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Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras
C A Schoenenberger1, A Zuk, D Kendall
1Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, Massachusetts 02115.
Abstract:
The effects of viral Kirsten ras oncogene expression on the polarized phenotype of MDCK cells were investigated. Stable transformed MDCK cell lines expressing the v-K-ras oncogene were generated via infection with a helper-independent retroviral vector construct. When grown on plastic substrata, transformed cells formed continuous monolayers with epithelial-like morphology. However, on permeable filter supports where normal cells form highly polarized monolayers, transformed MDCK cells detached from the substratum and developed multilayers. Morphological analysis of the multilayers revealed that oncogene expression perturbed the polarized organization of MDCK cells such that the transformed cells lacked an apical--basal axis around which the cytoplasm is normally organized. Evidence for selective disruption of apical membrane polarity was provided by immunolocalization of membrane proteins; a normally apical 114-kD protein was randomly distributed on the cell surface in the transformed cell line, whereas normally basolateral proteins remained exclusively localized to areas of cell contact and did not appear on the free cell surface. The discrete distribution of the tight junction-associated ZO-1 protein as well as transepithelial resistance and flux measurements suggested that tight junctions were also assembled. These findings indicate that v-K-ras transformation alters cell-substratum and cell-cell interactions in MDCK cells. Furthermore, v-K-ras expression perturbs apical polarization but does not interfere with the development of a basolateral domain, suggesting that apical and basolateral polarity in epithelial cells may be regulated independently.
Insights
Viral Kirsten ras oncogene expression disrupts epithelial cell polarity in MDCK cells, altering cell-substratum and cell-cell interactions. Apical polarization is perturbed, but basolateral domain development is unaffected, suggesting independent regulation.
Area of Science:
- Cell Biology
- Oncogenic Signaling
- Epithelial Biology
Background:
- MDCK cells are a standard model for studying epithelial cell polarity.
- Oncogene expression can significantly alter cell morphology and function.
- Understanding the role of Kirsten ras oncogene in cell polarity is crucial for cancer research.
Purpose of the Study:
- To investigate the impact of viral Kirsten ras (v-K-ras) oncogene expression on the polarized phenotype of Madin-Darby canine kidney (MDCK) cells.
- To determine how v-K-ras transformation affects cell-substratum and cell-cell interactions.
- To elucidate the specific effects of v-K-ras on apical and basolateral polarity development.
Main Methods:
- Generation of stable MDCK cell lines expressing v-K-ras using retroviral vectors.
- Culturing cells on plastic substrata and permeable filter supports to assess monolayer formation and polarization.
- Morphological analysis, immunolocalization of membrane proteins (apical and basolateral), and assessment of tight junction protein ZO-1 distribution.
- Transepithelial electrical resistance and flux measurements to evaluate tight junction integrity.
Main Results:
- Transformed MDCK cells formed multilayers on filter supports, detaching from the substratum, unlike normal polarized monolayers.
- v-K-ras expression disrupted the apical-basal axis organization within MDCK cells.
- Apical membrane proteins showed random distribution, while basolateral proteins remained localized to cell contact areas.
- Tight junctions, indicated by ZO-1 distribution and functional measurements, were assembled despite polarity disruption.
Conclusions:
- v-K-ras transformation significantly alters cell-substratum and cell-cell interactions in MDCK cells.
- v-K-ras expression selectively perturbs apical polarization.
- Apical and basolateral polarity in epithelial cells may be regulated independently, as basolateral domain development was not affected.