Real-time monitoring of the membrane-binding and insertion properties of the cholesterol-dependent cytolysin anthrolysin O from Bacillus anthracis.

Bacillus anthracis has recently been shown to secrete a potently hemolytic/cytolytic protein that has been designated anthrolysin O (ALO). In this work, we initiated a study of this potential anthrax virulence factor in an effort to understand the membrane-binding properties of this protein. Recombinant anthrolysin O (rALO35-512) and two N-terminally truncated versions of ALO (rALO390-512 and rALO403-512) from B. anthracis were overproduced in Escherichia coli and purified to homogeneity. The role of cholesterol in the cytolytic activity of ALO was probed in cellular cholesterol depletion assays using mouse and human macrophage-like lines, and also Drosophila Schneider 2 cells. Challenging the macrophage cells with rALO35-512, but not rALO390-512 or rALO403-512, resulted in cell death by lysis, with this cytolysis being abolished by depletion of the membrane cholesterol. Drosophila cells, which contain ergosterol as their major membrane sterol, were resistant to rALO-mediated cytolysis. In order to determine the molecular mechanism of this resistance, the interaction of rALO with model membranes comprised of POPC alone, or with a variety of structurally similar sterols including ergosterol, was probed using Biacore. Both rALO35-512 and rALO403-512 demonstrated robust binding to model membranes composed of POPC and cholesterol, with amount of protein bound proportional to the cholesterol content. Ergosterol supported greatly reduced binding of both rALO35-512 and rALO403-512, whereas other sterols tested did not support binding. The rALO403-512--membrane interaction demonstrated an equilibrium dissociation constant (KD) in the low nanomolar range, whereas rALO35-512 exhibited complex kinetics likely due to the multiple events involved in pore formation. These results establish the pivotal role of cholesterol in the action of rALO. The biosensor method developed to measure ALO recognition of cholesterol in a membrane environment could be extended to provide a platform for the screening of inhibitors of other membrane-binding proteins and peptides.

Radiation enhancement by gemcitabine-mediated cell cycle modulations.

The purpose of this study was to investigate the exact dose dependency and time dependency of the radiation-enhancing effect of gemcitabine (2',2'difluoro desoxycytidine [dFdC]) in in vitro experiments (HeLa cells: cancer of the uterine cervix, #4197 cells: oropharyngeal squamous cell carcinoma), and to correlate this effect with the underlying changes in cell cycle distribution. Cell viability was determined fluorometrically after exposure to dFdC (0-20.0 micro mol/l), irradiation (0-37.5 Gy), and both modalities. Combining both therapies, cells were exposed to dFdC (0-10.0 micro mol/l) for 24 hours before further treatment and irradiated (0-30 Gy) immediately afterwards with or without removal of dFdC. For cell cycle analysis by flow cytometry, cells were irradiated (0-40 Gy) or treated with dFdC (0.012-1.0 micro mol/l, 24-48 hours). Additionally, cells were exposed to dFdC (2.0 micro mol/l, 0-4 hours). Cell cycle kinetics were evaluated using bromodeoxyuridine (BrdU) (10 micro mol/l) S-phase labeling, given either 30 minutes before or in the last hour of dFdC treatment (2.0 micro mol/l, 0-6 hours). The fluorometric assay revealed that dFdC enhances radiation-induced cytotoxicity at marginally toxic or nontoxic concentrations (<37 nmol/l). Radiation resulted in the anticipated G2/M arrest already at 2 Gy. DFdC induced concentration and exposure time-dependent cell cycle changes that were better resolved using BrdU, demonstrating a pronounced S-phase arrest already at 12 nmol/l. BrdU-pulse labeling revealed that the cell cycle block occurred at the G1/S boundary. Our data reconfirm the already known radiation enhancement, the S-phase specific activities of dFdC, and the relevance of the synchronized progression of cells through the S-phase with regard to the radiosensitizing properties of low-dose dFdC. However, we could demonstrate that before progressing in the S-phase, cells were blocked and partially synchronized at the more radiosensitive G1/S boundary. Furthermore, cells progressing past the block might accumulate proapoptotic signals caused by both radiation and dFdC, which will also results in cell death.

[Combined radiotherapy and gemcitabine. Evaluation of clinical data based on experimental knowledge]. / Kombination von Radiotherapie und Gemcitabine. Bewertung der klinischen Daten auf der Basis experimenteller Erkenntnisse.

BACKGROUND: In experimental studies the nucleoside analog Gemcitabine (2',2' difluorodesoxycytidine) clearly demonstrates radiation enhancing properties. After describing the pharmacological Gemcitabine-related data and the clinical studies regarding combined radiochemotherapy and taking under consideration the in-vitro data and the results provided by animal models, this overview is aimed to draw clinically relevant conclusions, resulting in the improvement of treatment approaches. MATERIALS AND METHODS: The available literature data regarding the metabolism and the mechanism of action, the evaluation of possible schedules of administration, and combined radiochemotherapy including Gemcitabine has been reviewed. Publications reporting experimental data in vitro and in vivo as well as our own experimental results have been incorporated. RESULTS: In clinical phase I and II studies, the favorable tumor response is accompanied by a high incidence of grade III-IV toxicities whereby the maximum-tolerated dose (MTD) of the various schedules of administration used is always lower compared to the MTD of single-agent treatment. In in-vitro and in-vivo data addressing the description and the evaluation of the radiation enhancing mechanism (especially influence on cell cycle, depletion of the dATP pool, induction of apoptosis, inhibition of DNA synthesis, reduction of DNA repair) this effect is already observed with non and moderately toxic Gemcitabine concentrations and depends on drug concentration and exposure time. Independent of the fractionation effect of radiotherapy, the radiation enhancement is persistent for at most 72 hours after the end of drug exposure. Taking under consideration the single dose per day and the target volume, a prolonged infusion and/or a twice-weekly administration of Gemcitabine at low concentration each and simultaneous radiotherapy are presumably considered to resemble the experimental data. CONCLUSION: It is without doubt that data provided by clinical studies are of highest relevance for the evaluation of an optimized radiochemotherapy with Gemcitabine. However, although it is often difficult to transfer experimental data into the clinical situation, these data offer the possibility to develop an improved schedule of administration in patient treatment based on rational evidence in tumor biology.