Biomedical subjects

M Manfait

Publications and source records attributed to M Manfait.

At least 19 recordsLinked to original sources

PubMed1999

Characterization of intracellular pH gradients in human multidrug-resistant tumor cells by means of scanning microspectrofluorometry and dual-emission-ratio probes.

Multidrug-resistant cells are believed to contain a plasma-membrane-efflux pump which is hypothesized to expel anticancer drugs from the cytosol to the cell exterior. Many of these drugs are classified as weak bases whose binding to intracellular targets is pH-dependent. Slight alterations in intracellular pH gradients have been shown to affect accumulation, endocytosis and secretion of drugs. In this study, we developed a new method based on confocal spectral imaging analysis to determine intracellular pH gradients in sensitive and MDR tumor cells. Fluorescein isothiocyanate (FITC) and tetramethylrhodamine conjugated to dextran (FRD) and SNAFL-calcein-AM were used to determine pH in acidic compartments. Carboxy-SNARF1-AM was used to examine cytosolic pH. We observed that sensitive (HL60, K562, CEM and MCF7) cells exhibit lower acidity of the subcellular organelles than that corresponding to drug-resistant derivatives. Moreover, results obtained with carboxy-SNARF1-AM show that resistant cells display a more alkaline cytosolic pH. This results in a considerably larger pH gradient between the vesicular compartments and the cytosol of resistant cells than of sensitive cells. The lower pH gradient observed in sensitive cells may be related to a disruption in the organization of the trans-Golgi network (TGN). In drug-resistant cells, the organization of TGN appears compact. In addition, confocal microscopic analysis of cells labelled with FRD and SNAFL-calcein showed that sensitive cells contain a lower number of acidified vesicles. This suggest a diminished capacity of these cells to remove protonated drugs from the cytoplasm to secretory compartments followed by their secretion through the activity of the secretory and recycling pathways.

Breast Neoplasms↗

PubMed1999

A new UV-visible confocal laser scanning microspectrofluorometer designed for spectral cellular imaging.

With the aim to perform spectroscopic studies and spectral images inside living cells, a microspectrofluorometer has been designed for two-dimensional spectral imaging in the visible and in the near-UV region. The main advantage of the device relies on its ability to scan the laser beam along one direction of the sample. This scanning is optically coupled with one direction of a bidimensional detector, allowing an instantaneous recording of a one-dimensional spectral image. The overall scanning of the sample is achieved by means of submicrometric displacements of the stage in the perpendicular direction. The main characteristics and performances of the microspectrofluorometer in terms of sensitivity (detection of a few molecules), spatial resolution (0.5 x 0.5 x 1 microm), and spectral resolution (1 nm) are presented. Finally, applications of this new apparatus concerning in situ localization and spectral characterization of two dyes are shown with Drosophila salivary glands (ethidium bromide) and T47D tumor cells (Hoechst 33342).

Animals↗

PubMed1999

Evidence for functional discrimination between leukemic cells overexpressing multidrug-resistance associated protein and P-glycoprotein.

Multidrug-resistance (MDR), caused by overexpression of either P-glycoprotein (Pgp) or the multidrug-resistance associated protein (MRP), is characterised by a decreased cellular drug accumulation. One form of MDR is the sequestration of the drug inside cytoplasmic vesicles followed by an a exocytotic and/or efflux process. In some studies, increased intracellular glutathione (GSH) has been associated with MDR. In this study, we examined the effects of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole or NBD (a H(+)-ATPase pump inhibitor) and buthionine sulphoximine or BSO (an inhibitor of GSH biosynthesis) on the subcellular distribution of daunorubicin or DNR in two leukemic homoharringtonine-resistant K562 cell lines, overexpressing MRP (K-H30) and Pgp (K-H300). DNR nuclear accumulation was carried out using microspectrofluorometry. Our results show that DNR nuclear accumulation and sensitivity of K-H30 cells were increased by NBD and BSO whereas in K-H300 cells, NBD and BSO were unable to increase the DNR nuclear accumulation and sensitivity of these cells. This study demonstrates clearly that even if vesicular sequestration can happen in cells overexpressing MRP and Pgp proteins, only the MRP protein is able to extrude the drug through intracellular vesicles and efflux. In addition, GSH plays an important part in the pathway of drug transport in cells overexpressing MRP. Data entrain also the notion of functional discrimination between the MDR and MRP phenotype.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Two distinct modes of oncoprotein expression during apoptosis resistance in vincristine and daunorubicin multidrug-resistant HL60 cells.

Apoptosis is a genetically regulated cell death process which results in a variety of morphological changes like chromatin condensation and DNA fragmentation. The decision between survival or death in response to an apoptotic stimulus is determined and regulated in part by oncoproteins which include proteins of the Bcl-2 family (bcl-2, bax, bcl-xL) and bcr-abl. We investigated the effect of these proteins on the induction of this phenomenon in human promyelocytic leukemic HL60 cells and two multidrug resistant homologues selected respectively with vincristine (HL60/VCR) and daunorubicin (HL60R/DNR). We show that sensitive cells at 1 micron and HL60/VCR cells at DNR IC50 were able to undergo apoptosis while HL60R/DNR did not even at much higher concentration of DNR. However, treatment with synthetic C2-ceramide did not sensitize HL60/DNR cells to apoptosis. Cell death through apoptosis or necrosis was accompanied by acidification of the cytosol without mitochondrial membrane depolarization. Western blotting analysis shows that bax is expressed at slightly elevated level in HL60S/VCR in comparison with the other cells lines. Bcl-2 is overexpressed in HL60/VCR but not in HL60R/DNR. However, this cell line displayed a higher expression of bcl-xL. Interestingly, bcr-abl, a dysregulated tyrosine kinase was detected only in HL60R/DNR cells. DNR at the IC50, has no effect on expression of the oncoproteins. These data suggest that in addition of the multidrug resistance phenotype, bcr-abl translocation and bcl-xL overexpression could also account for the development of resistance to cell death induced by anthracyclines in leukemic cells.

Apoptosis↗

PubMed1998

Modulation in kinetics of lactone ring hydrolysis of camptothecins upon interaction with topoisomerase I cleavage sites on DNA.

The kinetics of hydrolysis of the alpha-hydroxylactone ring of anticancer agents belonging to the camptothecin (CPT) series has been followed using their fluorescence emission. Data obtained for CPT, CPT-11, and SN-38, either in their free form or in the presence of DNA and/or topoisomerase I (top1), have been compared. DNA was modeled using three types of double-strand oligonucleotides corresponding to top1 cleavage site enhanced in the presence of the drug (olg1), top1 site independent of CPT (olg2), and nonspecific synthetic oligonucleotide containing only AT and no GC base pairs (olg3). Cleavage assays indicated the absence of top1-mediated cleavage on olg3, both in the presence and in the absence of CPT. The kinetics data also showed ratio-dependent stabilization of the lactone forms of CPTs when in the presence of an excess of olg1 or olg2, but not of olg3. These observations correlate with the previously reported preferential binding of CPTs to guanines. Although lactone hydrolysis was not perturbed by top1 alone, this enzyme hindered lactone stabilization by specific oligonucleotides. After addition of top1 to CPT-olg1 or CPT-olg2 complexes, the lactone ring of the drug was destabilized. No lactone stabilization was observed when olg1 was added to CPT-top1 complexes or when olg1-top1 complexes were added to CPT.

Camptothecin↗

PubMed1998

Extracellular Mg2+ inhibits both histamine-stimulated Ca(2+)-signaling and exocytosis in human tracheal secretory gland cells.

The effects of extracellular Mg2+ concentration have been investigated on the histamine-stimulated exocytotic process of human tracheal secretory gland (HTG) cells. The exocytosis of secretory granules (SG) was observed concomitantly with dynamic changes of intracellular Ca2+ ([Ca2+]i) and Mg2+ concentrations ([Mg2+]i). The rate of SG exocytosis was appraised by the decrease of quinacrine fluorescence emission. Dynamic changes of [Mg2+]i and [Ca2+]i in HTG cells were determined by the combined use of UV-microspectrofluorometry with Mag-Indo-1 and Indo-1 probes, respectively. High Mg2+ medium significantly inhibited the histamine-stimulated secretion. The influence of the extracellular and intracellular Mg2+ concentrations on [Ca2+]i was analyzed. Basal [Mg2+]i increased from 0.8 mM in a Mg(2+)-free medium to 1.7 mM in 10 mM Mg2+ medium. Histamine induced a [Mg2+]i increase which is dependent on extracellular Mg2+ concentration. The histamine stimulated [Ca2+]i rise was reduced in the presence of elevated Mg2+ extracellular medium and inhibitory effects of extracellular Mg2+ were concomitant with changes in [Mg2+]i. Our data suggest that the inhibition by extracellular Mg2+ of stimulated exocytosis is dependent on both the increase of [Mg2+]i and the inhibition of cytosolic Ca2+ influx.

Calcium↗

PubMed1998

Resistance to apoptosis induced by topoisomerase I inhibitors in multidrug-resistant HL60 leukemic cells.

The induction of apoptosis by topoisomerase I inhibitors, camptothecin and SN38, was evaluated in drug-sensitive HL60 and multidrug-resistant (MDR) HL60-Vinc leukemic cells. MDR cells displayed a partial resistance to these apoptotic stimuli and this phenomenon was not modulated by verapamil. Basal free calcium concentrations were similar in both cell sublines and were not modified during treatment. Cytoplasmic pH was more acidic in sensitive cells than in MDR cells. Moreover, a significant acidification was obtained during the early stage of apoptosis in sensitive HL60 cells only. Basal Bcl-2 protein expression was found to be greater in MDR than in sensitive cells and was not modulated by apoptosis inducers. This increase of Bcl-2 in MDR cells could be due to the selection process as vincristine enhances Bcl-2 phosphorylation and expression in HL60 sensitive cells. MDR HL60-Vincristine cells therefore display a resistance to apoptosis induced by non-MDR drugs, possibly by Bcl-2 overexpression and inability of these drugs to mediate intracellular pH changes in these drug-resistant cells.

Antineoplastic Agents, Phytogenic↗

PubMed1998

Spectroscopic and biochemical characterisation of self-aggregates formed by antitumor drugs of the camptothecin family: their possible role in the unique mode of drug action.

We describe the effect strongly influencing the biological activity of some camptothecin (CPT) drugs, the inhibitors of DNA topoisomerase I (topo I), namely, the formation of J-type aggregates in an aqueous buffer solution. These aggregates were built up under certain dilution conditions of the stock DMSO solutions of 20-S-camptothecin (20(S)CPT), 10,11-methylenedioxy-CPT (10,11-CPT) and 7-ethyl-10-hydroxy-CPT (SN38). The aggregates were found to be stereospecific, not being detectable for the 20(R)-stereoisomer of CPT. They were formed by the stacking interaction between quinoline rings of CPT chromophores with the inverse position of the nitrogen atoms. The aggregates were stable at acidic and neutral pHs, but dissociated at basic pHs. Self-aggregation prevented hydrolysis of the lactone ring at neutral pHs, thus preserving the drugs in a biologically active form. Addition of BSA did not induce either disaggregation or hydrolysis of the lactone ring, whereas the monomeric form of the drugs was shown to undergo rapid conversion to an inactive carboxylate form in the presence of human serum albumin [5]. The drugs did not form the aggregates in the presence of topo I. Moreover, rapid dissociation of the aggregates was observed if a self-aggregated drug solution was added to topo I alone or to the DNA-topo I cleavage assay. Neither DNA alone nor oligonucleotides derived from the sequences of the CPT-enhanced or topo I-induced cleavage sites in SV40 plasmid DNA induces changes in the aggregation state of the drugs. These observations are indicative of interaction between the aggregates and topo I. The aggregates were found to penetrate within the cells with much higher efficiency than a monomeric form of the drugs. Cellular uptake of aggregated and nonaggregated species correlated well with cytotoxic effects produced by the drug. In this manner, CPT's self-aggregation should be regarded as a favourable phenomenon producing species with a more stable biologically active structure of the lactone ring and exhibiting enhanced cellular uptake levels relative to the monomeric forms of medications.

Antineoplastic Agents, Phytogenic↗

PubMed1998

Kinetics of lactone hydrolysis in antitumor drugs of camptothecin series as studied by fluorescence spectroscopy.

Potent antitumor activity exhibited by 20-S-camptothecin (CPT) and numerous derivatives is known to be lost upon opening of the alpha-hydroxy-lactone ring of these drugs, hydrolyzable at neutral and basic pH. To quantify in 'real time' the lactone hydrolysis reaction in CPTs under physiological conditions, we have applied a non-perturbing approach by fluorescence spectroscopy. CPT and a set of its derivatives (21-lactam-S-CPT, 10,11-(methylenedioxy)-CPT, CPT-11, SN-38, topotecan, tricyclic ketone-CPT) with antitumor activity varying from negligible to 10 times that of CPT have been studied. Prior to the kinetic measurements, the effects of substitutions, pH, polarity of molecular environment, lactone ring opening (lactone-carboxylate transition) have been investigated in terms of the UV-visible absorption and fluorescence emission spectra of CPTs. Then the determined parameters of the fluorescence emission spectra corresponding to the respective lactone and carboxylate forms have been used to estimate the residual lactone percentage as a function of time. The reproducibility of the obtained data demonstrates that the spectroscopic approach provides a satisfactory precision for this kind of measurements. For CPT at pH 7.3, the lactone half-life was 29.4 +/- 1.7 min and the lactone percentage at equilibrium was 20.9 +/- 0.3%. Within a series of derivatives with substitutions at quinoline rings, the lactone half-life varied from 29 to 32 min and the equilibrium lactone content varied from 15% to 23%. For each compound, even slight increase of pH from 7.1 to 7.3 or from 7.3 to 7.6 logically leads to a remarkable decrease of both lactone half-life and equilibrium lactone percentage.

Antineoplastic Agents, Phytogenic↗

PubMed1998

Surface-enhanced Raman scattering and fluorescence spectroscopy reveal molecular interactions of all-trans retinoic acid and RAR gamma ligand-binding domain.

Surface-enhanced Raman scattering and fluorescence were used to investigate the interactions of all-trans retinoic acid with the gamma-type retinoic acid receptor. Raman data revealed a significant attenuation in intensity of the bands originating from the retinoic acid polyenic chain upon receptor binding, with the spectrum being dominantly that of the beta-ionone ring. Fluorescence measurements supported the hydrophobic character of the ligand binding. These novel spectroscopic results are fully consistent with the published X-ray crystallographic data and suggest that these techniques may be valuable additional tools to characterize the interactions of agonists and antagonists with residues in the ligand-binding pockets of retinoid receptor homo- and heterodimers.

Binding Sites↗

PubMed1998

Characterization of island films as surface-enhanced Raman spectroscopy substrates for detecting low antitumor drug concentrations at single cell level.

Gold and silver vacuum-deposited island films were characterized by studying deposition variables such as film thickness, evaporation rate, and substrate temperature. For both metals, these parameters were correlated with the surface-enhanced Raman spectroscopy (SERS) effect and an increase in film thickness and low evaporation rates were shown to upshift the wavelength at maximum optical density (lambda max) and increase the optical density of the substrates. In contrast, pre- and postdeposition annealing of gold films led to the formation of substrates that exhibited a downshift of lambda max. Our spectral data also indicated that silver films are substrates that are more suited for SERS applications where high frequency visible excitations are used. Measurements on gold films classified them into two groups: thin Au films (10-50 A) well adapted for red excitations and thicker ones that are operative in the near infrared. SERS results, which were obtained from a single HL60 cell treated with micromolar drug quantities, placed on thin gold island films indicated that these island films could be future promising substrates for SERS imaging at the cellular level.

Antineoplastic Agents↗

PubMed1998

Characterization of H+-ATPase-dependent activity of multidrug resistance-associated protein in homoharringtonine-resistant human leukemic K562 cells.

Multidrug resistance (MDR), caused by overexpression of either P-glycoprotein or the multidrug resistance-associated protein (MRP), is characterized by a decreased cellular drug accumulation due to an enhanced drug efflux. Many studies on cells overexpressing MRP and/or Pgp, have shown a concentration of the drug inside cytoplasmic acidic vesicles followed by an exocytotic process. In this study, we examined the effects of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole or NBD (a H+-ATPase pump inhibitor), buthionine sulphoximine or BSO (an inhibitor of glutathione (GSH) biosynthesis) and verapamil or VPL (a calcium channel blocker) on the subcellular distribution of daunorubicin or DNR in K562 cells overexpressing MRP (K-H30) and Pgp (K-H300) and A549 cells overexpressing spontaneously MRP. Nucleo-cytoplasmic distribution of DNR was carried out using scanning confocal microspectrofluorometry. This technique allows determination of nuclear accumulation of anthracyclines. Our results show that nuclear accumulation of DNR in K-H30 and A549 cells was increased by NBD, BSO and VPL while in K-H300 cells, only VPL was able to increase nuclear accumulation of DNR. Similarly, NBD, BSO and VPL could reverse DNR resistance in K-H30 cells whereas, in K-H300 cells, only VPL increased the sensitivity of these cells. These data suggest a requirement for GSH in MRP-mediated resistance and suggest that even if vesicular sequestration can happen in cells overexpressing MRP and Pgp proteins, probably only the MRP protein is able to extrude the drug through intracellular vesicles and efflux. Finally, NBD and BSO might be a useful agents in facilitating discrimination between Pgp and MRP phenotypes and prognosis in patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Pirarubicin nuclear uptake does not correlate with its induced cell death effect during reversal of multidrug resistance by quinine in human K562 and CEM leukemic cells.

A number of small and lipophilic cations are able to reverse in vitro the resistance to anthracyclines and other natural products through their interaction with P-glycoprotein or P-gp. However, some modulators do not interact with P-gp. We have demonstrated in a previous a work, using confocal laser microspectrofluorometry, that quinine does not increase nuclear anthracycline uptake in multidrug-resistant Chinese hamster ovary LR73 cells. In this case the LR73 cells were transfected with the mdr1 gene. Moreover, quinine induced in these cells an increase of mdr1 gene expression. In the present study, we investigated verapamil and quinine for their ability to increase nuclear pirarubicin uptake in multidrug-resistant K562R and CEMR human leukemic cell lines. These two cell lines resist, respectively, to doxorubicin and vinblastine and both overexpress the P-gp. Verapamil was able to restore nuclear pirarubicin in both cell lines. On the other hand, quinine was unable to significantly increase nuclear pirarubicin uptake. Both modulators were able to restore pirarubicin sensitivity in both resistant cell lines. After treatment with quinine, mdr1 gene and P-gp expression was not significantly altered as observed previously in the LR73 cells. This suggest that the effect of quinine on mdr1 gene expression is dependent on the cell line studied. These data suggest that quinine could modify the molecular environment of anthracyclines and/or its binding to a possible cytoplasmic target, and that the mechanisms by which anthracyclines induce cell death, and ways by which chemotherapy fails in multidrug-resistant leukemic cells remain complex and are related to more than one target.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Neutrophil elastase promotes rapid exocytosis in human airway gland cells by producing cytosolic Ca2+ oscillations.

The molecular and ionic mechanisms responsible for the regulation of mucus exocytosis in human airway gland cells remain poorly defined. To determine whether dynamic changes of intracellular free Ca2+ concentration [Ca2+]i can promote different exocytotic responses, we monitored dynamic changes in [Ca2+]i and secretory granule (SG) exocytosis in individual human tracheal submucosal serous gland (HTG) cells. These changes were in response to exposure of the cells to three different secretagogues associated with airway inflammation and disease: human neutrophil elastase (HNE), histamine, and ATP. Dynamic changes in [Ca2+]i from single cells were determined with Indo-1/AM using quantitative UV laser microspectrofluorometry. The rate of SG exocytosis was measured in single cells by fluorescence videomicroscopy of SG degranulation and by the ELISA method. Exposure of HTG cells to a low concentration of HNE (1.0 microM) caused a high rate of SG exocytosis (52% decrease in the initial quinacrine fluorescence) during the first 8-min stimulation period compared with that observed following exposure of the cells to 100 microM histamine (10% decrease) or 100 microM ATP (6% decrease). In contrast to a rapid and transient rise in [Ca2+]i induced by histamine (1.0-100 microM) and ATP (10-100 microM), HNE (0.01-1 microM) generated asynchronous oscillations in [Ca2+]i over the first 8-min period. Depletion of internal Ca2+ stores with thapsigargin (500 nM) induced a significant reduction (P < 0.01) in the observed increases in [Ca2+]i upon addition of each of the secretagogues, but did not greatly affect the SG exocytotic responses. Interestingly, the removal of extracellular Ca2+ (+5 mM EGTA) significantly reduced (P < 0.01) both the [Ca2+]i increases and the rate of SG exocytosis following exposure to the secretagogues. We also demonstrate that the influx of extracellular Ca2+ and [Ca2+]i oscillations rather than the absolute level of [Ca2+]i regulate the rapid onset and extent of exocytotic responses to HNE in airway gland cells. Taken together, these results provide strong evidence that [Ca2+]i is a critical intracellular messenger in the regulation of exocytosis process in human airway gland cells.

Adenosine Triphosphatases↗

PubMed1998

Confocal scanning microspectrofluorometry reveals specific anthracyline accumulation in cytoplasmic organelles of multidrug-resistant cancer cells.

We used confocal microspectrofluorometry to investigate intracellular distribution of pirarubicin or THP-DOX in parental K562, CEM, and LR73 tumor cells and their corresponding multidrug-resistant (MDR) strains. Each spectrum of a recorded image was considered as a combination of cell autofluorescence and fluorescence of the drug. In the cytoplasm of parental K562, CEM, and LR73 cells, THP-DOX fluorescence emission profile was similar to that of free drug in aqueous buffer. The (I550nm/I600nm) ratio was 0. 50 +/- 0.1. However, in the cytoplasm of resistant cells the 550-nm band profile was modified. The I550nm/I600nm ratio was 0.85 +/- 0.2 in MDR K562 cells, which is significantly different from the ratio in sensitive cells (p<0.01). This appeared first to correspond to accumulation and self-oligomerization of THP-DOX in cytoplasmic organelles of MDR cells. Transfection of LR73 cells with the mdr1 gene conferred this characteristic on the resistant LR73R cells. Bodipy-ceramide, a trans-Golgi probe, was co-localized with the typical fluorescence emission peak at 550 nm observed in the cytoplasm of MDR cells. This organelle has been shown to be more acidic in MDR cells. Moreover, this specific pattern was similar to that observed when anthracycline is complexed with sphingomyelin. The typical fluorescence emission peak at 550 nm decreased in MDR cells incubated simultaneously in the presence of the drug and quinine, verapamil, or S9788. Growth inhibitory effect and nuclear accumulation of THP-DOX data obtained on LR73R and LR73D cell lines showed that only during reversion of resistance by verapamil and S9788 was an increase of nuclear THP-DOX accumulation observed. Our data suggest that characteristics of molecular environment, such as higher pH gradient or lipid structures, would be potential mechanisms of multidrug-resistance via the sequestration of anthracyclines.

Animals↗

PubMed1998

Uptake and intracellular distribution of 4-aminofluorescin-labelled poly(L-lysine citramide imide) in K562 cells.

4-Aminofluorescein (F1-NH2) was conjugated with various macromolecular carriers of the poly(L-lysine citramide)-type which were hydrophobised by ethyl (C2), heptyl (C7), and dodecyl (C12) alkyl groups attached to the pendent carboxyl of the lysine moieties present in repeating units. The dye was used to label the carriers and monitor their intracellular fate after introduction within the incubation medium of K562 cells. The labelled hydrophobised carriers formed multimolecular compacted aggregates stabilised by the balance of attractive hydrophobic interactions and repulsive electrostatic forces in the aqueous culture medium. The apparent molecular weights and the sizes of these aggregates were determined by Size Exclusion Chromatography (SEC) and by light scattering respectively. Comparison was made of the cell distribution of free and conjugated F1-NH2 in the cell cytoplasm and nucleus by using fluorescence microscopy and laser microspectrofluorometry. It was shown that cell uptakes resulted from adsorptive pinocytosis and depended on hydrophobicity and aggregation of the conjugates. The influence of physical entrapment of free-F1-NH2 within the hydrophobic microdomains formed by aggregates F1-NH2 conjugates was also discussed.

Chromatography, Gel↗

PubMed1998

Role of the vacuolar H+-ATPase in daunorubicin distribution in etoposide-resistant MCF7 cells overexpressing the multidrug-resistance associated protein.

Some multidrug-resistant cell lines efflux anticancer drugs but do not overexpress the well-known P-glycoprotein pump or Pgp. A 190 kDa or multidrug-resistant associated protein (MRP) has been identified and described as an MDR mediator. Many studies on cells overexpressing MRP and Pgp, show a concentration of the drug inside cytoplasmic vesicles followed by an exocytotic process. We studied daunorubicin (DNR) subcellular distribution in the presence of an H+-ATPase pump inhibitor 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD) and verapamil (VPL) in two human breast adenocarcinoma MCF7 etoposide-resistant and adriamycin-resistant cell lines, overexpressing respectively MRP (MCF7/VP) and Pgp (MCF7/ADR). Nucleo-cytoplasmic distribution of daunorubicin was carried out using scanning confocal microspectrofluorometry. This technique allows the determination of nuclear accumulation of anthracyclines. Our results show that NBD was able to increase the nuclear accumulation of DNR in MCF7/VP but not in MCF7/ADR cells. Similarly, NBD could reverse DNR resistance in MCF7/VP cells but had no effect on DNR cytotoxicity in MCF7/ADR cells. VPL caused a significant increase in nuclear accumulation of DNR in MCF7/VP and MCF7/ADR cells. Incubation of MCF7/VP and MCF7/ADR cells with VPL, increased the sensitivity of these cells. These data demonstrate clearly that even if vesicular sequestration can happen in cells overexpressing MRP and Pgp proteins, only the MRP protein is able to extrude the drug through intracellular vesicles and efflux. In cells overexpressing Pgp, drug efflux probably takes place directly at the membrane level.

4-Chloro-7-nitrobenzofurazan↗

PubMed1998

FT-IR spectroscopy as an emerging method for rapid characterization of microorganisms.

Statistical methods such as principal component analysis and cluster analysis were used to analyze ATR-FT-IR spectra obtained from bacterial whole cells. Both methods gave satisfactory results and are conclusive in showing that they can discriminate and classify bacterial strains of clinical origin exhibiting different resistance mechanisms. This approach places FT-IR spectroscopy at the forefront of those new potential techniques that could be used in the rapid screening of microorganisms.

Escherichia coli↗