Biomedical subjects

G Arancia

Publications and source records attributed to G Arancia.

At least 19 recordsLinked to original sources

PubMed2006

Cationic liposomes, loaded with m-THPC, in photodynamic therapy for malignant glioma.

In this study we investigated the feasibility of mixed liposomes formed by dimyristoyl-sn-glycero-phosphatidylcholine (DMPC) and cationic gemini surfactant (Gemini 1) loaded with the chlorin m-tetrahydroxyphenylchlorin (m-THPC), in photodynamic therapy (PDT) for glioma. To this aim, an in vitro study was carried out by employing various human glioblastoma cell lines (A172, DBTRG, LN229, U118). The following liposomal formulations were tested: (i) DMPC and Gemini 1; (ii) m-THPC in DMPC in the absence or (iii) in the presence of Gemini 1 in the molar ratio 8:2; 7:3, and 6:4. The presence of Gemini 1 significantly increased the intracellular uptake of chlorin in all cell tested although with a different extent: LN229>U118>A172>DBTRG. The cytotoxicity of chlorin-loaded liposomes was then tested by cloning efficiency performed on different cultures, before and after irradiation with laser light at 652nm, at a Fluence Rate of 200mW/s for 100s, with a total Fluence of 20J/cm(-2). In the absence of irradiation, the different liposomal formulations induced a cytotoxicity in less than 30% of glioblastoma cells. On the contrary, irradiation induced total destruction of all cultures treated with m-THPC/DMPC+Gemini 1 in the ratios 8:2, or 7:3, or 6:4.

Cell Line, Tumor↗

PubMed2006

Autophagy-mediated chemosensitizing effect of the plant alkaloid voacamine on multidrug resistant cells.

In our previous studies, voacamine, a bisindolic alkaloid extracted from Peschiera fuchsiaefolia, was examined for its possible capability of enhancing the cytotoxic effect of doxorubicin (DOX) on multidrug resistant (MDR) human osteosarcoma cells (U-2 OS-R). Voacamine induced in resistant cells a significant increase of drug retention and intranuclear location which became comparable to those observed in the parental sensitive counterparts (U-2 OS-WT). In the present study, the cell survival analysis and the electron microscopic observations confirmed the evident cytotoxicity of DOX on MDR cells after pre-treatment with the plant extract. Moreover, an increase of the reactivity of P-glycoprotein (P-gp) with the monoclonal antibody UIC2, which recognizes an epitope of the drug transporter in its functional conformation, was revealed, demonstrating that voacamine is a substrate of P-gp, thus acting as a competitive antagonist of the cytotoxic agent. Moreover, to investigate if the enhancement of the cytotoxic effect induced by voacamine could be due to an apoptotic process, we carried out the analysis of cell morphology after Hoechst staining and the quantification of apoptosis by Annexin V-FITC assay. These evaluations showed a very low rate of apoptosis in U-2 OS-R cells treated with voacamine and DOX given in association. In addition, the combined treatment induced ultrastructural modifications suggestive of autophagic cell death. In particular, transmission electron microscopy observations revealed the presence of numerous lysosomes and the formation of a large number of autophagosomes containing residual digested material. In conclusion, these findings seem to indicate that voacamine is capable of enhancing the cytotoxic effect of DOX on MDR cells by favouring a lethal autophagic process.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Caspase-independent apoptosis is activated by diazepam-induced mitotic failure in HeLa cells, but not in human primary fibroblasts.

DZ, a benzodiazepine known to affect centrosome separation at prophase, leads to a higher degree of mitotic arrest in HeLa cells than in primary human fibroblasts. In fact, differently from fibroblasts, which undergo a transient block in prophase-to-prometaphase transition, a high proportion of tumor cells attempt to escape from the DZ-imposed mitotic block, fail to undergo complete mitosis and die by mitotic failure. DZ-treated samples showed certain biochemical hallmarks of apoptosis, such as induction of the proapototic Bax protein, mitochondrial alterations assessed by JC-1 staining and TEM analysis, PARP cleavage, and DNA fragmentation. However, in DZ-treated cells, we observed a very low or absent caspase activation as shown by immunofluorescence and immunoblot experiments with antibodies directed to activated caspases and by staining with the pancaspase inhibitor FITC-VAD-FMK. Experiments on mitochondrial depolymerization and apoptosis induction carried out in the presence of specific inhibitors of caspase-2 and caspase-3/7 indicated a caspase-independent apoptotic process induced by DZ. Accordingly, TEM analysis of treated cells revealed ultrastructural features resembling those reported for caspase-independent apoptosis. In conclusion, we hypothesize that HeLa cells override the prophase block imposed by DZ, producing a high rate of aberrant pro-metaphases, which, in turn, activates caspase-independent, apoptosis-like mitotic catastrophe.

Apoptosis↗

PubMed2005

Glucan-associated protein modulations and ultrastructural changes of the cell wall in Candida albicans treated with micafungin, a water-soluble, lipopeptide antimycotic.

The composition of glucan-associated proteins (GAP) in the cell wall of Candida albicans was strongly affected by treatment with a sub-MIC yet beta-glucan synthesis inhibitory concentration (0.01 microg/ml) of FK463 (micafungin). Namely, a decrease in enzymes of glucose metabolism (mostly enolase and a novel 40 kDaltons component, here identified as the enzyme fructose-1,6-biphosphate aldolase) was observed, and this was coupled with an increase in two beta1-3 exo-glucanase isoforms (34 and 44 kDa, respectively). No GAP changes were detected in the same strain of the fungus made resistant to the drug, attesting to the specificity of the observed cell wall protein modulation. In addition, GAP changes were accompanied by marked ultrastructural alterations upon treatment with the sub-MIC dose of the drug, the majority of which was an aberrant cell surface morphology and a derangement of the normal layering of the cell wall. Our data demonstrate that sub-MIC doses of micafungin do critically affect not only the beta-glucan synthetic machinery but also protein composition and the whole cell wall structure of Candida albicans.

Antifungal Agents↗

PubMed2005

Invasive properties of multidrug resistant human melanoma cells.

Melanoma cells exhibit a high level of intrinsic or acquired resistance to the cytotoxic agents often associated with the over-expression of drug transporters such as P-glycoprotein (P-gp). In this in vitro study, we investigated the possible relationship between P-gp and CD44, the cell adhesion molecule involved in metastasis and tumor progression of melanoma cells. CD44 expression appeared to be similar in the parental sensitive M14 WT cells and in their resistant counterparts M14 ADR cells. Double-labeling of cryosectioned cells showed that P-gp and CD44 were transported from the synthesis loci to the cell periphery by different vesicles and began to coalesce in proximity of the plasma membrane; thus, P-gp and CD44 seemed to reach together the cell surface. Moreover, P-gp and CD44 appeared to be associated with ERM proteins. The invasive activities of both M14 WT and M14 ADR cells were analyzed by the "transwell chamber invasion" assay. M14 WT cells revealed low capacity to traverse the filters, both in the absence (motility) and in the presence (invasion) of a Matrigel coating. In comparison, M14 ADR cells displayed significantly higher motility and invasion. SEM observations showed that sensitive cells employed lamellar cytoplasmic extrusions to pass through the filter pores whereas resistant cells elongated along the hole through globular processes. In conclusion, the results herein reported suggest that drug resistance in melanoma cells appears associated with a more aggressive behaviour. P-gp and CD44 might cooperate to confer this more invasive phenotype.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Voacamine, an alkaloid extracted from Peschiera fuchsiaefolia, inhibits P-glycoprotein action in multidrug-resistant tumor cells.

Multidrug resistance (MDR) in tumor cells is generally associated with increased efflux of the cytotoxic compounds, due to the activation of mechanisms of intracellular transport and to the overexpression of surface proteins, such as P-glycoprotein (Pgp), which act as ATP-dependent molecular pumps. In a previous study, voacamine, a bisindolic alkaloid from Peschiera fuchsiaefolia, was examined for its possible capability of enhancing the cytotoxic effect of doxorubicin (DOX) on resistant human osteosarcoma cells. The effects of voacamine on the cell survival and on accumulation of DOX were investigated on both the parental cell line, U-2 OS-WT, and its resistant counterpart, U-2 OS-R. A differential effect between sensitive and resistant cells on the intracellular DOX concentration and distribution was revealed. In particular, voacamine induced a significant increase of drug retention and intranuclear location in resistant cells. Moreover, the cell survival analysis and the electron microscopic observations revealed an enhancement of the cytotoxic effect of DOX induced by the plant extract. In the present study, a panel of monoclonal antibodies (MAbs), recognizing different and specific structural and functional state of Pgp, was used. By flow cytometry and immunofluorescence confocal microscopy, a dose-dependent increase of the reactivity of Pgp with MAb UIC2, which specifically recognizes an epitope of the drug transporter in its functional conformation, was detected in voacamine-treated U-2 OS-R cells. Conversely, the expression of the epitope recognized by MAb MC57 was downregulated while MAb MM4.17 did not change its binding level to treated and untreated MDR cells. These data suggest that the plant extract reacts with Pgp producing conformational changes with consequent epitope modulation. Taken together, our observations seem to demonstrate that voacamine is a substrate for Pgp and, therefore, interferes with the Pgp-mediated drug export, acting as a competitive antagonist of cytotoxic agents.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2004

The biological functions of polyamine oxidation products by amine oxidases: perspectives of clinical applications.

The polyamines spermine, spermidine and putrescine are ubiquitous cell components. If they accumulate excessively within the cells, due either to very high extracellular concentrations or to deregulation of the systems which control polyamine homeostasis, they can induce toxic effects. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper containing amine oxidases. Polyamine concentrations are high in growing tissues such as tumors. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. These enzymes catalyze the oxidative deamination of biogenic amines and polyamines to generate the reaction products H2O2 and aldehyde(s) that are able to induce cell death in several cultured human tumor cell lines. H2O2 generated by the oxidation reaction is able to cross the inner membrane of mitochondria and directly interact with endogenous molecules and structures, inducing an intense oxidative stress. Since amine oxidases are involved in many crucial physiopathological processes, investigations on their involvement in human diseases offer great opportunities to enter novel classes of therapeutic agents.

Animals↗

PubMed2004

Tyrosine phosphatase activity in mitochondria: presence of Shp-2 phosphatase in mitochondria.

Tyrosine phosphorylation by unidentified enzymes has been observed in mitochondria, with recent evidence indicating that non-receptorial tyrosine kinases belonging to the Src family, which represent key players in several transduction pathways, are constitutively present in mitochondria. The extent of protein phosphorylation reflects a coordination balance between the activities of specific kinases and phophatases. The present study demonstrates that purified rat brain mitochondria possess endogenous tyrosine phosphatase activity. Mitochondrial phosphatases were found to be capable of dephosphorylating different exogenous substrates, including paranitrophenylphosphate, (32)P-poly(Glu-Tyr)(4:1) and (32)P-angiotensin. These activities are strongly inhibited by peroxovanadate, a well-known inhibitor of tyrosine phosphatases, but not by inhibitors of alkali or Ser/Thr phosphatases, and mainly take place in the intermembrane space and outer mitochondrial membrane. Using a combination of approaches, we identified the tyrosine phosphatase Shp-2 in mitochondria. Shp-2 plays a crucial role in a number of intracellular signalling cascades and is probably involved in several human diseases. It thus represents the first tyrosine phosphatase shown to be present in mitochondria.

Animals↗

PubMed2003

Mitochondrial alterations induced by serum amine oxidase and spermine on human multidrug resistant tumor cells.

Multidrug resistance (MDR) has been studied extensively because it is one of major problems in cancer chemotherapy. The MDR phenotype is often due to overexpression of P-glycoprotein (P-gp), that acting as an energy-dependent drug efflux pump exports various anticancer drugs out of cells. The major goal of our investigation is to establish whether bovine serum amine oxidase (BSAO), which generates the products H(2)O(2) and aldehyde(s), from the polyamine spermine, is able to overcome MDR of human cancer cells. The cytotoxicity of the products was evaluated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. A clonogenic cell survival assay demonstrated that LoVo DX cells were more sensitive than LoVo WT cells. Exogenous catalase protected cells against cytotoxicity mainly due to the formation of H(2)O(2). However, spermine-derived aldehyde(s) still induced some cytotoxicity. The cytotoxic effect was totally inhibited in the presence of both enzymes, catalase and NAD-dependent aldehyde dehydrogenase (ALDH). Transmission electron microscopy investigations showed that BSAO and spermine induced evident mitochondria alterations, more pronounced in MDR than in LoVo WT cells. The mitochondrial activity was checked by flow cytometry studies, labelling cells with the probe JC1, that displayed a basal hyperpolarized status of the mitochondria in multidrug-resistant cells. After treatment with amine oxidase in the presence of polyamine-spermine, the cells showed a marked increase in mitochondrial membrane depolarization higher in LoVo DX than in LoVo WT cells. Our findings suggest that toxic oxidation products formed from spermine and BSAO could be a powerful tool in the development of new anticancer treatments, mainly against MDR tumor cells.

Adenocarcinoma↗

PubMed2003

Voacamine, a bisindolic alkaloid from Peschiera fuchsiaefolia, enhances the cytotoxic effect of doxorubicin on multidrug-resistant tumor cells.

Multidrug-resistance (MDR) is largely caused by the efflux of therapeutics from the tumor cell by means of P-glycoprotein (P-gp), resulting in reduced efficacy of the chemotherapy. In order to overcome MDR, substances, such as verapamil and cyclosporin A (CsA), were employed. As these P-gp modulating agents did not seem promising in clinical practice, new compounds with a low degree of undesirable side effects, were introduced. In this study, bisindolic alkaloid voacamine was examined for its possible capability of enhancing the cytotoxic effect of doxorubicin (DOX) on drug resistant cells. Two different pairs of tumor cell lines were analyzed: the parental lymphoblastoid cell line CEM-WT and its MDR derivative CEM-R, the parental osteosarcoma cell line U-2 OS-WT and its resistant counterpart U-2 OS-R. These cell lines were characterized for their morphological features by scanning electron microscopy (SEM) and for the expression of the main drug transporters by flow cytometric analysis. The effects of voacamine on the cell survival and on both accumulation and efflux of DOX were then investigated. The intracellular distribution of DOX, given alone or in association with CsA or voacamine, was observed by laser scanning confocal microscopy. A differential effect of voacamine between sensitive and resistant cells on the intracellular DOX concentration and distribution was shown. In particular, voacamine induced a significant increase of drug retention and intranuclear location in resistant cells. The results of cell survival experiments revealed an enhancement of the cytotoxic effect of DOX induced by voacamine, confirmed by evident morphological changes observed by SEM. These findings suggest promising applications of this natural substance against MDR tumors.

Alkaloids↗

PubMed2002

Role of the lung resistance-related protein (LRP) in the drug sensitivity of cultured tumor cells.

Drug resistance, one of the major obstacle in the successful anticancer therapy, can be observed at the outset of therapy (intrinsic resistance) or after exposure to the antitumor agent (acquired resistance). To gain a better insight into the mechanisms of intrinsic resistance we have analyzed two human cell types derived from untreated tumors: MCF-7 breast cancer and A549 non small cell lung cancer (NSCLC). We have examined: the cytotoxic effect induced by doxorubicin (DOX); the time course of drug accumulation by flow cytometry and intracellular drug distribution by confocal microscopy; the expression and distribution of proteins related to anthracycline resistance, such as P-gp (P-glycoprotein), MRP1 (multidrug resistance-associated protein) and LRP (lung resistance-related protein). The cytotoxicity assays showed that A549 cells were less sensitive than MCF-7 cells to the DOX treatment in agreement with the different DOX uptake. Moreover, while in A549 cells DOX was mostly located in well defined intracytoplasmic vesicles, in MCF-7 cells it was mainly revealed inside the nuclei. The analysis of P-gp and MRP expression did not show significant differences between the two cell lines while a high expression of LRP was detected at the nuclear envelope and cytoplasmic levels in A549 cells. These findings suggest that the lower sensitivity to DOX treatment showed by lung carcinoma cells could be ascribed to drug sequestration by LRP inside the cytoplasmic compartments.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

What is the relationship between P-glycoprotein and adhesion molecule expression in melanoma cells?

A number of studies have reported that increased P-glycoprotein expression in drug-resistant tumour cells may be associated with decreased expression of a family of surface glycoproteins. However, despite its potential biological and clinical relevance, this phenomenon has not been extensively studied. In this study the phenotypic alterations that are associated with the acquisition of the multidrug-resistant phenotype in tumour cells, together with drug transporter overexpression, were investigated in human melanoma cells. The expression of cell adhesion molecules was analysed in a panel of multidrug-resistant melanoma cell lines (M14Dx) showing different degrees of resistance to doxorubicin and different levels of the expression of the drug transporter P-glycoprotein. In particular, expression of intercellular adhesion molecule-1 (ICAM-1), CD44, very late activation antigen (VLA)-5 and VLA-2 was determined by flow cytometry in the different resistant cell lines. A progressive downregulation of all the adhesion molecules examined was revealed in M14Dx cells, in parallel with an increasing level of expression of the drug transporter P-glycoprotein. The results obtained raise the question of the role of P-glycoprotein in the invasive and metastatic behaviour of tumour cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Subcellular detection and localization of the drug transporter P-glycoprotein in cultured tumor cells.

In vitro studies on the cellular location of P-glycoprotein (Pgp) are reported with the aim to clarify the relationship between its intracellular expression and the multidrug resistance (MDR) level of tumor cells. Pgp was found abnormally expressed on the plasma membrane of tumor cells with "classical" MDR phenotype. However, Pgp was also often detected on the nuclear envelope and on the membrane of cytoplasmic organelles. The hypothesis that this drug pump maintains a transport function when located in these compartments, is still under debating. Our results, together with those obtained by other researchers, demonstrate that cytoplasmic Pgp regulates the intracellular traffic of drugs so that they are no more able to reach their cellular targets. In particular, we revealed that in MDR breast cancer cells (MCF-7) a significant level of Pgp was expressed in the Golgi apparatus. A similar result was found in human melanoma cell lines, which never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane. A strict relationship between intracellular Pgp and intrinsic resistance was demonstrated in a human colon carcinoma (LoVo) clone, which did not express the drug transporter on the plasma membrane. Finally, a structural and functional association between Pgp and ERM proteins has been discovered in drug-resistant human T- lymphobastoid cells (CEM-VBL 100). Our findings strongly suggest a pivotal role of the intracytoplasmic Pgp in the transport of drugs into cytoplasmic vesicles, thus actively contributing to their sequestration and transport outwards the cells. Thus, intracellular Pgp seems to represent a complementary protective mechanism of tumor cells against cytotoxic agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Saposin D solubilizes anionic phospholipid-containing membranes.

Saposin (Sap) D is a late endosomal/lysosomal small protein, generated together with three other similar proteins, Sap A, B, and C, from the common precursor, prosaposin. Although the functions of saposins such as Sap B and C are well known (Sap B promotes the hydrolysis of sulfatides and Sap C that of glucosylceramide), neither the physiological function nor the mechanism of action of Sap D are yet fully understood. We previously found that a dramatic increase of Sap D superficial hydrophobicity, occurring at the low pH values characteristic of the late endosomal/lysosomal environment, triggers the interaction of the saposin with anionic phospholipid-containing vesicles. We have presently found that, upon lipid binding, Sap D solubilizes the membranes, as shown by the clearance of the vesicles turbidity. The results of gel filtration, density gradient centrifugation, and negative staining electron microscopy demonstrate that this effect is due to the transformation of large vesicles to smaller particles. The solubilizing effect of Sap D is highly dependent on pH, the lipid/saposin ratio, and the presence of anionic phospholipids; small variations in each of these conditions markedly influences the activity of Sap D. The present study documents the interaction of Sap D with membranes as a complex process. Anionic phospholipids attract Sap D from the medium; when the concentration of the saposin on the lipid surface reaches a critical value, the membrane breaks down into recombinant small particles enriched in anionic phospholipids. Our results suggest that the role played by Sap D is more general than promoting sphingolipid degradation, e.g. the saposin might also be a key mediator of the solubilization of intralysosomal/late endosomal anionic phospholipid-containing membranes.

Anions↗

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

1H NMR-visible mobile lipid domains correlate with cytoplasmic lipid bodies in apoptotic T-lymphoblastoid cells.

The presence of nuclear magnetic resonance (NMR)-visible mobile lipid (ML) domains in apoptotic lymphoblasts suggests alterations in neutral lipid metabolism and compartmentation during programmed cell death. The detection of similar ML signals in activated lymphocytes raises questions about common mechanisms of ML formation during apoptosis and upon lymphoblast stimulation. Structure and subcellular localization of ML domains were therefore investigated by NMR, fluorescence and electron microscopy in Jurkat T-lymphoblasts either induced to apoptosis (by anthracyclines or dexamethasone or by serum deprivation) or activated by phorbol myristate acetate (PMA) plus ionomycin. ML contents in drug-treated cells correlated linearly with apoptosis, irrespective of the specific inducer and cell cycle arrest phase (r = 0.993, P < 0.001). Similar ML levels were measured in drug-induced apoptotic cells (A approximately 30-40%) and in non-apoptotic PMA/ionomycin-treated lymphoblasts (72 h). Lower ML contents were instead formed in serum-deprived apoptotic cells, with respect to controls. Increases in ML signals were associated, in either apoptotic or activated cells, with the accumulation of cytoplasmic, osmophilic lipid bodies (diameter < or = 1.0 microm), surrounded by own membrane, possessing intramembrane particles. The results support the hypothesis that ML are formed in the cytoplasm of drug-induced apoptotic cells during an early, 'biochemically active' phase of programmed cell death.

Apoptosis↗

PubMed2001

Immunogold localisation of P-glycoprotein in supported lipid bilayers by transmission electron microscopy and atomic force microscopy.

In this study, purified P-glycoprotein molecules, a membrane drug pump responsible for the multidrug resistance phenomenon, were incorporated in model membranes deposited onto solid supports, according to the method described by Puu and Gustafson (1997). The insertion of proteins into planar supported model membranes is of interest, as the films are fundamental in biosensor applications and for the investigation of how proteins conform and aggregate in a lipid environment. In our investigation, two model membranes were prepared by transferring liposomes containing P-glycoprotein to different hydrophobic supports: (a) thin amorphous carbon films; (b) Langmuir-Blodgett lipid monolayers on mica. After the labelling of P-glycoprotein with two well-characterised monoclonal antibodies, MM4.17 and MRK-16, samples (a) were observed by transmission electron microscopy (TEM) and samples (b) by atomic force microscopy (AFM). The comparative analysis performed by TEM and AFM allowed us to demonstrate the successful insertion of P-glycoprotein in the model membranes and their stability under different environmental conditions (vacuum, air and water). P-glycoprotein appeared to maintain, after purification and insertion in lipid bilayers, a good part of its conformational features as shown by the P-glycoprotein segments bearing the specific monoclonal antibody epitopes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

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

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

Antineoplastic Agents↗