Study of the intracellular nanoparticle-based radiosensitization mechanisms in F98 glioma cells treated with charged particle therapy through synchrotron-based infrared microspectroscopy.

The use of nanoparticles (NP) as dose enhancers in radiotherapy (RT) is a growing research field. Recently, the use of NP has been extended to charged particle therapy in order to improve the performance in radioresistant tumors. However, the biological mechanisms underlying the synergistic effects involved in NP-RT approaches are not clearly understood. Here, we used the capabilities of synchrotron-based Fourier Transform Infrared Microspectroscopy (SR-FTIRM) as a bio-analytical tool to elucidate the NP-induced cellular damage at the molecular level and at a single-cell scale. F98 glioma cells doped with AuNP and GdNP were irradiated using several types of medical ion beams (proton, helium, carbon and oxygen). Differences in cell composition were analyzed in the nucleic acids, protein and lipid spectral regions using multivariate methods (Principal Component Analysis, PCA). Several NP-induced cellular modifications were detected, such as conformational changes in secondary protein structures, intensity variations in the lipid CHx stretching bands, as well as complex DNA rearrangements following charged particle therapy irradiations. These spectral features seem to be correlated with the already shown enhancement both in the DNA damage response and in the reactive oxygen species (ROS) production by the NP, which causes cell damage in the form of protein, lipid, and/or DNA oxidations. Vibrational features were NP-dependent due to the NP heterogeneous radiosensitization capability. Our results provided new insights into the molecular changes in response to NP-based RT treatments using ion beams, and highlighted the relevance of SR-FTIRM as a useful and precise technique for assessing cell response to innovative radiotherapy approaches.

A synchrotron-based infrared microspectroscopy study on the cellular response induced by gold nanoparticles combined with X-ray irradiations on F98 and U87-MG glioma cell lines.

The inclusion of nanoparticles (NP) in radiotherapy has been shown to increase the damaging effect on tumor cells. However, the mechanisms of action of NP combined with radiotherapy, and the influence of NP parameters and cell type on their radiosensitization capability at molecular and cellular levels still remain unclear. Gold NP (AuNP) have become particularly popular due to their multiple advantages. Within this context, our research work aimed to study the biochemical radiosensitization capacity of F98 and U87-MG glioma cell lines to 1.9 nm AuNP combined with X-ray irradiation. For this purpose, synchrotron-based infrared microspectroscopy (SR-FTIRM) was used as a powerful tool for biochemical composition and treatment response assessment of cells at a single-cell level. SR-FTIRM data, supported by multivariate analysis, revealed clear AuNP-induced changes in the DNA, protein and lipid spectral regions. The AuNP-related biochemical alterations appear prior to the irradiation, which gave us a first indication on the AuNP radiosensitization action. Biochemical modifications induced by the AuNP in the presence of radiotherapy irradiations include enhanced conformational changes in the protein secondary structures, variations in the intensity and position in the phosphodiester bands, and changes in the CH2 and CH3 stretching modes. These changes are better manifested at 24 hours post-irradiation time. SR-FTIRM results showed a clear heterogeneity in the biochemical cell response, probably due to the distinct cell-NP interactions and thus, to different DNA damage and cell death processes.

Cellular uptake of phosphorothioate oligonucleotide facilitated by cationic porphyrin: a microfluorescence study.

Cationic 5,10,15,20-tetrakis (1-methyl-4-pyridyl) porphyrin was tested as a delivery agent for oligonucleotides. By using fluorescence microimaging, it has been shown that complexation of the porphyrin to the phosphorothioate analog of dT(15) labeled by rhodamine enabled its nonendocytic penetration into the cell and regular distribution in the cytoplasm and preferentially into the nucleus. Time-resolved microfluorescence spectroscopy revealed that the oligonucleotide integrity was kept. A small fraction of the porphyrin molecules seems to undergo change of the binding mode after internalization, probably due to duplex formation between the oligonucleotide and its cellular target sequences, or due to dissociation of the porphyrin from the oligonucleotide and subsequent interactions in the cellular environment.

Anthrapyridones, a novel group of antitumour non-cross resistant anthraquinone analogues. Synthesis and molecular basis of the cytotoxic activity towards K562/DOX cells.

1. Multidrug resistance (MDR) to antitumour agents, structurally dissimilar and having different intracellular targets, is the major problem in cancer therapy. MDR phenomenon is associated with the presence of membrane proteins which belong to the ATP-binding cassette family transporters responsible for the active drug efflux leading to the decreased intracellular accumulation. 2. The search of new compounds able to overcome MDR is of prime importance. 3. Recently we have synthesized a new family of anthrapyridone compounds. The series contained derivatives modified with appropriate hydrophobic or hydrophylic substituents at the side chain. 4. The interaction of these derivatives with erythroleukemia K562 sensitive and K562/DOX resistant (overexpressing P-glycoprotein) cell lines has been examined. The study was performed using a spectrofluorometric method which allows to continuously follow the uptake and efflux of fluorescent molecules by living cells. 5. It was demonstrated that the increase in the lipophilicity of anthrapyridones favoured the very fast cellular uptake exceeding the rate of P-gp dependent efflux out of the cell. For these derivatives, very high accumulation (the same for sensitive and resistant cells) was observed and the in vitro biological data confirmed that these compounds exhibited comparable cytotoxic activity towards sensitive and P-gp resistant cell line. In contrast, anthrapyridones modified with hydrophylic substituents exhibited relatively low kinetics of cellular uptake. 6. For these derivatives decreased accumulation in resistant cells was observed and the in vitro biological data demonstrated that they were much less active against P-gp resistant cells in comparison to sensitive cells. 7. We also studied, using confocal microscopy, the intracellular distribution of anthrapyridones in NIH-3T3 cells. Our data showed that these compounds were strongly accumulated in the nucleus and lysosomes.

Transport of new non-cross-resistant antitumor compounds of the benzoperimidine family in multidrug resistant cells.

Multidrug resistance (MDR) phenotype in mammalian cells is often correlated with overexpression of P-glycoprotein or multidrug resistance-associated protein (MRP1). Both proteins are energy-dependent drug efflux pumps that efficiently reduce the intracellular accumulation and hence the cytotoxicity of many natural cytotoxins. The influx and efflux of drugs across the cell membrane are in large part responsible for their intracellular concentrations, and in the search for new compounds able to overcome MDR, it is of prime importance to determine the molecular parameters whose modification would lead to an increase in the kinetics of uptake and/or to a decrease in the pump-mediated efflux. Here, we studied three members of a new family of benzoperimidine antitumor compounds which exhibit comparable cytotoxicity towards resistant cells expressing P-glycoprotein, or MRP1, and sensitive cells. We used spectrofluorometric methods to determine the kinetics of the uptake and release of these three drugs in different cell lines: the erythroleukemia cell line K562 and the resistant K562/Adr expressing P-glycoprotein, the small-cell lung cancer cell line GLC4 and resistant GLC4/Adr expressing MRP1. We also studied, using confocal microscopy, the intracellular distribution of these drugs in NIH/3T3 cells. Our data show that (i) the kinetics for the uptake of these drugs is very rapid, higher than 2 x 10(-17) mole cell(-1) s(-1), (ii) the drugs are strongly accumulated in the nucleus and lysosomes, (iii) the three drugs are recognized and pumped out by both transporters, as shown by the inhibition of P-glycoprotein- and MRP1-mediated efflux of pirarubicin by benzoperimidine, with inhibitory constants of 1.5 and 2.1 microM for P-glycoprotein and MRP1, respectively, suggesting that benzoperimidine is transported by the two transporters with K(m) approximately 2 microM. In conclusion, the fast uptake kinetics of the benzoperimidines counterbalance their efflux by P-glycoprotein and MRP1.

Delivery systems for antisense oligonucleotides.

In vitro, the efficacy of the antisense approach is strongly increased by systems delivering oligodeoxyribonucleotides (ODNs) to cells. Up to now, most of the developed vectors favor ODN entrance by a mechanism based on endocytosis. Such is the case for particulate systems, including liposomes (cationic or non-cationic), cationic polyelectrolytes, and delivery systems targeted to specific receptors. Under these conditions, endosomal compartments may represent a dead end for ODNs. Current research attempts to develop conditions for escaping from these compartments. A new class of vectors acts by passive permeabilization of the plasma membrane. It includes peptides, streptolysin O, and cationic derivatives of polyene antibiotics. In vivo, the interest of a delivery system, up to now, has appeared limited. Development of vectors insensitive to the presence of serum seems to be a prerequisite for future improvements.

A cationic derivative of amphotericin B as a novel delivery system for antisense oligonucleotides.

A novel approach based on a plasma membrane permeability-disturbing agent was proposed as an antisense oligonucleotide delivery system. AMA, a derivative of the polyene antibiotic amphotericin B, formed a stable complex when mixed with phosphodiester oligodeoxynucleotides and enhanced the intracellular uptake of a 5' fluoresceinated anti-mdr1 20-mer into NIH-MDR-G185 cells. The nonlabeled phosphorothioate form of the oligodeoxynucleotide, complexed to AMA, inhibited P-glycoprotein expression with better efficiency and less nonspecific effects than when vectorized by Lipofectin. AMA may thus be a good agent for antisense strategy.

Size-dependent DNA mobility in cytoplasm and nucleus.

The diffusion of DNA in cytoplasm is thought to be an important determinant of the efficacy of gene delivery and antisense therapy. We have measured the translational diffusion of fluorescein-labeled double-stranded DNA fragments (in base pairs (bp): 21, 100, 250, 500, 1000, 2000, 3000, 6000) after microinjection into cytoplasm and nucleus of HeLa cells. Diffusion was measured by spot photobleaching using a focused argon laser spot (488 nm). In aqueous solutions, diffusion coefficients of the DNA fragments in water (D(w)) decreased from 53 x 10(-8) to 0.81 x 10(-8) cm(2)/s for sizes of 21-6000 bp; D(w) was related empirically to DNA size: D(w) = 4.9 x 10(-6) cm(2)/s.[bp size](-0.72). DNA diffusion coefficients in cytoplasm (D(cyto)) were lower than D(w) and depended strongly on DNA size. D(cyto)/D(w) decreased from 0.19 for a 100-bp DNA fragment to 0.06 for a 250-bp DNA fragment and was <0.01 for >2000 bp. Diffusion of microinjected fluorescein isothiocyanate (FITC) dextrans was faster than that of comparably sized DNA fragments of 250 bp and greater. In nucleus, all DNA fragments were nearly immobile, whereas FITC dextrans of molecular size up to 580 kDa were fully mobile. These results suggest that the highly restricted diffusion of DNA fragments in nucleoplasm results from extensive binding to immobile obstacles and that the decreased lateral mobility of DNAs >250 bp in cytoplasm is because of molecular crowding. The diffusion of DNA in cytoplasm may thus be an important rate-limiting barrier in gene delivery utilizing non-viral vectors.

Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus.

Fluorescence recovery after photobleaching (FRAP) was used to quantify the translational diffusion of microinjected FITC-dextrans and Ficolls in the cytoplasm and nucleus of MDCK epithelial cells and Swiss 3T3 fibroblasts. Absolute diffusion coefficients (D) were measured using a microsecond-resolution FRAP apparatus and solution standards. In aqueous media (viscosity 1 cP), D for the FITC-dextrans decreased from 75 to 8.4 x 10(-7) cm2/s with increasing dextran size (4-2,000 kD). D in cytoplasm relative to that in water (D/Do) was 0.26 +/- 0.01 (MDCK) and 0.27 +/- 0.01 (fibroblasts), and independent of FITC-dextran and Ficoll size (gyration radii [RG] 40-300 A). The fraction of mobile FITC-dextran molecules (fmob), determined by the extent of fluorescence recovery after spot photobleaching, was >>0.75 for RG << 200 A, but decreased to <<0.5 for RG >> 300 A. The independence of D/Do on FITC-dextran and Ficoll size does not support the concept of solute "sieving" (size-dependent diffusion) in cytoplasm. Photobleaching measurements using different spot diameters (1.5-4 micron) gave similar D/Do, indicating that microcompartments, if present, are of submicron size. Measurements of D/Do and fmob in concentrated dextran solutions, as well as in swollen and shrunken cells, suggested that the low fmob for very large macromolecules might be related to restrictions imposed by immobile obstacles (such as microcompartments) or to anomalous diffusion (such as percolation). In nucleus, D/Do was 0.25 +/- 0.02 (MDCK) and 0.27 +/- 0.03 (fibroblasts), and independent of solute size (RG 40-300 A). Our results indicate relatively free and rapid diffusion of macromolecule-sized solutes up to approximately 500 kD in cytoplasm and nucleus.

Evidence against defective trans-Golgi acidification in cystic fibrosis.

Defective organelle acidification has been proposed as a unifying hypothesis to explain the pleiotropic cellular abnormalities associated with cystic fibrosis. To test whether cystic fibrosis transmembrane conductance regulator (CFTR) participates in trans-Golgi pH regulation, intraluminal trans-Golgi pH was measured in stably transfected Swiss 3T3 fibroblasts (expressing CFTR or DeltaF508-CFTR) and CFTR-expressing and nonexpressing epithelial cells. trans-Golgi pH was measured by ratio-imaging confocal microscopy using a liposome injection procedure to label the lumen of trans-Golgi with fluid phase fluorescein and rhodamine chromophores (Seksek, O., Biwersi, J., and Verkman, A. S.(1995) J. Biol. Chem. 270, 4967-4970). Selective labeling of trans-Golgi was confirmed by colocalization of the delivered fluid phase fluorophores with N-(6-[(7-nitrobenzo-2-oxa-1, 3-diazol-4-yl)amino]caproyl)-sphingosine. In unstimulated fibroblasts in HCO3--free buffer, trans- Golgi pH was 6.25 +/- 0.04 (mean +/- S.E.; n = 80, vector control), 6.30 +/- 0.03 (n = 74, CFTR) and 6.23 +/- 0.06 (n = 60, DeltaF508) (not significant). After stimulation of plasma membrane Cl- conductance by 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), trans-Golgi pH was 6.42 +/- 0.07 (n = 22, control), 6.47 +/- 0.07 (n = 20, CFTR), and 6.35 +/- 0. 07 (n = 22, DeltaF508) (not significant). Similarly, significant pH differences were not found for control versus CFTR-expressing cells in 25 mM HCO3- buffer. In epithelial cells, which do not express CFTR, trans-Golgi pH was (in 25 mM HCO3-) 6.36 +/- 0.04 (n = 33) and 6.34 +/- 0.08 (n = 23, CPT-cAMP) in MDCK cells and 6.25 +/- 0.04 (n = 18) and 6.24 +/- 0.06 (n = 15, CPT-cAMP) in SK-MES-1 cells. In Calu-3 cells, which natively express CFTR, trans-Golgi pH was (in 25 mM HCO3-) 6.19 +/- 0.05 (n = 25) and 6.17 +/- 0.08 (n = 23, CPT-cAMP). To test whether CFTR expression affects pH in the endosomal compartment in HCO3- buffer, pH was measured by ratio imaging in individual endosomes labeled with fluorescein-rhodamine dextrans. Comparing control and CFTR-expressing fibroblasts, average endosome pH (range, 5.40-5.53 after 10 min; 4.79-4.89, 30 min) differed by <0.13 unit, both before and after cAMP stimulation. These results indicate that CFTR expression and activation do not influence pH in the trans-Golgi and endosomal compartments, providing direct evidence against the defective acidification hypothesis.

Nuclear pH gradient in mammalian cells revealed by laser microspectrofluorimetry.

Intracellular pH has been measured by laser microspectrofluorimetry, using the pH-sensitive dyes SNARF-1, SNARF-calcein and SNARF-1-dextran. By this technique it was possible to accurately determine pH in volumes as small as 0.5 x 0.5 x 1 microns 3. The probes were loaded into the cells either by diffusion of their acetoxymethylester derivatives (SNARF-1-AM, SNARF-calcein-AM) or by microinjection (SNARF-1-dextran). When the five types of cells were studied in RPMI medium, the nuclear pH was consistently found to be 0.3 to 0.5 units above that of the cytosol. Although the presence of pores in the nuclear membrane has been taken as evidence that free diffusion of ions and small molecules can occur in and out the nucleus, we conclude that the nuclear membrane of these cells presents a permeability barrier to H+. The pH gradient was not observed in cells suspended in PBS.

Identification of the structural elements of amphotericin B and other polyene macrolide antibiotics of the hepteane group influencing the ionic selectivity of the permeability pathways formed in the red cell membrane.

The selectivity of the transmembrane permeability induced by polyene antibiotics was studied in human erythrocytes and related to the hemolytic potency of the drugs. The selectivity induced was differently, dependent on the antibiotic structure in aromatic (vacidin A, gedamycin) and nonaromatic heptaenes (amphotericin B, candidin). Aromatic heptaenes were more effective than nonaromatic in inducing permeability to K+. For both groups of antibiotics, permeability to K+ was not affected by substitution at the carboxyl group but important differences in the induction of permeability to H+, OH- and Cl- were found. The strongly hemolytic aromatic heptaenes vacidin A and gedamycin exhibited much higher protonophoric activity than the nonaromatic ones: amphotericin B, and candidin. The protonophoric properties of aromatic heptaenes were related to the presence of a free carboxyl group in the antibiotic molecule. Indeed the esterification or amidation of the carboxyl group of vacidin A or gedamycin eliminated the ability of the antibiotic to increase H+ conductance and consequently diminished their hemolytic activity to an important extent. Both groups of antibiotics differed also in the efficiency of anion permeability induction. Only unsubstituted aromatic heptaenes, at high concentration, induced Cl-/OH- exchange and conductive flux of Cl- in a concentration-dependent manner. Substitution at the carboxyl group of vacidin A or gedamycin eliminated this property. Amphotericin B as well as its carboxyl-substituted derivatives formed a pathway characterized by low K+ over Cl- selectivity, whatever the concentration. The hemolytic activity, related to K+ permeability increased by heptaenes was dependent on simultaneous increase of the permeability to anions, and net KCl influx. Carboxyl-substituted derivatives of aromatic heptaenes presenting a remarkably high selectivity for K+, had consequently a very poor hemolytic activity.

Direct measurement of trans-Golgi pH in living cells and regulation by second messengers.

In the endocytic compartment, an acidic pH plays a key role in receptor and ligand sorting, vesicular transport, and protein degradation. In the secretory compartment, indirect estimates of trans-Golgi pH based on partitioning of weak bases and following viral infection suggest a mildly acidic pH of > 6.0. We developed a liposome microinjection method to introduce fluorescent indicators into the aqueous compartment of trans-Golgi in living cells. In the presence of ATP and at 37 degrees C, 70-nm diameter liposomes delivered their fluid-phase contents selectively into the trans-Golgi compartment as assessed by colocalization with the trans-Golgi stain N-[6-[(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-sphingosine (C6-NBD-ceramide). Liposome fusion was ATP- and temperature-dependent and blocked by N-ethylmaleimide but not by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). trans-Golgi pH in skin fibroblasts was 6.17 +/- 0.02 (S.E., n = 174) as measured by ratio imaging confocal microscopy using fluorescein and rhodamine-based indicators and an in vivo calibration procedure. trans-Golgi pH increased to 6.8 +/- 0.1 by cAMP agonists and to 6.5 +/- 0.1 by protein kinase C activation. These results provide the first direct measurement of trans-Golgi pH in living cells and demonstrate pH regulation by second messengers.

Polyene macrolide antibiotics: indirect stimulation of the Na+/H+ exchanger of BALB/c B lymphoid cell line, A20.

The fluorescent pH probe, 2'-7'-bis (carboxyethyl) 5-carboxyfluorescein, was used to follow changes in internal pH (pHi) induced by aromatic polyene antibiotics in the BALB/c lymphoid cell line A20. The antibiotics studied were vacidin, which contains a free carboxylic group in the position C18 of the macrolide ring, and vacidin glycyl methyl ester and perimycin, which are without free carboxylic groups. Although all of them induced transmembrane Na+ and K+ movements, only vacidin had protonophoric activity, as previously demonstrated for red blood cells [Cybulska B et al., Biochem Pharmacol 38: 1755-1762, 1989]. However, with all three antibiotics, pHi changes were observed in A20 cells. It was demonstrated that the transmembrane H+ movements resulted to different degrees, principally in the case of perimycin and vacidin glycyl methyl ester, or partially in the case of vacidin, from the stimulation of the Na+/H+ exchanger by the induced Na+ permeability. The non-aromatic polyene antibiotic amphotericin B had a low ability to increase proton permeability.

SNARF-1 as an intracellular pH indicator in laser microspectrofluorometry: a critical assessment.

The use of SNARF-1-AM (seminaphtorhodafluor-1-acetoxymethylester) to measure the internal pH of a single living cell by laser microspectrofluorometry has been analyzed with a lymphocyte murine B cell line A20. After incubation of the cells at 37 degrees C in the presence of 10 microM SNARF-1-AM, the internal concentration of SNARF-1 was approximately 200 microM. The enhancement of fluorescent intensity of the probe is concomitant with its leakage out of the cells. During the measurement period, this induces a continuous increase of the contribution of the external probe to the total fluorescence intensity. This prevented classical spectrofluorometry measurements, but did not preclude microspectrofluorometry measurements of internal pH. The ratio R was calculated from fluorescence intensities at 635 and 590 nm and used as an indicator of the intracellular pH. Calibration curves of the intracellular pH were obtained in the presence of nigericin and valinomycin. It appeared that both the fluorescence intensity and the ratio R were lower inside the cell than those values obtained in aqueous solutions. Possible interactions with the main biological macromolecules (i.e., DNA, proteins, membranes) were investigated as well as a possible compartmentation of the probe in cellular organelles. The modifications of probe characteristics inside the cells were attributed to the binding of the probe to cellular proteins. The intracellular pH of A20 cells, measured by SNARF-1 on 84 cells, was found to be 7.18 +/- 0.10 (with an external pH of 7.40 +/- 0.05), which corresponded with values obtained by conventional fluorometric methods.