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Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes
Published on: April 12, 2015
Different responses in transformation of MDCK cells in 2D and 3D culture by v-Src as revealed by microarray
Mira Töyli1, Linda Rosberg-Kulha, Janne Capra
1Biocenter Oulu and the Department of Pathology, Institute of Diagnostics, University of Oulu, Oulu, Finland.
Abstract:
Differentiation and transformation of untransformed and ts-Src-transformed canine kidney MDCK cells in 2D and 3D environment were investigated using microarray technique, RT-PCR, confocal microscopy and functional assays. Activated Src induced epithelial-mesenchymal transition (EMT) in 2D environment followed by translocation of junctional proteins to the cytoplasm, without significant changes in protein expression. In 3D environment untransformed MDCK cells formed cell cysts with apical domain facing a lumen, E-cadherin delineating the lateral membranes, ZO-1 at tight junctions and caspase-3 in apoptotic cells captured within the lumen. This was accompanied by reduced expression of an apoptosis inhibitor, survivin and vesicle transport effectors, rab 7 and 8, whereas rab 5 expression increased. In 3D environment activated Src induced changes in expression of over 100 genes as revealed by microarray analysis, mostly involved in cell signaling, division and energy metabolism. Only response in cytoskeletal components was decreased expression of actin and Arp2/3 by v-Src, whereas two p120catenin binding proteins Kaiso and Nanos increased their expression. Concomitantly, apoptosis was inhibited by v-Src resulting in formation of a sphere with epitheloid cells facing extracellular matrix and undifferentiated cells captured within the cluster. This was accompanied by increased expression of apoptosis inhibitor survivin, as revealed by western blotting. Mitochondrial membrane potential in untransformed MDCK cells was lower than in ts-Src-MDCK cells in early days of cluster formation correlating with the induction of apoptosis. Hence, v-Src activation in 3D environment did not induce EMT, but brought about inhibition of apoptosis and increased proliferation where increased expression of survivin and inhibition of the mitochondrial permeability have a role.
Insights
Activated Src in 3D environments inhibits apoptosis and increases proliferation in kidney cells. This involves increased survivin expression and reduced mitochondrial permeability, unlike 2D environments where it induces epithelial-mesenchymal transition.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- The Src protein kinase plays a crucial role in cell signaling, proliferation, and survival.
- Epithelial-mesenchymal transition (EMT) is a process implicated in cancer progression.
- Cellular behavior differs significantly between 2D and 3D culture environments.
Purpose of the Study:
- To investigate the effects of activated Src on Madin-Darby canine kidney (MDCK) cells in both 2D and 3D culture.
- To compare the cellular responses, including differentiation, transformation, and apoptosis, in different dimensional environments.
- To elucidate the molecular mechanisms underlying Src-induced cellular changes.
Main Methods:
- Microarray analysis to assess global gene expression changes.
- Reverse transcription-polymerase chain reaction (RT-PCR) for gene expression validation.
- Confocal microscopy for visualizing protein localization and cell morphology.
- Functional assays, including western blotting and mitochondrial membrane potential measurements.
Main Results:
- In 2D, activated Src induced EMT with junctional protein translocation.
- In 3D, untransformed cells formed cysts; Src-transformed cells formed spheres with inhibited apoptosis and increased proliferation.
- Src activation in 3D altered expression of over 100 genes, increased survivin, and reduced mitochondrial permeability.
Conclusions:
- Activated Src induces EMT in 2D but promotes proliferation and inhibits apoptosis in 3D kidney cell models.
- Survivin upregulation and mitochondrial permeability changes are key mechanisms in Src-driven 3D cell growth.
- The 3D microenvironment significantly modulates Src's oncogenic functions.
