The ability to accumulate deoxyuridine triphosphate and cellular response to thymidylate synthase (TS) inhibition

Thymidylate synthase (TS) is an important enzyme catalysing the reductive methylation of dUMP to dTMP that is further metabolized to dTTP for DNA synthesis. Loss of viability following TS inhibition occurs as a consequence of depleted dTTP pools and at least in some cell lines, accumulation of dUTP and subsequent misincorporation of uracil into DNA. The expansion in dUTP pools is largely determined by the expression of the pyrophosphatase, dUTPase. Our previous work has shown that following TS inhibition the ability to accumulate dUTP was associated with an earlier growth inhibitory effect. 3 human lung tumour cell lines and HT29 human colon tumour cells transfected with dUTPase have been used to investigate the relationship between loss of viability following TS inhibition and dUTP accumulation. Cell cycle arrest typical of TS inhibition was an early event in all cell lines and occurred irrespective of the ability to accumulate dUTP or p53 function. However, a large expansion of dUTP pools was associated with mature DNA damage (4 h) and an earlier loss of viability following TS inhibition compared to cells in which dUTP pools were not expanded. In A549 cells damage to mature DNA may have been exacerbated by significantly higher activity of the excision repair enzyme, uracil-DNA glycosylase. Consistent with results using different inhibitors of TS, transfection of dUTPase into HT29 cells significantly reduced the cytotoxicity of a 24 h but not 48 h exposure to ZD9331. Although loss of viability can be mediated through dTTP deprivation alone, the uracil misincorporation pathway resulted in an earlier commitment to cell death. The relevance of this latter pathway in the clinical response to TS inhibitors deserves further investigation. © 2001 Cancer Research Campaign http://www.bjcancer.co

Deoxyuridine triphosphatase (dUTPase) expression and sensitivity to the thymidylate synthase (TS) inhibitorD9331

Uracil DNA misincorporation and misrepair of DNA have been recognized as important events accompanying thymidylate synthase (TS) inhibition. dUTPase catalyses the hydrolysis of dUTP to dUMP, thereby maintaining low intracellular dUTP. We have addressed the relationship between dUTPase expression and cellular sensitivity to TS inhibition in four human lung tumour cell lines. Sensitivity (5-day MTT assay) to the growth inhibitory effects of the non-polyglutamatable, specific quinazoline TS inhibitor ZD9331, varied up to 20-fold (IC 50 3–70 nM). TS protein expression correlated with TS activity (r2= 0.88 P= 0.05). Intracellular concentrations of drug following exposure to ZD9331 (1 μM, 24 h) varied by ~2-fold and dTTP pools decreased by > 80% in all cell lines. No clear associations across the cell lines between intracellular drug concentrations, TS activity/expression, or TTP depletion could be made. dUTPase activity varied 17-fold and correlated with dUTPase protein expression (r2= 0.94 P= 0.03). There was a striking variation in the amount of dUTP formed following exposure to ZD9331 (between 1.3 and 57 pmole 10–6cells) and was in general inversely associated with dUTPase activity. A large expansion in the dUTP pool was associated with increased sensitivity to a 24-h exposure to ZD9331 in A549 cells that have low dUTPase activity/expression. dUTPase expression and activity were elevated (approximately 3-fold) in two variants of a human lymphoblastoid cell line with acquired resistance to TS inhibitors, further suggesting an important role for this enzyme in TS inhibited cells. © 2000 Cancer Research Campaig

Measurement of the critical DNA lesions produced by antibody-directed enzyme prodrug therapy (ADEPT) in vitro, in vivo and in clinical material

An antibody-directed enzyme prodrug therapy (ADEPT) system against CEA-positive tumours is currently in phase I clinical trials. It consists of a prodrug, 4-[N,N-bis(2-iodoethyl) amino] phenoxycarbonyl L -glutamic acid (ZD2767P) and a conjugate of the F(ab')2 anti-CEA antibody A5B7 and the bacterial enzyme carboxypeptidase G2 (CPG2). ZD2767P is converted by antibody-targeted CPG2 into an active bifunctional alkylating drug (ZD2767) at the tumour site. The IC 50 value of the prodrug against the human colorectal tumour LS174T cell line was 55 ± 9 μM following a 1 h exposure. In contrast, co-incubation of ZD2767P with CPG2 resulted in 229-fold increase in activity. Using a modified comet assay, DNA interstrand cross links (ISC) were detected within 1 h of ZD2767P + CPG2 treatment and were repaired by 24 h. A clear dose–response was seen between the level of ISC, growth inhibition and ZD2767 concentration. Administration of a therapeutic dose of ZD2767P 72 h after the F(ab′)2 A5B7 conjugate to mice bearing LS147T xenografts resulted in extensive ISC in the tumour after 1 h; repair was seen at 24 h. Tumour biopsies and peripheral lymphocytes were studied in 5 patients on the ADEPT phase I clinical trial. In 4 patients no ISC were detected. These patients also demonstrated poor localization of conjugate and no tumour response was seen. However a significant level of ISC was detected in one tumour biopsy, which also showed evidence of conjugate localization and clinical response. These studies demonstrate the application of the comet assay in the measurement of ISC in vitro and in clinical material and confirm that activation of ZD2767P results in the formation of DNA crosslinks. © 2001 Cancer Research Campaign http://www.bjcancer.co

Regeneration performance of metal–organic frameworks

Notions about the reception of print fiction as well as new media texts have a strong tendency to fall back upon the dichotomy between naïve and critical reading. It is presupposed that reception will be characterized by either the one or the other. We will try to critique this dichotomy on the basis of the hypothesis that media cultural change brings with it new and hybrid textual forms, ways of reading, and patterns of reception which not lend themselves to description in simple terms of naïve or critical. We make a case for the necessity of transgressing the dominant assumptions of transactional reception theory within literary studies and instead move in the direction of what we call creative reading and media-reflexivity

Evaluating the structure and magnitude of the ash plume during the initial phase of the 2010 Eyjafjallajökull eruption using lidar observations and NAME simulations

The Eyjafjallajökull volcano in Iceland erupted explosively on 14 April 2010, emitting a plume of ash into the atmosphere. The ash was transported from Iceland toward Europe where mostly cloud-free skies allowed ground-based lidars at Chilbolton in England and Leipzig in Germany to estimate the mass concentration in the ash cloud as it passed overhead. The UK Met Office's Numerical Atmospheric-dispersion Modeling Environment (NAME) has been used to simulate the evolution of the ash cloud from the Eyjafjallajökull volcano during the initial phase of the ash emissions, 14–16 April 2010. NAME captures the timing and sloped structure of the ash layer observed over Leipzig, close to the central axis of the ash cloud. Relatively small errors in the ash cloud position, probably caused by the cumulative effect of errors in the driving meteorology en route, result in a timing error at distances far from the central axis of the ash cloud. Taking the timing error into account, NAME is able to capture the sloped ash layer over the UK. Comparison of the lidar observations and NAME simulations has allowed an estimation of the plume height time series to be made. It is necessary to include in the model input the large variations in plume height in order to accurately predict the ash cloud structure at long range. Quantitative comparison with the mass concentrations at Leipzig and Chilbolton suggest that around 3% of the total emitted mass is transported as far as these sites by small (<100 μm diameter) ash particles

A phase I trial of antibody directed enzyme prodrug therapy (ADEPT) in patients with advanced colorectal carcinoma or other CEA producing tumours

Antibody-directed enzyme prodrug therapy is a targeted therapy in which a prodrug is activated selectively at the tumour site by an enzyme, which has been targeted to the tumour by an antibody (antibody-enzyme conjugate). Previous clinical trials have shown evidence of tumour response, however, the activated drug had a long half-life, which resulted in dose-limiting myelosuppression. Also, the targeting system, although giving high tumour to blood ratios of antibody-enzyme conjugate (10 000 : 1) required administration of a clearing antibody in addition to the antibody-enzyme conjugate. The purpose of this current study therefore was to attempt tumour targeting of the antibody-enzyme conjugate without the clearing antibody, and to investigate a new prodrug (bis-iodo phenol mustard, ZD2767P) whose activated form is highly potent and has a short half-life. Twenty-seven patients were treated with antibody-directed enzyme prodrug therapy using A5CP antibody-enzyme conjugate and ZD2767P prodrug, in a dose-escalating phase I trial. The maximum tolerated dose of ZD2767P was reached at 15.5 mg m−2×three administrations with a serum carboxypeptidase G2 level of 0.05 U ml−1. Myelosuppression limited dose escalation. Other toxicities were mild. Patients' quality of life was not adversely affected during the trial as assessed by the measures used. There were no clinical or radiological responses seen in the study, but three patients had stable disease at day 56. Human anti-mouse antibody and human anti-carboxypeptidase G2 antibody were produced in response to the antibody enzyme conjugate (A5CP). The antibody-enzyme conjugate localisation data (carboxypeptidase G2 enzyme levels by HPLC on tumour and normal tissue samples, and gamma camera analysis of I-131 radiolabelled conjugate) are consistent with inadequate tumour localisation (median tumour: normal tissue ratios of antibody-enzyme conjugate of less than 1). A clearance system is therefore desirable with this antibody-enzyme conjugate or a more efficient targeting system is required. ZD2767P was shown to clear rapidly from the circulation and activated drug was not measurable in the blood. ZD2767P has potential for use in future antibody-directed enzyme prodrug therapy systems

A randomised controlled study of the effectiveness of breathing retraining exercises taught by a physiotherapist either by instructional DVD or in face-to-face sessions in the management of asthma in adults.

BACKGROUND: Asthma control is suboptimal, resulting in quality of life (QoL) impairment and costs. Breathing retraining exercises have evidence of effectiveness as adjuvant treatment, but are infrequently used. OBJECTIVES: To transfer the contents of a brief (three-session) physiotherapist-delivered breathing retraining programme to a digital versatile disc (DVD) and booklet format; to compare the effectiveness of the self-guided intervention with that of 'face-to-face' physiotherapy and usual care for QoL and other asthma-related outcomes; to perform a health economic assessment of both interventions; and to perform a process evaluation using quantitative and qualitative methods. DESIGN: Parallel-group three-arm randomised controlled trial. SETTING: General practice surgeries in the UK. PARTICIPANTS: In total, 655 adults currently receiving asthma treatment with impaired asthma-related QoL were randomly allocated to the DVD (n = 261), physiotherapist (n = 132) and control (usual care) (n = 262) arms in a 2 : 1 : 2 ratio. It was not possible to blind participants but data collection and analysis were performed blinded. INTERVENTIONS: Physiotherapy-based breathing retraining delivered through three 'face-to-face' respiratory physiotherapist sessions or a self-guided programme (DVD plus our theory-based behaviour change booklet) developed by the research team, with a control of usual care. MAIN OUTCOME MEASURES: The primary outcome measure was asthma-specific QoL, measured using the Asthma Quality of Life Questionnaire (AQLQ). Secondary outcomes included asthma symptom control [Asthma Control Questionnaire (ACQ)], psychological state [Hospital Anxiety and Depression Scale (HADS)], hyperventilation symptoms (Nijmegen questionnaire), generic QoL [EuroQol-5 Dimensions (EQ-5D)], assessments of airway physiology (spirometry) and inflammation (exhaled nitric oxide) and health resource use and costs. Assessments were carried out at baseline and at 3, 6 and 12 months post randomisation. Patient engagement and experience were also assessed using quantitative and qualitative methods. RESULTS: Primary efficacy analysis was between-group comparison of changes in AQLQ scores from baseline to 12 months in the intention-to-treat population with adjustments for prespecified covariates. Significant improvements occurred in the DVD group compared with the control group [adjusted mean difference 0.28, 95% confidence interval (CI) 0.11 to 0.44; p < 0.001] and in the face-to-face physiotherapy group compared with the control group (adjusted mean difference 0.24, 95% CI 0.04 to 0.44; p < 0.05), with equivalence between the DVD and the face-to-face physiotherapy groups (adjusted mean difference 0.04, 95% CI -0.16 to 0.24). In all sensitivity analyses, both interventions remained significantly superior to the control and equivalence between the interventions was maintained. In other questionnaire outcome measures and in the physiological measures assessed, there were no significant between-group differences. Process evaluations showed that participants engaged well with both of the active interventions, but that some participants in the DVD arm would have liked to receive tuition from a professional. Asthma health-care costs were lower in both intervention arms than in the control group, indicating 'dominance' for both of the interventions compared with the control, with lowest costs in the DVD arm. The rate of adverse events was lower in the DVD and face-to-face physiotherapy groups than in the control group. CONCLUSIONS: Only 10% of the potentially eligible population responded to the study invitation. However, breathing retraining exercises improved QoL and reduced health-care costs in adults with asthma whose condition remains uncontrolled despite standard pharmacological therapy, were engaged with well by patients and can be delivered effectively as a self-guided intervention. The intervention should now be transferred to an internet-based platform and implementation studies performed. Interventions for younger patients should be developed and trialled. TRIAL REGISTRATION: Current Controlled Trials ISRCTN88318003. FUNDING: This project was primarily funded by the NIHR Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 21, No. 53. See the NIHR Journals Library website for further project information. Additional financial support was received from Comprehensive Local Research Networks