Expression of LIM kinase 1 is associated with reversible G1/S phase arrest, chromosomal instability and prostate cancer
© Davila et al; licensee BioMed Central Ltd. 2007
Received: 14 March 2007
Accepted: 08 June 2007
Published: 08 June 2007
LIM kinase 1 (LIMK1), a LIM domain containing serine/threonine kinase, modulates actin dynamics through inactivation of the actin depolymerizing protein cofilin. Recent studies have indicated an important role of LIMK1 in growth and invasion of prostate and breast cancer cells; however, the molecular mechanism whereby LIMK1 induces tumor progression is unknown. In this study, we investigated the effects of ectopic expression of LIMK1 on cellular morphology, cell cycle progression and expression profile of LIMK1 in prostate tumors.
Ectopic expression of LIMK1 in benign prostatic hyperplasia cells (BPH), which naturally express low levels of LIMK1, resulted in appearance of abnormal mitotic spindles, multiple centrosomes and smaller chromosomal masses. Furthermore, a transient G1/S phase arrest and delayed G2/M progression was observed in BPH cells expressing LIMK1. When treated with chemotherapeutic agent Taxol, no metaphase arrest was noted in these cells. We have also noted increased nuclear staining of LIMK1 in tumors with higher Gleason Scores and incidence of metastasis.
Our results show that increased expression of LIMK1 results in chromosomal abnormalities, aberrant cell cycle progression and alteration of normal cellular response to microtubule stabilizing agent Taxol; and that LIMK1 expression may be associated with cancerous phenotype of the prostate.
LIMK1 belongs to a family of unique LIM domain containing serine/threonine kinases (LIMK1 and LIMK2). It modulates actin dynamics by inactivating phosphorylation of cofilin, a member of the ADF (actin depolymerizing factor) family [1, 2]. LIMK1 is expressed predominantly in the brain but a modest expression of LIMK1 has been noted in other organs. Recently, a novel role for LIMK1 as an oncogene has been demonstrated in prostate and breast cancer cells [3, 4]. LIMK1 is upregulated in malignant prostate tissues compared to histologically normal or benign prostates . Biochemical and in vivo studies have indicated that LIMK1 is involved in promotion and maintenance of the invasive behavior in prostatic and breast cancer cells [3–5] and that when injected in nude mice, breast cancer cells overexpressing LIMK1 are capable of formation of osteolytic lesions in lower extremities and induction of tumor angiogenesis [4, 5]. Indirect evidence based on comparative genomic hybridization analysis have indicated a significant correlation between high-stage prostate cancers and chromosomal gains in the short arm of chromosome 7 (7q11.2) , which includes the region where LIMK1 is located (7q11.23).
The structural features of LIMK1 include two N-terminal LIM domains in tandem, a single PDZ domain and a C-terminal kinase domain [7, 8]. LIMK1 also contains a single nuclear localization signal and a single exit signal . Functional activation of LIMK1 requires phosphorylation at T508 mediated by p21-activated kinase PAK1 and ROCK [10, 11]. Activation of LIMK1 through ROCK promotes accumulation of F actin through inactivation of cofilin, a Rho phenotype, which results in formation of stress fibers . Importantly, LIMK1 is also activated by PAK1, which is the effector molecule of the Rac-induced lamellipodia formation or cell spreading .
LIMK1 undergoes a distinct pattern of activation during mitosis, becoming hyperphosphorylated and activated at the prometaphase and metaphase and gradually becoming inactivated during telophase and cytokinesis [13, 14]. Mitosis-specific activation and phosphorylation of LIMK1 is mediated by Cdk1  but not through active PAK or ROCK . The activation pattern of LIMK1 corresponds to the phosphorylation and dephosphorylation pattern of cofilin during mitosis, which is essential for proper cytokinesis . Thus, maintenance of optimum concentrations and activation of LIMK1 at specific mitotic phases are critical for normal cell cycle progression. Cdk inhibitor p57Kip interacts with LIMK1 and sequesters it to the nucleus  as a result of inhibition of Rho signaling by Cip/Kip family members. LIMK1 exhibits contrasting function of activation and inactivation of Cyclin D1 expression depending on the activation of Rac/Cdc42 or Rho-Rho kinase pathways . There also are conflicting reports on expression of Cdk inhibitors (p21Cip1 or p27Kip1) following inactivation of Rho-kinase or LIMK1 [18, 19]. To date, it is uncertain whether an active LIMK1 is essential for progression of cells through the G1/S phase. The other family member, LIMK2, also is activated through ROCK at T505 ; and it is involved in meiosis during maturation of Xenopus oocyte through phosphorylation of cofilin . Activation of ROCK also stimulates cell cycle progression partly through increased cyclin A levels via LIMK2 . Nonetheless, LIMK1 and LIMK2 exhibit different cellular functions and subcellular localizations [23, 24].
We have noted that LIMK1 but not LIMK2 is overexpressed in highly aggressive and metastatic PC3 prostate cancer cells and in prostate tumor tissues compared to benign prostatic hyperplasia (BPH-1) cells and normal prostatic epithelium. However, the precise role of LIMK1 in development of abnormal cellular processes, which might facilitate prostate tumor growth and behavior, is unclear. In this study, we demonstrate that increased expression of LIMK1 is associated with accumulation of chromosomal abnormalities, and development of cell cycle defects in cells that naturally express lower concentrations of LIMK1. We also show that expression of LIMK1 is higher in prostate tumors with higher Gleason Scores and incidence of metastasis.
Cell culture, antibodies and tumor samples
A benign prostatic hyperplasia cell line (BPH-1 cells) (generated by Hayward et al.  and initially obtained as a gift from P. Narayan, University of Florida) are routinely maintained in our laboratory in DMEM containing 10% FBS and antibiotic/antimycotic. BPH-1 cells were co-transfected with the ORFs of human LIMK1 or LacZ cloned in pIND vector and PVRxR plasmid (Invitrogen) to establish stable cell lines (BPHL and BPHLacZ) as described earlier . Stable cells resistant to G418 (Invitrogen) (500 mg/ml) and Zeocin (Invitrogen) (50 ng/ml), and expressing LIMK1 or LacZ upon induction with ponasterone A (ecdysone analog) were selected and maintained in DMEM/10%FBS/antibiotic/antimycotic. In some cases, cells were induced with ponasterone A (Invitrogen) (5 μ M) for 24 h prior to harvest. Anti-LIMK1 (Transduction Laboratories), anti-α-tubulin (Sigma) and anti-glyceraldehyde-3-phosphate dehydrogenase (Biogenesis) monoclonal antibodies, and anti-γ-tubulin (Sigma) polyclonal antibodies were used for immunoblotting, immunofluorescence and flow cytometry. Anti-GAPDH antibodies were used in immunoblots to monitor GAPDH expression as an internal control. A low-density tissue microarray (TMA) of prostate tumors containing 50 prostate tumors and 3 uninvolved prostate tissues was obtained from IMGENEX (Histo-Array, IMGENEX 2.0 mm core diameter) . The array provided by the manufacturer includes pathological reports and histories of metastasis for individual tumor tissues. The array was stained with anti-LIMK1 antibodies using immunohistochemistry. The anti-LIMK1 antibody is highly specific, does not cross react with LIMK2, and yields a 72 kD polypeptide band in western blots.
Immunoblot, immunofluorescence and immunohistochemistry
Total cell lysates were resolved in SDS-PAGE and subjected to immunoblotting using anti-LIMK1 and anti-GAPDH antibodies. For indirect immunofluorescence, BPHLacZ and BPHL cells were plated on poly-L-lysine coated glass coverslips and induced with ponasterone A for 24 h prior to staining and in some cases treated with Taxol (10 nM) for various time points. Cells were permeabilized and either singly or dually stained with combinations of Alexa Fluor 488-conjugated phalloidin (Molecular Probes), anti-α-tubulin and anti-γ-tubulin antobodies. Alexa Fluor 488 or Cy3-conjugated anti-mouse or anti-rabbit antibodies (Molecular Probes) were used singly or in combination as the secondary antibody. Nuclei were stained using 4–6-diamidino 2-phenylindole, dilactic (DAPI, Molecular Probes). Fluorescent images were visualized in an epifluorescent microscope (Nikon TE300). The paraffin-embedded TMA was subjected to immunohistochemistry (IHC) using a pepsin-based antigen retrieval protocol (BioGenex, San Ramon, CA) and anti-LIMK1 antibodies. A biotinylated multi-link goat anti-immunoglobulin for mouse, rabbit, guinea pig and rat was used as the secondary antibody. Nuclei were counterstained with Hematoxylin QS (Vector Laboratories). Positive signals were detected by HRP-conjugated streptavidin and DAB as the chromagen (BioGenex Multi-Link kit).
Immunostaining grade and statistical analysis
Immunostaining grades of the individual tumors in the array were assessed on the basis of a scale of 0 (no staining) to +4 (intense staining) . The percentage of cells at each scale was estimated first and then the decimal-equivalent of the percentage was multiplied by the appropriate intensity score to obtain a weighted average of the intensity score . This average, which will vary between 0 and 4, was referred to as the immunostaining score. To determine the discriminative ability of the cytoplasmic staining and nuclear staining scores, an approach based on receiver operating characteristic (ROC) curve analysis was used. ROC curves for the two measurements were calculated using the method of Pepe . The cut points were chosen based on the region where the slope of the ROC curve was the greatest. Based on that cut point, a chi-squared test was used to test for association between clinical outcome and if the score exceeded the threshold. The clinical outcome considered was the presence or absence of metastases. This approach was taken relative to a t-test because of the relatively limited sample size and the fact that a difference in discriminative ability would manifest in a way other as a difference in mean expression.
Cell synchronization and cell cycle analysis
BPHLacZ and BPHL cells were synchronized at the G1/S boundary using double thymidine (2 mM) treatment. Cells were treated with thymidine for 18 h, released from thymidine block for 8 h in fresh culture medium and blocked again with thymidine for 16 h. Cells were then released from the block in fresh medium containing bromodeoxyuridine (BrDU) (10 μ M). Ponasterone A was added 24 h before the last release of the thymidine block and cells were harvested at different times as specified. Cells were treated with nocodazole (0.1 μg/ml) for 18 h for synchronization at the G2/M boundary. Ponasterone A was added also at the same time. Cells were released from nocodazole block in fresh medium containing BrDU and collected at different time points as specified. Cells were reinduced every 24 h until they were harvested. Next, cells were stained with FITC-conjugated anti-BrDU antibodies and 7AAD using a BrDU labeling kit (Beckton Dickinson) according to manufacturer's specification and analyzed in a Flow cytometer (FACSCalibur, BD Biosciences). Raw data were analyzed using CellQuest and Modfit (BD Biosciences) software after elimination of aggregates. Cells present inside the gated area were used for calculation of percentage of cells in each phase.
Asynchronous BPHLacZ and BPHL cells were induced with Ponasterone A and treated with Taxol (10 nM) for 24 h and 48 h. Cell cycle progression and DNA synthesis were analyzed using BrDU incorporation and propidium iodide staining. BrDU was added to monitor DNA synthesis 16 h before harvesting. Cells were processed for flow cytometry as described before. Growth of Taxol-treated cells was monitored using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays which produce formazan crystals upon cleavage of tetrazolium rings by mitochondrial dehydrogenase from viable cells using the protocol published elsewhere . Values for MTT assays are presented as mean +SD. The significance of changes was determined by ANOVA (paired t-test) using Statview software (Abacus Concepts, Calabasas, CA). Statistical significance was established at P < 0.05.
Altered cell morphology and abnormal centrosome number were noted in cells overexpressing LIMK1
To study the morphology of mitotic cells, we monitored the distribution of α and γ tubulins in transfected cells as components of microtubules and centrosomes respectively, by immunofluorescence analysis. Our results showed abnormal centrosome duplication and spindle assembly, such as multipolar spindles and abnormal chromosomal alignment at the equatorial plate in mitotic BPHL cells. Appearance of abnormal/disorganized spindles and multiple centrosomes were noted in 12% of the cells undergoing mitosis (Figure 1C2 and 1C3). None of these abnormalities was noted in mitotic BPHLacZ cells (Figure 1C1). Furthermore, 9–10% of the BPHL cells exhibited smaller DAPI stained chromosomal masses presumably micronuclei (Figure 1C4 and 1C5). Presence of multinucleated cells (Figure 1C5) also was noted in BPHL cells (5%), which express increased amounts of LIMK1 (Figure 1D).
LIMK1 expression transiently arrested cells at G1/S phase
Expression of LIMK1 delayed progression of cells through G2/M phase
LIMK1 prevented paclitaxel-mediated G2/M arrest and multinuclearity
Immunofluorescence analysis of α tubulin and DAPI staining showed that a significant number of Taxol-treated BPHLacZ cells were with multiple nuclei (40%) (Figure 4F and 4G) and about 25% of the cells were arrested in metaphase (Figure 4F). No multinucleated BPHL cell was detected following Taxol treatment. Only a small percentage of these cells were in metaphase and anaphase (2% and 6%, respectively) (Figure 4F and 4G). MTT-based growth analysis indicated that Taxol treatment induced growth retardation in BPHLacZ cells within 24 h compared to untreated (vehicle) cells. No inhibition of growth of Taxol treated BPHL cells was observed at 24 h compared to DMSO treated cells. Growth patterns of untreated BPHLacZ and BPHL cells showed an overall slower proliferation rate of BPHL cells compared to BPHLacZ cells (Figure 4H).
Expression of LIMK1 was associated with prostate cancer
Distribution of tumors based on the Gleason Scores and history of metastasis with respect to nuclear and cytoplasmic staining of LIMK1
Incidence of metastasis
Cytoplasmic and Nuclear Staining
Cytoplasmic Staining Only
No Incidence of metastasis
Cytoplasmic and Nuclear Staining
Cytoplasmic Staining Only
In this study, the consequence of overexpression of LIMK1 in prostate epithelial cells, and any association of LIMK1 with prostate cancer have been assessed. Here we provide evidence that an elevated expression of LIMK1 induces abnormal centrosomal multiplication and generation of multipolar spindles in BPH-1 cells. Disorganization of spindles in BPHL cells may occur as a result of altered stability of microtubules. In a recent study, LIMK1 has been shown to be involved in microtubule destabilization . Appearance of multinucleated cells and micronuclei, markers for genetic instability, also were evident in response to increased expression of LIMK1. Although centrosomal defects that often occur at the advanced stages of cancer are believed to contribute to genetic instability by altering fidelity of chromosomal segregation during mitosis, centrosomal defects also occur concurrently with chromosomal abnormalities and cytological changes in early stages of cancer [33–36]. Pihan et al demonstrated that progressive dysfunction of centrosomes and misallocation of centrosomes occur during prostate cancer progression and such changes increase with increasing Gleason Score in invasive cancer . It is possible that the centrosome and spindle defects induced by increased expression of LIMK1 may play a role in phenotypic alterations in BPHL cells. Such changes may also cause BPHL cells to become highly invasive compared to naturally noninvasive BPH-1 parental cells. Our earlier studies showed that expression of LIMK1 made BPHL cells highly invasive compared to BPHLacZ cells .
Our results on cell cycle analysis indicated an altered cell cycle progression in BPHL cells, which showed a distinct but transient arrest of cells at the G1/S phase and delayed progression of cells through the G2/M phase. These findings are in concurrence with the idea that chromosomal abnormalities are intimately involved in generation of cell cycle defects, which frequently become a phenotypic characteristic of advanced cancers . Although LIMK1 expression led to a transient arrest in G1/S phase it did not inhibit cell proliferation as evident from BrDU incorporation, which is in contrast to the earlier reports on the inhibitory effect of LIMK1 on NIH3T3 cell proliferation . The transient nature of G1 arrest implies a temporary block that possibly depends on expression of one or more key proteins. It is possible that the block is released when concentrations of these proteins reach a threshold level.
Our experiments showing a delayed G2/M transition following increased expression of LIMK1 suggest the possibility of cytokinesis defects rendered by increased concentration of LIMK1. This observation supports the earlier report showing that expression of LIMK1 in HeLa cells induced cytokinesis defects [13, 14]. Nonetheless, this defect did not arrest cells permanently in the mitotic phase. G2/M synchronized cells progressed to G1/S and incorporated BrDU at a later time showing resumption of the cell cycle progression. This observation suggests that the concentration of LIMK1 needs to be tightly regulated for proper progression of mitosis and cytokinesis. These observations are supported by our MTT assay data, which showed an overall slower growth rate of BPHL cells compared to BPHLacZ cells. Expression of LIMK1 also promoted resistance to Taxol induced metaphase arrest, cell growth retardation and appearance of multinucleated cells as a possible predisposition for apoptosis . It is speculated that the microtubule destabilizing effect of LIMK1 interferes with the Taxol-induced microtubule stabilization and inhibits normal cellular response to Taxol. However, the exact mechanism whereby LIMK1 expression confers resistance to Taxol induced cellular responses is not clear and is a subject for further study.
Our studies on expression of LIMK1 in prostate tumors showed that all tumor samples were positive for weak to strong cytoplasmic expression of LIMK1 compared to no or very weak expression in the luminal cells of uninvolved prostate glands. Expression of LIMK1 was noted in basal cells of uninvolved tissues by IHC. Because basal cells are highly proliferative in nature, they may require expression LIMK1 for rapid cell growth. Furthermore, increased expression of LIMK1 in stromal cells in close proximity to the tumor areas in majority of the tumor samples may have a functional significance in phenotypic changes associated with advanced tumors. It has been documented that reciprocal interactions between stroma and tumor epithelium play important roles in acquisition of metastatic phenotypes by the prostate tumors [41, 42]. Our studies also indicated a possible correlation between the extent of nuclear or cytoplasmic expression of LIMK1 in the luminal cells and Gleason Scores. Analysis of immunostaining scores also showed an almost significant association between increased expression of nuclear LIMK1 and history of metastasis. Nonetheless, our study strongly suggests an association between expression of LIMK1 and prostate cancer.
To summarize, this study provides evidence that an elevated expression of LIMK1 generates chromosomal instability and cell cycle defects. This report also shows that overexpression of LIMK1 is associated with prostate cancer. The level of expression of LIMK1 achieved in our experiment was either parallel to or less than that noted in advanced prostate tumors and in PC3 prostate cancer cells; this suggests that over expression of LIMK1 in prostate tumors may suffice to elicit the biological effects noted here. Furthermore, overexpression of LIMK1 conferred resistance to Taxol-mediated mitotic arrest and multinucleated giant cell formation. Taxol, an agent widely used for treatment of cancer, works through the inhibition of cell cycle by activation of mitotic checkpoints. Expression of LIMK1 may deregulate mitotic checkpoints in cancer cells whereby it can promote development of resistance of advanced prostate cancer to taxenes. Further studies are required to assess the relevance of LIMK1- mediated deregulation of cell cycle in progression of prostate cancer.
LIMK1 is involved in regulation of actin cytoskeleton and microtubule dynamics and as a result, plays important roles in cell division and cell behavior. Our studies imply that the concentration of LIMK1 needs to be tightly regulated for proper cell cycle progression and that up regulation of LIMK1 may contribute to tumor progression through altered cell cycle pattern and chromosomal defects.
We thank Dr. Annette Khaled for her invaluable input in flow cytometry data analysis. This work was funded by a grant to R.C. from the Department of Defense (PCRP grant PC041048).
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