[Show abstract]ABSTRACT: Multicellular tumour spheroids are an important model system to investigate the response of tumour cells to radio- and chemotherapy. They share more properties with the original tumor than cells cultured as 2D monolayers do, which helps distinguish the intrinsic properties of monolayer cells from those induced during cell aggregation in 3D spheroids. The paper investigates some metabolic aspects of small tumour spheroids of breast cancer and their originating MCF-7 cells, grown as monolayer, by means of high resolution 1H NMR spectroscopy and MR microimaging before and after gamma irradiation. The spectra of spheroids were characterized by higher intensity of mobile lipids (ML), mostly neutral lipids, and glutamine (Gln) signals with respect to their monolayer cells counterpart, mainly owing to the lower oxygen supply in spheroids. Morphological changes of small spheroids after gamma-ray irradiation, such as loss of their regular shape, were observed by MR microimaging. Lipid signal intensity increased after irradiation, as evidenced in both MR localized spectra of the single spheroid and in high-resolution NMR spectra of spheroid suspensions. Furthermore, the intense Gln signal from spectra of irradiated spheroids remained unchanged, while the low Gln signal observed in monolayer cells increased after irradiation. Similar results were observed in cells grown in hypoxic conditions. The different behavior of Gln in 2D monolayers and in 3D spheroids supports the hypothesis that a lower oxygen supply induces both an upregulation of glutamine synthetase and a downregulation of glutaminases with the consequent increase in Gln content, as already observed under hypoxic conditions. The data herein indicate that 1H NMR spectroscopy can be a useful tool for monitoring cell response to different constraints. The use of spheroid suspensions seems to be a feasible alternative to localized spectroscopy since similar effects were found after radiation treatment.
Full-text available · Article · Apr 2016 · Frontiers in Oncology
[Show abstract]ABSTRACT: Glycosylation is the most abundant and diverse form of post-translational modification of proteins. Two types of glycans exist in glycoproteins: N-glycans and O-glycans often coexisting in the same protein. O-glycosylation is frequently found on secreted or membrane-bound mucins whose overexpression and structure alterations are associated with many types of cancer. Mucins have several cancer-associated structures, including high levels of Lewis antigens characterized by the presence of terminal fucose. The present study deals with the identification of MR signals from N-acetylgalactosamine and from fucose in HeLa cells by detecting a low-field signal in one-dimensional (1D) spectra assigned to the NH of N-acetylgalactosamine and some cross peaks assigned to fucose in two-dimensional (2D) spectra. The increase of Golgi pH by treatment with ammonium chloride allowed the N-acetylgalactosamine signal assignment to be confirmed. Behaviour of MR peak during cell growth and comparison with studies from literature taken together made it possible to have more insight into the relationship between aberrantly processed mucin and the presence of non-processed N-acetylgalactosamine residues in HeLa cells. Fucose signals, tentatively ascribed to residues bound to galactose and to N-acetylglucosamine, are visible in both intact cell and perchloric acid spectra. Signals assigned to fucose bound to galactose are more evident in ammonium chloride-treated cells where structural changes of mucin-related Lewis antigens are expected as a result of the higher Golgi pH. A common origin for the N-acetylgalactosamine and fucose resonances attributing them to aberrantly processed mucin can be inferred from the present results.
[Show abstract]ABSTRACT: The glycosylation process, through the addition of carbohydrates, is a major post-translational modification of proteins and glycolipids. Proteins may be glycosylated in either the secretory pathway leading to N-linked or O-linked glycoproteins or as nucleocytoplasmic glycosylation that targets only single proteins involving a single β-linked N-acetylglucosamine. In both cases, the key precursors are the uridine diphospho-N-acetylhexosamines synthesised by the hexosamine biosynthetic pathway. Furthermore, uridine diphospho-N-acetylglucosamine participates in the biosynthesis of sialic acid. In this work, we propose MRS for the detection of uridine diphospho-N-acetylhexosamines visible in high-resolution MR spectra of intact cells from different human tumours. Signals from the nucleotide and amino sugar moieties, including amide signals observed for the first time in whole cells, are assigned, also taking advantage of spectral changes that follow cell treatment with ammonium chloride. Finally, parallel changes in uridine diphospho-N-acetylhexosamines and glutamine pools, observed after pH changes induced by ammonium chloride in the different tumour cell lines, may provide more details on the glycosylation processes.
[Show abstract]ABSTRACT: A first prototype of optical scanner based on a CCD camera and the use of Fricke-agarose-XO has allowed us to reconstruct 3D dose maps, monitoring doses in the range 0.5–3 Gy. Measurements performed with laboratory tests and with clinical beams would indicate that the uncertainty involved in dose mapping are compatible with requirements of a 3D dosimetry for the verifications of treatment plans.
Article · Jun 2009 · Journal of Physics Conference Series
[Show abstract]ABSTRACT: Magnetic resonance spectroscopy studies are often carried out to provide metabolic information on tumour cell metabolism, aiming for increased knowledge for use in anti-cancer treatments. Accordingly, the presence of intense lipid signals in tumour cells has been the subject of many studies aiming to obtain further insight on the reaction of cancer cells to external agents that eventually cause cell death. The present study explored the relationship between changes in neutral lipid signals during cell growth and after irradiation with gamma rays to provide arrest in cell cycle and cell death. Two cell lines from human tumours were used that were differently prone to apoptosis following irradiation. A sub-G1 peak was present only in the radiosensitive HeLa cells. Different patterns of neutral lipids changes were observed in spectra from intact cells, either during unperturbed cell growth in culture or after radiation-induced growth arrest. The intensities of triglyceride signals in the spectra from extracted total lipids changed concurrently. The increase in lipid peak intensities did not correlate with the apoptotic fate. Modelling to fit the experimental data revealed a dynamic equilibrium between the production and depletion of neutral lipids. This is observed for the first time in cells that are different from adipocytes.
Full-text available · Article · Mar 2009 · FEBS Journal
[Show abstract]ABSTRACT: Signals attributable to amide protons and used in previous studies to measure intracellular pH were observed in the low-field region of the (1)H-MR spectra of four tumour cell lines: T98G, MCF-7, A172 and HeLa. The signals were more intense in the spectra of the two cell lines (T98G and MCF-7) characterised by higher concentrations of glutathione (GSH). After comparison with (1)H-MR spectra of GSH in solution at different pH values, the peaks were attributed to NHs of the Cys and Gly residues of GSH. Modification of the intracellular concentration of GSH by treatment with buthionine sulfoximine produced comparable decreases in the intensity of aliphatic signals of GSH and NHs under examination. The assignment was therefore confirmed.
[Show abstract]ABSTRACT: Although more advanced techniques such as intensity-modulated radiotherapy are rapidly spreading, 3D conformal radiotherapy (3D-CRT) remains the standard of treatment for many diseases. The authors outline essential indications to guarantee the quality of 3D-CRT treatments. Criteria for clinical indications and potential clinical advantages and disadvantages of 3D-CRT technology are presented. After briefly listing human and technological resources requirements, procedures for 3D-CRT and physical aspects peculiar to 3D-CRT are described. Medical physics support activities are also considered, including suggestions concerning quality control protocols. Difficulties in the application of correct quality procedures, particularly related to human and technological resources, procedures for patient positioning, imaging, contouring, treatment planning, in vivo dosimetry, set-up verification, follow-up, dose delivery are then discussed.
Full-text available · Article · Oct 2008 · Critical reviews in oncology/hematology
[Show abstract]ABSTRACT: In this paper we present the main outcomes of a wide collaborative effort (carried out, within the INFN project, "EPICA" and in part within the European projects "RISC-RAD" and "NOTE" and the ASI project MoMa-COUNT), both experimental and theoretical, devoted to the characterization and quantification of the induction of DNA-targeted and non-DNA-targeted molecular and cellular biological endpoints, following irradiation of human cells with different Charged particles The work was mainly aimed at reaching a better understanding of the mechanisms governing the physical and biophysical pathways leading from the initial energy deposition by radiation in matter to the induction of observable radiobiological damage, with particular focus on the role played by radiation quality. More, specifically we characterized the induction of DNA DSB within different fragment-size ranges outlining the effectiveness of high-LET radiation at inducing small fragments and thus clustered DNA breaks, which can evolve in terms of endpoints like chromosome aberrations (CAs). This was confirmed by the development and application of a model of CA induction based oil the assumption that only DNA breaks can lead to aberrations. Concerning non-DNA-targeted damage, we quantified the time-dependent induction of medium-mediated DNA damage in bystander cells and we characterized the time and dose dependence of cytokine concentration in the medium of sham-irradiated and irradiated cells, since medium-mediated bystander damage is thought to arise from molecular signalling between irradiated and unirradiated cells. The mechanisms governing such Signalling were investigated developing a model and a MC code simulating cytokine release, diffusion mid internalization, showing good agreement with experimental data. Non-DNA-targeted effects were further characterized by MRS investigation of the radiation effects on lipids and oxidative metabolism, which are particularly relevant also considering that, they may differently expressed in different tumors and ill normal tissues.
Full-text available · Article · Jan 2008 · Il Nuovo Cimento C
[Show abstract]ABSTRACT: The relationship between apoptosis induced by gamma radiation and glutathione in cells of two human cancer cell lines, HeLa from cervix carcinoma and MCF-7 from mammary carcinoma, was examined. MCF-7 cells appeared to be more radioresistant than HeLa cells, and radiation-induced apoptosis, which was monitored by assessing phosphatidylserine externalization, was observed in HeLa cells but not in MCF-7 cells. Glutathione levels monitored by (1)H MRS were higher in MCF-7 cells than in HeLa cells, while the opposite was true for the free glu signals. MCF-7 cells became more radiosensitive when treated with 0.1 mM buthionine sulfoximine, which inhibits GSH synthesis through inactivation of gamma-glutamylcysteine synthetase, with the concomitant appearance of radiation-induced apoptosis. We can thus reasonably associate, at least in part, the resistance of MCF-7 cells to apoptosis with a high level of glutathione and probably with a high activity of gamma-glutamylcysteine synthetase. A late decrease in glutathione concentration after irradiation was observed in MCF-7 cells, but not in HeLa cells and to a lesser degree in buthionine sulfoximine-treated MCF-7 cells. This would indicate that the radiation-induced decrease in glutathione concentration is not related to the onset of apoptosis, but it is more likely related to glutathione consumption as a result of detoxification reactions.
Full-text available · Article · Apr 2007 · Radiation Research
[Show abstract]ABSTRACT: In this paper we investigate Fricke-gels for their possible use as 3D dosimeters. Two series of experiments are presented. The first one is aimed at examining dosimetric performances of the gel and is relative to uniform dose distributions; the second one is aimed at comparing dose distributions obtained with the gel with those obtained with point dosimeters. The dosimetric performances of the Fricke gel examined indicated that they are very promising for mapping 3D dose distributions with a high spatial resolution.
Article · Mar 2007 · Journal of Physics Conference Series
[Show abstract]ABSTRACT: There is a widespread and increasing tendency to develop hospital performance indicators in the field of accreditation/certification systems and quality benchmarking. A study has been undertaken to develop a set of performance indicators for a typical radiotherapy Centre and to evaluate their ability to provide a continuous quality improvement.
A working group consisting of radiation oncologists, medical physicists and radiation technologists under the coordination of experts in health technology assessment has elaborated a set of general indicators able to monitor performances and the quality level of a typical radiotherapy Centre. The work has been carried out through four steps: a preliminary set of indicators was selected; data on these indicators were collected in a number of Italian radiotherapy Centres and medical physics Services; problems in collection and analysis of data were discussed; a final set of indicators was developed.
A final set of 13 indicators is here presented. They concern general structural and/or operational features, health physics activities and accuracy and technical complexity of the treatment.
The indicators tested in a few Italian Centres of radiotherapy and medical physics Services are now ready to be utilized by a larger community.
[Show abstract]ABSTRACT: The purpose of this work was to examine the dosimetric performances of the radiochromic Fricke-Agarose-Xylenol Orange gel
by optical measurements in order to perform dose reconstructions, in view of a future development for 3-D maps. Optical images
and dose–response curves of the gel were obtained by a CCD-based device, originally designed for reading radiochromic films,
that was modified to meet the optical properties of the dosemeter. With a resolution of 0.18 × 0.18 mm the optimum range of
doses in which per cent uncertainty is lower than 2% was 3–10 Gy. The minimum detectable dose, estimated as the absorbed dose
corresponding to 3 SD above background, was 0.1 Gy. With a resolution of 1.98 × 1.98 mm the optimum range of doses in which
per cent uncertainty is lower than 2% was 0.3–10 Gy. The minimum detectable dose, estimated as the absorbed dose corresponding
to 3 SD above background, was 0.015 Gy. The comparison with alanine dosemeters in the dose range 7–10 Gy showed agreement
within a few per cent and the same agreement was observed for the comparison with TLD in the range 1–3 Gy.
Article · Feb 2006 · Radiation Protection Dosimetry
[Show abstract]ABSTRACT: Inhibition of apoptosis in tumour cells may depend on intracellular reduced glutathione (GSH) level. In this work, GSH levels were studied by (1)H MRS in MCF-7 and HeLa cells, characterised by a different radiosensitivity. Annexin-V test showed that the fraction of apoptotic HeLa cells after irradiation is much higher than in control, although MCF-7 cells did not show a significant apoptosis. MRS signals from GSH (G) show lower intensity in HeLa with respect to MCF-7 cells; the opposite is true for free glutamic acid [glu (g)]. After irradiation, the G/g ratio decreases in MCF-7, although remaining approximately constant in HeLa cells. Buthionine sulfoximine (BSO) treated MCF-7 cells show an increase in the percentage of apoptotic cells; in parallel, G/g ratio behaves as in HeLa. This study indicates that GSH level may act as predictive marker of apoptosis by irradiation.
Article · Feb 2006 · Radiation Protection Dosimetry
[Show abstract]ABSTRACT: Much attention has been devoted in the past to monitor changes of mobile lipid (ML) 1H MRS signals in spectra of tumour cells. The purpose of this work is to exploit ML signals to provide information on cell
metabolism after irradiation, comparing tumour cells characterised by different radiosensitivity and relating MRS findings
to changes in cell proliferation and delays in cell cycle phases. Irradiated HeLa cells present less intense ML signals with
respect to controls. The opposite is true for MCF-7 cells. A G2 arrest is observed for both cell lines after irradiation. In HeLa cells, G1 decreases and S phase is maintained; a sub G1 peak is also visible. In MCF-7 cells, G1 is decreased and S phase is strongly reduced, while no sub G1 is present. The observed changes in ML are tentatively associated to cell cycle regulation of phospholipid synthesis. Mathematical
modelling of ML variations is in progress.
Full-text available · Article · Feb 2006 · Radiation Protection Dosimetry
[Show abstract]ABSTRACT: In Fricke-agarose gels, an accurate determination of the spatial dose distribution is hindered by the diffusion of ferric
ions. In this work, a model was developed to describe the diffusion process within gel samples of finite length and, thus,
permit the reconstruction of the initial spatial distribution of the ferric ions. The temporal evolution of the ion concentration
as a function of the initial concentration is derived by solving Fick's second law of diffusion in two dimensions with boundary
reflections. The model was applied to magnetic resonance imaging data acquired at high spatial resolution (0.3 mm) and was
found to describe accurately the observed diffusion effects.
Article · Feb 2006 · Radiation Protection Dosimetry