外源上皮钙粘着蛋白基因在人肺腺癌细胞中的表达及调节细胞悬浮生长

为了研究上皮钙粘着蛋白（E-cadherin)对人肺腺癌细胞间粘聚和细胞悬浮生长的影响。利用基因重组技术构建了含全长上皮钙粘着蛋白cDNA的真核表达载体。通过脂质体法转染到A549肺腺癌细胞株中,用RT-PCR和Western印迹鉴定并筛选上皮钙粘着蛋白高表达的细胞,株,并观察转染前后肿瘤细胞间粘聚能力的改变以及细胞悬浮培养下的生长状态,结果表明转染细胞间聚集力比对照细胞增强,上皮钙粘着蛋白能够促进细胞悬浮生长的速度。     

Effects of injecting cadherin gene dsRNA on growth and development in diamondback moth Plutella xylostella (Lep.: Plutellidae)

Two laboratory diamondback moth (DBM) strains were used to study the effects of injecting cadherin gene double‐stranded RNA (dsRNA) on the growth and development of Plutella xylostella (L.). Specifically, the susceptible strain named DBM.1Ac‐S and the low resistant strain DBM.1Ac‐R selected with Cry1Ac toxin were studied. The third larvae of the two strains were injected dsRNA of cadherin gene and their corresponding controls, DBM.1Ac‐RH and DBM.1Ac‐SH, were both injected diethypyrocarbonate (DEPC)‐treated water respectively. The basic biological properties such as death rate, hatching ratio, fecundity, weight of pupa and eclosion rate of the strains mentioned above were likewise studied. Meanwhile, the length and width of the egg and pupa were also measured. The results showed that the cadherin gene dsRNA injection resulted in a significant increase of the death rate and sex ratio. On the other hand, hatching ratio, fecundity, weight of pupa, eclosion rate and adult longevity for male and female of treatments decreased compared to their corresponding controls. As such, there was no significant difference on the length of egg and pupa in between treatments and the corresponding controls. However, their width increased inversely with their corresponding controls. Hence, the results suggest that cadherin gene dsRNA injection retarded the larval growth and development of P. xylostella. Also, these results can help reveal the function of cadherin gene through the RNA interference technique.     

Integrin α3β1 Engagement Disrupts Intercellular Adhesion

During tissue morphogenesis and tumor invasion, epithelial cells must undergo intercellular rearrangement in which cells are repositioned with respect to one another and the surrounding mesenchymal extracellular matrix. Using three-dimensional aggregates of squamous epithelial cells, we show that such intercellular rearrangements can be triggered by activation of β1 integrins after their ligation with extracellular matrices. On nonadherent substrates, multicellular aggregates (MCAs) formed rapidly via E-cadherin junctional complexes and over time became compacted spheroids exhibiting a more epithelial phenotype. After MCAs were replated on culture substrates, the spheroids collapsed to yield tightly arranged cell monolayers. Cell–cell contact induced rapid elevation in E-cadherin levels, which was due to an increase in the metabolic stability of junctional receptors. During MCA remodeling of cell–cell adhesions, and monolayer formation, their E-cadherin levels fell rapidly. Similar behavior was obtained regardless of which ECM ligand—collagen type I, fibronectin, or laminin 1—MCAs were seeded on. In contrast, when seeded onto a matrix elaborated by squamous epithelial cells, cells in the MCA attached, spread, lost cell–cell junctions, and dispersed. Analysis identified laminin 5 as the active ECM ligand in this matrix, and MCA dispersion required functional β1 integrin and specifically α3β1. Furthermore, substrate-immobilized anti-integrin antibody effectively reproduced the epithelial–mesenchymal-like transition induced by the laminin 5 matrix. During the early stages of aggregate rearrangement and collapse, cells on laminin 5 substrates, but not those on collagen I substrates, exhibited intense cortical arrays of F-actin, microspikes, and fascin accumulation at their peripheral surfaces. These results suggest that engagement of specific integrin–ligand pairs regulates cadherin junctional adhesions during events common to epithelial morphogenesis and tumor invasion.     

The path leading to the discovery of cadherin: In retrospect

Problems of adhesion between cells are undoubtedly one of the key major issues in developmental biology and its related field. It is little doubt that cell adhesion is one of the most fundamental mechanisms underlying the morphogenesis in multicellular animals and that it is intrinsically related to the metastasis of cancer cells as well. The historical source of adhesion studies can be traced to Wilson's work using sponges published in 1907. The present article starts to outline briefly the conceptual history up to a rise of cell adhesion in the 1950s as a problem for understanding morphogenesis in general. A crucial landmark to a recent burst of the interest to adhesion mechanisms in terms of adhesion molecules is the discovery of a group of major molecules functioning cell-to-cell adhesion, cadherins, from a research group at Kyoto University, Japan, which was initiated by myself and very successfully continued by Takeichi. A main part of the present review is to record the path leading to this important discovery based on my own personal experience, including some retrospective anecdotes. The path was initiated with a series of very simple experiments of a naïve question; that is, to examine whether or not different divalent cations were required for cell adhesion in qualitatively different manners.     

Adhesion of Drosophila imaginal disc cells in vitro

Summary We have used our imaginal disc cell lines to carry out in vitro studies on the cell-cell and cell-substrate adhesion of Drosophila leg and wing disc cells. Single cells were allowed to reaggregate in roller culture, and this process was found to be partially dependent on the presence of magnesium and calcium ions in the suspension medium. Varying rates of reaggregation were observed in cells from different stages of a passage, correlating with the pattern of morphogenesis which occurs during the passage. We have demonstrated that cloned cell lines can be produced showing certain selected characteristics, such as reduced cell adhesiveness.     

Numb regulates cell–cell adhesion and polarity in response to tyrosine kinase signalling

Epithelial‐mesenchymal transition (EMT), which can be caused by aberrant tyrosine kinase signalling, marks epithelial tumour progression and metastasis, yet the underlying molecular mechanism is not fully understood. Here, we report that Numb interacts with E‐cadherin (E‐cad) through its phosphotyrosine‐binding domain (PTB) and thereby regulates the localization of E‐cad to the lateral domain of epithelial cell–cell junction. Moreover, Numb engages the polarity complex Par3–aPKC–Par6 by binding to Par3 in polarized Madin‐Darby canine kidney cells. Intriguingly, after Src activation or hepatocyte growth factor (HGF) treatment, Numb decouples from E‐cad and Par3 and associates preferably with aPKC–Par6. Binding of Numb to aPKC is necessary for sequestering the latter in the cytosol during HGF‐induced EMT. Knockdown of Numb by small hairpin RNA caused a basolateral‐to‐apicolateral translocation of E‐cad and β‐catenin accompanied by elevated actin polymerization, accumulation of Par3 and aPKC in the nucleus, an enhanced sensitivity to HGF‐induced cell scattering, a decrease in cell–cell adhesion, and an increase in cell migration. Our work identifies Numb as an important regulator of epithelial polarity and cell–cell adhesion and a sensor of HGF signalling or Src activity during EMT.