BubR1 is required for a sustained mitotic spindle checkpoint arrest in human cancer cells treated with tubulin-targeting pyrrolo-1,5-benzoxazepines

Intrinsic or acquired resistance to chemotherapy is a major clinical problem that has evoked the need to develop innovative approaches to predict and ultimately reverse drug resistance. A prolonged G(2)M arrest has been associated with apoptotic resistance to various microtubule-targeting agents (MTAs). In this study, we describe the functional significance of the mitotic spindle checkpoint proteins, BubR1 and Bub3, in maintaining a mitotic arrest after microtubule disruption by nocodazole and a novel series of MTAs, the pyrrolo-1,5-benzoxazepines (PBOXs), in human cancer cells. Cells expressing high levels of BubR1 and Bub3 (K562, MDA-MB-231, and HeLa) display a prolonged G(2)M arrest after exposure to MTAs. On the other hand, cells with low endogenous levels of mitotic spindle checkpoint proteins (SK-BR-3 and HL-60) transiently arrest in mitosis and undergo increased apoptosis. The phosphorylation of BubR1 correlated with PBOX-induced G(2)M arrest in four cell lines tested, indicating an active mitotic spindle checkpoint. Gene silencing of BubR1 by small interfering RNA interference reduced PBOX-induced G(2)M arrest without enhancing apoptotic efficacy. Further analysis demonstrated that PBOX-treated BubR1-depleted cells were both mononucleated and multinucleated with a polyploid DNA content, suggesting a requirement for BubR1 in cytokinesis. Taken together, these results suggest that BubR1 contributes to the mitotic checkpoint induced by the PBOXs.

STI-571 (imatinib mesylate) enhances the apoptotic efficacy of pyrrolo-1,5-benzoxazepine-6, a novel microtubule-targeting agent, in both STI-571-sensitive and -resistant Bcr-Abl-positive human chronic myeloid leukemia cells

Interactions between the Bcr-Abl kinase inhibitor STI-571 (imatinib mesylate) and a novel microtubule-targeting agent (MTA), pyrrolo-1,5-benzoxazepine (PBOX)-6, were investigated in STI-571-sensitive and -resistant human chronic myeloid leukemia (CML) cells. Cotreatment of PBOX-6 with STI-571 induced significantly more apoptosis in Bcr-Abl-positive CML cell lines (K562 and LAMA-84) than either drug alone (P < 0.01). Cell cycle analysis of propidium iodide-stained cells showed that STI-571 significantly reduced PBOX-6-induced G2M arrest and polyploid formation with a concomitant increase in apoptosis. Similar results were obtained in K562 CML cells using lead MTAs (paclitaxel and nocodazole) in combination with STI-571. Potentiation of PBOX-6-induced apoptosis by STI-571 was specific to Bcr-Abl-positive leukemia cells with no cytoxic effects observed on normal peripheral blood cells. The combined treatment of STI-571 and PBOX-6 was associated with the down-regulation of Bcr-Abl and repression of proteins involved in Bcr-Abl transformation, namely the antiapoptotic proteins Bcl-x(L) and Mcl-1. Importantly, PBOX-6/STI-571 combinations were also effective in STI-571-resistant cells. Together, these findings highlight the potential clinical benefits in simultaneously targeting the microtubules and the Bcr-Abl oncoprotein in STI-571-sensitive and -resistant CML cells.

Identification of tubulin as the molecular target of proapoptotic pyrrolo-1,5-benzoxazepines

We have demonstrated previously that certain members of a series of novel pyrrolo-1,5-benzoxazepine (PBOX) compounds potently induce apoptosis in a variety of human chemotherapy-resistant cancer cell lines and in primary ex vivo material derived from cancer patients. A better understanding of the molecular mechanisms underlying the apoptotic effects of these PBOX compounds is essential to their development as antineoplastic therapeutic agents. This study sought to test the hypothesis that proapoptotic PBOX compounds target the microtubules. We show that a representative proapoptotic PBOX compound, PBOX-6, induces apoptosis in both the MCF-7 and K562 cell lines. An accumulation of cells in G2/M precedes apoptosis in response to PBOX-6. PBOX-6 induces prometaphase arrest and causes an accumulation of cyclin B1 levels and activation of cyclin B1/CDK1 kinase in a manner similar to that of two representative antimicrotubule agents, nocodazole and paclitaxel. Indirect immunofluorescence demonstrates that both PBOX-6 and another pro-apoptotic PBOX compound, PBOX-15, cause microtubule depolymerization in MCF-7 cells. They also inhibit the assembly of purified tubulin in vitro, whereas a nonapoptotic PBOX compound (PBOX-21) has no effect on either the cellular microtubule network or on the assembly of purified tubulin. This suggests that the molecular target of the pro-apoptotic PBOX compounds is tubulin. PBOX-6 does not bind to either the vinblastine or the colchicine binding site on tubulin, suggesting that it binds to an as-yet-uncharacterised novel site on tubulin. The ability of PBOX-6 to bind tubulin and cause microtubule depolymerization confirms it as a novel candidate for antineoplastic therapy.

The pyrrolo-1,5-benzoxazepine, PBOX-6, inhibits the growth of breast cancer cells in vitro independent of estrogen receptor status and inhibits breast tumour growth in vivo

Members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds have been shown to induce apoptosis in a number of human leukemia cell lines of different haematological lineage, suggesting their potential as anti-cancer agents. In this study, we sought to determine if PBOX-6, a well characterised member of the PBOX series of compounds, is also an effective inhibitor of breast cancer growth. Two estrogen receptor (ER)-positive (MCF-7 and T-47-D) and two ER-negative (MDA-MB-231 and SK-BR-3) cell lines were examined. The 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine reduction in cell viability. PBOX-6 reduced the cell viability of all four cell lines tested, regardless of ER status, with IC(50) values ranging from 1.0 to 2.3 microM. PBOX-6 was most effective in the SK-BR-3 cells, which express high endogenous levels of the HER-2 oncogene. Overexpression of the HER-2 oncogene has been associated with aggressive disease and resistance to chemotherapy. The mechanism of PBOX-6-induced cell death was due to apoptosis, as indicated by the increased proportion of cells in the pre-G1 peak and poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, intratumoural administration of PBOX-6 (7.5 mg/kg) significantly inhibited tumour growth in vivo in a mouse mammary carcinoma model (p=0.04, n=5, Student's t-test). Thus, PBOX-6 could be a promising anti-cancer agent for both hormone-dependent and -independent breast cancers.

Selective induction of apoptosis by the pyrrolo-1,5-benzoxazepine 7-[[dimethylcarbamoyl]oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine (PBOX-6) in Leukemia cells occurs via the c-Jun NH2-terminal kinase-dependent phosphorylation and inactivation of Bcl-2 and Bcl-XL

Overexpression of the Bcl-2 proto-oncogene in tumor cells confers resistance against chemotherapeutic drugs. In this study, we describe how the novel pyrrolo-1,5-benzoxazepine compound 7-[[dimethylcarbamoyl]oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine (PBOX-6) selectively induces apoptosis in Bcl-2-overexpressing cancer cells, whereas it shows no cytotoxic effect on normal peripheral blood mononuclear cells. PBOX-6 overcomes Bcl-2-mediated resistance to apoptosis in chronic myelogenous leukemia (CML) K562 cells by the time- and dose-dependent phosphorylation and inactivation of antiapoptotic Bcl-2 family members Bcl-2 and Bcl-XL. PBOX-6 also induces Bcl-2 phosphorylation and apoptosis in wild-type T leukemia CEM cells and cells overexpressing Bcl-2. This is in contrast to chemotherapeutic agents such as etoposide, actinomycin D, and ultraviolet irradiation, whereby overexpression of Bcl-2 confers resistance against apoptosis. In addition, PBOX-6 induces Bcl-2 phosphorylation and apoptosis in wild-type Jurkat acute lymphoblastic leukemia cells and cells overexpressing Bcl-2. However, Jurkat cells containing a Bcl-2 triple mutant, whereby the principal Bcl-2 phosphorylation sites are mutated to alanine, demonstrate resistance against Bcl-2 phosphorylation and apoptosis. PBOX-6 also induces the early and transient activation of c-Jun NH2-terminal kinase (JNK) in CEM cells. Inhibition of JNK activity prevents Bcl-2 phosphorylation and apoptosis, implicating JNK in the upstream signaling pathway leading to Bcl-2 phosphorylation. Collectively, these findings identify Bcl-2 phosphorylation and inactivation as a critical step in the apoptotic pathway induced by PBOX-6 and highlight its potential as an effective antileukemic agent.

Activation of the c-Jun N-terminal kinase (JNK) signaling pathway is essential during PBOX-6-induced apoptosis in chronic myelogenous leukemia (CML) cells

The mitogen-activated protein (MAP) kinase family is activated in response to a wide variety of external stress signals such as UV irradiation, heat shock, and many chemotherapeutic drugs and leads to the induction of apoptosis. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in chronic myelogenous leukemia (CML) cells, which are resistant to many chemotherapeutic agents. In this study we have delineated part of the mechanism by which a representative compound known as PBOX-6 induces apoptosis. We have investigated whether PBOX-6 induces activation of MAP kinase signaling pathways in CML cells. Treatment of K562 cells with PBOX-6 resulted in the transient activation of two JNK isoforms, JNK1 and JNK2. In contrast, PBOX-6 did not activate the extracellular signal-regulated kinase (ERK) or p38. Apoptosis was found to occur independently of the small GTPases Ras, Rac, and Cdc42 but involved phosphorylation of the JNK substrates, c-Jun and ATF-2. Pretreatment of K562 cells with the JNK inhibitor, dicoumarol, abolished PBOX-6-induced phosphorylation of c-Jun and ATF-2 and inhibited the induced apoptosis, suggesting that JNK activation is an essential component of the apoptotic pathway induced by PBOX-6. Consistent with this finding, transfection of K562 cells with the JNK scaffold protein, JIP-1, inhibited JNK activity and apoptosis induced by PBOX-6. JIP-1 specifically scaffolds JNK, MKK7, and members of the mixed-lineage kinase (MLK) family, implicating these kinases upstream of JNK in the apoptotic pathway induced by PBOX-6 in K562 cells.

Public Value as Performance: Politicians’ and Managers’ Perspectives on the Importance of Budgetary, Accruals and Non-Financial Information

Purpose
The study contributes to the literature on public value and performance examining politicians’ and managers’ perspectives by investigating the importance they attach to the different facets of performance information (i.e. budgetary, accrual based- and non-financial information (NFI)).

Design/methodology/approach
We survey politicians and managers in all Italian municipalities of at least 80,000 inhabitants.

Findings
Overall, NFI is more appreciated than financial information (FI). Moreover, budgetary accounting is preferred to accrual accounting. Politicians’ and managers’ preferences are generally aligned.

Research limitations/implications
NFI as a measure of public value is not alternative, but rather complementary, to FI. The latter remains a fundamental element of public sector accounting due to its role in resource allocation and control.

Practical implications
The preference for NFI over FI and of budgetary over accruals accounting suggests that the current predominant emphasis on (accrual-based) financial reporting might be misplaced.

Originality/value
Public value and performance are multi-faceted concepts. They can be captured by different types of information and evaluated according to different criteria, which will also depend on the category of stakeholders or users who assesses public performance. So far, most literature has considered the financial and non-financial facets of performance as virtually separate. Similarly, in the practice, financial management tends to be decoupled from non-financial performance management. However, this research shows that only by considering their joint interactions we can achieve an accurate representation of what public value really is.

Variations in the Processing of DNA Double-Strand Breaks Along 60-MeV Therapeutic Proton Beams

PURPOSE: To investigate the variations in induction and repair of DNA damage along the proton path, after a previous report on the increasing biological effectiveness along clinically modulated 60-MeV proton beams.

METHODS AND MATERIALS: Human skin fibroblast (AG01522) cells were irradiated along a monoenergetic and a modulated spread-out Bragg peak (SOBP) proton beam used for treating ocular melanoma at the Douglas Cyclotron, Clatterbridge Centre for Oncology, Wirral, Liverpool, United Kingdom. The DNA damage response was studied using the 53BP1 foci formation assay. The linear energy transfer (LET) dependence was studied by irradiating the cells at depths corresponding to entrance, proximal, middle, and distal positions of SOBP and the entrance and peak position for the pristine beam.

RESULTS: A significant amount of persistent foci was observed at the distal end of the SOBP, suggesting complex residual DNA double-strand break damage induction corresponding to the highest LET values achievable by modulated proton beams. Unlike the directly irradiated, medium-sharing bystander cells did not show any significant increase in residual foci.

CONCLUSIONS: The DNA damage response along the proton beam path was similar to the response of X rays, confirming the low-LET quality of the proton exposure. However, at the distal end of SOBP our data indicate an increased complexity of DNA lesions and slower repair kinetics. A lack of significant induction of 53BP1 foci in the bystander cells suggests a minor role of cell signaling for DNA damage under these conditions.

Impact of fractionation on out-of-field survival and DNA damage responses following exposure to intensity modulated radiation fields

To limit toxicity to normal tissues adjacent to the target tumour volume, radiotherapy is delivered using fractionated regimes whereby the total prescribed dose is given as a series of sequential smaller doses separated by specific time intervals. The impact of fractionation on out-of-field survival and DNA damage responses was determined in AGO-1522 primary human fibroblasts and MCF-7 breast tumour cells using uniform and modulated exposures delivered using a 225 kVp x-ray source. Responses to fractionated schedules (two equal fractions delivered with time intervals from 4 h to 48 h) were compared to those following acute exposures. Cell survival and DNA damage repair measurements indicate that cellular responses to fractionated non-uniform exposures differ from those seen in uniform exposures for the investigated cell lines. Specifically, there is a consistent lack of repair observed in the out-of-field populations during intervals between fractions, confirming the importance of cell signalling to out-of-field responses in a fractionated radiation schedule, and this needs to be confirmed for a wider range of cell lines and conditions.

Using Answer Set Programming for Solving Boolean Games

Boolean games are a framework for reasoning about the rational behaviour of agents, whose goals are formalized using propositional formulas. They offer an attractive alternative to normal-form games, because they allow for a more intuitive and more compact encoding. Unfortunately, however, there is currently no general, tailor-made method available to compute the equilibria of Boolean games. In this paper, we introduce a method for finding the pure Nash equilibria based on disjunctive answer set programming. Our method is furthermore capable of finding the core elements and the Pareto optimal equilibria, and can easily be modified to support other forms of optimality, thanks to the declarative nature of disjunctive answer set programming. Experimental results clearly demonstrate the effectiveness of the proposed method.

Imaging and radiation effects of gold nanoparticles in tumour cells

Gold nanoparticle radiosensitization represents a novel technique in enhancement of ionising radiation dose and its effect on biological systems. Variation between theoretical predictions and experimental measurement is significant enough that the mechanism leading to an increase in cell killing and DNA damage is still not clear. We present the first experimental results that take into account both the measured biodistribution of gold nanoparticles at the cellular level and the range of the product electrons responsible for energy deposition. Combining synchrotron-generated monoenergetic X-rays, intracellular gold particle imaging and DNA damage assays, has enabled a DNA damage model to be generated that includes the production of intermediate electrons. We can therefore show for the first time good agreement between the prediction of biological outcomes from both the Local Effect Model and a DNA damage model with experimentally observed cell killing and DNA damage induction via the combination of X-rays and GNPs. However, the requirement of two distinct models as indicated by this mechanistic study, one for short-term DNA damage and another for cell survival, indicates that, at least for nanoparticle enhancement, it is not safe to equate the lethal lesions invoked in the local effect model with DNA damage events.

Identification and validation of the dopamine agonist bromocriptine as a novel therapy for high-risk myelodysplastic syndromes and secondary acute myeloid leukemia

Myelodysplastic syndromes (MDS) represent a broad spectrum of diseases characterized by their clinical manifestation as one or more cytopenias, or a reduction in circulating blood cells. MDS is predominantly a disease of the elderly, with a median age in the UK of around 75. Approximately one third of MDS patients will develop secondary acute myeloid leukemia (sAML) that has a very poor prognosis. Unfortunately, most standard cytotoxic agents are often too toxic for older patients. This means there is a pressing unmet need for novel therapies that have fewer side effects to assist this vulnerable group. This challenge was tackled using bioinformatic analysis of available transcriptomic data to establish a gene-based signature of the development and progression of MDS. This signature was then used to identify novel therapeutic compounds via statistically-significant connectivity mapping. This approach suggested re-purposing an existing and widely-prescribed drug, bromocriptine as a novel potential therapy in these disease settings. This drug has shown selectivity for leukemic cells as well as synergy with current therapies.

Investigating the Implications of a Variable RBE on Proton Dose Fractionation Across a Clinical Pencil Beam Scanned Spread-Out Bragg Peak

Purpose: To investigate the clinical implications of a variable relative biological effectiveness (RBE) on proton dose fractionation. Using acute exposures, the current clinical adoption of a generic, constant cell killing RBE has been shown to underestimate the effect of the sharp increase in linear energy transfer (LET) in the distal regions of the spread-out Bragg peak (SOBP). However, experimental data for the impact of dose fractionation in such scenarios are still limited.

Methods and Materials: Human fibroblasts (AG01522) at 4 key depth positions on a clinical SOBP of maximum energy 219.65 MeV were subjected to various fractionation regimens with an interfraction period of 24 hours at Proton Therapy Center in Prague, Czech Republic. Cell killing RBE variations were measured using standard clonogenic assays and were further validated using Monte Carlo simulations and parameterized using a linear quadratic formalism.

Results: Significant variations in the cell killing RBE for fractionated exposures along the proton dose profile were observed. RBE increased sharply toward the distal position, corresponding to a reduction in cell sparing effectiveness of fractionated proton exposures at higher LET. The effect was more pronounced at smaller doses per fraction. Experimental survival fractions were adequately predicted using a linear quadratic formalism assuming full repair between fractions. Data were also used to validate a parameterized variable RBE model based on linear α parameter response with LET that showed considerable deviations from clinically predicted isoeffective fractionation regimens.

Conclusions: The RBE-weighted absorbed dose calculated using the clinically adopted generic RBE of 1.1 significantly underestimates the biological effective dose from variable RBE, particularly in fractionation regimens with low doses per fraction. Coupled with an increase in effective range in fractionated exposures, our study provides an RBE dataset that can be used by the modeling community for the optimization of fractionated proton therapy.