Maria Teresa Santini

Three-dimensional spheroid model in tumor biology.

It is becoming more and more apparent that monolayer cultures of tumor cells cannot completely represent the characteristics of three-dimensional solid tumors. Consequently, the multicellular tumor spheroid model, which is of intermediate complexity between in vivo tumors and monolayer cultures, was developed. In this review, the major similarities between spheroids and solid tumors are discussed. After a brief survey of the different spheroid culturing techniques, the general morphological and growth characteristics of these systems are examined and compared to solid tumors. Finally, selected studies regarding the use of tumor spheroids to examine cell response to antineoplastic agents and radiation, cell death including both necrosis and apoptosis and cell adhesion in spheroids are reviewed.

N-Acetylcysteine inhibits apoptosis and decreases viral particles in HIV-chronically infected U937 cells

Apoptosis or programmed cell death (PCD) is a type of death occurring in various physiological processes. Several data suggest that: (1) apoptosis may play a critical role in AIDS pathogenesis; (2) an increase of endocellular free radical levels can be associated with activation of previously latent HIV virus. Tumor necrosis factor (TNF), a cytokine capable of inducing oxygen free radicals and apoptosis, appears also to be involved in HIV activation. The present findings, which elucidate a relationship between the percentage of apoptotic cells, reduced glutathione (GSH) depletion and an increase of p24 antigenemia, suggest that pretreatment with N‐acetylcysteine (NAC) is capable of decreasing the above‐mentioned phenomena in HIV‐infected U937 cells.

Apoptosis, cell adhesion and the extracellular matrix in the three-dimensional growth of multicellular tumor spheroids

In the last few years, it has become increasingly apparent that cell survival and death, especially apoptosis, strongly depend on cell adhesion and the extracellular matrix. In addition, it has also become clear that the use of three-dimensional multicellular tumor spheroids, which mimick more closely solid tumors in vivo, are a realistic experimental model to investigate many aspects of tumor biology. In the present review, after a general overview of the current knowledge regarding apoptosis, cell adhesion and the extracellular matrix, the results obtained utilizing multicellular tumor spheroids in these types of studies are discussed. The main conclusion that may be drawn from a synthesis of the literature on these topics is that investigations with multicellular tumor spheroids yield much useful information that is sometimes in contradiction to that obtained with monolayer cultures, but is closer to that derived from in vivo studies. Consequently, the authors encourage that these three-dimensional systems be used in many studies in which cell death and adhesion are being examined.

Multicellular tumour spheroids in radiation biology.

PURPOSE Multicellular tumour spheroids are being used with increasing frequency in various aspects of tumour biology, including studies dealing with radiation biology. This review attempts to outline recent studies using these three-dimensional systems in radiation biology with particular reference made to papers testing radiotherapeutic protocols with spheroids. DEFINITIONS Multicellular tumour spheroids are three-dimensional structures composed of cancer cells. They are formed from monolayer tumour cells when these are grown by various in vitro methods (e.g. liquid-overlay, spinner flask and gyratory rotation systems). Because of the cellular organization in spheroids, they have been often shown to recreate in vivo tumours much more closely than two-dimensional in vitro models. CONCLUSIONS Because of their particular architectural characteristics, multicellular spheroids are demonstrated to be extremely useful in testing radiotherapeutic protocols, including dose rate and fractionation, radioimmunotherapy and the effects of combined treatments (e.g. radiation and anti-neoplastic drugs). Further studies should seek not only to continue testing these protocols, but also to investigate the more fundamental questions of radiation-induced apoptotic cell death, cell-cycle events, cell-cell interactions and cell adhesion phenomena.

Cellular effects of extremely low frequency (ELF) electromagnetic fields

Purpose: The major areas of research that have characterised investigation of the impact of extremely low frequency (ELF) electromagnetic fields on living systems in the past 50 years are discussed. In particular, selected studies examining the role of these fields in cancer, their effects on immune and nerve cells, and the positive influence of these ELF fields on bone and nerve cells, wound healing and ischemia/reperfusion injury are explored. Conclusions: The literature indicates that there is still no general agreement on the exact biological detrimental effects of ELF fields, on the physical mechanisms that may be behind these effects or on the extent to which these effects may be harmful to humans. Nonetheless, the majority of the in vitro experimental results indicate that ELF fields induce numerous types of changes in cells. Whether or not the perturbations observed at the cellular level can be directly extrapolated to negative effects in humans is still unknown. However, the myriad of effects that ELF fields have on biological systems should not be ignored when evaluating risk to humans from these fields and, consequently, in passing appropriate legislation to safeguard both the general public and professionally-exposed workers. With regard to the positive effects of these fields, the possibility of testing further their efficacy in therapeutic protocols should also not be overlooked.

Whole mouse nitroxide free radical pharmacokinetics by low frequency electron paramagnetic resonance

The in vivo uptake distribution and reduction of the oxygen-sensitive nitroxide spin label PCA in the mouse monitored by low frequency electron paramagnetic resonance (EPR) spectroscopy are reported. Spectra were obtained from the head and liver regions of pentobarbital anesthetized mice during different circulatory and ventilatory conditions. Identical clearances were found in these regions during normoxia. Moderate hypoxia (10% O2-90% N2) did not significantly affect the spin label reduction rate.

The Relationship between 1H- NMR mobile lipid intensity and cholesterol in two human tumor multidrug resistant cell lines (MCF-7 and LoVo)

The high resolution proton nuclear magnetic resonance (1H-NMR) spectra of two different cell lines exhibiting multidrug resistance (MDR) as demonstrated by the expression of the well-known energy-driven, membrane-bound 170 kDa P-glycoprotein pump known as Pgp were investigated. In particular, the mobile lipid (ML) profile, and the growth and biochemical characteristics of MCF-7 (human mammary carcinoma) and LoVo (human colon adenocarcinoma) sensitive and resistant tumor cells were compared. The results indicate that both MCF-7 and LoVo resistant cells have a higher ML intensity than their respective sensitive counterparts. However, since sensitive and resistant cells of each pair grow in the same manner, variations in growth characteristics do not appear to be the cause of the ML changes as has been suggested by other authors in non-resistant tumor cells. In order to investigate further the origin of the ML changes, lipid analyses were conducted in sensitive and resistant cell types. The results of these experiments show that resistant cells of both cell types have a greater amount of esterified cholesterol and saturated cholesteryl ester and triglyceride fatty acid than their sensitive counterparts. From a thorough analysis of the data obtained in this paper utilizing numerous techniques including biological, biophysical and biochemical ones, it is hypothesized that cholesterol and triglyceride play a pivotal role in inducing changes in NMR ML signals. The importance of these lipid variations in MDR is discussed in view of the controversy regarding the origin of ML signals and the paramount role played by the Pgp pump in resistance.

Lipid raft disruption protects mature neurons against amyloid oligomer toxicity.

A specific neuronal vulnerability to amyloid protein toxicity may account for brain susceptibility to protein misfolding diseases. To investigate this issue, we compared the effects induced by oligomers from salmon calcitonin (sCTOs), a neurotoxic amyloid protein, on cells of different histogenesis: mature and immature primary hippocampal neurons, primary astrocytes, MG63 osteoblasts and NIH-3T3 fibroblasts. In mature neurons, sCTOs increased apoptosis and induced neuritic and synaptic damages similar to those caused by amyloid beta oligomers. Immature neurons and the other cell types showed no cytotoxicity. sCTOs caused cytosolic Ca(2+) rise in mature, but not in immature neurons and the other cell types. Comparison of plasma membrane lipid composition showed that mature neurons had the highest content in lipid rafts, suggesting a key role for them in neuronal vulnerability to sCTOs. Consistently, depletion in gangliosides protected against sCTO toxicity. We hypothesize that the high content in lipid rafts makes mature neurons especially vulnerable to amyloid proteins, as compared to other cell types; this may help explain why the brain is a target organ for amyloid-related diseases.

Extremely low frequency (ELF) magnetic fields and apoptosis: a review

In recent years, there has been increasing evidence that extremely low frequency magnetic fields might be linked to tumours, particularly with childhood leukaemia. In the same period, the role of apoptosis in the tumour process has also gained increasing importance. It is the purpose of this review to describe the apoptotic process, discuss selected papers in which apoptosis is examined in cells exposed to magnetic fields and describe the possible biophysical mechanisms responsible for changes in the apoptotic process in exposed cells. Despite some differences, as a whole, the literature seems to demonstrate that magnetic fields induce changes in apoptosis in cells exposed to different experimental protocols. In addition, the important role of ions, particularly of Ca2 + , in the apoptotic process is also discussed, and one possible model for magnetic field action on apoptosis that brings together experimental observations of different nature is suggested and discussed.

The cesium-induced delay in myoblast membrane fusion is accompanied by changes in isolated membrane lipids

We have recently demonstrated that cesium ions delay the sharp decrease in both membrane conductivity and membrane permittivity of chick embryo myoblasts seen at fusion (Santini, M.T., Bonincontro, A., Cametti, C. and Indovina, P.L. (1988) Biochim. Biophys. Acta 945, 56-64). Analysis of the conductivity dispersion data (obtained in the radiowave frequency range) indicated that cesium delays fusion by about 30 h. We suggested that cesium is affecting both active ionic transport by blocking potassium channels as well as interfering with membrane lipid and/or protein charges. In the present study, we have investigated both the possible role of membrane lipids in myoblast fusion and the possible effects of cesium on these lipids. Our data indicate that lipid changes do occur in the isolated myoblast plasma membrane of controls during myogenic differentiation especially prior to fusion and that in cesium cultures these variations do not occur. These variations are in accordance with current membrane fusion theory. Specifically, there is a decrease in bilayer-stabilizing lipids (phosphatidylcholine) and an increase in bilayer-destabilizing ones (phosphatidylethanolamine and phosphatidic acid) and cholesterol during the fusion process. In addition, although slight, during fusion there appears to be a decrease in phosphatidylinositol which is believed to be involved in the inositol phosphate second messenger system. In cesium cultures, in which fusion is greatly delayed, the same lipid changes do not take place and those that are observed seem to reflect the fusion delay.