Glucocorticoid receptors and cortico-sensitivity in a human clonal monocytic cell line, CM-SM

CM-SM is a clonal line of human precursor mononuclear phagocytes inducible to macrophage differentiation in response to the tumor promoter phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Untreated CM-SM cells contain single class, high-affinity (KD = 4.0 X 10(-9) M) glucocorticoid-specific receptor sites (approximately 60,000 per cell), as measured by a whole cell assay, at 37 degrees C, using [3H]triamcinolone acetonide (TA). Exposure of CM-SM to dexamethasone (DEX) produced a progressive, dose- and time-related series of changes in CM-SM cell growth, saturation density, morphology, and functional properties, with half-maximal effects at about 10(-9) M for DEX. TA-receptor sites rapidly decreased (about 70%) after DEX treatment, without any apparent change in steroid specificity and affinity. After 5 days in culture with a saturating concentration (3.6 X 10(-8) M) of hormone, the cells reached a saturation density of about 9.0 X 10(6) viable cells/ml (about 4.0 X 10(6) viable cells/ml in the controls), while the modal volume of the resulting cell population was approximately 60%, as compared to the volume of untreated cells. DEX-treated cells appeared less differentiated than controls, as assessed by combined morphologic, antigenic, and cytoenzymatic analyses. DEX almost completely inhibited TPA activation of the following macrophage functions: adherency to the culture plate, expression of lysosomal enzymes, Fc and C3 receptors, and stimulation of phagocytosis. After removal of DEX, the cells, within a few passages, returned to a state apparently identical to the untreated controls and could be induced to macrophage differentiation in response to TPA.     

Culture of epithelial and stromal cells of guinea-pig endometrium and the effect of oestradiol-17 beta on the epithelial cells

Epithelial and stromal cells of guinea-pig endometrium were separated by enzymic digestion, isolated by successive centrifugation, and maintained in culture as pure cell types for 5 days on growth medium. On Day 5, ultrastructural studies were performed on the two cell types, demonstrating that epithelial cells can grow as a monolayer composed of cohesive groups of polygonal cells (1.3 X 10(5) cells/cm2), while stromal cells were mostly fibroblastic. The effect of hormones was studied on the epithelial cells in culture. The monolayer was cultured into harvest medium for 3 days to ensure the complete removal of endogenous steroids, then these cells were incubated with 2 X 10(-9) M-oestradiol-17 beta for 3 days. There was a rise in the progesterone receptor level, varying from 1.3 to 10.8 times. The three enzymes known to interfere with oestradiol-17 beta metabolism were present in the epithelial cells grown in our culture conditions. By incubation with oestrone sulphate for 3 days it was demonstrated that, in cultured epithelial cells, oestrone sulphate is converted into oestradiol-17 beta sulphate, and oestrogen sulphates are hydrolysed to active oestrogens.     

The enumeration of Leptospira interrogans serovar pomona by a bioluminescence ATP assay

A rapid method for enumerating viable Leptospira interrogans serovar pomona cells was investigated using a bacterial adenosine triphosphate (ATP) assay. The ATP was assayed by the luciferin-luciferase bioluminescence reaction. Samples of serovar pomona grown in liquid polysorbate 80-bovine albumin (P80-BA) medium for 1-3 days were analysed for ATP content, culture density (nephelometry), direct cell count and most probable number of viable cells (MPNVC) as determined by the dilution tube technique. A linear relationship was found between ATP content and the number of viable cells over the range of 4 X 10(8) to 8 X 10(9) leptospires/ml. Over this range the correlation coefficient for ATP content versus viable cells (0.96) was similar to the coefficient for culture density versus the number of viable cells. The coefficient for direct counts versus the number of viable cells was smaller. The bioluminescence assay of bacterial ATP is a promising method for enumerating viable leptospires in pure culture.     

Determination of the number of endothelial cells in culture using an acid phosphatase assay

A simple assay is described in which small numbers of endothelial cells in culture can be determined by measuring acid phosphatase activity. After removal of the growth medium from cells grown in 96-well culture plates, the cells are lysed in buffer containing the detergent Triton X-100 and the phosphatase substrate p-nitrophenyl phosphate. After 2 h at 37 degrees C, the reaction is stopped with sodium hydroxide, and color development is determined using a rapid multiwell plate reader. The assay detects 100 to 10,000 cells per well. The assay has been used to determine growth curves for endothelial cells in the presence and absence of endothelial cell growth factor from bovine hypothalamus and to monitor fractions during purification of the growth factor. Minor modifications in the assay allow it to be fully automated.     

Effect of thrombopoietin on in vitro production of megakaryocytes from fetal mouse liver cells

Plasma clots containing fetal mouse liver cells (FMLC) were used to study the effects of a thrombocytopoiesis-stimulating factor (TSF) from kidney cell culture medium on the proliferation and maturation of megakarocytes. Cells in the megakaryocytic series were identified by the presence of acetylcholinesterase (AChE) and by their morphological and ultrastructural characteristics. For these experiments, 1 X 10(3) to 1 X 10(5) FMLC were cultured for 1-7 days with 0-5 micrograms of TSF; control cultures were treated with production medium (PMC) in which kidney cells had not been grown. The number of AChE+ cells that were observed depended upon the number of cells plated, i.e., after 6 days of culture with 5 micrograms of TSF, an average of 187 AChE+ cells was found after plating 1 X 10(4) cells and 1020 AChE+ cells were observed after plating 1 X 10(5) cells. In dose-response experiments, the number of AChE+ cells rose with increasing doses of TSF. Significantly elevated numbers of AChE+ cells were observed after the addition of 1-5 micrograms of TSF. The optimum time of culture, based upon the number of AChE+ cells found, was 3-5 days. Ultrastructural analysis of megakaryocytes in plasma clots showed evidence of platelet shedding on Day 5. After the culture of FMLC with TSF, a larger number of AChE+ cells was formed from a given number of cells plated than in previous studies that used adult bone marrow cells. Therefore, because of its greater sensitivity, FMLC may be useful for the assay of low levels of TSF, and may be a valuable tool for studying the effects of megakaryocytic regulatory factors on megakaryocytopoiesis.     

Comparative studies of the binding and growth-supportive ability of mammalian transferrins in human cells

The ability of human-derived cells in culture to bind, remove iron from, and grow in the presence of transferrins (Tf) isolated from the sera of species commonly included in tissue culture medium was investigated. Kinetic studies on HeLa cells reveal apparent first-order association rate constants of 0.43 min-1 for human Tf and 0.15 min-1 for equine Tf. Labeled chicken ovo-Tf and fetal bovine Tf were not recognized by the HeLa cells. Competition experiments with HeLa cells that use either isolated Tf or parent serum confirm these findings. Equilibrium binding experiments performed on HeLa cells at 37 degrees C in the presence of 2,4-dinitrophenol to prevent iron removal indicate 1 X 10(6) Tf bound/cell with a dissociation constant (K'D) of 28 nM for human Tf and 182 nM for equine Tf. Equilibrium binding performed at 0 degrees C to prevent endocytosis reveals 4.1-6.7 X 10(5) Tf binding sites/cell with a K'D of 8.3 nM for human Tf and 41.5 nM for equine Tf. Parallel experiments in normal human diploid fibroblast-like MRC-5 cells indicate expression of 0.82-2.78 X 10(5) Tf binding sites/cell with a K'D of 8.2 nM for human and 39.1 nM for equine Tf. Thus, the results of equilibrium binding studies of a more differentiated cell type are consistent with those found for HeLa cells. Fetal bovine Tf was found to compete weakly with labeled human Tf for human receptor on HeLa cells in a soluble receptor assay, with an approximately 500-fold excess needed to reduce binding to half maximal. Iron uptake experiments show an iron donating hierarchy where human greater than horse greater than calf, suggesting that the rate of iron uptake depends on the affinity of receptor for transferrin. Growth experiments involving HeLa cells in chemically defined serum-free medium demonstrate that bovine Tf will support growth as well as human Tf, but at concentrations much higher than are required of human Tf.     

Maintenance of growth transformation with Epstein-Barr virus is mediated by secretion of autocrine growth factors in two serum-free B-cell lines.

The characteristics of two tamarin (Saguinus oedipus) B-cell lines (sfBIT and sfBT) growth-transformed by Epstein-Barr virus (EBV) that proliferate continuously in serum-free medium are described. sfBIT was established by selecting cells for growth in RPMI 1640 supplemented with insulin, transferrin, and selenium (J. E. Shaw, R. G. Petit, and K. Leung, J. Virol. 61:4033-4037, 1987). sfBT, a subline of sfBIT cells reported here for the first time, required transferrin as the only protein supplement for continuous growth in RPMI 1640. Growth of sfBT cells was linear with human transferrin at 10(-2) to 10 micrograms/ml. Transferrin at 5 micrograms/ml yielded a culture density of 5 X 10(5) to 1 X 10(6) cells per ml, a cell doubling time of 2 to 3 days, and a culture viability greater than 95%. sfBIT and sfBT cells released transforming virus during continuous growth in serum-free culture medium without EBV-inducing agents. The spent medium of both serum-free lines supported cell growth at low culture density (1 x 10(4) to 5 X 10(4) cells per ml), but growth was arrested at low culture density with fresh serum-free medium. A procedure to measure growth-promoting activity (GPA) was established, and it revealed that the GPA of spent medium was greater than that of fresh medium for both serum-free cell lines. When fresh and spent media were dialyzed (molecular weight cutoff, 3,500) and subsequently concentrated by lyophilization, only the GPA of spent medium increased. We conclude that maintenance of growth transformation of tamarin cells latently infected with EBV is mediated by growth factors that are entirely autocrine in origin.     

A rapid micro method for counting cells “in situ” using a fluorogenic alkaline phosphatase enzyme assay

Summary A new method has been developed to count cells “in situ”, based on a fluorogenic enzyme assay that measures the activity of alkaline phosphatase. Increasing cell number was shown to correlate closely with alkaline phosphatase activity and this relationship did not change with time in culture. The alkaline phosphatase assay (ALP assay) was able to estimate relative cell numbers over a range from about 10⁴ to 5×10⁵ for many cell types, including Hep-2, a derivative of HeLa, several human colorectal cell lines SW1222, SW837, LS174T and HT29, a normal human diploid cell strain MRC5 and a rodent line NIH-3T3. The ALP assay is rapid and efficient, making it a useful method for studying growth assays. Editor's Statement This paper describes a quick method for quantitation of cell number in microcultures. Such procedures are valuable for the many situations in which minimizing cells and medium volume is desirable, although somewhat specialized equipment is required for the procedure. An alternative procedure for quantitation of cells in microtiter culture appeared previously in this journal (McCaffrey, et al., 24∶247–252).     

Microcalorimetric measurements of tissue cells in vitro

A culture flask was designed for the microcalorimetric measurements of tissue cells by an MS 80 standard calvet microcalorimeter. Tissue cells cultured in this flask behaved in the same manner as in the common culture flask used in cytobiological studies. The thermograms of human adenocarcinoma gastric cells (SGc 7901) and HeLa cells were obtained. The heat output power of SGc 7901 cells continuously increased for 70 h with an initial cell number of 3.0 X 10(5). The thermogram was reproducible under strictly controlled conditions. The relationship between the heat output power and the number of SGc 7901 cells within 48 h was obtained. The heat output power was 40 pW/cell to 49 pW/cell when the cell number was in the range 4.5 X 10(5) to 10.4 X 10(5). It was 62.3 +/- 2.9 pW/cell for HeLa cells when the cell number was 6 X 10(5).     

Selectivity of hemoregulatory peptide (HP5b) action in culture

A synthetic analog of a hemoregulatory peptide associated with mature human granulocytes (HP5b) has been investigated for inhibitory effects on various cell types in culture as compared to inhibitory action on mouse and human myelopoietic colonies (CFU-gm), which occurs from 1 X 10(-13) to 1 X 10(-6) M in vitro. This includes colony formation by lymphoid T and B cells in capillary cultures, as well as mitogen activation of T, B and NK cells. At higher concentrations, i.e., above 1 X 10(-7) M, an inhibitory effect was found on colony formation. Neither the production of interleukin (IL) 3 by mitogen-activated T cells, nor the proliferation of the IL-3-dependent L/B cell line were affected by the peptide up to 1 X 10(-5) M. A slight inhibitory effect was found above 1 X 10(-9) M on mouse 3T3 fibroblasts. A series of malignant cell lines was also tested. No effect was seen between 1 X 10(-11) and 1 X 10(-7) M on human mammary carcinoma cells in culture. On Ehrlich ascites mouse mammary carcinoma cells a 30% inhibition was seen at 10(-6) M. On a human glioblastoma cell line (GaMg) no effect was seen, and on a rat glioma cell line (BT5C) an inhibitory effect was seen at 1 X 10(-7) M and above. No significant inhibition of cell growth was seen on SC1 mouse lymphoma cells from 1 X 10(-9) to 1 X 10(-5) M during 7 days of culture. The investigated normal and malignant cell types in culture were thus not inhibited in very low concentrations which act on CFU-gm. However, a variable inhibitory effect was found at higher concentrations where the inhibition of myelopoiesis was maximal and at concentrations where the inhibition is released. The hemoregulatory peptide thus seems to be a concentration-dependent selective inhibitor of myelopoiesis. The finding that various malignant cells do not respond at lower concentrations supports the possibility of using the peptide as a protector of normal cells during cancer chemotherapy.     

Enumeration of 6-thioguanine-resistant tumour cells using flow cytometry and comparison with a microtitration cloning assay

Measurement of mutant frequency in tumour specimens has been hampered by low cloning efficiency in soft agar. A method was developed to detect cell proliferation using the thymidine analogue 5-bromo-2'-deoxyuridine (BrUdR). BrUdR incorporation was monitored by immunofluorescent staining of fixed cells using a monoclonal antibody highly specific for this nucleoside analogue. The 6-thioguanine (6TG) exposure conditions which inhibited DNA synthesis, as measured by BrUdR incorporation, in wild-type cells while allowing proliferation of spontaneous hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutants were investigated using tumour cell lines. It was shown that exposure to 10(-5) M BrUdR for the equivalent of 1 cell cycle time did not affect growth of wild-type cells, nor did it affect the growth of HPRT- mutants in the presence of 6TG. Methods for rapid flow cytometric enumeration of BrUdR-labelled 6TG-resistant cells were developed using fluorescent microspheres as an internal standard. To validate the BrUdR mutation assay, the 6TG mutant frequency (MF) was measured in L1210 R/S, a mouse leukaemic cell line (BrUdR 6TG MF = 7.0 X 10(-5] and the results directly compared with those from a microtitration cloning assay (MF = 4.6 X 10(-5]. The results were similar and within the range reported for HPRT MF in mammalian cells.     

A method to quantify spontaneous and in vivo induced thioguanine-resistant mouse lymphocytes

A clonogenic assay to quantify thioguanine (TG)-resistant (TGr) spleen lymphocytes in the mouse has been developed to support studies of in vivo mutation affecting the hypoxanthine phosphoribosyltransferase (hprt) locus. Lymphocytes are cultured in 96-well microtiter plates for 9 days with proliferation initiated by the mitogen concanavalin A and supported thereafter by conditioned medium containing interleukin-2. Lymphocytes are plated at high densities (4-8 X 10(5)/well) with TG and irradiated L5178Y lymphoma cells (10(4)/well) to detect the presence of TGr cells. To determine the cloning efficiency without TG lymphocytes are plated at a low density (10/well) with irradiated L5178Y cells and irradiated lymphocytes (4-8 X 10(5)/well). Proliferation of cells is detected by [3H]thymidine incorporation and scintillation spectrometry. Spontaneous frequencies of TGr clones are independent of TG dose from 0.2 to 10 micrograms/ml and independent of cell density over the range cited. The TGr clones tested have less than 10% hypoxanthine incorporation in vivo relative to unselected clones and have stable phenotypes in the absence of selection. The spontaneous frequency of TGr cells ranged from 1 to 3 X 10(-6). In vivo treatment of mice intraperitoneally with ethylnitrosourea 15 days prior to in vitro culture resulted in a linear dose-related increase of TGr cells, with 70.2 mg/kg inducing a frequency of TGr cells of 2 X 10(-5).     

雌激素对兔子宫内膜细胞EGF受体和细胞周期的调控

 由受体放射配基结合分析证明家兔子宫内膜细胞的EGF受体Kd值为0.53nmol/L,每个细胞的最大结合容量为1.11×10~4结合位点。10~(-10)mol/L雌二醇处理24h,细胞的最大结合容量增至2.75×10~4结合位点数/细胞,而Kd值无明显变化,可是,当10~(-5)mol/L雌二醇处理24h,细胞的EGF受体结合率,DNA合成速度率均下降。G_0/G_1期细胞比值明显下降,而G_2+M期和S期细胞明显上升。     

Electrochemical treatment of human KB cells in vitro

Electrochemical treatment (ECT) of cancer is a promising new method by which direct current is delivered into tumor tissue to induce tumor regression. The purpose of this study is to evaluate the effectiveness of ECT on human cancer cells and to investigate the factors that affect ECT. The biological mechanisms of ECT in cancer treatment were also explored. Using human KB cells, ECT was found to delay cell growth by using 0.3 coulombs (C)/ml (1.5 C in 5 ml of culture medium; 3 V, 400 μA for 62.5 min). From the results of a colony‐forming assay, it was clearly demonstrated that increasing the ECT dose decreases tumor cell survival. A cytotoxicity study, in which a methylene blue assay was used, determined that, for 2.5 × 10⁵ cells in culture, the ID₅₀ was 0.68 C/ml. For a fixed dose of 0.6 C/ml (3 C in 5 ml), using higher current and shorter treatment time resulted in better cell survival. Time, therefore, is an important factor. When cell concentration was altered, the survival was higher for increased cell concentrations. A thymidine incorporation assay indicated that the amount of [³H]thymidine incorporated into DNA decreased as the ECT dose increased. After treatment with 1 C/ml (5 C in 5 ml; 3 V, 400 μA for 208.4 min), pH at the anode decreased to 4.53 and at the cathode increased to 10.46. These results indicate that ECT is effective for killing human KB cells in vitro and that the toxicity effect is related to charge, current, and treatment time. The effect of pH alteration on cells is one of the mechanisms of ECT. Bioelectromagnetics 20:34–41, 1999.© 1999 Wiley‐Liss, Inc.     

Simultaneous enumeration of T-cell subsets and macrophages in bronchoalveolar lavage fluids by immunoenzyme double staining. Comparison with conventional immunofluorescence

Bronchoalveolar lavage is a common research and clinical tool for the retrieval of cells from the lower respiratory tract. In addition to conventional morphologic study of these cells, the subtyping of T lymphocytes is often important for reaching a diagnosis of a disease or assessing its activity; subtyping is usually done by a standard immunofluorescence assay on cell suspensions requiring about 5 X 10(5) cells. Since the number of leukocytes in the lavage fluid from many patients is too small to obtain reliable information by this assay, a double immunoenzyme staining of T-lymphocyte subtypes on Cytospin preparations was utilized. This method, which requires a small number of cells, was compared with the standard immunofluorescence assay for the identification and quantitation of lymphocyte subtypes in the lavage fluids of patients with different disorders. Although the immunoenzyme double staining assay is somewhat more laborious, it provides important advantages: (1) simultaneous observation of two lymphocyte subsets and macrophages on the same slide; (2) a considerably smaller number of cells (2 X 10(4) instead of 5 X 10(5] is necessary; (3) the availability of permanent preparations; (4) the possibility of storing the Cytospin slides before staining; and (5) conventional light microscopy can be used. Since the reliability of both techniques appeared to be the same, the double staining assay for routine usage with bronchoalveolar lavage fluids appears to be preferable.     

Factors affecting the reproducibility of a spectrofluorimetric assay for the enumeration of human venous endothelium in culture

In an EDTA/Hoechst 33258 assay system, a linear increase in fluorescence with increase in cell number between 2 X 10(3) and 1 X 10(5) was obtained if a dye concentration of 800 ng/ml was used. For a given number of cells, the enhancement of fluorescence was found to be greater than that of a theoretically equivalent of DNA. A standard curve for the assay was derived by plotting enhancement of fluorescence against cell number. The effect of storage on the fluorescence of intact monolayers, cellular or commercial DNA, or dye-DNA complexes made it essential that the assay was carried out on fresh samples.     

Membrane receptors for murine leukemia viruses: characterization using the purified viral envelope glycoprotein, gp71.

The 71,000 dalton glycoprotein (gp71) purified from Rauscher murine leukemia virus (R-MuLV) by affinity chromatography specifically binds to murine but not other mammalian cells in culture. Binding is prevented by specific antiserum raides to gp71 (anti-gp71). The binding assay as described in this report can detect receptors on as few as 300 murine cells, and with 1 X 10(5) cells gives significant binding with 30 sec. The results show that the purified glycoprotein retians biologic activity and can form a stable complex with specific receptors on mouse cell membranes. The assay can therefore be used to characterize the nature of the cellular receptors that are essential for leukemia virus infection. Purified gp71 binding to mouse cells is prevented if the cells are actively producing related ecotropic type C viruses, presumably because the receptors are occupied and are not available to bind exogenously applied gp71. The binding of gp71 to murine cells is enhanced by the presence of calcium ions and low pH. Binding studies performed using an excess of 125I-gp71 indicate the NIH/3T3 cells bind approximately 5.3 X 10(5) molecules of 125I-gp71 per cell.     

Isolation and long-term serial cultivation of endothelial cells from the microvessels of the adult human dermis

A method to isolate and maintain microvascular endothelial cells from the cutaneous vessels of adult human skin in long-term culture has been developed. Endothelial cells lining the microvessels of the papillary dermis are released from surrounding tissue during a brief trypsin incubation (0.3% trypsin, 1% EDTA). Cells are plated onto a fibronectin substrate and maintained in Leibovitz (L15) culture medium containing pooled human serum (50%) and antibiotics. Proliferation is dependent upon the presence of several additional growth factors, cholera enterotoxin (1 X 10(-9) M), isobutyl methylxanthine (3.3 X 10(-5) M), and medium conditioned by explant culture of the mouse EHS sarcoma. Using this supplemented medium, cells proliferate readily and can be cultivated serially for more than 6 passages (3 months in vitro). These cells retain their characteristic endothelial cell morphology, stain positively for Factor VIII antigen, and contain Weibel-Palade bodies.     

Alpha-factor inhibition of the rate of cell passage through the "start" step of cell division in Saccharomyces cerevisiae yeast: estimation of the division delay per alpha-factor.receptor complex

A highly sensitive, kinetically unambiguous assay for alpha-factor-induced delay of cell passage through the "start" step of cell division in yeast is presented. The assay employs perfusion with periodic microscopy to monitor the bud emergence kinetics on the 20% of cells within an exponentially growing population which exist prior to the alpha-factor execution point of start. The t1/2 for cell passage through start by this population of cells is 31 min in the absence of alpha-factor. The inhibition constant, KI, represents the alpha-factor concentration which produces a 50% inhibition of this rate and is equal to 2 X 10(-10) M. A second assay for maximal cell division arrest by alpha-factor on whole populations of cells is presented. This assay shows a maximum cell division arrest time of 125 +/- 5 h at saturating alpha-factor, and a K50 (that is, an alpha-factor concentration which produces a half-maximal response) of 2.5 X 10(-8) M. Both assays were performed in the effective absence of alpha-factor inactivation. Values of the dissociation constant KD and total number of receptors per cell which specifically mediate cell division arrest or delay were estimated to be 2.5 X 10(-8) M and 10(4), respectively. These estimates, along with the quantitative dose-response data for division arrest which are presented here, are consistent with each receptor.alpha-factor complex which is present on the cell at equilibrium producing a 43 +/- 10 s delay of cell passage through start. Surprisingly, this number is constant within twofold over the entire range of cellular division arrest responses to alpha-factor, that is, from a 1.9-fold inhibition of the rate of cell passage through start at 0.17 nM alpha-factor to a 125 +/- 5 h maximum arrest at saturating alpha-factor concentrations of greater than 170 nM. The possible significance of this observation toward the mechanism of alpha-factor-induced cell division arrest is discussed.