Article

Role of E-cadherin in the induction of apoptosis of HPV16-positive CaSki cervical cancer cells during multicellular tumor spheroid formation.

Department of Veterinary Microbiology, University of Miyazaki, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki, Japan.
APOPTOSIS (Impact Factor: 3.61). 02/2008; 13(1):97-108. DOI: 10.1007/s10495-007-0132-2
Source: PubMed

ABSTRACT Multicellular tumor spheroids (MCTS) are three dimensional cell culture systems induced by suspension culture. MCTS are widely used in cancer research because of their similarity to solid tumors. CaSki cells are derived from a metastatic cervical cancer containing human papillomavirus 16 (HPV16). Cell death of CaSki cells in MCTS has been previously reported, and our model is used to better characterize the mechanisms of cell death of HPV16-positive keratinocytes. In this study, we found that apoptosis of CaSki cells was induced by suspension culture along with the formation of MCTS after 24 h of incubation. In suspended CaSki cells, monoclonal antibodies blocking E-cadherin function inhibited MCTS formation and suppressed suspension-induced apoptosis in a dose-dependent manner. Western blot for E-cadherin detected upregulation of the authentic 120 kDa band from MCTS of CaSki cells as well as a shorter 100 kDa band. Addition of EGF, whose receptor is known to form a complex with E-cadherin, abrogated apoptosis of suspended CaSki cells in a dose-dependent manner. These findings suggest that E-cadherin-dependent cell-cell contact, directly or indirectly, mediates the signal to undergo apoptosis of CaSki cells during MCTS formation, and thus provides new information on the role of E-cadherin in cervical cancer cell apoptosis.

Full-text

Available from: Takeshi Haga, Jul 13, 2014
0 Followers
 · 
105 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: A unique biomaterial-based system was developed to generate dynamic three-dimensional (3D) multicellular spheroids of mesenchymal stem cells (MSCs). MSCs were cultured on transparent membranes made of chitosan or those further grafted with hyaluronan (HA) in different densities. MSCs vigorously migrated and were self-assembled into highly mobile 3D spheroids with substrate-dependent upregulation of adhesion molecule N-cadherin. MSC spheroids showed increased expression of Wnt genes/proteins and substrate-dependent cell fate. The correlation of differentiation capacities with Wnt signaling and crosstalk with other pathways such as ERK1/2 or Smad2/3 were observed for MSC spheroids but not for the conventional 2D cultured cells. Wnt3a-mediated canonical Wnt signaling was more active for MSC spheroids derived on chitosan, which were prone to osteogenesis. Wnt5a-mediated non-canonical Wnt signaling was more active for MSC spheroids derived on HA-grafted chitosan, which were prone to chondrogenesis. In particular, the relative importance of Wnt5a-mediated non-canonical vs. Wnt3a-mediated canonical Wnt signals in determining the cell fate was controlled by the grafting density of HA on chitosan. Treatment with the inhibitor of canonical Wnt-associated signaling molecules suppressed the osteogenesis of MSC spheroids on chitosan. This study demonstrates that Wnt signaling of MSCs is distinct in 3D environment and is substrate-dependent. The convenient 3D platform may be used to examine the role of Wnt signaling in controlling MSC fate under different extracellular environments, and potentially applied to study stem cell behavior in regenerative medicine, normal development, and cancer.
    Biomaterials 04/2013; 34(20). DOI:10.1016/j.biomaterials.2013.03.031 · 8.31 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The envisioned clinical and industrial use of human pluripotent stem cells and their derivatives has given major momentum to the establishment of suspension culture protocols that enable cell mass production. Understanding molecular changes accompanying the transfer from adherent to suspension culture is of utmost importance because this information may directly impact on the development of optimized culture conditions. In this study we assessed gene expression of human embryonic stem cells and induced pluripotent stem cells grown in surface-adherent culture (2D) versus free-floating suspension culture spheroids (3D). We combined a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture (SILAC) with Deep-Sequencing-based transcriptomics. Cells in 3D culture showed reduced expression of proteins forming structural components of cell-cell and cell-extracellular-matrix junctions. However, fully unexpected, we found upregulation of secreted inhibitors of the canonical Wnt signaling pathway and, concomitantly, a reduction in the level of active β-catenin and in the expression of Wnt target genes. By western blot analysis the cysteine protease calpain was shown to cleave E-cadherin and β-catenin under 3D culture conditions. Our data allowed the development of a model in which calpain cleavage of E-cadherin induces the disintegration of focal cell contacts and generates a 100 kDa E-cadherin fragment required for the formation of three-dimensional cell-cell contacts in spheroids. The parallel release of β-catenin and its potential activation by calpain cleavage is counter-balanced by the overexpression of soluble Wnt pathway inhibitors. According to this model calpain holds a key function in the interplay between E-cadherin and β-catenin-mediated intercellular adhesion and the canonical Wnt signaling pathway. Supporting this model we show that pharmacological modulation of calpain activity prevents spheroid formation and causes disassembly of pre-existing spheroids into single cells, thereby providing novel strategies for improving suspension culture conditions for human pluripotent stem cells in the future.
    Molecular &amp Cellular Proteomics 01/2014; 13(4). DOI:10.1074/mcp.M113.033423 · 7.25 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A major obstacle in applying finite-state model checking to the verification of large systems is the combinatorial explosion of the state space arising when many loosely coupled parallel processes are considered. The problem also known as the *state- explosion problem* has been attacked from various sides. This paper presents a new approach based on *partial model checking*: Parts of the concurrent system are gradually removed while transforming the specification accordingly. When the intermediate specifications constructed in this manner can be kept small, the state-explosion problem is avoided. Experimental results with a prototype implemented in Standard ML, shows that for Milner's Scheduler - an often used benchmark - this approach improves on the published results on Binary Decision Diagrams and is comparable to results obtained using generalized Decision Diagrams. Specifications are expressed in a variant of the modal mu- calculus.
    Proceedings - Symposium on Logic in Computer Science 01/1995; DOI:10.1109/LICS.1995.523274