embryo ®broblast cells (REF) transformed by E1A+cHa-ras oncogenes and expressing wild type p53. Gamma-irradiation at a dosage of 6 Gy has been used to analyse the p53-dependent transactivation of the target p21 cip1/waf1 gene and the levels of activity of cyclin-dependent kinases. Our results show that the cell cycle inhibitors p21 Cip1/Waf1 and p27 KIP accumulate in response to irradiation both in REF and E1A+cHa-ras cells. In contrast to normal REF cells, the accumulation of p21 Cip1/Waf1 and p27 KIP inhibitors, however, does not lead to inhibition of Cdk2 and cyclins E, A-associated kinase activities and to a G1/S block in E1A+cHa-ras cells. It is unlikely that the lack of inhibitory function of p21 Cip1/Waf1 can be explained by its inability to bind Cdk2 and Cdk4 kinases or PCNA. Moreover, the p21 Cip1/Waf1-associated kinase activity is increased upon g-irradiation of E1A+cHa-ras cells. We suggest that inactivation of p21 Cip1/Waf1 may be accounted for by its interaction with E1A oncoproducts as the inhibitor is detected in immunoprecipitates using E1A-speci®c antibodies. During a temporary G2/M delay induced by g-irradiation, E1A+cHa-ras transformants continue DNA replication, which leads to accumulation of polyploid cells with lobulated nuclei and micronuclei. Thus, DNA damage of E1A+cHa-ras transformed cells, with a combination of functionally active wild type p53 and inactive p21 Cip1/Waf1 , contributes to formation of polyploid cells which then die due to apoptosis.