The human cervical cancer cell line HeLa was provided by the late Professor Masakatsu Horikawa, Faculty of Pharmaceutical Sciences, Kanazawa University (Kanazawa, Japan) (28). The human colon cancer cell lines HT-29, HCT116 and SW48 were purchased from ATCC (Rockville, MD). The human colon cancer cell lines DLD-1 and LoVo were purchased from the Human Science Research Resources Bank (Osaka, Japan). The human colon cancer cell lines SW480 and SW620 (29) were provided by Dr Ryuichi Yatani, Mie University School of Medicine (Mie, Japan). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (50 U/ml), and streptomycin (50 μg/ml) at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. Human microvascular endothelial cells (HMVEC) (Kurabo Industries Ltd., Osaka, Japan) were maintained in HuMedia-MvG in accordance with the supplier’s instructions. Immortal OKF keratinocytes (OKF6/TERT-2) were kindly provided by Dr J.G. Rheinwald (Harvard Medical School, Boston, MA) and were cultured in keratinocyte serum-free medium (K-sfm, BD Biosciences, San Diego, CA) with 30 μg/ml bovine pituitary extract, 0.1 ng/ml EGF and 0.4 mM Ca²⁺(30). This culture condition permits cells to form cadherin- and desmosome-mediated junctions, and for some cells to stratify.

Anti-RhoGDIα antibody (sc-360), anti-RhoGDIβ antibody (sc-6047) and the blocking peptide (sc-6047P) for the anti-RhoGDIβ antibody (sc-6047) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-GFP (ab290), anti-γ-tubulin (clone GTU-88) and anti-RhoGDIβ (556498) antibodies were purchased from Abcam (Cambridge, UK), Sigma-Aldrich (St. Louis, MO) and BD Biosciences (San Jose, CA), respectively. The antigen peptides for the anti-RhoGDIβ (sc-6047) and anti-RhoGDIβ (556498) antibodies were amino acid residues 175–194 and 4–21, respectively, of human RhoGDIβ. Anti-RhoGDIα/RhoGDIβ antibody (66586E) was purchased from Pharmingen (San Diego, CA). Peroxidase-conjugated anti-mouse and anti-rabbit IgG antibodies were purchased from DakoCytomation (Glostrup, Denmark). Peroxidase-conjugated anti-goat IgG antibody was purchased from Nichirei Corporation (Tokyo, Japan).

Six-week-old female ICR mice were obtained from Japan SLC Inc. (Shizuoka, Japan) and maintained under pathogen-free conditions. Seven-week-old mice were anesthetized with pento-barbital, then blood was gently drained from the inferior vena cava using a heparinized syringe equipped with a 22-gauge needle. Leukocytes and erythrocytes were isolated from blood using Lympholyte^®-Mammal (Cedarlane Laboratories Ltd., Burlington, Canada) according to the manufacturer’s instructions and were lysed in Laemmli buffer (31). After blood collection, organs were quickly removed, washed with ice-cold phosphate-buffered saline (PBS), minced into small pieces and lysed in Laemmli buffer. All experiments using mice were approved by the Committee on Experimental Animals at Kanazawa Medical University and conducted in accordance with their guidelines.

Protein concentrations of lysed cells and organs were measured using the Bradford ULTRA kit (Novexin Ltd., Cambridge, UK). Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes (Millipore, Billerica, MA). The membranes were then probed with a primary antibody, followed by incubation with a peroxidase-conjugated secondary antibody. Immunoreactive proteins were visualized using ECL Plus reagents (GE Healthcare UK Ltd., Little Chalfont, UK). In the immunoblot experiments, we used RhoGDIα as a loading control because of its universal expression. We applied the same amount of protein for immunoblot experiments. The samples from organs contain several kinds of extracellular matrices at various levels and the cell numbers contained in each samples was not constant, therefore the expression levels of RhoGDIα among various organs were not as constant as those among cultured cell lines.

For immunofluorescence staining, cells were grown on 35-mm culture dishes (BD Biosciences, San Jose, CA) or Lab-Tek II chamber slides (Nalge Nunc International, Naperville, IL). Cells were fixed with freshly prepared 4% paraformaldehyde for 2 min, permeabilized with 0.5% Triton X-100 for 10 min and fixed again with 4% paraformaldehyde for 10 min at room temperature. In some experiments, cells were simply fixed with 99.8% methanol for 30 min. After washing with PBS, cells were incubated with 0.5% bovine serum albumin (BSA) in PBS for 60 min at room temperature and then incubated overnight at 4°C with primary antibodies diluted in 0.5% BSA in PBS (1:2,000 for anti-γ-tubulin antibody or 1:200 for all other primary antibodies). After three washes with PBS, cells were incubated for 60 min at room temperature with secondary antibodies (Invitrogen, Carlsbad, CA) diluted 1:400 with 0.5% BSA in PBS containing 0.1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI). After washing with PBS, cells were mounted with Prolong Gold (Invitrogen). For the blocking experiment, anti-RhoGDIβ antibody (sc-6047) was incubated with 10-fold concentration of the blocking peptide for 60 min at room temperature before use. Images were obtained using an Axiovert 200 inverted fluorescence microscope or LSM710 confocal microscope (Carl Zeiss, Jena, Germany).

The entire sequence was amplified by PCR using pcDNA3.1-LyGDI, an expression vector for wild-type human RhoGDIβ (15). The product was then subcloned into pAcGFP1-C3 and used as pAcGFP-RhoGDIβ. Cells were transfected with the expression plasmids using Lipofectamine 2000 (Invitrogen). To obtain cell lines that stably expressed the introduced genes, G418-resistant cells were isolated in medium containing 800 μg/ml G418 (Nacalai Tesque Inc., Kyoto, Japan).

HeLa cells that stably expressed GFP-RhoGDIβ were cultured in 35-mm glass-bottomed dishes (Asahi Glass Co. Ltd., Tokyo, Japan). The cells were maintained at 37°C in a humidified atmosphere of 5% CO₂ and 95% air in an enclosed stage incubator (Incubator-XL, Carl Zeiss) built on top of an Axiovert 200 M inverted microscope. Time-lapse images of green fluorescence and differential interference contrast (DIC) were acquired on an Axiovert 200 M controlled by AxioVision image processing and analysis system 4.4 (Carl Zeiss). The onset of mitosis was considered to be the beginning of cell rounding, the onset of anaphase was defined as the beginning of chromosome segregation and the end of cytokinesis was recognized when the cleavage furrow maximally contracted. We analyzed the progression of mitosis temporally in cells in which these mitotic processes could be clearly recognized. The entire duration of mitosis was measured as the time from the beginning of cell rounding to the maximal contraction of the cleavage furrow.

Three different 25-mer Stealth RNAi duplexes targeting human RhoGDIβ and negative control Stealth RNAi were purchased from Invitrogen. The sequences of the three Stealth RNAi are as follows: no. 153, sense 5′-GAGCUGGACAGCAAGCUCAAUUAUA-3′, anti-sense 5′-UAUAAUUGAGCUUGCUGUCCAGCUC-3′; no. 469, sense 5′-GCCUGAAAUACGUUCAGCACACCUA-3′, anti-sense 5′-UAGGUGUGCUGAACGUAUUUCAGGC-3′; and no. 800, sense 5′-GGUCCCUCUUCAACACUGCCACAUU-3′, anti-sense 5′-AAUGUGGCAGUGUUGAAGAGGGACC-3′. Stealth RNAi duplexes were transfected into HeLa cells using Lipofectamine 2000 according to the manufacturer’s protocol.

Cells were fixed with 20% ethanol and incubated with 0.1% RNase (Type II-A, Sigma-Aldrich, St. Louis, MO) for 30 min at 37°C. The cells were stained with propidium iodide (50 μg/ml) and analyzed using a FACSort flow cytometer (BD Biosciences, San Jose, CA).

Differences between values were analyzed by the two-tailed Mann-Whitney U-test using the statistics function of KaleidaGraph (Version 4.1). P<0.05 was considered significant.

To confirm the previous observations showing the expression of RhoGDIβ in cells other than those in the hematopoietic cell lineage (13–17), we examined the levels of RhoGDIβ protein in various mouse organs (Fig. 1A, upper panel). RhoGDIβ was expressed in mouse brain, lung, trachea, esophagus, adrenals, bladder, blood vessels, small and large intestine, although the expression levels were much lower than in hematopoietic cells such as those from the thymus, spleen and leukocytes. Blood was thoroughly drained before the isolation of organs, but slight contamination of hematopoietic cells was unavoidable. Therefore, it could not be ruled out that very low expression of RhoGDIβ reflects contamination of hematopoietic cells. In contrast, RhoGDIα was expressed at various levels in all examined tissues (Fig. 1A, lower panel). Consistent with the expression of RhoGDIβ in the large intestine and in blood vessels, RhoGDIβ was expressed in cultured human colon cancer cells and microvascular endothelial cells (HMVEC) (Fig. 1B). RhoGDIβ was also expressed in other types of cultured epithelial cells, such as HeLa and OKF6/TERT-2 human keratinocytes (Fig. 1B).

We examined the subcellular localization of RhoGDIβ in cultured colon cancer cells by immunofluorescence staining. Simultaneous staining with anti-RhoGDIβ and anti-γ-tubulin antibodies showed the colocalization of them in DLD-1 human colon cancer cells (Fig. 2A, upper panels). Similar results were also obtained in HT-29, HCT116, LoVo, SW48, SW480 and SW620 human colon cancer cell lines. Representative images of simultaneous staining with anti-RhoGDIβ and anti-γ-tubulin antibodies in LoVo, HT-29 and HCT116 human colon cancer cells are shown in Fig. 2B. RhoGDIβ colocalized with γ-tubulin also in OKF6/TERT-2 human keratinocyte (Fig. 2C, upper panels) and HeLa cells (Fig. 2D, upper panels). During interphase, the colocalization of RhoGDIβ with γ-tubulin was not as clear as during metaphase. Such centrosome staining pattern is likely to be associated with centrosome maturation. The staining with anti-RhoGDIβ antibody was abolished by pretreatment of the antibody with antigen peptide (Fig. 2A, C and D, lower panels). Unlike RhoGDIβ, RhoGDIα did not colocalize with γ-tubulin in colon cancer cells and HeLa cells (data not shown).

We used GFP-RhoGDIβ to confirm the localization of RhoGDIβ to centrosomes. pAcGFP-RhoGDIβ was transfected into HeLa cells and the cells stably expressing the introduced genes were selected (Fig. 3A). Expression of GFP-RhoGDIβ did not affect the levels of endogenous RhoGDIβ protein (Fig. 3A, right panel). In living cells, localization of GFP-RhoGDIβ was distinct from localization of GFP only. Specifically, GFP-empty localized all over the cell, although it was brighter in the nuclei than in cell cytoplasm, while GFP-RhoGDIβ localized predominantly to the cytoplasm (Fig. 3B). It was difficult to observe centrosomal localization of GFP-RhoGDIβ in living cells because of its granular localization throughout the cytoplasm. To confirm the centrosomal localization of GFP-RhoGDIβ, cells were fixed and stained with both anti-GFP and anti-γ-tubulin antibodies. GFP-RhoGDIβ colocalized with γ-tubulin in both metaphase and interphase cells, while GFP-empty did not (Fig. 3C).

Our observations suggested that RhoGDIβ had a role related to centrosome function. To investigate this, we used time-lapse microscopy to observe the mitotic progression in HeLa cells stably expressing GFP-RhoGDIβ. The incidence of morphologically aberrant cytokinesis, in which the cell shape was distorted, increased about 4-fold in GFP-RhoGDIβ expressing cells (Fig. 4A). Representative images of aberrant cytokinesis of GFP-RhoGDIβ-expressing cell are shown (Fig. 4B). To examine the defects in cytokinesis in detail we analyzed the time-lapse images of the cells, in which the onset of mitosis, the onset of anaphase, and the end of cytokinesis could be clearly distinguished (Fig. 4C). In these cells, morphologically aberrant cytokinesis was observed in 70% (14/20) of cells expressing GFP-RhoGDIβ, but was not observed in cells expressing GFP-empty. Duration from the onset of anaphase and from the onset of anaphase to the end of cytokinesis was significantly increased in cells expressing GFP-RhoGDIβ compared with those of cells expressing GFP-empty (Fig. 4D). These observations indicated that GFP-RhoGDIβ affected the mitotic processes including anaphase and cytokinesis in HeLa cells, suggesting that RhoGDIβ plays a role in these mitotic processes.

We examined the effect of RhoGDIβ knockdown in HeLa cells using three different siRNAs. All siRNAs decreased the expression of RhoGDIβ by about 90% and did not decrease the expression of RhoGDIα 72 h after transfection (Fig. 5A). Knockdown of RhoGDIβ by all three siRNAs increased the incidence of monopolar spindle mitosis (Fig. 5B). In contrast with the appearance of monopolar mitotic cells, a slightly higher frequency of multiple centrosomes was observed only in cells that had RhoGDIβ knocked down by no. 469 siRNA (Fig. 5C). Representative images of monopolar spindle mitosis and multiple centrosomes mitosis are shown (Fig. 5D). Therefore, we concluded that RhoGDIβ knockdown induced inhibition of centrosome separation rather than multipolar mitosis. The increase of polyploid (8, 16c or more) cells after RhoGDIβ knockdown as shown in Fig. 5E were thought to mainly result from monopolar mitosis. Overall, our data show that both upregulation and downregulation of RhoGDIβ constitute key events leading to perturbed mitotic processes in cancer cells.