Mean Fluorescence Research Articles

Coumarin modulates the cell-cycle progression of an MTV-EJras cell line.

The effects of coumarin (1,2-benzopyrone) on ras oncogene expression during the cell cycle of an MTV-EJras cell line was determined by flow cytometry. ras oncogene expression in cells was induced by dexamethasone and increased fivefold during G1/G0 phase and threefold in S phase. Dexamethasone also increased the percentage of cells in S phase from 21% to 31%, compared to phosphate-buffered-saline-treated control cells (P < 0.01). Coumarin decreased the percentage of dexamethasone-treated cells in S phase from 31% to 19%, with a concomitant increase in the percentage of cells in G1/G0 compared to control levels. Coumarin also reduced ras expression (mean fluorescence) by 40% in G1/G0 phase and 30% in S phase. We conclude that coumarin significantly reduces the cell-cycle progression of MTV-EJras cells and decreases ras expression in both G1 and S phases. These findings suggest a novel approach to the selective control of proliferation of malignant cells.

Modulation by cytokines on the surface expression and shedding of intercellular adhesion molecule-1 (ICAM-1) in human liver cancer cell lines

Surface expression and shedding of intercellular adhesion molecule-1 (ICAM-1) in the absence and presence of cytokines were measured in seven human liver cancer cell lines. All cancer cell lines were positive for ICAM-1 on the surface of the cells, and the percentages of positive cells ranged from 60 to 97% as analyzed by flow cytometry. Mean fluorescence was increased in the presence of IL-1β, TNF-α, IFN-α or IFN-γ, and the effect was augmented in a dose-dependent fashion. In the culture medium of all cancer cell lines, a soluble form of ICAM-1 was detected even in the absence of cytokines by enzyme-linked immunosorbent assay, and the concentration was increased by IL-1β, TNF-α, or IFN-γ. In two of seven cancer cell lines, the patterns of modulation by cytokines on the surface expression of ICAM-1 and the shedding of the molecules were clearly distinct, suggesting that the mechanisms for these two phenomena might be different. Soluble ICAM-1 was recently reported to suppress non-MHC-restricted cytotoxicity mediated by natural killer and lymphokine-activated killer cells. Our present data suggest that human liver cancer cells may escape from host immune surveillance by shedding of ICAM-1.

Combined preoperative and postoperative immunotherapy for murine C1300 neuroblastoma

Preoperative treatment of murine C1300-neuroblastoma (C1300) with triple immunotherapy using low-dose cyclophosphamide (CY), retinyl palmitate (RP), and interleukin-2 (IL2), followed by tumor resection leads to significant initial tumor control and prolonged survival. However, because long-term tumor recurrence is 67%, the efficacy of continued postoperative immunotherapy is now evaluated. Thirty-two A J mice with 1 cm subcutaneous C1300 tumors were treated for 13 days with CY-100 mg/kg, intraperitoneally (IP), on day 2 of treatment then 25 mg/kg on day 9, RP-2500 IU IP 2 x/week, and IL2 1.6 × 10 5 U IP BID on days 4 to 9 and 11 to 13. On day 14, mice were divided into five treatment groups: (1) OP (operated-tumor resection, n = 6); (2) OP + CY (resection and postoperative CY, n = 7); (3) OP+CY+RP (resection and postoperative CY+RP, n = 7); (4) OP+CY+RP+IL2 (resection and postoperative CY+RP+IL2, n = 7); and (5) CY+RP+IL2 (continued CY+RP+IL2 with no resection, n = 5). Survival and postoperative tumor recurrence were followed for 60 days. The cure rates were group 1 33% ( 2 6 ), group 2 43% ( 3 7 ), group 3 29% ( 2 7 ), group 4 71% ( 5 7 ), and group 5 20% ( 1 5 ). After surgery, tumors that recurred did so in 8 to 22 days, with no statistical difference noted between groups. MHC class I antigenic expression of tumors resected on day 14 and recurrent tumors was determined with monoclonal antibodies and flow cytometry. In tumors resected on day 14, class I expression measured by mean fluorescence, was 374.8 ± 27.40. Recurrent tumors in all groups expressed significantly less class I antigen (230.7 ± 38.32) than the original tumors. Because class I expression is important for cell-mediated tumor lysis, the low expression in the recurrent tumors suggests that the mechanism of their recurrence and resistance to preoperative immunotherapy was through escape of host antitumor immunosurveillance. Because the best overall results, with 71% cures, were obtained with continued postoperative triple immunotherapy despite the presence of viable immunoresistant cells, the success of continuing the triple-drug regimen suggests that the mechanism of action of the regimen may not be purely immunotherapeutic but chemotherapeutic as well.

Confocal microscopy reveals coordinated calcium fluctuations and oscillations in synaptic boutons

Calcium ions are one of the main factors regulating quantal transmitter release and thus synaptic transmission in the nervous system. Using confocal microscopy, fluorescent imaging with the calcium indicator Rhod-2, and time series analysis, we show that the levels of calcium ions inside single synaptic boutons of the lizard neuromuscular junction are not constant at rest, but undergo coordinated fluctuations in the space domain, which cover a large fraction of the synaptic bouton. Furthermore, oscillations in intracellular calcium were frequently observed in the time domain. Control experiments showed no coordinated fluctuations or oscillations at locations outside the synaptic boutons. Edge detection analysis showed that the coordinated fluctuations and oscillations were not due to movement artifacts. No coordinated fluctuations and oscillations were seen when similar measurements and analyses were performed on artificial fluorescent beads. A variance analysis was performed on artificial fluorescent beads and on synaptic boutons. The variance of the fluorescent signal at the synaptic boutons was larger than the variance in artificial beads with the same mean fluorescence. This extra variance was greatly reduced when the extracellular calcium concentration was decreased from 2.0 mM to 0.4 mM. We conclude that the coordinated fluctuations and oscillations in the calcium-induced fluorescence at the synaptic boutons are genuine biological phenomena and may be of significance in the regulation of transmitter release.

Regulation of low affinity IgE receptor (CD23) expression on mononuclear phagocytes in normal and asthmatic subjects.

Mononuclear phagocytic cells contain low affinity receptors for IgE (Fc epsilon RII or CD23) which induce cellular activation in the presence of specific allergen. These studies were performed to quantify the expression by monocytes and alveolar macrophages of Fc epsilon RII in asthma and to determine biologic response modifiers that regulate Fc epsilon RII. Whereas 2.5 +/- 1.0% of the monocytes obtained from normal volunteers were Fc epsilon RII positive, this increased to 16.7 +/- 2.4% in asthma (p < 0.001). Stimulation of Fc epsilon RII expression on monocytes was shown to be an activity of IL-4 (24.5 +/- 5.9%), granulocyte-macrophage-CSF (28.1 +/- 5.2%), IFN-alpha (15.8 +/- 5.3%), IFN-gamma (10.4 +/- 3.7%), and macrophage-CSF (7.3 +/- 0.7%) but not of IL-2, IL-6, or TNF-alpha. Expression of Fc epsilon RII by these cytokines was associated with the induction of specific mRNA transcripts. Using Fc epsilon RII subtype specific primers in the polymerase chain reaction expansion of cDNA, cytokine-induced receptors were shown to be Fc epsilon RIIb. Alveolar macrophages from nonasthmatic subjects displayed minimal expression of Fc epsilon RII (3.2 +/- 1.2%); however, these receptors were present on 69.2 +/- 6.3% of asthmatic volunteers (p < 0.001). Induction of Fc epsilon RII appears specific for allergic asthma insofar as these receptors are also not expressed in subjects with interstitial lung disease (1.3 +/- 1.3%). As assessed by shift in mean fluorescence, instillation of allergen in the asthmatic's airway further up-regulated Fc epsilon RII on alveolar macrophages by 151 +/- 7%. Up-regulation of Fc epsilon RII in atopic individuals may therefore reflect allergen-induced exposure of mononuclear phagocytes to one or more of these cytokines. These studies suggest a mechanism by which an immunologic stimulus that leads to the production of these cytokines (e.g., allergen or viral infection) would contribute to the development or exacerbation of allergic disease.

Studies on the structure of actin gels using time correlation spectroscopy of fluorescent beads.

Fluorescence correlation spectroscopy (FCS) has been used to measure the diffusion of fluorescently labeled beads in solutions of polymerized actin or buffer. The results, obtained at actin concentrations of 1 mg/ml, show that small beads (0.09 micron in diameter) diffuse nearly as rapidly in the actin gel as in buffer, whereas the largest beads tested (0.5 micron in diameter) are immobilized. Measured autocorrelation times for motions of beads with intermediate sizes show that the diffusion is retarded (relative to buffer) and that the time behavior cannot be represented as a single diffusive process. In addition to the retarded diffusion observed over distances > 1 micron, 0.23-micron beads also show a faster motion over smaller distances. Based on the measured rate of this faster motion, we estimate that the beads may be constrained within a cage approximately 0.67 micron on a side, equal to a filament length of approximately 250 subunits. Fluorescence correlation spectroscopy measurements made in the same small spot (radius of 1.4 microns) of the gel vary over time. From the variations of both the autocorrelation functions and the mean fluorescence, we conclude that, corresponding to a spatial scale of 1.4 microns, the actin gel is a dynamic structure with slow rearrangement of the gel occurring over periods of 20-50 s at 21-22 degrees C. This rearrangement may result from local reorganization of the actin matrix. Data for the retardation of beads by the actin gel are consistent with a detailed theory of the diffusion of particles through solutions of rigid rods that have longitudinal diffusion coefficients much less than that of the particles (Ogston, A. G., B. N. Preston, and J. D. Wells. 1973. Proc. R. Soc. Lond. A. 333:297-316).

Flow cytometric analysis on perforin induction in peripheral blood mononuclear cells with interleukin-2 or OK-432.

Perforin is a protein present in the cytoplasmic granules of killer cells and is considered to be an important effector molecule. We assessed the perforin appearance via flow cytometry in human peripheral blood mononuclear cells stimulated in vitro for 3 days by recombinant interleukin-2 (rIL-2) or OK-432, a biological response modifier. The relationship between the lymphocyte subsets and perforin was investigated via two-color assay. CD4-positive cells had almost no perforin, and most of the CD16-positive cells did. Regarding the relationship with CD8, some of the bright positive cells (which were likely T cells) and most of the dull positive cells (likely NK cells) had perforin. Mean fluorescence was greatest in perforin-positive cells incubated with rIL-2, less in cells incubated with OK-432, and minimal in cells incubated in a medium without additives. Immunohistochemical staining with antiperforin antibody revealed that blast-transformed and enlarge cells were stained positively and that the intensity of staining of each cell alone was enhanced in cells incubated with OK-432 or rIL-2. If the fluorescence intensity of perforin-positive cells correlates with the amount of perforin in those cells, then the appearance of perforin was enhanced with OK-432, more enhanced with rIL-2, and consistent for cytotoxicity against K562 and Daudi cells. IL-2 was induced by OK-432, suggesting that the indirect effect of this IL-2 may play a role in OK-432-perforin induction. The results suggest that perforin may be an effector molecule in killer cells induced by rIL-2 or OK-432.

Dynamic measurements of the platelet membrane glycoprotein IIb-IIIa receptor for fibrinogen by flow cytometry. II. Platelet size-dependent subpopulations

Platelet aggregation has previously been shown to occur within 1 s of activation with 100 microM adenosine diphosphate (ADP) for both large (L) and small (S) platelet subpopulations, but L platelets were about twofold more sensitive and more rapidly recruited into microaggregates than were S platelets after correcting for differences in platelet surface area. Because platelet aggregation normally requires fibrinogen binding to glycoprotein IIb-IIIa receptors (FbR) expressed on the activated platelet surface, we wished to compare the kinetics and nature of FbR expression induced by ADP for L versus S platelets, and to measure size-dependent differences in FbR expression for platelets maximally activated with phorbol myristate acetate (PMA). We presented the theory and methodology in Part I (Frojmovic, M., T. Wong, and T. van de Ven. 1991. Biophys. J. 59:815-827) for measuring the rate of FbR expression (k1) and both the rate (k2) and efficiency (alpha) of binding of PAC1 to FbR as a function of activation conditions from the initial on-rate of FITC-PAC1 to FbR (V) and the maximal number of FbR expressed: these are measured, respectively, from the initial rate of increase in platelet-bound fluorescence (v) and the maximal increase in mean fluorescence (Flmax). We extended these analyses to L and S platelets, selected by electronic gating of forward scatter profiles (FSC), with corresponding fluorescence (Fl) histograms retrieved analytically. Platelet size (V) and surface area (SA), determined directly for cells separated with a cell sorter, were highly correlated with FSC, allowing v and Flmax values to be expressed per unit area of membrane for L:S comparisons. Surprisingly, ADP activation appeared to express all FbR within 1-3 s of ADP activation for both L and S platelets, whereas k1 was similar for PMA activation. In addition, L platelets maximally expressed two and three times more FbR per unit area than did S platelets when maximally stimulated, respectively, with ADP or PMA. Whereas k2 was independent of platelet size for a given activator, the efficiency of PAC1 binding (alpha), per unit area of membrane, was two times greater for L than for S platelets, for either ADP or PMA activation. Our data suggest that the FbR structure, its microenvironment, or its surface organization may vary with platelet size or activator type. Major reorganization of FbR and/or its environment appears to occur after approximately 5 min of ADP activation equally for both L and S platelets. A model is presented to account for size-dependent differences in FbR expression with implications for regulation of platelet aggregation.

Mechanism of action and clinical potential of the selective tumor radiosensitizer nicotinamide

We have shown recently that the vitamin nicotinamide (niacinamide) can enhance the radiation response of several mouse tumors with little or no increase in the radiosensitivity of normal tissues (Horsman et al., Radiat. Res. in press). Using an in vivolin vitro survival assay, performed 24 hours after -Frxiation with 250 kV X-rays, we found tGt?-Targe single dose of nicotinamide (1000 mg/Kg) injected 1-2 hours prior to irradiation produces enhancement ratios (ER's) of 1.4-1.7 in 3 tumor models (EMT6, Lewis Lung and RIF-1). For the EMT6 tumor these ER's were dependent on the administered nicotinamide dose, but even at doses as low as 25% of the LD50 value (LD50 = 2000 mg/Kg) an ER greater than 1.5 could be observed. In 2 normal tissue assays (jejunal crypt cell survival and mean skin reaction) ER's of less than 1.2 were obtained using 1000 mg/Kg nicotinamide. In the present study, we have attempted to understand the mechanism for this preferential tumor radiosensitization. Our data suggest it is not a direct effect of nicotinamide or its metabolites on the tumor cells. First, we have found no radiosensitization of hypoxic or aerobic EMT6 cell in vitro with concentrations of nicotinamide up to 10 mM (greater than measured tumor concentrations foTo_a dose of 1000 mg/Kg). Second, if a clamp is placed around the tumors 90 minutes after injecting nicotinamide and immediately prior to irradiation, no radiosensitization of the hypoxic tumors is observed. Tumor clamping does not affect the extent of radiosensitization with the electron-affinic radiosensitizer SR 2508. Third, no tumor radiosensitization is seen with the major in vivo metabolite nicotinic acid. These findings suggest an indirect effect of nicotinamide, and 0% studies suggest that radiosensitization is a consequence of improved tumor oxygenation and blood flow. The oxygenation status of tumors after nicotinamide treatment was measured using the binding of I4C-misonidazole (I4C-MISO) to tumor cells, since a good correlation exists between tumor radioresistance produced by hypoxia and 14C-MISO binding. Nicotinamide (1000 mg/Kg) reduces the binding of I4C-MISO in the EMT6 tumor by as much as 8056, suggesting that nicotinamide increases tumor oxygenation. We have also measured tumor blood flow following nicotinamide administration. With one technique we observed a 40-60% increase in mean tumor cell fluorescence from the fluorescent dye Hoechst 33342 following nicotinamide injection indicating increased tumor perfusion by this i.v. injected drug. We are also in the process of measuring the effect of nicotinamide on tumor blood flow using clearance of I33Xenon injected directly into the tumor. Nicotinamide has been administered to patients with a variety of disorders. A reasonably safe dose associated with few side effects is 6 g daily. This is approximately double the daily dose achievable with SR 2508, and at these concentrations, SR 2508 and nicotinamide produce equivalent tumor radiosensitization in mice. Thus, it would appear that if similar increases in tumor oxygenation and blood flow can be produced in human tumors by nicotinamide, this drug may prove a simple and useful sensitizer in radiotherapy. Supported by Grant CA 15201 from the National Cancer Institute, DHHS.

Relationships between reduced gases, nutrients, and fluorescence in surface waters off Baja California

Temperature, in vivo fluorescence, silicate, phosphate, dissolved hydrogen, and carbon monoxide were measured in surface waters while crossing the California Current system into San Diego. The measurements were made to define surface water variability and to help understand the causes of variations in H 2 and CO concentrations. Striking changes were observed crossing the current system, with the most dramatic effects occuring within 100 km of the coast. In an attempt to separate the effects of different water histories, the data were subdivided according to ‘water type,’ with four types identified on the basis of temperature. Relations between parameters were distinctly different in the different waters. Over the ‘oceanic’ part of the track all variables were relatively constant. Hydrogen was not correlated with CO or in vivo fluorescence except in the warm, coastal water, where the correlation between hydrogen and fluorescence was strong. Carbon monoxide showed a distinct daily cycle when data from different types were examined separately. The mean concentration of CO for each water type covaried with the mean in vivo fluorescence. Likewise the amplitude of daily CO variation was positively correlated with mean fluorescence. This lends support to the hypothesis that the CO abundance is related both to light intensity and to organic content of the water.

FMFPAK1: A program package for routine analysis of single parameter flow microfluorimetric data on a low cost mini-computer

A program package is presented for routine analysis, on a low cost minicomputer, of data obtained by flow microfluorimetry. The programs, with the aid of a graphics capability, provide calculations of various properties of cell populations: percentage of cells in various phases of the cell-cycle (G 1, S, G 2 + M); mean fluorescence per cell; mean narrow-angle scatter per cell, and an approximation to relative mean volume per cell from narrow-angle light scatter measurements. The system is designed to allow either direct analysis or for storage of raw data on tape files for analysis at a later time. Provision is also made for hard copy on a digital x-y plotter.