Gabriella Rainaldi

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.

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.

FLOW CYTOMETRIC ANALYSIS OF THE EARLY PHASES OF APOPTOSIS BY CELLULAR AND NUCLEAR TECHNIQUES

The early events occurring during apoptosis at the plasma membrane, chromatin, and mitochondrial levels were investigated in freshly isolated irradiated human lymphocytes, growth factor-deprived cultured human lymphocytes, and dexamethasone (DEX)-treated murine thymocytes. In intact, unfixed cells, evaluation of the light scatter properties and of DNA stainability with ethidium bromide (EB) allowed a cell subset suggestive for initial apoptosis to be identified. The apoptotic nature of these cells was confirmed by cell sorting in irradiated human lymphocyte model. EB could not be substituted for by propidium iodide, indicating that the nature of DNA probe used is of major importance for detecting initial apoptotic changes. Because mitochondria are thought to represent a primary target during apoptosis, we measured the uptake of mitochondria transmembrane potential sensitive (Rhodamine 123) and nonsensitive (10-nonyl-acridine-orange) probes concomitantly with EB uptake. Cells starting apoptosis had an enhanced incorporation of both mitochondria dyes, which in combination with EB identified several cell subsets. This suggests that complex alterations in mitochondrial structure and functioning occur in the early stages of apoptosis. To investigate phenomena occurring at the chromatin level in similar phases of apoptosis, irradiated human lymphocytes and DEX-treated murine thymocytes were disrupted and DNA stainability assessed in nuclear suspensions. A transient increase in DNA stainability, i.e., the appearance of distinct hyperdiploid peaks in the human model and a generalised upward shift of the G0/1 peak in the murine model, was observed in the early phases of apoptosis concomitantly with specific alterations in light scattering properties. These findings suggest that chromatin texture is altered in early apoptosis and affects DNA stainability.

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.

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.

Metabolomics Using 1H-NMR of Apoptosis and Necrosis in HL60 Leukemia Cells: Differences between the Two Types of Cell Death and Independence from the Stimulus of Apoptosis Used

Abstract Rainaldi, G., Romano, R., Indovina, P., Ferrante, A., Motta, A., Indovina, P. L. and Santini, M. T. Metabolomics Using 1H-NMR of Apoptosis and Necrosis in HL60 Leukemia Cells: Differences between the Two Types of Cell Death and Independence from the Stimulus of Apoptosis Used. Radiat. Res. 169, 170–180 (2008). High-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy was used to examine and compare the metabolic variations that occur in cells of the HL60 promyelocytic leukemia cell line after induction of apoptosis by ionizing radiation and the antineoplastic drug doxorubicin as well as after induction of necrosis by heating. Apoptosis and necrosis were confirmed by fluorescence microscopy using the chromatin stain Hoechst 33258, agarose gel electrophoresis of DNA, and determination of caspase 3 enzymatic activity. The 1H-NMR experiments revealed that the spectra of both samples containing apoptotic cells were characterized by the same trend of several important metabolites. Specifically, an increase in CH2 and CH3 mobile lipids, principally of CH2, decreases in glutamine and glutamate, choline-containing metabolites, taurine and reduced glutathione were observed. By contrast, the sample containing necrotic cells presented a completely different profile of 1H-NMR metabolites since it was characterized by a significant increase in all the metabolites examined, with the exception of CH2 mobile lipids, which remain unchanged, and reduced glutathione, which decreased. The results suggest that variations in 1H-NMR metabolites are specific to apoptosis independent of the physical or chemical nature of the stimulus used to induce this mode of cell death, while cells dying from necrosis are characterized by a completely different behavior of the same metabolites.

MG‐63 human osteosarcoma cells grown in monolayer and as three‐dimensional tumor spheroids present a different metabolic profile: a 1H NMR study

High resolution proton nuclear magnetic resonance (1H NMR) spectroscopy was used to determine if the same cell line (MG‐63 human osteosarcoma cells) grown in monolayer or as small (about 50–80 μm in diameter), three‐dimensional tumor spheroids with no hypoxic center has different metabolic characteristics. Consequently, the 1H NMR spectra were obtained from both types of cultures and then compared. The results indicate that the type of cellular spatial array determines specific changes in MG‐63 cells. In particular, small but significant differences in lactate and alanine indicating a perturbation in energy metabolism were observed in the two cell models. In addition, although variations in CH2 and CH3 groups were also seen, it is not possible at this time to establish if lipid metabolism is truly different in cells and spheroids.