Induction of GPR40 positively regulates cell motile and growth activities in breast cancer MCF-7 cells

Abstract

Free fatty acid (FFA) receptors belong to a member of G-protein-coupled receptors. GPCR 120 (GPR120) and GPR40 are identified as FFA receptors and activated via the binding of long- and medium-chain FFAs. The aim of this study was to assess the effects of GPR120 and GPR40 on cell motility and growth in breast cancer cells treated with tamoxifen (TAM). MCF-7 cells were continuously treated with TAM for approximately 6?months. The expression level of GPR40 gene was markedly higher in the long-term TAM treated (MCF-TAM) cells than in MCF-7 cells. In cell motility assay, MCF-TAM cells indicated the high cell motile activity, compared with MCF-7 cells. The cell motile activity of MCF-TAM cells was suppressed by a selective GPR40 antagonist, GW1100. To evaluate the effects of GPR40 on cell growth activity under estrogen-free conditions, cells were maintained in serum-free DMEM without phenol red for 2?days. In estrogen-free conditioned medium, the cell growth rate of MCF-TAM cells was significantly higher than that of MCF-7 cells. In addition, treatment of GW1100 reduced the cell growth rate of MCF-TAM cells. These results suggest that the cell motile and growth activities may be positively regulated through the induction of GPR40 by the long-term TAM treatment in MCF-7 cells.     

A method of direct-viewing comparative analysis of tumour cell motility in vitro; effect of pH and adhesion on cells of different malignancy

A novel method for the comparative analysis of cell motility by direct viewing was developed and used in a preliminary study of sarcoma cells. Three cell lines from the RPS family of sarcomas in inbred LEW/CUB rats which differ in the incidence of spontaneous metastasis were studied. A system of four different culture conditions, designed to mimic the stress within the tumour, was created by changing and combining two variables: the pH of the medium (with 2% calf serum only), which was either physiological at 7.4 or 6.6; and the adhesiveness of the culture substratum, which was either at a standard level or decreased. It was found that changing the pH from 7.4 to 6.6 in a single step slowed the cells down, and also changed the way in which they moved, from «walking« in random directions to moving in a more directional way. Making the culture surface less adhesive sped up cell locomotion. Decreasing the adhesiveness of the culture substratum and the pH simultaneously stimulated the migration of highly metastasizing cells preferentially. Thus we have found a way to distinguish between highly metastasizing A297Nb sarcoma cells and poorly metastasizing T15 and non-metastasizing K2 sarcoma cells, a distinction that could not be made by a simple examination of the cells. It is concluded that a comparison of cell motility by directly viewing the cells under different conditions may be useful when investigating the in vitro motility properties of malignant cells.     

Effects of cell motility and chemotaxis on microbial population growth.

A mathematical model is developed to elucidate the effects of biophysical transport processes (nutrient diffusion, cell motility, and chemotaxis) along with biochemical reaction processes (cell growth and death, nutrient uptake) upon steady-state bacterial population growth in a finite one-dimensional region. The particular situation considered is that of growth limitation by a nutrient diffusing from an adjacent phase not accessible to the bacteria. It is demonstrated that the cell motility and chemotaxis properties can have great influence on steady-state population size. In fact, motility effects can be as significant as growth kinetic effects, in a manner analogous to diffusion- and reaction-limited regimes in chemically reacting systems. In particular, the following conclusions can be drawn from our analysis for bacterial populations growing at steady-state in a confined, unmixed region: (a) Random motility may lead to decreased population density; (b) chemotaxis can allow increased population density if the chemotactic response is large enough; (c) a species with superior motility properties can outgrow a species with superior growth kinetic properties; (d) motility effects become greater as the size of the confined growth region increases; and (e) motility effects are diminished by significant mass-transfer limitation of the nutrient from the adjacent source phase. The relationships of these results for populations to previous conclusions for individual cells is discussed, and implications for microbial competition are suggested.     

An investigation of the diffusion-limited growth of animal cells around single hollow fibers

To compare modeling with experimental data of cell growth surrounding individual fibers, the growth profiles of hybridoma cells in the extracapillary space of single hollow fiber bioreactors were examined. Agarose was provided in the extracapillary space to provide support and minimize convection. By sacrificing bioreactors at various time intervals, the growth profiles of cells surrounding a single hollow fiber could be monitored with increasing time. Using photomicroscopy and viable staining, areas of viable and nonviable cell growth were examined at various stages of development ranging from initial seeding to stable growth conditions. Cells were found to act as nucleation sites for the growth of individual colonies within the agarose. Profiles at stable growth conditions resulted in a thick cell mass near the surface of the fiber wall followed by cell colonies of decreasing size with increasing radial distance. A simplified theoretical model for cell growth was developed using mass balance equations for substrate penetration into individual cell colonies as well as away from the wall of a single fiber. The resulting profiles derived from theory were compared with experiments and found to be in good agreement for entering oxygen concentrations of 5% and 20%. (c) 1992 John Wiley & Sons, Inc.     

Epidermal growth factor increased the expression of alpha2beta1-integrin and modulated integrin-mediated signaling in human cervical adenocarcinoma cells

Epidermal growth factor (EGF) receptor (EGFR) is involved in various basic biochemical pathways and is thus thought to play an important role in cell migration. We examined the effect of EGF on motility, migration, and morphology of a human adenocarcinoma cell line CAC-1. EGF treatment increased the motility of cervical adenocarcinoma cells and promoted migration of the cells on fibronectin and type IV collagen. EGF induced morphological changes with lamellipodia during EGFR-mediated motility. The results of an immunoprecipitation study showed that EGF up-regulated the expression of alpha2beta1-integrin in a dose-dependent manner. EGF-induced cell migration was blocked by alpha2beta1-integrin antibody. Our results also showed that EGF treatment stimulated the level of tyrosine dephosphorylation of FAK, which is required for EGF-induced changes in motility, migration, and cell morphology. A tyrosine kinase inhibitor (ZD1839) blocked EGF-induced changes in cervical adenocarcinoma cells. The results suggest that EGF promotes cell motility and migration and increases the expression of alpha2beta1-integrin, possibly by decreasing FAK phosphorylation.     

Influence of salinomycin treatment on division and movement of individual cancer cells cultured in normoxia or hypoxia evaluated with time-lapse digital holographic microscopy

Most studies on new cancer drugs are based on population-derived data, where the absence of response of a small population may pass unnoticed. Thus, individual longitudinal tracking of cells is important for the future development of efficient cancer treatments. We have used digital holographic microscopy to track individual JIMT-1 human breast cancer cells and L929 mouse fibroblast cultivated in normoxia or hypoxia. In addition, JIMT-1 cells were treated with salinomycin, a cancer stem cell targeting compound. Three-day time-lapse movies were captured and individual cells were analysed with respect to cell division (cell cycle length) and cell movement. Comparing population-doubling time derived from population-based growth curves and individual cell cycle time data from time-lapse movies show that the former hide a sub-population of dividing cells. Salinomycin treatment increased the motility of cells, however, this motility did not result in an increased distant migration i.e. the cells increased their local movement. MCF-7 breast cancer cells showed similar motility behaviour as salinomycin-treated JIMT-1 cells. We suggest that combining features, such as motility and migration, can be used to distinguish cancer cells with mesenchymal (JIMT-1) and epithelial (MCF-7) features. The data clearly emphasize the importance of longitudinal cell tracking to understand the biology of individual cells under different conditions.     

带培养室的倒置显微镜下人雪旺细胞形态和运动的观察

为了显示体外培养条件下三维结构上人雪旺细胞的形态和运动方式,将引产人胎儿坐骨神经的雪旺细胞培养在聚酯纤维上,用带培养室的显微镜观察。结果显示人雪旺细胞呈梭形,绕聚酯纤维作螺旋式迁移;有的细胞同时抓住两根纤维;在相对静止期,细胞虽无位置的迁移,但有频繁的形态变化;分裂后的子细胞有形态和运动时相的不同。     

Cell motility and SCAR localisation in axenically growing Dictyostelium cells

Dictyostelium is a popular experimental organism, in particular for studies of actin dynamics, cell motility and chemotaxis. We find that the motility of axenic cells is unexpectedly different from other strains during growth. In particular, vegetative AX3 cells do not show detectable localisation of SCAR and its regulatory complex to actin-rich protrusions such as filopodia and pseudopodia. Similarly, a range of different mutations, in particular knockouts of members of the SCAR complex and Ras proteins, cause different phenotypes during vegetative growth in different parental strains. Development reverses this unusual behaviour; aggregation-competent AX3 cells localise SCAR in the same way as cells of other strains and species. Studies on cell motility using vegetative cells should therefore be interpreted with caution.     

Stimulation of motility in cultured bovine capillary endothelial cells by angiogenic preparations

Several angiogenic preparations that have been shown to stimulate plasminogen activator (PA) and collagenase production by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate BCE cell motility in the phagokinetic track assay. Bovine retinal extract, medium conditioned by 3T3-F442A differentiated mouse adipocytes, SK HEP-1 human hepatoma cell lysate, mouse sarcoma 180 cell lysate, and medium conditioned by mouse sarcoma 180 cells stimulated motility 68.7%, 48.5%, 140.9%, 56.5%, and 102.1%, respectively, relative to untreated cells. The motility-stimulating activity of these preparations was dose dependent and linear over the 16-h assay period. Several hormones and growth factors were tested for BCE cell motility-stimulating activity, including insulin, vasopressin, fibroblast growth factor, and a partially purified preparation of sarcoma growth factor, and were found to be ineffective. 12-0-tetradecanoyl-phorbol-acetate (TPA), a potent stimulator of both PA and collagenase activities in BCE cells, also did not stimulate motility, indicating that protease production is not sufficient to stimulate BCE cell motility in this assay. Neither SK HEP-1 hepatoma cell lysate nor TPA was effective in stimulating motility in bovine aortic endothelial (BAE) cells. The inability of SK HEP-1 hepatoma cell lysate to stimulate movement in BAE cells is consistent with the observation that angiogenesis occurs by sprouting of capillaries, not large vessels.     

甲胎蛋白在体外对人肝癌细胞生长的影响

本文用MTT比色法观察了甲胎蛋白(AFP)在体外对人肝癌细胞生长的影响。结果表明,AFP能促进SMMC-7721人肝癌细胞的生长。当AFP与AFP抗体合用时,AFP抗体能减弱AFP对SMMC-7721细胞生长的促进作用;AFP抗体单用对此种细胞的生长亦有抑制作用。另一方面,在相同的实验条件下,AFP和AFP抗体对HL-60人白血病细胞的生长无明显影响;提示AFP的促生长作用具有一定的肿瘤细胞特异性,并非一种蛋白质对培养细胞的非特异性营养作用。此外,AFP亦能促进MCF-7人乳腺癌细胞的生长,AFP抗体对此种细胞的生长有抑制作用。由于MCF-7细胞存在功能性AFP受体,也能合成和分泌AFP。这就提示,人肝癌细胞中的AFP很可能与其受体特异性结合,产生促生长效应。确切机制尚待进一步阐明。     

Collective dynamics of cancer cells confined in a confluent monolayer of normal cells

Tumorigenesis often involves specific changes in cell motility and intercellular adhesion. Understanding the collective cancer cell behavior associated with these specific changes could facilitate the detection of malignant characteristics during tumor growth and invasion. In this study, a cellular vertex model is developed to investigate the collective dynamics of a disk-like aggregate of cancer cells confined in a confluent monolayer of normal cells. The effects of intercellular adhesion and cell motility on tumor progression are examined. It is found that the stresses in both the cancer cells and the normal cells increase with tumor growth, resulting in a crowded environment and enhanced cell apoptosis. The intercellular adhesion between cancer cells and normal cells is revealed to promote tumor growth and invasion. The tumor invasion dynamics hinges on the motility of cancer cells. The cancer cells could orchestrate into different collective migration modes, e.g., directional migration and rotational oscillations, dictated by the competition between cell persistence and local coordination. Phase diagrams are established to reveal the competitive mechanisms. This work highlights the role of mechanics in regulating tumor growth and invasion.     

The integrin-linked kinase regulates cell morphology and motility in a rho-associated kinase-dependent manner

The integrin-linked kinase (ILK) is a multidomain focal adhesion protein implicated in signal transmission from integrin and growth factor receptors. We have determined that ILK regulates U2OS osteosarcoma cell spreading and motility in a manner requiring both kinase activity and localization. Overexpression of wild-type (WT) ILK resulted in suppression of cell spreading, polarization, and motility to fibronectin. Cell lines overexpressing kinase-dead (S343A) or paxillin binding site mutant ILK proteins display inhibited haptotaxis to fibronectin. Conversely, spreading and motility was potentiated in cells expressing the "dominant negative," non-targeting, kinase-deficient E359K ILK protein. Suppression of cell spreading and motility of WT ILK U2OS cells could be rescued by treatment with the Rho-associated kinase (ROCK) inhibitor Y-27632 or introduction of dominant negative ROCK or RhoA, suggesting these cells have increased RhoA signaling. Activation of focal adhesion kinase (FAK), a negative regulator of RhoA, was reduced in WT ILK cells, whereas overexpression of FAK rescued the observed defects in spreading and cell polarity. Thus, ILK-dependent effects on ROCK and/or RhoA signaling may be mediated through FAK.     

The type I insulin-like growth factor receptor is a motility receptor in human melanoma cells

Insulin-like growth factors I and II (IGF-I and II) and insulin are chemotactic agents for the human melanoma cell line A2058. As shown in this report, the motility receptor mediating this response is the heterodimeric type I IGF receptor. These three factors are able to compete with 125I-labeled IGF-I for binding to the cell surface with IC50 values equal to approximately 2 (IGF-I), approximately 150 (IGF-II), and approximately 300 nM (insulin). Cross-linking of 125I-IGF-I to the cell surface with disuccinimidyl suberate followed by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography reveals a 130-kDa protein (reduced) consistent with the alpha component of a type I receptor and a 38-kDa protein which does not bind insulin, and thus could be another IGF-I cell surface binding protein. The anti-IGF-I receptor monoclonal antibody (alpha IR-3) also competes with labeled IGF-I in binding experiments. In contrast, a control monoclonal antibody, matched to alpha IR-3 with respect to IgG subclass, has no significant effect on IGF-I binding. While alpha IR-3 inhibits the motility induced by IGF-I, IGF-II, and insulin, pertussis toxin (0.01-1.0 micrograms/ml) has no significant effect on the motility induced by the insulin-like growth factors or insulin on this cell line. Therefore, the type I IGF receptor appears to mediate a highly potent pertussis toxin-insensitive motility response to IGF-I, IGF-II, and insulin. In contrast, motility induced by the autocrine motility factor, a cytokine produced by the A2058 cells, is not affected by alpha IR-3 but is extremely sensitive to pertussis toxin. When mixtures of autocrine motility factor and IGF-I are employed to induce chemotaxis, the resulting motility is greater than that induced by either agent alone. These data indicate that motility in this melanoma cell line can be initiated through multiple receptors that stimulate the cells by separate transduction pathways. This capability to respond to multiple stimuli could enhance the metastatic potential.     

胚胎干细胞向肝实质细胞体外定向诱导分化过程中的肝卵圆细胞

如果肝脏严重受损致使肝细胞大部分坏死,或由于某些原因 ( 肝毒性物质、致癌物质的作用 ) 抑制残存肝细胞增殖时,肝内前体／干细胞———肝卵圆细胞便被激活并分化生成肝细胞和胆管细胞等以参与肝修复 . 基于此理论,人们建立了啮齿类动物肝卵圆细胞诱导实验模型 . 但显然上述模型不适用于人类,所以有必要开发一种适用于人类的、高效的肝卵圆细胞的新诱导模型 . 选用小鼠胚胎干细胞,转成拟胚体分化 3 天后分组,诱导组添加肝细胞生长因子 (HGF) 、表皮生长因子 (EGF) 作定向诱导分化 . 其间用免疫细胞化学 (ICC) 检测肝卵圆细胞标志物 A6 等的表达,用流式细胞仪筛选肝卵圆细胞并行 RT-PCR 、透射电镜检测 . 所筛选的肝卵圆细胞进一步体外培养并进行 ICC 和 RT-PCR ,检测其分化生成成熟的肝细胞和胆管细胞的能力 . 研究证实胚胎干细胞体外定向诱导生成肝实质细胞的过程中,存在着有双向分化能力的肝卵圆细胞这个中间分化阶段 . 诱导组肝卵圆细胞分化率均显著地高于对照组,最高时可达 6.11% 左右 . HGF 和 EGF 能显著性诱导胚胎干细胞源性卵圆细胞的生成 . 流式细胞仪筛选 Sca-1+/CD34+ 细胞占总细胞数的 4.59% ,其中 A6 阳性肝卵圆细胞占 90.81% 左右 . 使用流式细胞仪可获得高富集的 A6+/Sca-1+/CD34+ 肝卵圆细胞 . 提供了一种可适用于人类的肝卵圆细胞的新诱导模型 .     

Motility of Bacillus subtilis during growth and sporulation.

The change of motility and the presence of flagella were followed throughout growth and sporulation in a standard sporulating strain and in 19 cacogenic sporulation mutants of Bacillus subtilis. For the standard strain, the fraction of motile cells decreased during the developmental period to less than 10% at T4. Motility was lost well before the cells lose their flagella. Conditions reducing the decrease of motility also reduced sporulation: motile cells never contained spores. The decrease of motility was not coupled with a decrease in the cellular concentration of adenosine 5'-triphosphate or a decline in oxygen consumption, but an uncoupling agent immediately destroyed motility at any time. Apparently, motility decreased during development because it became increasingly uncoupled from the energy generating systems of the cell. The motility of sporulation mutants decreased after the end of growth at the same time as or earlier than the motility of the standard strain; the early decrease of motility in an aconitase mutant, but not that in an alpha-ketoglurate dehydrogenase mutant, could be avoided by addition of L-glutamate. Sporulation or related events such as extracellular antibiotic or protease production were not needed for the motility decline.     

Cell size in Dictyostelium

Cellular slime mold amoebae have become a model system for the study of cell motility and the cytoskeleton. A basic problem which all cells face that involves the cytoskeleton is how to control their size. The varied ways in which cellular slime mold amoebae change their cell size--by changing the size at which division occurs, by cell fusion, and by control over cytokinesis--are reviewed. A model is presented which attempts to explain how the mechanisms affected in certain cytokinesis mutants in Dictyostelium discoideum known as phg mutants could be involved in control of cell size in the predatory slime mold Dictyostelium caveatum.     

Phospholipids and their derivatives as mitogen and motogen of budding tunicates

In order to discover novel invertebrate cytokines from the budding tunicate, Polyandrocarpa misakiensis, we treated the water-insoluble fraction of tunicate homogenates with trypsin. The extracts showed remarkable activities to promote the growth and motility of tunicate cells. The activities were heat-stable and proteinase K-resistant. After anion exchange chromatography, the activities were eluted with detergents such as 0.1% deoxycholic acid. The Fourier transform infrared spectrum indicated large amounts of fatty acids and phospholipids instead of polypeptides in the extracts. Consistently, the activities were extractable with organic solvents such as chloroform. Long chains of n-3 polyunsaturated free fatty acids (FFA), phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) were the major components in the lipid-soluble fraction. A cDNA for FFA-releasing enzyme phospholipase A(2) (PLA(2)) was cloned. The expression of this gene could be seen in epidermal cells during budding. The recombinant protein, as in the case of the authentic PLA2, preferred PC and PE as substrates, followed by PS and PI. The resultant FFAs only promoted cell growth, while the remaining lysophospholipids stimulated cell motility. The former contained unsaturated fatty acids (C18:1, C20:5, and C22:6) while the latter did not, suggesting that unsaturated fatty acids are responsible for mitogenic activity in tunicate cells. These results show for the first time that phospholipids and their derivatives are bio-mediators promoting cell growth and cell motility in invertebrates.     

Regulation of cell growth and motility by hepatocyte growth factor and receptor expression in various cell species.

Hepatocyte growth factor (HGF), a humoral mediator for regeneration of liver and kidney, possesses multiple biological activities. To investigate target cell specificity and to examine whether multiple actions of HGF are related to properties of the HGF receptor on target cells, we examined the effects of HGF on cell growth and motility and analyzed the HGF receptor in various species of cells. HGF stimulated growth and DNA synthesis of PAM212 (naturally immortalized mouse keratinocytes), Mv1Lu (mink lung epithelia), and A431 (human epidermoid carcinoma) cells, as well as mature hepatocytes, but inhibited those of IM-9 (human B-lymphoblasts). Conversely, HGF had a marked stimulatory effect on cell motility of MDCK (Mardin-Darby canine kidney epithelia) cells, but not on their growth. Also, HGF enhanced the motility of various species of cells, including A431, PAM212, HepG2 (human hepatoma), KB (human epidermoid carcinoma), and J-111 (human monocytes) cells. Scatchard analysis of 125I-HGF binding to hepatocytes indicated that the cells expressed both high- and low-affinity binding sites for HGF with Kd values of 23 and 260 pM, respectively. High-affinity HGF receptor with Kd values of 20-25 pM was detected at 40-720 sites/cell in MDCK, A431, PAM212, Lu99, and IM-9 cells, but not in fibroblasts and hematopoietic cells. In contrast, low-affinity binding sites were detected in all cell lines examined, even in those not responsive to HGF. Northern blots revealed that cells possessing a high-affinity HGF receptor expressed c-MET/HGF receptor mRNA. Therefore, HGF probably regulates both cell growth and motility of various types of epithelial cells and some types of mesenchymal cells. The multiple biological activities of HGF may be exerted through a high-affinity HGF receptor linked to multiple distinct intracellular signaling pathways.     

Nonrandom spatial distribution by mammalian cells in culture

Quadrat analysis was used to investigate the spatial distribution of seven mammalian cell lines in culture. The number of cells in replicate unit areas of the culture was determined, and the variance to mean ratio used as an index of random and nonrandom spatial distribution. Only mouse SV3T3 cells distributed themselves randomly throughout the entire culture growth cycle. The remaining six lines all assumed a nonrandom distribution at some point in their growth cycles. Mouse L929 cells displayed avoidance behavior, and spaced themselves at regular intervals in a uniform spatial distribution. The five remaining lines (mouse S180, rat C6, hamster CHO, canine MDCK, and human BeWo) formed multicellular clusters, and were distributed aggregatively rather than randomly. Random walk migration can account for the random distribution of SV3T3 cells. Random walk combined with contact inhibition of movement provides a satisfactory explanation for the uniform distribution of L929 cells. Random walk and contact inhibition are incompatible with cell clustering, however. Thus other mechanisms of motility or adhesiveness must contribute to cell clustering. It is possible that random walk and contact inhibition may be less common components of cell movement than generally assumed.