Antonella Rosi

RNA-lipid complexes released from the plasma membrane of human colon carcinoma cells

Cultured cells from human colon adenocarcinoma spontaneously release structures which display an intense 31P NMR signal from RNA and mobile phospholipids. Furthermore, the DPH probe in the cell supernatant shows an intense fluorescence, thus indicating its insertion in lipid vesicles. The total membranes, prepared from the same cells, also release similar structures. The fatty acid chain signals from the mobile lipids, observable in the H NMR spectrum, and the fluorescence polarization of the DPH probe are strongly affected by RNAase digestion, thus indicating an association between RNA molecules and lipids. The enzymatic marker cytochrome c reductase was assayed to rule out possible contamination from endoplasmatic reticulum. A high alkaline phosphatase activity was instead found in the supernatant samples, thus indicating that the shed material is released by the plasma membrane.

1H-MRS lipid signal modulation and morphological and ultrastructural changes related to tumor cell proliferation

Changes in signal intensity of 1H‐MRS mobile lipids, mostly triglycerides, have been observed in cultured HeLa cells during exponential growth. Signals were intense in the first days after seeding and almost disappeared after a few days in culture. Choline‐based metabolites were not affected by growth. Treatment with the antitumor drug lonidamine, which blocks cell proliferation and cell progression through cycle, increased lipid signal intensity. Morphological changes in the organization of the cell surface were detected by scanning electron microscopy during exponential growth, and were confirmed by freeze fracture analysis. The observed metabolic and structural modifications during cell growth were correlated to cell cycle progression of HeLa cells, as high‐intensity lipid signals were typical of cells with a high percentage of S‐ and G2 + M‐phases, while cells with a high frequency in G1‐phase were characterized by mobile lipid signals of very low intensity. Magn Reson Med 42:248–257, 1999.

Characterization of vesicles, containing an acylated oligopeptide, released by human colon adenocarcinoma cells: NMR and biochemical studies

RNA‐containing vesicles, recovered from the supernatant of high‐density cell samples of human colon carcinoma, produce a high‐resolution 1H NMR spectrum of lipids characterized by isotropic tumbling; these vesicles contain large amounts of triglycerides and cholesterol esters. Both findings have strict analogies to what is displayed by the proteolipid complexes isolated from the sera of tumor‐bearing patients [(1985) Proc. Natl. Acad. Sci. USA 82, 3455–3459; (1986) FEBS Lett. 203, 164–168]. Lipid analysis and enzymatic tests indicate that these vesicles are selected micromaps of plasma membranes, analogous to those that can be recovered from culture media in which tumor cells are grown [(1985) Dev. Biol. 3, 33–57]. Peculiar lipids, an acylated oligopeptide and a modified phospholipid, are also present in the vesicles.

Metabolic / Proteomic Signature Defines Two Glioblastoma Subtypes With Different Clinical Outcome

Glioblastoma (GBM) is one of the deadliest human cancers. Because of the extremely unfavorable prognosis of GBM, it is important to develop more effective diagnostic and therapeutic strategies based on biologically and clinically relevant subclassification systems. Analyzing a collection of seventeen patient-derived glioblastoma stem-like cells (GSCs) by gene expression profiling, NMR spectroscopy and signal transduction pathway activation, we identified two GSC clusters, one characterized by a pro-neural-like phenotype and the other showing a mesenchymal-like phenotype. Evaluating the levels of proteins differentially expressed by the two GSC clusters in the TCGA GBM sample collection, we found that SRC activation is associated with a GBM subgroup showing better prognosis whereas activation of RPS6, an effector of mTOR pathway, identifies a subgroup with a worse prognosis. The two clusters are also differentiated by NMR spectroscopy profiles suggesting a potential prognostic stratification based on metabolic evaluation. Our data show that the metabolic/proteomic profile of GSCs is informative of the genomic/proteomic GBM landscape, which differs among tumor subtypes and is associated with clinical outcome.

1H NMR detects different metabolic profiles in glioblastoma stem-like cells

The metabolic profiles of glioblastoma stem‐like cells (GSCs) growing in neurospheres were examined by 1H NMR spectroscopy. Spectra of two GSC lines, labelled 1 and 83, from tumours close to the subventricular zone of the temporal lobe were studied in detail and compared with those of neural stem/progenitor cells from the adult olfactory bulb (OB‐NPCs) and of the T98G glioblastoma cell line. In both GSCs, signals from myoinositol (Myo‐I), UDP‐hexosamines (UDP‐Hex) and glycine indicated an astrocyte/glioma metabolism. For line 1, the presence of signals from N‐acetyl aspartate, GABA and creatine pointed to a neuronal fingerprint. These metabolites were almost absent from line 83 spectra, whereas lipid signals, absent from normal neural lineages, were intense in line 83 spectra and remained low in those of line 1, irrespective of apoptotic fate. Spectra of OB‐NPC cells displayed strong similarities with those from line 1, with low lipid signals and clearly detectable neuronal signals. In contrast, the spectral profile of line 83 was more similar to that of T98G, displaying high lipids and nearly complete absence of the neuronal markers. A mixed neural–astrocyte metabolic phenotype with a strong neuronal fingerprint was therefore found in line 1, while an astrocytic/glioma‐like metabolism prevailed in line 83. We found a signal assigned to the amide proton of N‐acetyl galactosamine in GSC lines and in OB‐NPC spectra, whereas it was absent from those of T98G cells. This signal may be related to a stem‐cell‐specific protein glycosylation pattern and is therefore suggested as a marker of cell multipotency.

Characterization of 1H NMR detectable mobile lipids in cells from human adenocarcinomas

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.

Multiexponential relaxation in Fricke agarose gels: implications for NMR dosimetry

NMR relaxation times T1 and T2 of agarose and Fricke agarose gels have been measured in the range 17-51 MHz. The analysis of the spin echo curves indicates a multiexponential behaviour, characterized by three components, at all the examined frequencies. The relative T2 values, ranging from a few to a hundred milliseconds, can be attributed to different species of water molecules present in the gel. Two of these components are characterized by relaxation rates R2a and R2b, more sensitive than R1 to gamma irradiation, the sensitivity S being S(R1) = 0.066 s-1 Gy-1, S(R2a) = 0.088 s-1 Gy-1, S(R2b) = 0.17 s-1 Gy-1. The three T2 values decrease as a function of frequency, but no gain in dose sensitivity is obtained by changing the working frequency in the examined range. The relaxivity of agarose gels containing ferrous or ferric ions has also been measured and found to be different from those of the corresponding solutions in the absence of agarose. Thus it was possible to estimate the irradiation yield from three independent parameters, R1, R2a and R2b. No effect of the dose rate or of the source energy has been observed for any of these parameters.

Radiation effects on soluble metabolites in cultured HeLa cells examined by 1H MRS: Changes in concentration of glutathione and of lipid catabolites induced by gamma rays and proton beams

Cultured HeLa cells were irradiated with a single acute dose of either gamma rays (40 Gy) or low‐energy proton beams (20 Gy). 1H magnetic resonance spectra of intact cells harvested at different times after irradiation and of the correspondent perchloric acid (PCA) extracts prepared at different times after irradiation were run. Selected signals from glutathione and lactate were examined with the aim of investigating effects of irradiation on antioxidative stores and on mitochondrial activity. An increase of signal intensity of glutathione (GSH) takes place at 15 and 24 hr after irradiation, while a decrease of its signal intensity, accompanied by an increase of that of free glutamate, starts appearing 48 hr after irradiation. Lactate signal increases 48 hr after irradiation. Signals from lipid catabolites were also examined to explore their sensitivity in predicting the response to radiotherapy. Intensity ratios of signals of glycerophosphorylcholine and choline to that of phosphorylcholine increase with time after irradiation. Irradiating cells with gamma rays or proton beams at half a dose produces effects comparable to the metabolic variations presented here. The present experiments allow more insight into the complex pattern of the changes of GSH by irradiation and indicate that magnetic resonance spectroscopy signals from GSH, glutamate, lactate, and lipid catabolites are affected by irradiation. Finally, these data represent a first indication that the relative biological efficiency for some metabolic damage of low‐energy proton beams with respect to gamma rays can reach a value of 2.

Changes in cell volume and internal sodium concentration in HeLa cells during exponential growth and following lonidamine treatment.

Cell volumes decreased in HeLa cells as a function of time after seeding during exponential growth. Cell volume distributions revealed the presence of two cell populations in all stages of growth. When cells approached confluence, the ratio of the two populations abruptly shifted towards that characterised by the smallest volume. Percentages of G1-, S- and G2 + M-phase cells were also measured and it was found that G1 frequency increased as a function of cell density during exponential growth. Intracellular sodium concentration, [Na]i was monitored by 23Na NMR in the presence of 5 mM dysprosium (III) tripolyphosphate. [Na]i increased from 22.8 to 59.0 mM in cells from the second to the seventh day after seeding. Treatment with lonidamine, an antitumoral drug that it is known to slow down cell growth by affecting aerobic glycolysis, produced a complete block of cell progression after a few days of treatment. The progression of cell volume distributions towards smaller volumes and the increase in internal sodium concentration as a function of time after seeding were also affected by the drug. These phenomena were related to the existence of a subpopulation of mitotically inactive G1-phase cells during exponential growth, pointing out that a density-dependent cellular mechanism regulates the cell cycling in HeLa cells.

Dose distribution of proton beams with NMR measurements of Fricke-agarose gels.

Fricke-agarose gels have been irradiated with a proton beam. Then samples have been extracted at different depths with respect to the beam penetration distance, corresponding to different irradiation doses. Relaxation times T1 and T2, measured at 17 MHz, appear sensitive to this kind of radiation. In particular, T2 exhibits three components T2a, T2b and T2c, the first two being sensitive to proton irradiation. At 1% agarose concentration, the relaxation rates R1 = 1/T1, R2a = 1/T2a and R2b = 1/T2b of samples irradiated with both modulated and unmodulated beams, increase with the dose, irrespective of the beam energy. The yield G of Fe3+ ions per 100 eV of absorbed energy is always higher than that obtained for gamma irradiated samples.