Cyclin-dependent kinase 2/cyclin E complex is involved in p120 catenin (p120ctn)-dependent cell growth control: a new role for p120ctn in cancer

Abstract

Depending on its cellular localization, p120 catenin (p120ctn) can participate in various processes, such as cadherin-dependent cell-cell adhesion, actin cytoskeleton remodeling, and intracellular trafficking. Recent studies also indicate that p120ctn could regulate cell proliferation and contact inhibition. This report describes a new function of p120ctn in the regulation of cell cycle progression. Overexpression of the p120ctn isoform 3A in human colon adenocarcinoma cells (HT-29) results in cytoplasmic accumulation of the protein, as observed in many tumors. This cytoplasmic increase is correlated with a reduction in proliferation and inhibition of DNA synthesis. Under these conditions, experiments on synchronized cells revealed a prolonged S phase associated with cyclin E stabilization. Both confocal microscopy and biochemical analysis showed that cyclin E and cyclin-dependent kinase 2 colocalized with p120ctn in centrosomes during mitosis. These proteins are associated in a functional complex evidenced by coimmunoprecipitation experiments and the emergence of Thr199-phosphorylated nucleophosmin/B23. Such post-translational modification of this centrosomal target has been shown to trigger the initiation of centrosome duplication. Therefore, p120ctn-mediated accumulation of cyclin E in centrosomes may participate in abnormal amplification of centrosomes and the inhibition of DNA replication, thus leading to aberrant mitosis and polyploidy. Because these modifications are often observed in cancer, p120ctn may represent a new therapeutic target for future therapy.

Figure 1

Overexpressed p120ctn accumulates in the cytoplasm. A, WT HT-29 cells (WT) were transfected with pEF6-GFP (GFP) or pEF6-GFP-p120ctn (GFP-p120ctn). Expression levels of p120ctn, GFP, and E-cadherin were analyzed by Western blotting of whole-cell lysates. Actin blotting was used as a loading control. B, GFP or GFP-p120ctn HT-29 cells cultured for 48 h on coverslips were fixed in 3% paraformaldehyde before confocal microscopy analysis. Bar, 50 μm. C, total GFP or GFP-p120ctn cell lysates were separated into cytoplasmic (C) and membrane-associated (M) fractions and detection of endogenous and exogenous p120ctn was achieved by Western blotting.

Figure 2

Overexpression of p120ctn slows down HT-29 cell proliferation. A, HT-29 GFP and HT-29 GFP-p120ctn cells were cultured for 120 h and observed under phase-contrast microscopy. Bar, 50 μm. B, HT-29 GFP and HT-29 GFP-p120ctn cells were seeded at 2 × 10⁴ cells per well, grown for the indicated times and living cells, then counted using trypan blue solution. Points, mean of three independent experiments; bars, SE. C, BrdUrd incorporation assay. BrdUrd (5 μmol/L) was added for 15 min to the culture medium of 24-h precultured HT-29 GFP (dark gray line) or HT-29 GFP-p120ctn (gray line) cells. After fixation and BrdUrd immunostaining, positive cells were counted using a Becton Dickinson Facstar flow cytometer. Control cells non–treated with BrdUrd were used as negative control (black line). D, top, HT-29 GFP (left) or HT-29 GFP-p120ctn (right) cells were synchronized at G₁-S transition with aphidicolin (5 μg/mL, 24 h) and released in fresh medium for the indicated time. DNA content was then analyzed by FACS after propidium iodide staining. Bottom, histograms indicate the percentage of cells in G₁, S, and G₂-M phases. Asy, asynchronized.

Figure 3

Cyclin E protein is stabilized by p120ctn overexpression. A, top, asynchronized HT-29 GFP or HT-29 GFP-p120ctn cells or cells synchronized at G₁-S transition with aphidicolin blockade and released in fresh medium for indicated times were lyzed. Total lysates were analyzed for cyclin E, cdk2, p120ctn, and actin expression by Western blotting. Bottom, histograms represent the densitometric ratio of cyclin E to actin levels. Columns, mean of three independent experiments; bars, SE. B, in parallel, the cells were immunostained for cyclin E and analyzed by confocal microscopy. Bar, 50 μm. C, G₂-M–enriched GFP cells were treated or not with the indicated concentrations of proteasome inhibitor MG132. Total cell lysates were then analyzed for cyclin E and cdk2 expression by Western blotting. Actin staining was used as a loading control.

Figure 4

p120ctn localizes to the mitotic furrow and the centrosomes in mitotic HT-29 cells. A, HT-29 GFP (top) and HT-29 GFP-p120ctn (bottom) cells were grown for 48 h on coverslips, fixed with 3% paraformaldehyde, and immunostained for β-tubulin before observation under a confocal microscope DNA was stained blue with 4′,6-diamidino-2-phenylindole (DAPI). Chosen fields are representative of mitotic cells in each condition. Bar, 10 μm. B, centrosome fractions were enriched from mitotic HT-29 GFP-p120ctn cells using a discontinuous sucrose gradient (see Material and Methods). Antibodies against p120ctn and Aurora A were used for Western blotting analyses of each collected fraction. C, left, centrosome- and cell membrane–enriched fractions obtained from the previously described discontinuous sucrose gradient were analyzed by Western blotting using antibodies against p120ctn, cyclin E, cdk2, Aurora A, E-cadherin, and actin. Right, histograms represent the densitometric ratio of p120ctn, cyclin E, and cdk2 to Aurora A levels in centrosomal fractions. Columns, mean of three independent experiments; bars, SE.

Figure 5

p120ctn interacts with the cdk2/cyclin E complex. HT-29 GFP-p120ctn cells were grown for 48 h on coverslips, fixed with 3% paraformaldehyde, and immunostained for cyclin E and cdk2 (A) or phosphorylated nucleophosmin (P-nucleophosmin; C) before confocal microscopic analysis. DNA was stained blue with DAPI. Bar, 10 μm. B, left, HT-29 GFP or GFP-p120ctn cells were synchronized at G₁-S transition with aphidicolin and released in fresh medium for 6 h. cdk2 was immunoprecipitated (IP) from total cell lysates and levels of p120ctn, cyclin E, and cdk2 were detected by Western blotting. Right, histograms represent the densitometric ratio of cyclin E and p120ctn to immunoprecipitated cdk2 levels. This experiment was reproduced at least thrice. D, centrosomeenriched and total fractions from HT-29 GFP and GFP-p120ctn cells were analyzed by Western blotting using antibodies against nucleophosmin phosphorylated on Thr¹⁹⁹ (top). Coomassie blue staining of the transferred membrane was used as a loading control (bottom).

Figure 6

p120ctn overexpression leads to overduplication of centrosomes and polyploidy. A, HT-29 GFP-p120ctn cells were grown for 48 h on coverslips, fixed with 3% paraformaldehyde, and immunostained for γ-tubulin before confocal microscopic observation. Bar, 10 μm. B, the number of centrosomes per mitotic cell was then quantified under confocal microscopy. Multipolar mitotic cells were represented as the percentage of total mitotic cells (n = 200). *, P < 0.05, Student’s t test (left). Histogram showing the repartition of HT-29 GFP-p120ctn cells with supernumerary centrosomes (n = 200; right). C, polynuclear HT-29 GFP-p120 cells were observed under phase-contrast microscopy after 48 h of culture. D, caspase-3/caspase-7 activity was analyzed using Apo-One homogeneous assay in HT-29 GFP or HT-29 GFP-p120ctn cells according to the manufacturer’s instructions. Fluorescence emission at 527 nm was quantified using a Fluoroskan Ascent. All experiments were done in triplicate.