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Cloning and characterization of phorbol ester differentiation-resistant U937 cell variants
S C Kiley1, P D Adams, P J Parker
1Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, London, United Kingdom.
Cell Growth & Differentiation : the Molecular Biology Journal of the American Association for Cancer Research
|February 1, 1997
Summary
Differentiation-resistant U937 cells show altered protein kinase C (PKC) beta 2 localization and impaired beta 2-integrin transport. These changes prevent cell surface expression of adhesion molecules following 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- U937 human promonocytic leukemia cells are a model for studying cellular differentiation.
- 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inducer of differentiation in U937 cells.
- Protein kinase C (PKC) and beta 2-integrin adhesion molecules play crucial roles in cell differentiation and adhesion.
Purpose of the Study:
- To investigate the molecular mechanisms underlying TPA-induced differentiation resistance in U937 cells.
- To identify differences in protein kinase C (PKC) signaling and beta 2-integrin trafficking between wildtype and differentiation-resistant U937 cells.
Main Methods:
- Derivation of differentiation-resistant U937 cell lines by prolonged TPA exposure.
- Analysis of protein kinase C (PKC) isozyme expression, activation, and subcellular localization.
- Assessment of beta 2-integrin (cd11b and cd11c) expression and localization using flow cytometry and intracellular vesicle analysis.
Main Results:
- Differentiation-resistant U937 cells exhibited normal PKC expression and activation but altered subcellular localization of PKC beta 2.
- Resistant cells failed to express beta 2-integrin adhesion molecules on the cell surface after TPA treatment.
- TPA-induced translocation of cd11b and cd11c-containing vesicles to the cell surface was impaired in resistant cells.
Conclusions:
- PKC beta 2's colocalization with microtubules is essential for TPA-induced beta 2-integrin transport and cell surface expression.
- Events downstream of PKC activation, potentially involving cytoskeletal reorganization, are critical for U937 cell differentiation.
- Defects in these downstream pathways lead to differentiation resistance.