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Apoptosis was induced by treating L1210 leukaemia cells with mechlorethamine, and SW620 colorectal cells with doxorubicin. The onset and progression of apoptosis were monitored by assessing caspase activation, mitochondrial transmembrane potential, phosphatidylserine externalization, DNA fragmentation and cell morphology. In parallel, 31P magnetic resonance (MR) spectra of cell extracts were recorded. In L1210 cells, caspase activation was detected at 4 h. By 3 h, the MR spectra showed a steady decrease in NTP and NAD, and a significant build-up of fructose 1,6-bisphosphate (F-1,6-P) dihydroxyacetonephosphate and glycerol-3-phosphate, indicating modulation of glycolysis. Treatment with iodoacetate also induced a build-up of F-1,6-P, while preincubation with two poly(ADP-ribose) polymerase inhibitors, 3-aminobenzamide and nicotinamide, prevented the drop in NAD and the build-up of glycolytic intermediates. This suggested that our results were due to inhibition of glyceraldehyde-3-phosphate dehydrogenase, possibly as a consequence of NAD depletion following poly(ADP-ribose) polymerase activation. Doxorubicin treatment of the adherent SW620 cells caused cells committed to apoptosis to detach. F-1,6-P was observed in detached cells, but not in treated cells that remained attached. This indicated that our observations were not cell line- or treatment-specific, but were correlated with the appearance of apoptotic cells following drug treatment. The 31P MR spectrum of tumours responding to chemotherapy could be modulated by similar effects.

Purpose: To develop a sensitive analytical method to quantify gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and its metabolites 2',2'-difluorodeoxyuridine (dFdU) and 2',2'-difluorodeoxycytidine-5'-triphosphate (dFdCTP) simultaneously from tumour tissue.Methods: Pancreatic ductal adenocarcinoma tumour tissue from genetically engineered mouse models of pancreatic cancer (KP ( FL/FL ) C and KP ( R172H/+) C) was collected after dosing the mice with gemcitabine. (19)F NMR spectroscopy and LC-MS/MS protocols were optimised to detect gemcitabine and its metabolites in homogenates of the tumour tissue.Results: A (19)F NMR protocol was developed, which was capable of distinguishing the three analytes in tumour homogenates. However, it required at least 100 mg of the tissue in question and a long acquisition time per sample, making it impractical for use in large PK/PD studies or clinical trials. The LC-MS/MS protocol was developed using porous graphitic carbon to separate the analytes, enabling simultaneous detection of all three analytes from as little as 10 mg of tissue, with a sensitivity for dFdCTP of 0.2 ng/mg tissue. Multiple pieces of tissue from single tumours were analysed, showing little intra-tumour variation in the concentrations of dFdC or dFdU (both intra- and extra-cellular). Intra-tumoural variation was observed in the concentration of dFdCTP, an intra-cellular metabolite, which may reflect regions of different cellularity within a tumour.Conclusion: We have developed a sensitive LC-MS/MS method capable of quantifying gemcitabine, dFdU and dFdCTP in pancreatic tumour tissue. The requirement for only 10 mg of tissue enables this protocol to be used to analyse multiple areas from a single tumour and to spare tissue for additional pharmacodynamic assays. [ABSTRACT FROM AUTHOR]

Background: 5-Fluorouracil remains widely used in colorectal cancer treatment more than 40 years after its development. 19F magnetic resonance spectroscopy can be used in vivo to measure 5FU's half-life and metabolism to cytotoxic fluoronucleotides. Previous studies have shown better survival associated with longer 5FU tumour half-life. This work investigated 5FU pharmacokinetics in liver metastases of colorectal cancer.Methods: A total of 32 subjects with colorectal cancer undergoing 5FU treatment, 15 of whom had liver metastases, were examined in a 1.5T MRI scanner, using a large coil positioned over the liver. Non-localised spectra were acquired in 1-min blocks for 32 min after injection of a 5FU bolus. The 5FU half-life was measured in each subject, and averaged spectra were examined for the presence of fluoronucleotides. Associations with progression-free survival were assessed.Results: No association was observed between 5FU half-life, tumour burden and survival. Half-lives were all shorter than those associated with improved survival in the literature. Remarkably, in the group with liver metastases, high levels of fluoronucleotides were associated with poorer survival; this counterintuitive result may be due to the higher levels of fluoronucleotides (whose level is higher in tumour tissue than in normal liver) in patients with higher tumour burdens.Conclusions: It is recommended that future studies use chemical shift imaging at higher field strengths to better resolve tumour from normal liver. Non-localised spectroscopy retains prognostic potential by enabling straightforward detection of fluoronucleotides, which are present at very low concentrations distributed throughout the tissue. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the accuracy of (1)H-MR spectroscopy ((1)H-MRS) as an intervention limiting diagnostic tool for glioblastoma multiforme. GBM is the most common and aggressive primary brain tumor, with mean survival under a year. Oncological practice currently requires histopathological diagnosis before radiotherapy. Materials and Methods: Eighty-nine patients had clinical computed tomography (CT) and MR imaging and 1.5T SV SE (1)H-MRS with PRESS localization for neuroradiological diagnosis and tumor classification with spectroscopic and automated pattern recognition analysis (TE 30 ms, TR 2000 ms, spectral width 2500 Hz and 2048 data points, 128-256 signal averages were acquired, depending on voxel size (8 cm(3) to 4 cm(3)). Eighteen patients from a cohort of 89 underwent stereotactic biopsy. Results: The 18 stereotactic biopsies revealed 14 GBM, 2 grade II astrocytomas, 1 lymphoma, and 1 anaplastic astrocytoma. All 14 biopsied GBMs were diagnosed as GBM by a protocol combining an individual radiologist and an automated spectral pattern recognition program. Conclusion: In patients undergoing stereotactic biopsy combined neuroradiological and spectroscopic evaluation diagnoses GBM with accuracy that could replace the need for biopsy. We do not advocate the replacement of biopsy in all patients; instead our data suggest a specific intervention limiting role for the use of (1)H-MRS in brain tumor diagnosis. [ABSTRACT FROM AUTHOR]

Background: Local anaesthetic agents are commonly encountered in the Emergency Department (ED). Local anaesthetic toxicity leading to cardiorespiratory arrest is a rare, but potentially fatal, complication of an overdose of these agents. A recent innovation in the treatment of severe local anaesthetic toxicity has been the introduction of intravenous lipid emulsion therapy (Intralipid 20%). The aim of this study was to gauge the current level of knowledge surrounding the administration and complications associated with commonly used local anaesthetic agents. Methods: Questionnaires were distributed amongst the training grade doctors working in four Emergency Departments. Results were divided into two groups for ease of analysis. Core Trainees (CT) and Foundation Year 2 (F2) doctors were placed in one group. Specialist Registrars (SPR), Speciality Registrars (StR) and Staff Grades (SG) form the other group. Results: The results showed that less than half of the CT/F2 group knew the maximum dose of lignocaine 1%. 80% of these doctors were unable to calculate the maximum dose of lignocaine 1% for an 80 kg man, and nearly one-third would administer a toxic dose. In addition, only one out of 30 in the CT/F2 group were aware of lipid emulsion therapy. Conclusions: Those using local anaesthetic should also be able to recognise the signs and symptoms of toxicity should this occur and act accordingly. The lack of knowledge amongst the more junior staff, as demonstrated by this project, highlights failings in teaching the basics of safe practices in the ED. [ABSTRACT FROM AUTHOR]

This prospective study aims to evaluate the feasibility of DWI at 3 Tesla in patients with advanced ovarian cancer and investigate the differences in vascular signal fraction (VSF) and apparent diffusion coefficient (ADC) values between primary ovarian mass and metastatic disease. Twenty patients with suspected advanced ovarian carcinoma were enrolled in the study. High-resolution T2W FRFSE images were used to confirm the position of three marker lesions: primary ovarian mass, omental cake and peritoneal deposit. Multislice DWI was acquired in a single breath-hold using multiple b-values. The three marker lesions were outlined by an experienced radiologist on ADC and VSF maps. Ovarian lesions showed the highest ADC values. The mean ADC value for peritoneal deposits was significantly lower than for both ovarian lesions (p = 0.03) and omental cake (p = 0.03). The VSF for omental cake was significantly higher than for ovarian lesions (p = 0.01) and peritoneal deposits (p = 0.04). There was a significant positive correlation between ADC and VSF for peritoneal deposits (p = 0.04). DWI in advanced ovarian cancer is feasible at 3 T. There are significant differences in baseline ADC and VSF values between ovarian cancer, omental cake and peritoneal deposits that may explain the mixed treatment response that occurs at different disease sites. [ABSTRACT FROM AUTHOR]

Mutations affecting the Schizosaccharomyces pombe cAMP phosphodiesterase (PDE) gene cgs2+ were identified in a screen for suppressors of mutant alleles of the adenylate cyclase gene (git2+/eyr1+), which encode catalytically active forms of the enzyme that cannot be stimulated by extracellular glucose signaling. These mutations suppress both the g/t2- mutant alleles used in the suppressor selection and mutations in git1+, git3+, git5+, git+, git10+, and git11+, which are all required for adenylate cyclase activation. Notably, these cgs2 mutant alleles fail to suppress mutations in gpa2+, which encodes the Get subunit of a heterotrimeric G protein required for adenylate cyclase activation, although the previously identified cgs2-2allele does suppress loss of gpa2+. Further analysis of the cgs2-s1 allele reveals a synthetic interaction with the gpa2R176H-activated allele, with respect to derepression of fbp1-lacZ transcription in glucose-starved cells. In addition, direct measurements of cAMP levels show that cgs2-s1 cells maintain normal basal cAMP levels, but are severely defective in feedback regulation upon glucose detection. These results suggest that PDE activity in S. pombe may be coordinately regulated with adenylate cyclase activity as part of the feedback regulation mechanism to limit the cAMP response to glucose detection. [ABSTRACT FROM AUTHOR]