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PubMed1997

Development of the FUN-1 family of fluorescent probes for vacuole labeling and viability testing of yeasts.

A new family of fluorescent probes has been developed for assessing the viability and metabolic activity of yeasts. This class of halogenated unsymmetric cyanine dyes is exemplified by the FUN-1 [2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)- methylidene)-1-phenylquinolinium iodide] stain, a membrane-permeant nucleic acid-binding dye that has been found to give rise to cylindrical intravacuolar structures (CIVS) in Saccharomyces cerevisiae. Biochemical processing of the dye by active yeasts yielded CIVS that were markedly red shifted in fluorescence emission and therefore spectrally distinct from the nucleic acid-bound form of the dye. The formation of CIVS occurred under both aerobic and anaerobic conditions and was highly temperature dependent. Treatment of yeasts with the nonmetabolizable glucose analog 2-deoxy-D-glucose reduced cellular ATP levels approximately 6-fold and completely inhibited CIVS formation. Under aerobic conditions, the formation of CIVS was abrogated by the cytochrome oxidase inhibitors azide and cyanide; however, the H+ transport uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited CIVS formation under both aerobic and anaerobic conditions. Depletion of cellular thiols, including glutathione, with millimolar concentrations of N-ethylmaleimide, iodoacetamide, or allyl alcohol completely inhibited CIVS production. Marked reduction in the formation of CIVS by ethacrynic acid and sulfobromophthalein, inhibitors of glutathione S-transferase, suggested that dye processing can involve enzyme-mediated formation of glutathione conjugates. The conversion of FUN-1 by S. cerevisiae was studied quantitatively by using several techniques, including fluorometry, flow cytometry, and wide-field and confocal laser scanning fluorescence microscopy.

1-Propanol↗
PubMed1996

Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color.

Selective DNA strand break induction by photolysis (SBIP) at sites that contain incorporated halogenated nucleotides has been recently proposed as a means of analyzing DNA replication and detecting proliferating cells. The presence of numerous in situ DNA strand breaks is also an inherent feature of apoptotic cells. The aim of the present study was to differentially label DNA strand breaks in apoptotic cells vs photolysis-induced breaks in BrdUrd incorporating cells. This would allow one, by multicolor staining, to identify these respective cells in the same sample preparation. Toward this end, exponentially growing HL-60 cells were pulse labeled with BrdUrd and then were subjected to hyperthermia or treated with DNA topoisomerase I inhibitor camptothecin to induce apoptosis. DNA strand breaks in apoptotic cells were first labeled directly with fluorochrome-conjugated dUTP or dCTP, followed by dideoxynucleotide (to terminate chain elongation), in a reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The cells were subsequently exposed to UV light to photolyze DNA containing the incorporated BrdUrd. The photolysis-induced DNA strand breaks were, in turn, labeled with digoxygenin- or biotin-conjugated dUTP followed by digoxygenin antibody or avidin, respectively, conjugated with fluorochrome of another color. Alternatively, DNA strand breaks were labeled with BrdUTP which was then detected by FITC-conjugated anti-BrdUrd MoAb. Following counterstaining of cellular DNA with a fluorochrome of a third color it was possible to identify apoptotic cells, cells incorporating BrdUrd, and cells having no DNA strand breaks. Cell fluorescence was measured either by flow cytometry or with the microscope-based laser scanning cytometer. The SBIP approach also offers a possibility to study a colocalization of the immunocytochemically detectable cell constituents at the DNA replication points by microscopy. Using this approach the presence of the proliferating cell nuclear antigen at the DNA replication sites was revealed in MCF-7 breast carcinoma cells.

Antibodies, Monoclonal↗
PubMed2003

Apoptosis in glioma cells: review and analysis of techniques used for study with focus on the laser scanning cytometer.

Traditional approaches to the treatment of brain tumors are based on the hypothesis that tumors arise and grow because of the disordered regulation of cell proliferation. More recently, it has become apparent that tumor growth depends not only on the rate of cell proliferation but also on the rate of apoptosis (programmed cell death). Genomic alterations that occur in malignancy may limit the cell's ability to undergo apoptosis. Many new treatment strategies for gliomas stem from the use of techniques aimed at manipulating apoptosis. Being able to assess the efficacy of experimental treatments with refined techniques and being able to use instruments that can provide accurate measurements of the apoptotic markers will open the door for discovering novel strategies with the potential to induce effective and selective cytotoxicity. We discuss here in detail the major traditional techniques of assessing apoptosis. We provide an overview of cytometric techniques, including flow cytometry (FC), and will compare it with the laser scanning cytometer (LSC). This is a powerful new tool with potential for obtaining a fast and objective analysis of apoptosis through multiple mechanisms, as well as for assessing proliferation and DNA ploidy in solid malignant tumors.

Apoptosis↗
PubMed1999

Flow cytometry and other techniques show that Staphylococcus aureus undergoes significant physiological changes in the early stages of surface-attached culture.

The techniques of flow cytometry, scanning and transmission electron microscopy, and confocal scanning laser microscopy were used to study the physiology of Staphylococcus aureus in the early stages of surface-attached culture, and to make direct comparisons with planktonic bacteria grown under the same conditions. Attached bacteria growing in nutrient-rich batch culture were found to go through the same growth phases as equivalent planktonic cultures, but with an exponential growth rate of about half that of the planktonic bacteria. Viability of attached bacteria was very high (around 100%) throughout the first 24 h of growth. The size and protein content of attached bacteria varied with growth phase, and both measurements were always smaller than in planktonic bacteria at equivalent growth phases. Respiratory activity per bacterium, as measured by flow cytofluorimetry, and corrected for cell volume, peaked very early in attached cultures (before the first cell division) and declined from then on, whereas in planktonic bacteria it peaked in late exponential phase. Attached and planktonic bacteria showed thicker cell walls in stationary phase than in exponential phase. Membrane potentials of planktonic and attached bacteria were similar in stationary phase, but were much lower in exponential-phase attached cells than in the equivalent planktonic cells. It is apparent that a range of significant physiological adaptations occur during the early phases of attached growth.

Bacterial Adhesion↗
PubMed2005

Rheumatoid arthritis and pigmented villonodular synovitis: comparative analysis of cell polyploidy, cell cycle phases and expression of macrophage and fibroblast markers in proliferating synovial cells.

AIMS: Rheumatoid arthritis (RA) and pigmented villonodular synovitis (PVNS) are aggressive diseases with progressive joint destruction. The present study aims to define cell cycle phases, polyploidy and the immunophenotype of proliferating synovial cells in both diseases. METHODS AND RESULTS: Synovial tissues from patients with proliferative-active RA, localized and diffuse PVNS were analysed by DNA flow cytometry, immunohistochemistry and double immunofluorescence with confocal laser scan microscopy. Expression of macrophage markers (CD68/CD163), fibroblast markers (h4Ph/CD55) and Ki67 antigen was examined. Synovial cells positive for either macrophage or fibroblast markers as well as double-labelled cells were found in both RA and PVNS. In RA, CD68/CD163+ synoviocytes were preferentially located in the vicinity of the synovial lining layer, while they were more randomly distributed in PVNS. Of cases with diffuse PVNS, 20% showed an aneuploid cell pattern. All samples of localized PVNS and RA were diploid. Proliferative activity was significantly higher in aneuploid PVNS. CONCLUSIONS: In spite of their histologically homogeneous appearance, proliferating synovial cells display a heterogeneous immunophenotype in both RA and PVNS, indicating functional properties of both macrophages and fibroblasts. Aneuploidy seems to be a special feature of diffuse PVNS.

Antigens, CD↗
PubMed2004

Quantification of Saccharomyces cerevisiae viability using BacLight.

Yeast viability can be accurately quantified using BacLight, a kit which so far has been used only for bacterial analysis. Upon staining, viable cells can be differentiated from non-viable ones by either confocal laser scanning microscopy (CLSM), epifluorescence microscopy, or flow cytometry. Using Saccharomyces cerevisiae as a model, viabilities quantified by CLSM deviated an average of 1.7% from the actual data, and those determined by flow-cytometry by 1.4%.

Cell Culture Techniques↗
PubMed1995

Green fluorescent protein as a marker for gene expression and cell biology of mycobacterial interactions with macrophages.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria offers certain advantages over other bioluminescence systems because no exogenously added substrate or co-factors are necessary, and fluorescence can be elicited by irradiation with blue light without exposing the cells producing GFP to invasive treatments. A mycobacterial shuttle-plasmid vector carrying gfp cDNA was constructed and used to generate transcriptional fusions with promoters of interest and to examine their expression in Mycobacterium smegmatis and Mycobacterium bovis BCG grown in macrophages or on laboratory media. The promoters studied were: (i) ahpC from Mycoosis and Mycobacterium leprae, a gene encoding alkyl hydroperoxide reductase which, along with the divergently transcribed regulator oxyR, are homologues of corresponding stress-response systems in enteric bacteria and play a role in isoniazid sensitivity; (ii) mtrA, an M. tuberculosis response regulator belonging to the superfamily of bacterial two-component signal-transduction systems; (iii) hsp60, a previously characterized heat-shock gene from M. bovis; and (iv) tbprc3, a newly isolated promoter from M. tuberculosis. Expression of these promoters in mycobacteria was analysed using epifluorescence microscopy, laser scanning confocal microscopy, fluorescence spectroscopy, and flow cytometry. These approaches permitted assessment of fluorescence prior to and after macrophage infection, and analyses of promoter expression in individual mycobacteria and its distribution within populations of bacterial cells. Bacteria expressing GFP from a strong promoter could be separated by fluorescence-activated cell sorting from cells harbouring the vector used to construct the fusion. In addition, the stable expression of mtrA-gfp fusion in M. bovis BCG facilitated localization and isolation of phagocytic vesicles containing mycobacteria. The experiments presented here suggest that GFP will be a useful tool for analysis of mycobacterial gene expression and a convenient cell biology marker to study mycobacterial interactions with macrophages.

Animals↗
PubMed2002

[Antisense inhibition of gene expression in human dendritic cells by peptide nucleic acid against CD86].

OBJECTIVE: Dendritic cells (DCs) are the most potent antigen presenting cells (APCs) of the immune system. We intent to block the expression of CD86 in DCs using antisense peptide nucleic acids (PNA), a novel synthetic structural DNA mimic, and interrupt the second signal transmission so that a suppression of corresponding T cell function can be achieved. METHODS: Human DCs grown up from peripheral blood monocytes in GM-CSF and IL-4 were collected. We investigated antisense PNA internalization with laser scan confocal microscope (LSCM). Fluorescence immunocytochemistry, flow cytometry and RT-PCR were used to determine the expression of CD86 protein and mRNA in DCs. RESULTS: LSCM proved that cultured immature DCs could internalized PNA efficiently, according to the specific internalization property of the immature DCs. Antisense PNA DC exhibited striking reductions in cell surface staining for CD86, but not MHC class II, and were poor stimulators of T cell proliferation. RT-PCR found that PNA depressed the amounts of CD86 mRNA in DCs. CONCLUSION: Antisense PNA against CD86 could inhibit the expression of CD86 mRNA and protein in DCs. The blockade of B7/CD28 pathway may increase the potential of costimulatory molecule-deficient antisense PNA DCs of donor origin to induce long-lasting allograft survival.

Antigens, CD↗
PubMed2003

[Effects of niflumic acid on the proliferation of human hepatoma cells].

The purpose of this work was to investigate the effects of niflumic acid (NFA), a chloride channel blocker, on the proliferation of human hepatoma cell line (HHCC). Cell proliferation was analyzed by cell count and MTT assay. Cell cycle analysis was carried out by flow cytometry. [Ca(2+)](i) was determined by laser scanning confocal system. It was found that NFA decreased significantly the cell number and the MTT optical density (OD) of HHCC cells, and that the OD value was reversed after washout of NFA. Compared with control, NFA blocked cell cycle progression in G(1) phase. Extracellular application of NFA (100 micromol/L) induced a rapid decrease in [Ca(2+)](i). These findings demonstrate that blockage of chloride channels by NFA induces growth arrest of HHCC in G(1) phase, which may be due to the inhibition of Ca(2+)/CaM-dependent signaling pathways.

Calcium↗
PubMed2006

[Detection of cyclin E in lymph node metastases of laryngeal squamous cell carcinomas by laser scanning confocal microscopy].

OBJECTIVE: To study the expression of Cyclin E proteins in carcinogenesis and development of the metastasis of laryngeal squamous cell carcinomas (LSCC). METHOD: The expression of Cyclin E protein were detected by laser scanning confocal microscopy (LSCM) and multiparameter flow cytometry (FCM) after immunofluorescence staining method in twenty-one surgically resected primary LSCC (7 LSCC with cervical lymph node metastasis, 14 LSCC without cervical lymph node metastasis). RESULT: The fluorescence intensity was 78.12 +/- 6.54 in LSCC with cervical lymph node metastasis and 74.93 +/- 8. 82 in LSCC without cervical lymph node metastasis (P > 0. 05); The fluorescence intensity in cervical lymph node metastasis was 93.65 +/- 7.48, it's more higher than that in cervical lymph node negative metastasis (P < 0.01) and both LSCC with cervical lymph node metastasis and without cervical lymph node metastasis (P < 0.05). CONCLUSION: It is suggested that Cyclin E proteins may play an important role in the carcinogenesis and development of the lymph node metastases of LSCC. Detection of Cyclin E maybe becomes an indication to evaluating the lymph node metastases of LSCC.

Carcinoma, Squamous Cell↗
PubMed2005

Implementation of accurate and fast DNA cytometry by confocal microscopy in 3D.

BACKGROUND: DNA cytometry is a powerful method for measuring genomic instability. Standard approaches that measure DNA content of isolated cells may induce selection bias and do not allow interpretation of genomic instability in the context of the tissue. Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. Because the technique is technically challenging and time consuming, only a small number of usually manually selected nuclei were analyzed in different studies, not allowing wide clinical evaluation. The aim of this study was to describe the conditions for accurate and fast 3D CLSM cytometry with a minimum of user interaction to arrive at sufficient throughput for pilot clinical applications. METHODS: Nuclear DNA was stained in 14 microm thick tissue sections of normal liver and adrenal stained with either YOYO-1 iodide or TO-PRO-3 iodide. Different pre-treatment strategies were evaluated: boiling in citrate buffer (pH 6.0) followed by RNase application for 1 or 18 hours, or hydrolysis. The image stacks obtained with CLSM at microscope magnifications of x40 or x100 were analyzed off-line using in-house developed software for semi-automated 3D fluorescence quantitation. To avoid sectioned nuclei, the top and bottom of the stacks were identified from ZX and YZ projections. As a measure of histogram quality, the coefficient of variation (CV) of the diploid peak was assessed. RESULTS: The lowest CV (10.3%) was achieved with a protocol without boiling, with 1 hour RNase treatment and TO-PRO-3 iodide staining, and a final image recording at x60 or x100 magnifications. A sample size of 300 nuclei was generally achievable. By filtering the set of automatically segmented nuclei based on volume, size and shape, followed by interactive removal of the few remaining faulty objects, a single measurement was completely analyzed in approximately 3 hours. CONCLUSIONS: The described methodology allows to obtain a largely unbiased sample of nuclei in thick tissue sections using 3D DNA cytometry by confocal laser scanning microscopy within an acceptable time frame for pilot clinical applications, and with a CV small enough to resolve smaller near diploid stemlines. This provides a suitable method for 3D DNA ploidy assessment of selected rare cells based on morphologic characteristics and of clinical samples that are too small to prepare adequate cell suspensions.

Adrenal Glands↗
PubMed2004

Expression of green fluorescent protein and its application in pathogenesis studies of serotype 2 Streptococcus suis.

We investigated the interaction between type 2 Streptococcus suis and swine phagocytes during infection of the natural host, by using green fluorescent protein (GFP) as a specific marker to observe the challenge organism. We compared the strength of the S. suis sly promoter (SP332) and the synthetic promoter (CP25) in driving GFP expression. Two GFP alleles, gfpP11 and gfpmut3*, were also compared. The two promoters and two alleles were efficiently compared using three different promoter-GFP gene combinations on a shuttle vector, which were transformed into S. suis strains SX332, SX932 or M2. Plasmid pSL6.81 has SP332 with gfpP11, pSL5.24 has SP332 with gfpmut3*, and pSL5.28 has CP25 with gfpmut3*. The transformants were fluorescent with green light when viewed with an epifluorescence microscope or during flow cytometry. The signal was also detected using a laser scanning confocal microscope. The GFP expression level varied and CP25 with gfpmut3* led to greatest expression. For optimizing GFP detection, fluorescence-based cell sorting was applied to SX332(pSL5.28) and the mean fluorescence intensity increased 25.9% after optimization. Fluorescence activated cell sorting (FACS)-based phagocytosis assay showed that, without opsonization, phagocytosis rates of SX332, SX932 and M2 by both neutrophils and monocytes were similar and low. After opsonization, the phagocytosis of M2 increased 10-fold while phagocytosis of SX332 and SX932 did not change. GFP-labeled S. suis was identified in fresh pig tonsil tissue 18 h after infection. The results of this study indicated that GFP was expressed in type 2 S. suis and GFP labeled S. suis could be used in phagocytosis and pathogenesis studies.

Alleles↗
PubMed2006

Immunological and ultrastructural characterization of endothelial cell cultures differentiated from human cord blood derived endothelial progenitor cells.

The replacement of endothelium by endothelial progenitor cells (EPCs) for therapeutic use in order to ameliorate the vascular status of ischemic organs is now in the focus of vascular research. The aim of our studies was to investigate whether EPCs derived from peripheral blood mononuclear cells (PBMNCs-derived EPCs) or EPCs propagated from CD34(+) hematopoietic stem cells (HSCs-derived EPCs), both isolated from human cord blood, are able to differentiate into early mature endothelial cells (ECs) under certain in vitro conditions. We characterized both cell populations by flow cytometry, phase contrast microscopy, fluorescence microscopy and confocal laser scanning microscopy as well as ultrastructurally using transmission and scanning electron microscopy. While PBMNCs gave rise to clusters of spindle-like EPCs after few days but did not further mature under in vitro conditions, mature ECs could only be successfully propagated from a starting population of isolated HSCs. Both, PBMNCs- and HSCs-derived EPCs, took up Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL) and could be positively stained for CD31, CD105, the vascular endothelial growth factor receptor 2 (VEGFR-2, KDR) and ulex europaeus agglutinin 1 (UEA-1) at the cell surface. EPC showed surface expression of CD54 and CD106. However, only a small portion of HSCs-derived EPCs was positive for CD54 but negative for CD106. Intracellular staining for von Willebrand factor (vWF) provided a homogenous stain in PBMNC-derived EPCs while in HSCs-derived EPCs, during cultivation for 2-3 weeks, more and more a typical punctuated staining pattern related to Weibel-Palade bodies (WPBs) was visible. By phase contrast and scanning electron microscopy, an arrangement of PBMNCs-derived EPCs in cord-like structures could be demonstrated. In these formations, cells showed parallel alignment but exhibited only few cell contacts. Well-developed WPBs could never be found in PBMNCs-derived EPCs. In contrast, differentiating HSCs-derived EPCs developed adherence junctions, interdigitating junctions as well as syndesmos. During maturation, spindle-like cell types appeared with abundant WPBs as well as cobblestone-like cell types with a fewer content of these organelles. WPBs, in the spindle-like cell types displayed conspicuous shapes and were concentrated in close proximity to mitochondria-rich areas. HSCs-derived EPCs exhibited signs of high synthetic activity such as a well-developed rough endoplasmic reticulum (RER) and multiple Golgi complexes. In the trans-Golgi network (TGN), close to the Golgi complex, a new formation of WPBs could be observed. These morphological features correlated well with a high growing capacity. Although it was not possible to demonstrate the complete differentiation line from HSCs to early matured ECs by immunologic markers because of the limited number of cells available for such investigations, distinct morphologic maturation stages could be shown at light and electron microscopical levels. In conclusion, the study presented here characterizes not only the different cell populations involved in the differentiation of early EPCs into mature ECs but also the transition stage where the maturation step takes place by demonstration of the new formation of WPBs. In this respect, these investigations provide new insights into the in vitro differentiation which could have some in vivo correlation.

Antigens, CD34↗
PubMed2005

Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model.

Current vaccine development includes optimization of antigen delivery to antigen presenting cells, such as dendritic cells (DC). Particulate systems have attracted increasing attention in the development of vaccine delivery systems. In the present study, we investigated DC uptake of model fluorescent polystyrene particles with a broad size range and variable surface properties. Localization of particles was investigated using confocal laser scanning microscopy and uptake was quantified by flow cytometry. Immature DC were generated from mononuclear cells isolated from human blood. The polystyrene particles interacted with the DC throughout the tested diameter range of 0.04-15 microm in a time- and concentration-dependent manner. The optimal particle diameter for fast and efficient acquisition by a substantial percentage of the DC was 0.5 microm and below. The surface of 1 and 0.1 microm polystyrene particles was covalently modified with different polyaminoacids/proteins, yielding particles with varying surface charge. Uptake of 1 microm particles was greatly enhanced when particles displayed a positive surface charge. In general, the present findings establish that particle diameters of 0.5 microm and below were optimal for DC uptake; however uptake of larger particles could be greatly enhanced by rendering the particle surface positive. Whether increased particle uptake is correlated with increased immune responses, remains to be established.

Chemical Phenomena↗
PubMed2003

Laser-light scattering, a new method for continuous monitoring of platelet activation in circulating fluid.

We evaluated a novel technique of laser-light scattering (LLS) to detect platelet-volume changes continuously, reflecting platelet aggregation in circulating fluid. Carotid arteries from 20 dogs were mounted in a dual perfusion chamber. Balloon angioplasty (BA) was performed and arteries perfused with platelet-rich plasma (PRP). A He-Ne laser beam was passed through cuvettes connected to tubing draining the arteries. From the angle of incidence, the average volume of aggregates was measured by the ratio of scattering light at 1 to 5 degrees' spread on the diode array of a multichannel analyzer. Platelet volume varied linearly with the scattered light ratio at 1 to 5 degrees (y = -24.2 + 27.6 x [y = particle size, microm(3); x = scattered light ratio at 1/5 degrees]). For comparison, we used an electronic particle counter (Coulter counter) to measure platelet volume. P-selectin expression was measured to confirm platelet activation. Comparing 10 uninjured and 10 BA-injured arteries, we found that platelet volume as measured with LLS increased from 21.6 +/- 4.1 to 52.1 +/- 12.5 microm(3) (P < .003); as measured with the Coulter counter, it increased from 29.9 +/- 2.4 to 62.3 +/- 7.0 microm(3) (P < .005). Six BA-injured arteries perfused with PRP and aspirin (0.2 mg/mL) were compared with six arteries treated with BA alone. The aspirin decreased platelet volume as measured with LLS from 56.2 +/- 11.8 to 40.2 +/- 12.7 microm(3) (P < .01); the Coulter counter revealed a decrease from 51.9 +/- 18.5 to 38.8 +/- 14.2 microm(3) (P < .001). Coulter counter and LLS results were correlated: r = 0.74, P < .05. The peak of P-selectin expression coincided with peak platelet volume. These data demonstrate that increases in circulating-platelet size stimulated by endovascular injury can be reliably and continuously monitored with the use of LLS.

Angioplasty, Balloon↗
PubMed2002

Subcellular daunorubicin distribution and its relation to multidrug resistance phenotype in drug-resistant cell line SMMC-7721/R.

AIM: To investigate the correlation between subcellular daunorubicin distribution and the multidrug resistance phenotype in drug-resistant cell line SMMC-7721/R. METHODS: The multidrug resistant cell line SMMC-7721/R, a human hepatocellular carcinoma cell line, was established. Antisense oligonucleotides (AS-ODN) were used to obtain different multidrug resistance phenotypes by inhibiting the expression of mdr1 gene and/or multidrug resistance-related protein gene(mrp) using Lipofectamine as delivery agent. Expression of mdr1 and mrp genes was evaluated by RT-PCR and Western blotting. Intracellular daunorubicin (DNR) concentration was measured by flow cytometry. Subcellular DNR distribution was analyzed by confocal laser scanning microscopy. Adriamycin (ADM) and DNR sensitivity was examined by MTT method. RESULTS: Low level expression of mdr1 and mrp mRNAs and no expression of P-Glycoprotein(P-gp) and multidrug resistance-related protein (P(190)) were detected in parental sensitive cells SMMC-7721/S, but over-expression of these two genes was observed in drug-resistant cell SMMC-7721/R. The expression of mdr1 and mrp genes in SMMC-7721/R cells was down-regulated to the level in the SMMC-7721/S cells by AS-ODN. Intracellular DNR concentration in SMMC-7721/S cells was 10 times higher than that in SMMC-7721/R cells. In SMMC7721/S cells intracellular DNR distributed evenly in the nucleus and cytoplasm, while in SMMC-7721/R cells DNR distributed in a punctate pattern in the cytoplasm and was reduced in the nucleus. DNR concentration in SMMC-7721/R cells co-transfected with AS-ODNs targeting to mdr1 and mrp mRNAs recovered to 25 percent of that in SMMC7721/Scells. Intracellular DNR distribution pattern in drug-resistant cells treated by AS-ODN was similar to drug-sensitive cell, and the cells resistance index (RI) to DNR and ADM decreased at most from 88.0 and 116.0 to 4.0 and 2.3, respectively. Co-Transfection of two AS-ODNs showed a stronger synergistic effect than separate transfection. CONCLUSIONS: P-gp and P(190) are two members mediating MDR in cell line SMMC7721/R. Intracellular drug concentration increase and subcellular distribution change are two important factors in multidrug resistance (MDR) formation. The second factor, drugs transport by P-gp and P(190) from cell nucleus to organell in cytoplasm, may play a more important role.

Antibiotics, Antineoplastic↗
PubMedDate not supplied

Fluorescence-based assessment of LRP activity: a comparative study.

BACKGROUND: Broad resistance to anticancer drugs is a major cause of failure in cancer treatment. The Lung Resistance-related Protein (LRP) is a protein associated with drug resistance, which is involved in nucleo-cytoplasmic transport and is known to predict a poor response to chemotherapy in acute myeloid leukaemia. The only method allowing the detection of LRP activity is based on radio-labelled daunorubicin incorporation. Our goal was to develop a fluorescence-based assay to analyse LRP function. MATERIALS AND METHODS: We used human colon carcinoma cell lines treated with sodium butyrate (NaB) in order to induce LRP expression. Daunorubicin efflux in isolated nuclei was measured by flow cytometry, the localization and quantification of Daunorubicin analysed by confocal laser scanning microscopy (CLSM) and the diffusion coefficient of this drug estimated by Fluorescence Correlation Spectrometry (FCS). RESULTS: According to the method using [14C] Doxorubicin cells incubated with NaB displayed an efflux of Daunorubicin out of isolated nuclei demonstrated by flow cytometry or CLSM. The FCS method was able to evaluate kinetics of Daunorubicin molecules in nucleus and cytoplasm and showed a higher dispersion of Daunorubicin kinetics with cells previously NaB-treated. This argument is in favour of an increase of nucleo-cytoplasmic exchange. CONCLUSION: Using CLSM we showed that LRP was able to modify anticancer drug repartition in the cells. LRP activity assessment needs either isolated nuclei if flow cytometry is employed, or FCS, and only a few cells may be analysed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

A novel approach of low-frequency ultrasonic naked plasmid gene delivery and its assessment.

OBJECTIVE: To deliver the naked genes into cells through the bioeffects of cell membrane porous produced by low-frequency ultrasound (US) and to investigate the safety by determining the threshold of cell damage and membrane permeability. METHODS: The suspension of red cells from chickens, rabbits, rats, and S180 cells was exposed to calibrated US field with different parameters in still and flowing state. Laser scanning confocal microscopy, fluorescent microscopy, scanning electron microscopy, flow cytometry and spectrophotometry were used to examine cell morphology, membrane permeability, enzymes, free radicals, naked gene expression efficiency, threshold of cell damage and cell viability. RESULTS: The plasmid of green fluorescent protein (GFP) as a reporter gene was delivered into S180 cells under optimal conditions without cell damage and cytotoxicity. The transfection rate was (35.83 +/- 2.53)% (n = 6) in viable cells, and the cell viability was (90.17 +/- 1.47)% (n = 6). Also, malondialdehyde, hydroxyl free radical, alkaline phosphatase, and acid phosphatase showed a S-shaped growth model (r = 0.98 +/- 0.01) in response to the permeability change and alteration of cell morphology. The constant E of energy accumulation in US delivery at 90% cell viability was an optimal control factor, and at 80% cell viability was the damage threshold. CONCLUSION: US under optimal conditions is a versatile gene therapy tool. The intensity of GFP expression in US group has a higher fluorescent peak than that in AVV-GFP group and control group (P < 0.001). The optimal gene uptakes, expression of gene and safety depend on E, which can be applied to control gene delivery efficiency in combination with other parameters. The results are helpful for development of a novel clinical naked gene therapeutic system and non-hyperthermia cancer therapeutic system.

Animals↗