UCN-01: a Potent Abrogator of G2 Checkpoint Function in Cancer Cells With Disrupted p53 (original) (raw)
Related papers
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
Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation
Archives of Biochemistry and Biophysics, 2003
Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.
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.
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.
Cancer chemotherapy and …, 2000
To determine the ability of UCN-01 to abrogate the cell cycle arrest induced by camptothecin (CPT) in tumor cells that lack p53 function, and therefore enhance the cytotoxicity of CPT in these cells in relation to normal cells with wild-type p53. Methods: The responses of MDA-MB-231 and GI 101A breast cancer cells were compared to those of normal bovine endothelial cells. Cytotoxicity was assessed by the MTT assay, and the resulting data were modeled using median-eect analysis. Inhibition of DNA synthesis was determined by loss of [ 3 H]thymidine incorporation, and cell cycle status was determined by¯ow cytometric analysis of propidium-iodide-stained nuclei. Results: UCN-01, a speci®c inhibitor of protein kinase C (PKC) presently in clinical trials, abrogated CPT-induced activation of S and G 2 checkpoints in human MDA-MB-231 and GI 101A breast carcinoma cells, both of which are mutants for the p53 gene. This abrogation occurred with the use of sublethal doses (100 nM) of UCN-01 and correlated with the enhancement of CPT-induced cytotoxicity. Median-eect analysis showed that synergistic cytotoxic interactions existed between CPT and UCN-01 against these tumor cells. In normal cells, however, abrogation of the S phase arrest caused accumulation in G 0 /G 1 phase, perhaps by the presence of wild-type p53 activity, with no change in CPT-induced cytotoxicity. Conclusion: We have shown previously that the cytotoxicity of CPT is correlated with cell cycle response in normal and tumor cells. Low doses of CPT arrest cells in the G 2 /M phase and inhibit DNA synthesis, but higher doses cause arrest of cells in S phase. Thus modulation of events at the S and G 2 checkpoints may provide an opportunity to enhance CPT-induced cytotoxicity in tumor cells. The results of this study indicate that UCN-01 enhances the progression of tumor cells through S phase thus greatly increasing CPT-induced cytotoxicity. Normal cells, however, are able to arrest in G 0 /G 1 and thus avoid the increased toxicity induced by CPT. Our ®ndings suggest potential usefulness of combining UCN-01 in topoisomerase I inhibitor-based drug therapy for the treatment of breast cancer with a dysfunctional p53 gene. Key words Camptothecin á UCN-01 á Cell cycle á Drug synergism á Breast cancer á Endothelial cells Abbreviations BVEC bovine venular endothelial cells á cdk cyclin-dependent kinase á CI combination index á CPT camptothecin á DMSO dimethyl sulfoxide á Fa fraction aected á IC 50 concentration causing 50% inhibition of cell growth á MTT 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (thiazolyl blue) á PBS phosphate-buered saline á PKC protein kinase C á topo I DNA-topoisomerase I á UCN-01 7-hydroxystaurosporine
Determinants of Mitotic Catastrophe on Abrogation of the G2 DNA Damage Checkpoint by UCN-01
Molecular Cancer Therapeutics, 2011
Genotoxic stress such as ionizing radiation halts entry into mitosis by activation of the G2 DNA damage checkpoint. The CHK1 inhibitor 7-hydroxystaurosporine (UCN-01) can bypass the checkpoint and induce unscheduled mitosis in irradiated cells. Precisely, how cells behave following checkpoint abrogation remains to be defined. In this study, we tracked the fates of individual cells after checkpoint abrogation, focusing in particular on whether they undergo mitotic catastrophe. Surprisingly, while a subset of UCN-01–treated cells were immediately eliminated during the first mitosis after checkpoint abrogation, about half remained viable and progressed into G1. Both the delay of mitotic entry and the level of mitotic catastrophe were dependent on the dose of radiation. Although the level of mitotic catastrophe was specific for different cell lines, it could be promoted by extending the mitosis. In supporting this idea, weakening of the spindle-assembly checkpoint, by either depleting M...
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.
Cancer research, 1997
7-Hydroxystaurosporine (UCN-01) is a selective protein kinase C inhibitor in clinical trial for cancer treatment. In this study, we found that nanomolar concentrations of camptothecin (CPT), a topoisomerase I inhibitor, arrest or delay cell cycle progression during the S and G2 phases in p53 mutant human colon carcinoma HT29 cells and that UCN-01 abrogates the S-phase arrest or delay induced by CPT. Under these conditions, CPT increased cyclin A levels and cyclin A/cyclin-dependent kinase 2 activity. UCN-01 prevented the increase of cyclin A/cyclin-dependent kinase 2 activity induced by CPT and enhanced Cdc2 kinase activity. Replication protein A (RPA2) was hyperphosphorylated after CPT treatment, and this effect was also abrogated by UCN-01. UCN-01 potentiated the cytotoxicity of CPT and reduced by 6-fold the concentration of CPT required to kill 50% of the HT-29 cells, as determined by clonogenic assays. This effect was observed at concentrations of UCN-01 that alone were not cyto...
DNA damage and p53-mediated cell cycle arrest: A reevaluation
Proceedings of the National Academy of Sciences, 1996
Most mammalian cells exhibit transient delays in the G 1 and G 2 phases of the cell cycle after treatment with radiation or radiomimetic compounds. p53 is required for the arrest in G 1 , which provides time for DNA repair. Recently, a role of p53 in the G 2 ͞M transition has also been suggested. However, it has been reported that the presence of functional p53 does not always correlate with the induction of these checkpoints. To precisely assess the role of p53 in activating cell cycle checkpoints and in cell survival after radiation, we studied the response of two isogenic human fibrosarcoma cell lines differing in their p53 status (wild type or mutant). We found that when irradiated cells undergo a wild-type p53-dependent G 1 arrest, they do not subsequently arrest in G 2. Moreover, wild-type p53 cells irradiated past the G 1 checkpoint arrest in G 2 but do not delay in the subsequent G 1 phase. Furthermore, in these cell lines, which do not undergo radiation-induced apoptosis, the wild-type p53 cell line exhibited a greater radioresistance in terms of clonogenic survival. These results suggest that the two checkpoints may be interrelated, perhaps through a control system that determines, depending on the extent of the damage, whether the cell needs to arrest cell cycle progression at the subsequent checkpoint for further repair. p53 could be a crucial component of this control system.