Intrapituitary regulatory system of mammotrophs in the mouse

Estrogen stimulates the proliferation of pituitary cells, in particular mammotrophs. The present study was designed to clarify involvement of transforming growth factor alpha (TGF-alpha) in the estrogen-induced growth of mouse pituitary cells in vitro. Anterior pituitary cells obtained from ICR male mice were cultured in a primary, serum-free culture system. Proliferation of pituitary cells was detected by monitoring the cellular uptake of a thymidine analogue, bromodeoxyuridine. Secretory cell types were immunocytochemically determined. Treatment with TGF-alpha (0.1 and 1 ng/ml) for 5 days stimulated cell proliferation. Since TGF-alpha binds to the epidermal growth factor (EGF)-receptor, this action may be exerted through this receptor. Estradiol-17beta (E2, 10(-9) M) stimulated proliferation of mammotrophs. RG-13022, an EGF receptor inhibitor, reduced the cell proliferation induced by EGF or E2, showing that the EGF receptor was involved in this induction of mammotroph growth. Treatment with TGF-alpha antisense oligodeoxynucleotide (ODN) inhibited the cell proliferation induced by E2, but treatment with EGF antisense ODN did not. Dual detection of TGF-alpha mRNA and growth hormone by in situ hybridization and fluorescence-immunocytochemistry demonstrated that TGF-alpha mRNA was detected in most somatotrophs. Our recent RT-PCR analysis revealed that E2 stimulated TGF-alpha-mRNA and EGF-receptor mRNA expression. These results indicate that TGF-alpha produced in somatotrophs mediates the stimulatory effect of estrogen on pituitary cell proliferation in a paracrine manner, and that EGF-receptor expression is stimulated by estrogen. These findings indicate that intrapituitary cell-to-cell interaction plays an important role in the control of pituitary secretory cells.     

Effect of low-density lipoprotein on the expression of high affinity Fc gamma receptors

A substrain of the human monocyte-like cell line U937, which is a cholesterol auxotroph, was used to study the effect of cellular cholesterol depletion on the expression of the type I Fc receptor for IgG (Fc gamma RI). Measurement of Fc gamma RI expression was performed by immunofluorescence and flow cytometry using the monoclonal antibody (mAb) 32.2, which is specific for an epitope on Fc gamma RI, and monomeric IgG2a, which binds to the ligand binding site of Fc gamma RI. Incubation of these cells for 24 h in growth medium containing delipidated fetal calf serum depletes cellular cholesterol without affecting growth or viability. While incubation of U937 cells with human interferon-gamma (IFN-gamma) increased Fc gamma RI expression, cholesterol depletion after cell growth in media containing delipidated serum and IFN-gamma resulted in reduced binding of both mAb 32.2 and IgG2a. A significant decrease in the number of cell surface binding sites, as measured by mean fluorescence intensity, was observed after cholesterol depletion. Supplementation of the delipidated serum medium with pure cholesterol in an ethanol/bovine serum albumin mixture, which replenished cellular cholesterol and supported growth, failed to restore antibody binding significantly. In contrast, low-density lipoprotein (LDL) which also delivered cholesterol to the cells restored binding both in terms of the number of the reactive cells and cell surface receptor density. High-density lipoprotein (HDL3), which does not deliver cholesterol to the cells, showed results similar to those obtained with pure cholesterol. This indicates that either LDL cholesterol is better utilized for membrane synthesis than pure cholesterol or that LDL provides another component, in addition to cholesterol, which is required for expression of Fc gamma RI, but not for growth. These studies indicate a role for LDL in regulating the expression of Fc gamma RI on the cell surface.     

Differential effects of ETYA, a PUFA-analogue, on prostate (PC3) and monoblastoid U937 ultrastructure; lack of correlation with reduced proliferation.

ETYA (5,8,11,14-eicosatetraynoic acid), a polyunsaturated fatty acid analogue, inhibits proliferation of PC3 and U937 cells and induces a limited differentiation in U937 cells. Human prostate PC3 cells cultured for 72 h with 40 microM ETYA in fetal calf serum contained putative lipofuscin bodies, myelin figures and mitochondria with damaged cristae and matrices. These changes were absent from human U937 monoblastoid cells incubated with ETYA in CPSR3, a semipurified serum replacement. U937 cells cultured with ETYA in fetal calf serum contained occasional lipofuscin bodies, while PC3 cells cultured in CPSR3 exhibited all of the changes described. ETYA reduced the oxygen consumption of both cell lines. Therefore we conclude: (a) The response to ETYA by cells of dissimilar developmental origin is not identical; (b) unidentified serum components can augment potential ETYA-induced oxidative stress-responses of cells; (c) inhibition of U937 proliferation by ETYA does not depend upon the morphologic changes seen in PC3 cells, which resemble sequelae of oxidative stress with excess free radicals; and (d) rapid ETYA-induced inhibition of oxygen consumption in both cell lines implies a reduced synthesis of ATP that could contribute to the reversible impairment of cellular proliferation.     

Effect of epidermal growth factor on the sheet formation of the human keratinocyte in cell culture

Epidermal growth factor is an important element in maintaining keratinocyte proliferation and maturation. To evaluate its effect on keratinocyte growth in vitro, human foreskins were cultured. The epidermal keratinocyte growth in culture was separated into two stages by a conditional medium: the proliferation stage, in which the cells were maintained in a monolayer; and the differentiation stage, in which the cells grew to stratification and keratinization. The keratinocytes were cultured in various concentrations of epidermal growth factor, and their morphology and growth behavior were closely observed. Our results demonstrated that cultured keratinocytes grew in a confluent layer under the influence of epidermal growth factor. In contrast, in a culture without epidermal growth factor, the proliferation rate of cultured keratinocytes slowed down and eventually the cells stopped growing. During serum stimulation, with or without additional exogenous epidermal growth factor, the cultured keratinocytes grew continuously to the normal terminal differentiation. Under this two-stage culture model, the cultured keratinocytes could grow into an intact sheet of graftable epidermis.     

Antitumor activity of combinations of antibodies directed against different epitopes on the extracellular domain of the human EGF receptor

The receptor (EGFR) for epidermal growth factor (EGF) and transforming growth alpha (TGFα) is often overexpressed in certain types of human malignancy and high levels of expression of the receptor and/ or coexpression of growth factors. EGF and TGFα have also been correlated with poor prognosis in many patients. We have produced a number of rat monoclonal antibodies (MAbs) against four distinct epitopes on the external domain of the EGF receptor and are currently evaluating their potential for therapeutic use. Nine of these of MAbs were found to inhibit the binding of TGF and EGF to the receptor on tumor cells and these MAbs were able to inhibit the growth in vitro and in vivo of tumor cells that overexpress the EGF receptor. Here, we describe the results of experiments to determine the antitumor activity of combination treatment with two antibodies directed against separate epitopes on the external domain of human EGFR. Our results showed that treatment of human tumor xenografts with a combination of two anti-EGFR MAbs that bind to two distinct epitopes on the external domain of EGF receptor was not as effective as treatment with ICR62 alone, which binds to epitope C on the EGFR and is of IgG2b isotype. A phase I clinical trial with antibody ICR62 is currently underway in Royal Marseden Hospital (UK) in patients with head and neck and lung carcinomas.     

Glucose regulates insulin mitogenic effect by modulating SHP-2 activation and localization in JAr cells

The glucose effect on cell growth has been investigated in the JAr human choriocarcinoma cells. When JAr cells were cultured in the presence of 6 mm glucose (LG), proliferation and thymidine incorporation were induced by serum, epidermal growth factor, and insulin-like growth factor 1 but not by insulin. In contrast, at 25 mm glucose (HG), proliferation and thymidine incorporation were stimulated by insulin, serum, epidermal growth factor, and insulin-like growth factor 1 to a comparable extent, whereas basal levels were 25% lower than those in LG. HG culturing also enhanced insulin-stimulated insulin receptor and insulin receptor substrate 1 (IRS1) tyrosine phosphorylations while decreasing basal phosphorylations. These actions of glucose were accompanied by an increase in cellular tyrosine phosphatase activity. The activity of SHP-2 in HG-treated JAr cells was 400% of that measured in LG-treated cells. SHP-2 co-precipitation with IRS1 was also increased in HG-treated cells. SHP-2 was mainly cytosolic in LG-treated cells. However, HG culturing largely redistributed SHP-2 to the internal membrane compartment, where tyrosine-phosphorylated IRS1 predominantly localizes. Further exposure to insulin rescued SHP-2 cytosolic localization, thereby preventing its interaction with IRS1. Antisense inhibition of SHP-2 reverted the effect of HG on basal and insulin-stimulated insulin receptor and IRS1 phosphorylation as well as that on thymidine incorporation. Thus, in JAr cells, glucose modulates insulin mitogenic action by modulating SHP-2 activity and intracellular localization.     

Plant heterotrimeric G protein beta subunit is associated with membranes via protein interactions involving coiled-coil formation

The new functional role of activated protein C (APC) in the regulation of tissue factor (TF) expression was investigated using the cultured human monoblastic leukemia U937 cell line. A flow cytofluorometric analysis demonstrated that treatment with APC resulted in time- and dose-dependent decrease in TF expression in unstimulated and phorbol ester-stimulated cells. The effect was antagonized by the monoclonal antibody (mAb) to endothelial protein C/APC receptor (EPCR), 252, which strongly inhibited the interaction between APC and EPCR. In contrast, mAbs 49 and 379, which bind to EPCR without blocking APC binding, had no or only a modest effect. It is concluded that culturing U937 cells in the presence of APC caused down-regulation of TF expression through the EPCR-dependent mechanism, independent of whether induction was triggered by phorbol ester.     

Synthetic low-density lipoprotein, a novel biomimetic lipid supplement for serum-free tissue culture

Lipid supplementation in serum-free tissue culture employs solubilization techniques to permit the addition of lipids, but these systems are potentially cytotoxic and do not present lipid in a natural form. In this research a simplified preparation method for synthetic low-density lipoprotein (sLDL) has been developed that involves microfluidization of a solvent lipid solution in a simple aqueous solution. This produces material with size and zeta potential characteristics similar to those of native LDL. sLDL supplementation in tissue culture media provides cholesterol concentrations higher than those achieved by 10% serum supplementation and existing chemically defined lipid supplements. sLDL stimulates NS0 and U937 cellular proliferation in completely serum-free media, the former in a lipid concentration dependent manner that is also related to both the receptor peptide structure employed and its concentration on the particle. The greatest NS0 cellular proliferation was obtained at the highest cholesterol concentration tested (0.5 mg/mL), which was 10 times higher than the cholesterol concentration achieved by standard 10% serum supplementation. U937 cellular proliferation was influenced by variation of sLDL's fatty acid constituents with a natural mixture producing maximal effect. Cell uptake studies in NS0 with fluorescently labeled sLDL indicated that assimilation is reduced by competition from native LDL. The planktonic nature of NS0 cell growth meant that cell binding and uptake experiments were difficult to conduct because of cellular aggregation. However, sLDL-induced U937 proliferation is ablated by the presence of an anti-LDL receptor antibody. The results indicate that sLDL uptake is via the LDL receptor and that sLDL can function as a lipid supplement for serum-free media capable of supplementation to cholesterol concentrations up to 0.5 mg/mL. Cellular uptake studies also suggest that sLDL will be useful for the targeting and delivery of materials to cells. sLDL therefore represents a new and promising synthetic biomimetic alternative to native LDL with multiple applications.     

Effect of biosynthetic human epidermal growth factor on the synthesis and secretion of mucin glycoprotein from primary culture of rabbit fundal mucosa cells

Summary Biosynthetic human epidermal growth factor (Bh-EGF) induced dose-dependent synthesis and secretion of neutral mucin glycoprotein when the fundal cells isolated from rabbit stomach were cultured in serum-free medium containing Bh-EGF at concentrations as high as 10 to 100 ng/ml. At these high concentrations, Bh-EGF had no effect on the cell growth. In marked contrast, much lower concentrations from 0.1 to 1.0 ng/ml of Bh-EGF failed to stimulate mucin synthesis, but enhanced proliferation of the cells. Electrophoretic pattern of the mucin secreted from the cultured mucosal cells was very similar to that of the authentic mucin obtained from rabbit stomach. Maximal secretion of the mucin from the cells was observed at Hour 96 of the culture. Although fetal bovine serum (5%) and insulin (0.5 μg/ml) also stimulated the mucosal cells, both in growth and in mucin synthesis and release, the enhancing activity of the mucin synthesized and released by Bh-EGF at a concentration of 100 ng/ml per microgram DNA of cultured cells was far superior to that of 5% fetal bovine serum and 0.5 μg/ml insulin.     

CD147对白血病细胞U937生长和肿瘤形成的影响

目的：研究CD147对白血病细胞U937生长和肿瘤形成的影响。方法：分别采用脂多糖（LPS）或CD147单克隆抗体处理U937细胞;用RT-PCR和流式细胞术分别在mRNA和蛋白水平检测各组中CD147的表达情况;用流式细胞术检测在LtX3和CD147单克隆抗体作用下U937细胞周期的变化;用MTT法对各组细胞的生长状况进行分析;将细胞经皮下接种于裸鼠体内,对各组间肿瘤生长速度、肿瘤体积及裸鼠存活时间进行统计分析。结果：LPS在体外能够诱导自血病细胞U937表面CD147的表达,同时细胞增殖旺盛,但细胞凋亡数增加;使用CD147抗体阻断CD147后,能够将细胞周期阻断在G0／G1期,细胞活力下降,并诱导细胞凋亡;CD147抗体体外预处理能够抑制U937细胞在裸鼠体内的生长,使小鼠存活时间延长。结论：LPS可诱导U937细胞表面CD147分子表达增加,从而促进U937细胞的生长和肿瘤形成。     

家蚕表达人表皮生长因子gp67信号肽融合蛋白及生物活性的研究

人表皮生长因子(hEGF), 一种由53个氨基酸残基组成的单链多肽, 具有广阔的应用前景。本文主要探讨家蚕表达人表皮生长因子gp67信号肽融合蛋白的生物活性。采用家蚕杆状病毒表达系统来表达该信号肽融合蛋白。构建了重组质粒pBacPAKS-hEGF, 将该重组质粒与线性化病毒Bm-BacPAK6 DNA共转染家蚕细胞, 筛选获得重组病毒vBacPAK-SEGF, 用vBacPAK-SEGF感染家蚕BmN细胞和五龄蚕, Western blot检测表明在家蚕细胞、五岭幼虫的血淋巴和蛹中均有约12 kD的目的蛋白表达。ELISA检测发现在家蚕细胞中的表达量为23 mg/ 106细胞, 五龄幼虫中的表达量可达到82 mg/mL血淋巴。利用小鼠成纤维细胞Balb/c3T3分析家蚕表达的hEGF信号肽融合蛋白的生物活性, 结果表明表达产物能显著促进Balb/c3T3细胞的增值。另外, 研究还发现hEGF信号肽融合蛋白可使新生ICR小鼠体重增 加, 睁眼和萌齿时间提前。本研究为进一步开发利用家蚕表达的hEGF提供理论基础。     

Effects of growth factors and kinase inhibitors on the properties of human adipose-stromal cells in different culture conditions

The best-fit environments for the proliferation and differentiation of human adipose tissue-derived stromal cells (hADSCs) may require specific media and stimuli. The characteristics of hADSCs cultured in different media might be different. We evaluated the effects of growth factors on the proliferation and differentiation of hADSCs and compared the effects of these growth factors on hADSCs cultured in different serum-contained media. The effects of kinase inhibitors on the proliferation of hADSCs were also examined. The combination of insulin-transferrin-selenium (ITS) and fibroblast growth factor (FGF) induced the greatest increase in proliferation, while the combination of ITS and epidermal growth factor (EGF) showed the second-greatest effect in the human serum (HS)-contained media. By contrast, in the fetal bovine serum (FBS)-contained media, ITS had little effect while FGF had better results than the combination of ITS and EGF. The inhibitory potency of the kinase inhibitors was different in the two different media. These results suggest that hADSCs may have different characteristics according to the culture conditions, and that extrapolations of results from animal studies, using FBS media, may not correlate to findings in human studies using HS.     

A synthetic low density lipoprotein particle capable of supporting U937 proliferation in vitro.

A synthetic LDL (sLDL) has been prepared by combining a lipid microemulsion with amphipathic peptides containing the apoprotein B receptor domain. The biological properties of sLDL have been investigated using the U937 in vitro cell proliferation assay. sLDL exhibits a concentration dependent and saturable stimulation of U937 proliferation. By utilizing different amphipathic peptides, variable proliferation is achieved, indicating a specific interaction between sLDL and the U937 LDL receptor are possible. U937 proliferation is reduced by the addition of an anti-LDL receptor antibody, indicating that sLDL is assimilated via the LDL receptor pathway.The behavior of sLDL mimics that of native LDL, and this approach represents a viable technique for the production of an sLDL particle on a large scale for research and general application.     

Hyaluronic acid of high molecular weight inhibits proliferation and induces cell death in U937 macrophage cells

Hyaluronic acid (HA), a major glycosaminoglycan component of the extracellular matrix, has regulatory influences on cells and cellular activities. To explore the effects of a high concentration (1 mg/mL) of high molecular weight HA (500-730 kD) on U937 macrophage growth dynamics, three factors that influence overall cellular growth, namely proliferation, apoptosis, and cell death, were examined. Cells were cultured with HA and were analyzed by flow cytometry every 24 hours during a 168-hour period for proliferation and the presence of apoptotic and dead cells. These analyses demonstrated that HA inhibits U937 macrophage proliferation in a time-dependent manner. Through the first 72 hours, cells exhibited slowed proliferation. However, no evidence of cell division arrest or reduced cell viability was observed. Thereafter, HA continued to diminish proliferation, but induced apoptosis. This data is consistent with regulatory influences secondary to HA binding to CD44 and/or RHAMM cell surface receptors, both of which were shown to be expressed on U937 macrophages. This study demonstrates that a high concentration of high molecular weight HA greatly inhibits macrophage population growth by the dual actions of impeding cell proliferation and inducing apoptosis.     

P^53基因在U937细胞生长和分化过程中的调节作用

The expression of p53 gene has been found to be regulated during the induction of differentiation of U937 leukemic cells into mature macrophages by recombinant human granulocyte- macrophage colony stimulating factors (rhGM-CSF) We showed here that the increased expression of p53 seemed to be necessary for the differentiation of U937 cells induced by rh-GM-CSF. The inhibition of p53 expression by a p53 antisense oligodeoxynucleotide lead to the significant decrease of formation of mature macrophages from U 937 cells in the presence of rhGM-CSF. By contrast, the p53 sense oligodeoxynucleotide had no any effect. Furthermore, we have analysed the growth of U937 cells in the presence or absence of rhGM-CSF. The results showed that rhGM-CSF dramatically inhibited the growth of U 937 cells in the cultures. At the same time, the antisense inhibition experiment demonstrated that the inhibition of p53 expression partially diminished the growth-inhibitory effect of rhGM-CSF on U 937 cells. These results suggested that the p53 was required for the initiation of rhGM-CSF-induced differentiation of U 937 cells on one hand, and the inhibition of cell growth on the other hand. Thus we deduce that the increased expression of p53 induced by rhGM-CSF may be a coupling event of switch of U 937 cells from growth into differentiation.     

Binding of recombinant HIV coat protein gp120 to human monocytes

Inasmuch as the exact level of CD4 Ag expression on macrophages is controversial and because HIV may interact with macrophages in a manner different from that on T cells, we analyzed the binding of gp120 to freshly isolated and cultured monocytes. rgp120 was iodinated using the lactoperoxidase method to a sp. act. of 600 Ci/mmol. Highly purified monocytes (greater than 90%) were isolated from the leukapheresed blood of normal volunteers by Ficoll-Hypaque sedimentation followed by countercurrent centrifugal elutriation and cultured 7 days in DMEM supplemented with 1000 U/ml macrophage CSF in 10% human serum. Whereas MOLT/4 cells consistently bound freshly prepared 125I-rgp120 at 80% specificity with 5100 +/- 700 mol/cell, MCSF cultured monocytes bound rgp120 at only 0 to 20% specificity and 420 +/- 200 mol/cell. Most of the radioactivity bound by these cells could not be blocked by the addition of unlabeled rgp120. In contrast, the U937 myeloid cell line bound rgp120 with 50% specificity and about 2500 mol/cell. Whereas the antibody OKT4a (anti-CD4) blocked 80% of the binding on MOLT/4 cells and 50% on U937 cells, binding was only inhibited on the average of 6% on cultured monocytes. When soluble rCD4 was used as an inhibitor, binding to MOLT/4 cells was blocked by 80%. In contrast, binding to cultured monocytes was inhibited by 28%. HIV infectivity was blocked by similar concentrations of OKT4a. These observations suggest that although most binding of gp120 to cultured monocytes is not to the CD4 determinant, several hundred molecules do bind to a CD4-like molecule which promotes virus entry and replication.     

Human alpha-thrombin stimulates proliferation of interferon-gamma differentiated,growth-arrested U937 cells,overcoming differentiation-related changes in expression of p21CIP1/WAF1 and cyclin D1

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is a growth factor for a variety of cell types. We recently demonstrated that interferon-gamma (IFNgamma)-differentiated U937 cells show increased expression of the proteolytically activated receptor for thrombin (PAR-1) relative to undifferentiated U937. In the present study we show that cell proliferation is inhibited in IFNgamma-differentiated cells relative to undifferentiated U937. Addition of thrombin to the differentiated cells, however, overcomes the inhibition and restores the cells to a highly proliferative state. Ribonuclease protection assays indicate that the IFNgamma-induced growth arrest is associated with an increased expression of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) and downregulation of cyclin D(1). Treatment of cells with thrombin downregulates p21(CIP1/WAF1) expression in these cells and upregulates cyclin D(1) mRNA expression, thus overcoming the differentiation-related effects in a coordinated manner. Treating differentiated cells with the PAR-1 activation peptide, SFLLRN, stimulates proliferation and has effects similar to those of thrombin on expression of p21(CIP1/WAF1). Thus, it appears that these thrombin stimulated proliferative effects are mediated through activation of PAR-1. These results may help explain how thrombin can overcome growth arrest in normal tissue to initiate tissue repair and why thrombin and thrombin-like enzymes may contribute to unrestricted proliferation observed in certain malignancies.     

Induced autocrine signaling through the epidermal growth factor receptor contributes to the response of mammary epithelial cells to tumor necrosis factor alpha

In contrast to the well known cytotoxic effects of tumor necrosis factor (TNF) alpha in many mammary cancer cells, we have found that TNF stimulates the proliferation and motility of human mammary epithelial cells (HMECs). Since the response of HMECs to TNF is similar to effects mediated by epidermal growth factor receptor (EGFR) activation, we explored the potential role of cross-talk through the EGFR signaling pathways in mediating cellular responses to TNF. Using a microarray enzyme-linked immunoassay, we found that exposure to TNF stimulated the dose-dependent shedding of the EGFR ligand transforming growth factor alpha (TGFalpha). Both proliferation and motility of HMECs induced by TNF was prevented either by inhibiting membrane protein shedding with a metalloprotease inhibitor, by blocking epidermal growth factor receptor (EGFR) kinase activity, or by limiting ligand-receptor interactions with an antagonistic anti-EGFR antibody. EGFR activity was also necessary for TNF-induced release of matrix metalloprotease-9, thought to be an essential regulator of mammary cell migration. The cellular response to TNF was associated with a biphasic temporal pattern of extracellular signal-regulated kinase (ERK) phosphorylation, which was EGFR-dependent and modulated by inhibition of metalloprotease-mediated shedding. Significantly, the late phase of ERK phosphorylation, detectable within 4 h after exposure, was blocked by the metalloprotease inhibitor batimastat, indicating that autocrine signaling through ligand shedding was responsible for this secondary wave of ERK activity. Our results indicate a novel and important role for metalloprotease activation and EGFR transmodulation in mediating the cellular response to TNF.     

Is epidermal growth factor present in human blood? Interference with the radioreceptor assay for epidermal growth factor

Human whole blood serum reduces the binding of [125I]labeled mouse epidermal growth factor to cultured human fibroblasts as well as does 70-100 ng/ml mouse epidermal growth factor. However, at most 1-2% of the binding inhibitor detected in human whole blood serum is related to epidermal growth factor--the remaining consists of other factors released during preparation of serum, predominantly the platelet-derived growth factor, which are capable of altering the binding properties of the epidermal growth factor receptor. This accounts for much of the differences between values reported for epidermal growth factor concentration in blood by investigators using different assay procedures.