Expression of procoagulant activity in a human monocyte-like cell line

Peripheral blood monocytes generate the potent membrane-bound procoagulant TF in response to a number of immune-related stimuli. Although the contribution of the monocyte/macrophage and its soluble mediators to the immune response has been recognized, the role of macrophage procoagulant in the pathogenesis of this response is less certain. Previous studies have suggested that MTF generation is important in the pathogenesis of fibrin deposition in the inflammatory response. In order to pursue this relationship, we have studied the activation of a procoagulant in a human monocyte-like cell line, the U937. The U937 procoagulant has been characterized as TF by the following criteria: the PCA requires factors VII and X for expression; the PCA is not due to serine protease activity; PCA is neutralized by a monospecific antibody to purified bovine TF. The expression of TF in these cells was amplified after stimulation with LPS, a potent activator of peripheral blood MTF expression, and was inhibited by actinomycin D and cycloheximide. Cytosine arabinoside, an inhibitor of cell division, failed to affect U937 TF generation. The U937 cell line appears to be a useful in vitro model for the study of the activation of MTF.     

3H]gemcitabine uptake by nucleoside transporters in a human head and neck squamous carcinoma cell line

Cellular uptake of many chemotherapeutic nucleoside analogs is dependent on the activity of a family of nucleoside transport proteins located in the cell plasma membrane. In the present study, we examined the role of these transporters in the accumulation of gemcitabine by a human head and neck squamous carcinoma cell line. The uptake of [3H]gemcitibine was compared with that of [3H]uridine and [3H]formycin B in the parent cell line (HN-5a) and in a gemcitabine-resistant variant (GEM-8e). The HN-5a and GEM-8e cells were similar in their transport characteristics and expressed predominantly the es (equilibrative, inhibitor-sensitive) transporter subtype; less than 10% of the influx of [3H]formycin B or [3H]uridine was mediated by the ei (equilibrative inhibitor-resistant) system, and there was no evidence for Na+-dependent nucleoside transporters. [3H]Gemcitabine (10 microM) entered these cells via both the es and ei transporters with an initial rate of uptake similar to that seen with the use of [3H]formycin B or [3H]uridine. In addition, ATP-replete cells accumulated significantly less [3H]gemcitabine than did ATP-depleted cells, which is indicative of an active efflux mechanism for gemcitabine. These results show that gemcitabine is a substrate for both the es and ei nucleoside transporters of HN-5a and GEM-8e cells and that gemcitabine resistance of the GEM-8e cells cannot be attributed to changes in transporter activity. Further studies to define the characteristics of the putative efflux mechanism are clearly warranted because this system has the potential to significantly affect the clinical efficacy of gemcitabine.     

地塞米松对人单核细胞糖皮质激素受体蛋白表达的影响

目的研究不同浓度的地塞米松,在不同的刺激时间下对人单核细胞糖皮质激素受体蛋白(GRα,GRβ)表达的影响。方法对培养的人单核细胞株THP1给予不同浓度的地塞米松,在不同的刺激时间下,采用Westernblotting检测细胞中GRα、CRβ蛋白表达。结果人的单核细胞中存在GRQ、GRB受体。GRQ表达量在相同浓度地塞米松刺激下,随刺激时间的延长表现出下调;GRB表达量随地塞米松刺激浓度的增加以及刺激时间的延长而上调。结论GRα表达量随地塞米松刺激时间的延长表现出下调。GRβ表达量随地塞米松浓度的增加、刺激时间的延长而上调,具有一定的剂量和时间依赖性。GRα、GRβ的表达受糖皮质激素的调节。     

alpha(2)-adrenergic receptors stimulate oligopeptide transport in a human intestinal cell line

Di- and tripeptides, as well as peptidomimetic drugs such as cephalexin (CFX), are absorbed by enterocytes via the oligopeptide transporter PepT1. We recently showed that the alpha(2)-adrenergic agonist clonidine increases CFX absorption in anaesthetized rats. Herein, we investigated whether alpha(2)-adrenergic receptors can directly affect PepT1 activity in a clone of the differentiated human intestinal cell line Caco-2 (Caco-2 3B) engineered to stably express alpha(2A)-adrenergic receptors at a density similar to that found in normal mucosa. Measurement of CFX fluxes across cell monolayers cultured on transwell filters demonstrated that the alpha(2)-agonists clonidine and UK14304 caused a 2-fold increase of CFX transport in Caco-2 3B cells, but not in Caco-2 (expressing PepT1 but not alpha(2)-adrenergic receptors) or in the HT29 19A clone (expressing alpha(2)-adrenergic receptors but not PepT1). The stimulatory effect of clonidine was abolished by glycyl-sarcosine (a competitor for the transporter) and blocked by yohimbine or RX821002 (alpha(2)-antagonists). Analysis of the kinetics of CFX transport in control and clonidine-treated Caco-2 3B cells showed that clonidine increased V(max) of CFX transport without changing K(m). Clonidine action was abolished by colchicine but not altered by amiloride, demonstrating that microtubule integrity but not Na(+)/H(+) exchanger activity is necessary for the effect of alpha(2)-agonists to occur. In conclusion, clonidine can directly activate alpha(2)-adrenergic receptors located on epithelial cells. The precise molecular mechanisms whereby these receptors modulate PepT1 activity remain to be elucidated but an increased translocation to the apical membrane of preformed cytoplasmic transporter molecules is likely to be involved.     

In vivo and in vitro induction of human cytochrome P4503A4 by dexamethasone

PURPOSE: The aims of these experiments were to determine the effect of a therapeutic regimen of dexamethasone on cytochrome P4503A4 (CYP3A4) activity in healthy volunteers; and the concentration-effect relationship between dexamethasone and CYP3A4 activity in primary human hepatocyte cultures. METHODS: The effect of dexamethasone (8 mg administered by mouth two times a day for 5 days) on CYP3A4 activity in 12 healthy volunteers was assessed with the erythromycin breath test and urinary ratio of dextromethorphan to 3-methoxymorphinan. Concentration-effect of dexamethasone on CYP3A4-dependent testosterone 6-beta-hydroxylation was determined in human hepatocytes treated with 2 to 250 micromol/L dexamethasone. RESULTS: The percent of erythromycin metabolized per hour increased from 2.20% +/- 0.60% (mean +/- SD) at baseline to 2.67% +/- 0.55% on day 5 of dexamethasone (mean increase in hepatic CYP3A4 activity 25.7% +/- 24.6%; P = .004). The mean urinary ratio of dextromethorphan to 3-methoxymorphinan was 28 (4.8 to 109) and 7 (1 to 23) at baseline and on day 5 of dexamethasone (mean decrease = 49%; P = .06). Substantial intersubject variability was observed in the extent of CYP3A4 induction. The extent of CYP3A4 induction was inversely correlated with baseline erythromycin breath test (r2 = 0.58). In hepatocytes, dexamethasone 2 to 250 micromol/L resulted in an average 1.7-fold to 6.9-fold increase in CYP3A4 activity, respectively. The extent of CYP3A4 induction with dexamethasone in hepatocyte preparations was inversely correlated with baseline activity (r2 = 0.59). CONCLUSIONS: These data demonstrate that dexamethasone at doses used clinically increased CYP3A4 activity with extensive intersubject variability and that the extent of CYP3A4 induction was, in part, predicted by the baseline activity of CYP3A4 in both healthy volunteers and human hepatocyte cultures.     

Microarray-based detection and expression analysis of ABC and SLC transporters in drug-resistant ovarian cancer cell lines

Multiple drug resistance of cancer cells is multifactorial. A microarray technique may provide information about new candidate genes playing a role in drug resistance. Drug membrane transporters from ABC and SLC families play a main role in this phenomenon. This study demonstrates alterations in ABC and SLC gene expression levels in methotrexate, cisplatin, doxorubicin, vincristine, topotecan and paclitaxel-resistant variant of W1 ovarian cancer cell line. Resistant W1 cell lines were derived by stepwise selection of cells in increasing concentration of drugs. Affymetrix GeneChip^® Human Genome U219 Array Strip was used for hybridizations. Statistical significance was determined by independent sample t-test. The genes having altered expression levels in drug-resistant sublines were selected and filtered by scater plot. Genes up/downregulated more than threefolds were selected and listed. Among ABC genes, seven were upregulated and three were downregulated. Three genes: ABCB1, ABCB4 and ABCG2 were upregulated very significantly (over tenfold). One ABCA8 was significantly downregulated. Among 38 SLC genes, 18 were upregulated, 16 were downregulated and four were up- or downregulated dependent on the cell line. Expression of 10 SLC genes was changed very significantly (over tenfold). Four genes were significantly increased: SLC6A1, SLC9A2, SLC12A1, SLC16A6 and six genes were significantly decreased: SLC2A14, SLC7A3, SLC7A8, SLC7A11, SLC16A14, SLC38A9. Based on the expression profiles, our results provide a preliminary insight into the relationship between drug resistance and expression of membrane transporters involved in drug resistance. Correlation of specific drug transporter with drug resistance requires further analysis.     

Expression of interleukin 2 receptors by monocytes from patients with acquired immunodeficiency syndrome and induction of monocyte interleukin 2 receptors by human immunodeficiency virus in vitro.

A population of circulating mononuclear cells from patients with AIDS was identified which expressed interleukin 2 receptors (IL-2R). By dual-fluorescence flow microfluorometry, the patients' IL-2R+ cells were further identified as Leu M3+ monocytes (29.4 +/- 5.2% of the Leu M3+ cells were IL-2R+, n = 15), whereas Leu M3+ monocytes from normal subjects were IL-2R negative (2.0 +/- 0.42%; P less than 0.001). By Northern analysis, monocytes from AIDS patients, but not control subjects, constitutively expressed steady-state levels of IL-2R mRNA. Functionally, the IL-2R+ monocytes were capable of depleting IL-2 from culture supernatants, suggesting a mechanism for the reduced IL-2 levels commonly seen in AIDS patients. IL-2R+ monocytes also expressed increased levels of surface HLA-DR which may favor monocyte T-cell interactions and the transmission of human immunodeficiency virus (HIV). In additional studies, normal monocytes were infected with a macrophage-tropic HIV isolate in vitro and monitored for IL-2R and HLA-DR expression. Within 24-48 h after exposure to HIV in vitro, but before evidence of productive infection, greater than 25% of the monocytes became IL-2R+ with increasing numbers of IL-2R+ cells and HLA-DR levels through day 6. These early signaling effects of HIV could be mimicked by adding purified HIV envelope glycoprotein gp120 to the monocytes. This stimulation of monocytes before or independent of productive infection of the cells by HIV is consistent with in vivo observations of activated and/or abnormal functions by monocytes that do not appear to be infected with HIV in AIDS patients.     

人骨肉瘤细胞株MG63功能性N-甲基-D-天冬氨酸受体的表达

目的:研究人骨肉瘤细胞株MG63上功能性N-甲基-D-天冬氨酸(NMDA)受体的表达。方法:利用钙离子成像系统检测NMDA或谷氨酸对MG63细胞内游离钙离子浓度([Ca2+]i)的影响。结果:仅6.4%MG63细胞对NMDA产生[Ca2+]i升高反应,对谷氨酸则没有反应。多数MG63细胞呈现自发性的钙离子震荡现象,且该现象不能被MK801所阻断。结论:MG63细胞表达的功能性NMDA受体很少。     

Development of specific receptors for N-formylated chemotactic peptides in a human monocyte cell line stimulated with lymphokines

A human monocyte-like cell line, U937, when grown in continuous culture, does not secrete lysosomal enzymes or migrate towards chemotactic factors. When the cells are stimulated by lymphokines, however, they develop the ability both to migrate directionally and to secrete enzymes in response to several types of chemoattractants. The development, by stimulated cells, of chemotactic and secretory responses to one class of chemoattractants, the N- formylated peptides, is accompanied by the appearance on the cells of specific binding sites for these substances. Using tritiated N-formyl- methionyl-leueyl-phenylalanine (fMet-Leu-[(3)H]Phe) as a ligand, it was determined that unstimulated U937 cells possess no detectable binding sites. However, after stimulation with lymphocyte culture supernates for 24, 48, and 72 h, they developed 4,505 (+/-) 1,138, 22,150(+/-) 4,030, and 37,200 (+/-) 8,000 sites/cell, respectively. The dissociation constants for the interaction of fMet-Leu-[SH]Phe with the binding sites were approximately the same regardless of stimulation time and ranged between 15 and 30 nM. The binding of fMet-Leu-[(3)H]Phe by stimulated U937 cells was rapid and readily reversed by the addition of a large excess of unlabeled peptide. The affinity of a series of N-formylated peptides for binding to U937 cells exactly reflected the potency of the peptides in inducing lysosomal enzyme secretion and chemotaxis. The availability of a continuous human monocytic cell line that can be induced to express receptors for N-formylated peptides will provide a useful tool not only for the characterization of such receptors but also for the delineation of regulatory mechanisms involved in cellular differentiation and the chemotactic response.     

STAT3在人肝癌细胞株SMMC-7721细胞内的表达

目的研究信号传导及转录活化因子STAT3在人肝癌细胞内的表达及其表达产物STAT3酪氨酸磷酸化活化情况。方法分别采用western blotting、流式细胞术检测人肝癌细胞株SMMC-7721细胞内STAT3蛋白和磷酸化STAT3(P-STAT3)蛋白的表达。结果人肝癌细胞株SMMC-7721细胞内有STAT3的表达和酪氨酸(Y705)磷酸化。结论STAT3的异常表达和活化可能在肝癌的发生发展中起重要作用。     

全反式维甲酸对卵巢上皮腺癌细胞株抑制作用的实验研究

目的 探讨全反式维甲酸（ATRA）对卵巢上皮性腺癌细胞株（COC2）增殖的抑制作用.方法 用不同浓度的ATRA处理体外培养的COC2,采用直接细胞计数法计算一定时期内的细胞密度情况;利用倒置显微镜和电镜观察细胞形态学改变及细胞结构变化.结果 ATRA能够抑制COC2增殖,在1 ～20 μmol/L之间,具有浓度依赖性,在30 μmol/L时几乎未见存活细胞;倒置显微镜和电镜观察到使用ATRA处理后,细胞形态及结构受到破坏.结论 在一定浓度范围内,ATRA对卵巢上皮性腺癌细胞株（COC2）有抑制作用.     

Concomitant induction of the cell surface expression of Ia determinants and accessory cell function by a murine macrophage tumor cell line

This study demonstrates that an uncharacterized soluble factor produced in concanavalin A-induced rat spleen cell suspensions has the capacity to induce the increased expression of cell surface H-2K and H-2D molecules and the expression of I-region gene products on murine monocyte-macrophage lineage tumors that are not Ia positive in the absence of the factor. In parallel with induction of serologically defined Ia specificities, Ia-induced WEHI-3 macrophage tumor cells are capable of providing accessory cell function in stimulating IL-2 production by T-T hybridomas that are activated in a major histocompatibility complex-restricted, antigen-dependent fashion. The uninduced Ia-negative WEHI-3 tumor cells do not trigger a comparable response in this assay system.     

Vitamin D-binding protein synthesized by a human hepatocellular carcinoma cell line

The binding protein for 25-hydroxycholecalciferol was studied in the medium spent for the culture of HuH-7 cells, which were originally derived from a human hepatocellular carcinoma tissue. The binding protein for 25-hydroxycholecalciferol synthesized by HuH-7 cells was immunologically similar to vitamin D-binding protein in human serum and had an inter-alpha mobility. A sedimentation coefficient of 4.1 S was found on sucrose density gradient analyses. The molecular weight was estimated to be approximately 58,000 by gel filtration on a standardized column of Sephadex G-150. When mixed with filamentous actin purified from rabbit skeletal muscle, it depolymerized filamentous actin and bound to monomeric, globular actin to make a 5.5 S 1:1 molar complex with a molecular weight of approximately 100,000. These results support the conclusion that HuH-7 cells produce a functional vitamin D-binding protein.     

人乳腺癌细胞株KLK5 mRNA的表达及雌激素的调节作用

目的 探讨雌激素对雌激素受体阳性人乳腺癌细胞株MCF-7和B37组织激肽释放酶5（KLK 5）mRNA的表达水平,以及雌激素对KLK5 mRNA表达水平的影响。方法 分别取生长良好的MCF-7和B37细胞,在不同浓度的雌激素（17-βE2）存在下培养72小时后,收集细胞,提取总RNA,采用荧光定量逆转录-聚合酶链反应,检测KLK5 mRNA表达水平及变化。结果 在未经刺激的乙醇对照组,MCF-7细胞KLK5 mRNA的相对表达水平（KLK5 mRNA/GAPDH mR-NA）为（3.23±0.51）×10^-5,B37细胞KLK5 mRNA表达水平为（3.13±0.62）×10^-4。B37细胞KLK5 mRNA表达水平高于MCF-7细胞。当17-βE2浓度10^-11mol/L以下时,MCF-7和B37细胞中KLK5 mRNA表达水平与乙醇对照组无明显差异（P〈0.05）。随17-βE2浓度的增高,MCF-7和B37细胞中KLK5 mRNA的表达呈递增趋势,各组与乙醇对照组相比差异有显著意义（P〈0.01）。结论 雌激素对人乳腺癌细胞株MCF-7和B37细胞的KLK5 mRNA表达有剂量依赖的上调作用。     

吴茱萸碱抑制胃癌SGC-7901细胞生长及诱导凋亡作用的研究

目的观察吴茱萸碱对胃癌SGC-7901细胞凋亡的生长抑制作用,并探讨可能的分子机制。方法体外培养人胃癌SGC-7901细胞,分别用0.5、1.0、1.5μmol/L吴茱萸碱及吴茱萸碱+20μmol/L胱天蛋白酶抑制剂(Z-VAD-FMK)作用于SGC-7901细胞12、24和36 h。四唑盐(MTT)比色法观察吴茱萸碱对SGC-7901细胞增殖活性的影响。采用膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/PI)双染细胞,流式细胞仪检测SGC-7901细胞凋亡的情况。结果吴茱萸碱能抑制SGC-7901细胞增殖,MTT结果显示具有时间和剂量的依赖性;流式细胞仪结果显示吴茱萸碱可诱导SGC-7901细胞凋亡,且随剂量和时间的增加作用增强;Z-VAD-FMK可部分抑制吴茱萸碱诱导该细胞的凋亡作用。结论吴茱萸碱能抑制人胃癌SGC-7901细胞增殖并诱导其凋亡,且在加入Z-VAD-FMK后吴茱萸碱仍可诱导其凋亡,但作用减弱。该研究结果提示吴茱萸碱诱导胃癌SGC-7901细胞凋亡除胱天蛋白酶途径外,还存在其他的诱导凋亡途径。     

Expression of red cell antigens by K562 human leukemia cells before and after induction of hemoglobin synthesis by hemin

We studied the red cell antigens present on K562 human leukemia cells before and after induction of hemoglobin synthesis by hemin. The fetal antigens i, IF, and IT were detected on uninduced cells. While expression of both i and IT antigens increased after hemin induction, expression of IT was closely related to fetal hemoglobin synthesis as determined in experiments in which the induction was reversed. The EnaFR, NVg, and T antigens of glycophorin A were also present on uninduced cells. In contrast, the M and Pra antigens of glycophorin A, the Kell system antigens, and the P1 antigen became detectable only after hemin induction. Antigens of other major red cell systems were not detected.     

吴茱萸碱抑制胃癌SGC-7901细胞生长及诱导凋亡作用的研究

目的观察吴茱萸碱对胃癌SGC-7901细胞凋亡的生长抑制作用,并探讨可能的分子机制。方法体外培养人胃癌SGC-7901细胞,分别用0.5、1.0、1.5μmol/L吴茱萸碱及吴茱萸碱＋20μmol/L胱天蛋白酶抑制剂（Z-VAD-FMK）作用于SGC-7901细胞12、24和36 h。四唑盐（MTT）比色法观察吴茱萸碱对SGC-7901细胞增殖活性的影响。采用膜联蛋白V-异硫氰酸荧光素/碘化丙啶（Annexin V-FITC/PI）双染细胞,流式细胞仪检测SGC-7901细胞凋亡的情况。结果吴茱萸碱能抑制SGC-7901细胞增殖,MTT结果显示具有时间和剂量的依赖性;流式细胞仪结果显示吴茱萸碱可诱导SGC-7901细胞凋亡,且随剂量和时间的增加作用增强;Z-VAD-FMK可部分抑制吴茱萸碱诱导该细胞的凋亡作用。结论吴茱萸碱能抑制人胃癌SGC-7901细胞增殖并诱导其凋亡,且在加入Z-VAD-FMK后吴茱萸碱仍可诱导其凋亡,但作用减弱。该研究结果提示吴茱萸碱诱导胃癌SGC-7901细胞凋亡除胱天蛋白酶途径外,还存在其他的诱导凋亡途径。     

Inhibition of ABC transporters abolishes antimony resistance in Leishmania Infection

The emergence of antimony (Sb) resistance has jeopardized the treatment of visceral leishmaniasis in various countries. Previous studies have considered the part played by leishmanial parasites in antimony resistance, but the involvement of host factors in the clinical scenario remained to be investigated. Here we show that unlike infection with Sb-sensitive (Sbs) Leishmania donovani, infection with Sb-resistant (Sb r) L. donovani induces the upregulation of multidrug resistance-associated protein 1 (MRP1) and permeability glycoprotein (P-gp) in host cells, resulting in a nonaccumulation of intracellular Sb following treatment with sodium antimony gluconate (SAG) favoring parasite replication. The inhibition of MRP1 and P-gp with resistance-modifying agents such as lovastatin allows Sb accumulation and parasite killing within macrophages and offers protection in an animal model in which infection with Sb r L. donovani is otherwise lethal. The occurrence of a similar scenario in clinical cases is supported by the findings that unlike monocytes from SAG-sensitive kala-azar (KA) patients, monocytes from SAG-unresponsive KA patients overexpress P-gp and MRP1 and fail to accumulate Sb following in vitro SAG treatment unless pretreated with inhibitors of ABC transporters. Thus, the expression status of MRP1 and P-gp in blood monocytes may be used as a diagnostic marker for Sb resistance and the treatment strategy can be designed accordingly. Our results also indicate that lovastatin, which can inhibit both P-gp and MRP1, might be beneficial for reverting Sb resistance in leishmaniasis as well as drug resistance in other clinical situations, including cancer.     

Characterization of a soluble suppressor of human B cell immunoglobulin biosynthesis produced by a continuous human suppressor T cell line

A human suppressor T cell maintained in long-term culture with conditioned medium containing interleukin 2 elaborates a suppressor factor(s) that specifically inhibits human polyclonal B cell immunoglobulin biosynthesis. This soluble immune suppressor supernate of immunoglobulin production (CTC-SISS-B) shares a number of features with the previously described suppressive mediator elaborated by concanavalin A-activated human peripheral T cells (SISS-B) including: (a) the inhibition by a noncytotoxic mechanism, (b) the suppression of immunoglobulin biosynthesis either through direct action on the B cell or indirect action via the monocyte, (c) the loss of inhibition in the presence of the monosaccharide L-rhamnose, (d) the elaboration by cells irradiated with 500 ro 2,000 rad, and (e) molecular weights of 60,000-- 90,000. Furthermore, the suppression by this mediator appears to be specific for B cell immunoglobulin production in that CTC-SISS B has no effect on T cell proliferation to mitogens, antigens, an allogeneic cells or on T cell-mediated cytotoxicity. These data indicate that one possible mechanism of suppressor T cell inhibition of human immunoglobulin production is via the generation of a lectinlike suppressor lymphokine that interacts with defined saccharide determinants on the cell surface of either the B cell or monocyte.