Inhibitor 7-hydroxystaurosporine Research Articles

Abstract 4487: Design, synthesis, and biological activity of N-phenyl ureidobenzenesulfonates (PUB-SOs) as new and innovative small-molecule drugs inhibiting proteins involved in DNA repair/replication mechanisms

Abstract Our research group has developed a new family of promising anticancer agents designated as N-phenyl ureidobenzenesulfonates (PUB-SOs). PUB-SOs are based on a genuinely new molecular scaffold and pharmacophore constituted by two key aromatic moieties linked by a sulfonate bridge. PUB-SOs block the cell cycle progression in the S-phase and cause DNA double-strand breaks as confirmed by the phosphorylation of H2AX. In addition, PUB-SOs exemplified by prototypical PUB-SO referred to as SFOM-0046 activate selectively ATR-Chk1 pathway in all cell lines studied while it does not activate the ATM-Chk2 pathway. Using immunofluorescence, cell cycle analysis and cell survival assays, we showed that the combination of the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01) and SFOM-0046 provide a proof-of concept that the cytotoxicity of PUB-SOs can be synergized by relevant anticancer drugs. Accordingly, we hypothesized that PUB-SOs inhibit proteins or enzymes involved in critical DNA repair/replication mechanisms. In such a context, we designed a new structure-activity relationship study to assess the effects of the nature and position of different substituents on the aromatic ring B. More than 50 new PUB-SO derivatives were prepared so far and they exhibit antiproliferative activity on the low micromolar range on HT-1080 fibrosarcoma, HT-29 colon carcinoma, M21 skin melanoma and MCF7 breast carcinoma. They block also the cell cycle progression in the S-phase. These results show that substituents on ring B can be modulated to optimize both the antiproliferative and as well as biopharmaceutical properties. In conclusion, PUB-SOs are easily synthesized, purified and pharmacomodulated, and they are therefore promising new and innovative small-molecule drugs inhibiting proteins involved in DNA repair/replication mechanisms through a genuinely innovative molecular scaffold and pharmacophore. Citation Format: Sébastien Fortin, Hanane Moussa, Mathieu Gagné-Boulet, Jacques Lacroix, Marie-France Côté, Denis Velic, Joris Pauty, Jean-Yves Masson. Design, synthesis, and biological activity of N-phenyl ureidobenzenesulfonates (PUB-SOs) as new and innovative small-molecule drugs inhibiting proteins involved in DNA repair/replication mechanisms. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4487. doi:10.1158/1538-7445.AM2015-4487

Caspase-9 mediates Puma activation in UCN-01-induced apoptosis.

The protein kinase inhibitor 7-hydroxystaurosporine (UCN-01) is one of the most potent and frequently used proapoptotic stimuli. The BH3-only molecule of Bcl-2 family proteins has been reported to contribute to UCN-01-induced apoptosis. Here we have found that UCN-01 triggers Puma-induced mitochondrial apoptosis pathway. Our data confirmed that Akt-FoxO3a pathway mediated Puma activation. Importantly, we elucidate the detailed mechanisms of Puma-induced apoptosis. Our data have also demonstrated that caspase-9 is a decisive molecule of Puma induction after UCN-01 treatment. Caspase-9 mediates apoptosis through two kinds of feedback loops. On the one hand, caspase-9 enhances Puma activation by cleaving Bcl-2 and Bcl-xL independent of caspase-3. On the other hand, caspase-9 directly activated caspase-3 in the presence of caspase-3. Caspase-3 could cleave XIAP in an another positive feedback loop to further sensitize cancer cells to UCN-01-induced apoptosis. Therefore, caspase-9 mediates Puma activation to determine the threshold for overcoming chemoresistance in cancer cells.

Abstract 568: Analysis of p53 oligomerization and sensitivity to checkpoint abrogation of neuroblastoma.

Abstract Cell cycle checkpoints prevent cells from dividing with damaged DNA by causing cell cycle arrest or apoptosis. DNA damage-induced cell cycle arrest resulting from treatment with the topoisomerase I inhibitor SN38 can be abrogated specifically in p53-defective cells by treatment with the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). However, some p53 wild-type tumor cell lines have also been shown to be sensitive to checkpoint abrogation, suggesting that these cells have other defects in the p53 pathway that can be exploited for therapeutic purposes. Our recent studies revealed that cells that are sensitive to checkpoint abrogation lack p53 dimers and tetramers. Thus, one possible explanation for the sensitivity of p53 wildtype tumors to checkpoint abrogation is a defect in oligomerization. In the current study, we investigate the DNA damage response and UCN-01 sensitivity of three p53 wildtype neuroblastoma cell lines: SK-N-SH, SH-SY5Y, and IMR32. In order to determine the responses of these cells to DNA damage, the cells were treated with concentrations of SN38 ranging from 0-30 ng/ml. All three cell lines arrested in G2, S, or G1 depending upon SN38 concentration and displayed an increase in p53 levels with increasing SN38. Additionally, p53 was phosphorylated on serine 15 following SN38 treatment in all three cell lines, suggesting that p53 is active. In order to determine whether these cells are susceptible to UCN-01-mediated abrogation of cell cycle arrest, cell were treated with 3 ng/ml SN38 for 24 hours, followed by 25-100 nM UCN-01 for 6 and 24 hours. The SK-N-SH and IMR32 showed only slight sensitivity to the highest concentration of UCN-01 treatment as evidenced by partial abrogation of S and G2 arrest. The SH-SY5Y, however, abrogated S arrest after 6 hours at the lowest concentration of UCN-01 and abrogated G2 arrest after 24 hours. We also analyzed the oligomerization status of p53 using glutaraldehyde crosslinking. The SK-N-SH cells possess levels of p53 dimers and tetramers similar to what has previously been reported in p53 wildtype MCF10A cells. The IMR32 and SH-SY5Y, however, had extremely low levels of dimers and tetramers. Consistent with this, only SK-N-SH showed activation of p21waf1 in response to SN38 treatment. Previous studies have reported cytoplasmic sequestration as a mechanism of p53 inactivation p53 wildtype neuroblastomas, although more recent studies refute these reports. In order to determine the sub-cellular distribution of p53, we prepared nuclear and cytoplasmic extracts. Both the SK-N-SH and SH-SY5Y had primarily nuclear p53, while in IMR32, p53 was evenly distributed between the nucleus and cytoplasm. The results of this study suggest that oligomerization status alone is insufficient as an indicator of sensitivity of p53 wildtype tumors to the therapeutic combination of DNA damage agent and checkpoint inhibitor. Citation Format: Robert Lipski, Aime A. Levesque. Analysis of p53 oligomerization and sensitivity to checkpoint abrogation of neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 568. doi:10.1158/1538-7445.AM2013-568

P53 Dimers Associate with a Head-to-Tail Response Element to Repress Cyclin B Transcription

DNA damage induced by the topoisomerase I inhibitor SN38 activates cell cycle checkpoints which promote cell cycle arrest. This arrest can be abrogated in p53-defective cells by the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). Previously, we compared p53 wild-type MCF10A cells with derivatives whose p53 function was inhibited by over-expression of the tetramerization domain (MCF10A/OD) or expression of shRNA against p53 (MCF10A/Δp53). Treatment of SN38-arrested MCF10A/OD cells with UCN-01 abrogated S, but not G2 arrest, while the MCF10A/Δp53 cells abrogated both S and G2 arrest. The MCF10A/OD cells had reduced levels of cyclin B, suggesting that tetramerization of p53 is not required for repression of cyclin B gene expression. In the present study, we analyzed p53 oligomerization status using glutaraldehyde cross-linking. Following SN38 treatment, MCF10A cells contained oligomeric forms of p53 with molecular weights approximating monomers, dimers, trimers, and tetramers. However, MCF10A/OD cells possessed only monomers and dimers suggesting that these complexes may be involved in repression of cyclin B. While genes transcriptionally activated by p53 contain a consensus sequence with elements repeated in a head-to-head orientation, the cyclin B promoter contains similar elements oriented head-to-tail. Chromatin immunoprecipitation (ChIP) assays revealed that p53 associates with this head-to-tail element in both MCF10A and MCF10A/OD. Electrophoretic mobility shift assays (EMSA) using a biotin-labeled probe containing the head-to-tail element showed a shift in mobility consistent with the molecular weight of tetramers and dimers in MCF10A nuclear extract, but only the dimer in MCF10A/OD nuclear extract. Taken together, these results suggest a novel mechanism whereby p53 dimers associate with the head-to-tail element to repress cyclin B transcription.

Abstract 2220: p53 dimers associate with a head-to-head element to repress cyclin B transcription

Abstract DNA damage activates cell cycle checkpoints to promote cell cycle arrest. p53 is a major player in checkpoints by activating expression of the cdk inhibitor p21 and repressing expression of cyclin B. DNA damage-induced S and G2 arrest following treatment with the topoisomerase I inhibitor SN38 is abrogated in p53-defective cells by treatment with the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). Previously, we compared p53 wildtype MCF10A cells with derivatives whose p53 function is inhibited by overexpression of the tetramerization domain (MCF10A/OD) or overexpression of shRNA against p53 (MCF10A/αp53) (Levesque et al, Oncogene 24, 3786, 2005). Treatment of MCF10A/OD with SN38 followed by UCN-01 results in abrogation of S, but not G2, arrest, while the MCF10A/αp53 abrogated both S and G2 arrest. The S abrogation correlated with reduced p21, consistent with the requirement of p53 tetramerization for activation of p21 expression. Failure of G2 abrogation correlated with reduced cyclin B, suggesting that tetramerization of p53 is not required for repression of cyclin B expression. In the present study, we confirmed that the p53-dependent repression of cyclin B in response to DNA damage occurs at the transcriptional level by analyzing cyclin B mRNA levels. Treatment of MCF10A and MCF10A/OD with SN38 resulted in reduction of cyclin B mRNA levels whereas treatment of MCF10A/αp53 with SN38 did not. This reflects the change in protein levels following SN38 treatment. We next analyzed p53 oligomerization using glutaraldehyde crosslinking. Following SN38 treatment, MCF10A contained oligomeric forms of p53 with molecular weights approximating monomers, dimers, trimers, and tetramers. MCF10A/OD, however, possessed monomers and dimers, but lacked trimers and tetramers. These results suggest that p53 monomers or dimers are involved in repression of cyclin B. One model to explain the p53-mediated repression of cyclin B in MCF10A/OD is that p53 dimers, rather than tetramers, bind to a p53 response element in the cyclin B promoter. While genes transcriptionally activated by p53 contain a consensus sequence with elements repeated in a head-to-tail orientation, the cyclin B promoter contains similar elements oriented head-to-head. We performed both chromatin immunoprecipitation (ChIP) assays and electrophoretic mobility shift assays (EMSA) to determine whether p53 binds to this head-to-head element. ChIP assays revealed that p53 associates with this element in MCF10A and MCF10A/OD but not in MCF10A/αp53. We performed EMSA using a biotin-labeled probe containing the head-to-head element and observed shifts in mobility consistent with the molecular weight of tetramers and dimers using MCF10A nuclear extract (NE), but observed only the dimeric shift with MCF10A/OD NE, and no shift with MCF10A/αp53 NE. Taken together, these results suggest p53 dimers associate with the head-to-head element to repress cyclin B transcription. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2220. doi:1538-7445.AM2012-2220

Determinants of Mitotic Catastrophe on Abrogation of the G2 DNA Damage Checkpoint by UCN-01

Genotoxic stress such as ionizing radiation halts entry into mitosis by activation of the G(2) 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 G(1). 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 MAD2 or overexpressing the MAD2-binding protein p31(comet), suppressed mitotic catastrophe. Conversely, delaying of mitotic exit by depleting either p31(comet) or CDC20 tipped the balance toward mitotic catastrophe. These results underscore the interplay between the level of DNA damage and the effectiveness of the spindle-assembly checkpoint in determining whether checkpoint-abrogated cells are eliminated during mitosis.

Defective p53 signaling in p53 wild-type tumors attenuates p21waf1 induction and cyclin B repression rendering them sensitive to Chk1 inhibitors that abrogate DNA damage-induced S and G2 arrest

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 G(2) 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 p21(waf1) and repression of cyclin B prevent abrogation of S and G(2) 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 p21(waf1) in MCF10A cells overcame this transcriptional inhibition, and the S-phase-arrested cells became sensitive to UCN-01, although they now arrested in G(2) 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, U87MG, and SUM102, SN38 induced p21(waf1) and the cells were resistant to UCN-01. In contrast, HCT116 and MCF7 cells had markedly attenuated induction of p21(waf1) and failed to repress cyclin B. Accordingly, these cells were susceptible to UCN-01-mediated abrogation of both S and G(2) arrest. SN38 induced expression of another p53-inducible gene, 14-3-3sigma, suggesting selective dysregulation of p53 response genes. In summary, several cell lines commonly considered wild-type for p53 appear to have defects in expression of selected p53 response genes following DNA damage, and this makes them sensitive to the combination of DNA damage plus Chk1 inhibitor.

Cellular DNA repair cofactors affecting hepatitis B virus infection and replication

To investigate whether hepatitis B virus (HBV) infection activates DNA damage response and DNA repair cofactors inhibit HBV infection and replication. Human hepatocyte cell line HL7702 was studied. Immunoblotting was performed to test the expression of ataxia telangiectasia-mutated (ATM)-Rad3-related protein (ATR), p21 and the level of phosphorylation of Chk1, p53, H2AX, ATM in HBV-infected or non-infected-cells. Special short RNAi oligos was transfected to induce transient ATR knockdown in HL7702. ATR-ATM chemical inhibitors caffeine (CF) and theophylline (TP), or Chk1 inhibitor 7-hydroxystaurosporine (UCN01) was studied to determine whether they suppress cellular DNA damage response and MG132 inhibits proteasome. The ATR checkpoint pathway, responding to single-strand breaks in DNA, was activated in response to HBV infection. ATR knockdown cells decreased the HBV DNA yields, implying that HBV infection and replication could activate and exploit the activated DNA damage response. CF/TP or UCN01 reduced the HBV DNA yield by 70% and 80%, respectively. HBV abrogated the ATR-dependent DNA damage signaling pathway by degrading p21, and introduction of the p21 protein before HBV infection reduced the HBV DNA yield. Consistent with this result, p21 accumulation after MG132 treatment also sharply decreased the HBV DNA yield. HBV infection can be treated with therapeutic approaches targeting host cell proteins by inhibiting a cellular gene required for HBV replication or by restoring a response abrogated by HBV, thus providing a potential approach to the prevention and treatment of HBV infection.

H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation

Gemcitabine is a nucleoside analogue that is incorporated into replicating DNA, resulting in partial chain termination and stalling of replication forks. The histone variant H2AX is phosphorylated on Ser(139) (gamma-H2AX) and forms nuclear foci at sites of DNA damage. Here, we characterize the concentration- and time-dependent phosphorylation of H2AX in response to gemcitabine-induced stalled replication forks. The number of gamma-H2AX foci increased with time up to 2 to 6 h after exposure to gemcitabine, whereas longer exposures did not cause greater phosphorylation or increase cell death. The percentage of gamma-H2AX-positive cells increased with concentrations of gemcitabine up to 0.1 micromol/L, and gamma-H2AX was most evident in the S-phase fraction. Phosphorylation of ataxia-telangiectasia mutated (ATM) on Ser(1981) was also associated with S-phase cells and colocalized in the nucleus with phosphorylated H2AX foci after gemcitabine exposure. Chemical inhibition of ATM, ATM- and Rad3-related, and DNA-dependent protein kinase blocked H2AX phosphorylation. H2AX and ATM phosphorylation were associated with inhibition of DNA synthesis, S-phase accumulation, and activation of the S-phase checkpoint pathway (Chk1/Cdc25A/cyclin-dependent kinase 2). Exposure of previously gemcitabine-treated cultures to the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01) caused a 10-fold increase in H2AX phosphorylation, which was displayed as an even pan-nuclear staining. This increased phosphorylation was not due to apoptosis-induced DNA fragmentation and was associated with the S-phase fraction and decreased reproductive viability. Thus, H2AX becomes phosphorylated and forms nuclear foci in response to gemcitabine-induced stalled replication forks, and this is greatly increased upon checkpoint abrogation.

P21CDKN1A allows the repair of replication-mediated DNA double-strand breaks induced by topoisomerase I and is inactivated by the checkpoint kinase inhibitor 7-hydroxystaurosporine

This study provides evidence for the importance of p21(CDKN1A) for the repair of replication-mediated DNA double-strand breaks (DSBs) induced by topoisomerase I. We report that defects of p21(CDKN1A) and p53 enhance camptothecin-induced histone H2AX phosphorylation (gammaH2AX), a marker for DNA DSBs. In human colon carcinoma HCT116 cells with wild-type (wt) p53, gammaH2AX reverses after camptothecin removal. By contrast, gammaH2AX increases after camptothecin removal in HCT116 cells deficient for p53 (p53-/-) or p21(CDKN1A) (p21-/-) as the cells reach the late-S and G2 phases. Since p21-/- cells exhibit similar S-phase arrest as wt cells in response to camptothecin and aphidicolin does not abrogate the enhanced gammaH2AX formation in p21-/- cells, we conclude that enhanced gammaH2AX formation in p21-/- cells is not due to re-replication. The cell cycle checkpoint abrogator and Chk1/Chk2 inhibitor 7-hydroxystaurosporine (UCN-01) also increases camptothecin-induced gammaH2AX formation and inhibits camptothecin-induced p21(CDKN1A) upregulation in HCT116 wt cells. TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) assays demonstrate that gammaH2AX formation in late S and G2 cells following CPT treatment corresponds to DNA breaks. However, these breaks are not related to apoptotic DNA fragmentation. We propose that p21(CDKN1A) prevents the collapse of replication forks damaged by stabilized topoisomerase I cleavage complexes.

Phase I Trial of the Cyclin-Dependent Kinase Inhibitor and Protein Kinase C Inhibitor 7-Hydroxystaurosporine in Combination With Fluorouracil in Patients With Advanced Solid Tumors

Preclinical studies indicate that the cyclin-dependent kinase and protein kinase C inhibitor 7-hydroxystaurosporine (UCN-01) potentiates the cytotoxic effects of fluorouracil (FU). We designed a phase I clinical trial of FU in combination with UCN-01. FU was administered as a weekly 24-hour infusion. Doses were escalated in successive cohorts according to a modified Fibonacci design. UCN-01 was administered once every 4 weeks, immediately after disconnection from FU, at a dose of 135 mg/m(2) over 72 hours in cycle 1 and 67.5 mg/m(2) over 36 hours in subsequent cycles. FU and UCN-01 pharmacokinetics were obtained on all patients, and thymidylate synthetase (TS) activity was measured in peripheral-blood mononuclear cells by reverse-transcriptase polymerase chain reaction. We escalated the weekly FU dose to 2,600 mg/m(2) in combination with once a month infusions of UCN-01. Dose-limiting toxicity included arrhythmia and syncope. Other toxicities included hyperglycemia, headache, and nausea and vomiting. The mean maximal plasma concentration of UCN-01 was 33.5 micromol/L. There was significant interpatient variability, which correlated with plasma concentrations of alpha-1 acid glycoprotein. FU was rapidly cleared and the dose had no effect on the area under the curve of UCN-01. Changes in TS expression were detectable in peripheral-blood mononuclear cells after administration of UCN-01 but did not correlate with toxicity or activity. We observed no objective response, although seven patients had stable disease, six of whom had received prior fluoropyrimidines. The combination of weekly infusions of FU and monthly UCN-01 can be administered safely and warrants further study in phase II trials. The recommended phase II dose of FU in combination with monthly UCN-01 is 2,600 mg/m(2).

The broad-range cyclin-dependent kinase inhibitor UCN-01 induces apoptosis in colon carcinoma cells through transcriptional suppression of the Bcl-x(L) protein.

The broad-range cyclin-dependent kinase inhibitor 7-hydroxystaurosporine (UCN-01) is known to induce both a G1 cell cycle arrest and apoptosis. The mechanism of UCN-01-induced apoptosis is largely unknown. We analysed the mechanism of cytotoxicity of UCN-01 in four established colon carcinoma cell lines. The cell lines SW48 and LS513 responded to UCN-01 treatment by undergoing apoptosis in a concentration-dependent manner while the cell lines HT-29 and WiDr were completely resistant. Apoptosis in LS513 and SW48 cell lines was concomitant with the suppression of Bcl-x(L) on mRNA and protein level. In contrast, in the apoptosis-resistant cell lines, Bcl-x(L) expression was not affected by UCN-01. Stable overexpression of the Bcl-x(L) protein abrogated UCN-01-triggered apoptosis, but only partially restored growth, indicating that both cell cycle arrest and apoptosis exert the anticancer effect in a coordinated manner. The inhibition of Akt phosphorylation did not correlate with the apoptotic phenotype. UCN-01 inhibited the activating STAT3 phosphorylations on Ser727 and, notably, on Tyr705, but STAT3 did not contribute to Bcl-x(L) expression in colon carcinoma cells. Moreover, we show for the first time that UCN-01 induces apoptosis by suppression of Bcl-x(L) expression. The inhibition of this pathway is a new aspect of cytotoxic and modulatory potential of UCN-01.

Cell cycle-dependent potentiation of cisplatin by UCN-01 in non-small-cell lung carcinoma.

We evaluated the combination of UCN-01 plus cisplatin and sought to determine how the cell cycle effects of each agent affected the combined response. Cisplatin-induced DNA damage results in cell cycle arrest, primarily at the S and G(2) checkpoints, providing the opportunity for DNA damage repair prior to mitosis. Thus, strategies to enhance cisplatin cytotoxicity include attenuation of DNA damage-induced checkpoints. The cyclin-dependent kinase inhibitor 7-hydroxystaurosporine (UCN-01) can potentiate cisplatin activity, likely via abrogation of the S and G(2) checkpoints. UCN-01 has additional effects on cell cycling, including induction of an RB-associated G(1) arrest. NSCLC cell lines A549 (wt p53, wt RB), Calu1 ( p53-null, wt RB) and H596 (mt p53, RB-null) were treated with UCN-01 and/or cisplatin with two-drug treatments delivered in alternate sequences. Effects of drug treatment on cell growth, cell cycling, apoptosis and levels and phosphorylation of cell cycle-associated proteins were evaluated. The interaction between the two drugs was assessed using median effect analysis. When UCN-01 preceded cisplatin, growth inhibition was additive or less than additive, as assessed by median effect analysis. In contrast, when NSCLC cells were treated with cisplatin followed by UCN-01, the combination was synergistic. In this treatment sequence, a decrease in the proportion of cells at the G(2) checkpoint was confirmed by reduced expression of cyclins A and B and activation of Cdk1. Abrogation of the G(2) DNA damage checkpoint and apoptosis were prevalent only in cell populations treated with cisplatin followed by UCN-01 and was markedly enhanced in the cell lines with disrupted p53. These studies indicate that timing of drug administration strongly influences response to cisplatin plus UCN-01 in NSCLC cells, and this is related to the cell cycle-modulatory effects of these agents. Furthermore, this sequence combination was more effective in cell lines with dysfunctional p53. These findings support the hypothesis that checkpoint abrogation is the major mechanism of UCN-01-mediated potentiation of cisplatin cytotoxicity.

Bryostatin 1 and UCN-01 potentiate 1-beta-D-arabinofuranosylcytosine-induced apoptosis in human myeloid leukemia cells through disparate mechanisms.

The effects of the PKC activator and down-regulator bryostatin 1 and the PKC and Chk1 inhibitor 7-hydroxystaurosporine (UCN-01) were compared with respect to potentiation of 1-beta-D-arabinofuranosylcytosine (ara-C)-induced apoptosis in human myelomonocytic leukemia cells (U937). Whereas bryostatin 1 and UCN-01 both markedly enhanced ara-C-induced mitochondrial injury (e.g., cytochrome c and Smac/DIABLO release, loss of mitochondrial membrane potential), caspase activation, and apoptosis, ectopic expression of an N-terminal loop-deleted Bcl-2 mutant protein protected cells from ara-C/UCN-01- but not ara-C/bryostatin 1-mediated lethality. Conversely, ectopic expression of CrmA or dominant-negative caspase-8 abrogated potentiation of ara-C-mediated apoptosis by bryostatin 1 but not by UCN-01. Exposure of cells to ara-C and bryostatin 1 (but not UCN-01) resulted in sustained release of tumor necrosis factor (TNF) alpha; moreover, potentiation of ara-C lethality by bryostatin 1 (but not by UCN-01) was reversed by coadministration of TNF soluble receptors or the selective PKC inhibitor bisindolylmaleimide (1 microM). Finally, similar events were observed in the human promyelocytic leukemia cell line HL-60. Together, these findings suggest that potentiation of ara-C lethality in human myeloid leukemia cells by bryostatin 1 but not UCN-01 involves activation of the extrinsic, receptor-mediated apoptotic pathway, and represents a consequence of bryostatin 1-mediated release of TNF-alpha. They also argue that the mechanism by which bryostatin 1 promotes ara-C-induced mitochondrial injury, caspase activation, and apoptosis involves factors other than or in addition to PKC down-regulation or modulation of Bcl-2 phosphorylation status.

Inhibition of cyclin-dependent kinase 2 by the Chk1-Cdc25A pathway during the S-phase checkpoint activated by fludarabine: dysregulation by 7-hydroxystaurosporine.

Human myeloid leukemia ML-1 cells responded to cytostatic concentrations of fludarabine nucleoside (F-ara-A) by instituting an arrest in S-phase that involved the inhibition of cyclin-dependent kinase 2 (Cdk2). This seemed to be mediated by 1) persistent phosphorylation on the Tyr(15) residue of Cdk2 and 2) an increased association of Cdk2 with p21. S-phase arrest was also associated with an increase in Chk1 kinase activity. Concomitantly, the activity of Cdc25A phosphatase was decreased. Immunoprecipitation studies demonstrated complexes of Cdk2, Cdc25A, and Chk1. The addition of the Chk1 kinase inhibitor 7-hydroxystaurosporine (UCN-01) to F-ara-A-arrested S-phase cells resulted in a rapid decrease in the fraction of cells with an S-phase DNA content and a corresponding increase in the fraction of apoptotic cells. Under these conditions, the kinase activity of Chk1 was reduced, Cdc25A phosphatase activity was increased, the level of Tyr(15) phosphorylation of Cdk2 was reduced, and the kinase activity associated with immunoprecipitates of Cdk2 and cyclin A was reactivated. UCN-01 also had no effect on the association of p21 with Cdk2. Lastly, cells incubated with UCN-01 before F-ara-A addition did not arrest in S-phase. Thus, the DNA damage induced by F-ara-A initiated a hierarchical regulatory cascade through Chk1 and Cdc25A that resulted in Cdk2 inhibition, affecting an S-phase checkpoint that was dysregulated by UCN-01. These results suggest a mechanism by which UCN-01 enhances the cytotoxicity of agents that cause an S-phase arrest.

Potentiation of 1-β- d-arabinofuranosylcytosine-mediated mitochondrial damage and apoptosis in human leukemia cells (U937) overexpressing Bcl-2 by the kinase inhibitor 7-hydroxystaurosporine (UCN-01)

Antileukemic interactions between the nucleoside analog 1-β- d-arabinofuranosylcytosine (ara-C) and the kinase inhibitor 7-hydroxystaurosporine (UCN-01) have been examined in relation to Bcl-2 expression/phosphorylation, mitochondrial damage, caspase activation, and loss of clonogenic potential. Subsequent exposure of ara-C-pretreated U937 cells (1 μM; 6 hr) to UCN-01 (300 nM; 24 hr) resulted in marked potentiation of pro-caspase-3 and -9 cleavage/activation, poly(ADP-ribose)polymerase degradation, diminished mitochondrial membrane potential (Δψ m), enhanced cytochrome c release, reduction in the S-phase fraction, and induction of classic apoptotic morphologic features. Enforced expression of full-length Bcl-2 significantly protected cells (at 24 hr) from ara-C/UCN-01-induced caspase activation and apoptosis, but was ineffective in preventing loss of Δψ m and cytochrome c release. Ectopic expression of a Bcl-2 N-terminal phosphorylation loop-deleted protein (Bcl-2Δ 32–80) was more potent than its full-length counterpart in blocking drug-induced loss of Δψ m, caspase activation, and apoptotic morphology, but not cytochrome c release. Examination of cells at later intervals revealed that ectopic expression of Bcl-2 or Bcl-2Δ 32–80 could only delay, but not prevent, mitochondrial damage, caspase activation, and cell death induced by ara-C/UCN-01 treatment. Despite their initial ability to inhibit apoptosis, neither full-length nor truncated Bcl-2 protein restored clonogenic potential to drug-treated cells. These findings indicate that subsequent exposure of ara-C-pretreated human leukemia cells to UCN-01 potently triggers mitochondrial damage and apoptosis, and that these events are postponed but not prevented by ectopic expression of Bcl-2 or its phosphorylation loop-deleted counterpart.

Lamin B Phosphorylation by Protein Kinase Cα and Proteolysis during Apoptosis in Human Leukemia HL60 Cells

Protein phosphorylation plays an important role in signal transduction, but its involvement in apoptosis still remains unclear. In this report, the p53-null human leukemia HL60 cells were used to investigate phosphorylation and degradation of lamin B during apoptosis. We found that lamin B was phosphorylated within 1 h after addition of the DNA topoisomerase I inhibitor, camptothecin, and that lamin B phosphorylation preceded lamin B degradation and DNA fragmentation. Using a cell-free system we also found that cytosol from camptothecin-treated cells induced lamin B phosphorylation and degradation in isolated nuclei from untreated HL60 cells. Lamin B phosphorylation was prevented by the protein kinase C (PKC) inhibitor 7-hydroxystaurosporine (UCN-01) but not by the Cdc2 inhibitor, flavopiridol. Phosphorylation of lamin B was inhibited by immunodepletion of PKCalpha from activated cytosol and was restored by addition of purified PKCalpha. PKCalpha activity also increased rapidly as lamin B was phosphorylated after initiation of the apoptotic response in HL60 cells. These data suggest that lamin B is phosphorylated by PKCalpha and proteolyzed before DNA fragmentation in HL60 cells undergoing apoptosis.

Activation of PKCα Downstream from Caspases during Apoptosis Induced by 7-Hydroxystaurosporine or the Topoisomerase Inhibitors, Camptothecin and Etoposide, in Human Myeloid Leukemia HL60 Cells

We previously demonstrated that the anticancer agent and protein kinase C (PKC) inhibitor 7-hydroxystaurosporine (UCN-01) induces apoptosis independently of p53 and protein synthesis in HL60 cells. We now report the associated changes of PKC isoforms. PKCalpha, betaI, betaII, delta, and zeta activities were measured after immunoprecipitation of cytosols from UCN-01-treated HL60 cells. UCN-01 had no effect on PKCzeta and inhibited kinase activity of PKCbetaI, betaII, and delta. PKCalpha activity was initially inhibited at 1 h, and subsequently increased as cells underwent apoptosis 3 h after the beginning of UCN-01 treatment. Camptothecin (CPT) and etoposide (VP-16) also markedly enhanced PKCalpha activity during apoptosis in HL60 cells. However, CPT did not affect PKCbetaI, betaII and zeta, and activated PKCdelta. PKCalpha activation was not due to increased protein levels or proteolytic cleavage but was associated with PKCalpha autophosphorylation in vitro and increased phosphorylation in vivo. We also found that not only PKC delta but also PKC betaI was proteolytically activated in HL60 cells during apoptosis. The PKCalpha activation and hyperphosphorylation were abrogated by N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk) under conditions that abrogated apoptosis. z-VAD-fmk also prevented PKCdelta and betaI proteolytic activation. Together these findings suggest that caspases regulate PKC activity during apoptosis in HL60 cells. At least two modes of activation were observed: hyperphosphorylation for PKCalpha and proteolytic activation for PKC delta and betaI.

The Gastrin-releasing Peptide Receptor Is Rapidly Phosphorylated by a Kinase Other Than Protein Kinase C After Exposure to Agonist

Several guanine nucleotide-binding protein-coupled receptors are known to be rapidly phosphorylated after agonist exposure. In this study we show that the gastrin-releasing peptide receptor (GRP-R) is rapidly phosphorylated in response to agonist exposure. When [32P]orthophosphate-labeled cells were exposed to bombesin, the receptor was maximally phosphorylated on serine and threonine residues within 1 min. Although addition of 12-O-tetradecanoylphorbol 13-acetate also resulted in phosphorylation of the GRP-R, elimination of protein kinase C activity using the inhibitor 7-hydroxystaurosporine did not prevent bombesin-induced GRP-R phosphorylation. We conclude that a kinase other than protein kinase C is principally responsible for the rapid, agonist-induced phosphorylation of the GRP-R.