Biomedical subjects

M T Santini

Publications and source records attributed to M T Santini.

At least 19 recordsLinked to original sources

PubMed2005

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.

Apoptosis↗

PubMed2005

A 700 MHz 1H-NMR study reveals apoptosis-like behavior in human K562 erythroleukemic cells exposed to a 50 Hz sinusoidal magnetic field.

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) were 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.

Apoptosis↗

PubMed2001

Modulation of osteosarcoma cell growth and differentiation by silane-modified surfaces.

The effects of growing the Saos-2 human osteosarcoma cell line onto surfaces containing -CH(3), -OH, -COOH, -NH(2), 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 (CH(3)), hydroxyl (OH), and phenyl (C6H5) functional groups, their proliferation is slowed down while growth of these cells on glass surfaces modified with amino (NH(2)) 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 CH(3) 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.

Apoptosis↗

PubMed2001

An Escherichia coli cytotoxin increases superoxide anion generation via rac in epithelial cells.

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.

Bacterial Toxins↗

PubMed2001

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Fibronectin facilitates adhesion of K562 leukemic cells normally growing in suspension to cationic surfaces.

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.

Adsorption↗

PubMed2000

A time-domain algorithm for NMR spectral normalization.

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.

Algorithms↗

PubMed1999

Forced adhesive growth of K562 leukemic cells that normally grow in suspension induces variations in membrane lipids and energy metabolism: a proton NMR study.

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 gamma-glutamate, a 15% increase in beta-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.

Cell Adhesion↗

PubMed1999

A new algorithm for NMR spectral normalization.

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.

Algorithms↗

PubMed1999

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.

Animals↗

PubMed1998

The new EPR molecular oxygen probe fusinite is not toxic to cells.

The possible cytotoxic effects of fusinite, a new charcoal-like electron paramagnetic resonance (EPR) oxygen probe, were evaluated in three cell types with very different characteristics and growth features: K562 (an erythroleukemic cell line which grows in suspension), A431 (an epidermal carcinoma cell line which grows in monolayer) and primary cultures of murine fibroblasts (which also grow in adhesion culture) utilizing morphological and functional studies as well as growth analyses. Scanning and transmission electron microscopy as well as fluorescence microscopy were used for the morphological analyses while conductometric relaxation studies in the radiowave frequency range, membrane resistance measurements and adenine nucleotide levels were utilized for the more subtle functional evaluation of cell parameters. The results show that the presence of fusinite particles, even after long internalization times, does not induce any cytotoxic effects in the cells studied. Thus, from these results, it can be deduced that fusinite is non-toxic as well as highly stable, inert and very sensitive to oxygen, and can be used with great success for cell studies where determination of oxygen concentration is important.

Adenine Nucleotides↗

PubMed1998

Effects of hyaluronan viscous materials on cell membrane electrical properties.

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.

Cell Membrane↗

PubMed1998

Positively charged polymer polylysine-induced cell adhesion molecule redistribution in K562 cells.

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.

Journal Article↗

PubMed1998

Menadione induces changes in the membrane electrical properties associated with downregulation of insulin receptors in human erythrocytes.

We have demonstrated using dielectric relaxation measurements in the radiofrequency range that the electrical properties of human erythrocytes (membrane conductivity and permittivity as well as the conductivity of the cytosol) undergo a steady decrease over time during insulin-induced downregulation of insulin receptors (Santini et al., Experimental Hematology 22:40-44, 1994). In the present paper, we show that exposure of these erythrocytes to the strong oxidizing agent menadione before addition of insulin blocks insulin receptor internalization as well as the decrease in electrical parameters associated with this process. We hypothesize that menadione blocks internalization of these receptors by inducing oxidative damage on the erythrocytes and, consequently, disrupting the normally occurring variations in electrical parameters. This postulate was confirmed by addition of the antioxidant N-acetylcysteine to menadione-treated cells, in which a return to the original changes in electrical parameters noted in untreated cells was observed.

Down-Regulation↗

PubMed1997

Polylysine induces changes in membrane electrical properties of K562 cells.

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.

Cell Division↗

PubMed1996

Probucol reduces hepatic cholesterol secretion in hyperlipidemic Yoshida rats.

In this study, perfused livers from Yoshida rats, either on a normal diet or on a diet with 0.3% probucol, were examined. The analysis of liver lipid content and of bile and lipoprotein secretion changes showed that probucol had a relevant effect on liver lipid biosynthesis. In particular, it reduced the production of triacylglycerols and, to a much greater extent that of cholesterol. In addition, probucol reduced plasma cholesterol concentration by decreasing esterified cholesterol in HDL1 and HDL2 fractions. Furthermore, HDL1 composition of both hepatic neosynthetized and circulating particles was strongly modified by probucol. Finally, probucol did not appear to induce significant differences in lipid bile secretion while phospholipid secretion from perfused livers was increased. These facts suggest that the hypolipidemic action of probucol is not mediated by an increase in bile steroid secretion, but rather by a direct reduction in hepatic lipoprotein cholesterol secretion. This secretion induces a modified plasma profile of HDL particles such that these variations are advantageous in terms of reverse cholesterol transport.

Animals↗

PubMed1996

The oxidizing agent menadione induces an increase in the intracellular molecular oxygen concentration in K562 and A431 cells: direct measurement using the new paramagnetic EPR probe fusinite.

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.

Calibration↗

PubMed1995

Junctional sites of erythrocyte skeletal proteins are specific targets of tert-butylhydroperoxide oxidative damage.

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 tertbutylhydroperoxide. 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 malonidialdehyde 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, tertbutylhydroperoxide 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.

Ankyrins↗