Search

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.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 metabolic changes that occur in MG-63 osteosarcoma three-dimensional tumor spheroids exposed to 2 Gy of ionizing radiation, a dose that is comparable to radiation therapy, were studied using high-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. Specifically, the (1)H-NMR spectra of control and exposed MG-63 spheroids were compared. Small spheroids (about 50-80 microm in diameter) with no hypoxic center were used. The spectra of whole MG-63 spheroids as well as the perchloric acid extracts of these systems were evaluated. Cell damage was also examined by lactate dehydrogenase release and changes in cell growth. No cell damage was observed, but numerous metabolic changes took place in spheroids after exposure to ionizing radiation. In particular, significant increases in both CH(2) and CH(3) mobile lipids, considered by many authors as markers of apoptosis and also present in MG-63 spheroids undergoing overt apoptosis, were observed in spheroids irradiated with 2 Gy. However, the chromatin dye Hoechst 33258 and DNA fragmentation assays showed no overt apoptosis up to 7 days after irradiation with this low dose. Thus it is evident that increases in mobile lipids do not always indicate actual cell death. A detailed analysis of the other metabolic changes observed appears to suggest that the cell death program was initiated but not completed. In fact, the completely different behavior of two important cellular defense mechanisms, reduced glutathione and taurine, in spheroids irradiated with 2 Gy and in those undergoing overt apoptosis seems to indicate that these systems are protecting spheroids from actual cell death. In addition, these data also suggest that (1)H-NMR can be used to examine the effects of low doses of ionizing radiation in spheroids, a cell model of great complexity that closely resembles tumors in vivo. The importance of this possibility in relation to reaching the ultimate goal of a better evaluation of the outcome of radiotherapy protocols should not be ignored.

The effects of chemically induced hypoxia and ionizing radiation on the adhesive properties of MG-63 human osteosarcoma three-dimensional spheroids were investigated. Hypoxia was induced by addition of CoCl2 to small, nonhypoxic spheroids and verified by HIF-1alpha expression. In addition, the possible role of important cell adhesion molecules involved in tumor dissemination in inducing adhesive changes were also studied. In particular, two key integrins (i.e., the alpha chain of the fibronectin receptor, alpha5, and the alpha chain of the collagen receptor, alpha2), an important member of the immunoglobulin superfamily (CD54 or ICAM-1) and the strategic molecule CD44 (H-CAM, the principal receptor for hyaluronan) were examined. Because of the important role of fibronectin in adhesive processes, variations in this extracellular matrix component were also examined. The results seem to indicate that CoCl2-induced hypoxia greatly increases adhesion of MG-63 spheroids to both tissue culture plates and plates coated with fibronectin or collagen when compared to controls, while ionizing radiation induces a great decrease in this attachment. Furthermore, chemically induced hypoxia also partially inhibits the effects of ionizing radiation. The data also show that these adhesive changes are accompanied by concomitant variations in the expression of alpha5 and alpha2 integrins, CD44, and CD54 and fibronectin.

The possibility that a sinusoidal 50 Hz magnetic field with a magnetic flux density of 0.5 mT can induce variations in the expression of cell adhesion molecules (CAMs) in two human osteosarcoma cell lines (MG-63 and Saos-2) was investigated. In particular, the expression of two important integrins, VLA-2, the receptor for collagen, and VLA-5, the receptor for fibronectin, as well as CD44, were examined in both cell lines after these had been exposed for 7 and 14 days to a 50 Hz, 0.5 mT field. Cell surface morphology (scanning electron microscopy), cell growth characteristics (growth curves and cell cycle phase distribution), and cell death (necrosis and apoptosis) were also examined. The results demonstrate that no variations in surface morphology and cell death occurred between control and exposed cells in both MG-63 and Saos-2 cells, while significant changes were noted in cell growth and fibronectin and CD44 expression in MG-63 cells. The results are discussed in view of the important role that CAMs play in controlling various cancer cell functions, particularly proliferation and metastasis.

Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin from Escherichia coli that induces the activation of Rho, Rac, and Cdc42 GTPases, all involved in actin reorganization. Rac plays a further role in oxidase function. In epithelial cells, CNF1 has been reported to induce a phagocytic-like behavior in terms of a ruffle-driven ingestion of large material. We herein show that CNF1-activated epithelial cells may exert additional cell responses typical of professional phagocytes following stimulation, i.e., an increase in oxygen consumption and the generation of superoxide anions. Such effects were triggered by the contact of latex beads with epithelial cells and were significantly augmented by CNF1-induced Rac activation. Altogether our data indicate that Rac, one of the targets of CNF1, plays a pivotal role in these phenomena, suggesting the involvement in epithelial cells of a Rac-dependent NADPH-oxidase complex similar to that employed by professional phagocytes.

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.

The effects of growing the Saos-2 human osteosarcoma cell line onto surfaces containing -CH3, -OH, -COOH, -NH2, and C6H5 groups obtained by silane modification were examined. These cells were used because of the great importance of bone cells in many aspects of biomaterials research. Silane-modified surfaces were characterized by contact angle measurements and, subsequently, surface energies were calculated. Cells grown on clean glass, as well as those grown on glass surfaces containing the functional groups cited above, were examined by light and scanning electron microscopy and assessed for their growth characteristics (i.e., determination of cell number and Ki67 antigen expression). The data presented seemed to indicate that if Saos-2 cells are grown on silane-modifed surfaces containing the methyl (CH3), hydroxyl (OH), and phenyl (C6H5) functional groups, their proliferation is slowed down while growth of these cells on glass surfaces modified with amino (NH2) and carboxyl (COOH) groups did not significantly affect growth. Once it was demonstrated that these three functional groups induce significant variations in proliferation, cells grown on these surfaces were also tested for apoptosis and expression of important markers of bone cell differentiation (i.e., osteonectin and osteopontin) by flow cytometry and eventual rearrangement of these markers by fluorescence microscopy. The data suggested that growth of Saos-2 cells on CH3 induces the most evident morphological changes while growth of these cells on OH and C6H5 brings about the greater variations in osteonectin and osteopontin. We hypothesized that these changes are indicative of an increase in differentiation of Saos-2 cells when grown on the OH and C6H5 groups. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 55: 338–349, 2001

La spettroscopia con risonanza magnetica nucleare è utilizzata, in modo sempre crescente, per studi sia in vivo che in sistemi biologici in vitro, per esaminare variazioni indotte dall'azione di agenti chimici, fisici e biologici. In questo tipo di studi si effettua, in genere, un confronto tra le intensità dei segnali di campioni controllo con campioni trattati per poter ricavare delle informazioni sull'azione dell'agente. I metodi di confronto finora adottati consistono nel quantificare, mediante l'utilizzo di una sostanza di riferimento interna od esterna ai campioni, i segnali nei singoli spettri e nel confrontarne i valori. In questo lavoro, viene presentato un nuovo metodo di confronto, che consiste nel normalizzare gli spettri mediante un nuovo algoritmo. Esso fa riferimento ai segnali nella loro totalità e non richiede, per ottenere informazioni quantitative sulle variazioni relative, di alcuna sostanza di riferimento ( standard). In particolare, l'algoritmo è fondato sulla massimizzazione, mediante una opportuna misura a segno variabile, delle regioni di sovrapposizione degli spettri. L'algoritmo è stato verificato con simulazioni Monte Carlo e con esperimenti di laboratorio, che ne dimostrano l'affidabilità, la precisione e la sensibilità. Infine, è stato applicato a spettri relativi a campioni cellulari per dimostrarne l'applicabilità a campioni biologici reali.

There is increasing use of high-resolution NMR spectroscopy to examine variations in cell metabolism and/or structure in response to numerous physical, chemical, and biological agents. In these types of studies, in order to obtain relative quantitative information, a comparison between signal intensities of control samples and treated or exposed ones is often conducted. The methods thus far developed for this purpose are not directly related to the overall intrinsic properties of the samples, but rather to the addition of external substances of known concentrations or to indirect measurement of internal substances. In this paper, a new method for quantitatively comparing the spectra of cell samples is presented. It depends on a normalization algorithm which takes into consideration all cell metabolites present in the sample. In particular, the algorithm is based on maximizing, by an opportune sign variable measure, the spectral region in which the two spectra are superimposed. The algorithm was tested by Monte Carlo simulations as well as experimentally by comparing two samples of known contents with the new method and with an older method using a standard. At the end, the algorithm was applied to real spectra of cell samples to show how it could be used to obtain qualitative and quantitative biological information.

We have recently demonstrated that if human K562 erythroleukaemic cells, which normally grow in suspension, are grown on a positively-charged surface composed of polylysine, a transient reorganization of CD54 (ICAM-1), CD58 (LFA-3) and ανβ3 (vitronecin receptor), three important CAMs located on the cell membrane, takes place. In addition, changes of longer duration in membrane conductivity (ionic transport across the cell membrane) and membrane permittivity (static distribution of charges across the cell membrane), indicating more permanent structural as well as functional alterations in the cell membrane, were also observed [2]. Because of the close interrelationship which exists between the cell membrane, CAMs and the cytoskeleton, changes in this intracellular network as well as in the surface morphology of K562 cells grown on the positively-charged polymer, polylysine, were examined. In particular, actin and tubulin were investigated qualitatively and quantitatively by immunofluorescence microscopy and flow cytometry, respectively, while the cell surface was studied by scanning electron microscopy (SEM). The data indicate that when K562 cells are grown onto polylysine no quantitative changes occurred to the cytoskeletal elements even if these were rearranged and that the cell membrane surface is also greatly altered. These results are discussed in light of the pivotal role played by CAMs and the cell cytoskeleton in transducing environmental stimuli, in this case those provided by a positive charge, from the cell membrane to the inside of the cell. © 1999 Kluwer Academic Publishers

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.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.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.

Hyaluronan [hyaluronic acid (HA)] has been implicated in various cellular processes such as proliferation, adhesion, migration, and differentiation. The secondary and tertiary structures of HA give it very important and unique viscoelastic properties. HA-composed materials are currently used intraocularly during ophthalmological surgery to facilitate surgical procedures and prevent tissue damage. To examine the effects of three viscous biomaterials composed of hyaluronan (Healon, IAL, and Biolon) used in ophthalmological surgery, the membrane electrical properties of the erythroleukemic K562 cell line exposed to these materials were investigated. Membrane conductivity, membrane permittivity, and the conductivity of the cytosol were evaluated using dielectric relaxation measurements in the radiofrequency range and fitting the experimental results to the general equations of the Maxwell-Wagner effect. The results demonstrate that while membrane permittivity and the conductivity of the cytosol are not significantly altered, the membrane conductivity of K562 cells exposed to all three biomaterials increases substantially and in a time-dependent manner with respect to untreated cells. These observations seem to indicate that hyaluronan perturbs ionic transport while it does not vary the type, quantity, or distribution of membrane components. In addition, the variations induced by these substances on the cell membrane are not dependent upon the molecular weight or on the biological origin of hyaluronan. These results may aid in elucidating the mechanisms involved in hyaluronan/cell membrane interaction and thus may provide a deeper understanding of the complications related to their use in ophthalmological surgery.

Oxidative enzymes (laccases and peroxidases) isolated from the culture media of different fungi are involved in the basic mechanism of ligninolysis via radical intermediates. However, experiments aimed at reproducing natural biodegradation in vitro have been unsuccessful so far since the single biocatalysts alone are not able to solubilize lignins because of the simultaneous recondensation of these intermediates. FAD oxidases can prevent this side reaction in lignin depolymerization by reducing quinonoids and radical compounds. This study investigates the possible role of a laccase and a FAD-dependent aryl alcohol oxidase (veratryl alcohol oxidase, VAO) excreted by the basidiomycete Pleurotus ostreatus. In fact, we found that VAO is able to reduce synthetic quinones, laccase-generated quinonoids, and phenoxy radicals with concomitant oxidation of veratryl alcohol to veratryl aldehyde. This cooperative action of laccase and VAO also prevented the polymerization of phenolic compounds and reduced the molecular weight of soluble lignosulfonates to a significant extent.

The cell membrane appears to be one of the major targets of extremely low frequency (ELF) magnetic fields. To determine whether the membrane electrical properties of K562l leukaemic cells can be affected by ELF fields, these cells were exposed to a sinusoidal 50 Hz 2.5 mT magnetic field for 48 h. It should be recalled that the field intensity used is three or four orders of magnitude greater than the values found in homes and in many workplaces. Analyses of dielectric relaxation measurements in the r.f. range demonstrate that both the membrane electrical conductivity and permittivity of K562 cells decrease considerably after exposure of these cells to ELF fields, whereas the conductivity of the cytosol does not vary. Since both membrane conductivity (a measure of the dynamic ionic movement across cell membranes) and membrane permittivity (an indicator of the ionic charges present on cell membranes) vary, these data seem to suggest that exposure of K562 leukaemic cells to ELF fields affects both static and dynamic properties of these cells.

The feasibility of using EPR and the paramagnetic derivative of coal 'fusinite' to measure intracellular oxygen concentration in cultured cells in which this substance was internalized in the cytoplasm was examined. First, the possible cytotoxic effects of fusinite on cultured cells were ruled out by both morphological as well as by growth characteristics analyses. After construction of a calibration curve in which the EPR spectral linewidth of this substance was measured in response to known oxygen concentrations, the efficacy of using fusinite in the determination of intracellular oxygen concentration in cells was also tested by flowing different known oxygen gas mixtures outside cultured cells. The results indicate that fusinite is able of measuring the variations in cytoplasmic oxygen concentration that exist in response to the different gas mixtures. In addition, as an example of a possible use of fusinite, data are also presented demonstrating a decrease in cytoplasmic oxygen concentration during respiration in cells with a limited supply of oxygen. In fact, as the oxygen is consumed by the cells, the linewidth of fusinite narrows giving an intracellular oxygen concentration corresponding to zero. From the results obtained, fusinite appears to represent a new extremely precise biophysical cellular oxygen probe which may prove useful in the understanding of the complex interrelationships between oxygen and normal cell physiology and/or pathology.

It has been hypothesized that reactive oxygen intermediates (ROI) can activate human immunodeficiency virus (HIV) replication and that HIV can trigger programmed cell death (PCD). In this work, we studied PCD in U937 cultured cells chronically infected with HIV and exposed to tumor necrosis factor alpha (TNFα). This cytokine has been shown to induce apoptosis in some cell types and to produce intracellular free radical species including ROI. In addition, it was also demonstrated that HIV-induced PCD observable in U937 infected cells can be favored by TNF exposure. In one of our recent works, evidence was presented that the thiol supplier N-acetylcysteine (NAC) can 'protect', at least partially, HIV-infected cells from PCD and determine a significant decrease in viral progeny. In the present work, we demonstrate (a) that apoptosis can be easily induced by TNF only in infected U937 cells and not in control wild-type cells, (b) that daily treatment of TNF-exposed cells with low concentrations of NAC is able to impair viral progeny formation as early as 24 h, (c) that the mitochondrial damage induced by TNF is counteracted by preexposure to NAC, and (d) that NAC alone exerts changes in mitochondria which may be responsible for the protective effects exerted by this compound. Because of the radical producing capacity of TNF, these results seem to indicate that the protective effects of NAC may be due to the specific antioxidant nature of this substance which appears to be capable of impairing both the apoptotic machinery and viral replication by an intracellular mechanism involving mitochondrial integrity and function.

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.

Insulin-induced down-regulation of erythrocyte insulin receptors is a simplified model that can provide useful information on the cell surface regulative phenomena and on role of the plasma membrane and cytoskeleton in such physiological processes. Oxidative imbalance was examined since it was shown to play an important role in numerous cellular pathologies as well as in cell aging. Specifically, the free radical inducer menadione was used in order to evaluate if this compound is able to modify (and in which manner) the down-regulation process. Biochemical, biophysical, and ultrastructural approaches were used. The results obtained seem to indicate that menadione-induced oxidative damage was able to decrease the insulin-induced down-regulation process, as measured by binding assays. This effect was accompanied by slight alterations in plasma membrane ultrastructure and insignificant variations in plasma membrane lipid composition. In addition, the decrease in membrane order, measured by electron paramagnetic resonance, which was shown to usually occur during the process of down-regulation, was not observed. In contrast, cytoskeletal protein assembly, as previously shown in other in vitro systems, appeared to be remarkably altered. Such changes in specific cytoskeletal elements could lead to the decrease of down-regulation phenomenon induced by menadione. Changes in electrophoretic pattern of some cytoskeletal proteins (e.g., spectrin) reenforce this hypothesis. Considering the importance of free radicals in cell injury, data reported here could represent a specific example of a general mechanism by which cell surface receptor expression and recycling can be modified by changes in some intracellular molecule redox status and cell ionic homeostasis.

Plasma membrane modifications have been widely recognized as crucial factors in cell injury and death. One of these modifications, surface blebbing, has been considered as an injury-marker associated with a series of biochemical and physiological modifications. Our study focused on the different effects of free radical-induced cell damage by quinone menadione (2-methyl-1,4-naphthoquinone) and by hyperthermic shock (45 degrees C) on the erythroleukemic cell line K562. Different techniques including immunofluorescence, freeze-fracturing, and electron paramagnetic resonance spectroscopy were employed. Menadione induced the formation of surface blebs, accompanied by a rearrangement of the microfilament system and changes in the distribution of plasma membrane proteins. In contrast, heat-shocked cells showed neither blebbing nor important cytoskeletal changes. Finally, the electron paramagnetic resonance results showed an increase in membrane order not specifically related to the type of free radical-induced stress. These cell death features appear to suggest the existence of two different types of pathways for necrotic cell death: both treatments induce cell injury and eventual death by modifying plasma membrane integrity and function. However, one involves cytoskeleton-dependent surface blebbing, whereas the other does not.

The exposure of the epidermoid cell line A431 to different concentrations of CsCl was assessed using different methodological approaches. Two different effects were detected depending upon the concentration of the agent: at low concentrations, cell modification was represented mainly by a very pronounced cell flattening and an alteration of the cell-to-cell contacts, interpreted as an increase in cell adhesion. At higher concentrations, a clear pathogenic effect was observed that allowed the formulation of the hypothesis that specific mechanisms of toxicity at the subcellular level involving mitochondrial and cytoskeletal function can exist. In addition, membrane order parameters, as detected by electron paramagnetic resonance (EPR) spectroscopy, displayed a dose-dependent increase in membrane rigidity. Results reported here seem to suggest that cesium ions can enter the cell, modify plasma membrane integrity and alter some specific cytoplasmic components, e.g. the cytoskeleton. Considering that environmental contamination by cesium as a result of radioactive fallout is of major importance and that few data are available thus far on this matter, this study provides evidence for the possible mechanisms of action of the non-radioactive form of this ion in cells.

The effects of a 50-Hz extremely low frequency magnetic field on cultured K562 cells growing in suspension were studied by means of scanning electron microscopy and electron paramagnetic resonance spectroscopy. Exposure of K562 cells at 2.5 mT for periods to 96 hours induced significant changes in cell-surface structure and physiology without modification of proliferative capability as indicated by quantitative analysis. Thus extremely low frequency fields seem able to induce injurious, sublethal cell alterations, and the plasma membrane seems to play an important role in this effect. © 1993 Wiley-Liss, Inc.

The morphological (using scanning electron microscopy) and biophysical (determination of membrane order by electron paramagnetic resonance spectroscopy) changes occurring in the erythroleukemic cell line K562 were determined after heat-shock (physical) and gliotoxin treatment (chemical) stress. The data seem to indicate that although similarities in the response of these cells to different types of stress agents exist, there are profound differences in the mechanisms of cell death. Specifically, gliotoxin seems to induce free-radial cytoskeletal cross-linking of actin microfilaments and subsequent surface blebbing and increases in membrane order while heat-shock appears to bring about an increase in membrane order due to changes in membrane proteins.

Ions play a vital role in numerous biological processes including myoblast membrane fusion. 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 [M.T. Santini, A. Bonincontro, C. Cametti and P.L. Indovina, Biochim. Biophys. Acta, 945 (1988) 56–64]. Analysis of the conductivity dispersion data (obtained in the radio-frequency range) indicated that cesium delays fusion by ca. 30 hr. Since this ion is known to block potassium channels, the fusion delay appears to be due to interference with the sodium/potassium equilibrium. In the present study, we have concentrated our efforts on trying to understand the role of ions during myoblast fusion in a more detailed manner. Specifically, the effects of sodium, potassium and various concentrations of cesium on myoblast fusion were investigated. Our data indicate that addition of sodium has no effect on membrane fusion, whereas potassium delays this process by about 10 hr. In addition, different concentrations of cesium delay myoblast membrane fusion in a concentration-dependent manner. We discuss the possible mechanisms by which these ions perturb the sodium/potassium equilibrium and, consequently, the fusion process.

Primary chick embryo myoblasts can be a useful tool for studying the developmental events which accompany myoblast differentiation, particularly myoblast membrane fusion. To determine whether the electrical properties and/or fusion in these systems are affected by 50 Hz magnetic fields, chick embryo myoblast cultures were exposed to B-field intensities ranging from 1 to 10 mT. The electrical parameters of the myoblasts, i.e. membrane conductivity, membrane permittivity and the conductivity of the cell interior (cytosol) were determined by the analysis of conductivity dispersion data in the radio frequency range (10 kHz-100 MHz). Preliminary results indicate that the time of fusion (60 h) is not affected by these fields, but that the absolute values of the two membrane electrical parameters are affected. In particular, a B-field intensity-dependent decrease was observed. The maximum effect resulted after a 1 h exposure to a magnetic flux density of about 5 mT. The conductivity of the cytosol remained unchanged. These data seem to indicate that exposure to 50 Hz magnetic fields affects both static and dynamic membrane properties in primary chick embryo myoblasts.

We have recently demonstrated that the delay in myoblast membrane fusion induced by cesium is accompanied by changes in isolated membrane lipids (Santini, M.T., Indovina, P.L., Cantafora, A. and Blotta, I. (1990) Biochim. Biophys. Acta 1023, 298–304). In the present study, we have investigated changes in the lipid profile of total cell homogenates and microsomal membrane fractions during myoblast membrane fusion as well as the effects that addition of cesium may have on these lipid variations in order to try to understand the production and translocation of lipids during this myogenic process. The data presented here indicate that the lipid composition of cell homogenates and microsomes varies in a different manner from isolated plasma membranes during myogenic fusion. In addition, cesium affects, in a different manner, the normally-occurring lipid production and distribution which takes place in each subcellular fraction.

The intravenous administration of dimethylethanolamine in the rat promotes a selective enrichment of liver membranes with polyunsaturated phosphatidylcholines. The effect of dimethylethanolamine pretreatment on cholestasis induced by estradiol 17 beta-D-glucuronide, a potent cholestatic agent, was assessed in this study. Dimethylethanolamine, dissolved in sodium-taurocholate was infused intravenously (0.3 mg/kg/min) for 15 hr. One group of control rats (estradiol 17 beta-D-glucuronide controls) received the bile salt only. An estradiol 17 beta-D-glucuronide bolus was then injected intravenously (10.4 mg/kg) into dimethylethanolamine-pretreated and estradiol 17 beta-D-control rats, and its effect on bile flow and biliary lipid secretion was compared for 3 hr. The estradiol 17 beta-D-glucuronide inhibitory effect on bile flow and biliary lipid secretion was significantly antagonized by dimethylethanolamine pretreatment. The maximum inhibition of bile flow was found 30 min after estradiol 17 beta-D-glucuronide administration, when it decreased from 3.5 +/- 0.4 microliters/min/100 gm (basal) to 0.9 +/- 0.3 microliters/min/100 gm in estradiol 17 beta-D-glucuronide controls, whereas in dimethylethanolamine-pretreated rats this decreased only from 3.2 +/- 0.4 (basal) to 2.3 +/- 0.4 microliters/min/100 gm. Bile flow and the biliary secretion of cholesterol, phosphatidylcholine and bile salts were significantly higher in the dimethylethanolamine-pretreated rats than in estradiol 17 beta-D-glucuronide controls (p less than 0.02) during the cholestatic phase. The inhibitory effect of estradiol 17 beta-D-glucuronide on bile flow was associated with a marked decrease of membrane fluidity (p less than 0.001) assessed by 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy and with a cholesterol enrichment of microsomes, sinusoidal and canalicular liver plasma membranes and inhibition of sinusoidal Na+,K(+)-ATPase activity (p less than 0.05). These membrane alterations persisted 180 min after estradiol 17 beta-D-glucuronide administration despite complete normalization of bile flow. Dimethylethanolamine pretreatment significantly counteracted the reduction of membrane fluidity (p less than 0.001), the cholesterol enrichment and the inhibition of Na+,K(+)-ATPase (p less than 0.05) promoted by estradiol 17 beta-D-glucuronide administration in all membrane subfractions 30 and 180 min after administration. In addition, dimethylethanolamine-pretreated rats had more polyunsaturated fatty acids in membrane phosphatidylcholine with respect to the control groups. Dilatation of canaliculi and loss of microvilli were evident in estradiol 17 beta-D-glucuronide controls 180 min after estradiol 17 beta-D-glucuronide administration.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of hypoxia on adhesion and spreading of MG-63 human osteosarcoma spheroids were investigated. Hypoxia was induced in 2-day-old, small spheroids and verified by HIF-1alpha expression. Changes in adhesion were examined on both tissue culture plates and plates coated with fibronectin or collagen while spreading was analyzed in cocultures of MG-63 spheroids seeded on primary fibroblasts grown as a monolayer. In order to better distinguish the two different cell types, MG-63 cells were previously stably transfected with the green fluorescent protein EGFP-vector. Changes in the expression of molecules involved in tumor adhesion and spreading, such as two key integrins (fibronectin receptor, alpha5, and collagen receptor, alpha2) and fibronectin were also examined. The results indicate that hypoxia increases adhesion of spheroids and enhances their ability to spread into the surrounding fibroblast cell culture. These changes in adhesion and spreading are accompanied by concomitant variations in the expression of alpha5 and alpha2 integrins and fibronectin.

High-resolution proton nuclear magnetic resonance ((1)H-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.

Insulin receptor internalization in response to insulin incubation (down-regulation) has been shown to occur in human erythrocytes as well as in human erythrocyte ghosts. It is also known that changes in cell membrane events can be detected with electron paramagnetic resonance (EPR) spectroscopy using spin labels. In the present study, changes in erythrocyte membrane order during down regulation as measured by the 2 T ′ II parameter were investigated using EPR. The spin label, 5-nitroxystearate, which inserts into the lipid bilayer of cell membranes, was used. Changes in 2 T ′ II at 37° C were followed over a 3 h time period. A transient decrease in erythrocyte membrane order began within 30 min of the start of insulin incubation and reached a minimum level of 52.5 Gauss (G) within 90 min. This represented nearly a 2 G decrease from the zero incubation time value. Membrane order returned to the initial value by 2.5 h. These time-related changes in membrane order corresponded well with the insulin receptor internalization process as followed by surface binding assays. Surface insulin binding began to decrease within 30 min of the start of insulin incubation, and was reduced to 30% of control values within 2 h. Similar correlations between membrane order and receptor internalization were observed at 23° C. Erythrocytes incubated with denatured insulin, and ATP-depleted erythrocytes incubated with native insulin, did not down-regulate their insulin receptors. Under these conditions, these erythrocytes also did not exhibit the transient decreases in membrane order. These findings are consistent with the hypothesis that an increase in membrane disorder is part of the mechanism of insulin receptor down-regulation.

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.

Hypoxia through HRE (hypoxia-responsive element) activity in MG-63 human osteosarcoma cells grown in monolayer and as very small, three-dimensional tumor spheroids was investigated using molecular imaging techniques. MG-63 cells were stablv transfected with a vector constructed with multiple copies of ibe HRE sequence of the human vascular endothelial growth factor (VEGF) gene and with the enhanced green fluorescent protein (EGFP) coding sequence. During hypoxia when HIF-1 alpha (hypoxia-inducible factor-1 alpha) is stabilized, the binding of HIEF-1 to the HRE sequences of the vector allows the transcription of EGFP and the appearance of fluorescence. Transfecled monolayer cells were characterized by flow cytometric analysis in response to various hypoxic conditions and HIF-1 alpha expression in these cells was assessed by Western blotting. Two-photon excitation (TPE) microscopy was then used to examine both MG-63-transfected monolayer cells and spheroids at 2 and 5 days of growth in normoxic conditions. Monolayer cells reveal almost no fluorescence, whereas even very small spheroids (< 100 mu m) after 2 days of growth contain regions of high fluorescence. For the first time in the literature, at least to our knowledge, it is demonstrated, using highly sensitive and non-perturbing molecular imaging techniques, that three-dimensional cell organization leads to almost immediate HRE activation. This activation of the HRE sequences, which control a wide variety of genes, suggests that monolayer cells and spheroids of the MG-63 cell line have different genes activated and thus diverse functional activities. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The metabolic changes that occur as a function of time in MG-63 osteosarcoma three-dimensional tumor spheroids undergoing radiation-induced apoptosis were studied using high-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. Specifically, the (1)H-NMR spectra of MG-63 spheroids collected at 24, 48 and 72 h after exposure to 5 Gy of ionizing radiation were compared to the spectra of their respective controls. Small spheroids (about 50-80 microm in diameter) with no hypoxic center were used. Apoptosis was verified by both staining of spheroid DNA with the Hoechst 33258 dye and determination of caspase 3 enzyme activity at the three times examined. The results demonstrate that, as the percentage of apoptosis rises with time after exposure to ionizing radiation, the metabolic changes that take place in MG-63 spheroids follow very precise temporal dynamics. In particular, significant time-related increases in both CH(2) and CH(3) mobile lipids, considered by many authors as markers of apoptosis, were observed. In addition, temporal variations were also observed in choline-containing metabolites, reduced glutathione (GSH), glutamine/glutamate, taurine, alanine, creatine/phosphocreatine and lactate. These data show that in addition to CH(2) and CH(3) lipids, other metabolites can also be extremely useful in a deeper understanding of the temporal dynamics of radiation-induced apoptosis. This comprehension is particularly important in spheroids, a cell model of great complexity that resembles in vivo tumors much more closely than monolayer cultures. Ultimately, it is hoped that such studies can help to evaluate the outcome of radiotherapy protocols more accurately.

High resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy was used to examine the response of the MG-63 osteosarcoma cell line grown in monolayer and as 3-dimensional tumor spheroids to the same low dose (2 Gy) of ionizing radiation. The MG-63 cells and spheroids were irradiated at 24 h of growth and the 1H-NMR spectra of whole control and irradiated monolayer cells and of whole control and irradiated multicellular spheroids collected after another 24 h were compared. The 1H-NMR spectra of the perchloric acid extracts as well as the 2-dimensional 1H-NMR spectra of both pairs of cell systems were also obtained. Possible radiation-induced cell damage was determined by lactate dehydrogenase (LDH) release and variations in cell growth, while cell death was evaluated by chromatin dye Hoechst staining and DNA fragmentation assays. The results demonstrated that no cell damage took place, but that significant variations in numerous metabolites occured in both the monolayer cells and the spheroids after irradiation. Most of the changes observed were very similar in nature. In fact, significant increases in lactate, alanine, creatine and phosphocreatine and choline-containing metabolites and a significant decrease in glutathione (GSH) were observed in both cells and spheroids. However, while significant increases in CH2 and CH3 mobile lipids, glutamine/glutamate, taurine and inositol were seen in the spheroids, no variations in CH2 or CH3 lipids, glutamine/glutamate or taurine were recorded in the MG-63 cells grown in monolayer after irradiation. In addition, a significant decrease rather than a significant increase in inositol was also noted in the monolayer cells. The data presented seem to suggest that, although neither monolayer cells nor spheroids show apparent signs of damage after exposure to the same dose of ionizing radiation, very different cell death responses as well as very diverse antioxidant/osmoregulatory reactions were triggered by this stressing agent.

One of the most important questions in tumor biology is the understanding of the mechanisms responsible for the resistance of cancer cells to radiotherapy. In the present study, the possible role played by cell-cell interactions in determining the response of tumor cells to ionizing radiation was investigated. HT-29 colon adenocarcinoma spheroids were irradiated with a dose of 15 Gy in two different stages of growth characterized by diverse degrees of compaction: loosely organized spheroids (early spheroids) and compacted spheroids (late spheroids). Morphological analyses demonstrated that late spheroids were less damaged than early spheroids. Moreover, analyses of the cell cycle and cell death showed that ionizing radiation induced necrosis in early spheroids and apoptosis in late ones. From these results it can be concluded that late, compacted spheroids are more resistant to ionizing radiation than early, loose spheroids. In order to understand the mechanisms regulating this compaction-induced resistance of late spheroids, E-cadherin/beta-catenin complex expression and distribution were analyzed. In late spheroids, E-cadherin/beta-catenin complexes were shown to be tethered to the cytoskeleton, and since this organization has been demonstrated to strengthen cell-cell adhesion in other systems, it can be postulated that the same is true in HT-29 spheroids. In conclusion, it can be hypothesized that compaction of HT-29 spheroids is mediated by the reorganization of E-cadherin/beta-catenin complexes on the plasma membrane and that this compaction may be responsible for the increase in resistance of HT-29 spheroids to ionizing radiation.

The possibility that a sinusoidal 50 Hz magnetic field with a magnetic flux density of 1 mT can damage MG-63 osteosarcoma spheroids and induce variations in the invasive properties of these three-dimensional model systems after 2 days of exposure was investigated. Specifically, possible damage induced by these fields was examined by determining changes in spheroid surface morphology (light microscopy), growth (spheroid diameter and protein content determination), lactate dehydrogenase release, and reduced glutathione amount. Possible changes in the invasive properties were studied by invasion chambers. The results show no induction of cell damage by ELF fields while invasion chamber assays demonstrate a significant increase in the invasive potential of exposed spheroids. In order to determine if the fibronectin or hyaluronan receptors are involved, Western blot analysis was conducted on these two proteins. No significant variations were observed in either receptor in MG-63 multicellular tumor spheroids.

Purpose: To study cell damage and possible apoptosis in K562 human erythroleukemic cells exposed for 2?h to an extremely low frequency (ELF) 50 Hz sinusoidal magnetic field with a magnetic induction of either 1 or 5 mT using high resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy. Materials and methods: One-dimensional 1H-NMR spectra were obtained on whole K562 cells and perchloric acid extracts of these cells. In addition, two-dimensional 1H-NMR spectra were also acquired. Cell damage was examined by lactate dehydrogenase release and changes in cell growth were monitored by growth curve analyses, bromodeoxyuridine incorporation, and Ki67 antigen localization. Cell death (necrosis and apoptosis) was also studied by using the chromatin dye Hoechst 33258. Results: The variations in numerous metabolites observed with 1H-NMR reveal apoptosis-like behavior in response of K562 cells to ELF fields. Conclusion:1H-NMR can be extremely useful in studying the effects of ELF fields on cells. In particular, the variations in metabolites, which suggest apoptosis-like behavior, occur when the cells are not identifiable as apoptotic by more traditional techniques.

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.

We have recently demonstrated that if human K562 erythroleukemic cells, which normally grow in suspension, are grown in polylysine-coated culture flasks for 48 h, they adhere to these flasks and grow in an anchorage-dependent like manner. Important changes in both membrane conductivity (ionic transport across the cell membrane) and membrane permittivity (static distribution of charges across the cell membrane) were also observed, indicating perturbations in membrane lipids, proteins and polysaccharides. In order to better understand the changes occurring in K562 cells exposed to polylysine and because of the important role played by cell adhesion molecules (CAMs) in cell/cell and cell/substratum interactions, and in cellular adaptation to the surrounding environment, the possible redistribution of these molecules after exposure to polylysine were investigated. In particular, the CD54 (ICAM-1), CD58 (LFA-3) and alphaVbeta3 (vitronectin receptor) molecules were investigated at different times of growth both quantitatively and qualitatively utilizing flow cytometry and immunofluorescence microscopy, respectively. The data indicate that there were no significant quantitative variations in the CAMs examined at all the times tested. In addition, no qualitative changes were observed at 48 h (as well as 24 h) of exposure. However, shorter treatment times (30 min, 1 and 2 h) did induce important CAM reorganization. The results seem to demonstrate that this cycle of CAM redistribution may, in part, be responsible for cellular adaptation to the new growth environment of K562 cells and for the variations in membrane electrical properties observed.

High resolution proton nuclear magnetic resonance ((1)H 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 microm in diameter), three-dimensional tumor spheroids with no hypoxic center has different metabolic characteristics. Consequently, the (1)H 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 CH(2) and CH(3) groups were also seen, it is not possible at this time to establish if lipid metabolism is truly different in cells and spheroids.

This study investigates cell growth and death (apoptosis and necrosis) in actively proliferating MG-63 osteosarcoma spheroids in response to two doses of ionizing radiation (5 and 30 Gy). Cell growth was examined by growth curves and by cell cycle analyses utilizing flow cytometry. Death was examined in both disaggregated and whole spheroids by the chromatin dye Hoechst and by Western blot analysis of the bcl-2 family of proteins known to be involved in the apoptotic process. The results indicate that after exposure to 5 Gy MG-63 spheroids die by apoptosis, while after exposure to 30 Gy they die by necrosis. The analysis of the bcl-2 family of proteins demonstrates that bcl-2, bax and bcl-XL are involved in triggering apoptosis in spheroids exposed to 5 Gy. The possibility of studying the mechanisms responsible for radiation-induced cell death in three-dimensional spheroids (which represent much more closely in vivo tumors) is extremely important if the foundations for more selective and controllable clinical radiotherapeutic protocols are to be laid.

The role of protein adsorption in the forced adhesive growth of K562 leukemic cells onto a cationic surface composed of polylysine was investigated. Numerous studies have demonstrated that adhesion in anchorage-dependent cells is mediated in vitro by adsorption of serum proteins [particularly proteins of the extracellular matrix (ECM) such as fibronectin and vitronectin] present in the growth medium. Specifically, adhesion has been shown to occur when ECM proteins attach to the substratum and act as ligands for specific receptors located on the surface of cells. K562 cells are human erythroleukemic cells that normally grow in suspension. These cells are not involved in the same cell adhesion processes as anchorage-dependent cells and do not need to be attached to ECM proteins in order to survive and grow. Thus, with these systems, it is possible to better determine the role of protein adsorption in the adhesion of cells, growing in suspension such as blood cells, onto charged surfaces. The results presented show that adhesion of K562 cells onto the positively charged polylysine surface in the presence of serum is mediated through specific interactions between fibronectin receptors present on K562 cells and fibronectin adsorbed onto that cationic surface. Specifically, determination of cell adhesion under different experimental conditions indicates that nonspecific charge interactions do not take place directly between the cells and polylysine, but rather take place between polylysine and fibronectin, which adsorbs onto the cationic polymer. In addition, flow cytometric analyses reveal that only fibronectin receptors are present on these cells and, consequently, only fibronectin can be responsible for the actual adhesion of these cells onto the cationic surface. In view of the data presented, the possibility should be considered that ECM components adsorbed onto surfaces with specific charges and/or belonging to certain functional groups are involved in structural and functional modifications in cells. These cells grow in suspension and are normally not involved in adhesion phenomena, though these components should be considered. These considerations should be made especially when designing biomaterials that can modulate the response of cells growing in suspension, such as blood cells, and also in tissue engineering of blood substitutes.

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.

Recently, a new method for quantitatively comparing NMR spectra of control and treated samples, in order to examine the possible occurring variations in cell metabolism and/or structure in response to numerous physical, chemical, and biological agents, was proposed. This method is based upon the utilization of the maximum superposition normalization algorithm (MaSNAl) operative in the frequency domain and based upon maximizing, by an opportune sign variable measure, the spectral region in which control and treated spectra are superimposed. Although the frequency-domain MaSNAl algorithm was very precise in normalizing spectra, it showed some limitations in relation to the signal-to-noise ratio and to the degree of diversity of the two spectra being analyzed. In particular, it can rarely be applied to spectra with a small number of visible signals not buried in the noise such as generally in vivo spectra. In this paper, a time-domain normalization algorithm is presented. Specifically, it consists in minimizing the rank of a Hankel matrix constructed with the difference of the two free induction decay signals. The algorithm, denoted MiRaNAl (minimum rank normalization algorithm), was tested by Monte Carlo simulations as well as experimentally by comparing two samples of known contents both with the new algorithms and with an older method using a standard. Finally, the algorithm was applied to real spectra of cell samples showing how it can be used to obtain qualitative and quantitative biological information.

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.

The mechanisms responsible for the adhesion of cells onto a material's surface and the effects that that adhesion may have on cell structure and function are fundamental questions in biomaterials research. We recently demonstrated that the erythroleukemic cell line K562, which normally grows in suspension, can be induced to grow attached to a polylysine-coated solid surface in an anchorage-dependent manner. In this study, the effects of the growth of K562 cells onto polylysine were further investigated utilizing 500 MHz 1H-NMR spectroscopy. The NMR results showed that when K562 cells are grown attached to a positively-charged polylysine surface, there are alterations in lipids and energy metabolism. In particular, there was a 31% increase in phosphatidylcholine and a 15% decrease in each of its two precursors, glycerophosphatidylcholine and choline, as well as a 20% increase in CH2 lipids and a 7% decrease in CH3 lipids in treated cells compared to the controls. These results suggest that adhesive growth can induce strong variations in membrane structure, including the membrane fluidity of K562 cells. In addition, in cells attached to polylysine there was about a 10% decrease in creatine (together with phosphocreatine), a 20% increase in γ-glutamate, a 15% increase in β-glutamate, and a 24% decrease in lactate. This second set of results, which is closely related to energy metabolism, indicates that not only does adhesive growth induce changes in K562 cell membrane structure, but also in the utilization of energy in these cells. The data are discussed in view of the possible role played by surface charge in affecting cell structure and function in cells that come into direct contact with charged biopolymers. © 1999 John Wiley & Sons, Inc. J Biomed Mater Res, 46, 171–178, 1999.

Chronic obstructive pulmonary disease (COPD) is characterized by inflammation of deep lung and pulmonary hypoxemia. In order to investigate if the clinical manifestations of this disease can be correlated to specific alterations in red blood cell (RBC) morphology, the erythrocytes from 12 COPD patients and 12 control subjects were obtained and examined by scanning electron microscopy (SEM), fluorescence microscopy and electron paramagnetic resonance (EPR) spectroscopy. The results demonstrate that the RBCs from COPD patients are greatly altered with respect to control erythrocytes. Specifically, SEM analysis revealed important shape changes while light fluorescence microscopy demonstrated microfilament network (actin and spectrin) redistribution. Finally, EPR spectroscopy, using the paramagnetic spin label 5-nitroxystearate, revealed an increase in membrane order (rigidity) in the erythrocytes of COPD patients with respect to controls. When taken together and when compared to the morphological variations present in the RBCs of other ill patients (i.e., diabetics), the data presented in this report seem to suggest that changes in erythrocyte shape and rheological properties play a key role in RBC dysfunction in the course of COPD.

The ionic environment of the cell membrane is of extreme importance in maintaining cell integrity and the numerous functions necessary for cell growth, differentiation, etc., as well as in cell-biomaterial interactions. In this study, the effects of polylysine (a basic poly-amino acid with a net positive charge which is often used to coat biomaterials surfaces) on the erythroleukemic K562 cell membrane were investigated. In particular, the effects of this polycation were evaluated using dielectric relaxation studies in the radiofrequency range with which it is possible to measure both active ionic transport across the cell membrane (membrane conductivity) and the static charge distribution present on the cell surface due to the structural components of the cell membrane (membrane permittivity). The conductivity of the cytosol can also be determined. The results demonstrate that while the conductivity of the cytosol is not significantly altered, both the conductivity and permittivity of the K562 cell membrane are varied by exposure of these cells to polylysine. These observations indicate that both active ionic transport and the type, quantity, or distribution of membrane components such as lipids, proteins, and polysaccharides may also be altered. Although the precise mechanisms by which these variations in K562 cells occur are unknown, it can be hypothesized that changes in the growth characteristics of these cells may be in part responsible. In particular, as demonstrated by light microscopic examination of K562 cells directly in the culture flasks, the cells in polylysine-coated flasks do not grow in suspension as do the controls, but rather show anchorage-dependent-like behavior. It is this important change from suspension to monolayer growth induced by polylysine that may be responsible for the changes in membrane electrical parameters.

The intracellular molecular oxygen concentration in control and menadione-treated K562 (an erythroleukemic cell line that grows in suspension) and A431 (an epidermal carcinoma that grows in monolayer) cells was measured directly by using the new electron paramagnetic resonance (EPR) probe fusinite. Because the oxidizing agent menadione is known to damage mitochondria and the cytoplasmic membrane in other cell systems, before conducting measurements of oxygen concentration in K562 and A431 cells, it was necessary to establish injury in these systems as well. Consequently, morphological and flow cytometric analyses were conducted after menadione treatment. The data presented here show that the two cell lines are heavily damaged by menadione. Once this menadione-induced injury was demonstrated, measurements of oxygen concentration were carried out in both K562 and A431 cells. Treatment with this quinone induces a sharp increase in intracytoplasmic molecular oxygen in both cell lines (from about 1% to about 10 and 15% in K562 and A431 cells, respectively). In addition, to gain a more complete understanding of the effects of menadione on cells, the extracellular molecular oxygen concentration and the oxygen consumption rate were also measured in control and menadione-treated K562 cells. These measurements demonstrate that menadione treatment results in an increase in the extracellular oxygen concentration (from about 5% in controls to 15% in treated cells) as well as a decrease in the oxygen consumption rate (from about 10 ng O/min/10(6) cells in controls to 3 ng O/min/10(6) cells after menadione exposure). The importance of the new EPR probe fusinite in monitoring directly cellular functions in which oxygen is involved and the effects of menadione on cellular oxygen balance are discussed.

The oxidative denaturation of the erythrocyte membrane, which is considered a major cause of the haemolytic process, 'was evaluated upon ‘in vitro’ oxidative stress with tert -butylhydroperoxide. Biochemical and ultrastructural analyses were performed to point out the effect of this substance on the skeletal network, which is mainly responsible for red cell shape and viability. Moreover, cell morphology was observed by scanning electron microscopy and membrane rigidity assessed by EPR measurements. The most relevant features of the membrane denaturation were, (i) lipid peroxidation, as assessed by malonildialdehyde production, (ii) spectrin and ankyrin degradation with simultaneous globin binding to the membrane, as evidenced by electrophoretic pattern of red cell ghosts. These phenomena were related to the drug concentration in the incubation medium, and accompanied by depletion of intracellular reduced glutathione. The denaturation of protein components hindered the release of spectrin in a hypotonic extraction medium and could be only partially reversed by dithiothreitol. The extensive membrane protein and lipid degradation, at high drug concentration, was coherent with a marked increase of membrane order (membrane ‘rigidity’). No clustering of intramembrane proteins was shown by the transmission electron microscopy images. At the same time scanning electron microscopy demonstrated shrinking and disco-stomatocytic deformation of erythrocytes. Ultrastructural analysis of the membrane skeleton by fluorescence-labelling of spectrin and actin, allowed to point out that exposure to t-BHP caused the marginalization of spectrin and the rearrangement of actin molecules with formation of micro aggregates, so that a detachment of actin from the spectrin network was suggested. In addition to the generalized damage of red cell membrane, tert -butylhydroperoxide was found to induce a specific alteration of the skeletal network at the horizontal junction sites involving spectrin, actin, and protein 4.1 and thus to modify the cytoskeletal assembly. This effect on the membrane skeletal components was consistent with the hypothesis that oxidative stress plays a key role in the haemolytic process.