A recessive cellular mutation in v-fes-transformed mink cells restores contact inhibition and anchorage-dependent growth.

A contact-inhibited revertant of mink cells transformed by the Gardner-Arnstein strain of feline sarcoma virus was isolated by fluorescence-activated sorting of cells stained with the mitochondria-specific dye rhodamine 123. The revertant cell line exhibited a decrease in its proliferative rate and saturation density and a complete loss of its capacity for anchorage-independent growth, but it remained tumorigenic when inoculated into nude mice. The revertant cells retained a rescuable Gardner-Arnstein feline sarcoma provirus, expressed high levels of the v-fes oncogene product and its associated tyrosine kinase activity, manifested elevated levels of phosphotyrosine-containing cellular proteins similar to those observed in v-fes-transformed cells, and were refractory to retransformation by retroviruses containing the v-fes, v-fms, and v-ras oncogenes. Fusion of the revertant and parental cells generated somatic cell hybrids which formed colonies in semisolid medium, indicating that the block in transformation was recessive. These data together with the observation that the revertant phenotype is unstable in continuous culture suggest that the loss of transformation is due to the presence of limiting quantities of a gene product which functions downstream of the v-fes-coded kinase in the mitogenic pathway.     

In archaebacteria, there is a doxorubicin efflux pump similar to mammalian P-glycoprotein.

We selected for study an anthracycline-resistant mutant from the archaebacteria Haloferax volcanii. This resistance was reversed by a Ca(2+)-channel antagonist, nifedipine (NDP). This resistance and its reversal by NDP suggest P-glycoprotein (Pgp) to be responsible for maintaining an anticancer drug concentration below the cytotoxic level. Using rhodamine 123 (RH123) as a substrate for Pgp, we then examined whether the resistance to anthracyclines in this bacteria might involve a Pgp-like anthracycline efflux pump. RH123 accumulation by the bacteria was determined with flow cytometry. A steady-state RH123 accumulation by the resistant cells revealed approx. one-fifteenth of that by the wild-type cells, which could be remarkably enhanced by NDP. The other modulators of Pgp, diltiazem and verapamil, also enhanced RH123 accumulation in resistant cells. The uncoupler FCCP completely restored RH123 accumulation in resistant cells to the wild-type cell level. RH123 unidirectional efflux from resistant cells after its preloading revealed much greater than that from wild-type cells, which was remarkably inhibited by FCCP. These confirmed that RH123 low accumulation involves its active efflux mechanism. Taken together, the present study indicated that lower evolutionary archaebacteria might also express a Pgp-like protein very similar to mammalian Pgp.     

In archaebacteria,there is a doxorubicin efflux pump similar to mammalian P-glycoprotein

We selected for study an anthracycline-resistant mutant from the archaebacteria Haloferax volcanii. This resistance was reversed by a Ca²⁺-channel antagonist, nifedipine (NDP). This resistance and its reversal by NDP suggest P-glycoprotein (Pgp) to be responsible for maintaining an anticancer drug concentration below the cytotoxic level. Using rhodamine 123 (RH123) as a substrate for Pgp, we then examined whether the resistance to anthracyclines in this bacteria might involve a Pgp-like anthracycline efflux pump. RH123 accumulation by the bacteria was determined with flow cytometry. A steady-state RH123 accumulation by the resistant cells revealed approx. one-fifteenth of that by the wild-type cells, which could be remarkably enhanced by NDP. The other modulators of Pgp, diltiazem and verapamil, also enhanced RH123 accumulation in resistant cells. The uncoupler FCCP completely restored RH123 accumulation in resistant cells to the wild-type cell level. RH123 unidirectional efflux from resistant cells after its preloading revealed much greater than that from wild-type cells, which was remarkably inhibited by FCCP. These confirmed that RH123 low accumulation involves its active efflux mechanism. Taken together, the present study indicated that lower evolutionary archaebacteria might also express a Pgp-like protein very similar to mammalian Pgp.     

Species-specific differences in the toxicity of rhodamine 123 towards cultured mammalian cells

The toxicity of cationic fluorescent dye, rhodamine 123, towards a number of independently established cell lines from three different species, namely human, mouse, and Chinese hamster, has been examined. All of the cell lines from any one species that were examined were found to exhibit similar sensitivities towards rhodamine 123 and no appreciable differences were observed between the normal and transformed cell types. However, in comparison to the cells of human origin, mouse and Chinese hamster cell lines exhibited about 10-fold and 70-fold higher resistance, respectively, and these differences appeared to be species related. In contrast to rhodamine 123, no differences in relative toxicities for these cell lines were observed for the structurally related neutral dye, rhodamine B. Fluorescence studies with rhodamine 123 show that in comparison to mouse and Chinese hamster cells, the more sensitive human cells show much higher uptake/binding of the drug, and a good correlation was seen in these studies between the extent of dye uptake/binding and the relative sensitivities of cell lines to rhodamine 123. These results provide evidence that the observed species-related differences in cellular toxicities are due to differences in the cellular uptake/binding of the dye.     

Isolation of spleen-colony forming cells (CFU-s) using wheat germ agglutinin and rhodamine 123 labeling

Mouse pluripotent hemopoietic stem cells could be enriched 100 to 200-fold by a procedure consisting of three steps: 1) equilibrium density centrifugation, 2) light-activated cell sorting on the basis of light scatter characteristics and fluorescence due to wheat germ agglutinin binding, 3) cell sorting after subsequent rhodamine 123 staining. The new isolation procedure does not make use of antibodies with mouse-strain restricted applicability, which were employed in earlier described methods. Therefore, it is more versatile. It is also faster due to diminished incubation time. Rhodamine 123 can also be used as a photosensitizer. The experimental conditions were, however, designed to prevent this action of the dye. Between 80% and 100% of the selected spleen-colony forming cells survived the labeling and sorting treatments. The procedure enriches for two types of stem cells. The rhodamine-dull fraction contains stem cells that form spleen colonies in lethally irradiated mice at 12-16 days and no spleen colonies at 8 days after transplantation. The rhodamine-bright fraction contains stem cells that give day-8 and day-12 spleen colonies. These latter cells, however, have a low radioprotective capacity and it can be argued that these are not self-renewing pluripotent stem cells. The heterogeneity of day-12 CFU-s (colony-forming unit spleen) that can be detected after labeling with rhodamine 123 has been observed earlier after treatment of bone marrow donor mice with 5-fluorouracil, and has led to the postulation of pre-CFU-s and a "generation-age" hypothesis for stem cells. Our presently sorted rhodamine dull cells resemble such pre-CFU-s.     

Functional impairment induced by lipophilic cationic compounds on mitochondria

Lipophilic cations, such as rhodamine 123, have selective anticarcinoma activity both in epithelial-derived tumor cells and in tumor cells injected into mice. The mechanism by which rhodamine 123 and safranin have their effect on mitochondrial function was examined. Rhodamine 123 and safranin inhibit the stimulation of mitochondrial respiration by ADP in a similar concentration range. This inhibition occurs whether the mitochondria are respiring on succinate as a substrate or on ascorbate plus tetramethylphenylenediamine. ATP hydrolysis was stimulated twofold by high lipophilic cation concentration. These results demonstrate that rhodamine 123 and safranin affect oxidative phosphorylation in a similar fashion.     

Impact of proliferative activity and tumorigenic conversion on mitochondrial function of fibroblasts in 2D and 3D culture

The purpose of the present study was to examine mitochondrial function in differently transformed cells relative to their tumorigenic state and proliferative activity in vitro. An established two-step carcinogenesis model consisting of immortal and tumorigenic rat embryo fibroblasts that can be cultured as monolayers and multicellular spheroids was investigated. Flow cytometric measurements were carried out using the two mitochondrial-specific fluorochromes rhodamine 123 (Rh123) and 10-N-nonyl acridine orange (NAO), in combination with the DNA dye Hoechst 33342 for simultaneous cell cycle analysis. Since the accumulation of Rh123 depends on mitochondrial membrane potential, Rh123 fluorescence intensity gives an estimate of mitochondrial activity per cell, as determined by both overall mitochondrial function and mass. In contrast, NAO uptake reflects mitochondrial mass only, as it binds to cardiolipin in the inner mitochondrial membrane independently of membrane potential. Aliquots of cell suspensions derived from exponential monolayer, confluent monolayer, and a range of sizes of multicellular spheroids were stained with either Rh123 or NAO and Hoechst 33342, then mitochondrial mass and activity per unit cell volume and cellular DNA content were measured by flow cytometry. Differences in the average mitochondrial activity per cell in different cell lines and culture conditions were primarily due to alterations in cell volume. Importantly, tumorigenic conversion by ras-transfection did not consistently change mitochondrial activity per unit cell volume. The mitochondrial mass per unit cell volume increased for all cells when cellular quiescence was induced, either in monolayers or spheroids. However, mitochondrial function (activity/mass) decreased when cells became quiescent, resulting in a positive correlation between mitochondrial function and S-phase fraction, independent of transformation status or culture condition. We conclude that mitochondrial function reflects proliferative activity rather than tumorigenic conversion.     

Rhodamine 123 inhibits bioenergetic function in isolated rat liver mitochondria

Rhodamine 123 accumulates in the mitochondria of living cells and exhibits selective anticarcinoma activity. The biochemical basis of toxicity was investigated by testing the effect of the dye on isolated rat liver mitochondria. Much lower concentrations of rhodamine 123 were required to inhibit ADP-stimulated respiration and ATP synthesis in well-coupled energized mitochondria than were required to inhibit uncoupled respiration and uncoupler-stimulated ATP hydrolysis. The amount of rhodamine 123 associated with the mitochondria was several-fold greater under energized as compared to non-energized conditions, which may explain why coupled functions appeared to be more sensitive than uncoupled functions to inhibition at low concentrations of rhodamine 123. It was concluded that the site of rhodamine 123 inhibition is most likely the F0F1 ATPase complex and possibly electron transfer reactions as well.     

Metabolic toxicity of fluorescent stains on thawed cryopreserved bovine sperm cells

Several fluorescent probes, including derivatives of carboxyfluorescein, carbocyanine, ethidium, and rhodamine, have been used to assess sperm viability. However, the effects of these fluorescent dyes on the metabolic activity of sperm cells have not been systematically examined. This study was conducted to determine the effect of specific fluorescent stains on the metabolic processes of sperm. Cryopreserved bovine sperm cells were thawed, fluorescently stained, and examined using metabolic and flow cytometric techniques. Sperm were stained with either rhodamine 123 (Rhod-123), the aliphatic cell-tracking compound PKH2-GL, dihydro-ethidium (HED), the bisbenzimide stain Hoechst 33342 (Ho33342), or left unstained. The stained samples were compared for metabolic activity, cell staining pattern, and fluorescent intensity over a 180-min period. Samples stained with HED, Ho33342, and PKH2-GL had less oxygen uptake when compared with the unstained sperm samples (p greater than 0.05). Unstained samples and samples stained with Rhod-123 had similar oxygen consumption. The carbon dioxide produced during the 180 min was not different between controls and stained samples. Therefore, some fluorescent probes inhibit the oxygen metabolism of thawed, cryopreserved bovine sperm cells.     

Identification of cells with lowered sensitivity to cytostatic agents by the use of fluorescent dyes

With a help of stepwise increase of vincristine concentrations in culture medium several lines of mouse myeloma X63 Ag 8.863 cells resistant to low concentrations of vincristine (6-35-fold) were selected. Rhodamine 123 stained resistant cells and wild-type cells with an equal intensity. However, resistant cells differ significantly from the sensitive ones by the rate of rhodamine efflux. The rate of the efflux was in proportion to the degree of resistance. The efflux of the dye could be blocked by the addition to reserpine, the inhibitor of multidrug resistance. Thus, fluorescent dyes can be used for the detection of cells with low levels of multidrug resistance.     

Sensitivity of Ewing's sarcoma to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to kill transformed cells. We have studied the expression and functionality of the TRAIL apoptotic pathway in Ewing's sarcoma. We demonstrate that tumors from patients with Ewing's sarcoma express receptors TRAIL-R1 and -R2. Using a panel of nine Ewing's sarcoma cell lines TRAIL could induce apoptosis in seven cell lines. Preincubation with interferon-gamma rendered the two resistant cell lines sensitive. TRAIL was the most potent inducer of apoptosis when compared to Fas ligand or TNF. TRAIL-mediated apoptosis could be inhibited by various caspase-inhibitors. No difference in the surface expression of TRAIL-receptors was observed between sensitive and resistant cell lines. Also, all cell lines had similar levels of expression of Flice-like inhibitory protein (FLIP) on immunoblot. However, the two resistant cell lines had only very low level expression of caspase 8 on RNA and protein level. In summary, we show that Ewing's sarcoma expresses receptors for TRAIL, and that cells are exquisitely sensitive to TRAIL-mediated apoptosis. These results may warrant clinical trials with TRAIL in Ewing's sarcoma once the safety of TRAIL for humans has been established.     

Some effects of chlorambucil on nuclear protein phosphorylation in the yoshida ascites sarcoma

The ability of nuclei, isolated from Yoshida ascites sarcoma cells, to phosphorylate nuclear proteins in the presence of [γ-³²P] ATP has been investigated. Comparisons were made between a strain sensitive to the effects of the alkylating agent, chlorambucil, with a corresponding resistant strain both before and after drug-treatment of tumour-bearing animals.There was no gross quantitative differences between the drug-sensitive and -resistant untreated cells but treatment resulted in increased levels in the sensitive strain.Qualitative differences were seen before treatment in the phosphorylation pattern of the tris-saline-soluble nuclear sap fraction. The high molecular weight species in the fraction from sensitive cells showed phosphorylations which were absent, or present at very low levels, in the corresponding fraction from drug-resistant cells.Changes were observed in the tris-saline-soluble and non-histone protein fractions from drug-sensitive cells following treatment of tumour-bearing animals. Only minor alterations in patterns of phosphorylation were seen in fractions from drug-resistant cells.     

Cholesterol modulates P-glycoprotein activity in human peripheral blood mononuclear cells

P-glycoprotein (P-gp) is expressed in a wide range of cell types including peripheral blood mononuclear cells (PBMCs) where it may restrict intracellular accumulation of substrates like antineoplastic agents, HIV protease inhibitors, or rhodamine123. P-gp is known to be located in membrane microdomains, whose structure and function are susceptible to cholesterol alterations. This study evaluated the effect of cholesterol alteration in human PBMCs on P-gp activity. Whereas cholesterol depletion had no effect, cholesterol repletion of depleted cells significantly decreased intracellular rhodamine123 concentrations in lymphocytes to 32.2%+/-2.7 (p<0.001) and to 41.9%+/-3.5 (p<0.001) in monocytes. After cholesterol saturation of native cells intracellular rhodamine123 fluorescence decreased to 12.4%+/-1.6 (p<0.001) in lymphocytes and 12.9%+/-3.5 (p<0.001) in monocytes. These data demonstrate that elevated cellular cholesterol levels can markedly increase P-gp activity in human PBMCs.     

Compositions and Anti-Tumor Activity of Pyropolyporus fomentarius Petroleum Ether Fraction In Vitro and In Vivo

The chemical compositions and anti-tumor activities of the petroleum ether fraction (PE), from mushroom Pyropolyporus fomentarius, were studied. Upon gas chromatography–mass spectrometry (GC–MS) analysis, nine major constituents were identified in the fraction. In vitro, the PE showed cytotoxic activity against murine sarcoma S180 (S180) cells in a dose- and time-dependent manner, and the cytotoxic effects were associated with apoptosis. The mitochondrial membrane potential loss and the intracellular ROS generation were greatly increased in the Pyropolyporus fomentarius PE treated group, suggesting cell apoptosis, induced by the PE in S180 cells, might be mitochondria dependent and ROS mediated. Consistent with in vitro findings, the in vivo study showed that the Pyropolyporus fomentarius PE was also effective in inhibiting the tumor growth induced by S180 cells and had lower immune organ toxicity. We found that the Pyropolyporus fomentarius PE has significant anti-tumor activity and great potential in screening anti-tumor drugs.     

Cytotoxic and antitumor activity of a soluble fraction of Streptococcus pyogenes against S180 sarcoma cells

A fraction (60F) having cytotoxic and antitumor activities was obtained from cell-free extract of group A Streptococcus pyogenes by precipitating with 50% to 60% saturated ammonium sulfate. 60F showed cytotoxic activity inhibiting the uptake of 3H-thymidine by S180 sarcoma cells and enhancing 51Cr-release from 51Cr-labeled cells. 60F showed also antitumor activity, depressing tumor growth and prolonging lives of mice bearing S180 sarcoma cells.     

Characterization of a membrane-associated protein kinase of multidrug-resistant HL60 cells which phosphorylates P-glycoprotein

Cells containing increased levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug-resistant phenotype. In the present study we have analyzed protein kinases capable of phosphorylating P-glycoprotein in membranes of HL60 cells isolated for resistance to vincristine. Analysis of this system demonstrates that in isolated membranes the protein kinase inhibitor staurosporine greatly reduces P-glycoprotein phosphorylation. In contrast, the kinase inhibitor H-7 does not affect this reaction. Fractionation of solubilized membrane proteins from sensitive and resistant cells on DEAE-cellulose reveals a major protein kinase (PK-1) which exhibits optimal activity in the presence of Mn2+ and histone H1. This enzyme fraction does not contain detectable levels of protein kinase C or cAMP-dependent protein kinase. PK-1 phosphorylation of two endogenous proteins is, however, greatly enhanced in the presence of phosphatidylserine or phosphatidyl-inositol. In reaction mixtures containing Mg2+ or Mn2+ in the absence of phospholipid, PK-1 from resistant cells phosphorylates an endogenous protein of 180 kilodaltons (P180), which exhibits an electrophoretic mobility identical to P-glycoprotein. In parallel experiments with PK-1 from sensitive cells there is no detectable phosphorylation of a P180 protein. P180 phosphorylated by PK-1 from resistant cells is immunoprecipitated by antibody against P-glycoprotein. Additional studies demonstrate that PK-1 is capable of phosphorylating specific synthetic peptides which correspond to the sequence of P-glycoprotein. Peptide phosphorylation occurs at both serine and threonine residues. These studies thus identify a novel membrane-associated protein kinase in HL60 cells which is capable of phosphorylating P-glycoprotein. This enzyme may have an important role in regulating levels of multidrug resistance.     

Evidence of multifactorial mechanisms in an adriamycin-resistant HL-60 promyelocytic leukemia cell line

HL-60/AR leukemia cells, which were 60-fold resistant to the growth inhibitory activity of adriamycin, remained sensitive to the antiproliferative and differentiation-inducing activities of aclacinomycin A. The replication of HL-60/AR and of adriamycin sensitive parental HL-60 cells was inhibited by greater than 80% by 30 nM aclacinomycin A and the majority of cells (about 60 to 70%) of each line underwent granulocytic differentiation when treated with this agent, as assessed by the reduction of nitroblue tetrazolium. Measurement of the initial rates of uptake of daunorubicin and steady-state levels of adriamycin in sensitive and resistant lines indicated that transport differences do not fully account for the insensitivity of HL-60/AR cells to these anthracyclines. Furthermore, 30-fold greater levels of cell-associated adriamycin were required in HL-60/AR cells for toxic effects equivalent to those occurring in parental HL-60 cells. Analysis of DNA histograms of adriamycin treated HL-60 cells indicated that cell-cycle progression was blocked in G2-M, while this antibiotic blocked progression of resistant HL-60/AR cells in the S phase. These results suggest that, in addition to alterations in membrane permeability, differential sensitivity of multiple biochemical targets may be important in the toxicity and the development of resistance to anthracyclines. Furthermore, the finding that HL-60/AR cells do not exhibit cross-resistance to aclacinomycin A indicates that this oligosaccharide-containing anthracycline may have utility in the treatment of adriamycin resistant neoplasms.     

Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug selection

Multidrug-resistant (MDR) cells demonstrate the increased activity of the membrane transport system performing efflux of diverse lipophylic drugs and fluorescent dyes from the cells. In order to detect MDR cells we have developed a simple test consisting of three steps: staining of the cells with fluorescent dye rhodamine 123, incubation in the dye-free medium and, finally, detection by fluorescence microscopy of the cells that have lost accumulated dye. The experiments with B-lymphoma cell lines with different degrees of MDR have shown that the cell fluorescence after the poststaining incubation is indeed inversely proportional to the degree of resistance. Application of this testing procedure to normal human or mouse leukocytes revealed the presence of the cells rapidly losing the dye in these populations. Cell fractionation experiments have shown that there are T-lymphocytes (most T-killers/suppressors and a part of T-helpers) that demonstrate rapid efflux of rhodamine 123. This characteristic was detected also in T-killer clones and cell line and in some T-lymphomas. The inhibitors of the MDR transport system, reserpine and verapamil, blocked the efflux of the dye from these cells. Rhodamine-losing T-lymphoma contained large amounts of the mRNA coding P-glycoprotein, the MDR efflux pump, and demonstrated increased resistance to rhodamine 123, gramicidin D, colchicine, and vincristine, the drugs belonging to the cross-resistance group for the MDR cells. The role of the increased activity of the MDR membrane transport system in T-lymphocytes is discussed.     

Rhodamine 123 as a probe of in vitro toxicity in MDCK cells

The effect of mercuric chloride on Madin-Darby Canine Kidney (MDCK) cells grown in culture was assayed by the mitochondrial-specific fluorescent probe, rhodamine 123. Treatment of cells with mercuric chloride resulted in a dissipation of rhodamine fluorescence from the mitochondria into the cytoplasm, followed by a release into the medium bathing the cells. Toxicity was assayed either by determining the proportion of cells with delocalized rhodamine fluorescence, or by measuring the rhodamine released from or retained in the cells. Quantifying the release or retention of rhodamine 123 is semi-automated and represents a highly sensitive method of using a vital fluorescent dye for in vitro toxicity analysis.