Study of the X-ray radiation interaction with a multislit collimator for the creation of microbeams in radiation therapy.

Microbeam radiation therapy (MRT) is a developing radiotherapy, based on the use of beams only a few tens of micrometres wide, generated by synchrotron X-ray sources. The spatial fractionation of the homogeneous beam into an array of microbeams is possible using a multislit collimator (MSC), i.e. a machined metal block with regular apertures. Dosimetry in MRT is challenging and previous works still show differences between calculated and experimental dose profiles of 10-30%, which are not acceptable for a clinical implementation of treatment. The interaction of the X-rays with the MSC may contribute to the observed discrepancies; the present study therefore investigates the dose contribution due to radiation interaction with the MSC inner walls and radiation leakage of the MSC. Dose distributions inside a water-equivalent phantom were evaluated for different field sizes and three typical spectra used for MRT studies at the European Synchrotron Biomedical beamline ID17. Film dosimetry was utilized to determine the contribution of radiation interaction with the MSC inner walls; Monte Carlo simulations were implemented to calculate the radiation leakage contribution. Both factors turned out to be relevant for the dose deposition, especially for small fields. Photons interacting with the MSC walls may bring up to 16% more dose in the valley regions, between the microbeams. Depending on the chosen spectrum, the radiation leakage close to the phantom surface can contribute up to 50% of the valley dose for a 5 mm × 5 mm field. The current study underlines that a detailed characterization of the MSC must be performed systematically and accurate MRT dosimetry protocols must include the contribution of radiation leakage and radiation interaction with the MSC in order to avoid significant errors in the dose evaluation at the micrometric scale.

A commercial treatment planning system with a hybrid dose calculation algorithm for synchrotron radiotherapy trials.

Synchrotron Radiotherapy (SyncRT) is a preclinical radiation treatment which delivers synchrotron x-rays to cancer targets. SyncRT allows for novel treatments such as Microbeam Radiotherapy, which has been shown to have exceptional healthy tissue sparing capabilities while maintaining good tumour control. Veterinary trials in SyncRT are anticipated to take place in the near future at the Australian Synchrotron's Imaging and Medical Beamline (IMBL). However, before veterinary trials can commence, a computerised treatment planning system (TPS) is required, which can quickly and accurately calculate the synchrotron x-ray dose through patient CT images. Furthermore, SyncRT TPS's must be familiar and intuitive to radiotherapy planners in order to alleviate necessary training and reduce user error. We have paired an accurate and fast Monte Carlo (MC) based SyncRT dose calculation algorithm with EclipseTM, the most widely implemented commercial TPS in the clinic. Using EclipseTM, we have performed preliminary SyncRT trials on dog cadavers at the IMBL, and verified calculated doses against dosimetric measurement to within 5% for heterogeneous tissue-equivalent phantoms. We have also performed a validation of the TPS against a full MC simulation for constructed heterogeneous phantoms in EclipseTM, and showed good agreement for a range of water-like tissues to within 5%-8%. Our custom EclipseTM TPS for SyncRT is ready to perform live veterinary trials at the IMBL.

High resolution radiochromic film dosimetry: Comparison of a microdensitometer and an optical microscope.

PURPOSE: Microbeam radiation therapy is a developing technique that promises superior tumour control and better normal tissue tolerance using spatially fractionated X-ray beams only tens of micrometres wide. Radiochromic film dosimetry at micrometric scale was performed using a microdensitometer, but this instrument presents limitations in accuracy and precision, therefore the use of a microscope is suggested as alternative. The detailed procedures developed to use the two devices are reported allowing a comparison. METHODS: Films were irradiated with single microbeams and with arrays of 50 µm wide microbeams spaced by a 400 µm pitch, using a polychromatic beam with mean energy of 100 keV. The film dose measurements were performed using two independent instruments: a microdensitometer (MDM) and an optical microscope (OM). RESULTS: The mean values of the absolute dose measured with the two instruments differ by less than 5% but the OM provides reproducibility with a standard deviation of 1.2% compared to up to 7% for the MDM. The resolution of the OM was determined to be ~ 1 to 2 µm in both planar directions able to resolve pencil beams irradiation, while the MDM reaches at the best 20 µm resolution along scanning direction. The uncertainties related to the data acquisition are 2.5-3% for the OM and 9-15% for the MDM. CONCLUSION: The comparison between the two devices validates that the OM provides equivalent results to the MDM with better precision, reproducibility and resolution. In addition, the possibility to study dose distributions in two-dimensions over wider areas definitely sanctions the OM as substitute of the MDM.

Absorbed dose-to-water protocol applied to synchrotron-generated x-rays at very high dose rates.

Microbeam radiation therapy (MRT) is a new radiation treatment modality in the pre-clinical stage of development at the ID17 Biomedical Beamline of the European synchrotron radiation facility (ESRF) in Grenoble, France. MRT exploits the dose volume effect that is made possible through the spatial fractionation of the high dose rate synchrotron-generated x-ray beam into an array of microbeams. As an important step towards the development of a dosimetry protocol for MRT, we have applied the International Atomic Energy Agency's TRS 398 absorbed dose-to-water protocol to the synchrotron x-ray beam in the case of the broad beam irradiation geometry (i.e. prior to spatial fractionation into microbeams). The very high dose rates observed here mean the ion recombination correction factor, k s , is the most challenging to quantify of all the necessary corrections to apply for ionization chamber based absolute dosimetry. In the course of this study, we have developed a new method, the so called 'current ramping' method, to determine k s for the specific irradiation and filtering conditions typically utilized throughout the development of MRT. Using the new approach we deduced an ion recombination correction factor of 1.047 for the maximum ESRF storage ring current (200 mA) under typical beam spectral filtering conditions in MRT. MRT trials are currently underway with veterinary patients at the ESRF that require additional filtering, and we have estimated a correction factor of 1.025 for these filtration conditions for the same ESRF storage ring current. The protocol described herein provides reference dosimetry data for the associated Treatment Planning System utilized in the current veterinary trials and anticipated future human clinical trials.

Synchrotron X-ray microbeams: A promising tool for drug-resistant epilepsy treatment.

Epilepsy is one of the most important neurological diseases. It concerns about 1% of the population worldwide. Despite the discovery of new molecules, one third of epileptic patients are resistant to anti-epileptic drugs and among them only a few can benefit from resective surgery. In this context, radiotherapy is an interesting alternative to the other treatments and several clinical devices exist (e.g., Gamma Knife(®)). The European Synchrotron Radiation Facility offers the possibility to develop new methods of radiosurgery and to study their antiepileptic effects. Here, we discuss several studies that we performed recently to test and try to understand the antiepileptic effects of X-ray synchrotron microbeams in different animal models of epilepsy. We showed a decrease of seizures after Interlaced Microbeam Radiotherapy (IntMRT) of the somatosensory cortex, known as the seizure generator, in a genetic model of absence epilepsy. These antiepileptic effects were stable over 4 months and with low tissular and functional side-effects. The irradiated pyramidal neurons still displayed their physiological activity but did not synchronize anymore. We also obtained a lasting suppression of seizures after IntMRT of the dorsal hippocampus in a mouse model of mesiotemporal lobe epilepsy. However, an important variability of antiepileptic efficiency was observed probably due to the small size of the targeted structure. Despite these encouraging proofs-of-concepts, there is now a need to adapt IntMRT to other models of epilepsy in rodents which are close to refractory forms of epilepsy in human patients and to implement this approach to non-human primates, before moving to clinical trials.

Pharmacological characterization of designer cathinones in vitro.

BACKGROUND AND PURPOSE: Designer ß-keto amphetamines (e.g. cathinones, 'bath salts' and 'research chemicals') have become popular recreational drugs, but their pharmacology is poorly characterized. EXPERIMENTAL APPROACH: We determined the potencies of cathinones to inhibit DA, NA and 5-HT transport into transporter-transfected HEK 293 cells, DA and 5-HT efflux from monoamine-preloaded cells, and monoamine receptor binding affinity. KEY RESULTS: Mephedrone, methylone, ethylone, butylone and naphyrone acted as non-selective monoamine uptake inhibitors, similar to cocaine. Mephedrone, methylone, ethylone and butylone also induced the release of 5-HT, similar to 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) and other entactogens. Cathinone, methcathinone and flephedrone, similar to amphetamine and methamphetamine, acted as preferential DA and NA uptake inhibitors and induced the release of DA. Pyrovalerone and 3,4-methylenedioxypyrovalerone (MDPV) were highly potent and selective DA and NA transporter inhibitors but unlike amphetamines did not evoke the release of monoamines. The non-ß-keto amphetamines are trace amine-associated receptor 1 ligands, whereas the cathinones are not. All the cathinones showed high blood-brain barrier permeability in an in vitro model; mephedrone and MDPV exhibited particularly high permeability. CONCLUSIONS AND IMPLICATIONS: Cathinones have considerable pharmacological differences that form the basis of their suggested classification into three groups. The predominant action of all cathinones on the DA transporter is probably associated with a considerable risk of addiction.

Intranasal midazolam: pharmacokinetics and pharmacodynamics assessed by quantitative EEG in healthy volunteers.

The pharmacokinetics and pharmacodynamics of a highly concentrated cyclodextrin-based intranasal (i.n.) midazolam formulation containing the absorption-enhancer chitosan were studied in 12 healthy volunteers and compared with intravenous (i.v.) midazolam. The pharmacodynamic (PD) effects were assessed using quantitative electroencephalography (EEG). Maximal plasma concentrations of 63 and 110 ng/ml were reached at 8.4 and 7.6 min after 3 and 6 mg i.n. midazolam, respectively. After 5 mg i.v. and 6 and 3 mg i.n. midazolam, the times to onset of significant EEG effects in the ß2 band (18-25 Hz) were 1.2, 5.5, and 6.9 min, respectively, and the times to loss of response to auditory stimuli were 3.0, 8.0, and 15.0 min, respectively. A sigmoid maximum-effect (E(max)) model indicated disequilibrium between plasma and effect-site concentrations, with equilibration half-lives of 2.1-4.8 min. The observed pharmacokinetic-PD (PK-PD) properties suggest that i.n. midazolam deserves to be evaluated as an easy and noninvasive method of administering a first benzodiazepine dose, e.g., in out-of-hospital emergency settings with no immediate i.v. access.

Carvedilol inhibits the cardiostimulant and thermogenic effects of MDMA in humans.

BACKGROUND AND PURPOSE: The use of ± 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') is associated with cardiovascular complications and hyperthermia. EXPERIMENTAL APPROACH: We assessed the effects of the α(1) - and ß-adrenoceptor antagonist carvedilol on the cardiostimulant, thermogenic and subjective responses to MDMA in 16 healthy subjects. Carvedilol (50 mg) or placebo was administered 1 h before MDMA (125 mg) or placebo using a randomized, double-blind, placebo-controlled, four-period crossover design. KEY RESULTS Carvedilol reduced MDMA-induced elevations in blood pressure, heart rate and body temperature. Carvedilol did not affect the subjective effects of MDMA including MDMA-induced good drug effects, drug high, drug liking, stimulation or adverse effects. Carvedilol did not alter the plasma exposure to MDMA. CONCLUSIONS AND IMPLICATIONS: α(1) - and ß-Adrenoceptors contribute to the cardiostimulant and thermogenic effects of MDMA in humans but not to its psychotropic effects. Carvedilol could be useful in the treatment of cardiovascular and hyperthermic complications associated with ecstasy use.

Alterations of ubiquitin ligases in human cancer and their association with the natural history of the tumor.

Protein ubiquitination is critical for many cellular processes, through its ability to regulate protein degradation and various signaling mechanisms. In the ubiquitin (Ub) system, substrate specificity is achieved through the E3 family of Ub ligases. Because alterations of the ubiquitination machinery have been reported in human cancers, the selective interference with Ub ligases might represent a powerful therapeutic tool. Here, we report the first wide survey of misregulation of Ub ligases in cancer. We analysed 82 Ub ligases in nine types of cancer by in situ hybridization on tissue microarrays. We found 27 instances in which an Ub ligase was altered in a given type of tumor, when compared with normal tissues: 21 cases of overexpression and 6 cases of underexpression. We further analysed selected Ub ligases in large cohorts of breast and non-small-cell lung carcinomas. In five, of six, of these extended analyses (HUWE1, CCNB1IP1, SIAH1 and SIAH2 in breast cancer and CCNB1IP1 in lung cancer), we found that the levels of Ub ligases correlated significantly with relevant prognostic factors, and with clinical outcome. Our findings show that the alteration of Ub ligases is a frequent event in cancer and identify candidate targets for molecular therapies.

Different effects of methotrexate on DNA mismatch repair proficient and deficient cells.

Antifolates exert their antiproliferative activity through the inhibition of dihydrofolate reductase and, as a consequence, of thymidylate synthesis, thereby inducing nucleotide misincorporation and impairment of DNA synthesis. We investigated the processes involved in the repair of antifolate-induced damage and their relationship with cell death. Since misincorporated bases may be removed by DNA mismatch repair (MMR), the study was carried out on the MMR-proficient human cell lines HeLa and HCT116+chr3, and, in parallel, on the MMR-deficient cell lines HeLa cell-clone12, defective in the protein hPMS2, and HCT116, with an inactive hMLH1. After treatment with methotrexate (MTX), we observed that DNA repair synthesis occurs independently of the cellular MMR function. Clear signs of apoptosis such as nuclear shrinkage, chromatin condensation and degradation, DNA laddering, and poly (ADP-ribose) polymerase (PARP) proteolysis, were visible in both MMR(+) and MMR(-) cells. Remarkably, cell viability was lower and the apoptotic process was triggered more efficiently in the MMR-competent cells.

Apoptosis-prone phenotype of human colon carcinoma cells with a high level amplification of the c-myc gene.

Although apoptosis can be induced by the enforced expression of exogenously introduced c-myc genes, it is not clear whether overexpression resulting from the amplification of the resident c-myc gene in tumor cells is sufficient to induce apoptosis. We have investigated the relationship between c-myc gene amplification and the propensity of tumor cells to undergo apoptosis, using the SW613-12A1 and SW613-B3 cell lines, which are representatives, respectively, of tumorigenic and non-tumorigenic clones isolated from the SW613-S human colon carcinoma cell line. Tumorigenic clones are characterized by a high level of amplification and expression of the c-myc gene, whereas cells of non-tumorigenic clones have a small number of copies and a lower level of expression of this gene. Analysis of c-myc mRNA level in cells cultured under low serum conditions indicated that the expression of the gene is tightly regulated by serum growth factors in non-tumorigenic B3 cells, whereas it is poorly regulated in tumorigenic 12A1 cells, the level of mRNAs remaining relatively high in serum-starved 12A1 cells. Under these conditions, 12A1 cells showed clear evidence of apoptosis, whereas B3 cells were completely refractory to the induction of apoptosis. Moreover, the study of cell lines derived from non-tumorigenic apoptosis-resistant clones following the introduction by transfection of exogenous c-myc gene copies showed that they have acquired an apoptosisprone phenotype. Altogether, our results strongly suggest that deregulated c-myc expression due to high-level amplification confers an apoptosis-prone phenotype to tumor cells. The possible consequences of these observations for cancer therapy are discussed.

Multiparametric staining to identify apoptotic human cells.

To analyze relevant features of HeLa and HL60 cells driven into apoptosis by etoposide, we have developed a new "tricolor" assay, based on the simultaneous analysis in the single cell of chromatin condensation, DNA degradation, and cellular poly(ADP-ribose) synthesis. The latter reaction is catalyzed by poly(ADP-ribose)-polymerase (E.C. 2.4.2.30), an enzyme which is activated by the presence of DNA free ends. The protocol consists in the visualization of apoptotic cells by Hoechst staining, TUNEL assay, and immunoreaction with anti-poly(ADP-ribose) antibody. We thus provide the first evidence that endogenous poly(ADP-ribose) production is indeed stimulated in cells undergoing apoptosis after treatment with antitumoral drugs, and that the monitoring of this endogenous enzymatic reaction, combined with morphological and other biochemical parameters, should facilitate the detection of apoptotic cells.

Poly(ADP-ribose) synthesis: a useful parameter for identifying apoptotic cells.

Cell death by apoptosis was analysed in HeLa cells either treated with the antitumoral drug bleomycin or depleted of growth factors by long-term culture without medium change. The interference of apoptosis with normal cell cycle progression was followed by flow cytometry in cells stained with propidium iodide and with antibody to S-phase-related PCNA protein. Bleomycin-treated cells showed a net accumulation in G2/M phase paralleled by the appearance of material with a subdiploid DNA content. Cells with a subdiploid DNA content were also present in growth factor-depleted cultures and were shown to derive from all the cell cycle phases. To identify apoptotic features in HeLa cell cultures, we applied a recently developed assay based on the simultaneous analysis in the single cell of three parameters, namely chromatin condensation, DNA degradation and poly(ADP-ribose) synthesis. Apoptotic cells were visualized by sequential reactions: Hoechst staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling assay and immunoreaction with anti-poly(ADP-ribose) monoclonal antibody. Positive reactions were obtained for cells at different stages of the apoptotic programme showing condensed nuclei, fragmented chromatin and apoptotic bodies.

Sequence of events leading to apoptosis in long term cultured HeLa cells.

We have maintained HeLa cells in culture in the original medium for increasing times to induce growth arrest. Cell viability was evaluated by trypan blue dye exclusion assay. We observed that when cells are maintained in culture for several days, morphological hallmarks of apoptosis become evident. DNA synthesis rate, followed by (3)H-thymidine incorporation slowed down in long term cultured cells. This evidence was supported by the analysis of cell cycle progression determined by proliferating cell nuclear antigen (PCNA) immunostaining. Apoptotic cells have been characterized with respect to the sequential appearance of high molecular weight and internucleosomal DNA fragmentation. We have provided evidence that in this experimental model the first step in DNA degradation is represented by the formation of high molecular weight fragments, whereas nucleosomal DNA ladder is visible later on. The activation of the enzyme poly(ADP-ribose)polymerase, considered a marker of apoptotic death, has been observed. The results suggest that long term culture conditions activate the apoptotic programme.

Activation of poly(ADP-ribose)polymerase in apoptotic human cells.

We have studied the effect of the chemotherapeutic drug VP-16 (etoposide) on the metabolism of HeLa cells by analysing different cellular parameters considered as markers of apoptosis. Typical features such as chromatin condensation and internucleosomal DNA cleavage are visible in HeLa cells exposed to VP-16. We investigated whether the appearance of small-sized DNA fragments could regulate the ADP-ribosylation process. To this purpose, we have analysed, by means of the activity gel technique; the structural and catalytical properties of poly(ADP-ribose)polymerase. In extracts from cells where etoposide-induced DNA fragmentation occurred, we have shown that the label of the autoribosylated form of the enzyme is greatly increased even if the amount of the protein remains constant. This phenomenon is completely abolished in cells preincubated with poly(ADP-ribose)polymerase inhibitor, 3-aminobenzamide. After VP-16 administration, we have observed that the level of NAD is not heavily decreased. It is widely agreed that zinc exerts an inhibitory effect on the endonuclease(s) responsible for the fragmentation of DNA during apoptosis. After incubation of cells with zinc/VP-16 we have found the occurrence of apoptotic parameters even in the absence of internucleosomal DNA cleavage. The inhibition of DNA fragmentation prevents the activation of poly(ADP-ribose)polymerase activity. These results indicate that the activation of the enzyme towards the automodification reaction is strictly dependent on the appearance of DNA internucleosomal fragments and could represent a way to control enzyme activity.

Induction of apoptotic cell death by DNA topoisomerase II inhibitors.

We have analyzed the interference of antitumoral drugs acting through the inhibition of DNA topoisomerase II on the human HeLa cell metabolism. Different compounds characterized by a diverse mechanism of action have been used, namely m-amsacrine, an intercalative drug, etoposide, which does not intercalate DNA, and suramin, which exerts its effect through an unknown mechanism. In HeLa cells treated with increasing doses of these drugs, we have examined cell viability and DNA synthesis capacity, and we have evaluated topoisomerase II activity. Cellular morphology and DNA integrity have been studied in order to characterize the mechanism of cell death. The results we have obtained clearly indicate that topoisomerase II poisons induce cell death by apoptosis. These observations suggest a role of the inhibition of topoisomerase II activity in the apoptotic program.

Analysis of phosphorylation level of DNA topoisomerase-ii-alpha and topoisomerase-ii-Beta in human hela-cells.

We have studied the phosphorylation of DNA topoisomerases II in HeLa cells focusing on the beta isoform of the enzyme which is very difficult to analyze because of its instability. In proliferating cells, we observed that both the a and beta isozymes are labeled after cell incubation with (32)p. The phosphorylation of beta enzyme occurs to a low extent, thus reflecting the expression of topoisomerases II during cell cycle. In cells treated with etoposide, the activity of topoisomerase II is inhibited and the level of phosphorylation decreases, suggesting a possible cooperation between this modification and drug response.

Dose-dependent zinc inhibition of DNA ladder in apoptotic HeLa cells regulates the activity of poly(ADP-ribose) polymerase and does not protect from death induced by VP-16.

Zinc ions exert an inhibitory effect on Ca(2+)Mg(2+)-dependent endonuclease which is supposed to be responsible for the fragmentation of DNA during apoptosis. In the experimental system we used, that is HeLa cells treated with VP-16, the protection from internucleosomal DNA degradation is modulated by Zn concentration and appears to be dependent on the time after treatment. This effect does not prevent cell death or occurrence of apoptotic parameters, suggesting that DNA ladder appearance is not a crucial event in apoptosis. The activation of poly(ADP-ribose)polymerase following the administration of VP-16, is not observed in cells in which DNA fragmentation has been abolished by zinc, supporting the hypothesis that this event is regulated by the appearance of small-sized DNA fragments.