Divergent impact of actin isoforms on cell cycle regulation

Abstract

We have shown that cytoplasmic actin isoforms play different roles in neoplastic cell transformation. β-Cytoplasmic actin acts as a tumor suppressor, affecting epithelial differentiation, cell growth, cell invasion and tumor growth of colon and lung carcinoma cells. In contrast, γ-cytoplasmic actin enhances malignant features of tumor cells whose actin network regulation is carried out via the γ-actin isoform. The goal of this study was to describe the role of cytoplasmic actins in cell cycle regulation of breast cancer cell lines MCF-7 and MDA-MB-231. The distinct roles of each cytoplasmic actin in the cell cycle driving were observed. β-Actin as well as γ-actin down-regulation inhibited proliferation of breast cancer cells, but only down-regulation of β-actin induced a significant decrease in diploid cell population and accumulation of tetraploid cells. Down-regulation of β-actin stimulated cyclin A2, B1 and D3 expression, whereas down-regulation of γ-actin reduced expression of these cyclins in both cell lines. Moreover, cyclin B1 and γ-actin were co-localized in mitotic control and β-actin-deficient cells. In mitotic MCF-7 cells down-regulation of β-actin caused an enrichment of prophase/metaphase population compared with control. γ-Actin down-regulation induced telophase enrichment. ERK1/2 and γ-actin co-localization and possible selective binding were revealed in MCF7 cells. β-Actin down-regulation induced ERK1/2 activation, while γ-actin down-regulation led to reduction of p-ERK1/2. A direct interaction of ERK1/2 with γ-actin and cyclin A2 in the same protein complex was also discovered. We suggest that γ-actin down-regulation leads to decrease of cyclin A2 level, inhibits ERK1/2 signaling and deceleration of breast cancer cells proliferation.

Keywords: Cancer; ERK1/2; actin isoforms; cyclins.

Figure 1.

Down-regulation of β- or γ-actin expression by corresponding shRNAs in breast cancer cells leads to phenotypical changes; (a, b). Immunofluorescent staining of MCF-7 (a) and MDA-MB-231 (b) cells with β- or γ-actin depletion by corresponding shRNAs. Both β- and γ-actin depletion induce major and different morphological changes. Bar, 10 µm; (c, d) Down-regulation of cytoplasmic actins in MCF-7 (c) and MDA-MB-231 (d) cells. WB analysis. Graphs represent relative actins expression (Mean ± SEM).

Figure 2.

The effects of β/γ-actin down-regulation on cell growth and cell cycle in vitro. (a, b) MCF-7 and MDA-MB-231 cells with β- or γ-actin depletion by corresponding shRNAs were cultured after 2 d of infection for the time indicated to analyze the effect on cell growth. (c) After 4 d of β- or γ-actin depletion by corresponding shRNAs the MCF-7 cells were stained with propidium iodide, and cell cycle distributions were analyzed by flow cytometry. (d) High content analysis of cell cycle distributions of flow cytometric data was performed after 6 d of β- or γ-actin depletion by corresponding shRNAs in the MCF-7 cells. (e) The effects of β- or γ-actin depletion on mitotic phases were also analyzed. The Y-axis indicates the percentage of cells in different phases of mitosis.

Figure 3.

Cyclins are regulated by β-/γ-actins level. (a, b) WB analysis of MCF-7 (a) and MDA-MB-231 (b) cells with down-regulated β- or γ-actins. (c) Subcellular localization of Cyclin A2 (red) and cyclin B1 (green) by immunofluorescence staining of MDA-MB-231 cells with down-regulated β- or γ-actins. (d) Cyclin B1/γ-actin PLA analysis of MCF-7 cells with down-regulated β- or γ-actins. Graph represents relative fluorescence intensity (Mean ± SEM). (e) Cyclin D3 (red) immunofluorescence images of MDA-MB-231 cells with down-regulated β- or γ-actins. F. Cyclin H (green) and β-actin (red) immunofluorescence images of MCF-7 cells with down-regulated β- or γ-actins. (g) Cyclin H (green) immunofluorescence images of MDA-MB-231 cells with down-regulated β- or γ-actins. DAPI/DNA staining (blue). Bar, 10 μm.

Figure 4.

ERK1/2 activity is regulated by γ-actin. (a) Laser Scanning Microscopy (LSM) of MCF-7 cells with down-regulated β- or γ-actins with β-actin (green), γ-actin (purple) or p-ERK1/2 (red) immunofluorescent staining. DAPI/DNA staining (blue). Scale bars represent 10 µm. (b)WB analysis of p-ERK1/2 in MCF-7 cells with down-regulated β- or γ-actins by corresponding shRNAs. (c) p-ERK1/2 immunoprecipitation analysis of MCF-7 cells with down-regulated β- or γ-actins. (d) p-ERK1/2/γ-actin PLA analysis of MCF-7 and MDA-MB-231 cells with down-regulated β- or γ-actins. Immunofluorescence images of p-ERK1/2/γ-actin PLA dots at nuclear (green) and lamellar (red) z-levels in MCF-7 (upper panel) and MDA-MB-231(lower panel) cells with down-regulated β- or γ-actins. Bar, 10 µm. Graphs represent relative amount of PLA dots at nuclear (green) and lamellar (red) z-levels (Mean ± SEM).