Cell cycle regulated phosphorylation of LIMD1 in cell lines and expression in human breast cancers

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Cancer Letters 267 (2008) 55–66 www.elsevier.com/locate/canlet

Cell cycle regulated phosphorylation of LIMD1 in cell lines and expression in human breast cancers Christopher Jack Huggins a,b, Irene L. Andrulis a,c,* a

Fred A. Litwin Centre for Cancer Genetics, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ont., Canada M5G 1X5 b Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ont., Canada c Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ont., Canada Received 21 August 2007; received in revised form 29 February 2008; accepted 3 March 2008

Abstract LIMD1 is a member of the ZYXIN family of related proteins which includes AJUBA, TRIP6, LPP, WTIP, migfilin, and ZYXIN. The LIMD1 locus, 3p21.3, has been shown to undergo loss of heterozygosity in neoplastic tissues, suggesting potential tumor suppressor function. To further understand the role of LIMD1 in cancer, we have characterized endogenous expression of the LIMD1 protein and evaluated LIMD1 RNA expression in primary human breast tumors. LIMD1 levels were found to be constant throughout the cell cycle, but LIMD1 is phosphorylated during mitosis in HeLa cells. In addition, we observed colocalization of endogenous LIMD1 with vinculin at focal adhesions. In the MDA–MB435 cell line, which lacks LIMD1 expression, we detected methylation of the putative promoter region. LIMD1 mRNA expression was found to vary among primary human breast tumors; however, differences in LIMD1 expression in human breast cancers were not associated with DNA methylation of the predicted promoter region. These results suggest that some breast tumors have altered expression of LIMD1 RNA and that LIMD1 may be involved in cell anchoring via focal adhesions and in the cell cycle, particularly during mitosis. Ó 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: LIM domain containing gene 1 (LIMD1); Breast cancer; DNA methylation; Focal adhesions

1. Introduction LIMD1, a member of the ZYXIN family of proteins, was originally isolated in a functional mouse assay designed to identify genomic regions of loss [1]. The LIMD1 gene encodes a 676 amino acid pro* Corresponding author. Address: Fred A. Litwin Centre for Cancer Genetics, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ont., Canada M5G 1X5. Tel.: +416 586 8256; fax: +416 586 8663. E-mail address: [email protected] (I.L. Andrulis).

tein, with a leucine-rich nuclear export signal located in the Pre-LIM domain, and a C-terminus that harbors three LIM domains. LIM domains contain tandem zinc fingers and have roles in directing protein–protein interactions. Initial work has shown that LIMD1 is ubiquitously expressed in human and mouse tissues [1]. A common feature among ZYXIN family members is the ability to transit between sites of focal adhesions/cell–cell contacts and the nucleus. Although the exact reason for cycling is not known, it has been speculated to involve transducing signals

0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.03.015

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from the sites of attachment to the nucleus. Some family members (e.g. AJUBA and ZYXIN) can interact with mitotic regulators, such as Aurora-A kinase and h-warts/LATS1 [2,3]. The interaction between Aurora-A kinase and AJUBA is required for Aurora-A kinase activation enabling a cell to transit the G2/M phase of the cell cycle. This indicates that some members may have a role in the proper completion of mitosis. LIMD1 was previously shown to directly interact with Retinoblastoma (RB) and inhibit E2F-mediated transcription [4]. It was further demonstrated that overexpression of LIMD1 blocked tumor growth, both in vitro and in vivo, indicating potential tumor suppressing function. Recent work has suggested that Limd1 plays an important role in osteoclast development through an interaction with Traf6 during times of stress [5]. Limd1/ osteoclast precursor cells were shown to be deficient in the activation of AP-1 transcription complex, known to be critical for osteoclast development [5]. The LIMD1 gene is found at chromosome 3p21.3, a locus previously reported to undergo loss of heterozygosity in neoplastic tissues, suggesting potential tumor suppressor function [6–8]. We previously screened the LIMD1 gene for mutations in breast tumors and identified a rare variant associated with sporadic cases but found no definitive tumor-associated mutations [9]. To further understand the role of LIMD1 in cancer, we have characterized endogenous expression of LIMD1 and evaluated LIMD1 expression in primary human breast tumors. 2. Materials and methods 2.1. Cell culture, transfections, and synchronizations Human embryonic adenovirus-transformed kidney 293T/17, breast cancer MCF-7, and lung cancer A549 cell lines were propagated using DMEM high glucose supplemented with antibiotics and 10% FBS (Cansera). The human pancreatic Capan-1 cell line was grown in Iscoves media supplemented with antibiotics, 1.5 g/L bicarbonate, and 20% FBS. The human cervical HeLa cell line was grown in MEM F15 supplemented with antibiotics, 1.5 g/L bicarbonate, 1 mM sodium pyruvate (Sigma), and 10% FBS. Human MDA–MB435 and MDA– MB435‘‘S” cell lines were propagated using aMEM supplemented with antibiotics and 10% FBS. All mammalian cell lines were purchased from ATCC (Virginia, USA) except MDA–MB435, which was a gift from Dr. Susan Done (Princess Margaret Hospital, Canada). All growth

media were purchased from PMH media (PMH, Canada). Fugene (Roche) was used to transfect 293T cells for 24– 48 h. To synchronize cells in Go using a double-thymidine block, 100 mm plates of HeLa cultures were grown to 40% confluency and incubated with 2 mM thymidine (Sigma) for 18 h. Fresh media was then added for 8 h, removed and replaced with media containing 2 mM thymidine for an additional 12–16 h. Fresh media was then added and cells were harvested every hour for the next 12 h. To enrich cells for mitosis, growing 150 mm plates of HeLa cultures were incubated with 100 ng/mL of nocodazole (Sigma) for 18 h. Plates were tapped to release loosely attached mitotic cells. 2.2. PCR and expression constructs mRNA was isolated from cell lines with RNeasy kits (Qiagen) and transcribed to cDNA using reverse transcriptase and primers (random hexamers) of Moloney Murine Leukemia Virus (MMLV) kit (Invitrogen) according to the manufacturer’s instructions. The primer pairs, LIMD1-RT-F 50 -GACTTCCTGTACTCTGG-30 and LIMD1-RT-R 50 -GTAGATCTTGTTCTCTGAG30 amplify a 210 base pair (bp) fragment of LIMD1 cDNA, whereas AS1 50 -ACATTGAAGCACTCCGCG AC-30 and AS4 50 -CCTGAGGTTGTTCTTCACAG-30 amplify a 180 bp fragment of control gene AS cDNA. PCR amplifications were carried out with Hot Star Taq Polymerase (Qiagen) according to conditions for 35 cycles. PCR conditions to amplify human LIMD1 were as follows: 3 lL 10 HiFidelity Buffer (Invitrogen), 0.9 lL of 50 mM MgSO4 (Invitrogen), 2 lL of 10 mM dNTPs (Invitrogen), 0.6 lL of 30 lM forward primer (50 -CTGGT TGAATTCATGGATAAGTATGAC-30 ), 0.6 lL of 30 lM reverse primer (5’-GTTGAGCTCGAGCTAGAAGTG GTGCTGGTG-3’), 50 ng of pooled cell line cDNA, 0.3 lL of HiFi Taq Polymerase (5 U/lL, Invitrogen), and 22.6 lL of ddH2O. Cycle parameters were as follows; an initial incubation for 3 min at 95 °C; a three-step 35 cycle of 30 s at 95 °C, 20 s at 56 °C, 2 min at 72 °C; and a final extension for 10 min at 72 °C. Full-length LIMD1 (a.a. 1–676) was ligated into pCMV–FLAG vector (2B) (Stratagene). LIMD1 (a.a. 1–200 and 200–400) PCR products were generated using LIMD1–FLAG plasmid and ligated into the GST expression vector pGEX-4T-2 (Amersham Biosciences) to create GST–LIMD1 fusion proteins. All plasmids were confirmed by automated sequencing. 2.3. Antibody production GST–LIMD1 a.a. 1–200 and 200–400 plasmids were transformed into BL21 bacteria and expression was induced with 1.0 mM isopropyl-b-D-thiogalactopyranoside (IPTG). Cultures were lysed and incubated with

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50% glutathione-S-transferase (GST) slurry overnight. LIMD1 a.a. 1–200 (#2027) and 200–400 (#2023) peptides were cleaved using 50 mM glutathione (GSH) (Sigma), concentrated with Bioman-5 Membranes (Millipore), mixed with 500 lL of Freunds Complete Adjuvant (Difco) and injected into rabbits to raise polyclonal antiserums. Serums were affinity-purified using immobilized antigen according to protocol (Amersham Pharmacia Biotech). Rabbits were maintained in the Mt. Sinai animal facility. Anti-b-Actin antibody was purchased from Sigma–Aldrich and anti-cyclinB1 antibody was purchased from Santa Cruz. 2.4. Western blotting and phosphorylation Cells were lysed in 1 mL NETN (0.5% NP40, 1 mM EDTA, 50 mM Tris, and 150 mM NaCl) buffer supplemented 1 mM PMSF and 1 protease and phosphatase inhibitors. Immunoprecipitates were washed 3 with 1 mL of lysis buffer plus inhibitors. Bound proteins were eluted in 6 SDS–PAGE loading buffer and separated on a 10% SDS–PAGE. Proteins were blotted using M2 mouse anti-FLAG (1:5000; Sigma) or anti-LIMD1 antibodies (1:5000 to 1:10,000). Secondary conjugates, HRP-Donkey anti-mouse or HRP-Donkey anti-rabbit (Jacksons Immunochemicals) were incubated for 1 h at a 1:5000 dilution. Protein bands were visualized by autoradiography. Mitotic cells were collected and lysed in NETN supplemented with inhibitors. Cell lysate was incubated with 5 lL of affinity-purified anti-LIMD1 antibody for 2 h. Fifty microliters of Protein A–Agarose (Santa Cruz) was added for an additional 2 h and washed 2 with 1 mL NETN supplemented inhibitors without phosphatase inhibitors. A third wash was carried out in a 50 mM Tris (pH 7.5) and 150 mM NaCl buffer. Protein beads were split equally in two and incubated at 37 °C with or without 10 U of calf intestinal alkaline phosphatase (CIP, Promega). All lysis and protocols thereafter were carried out at 4 °C. 2.5. Immunofluorescence microscopy Cells were cultured on glass coverslips for 24 h, fixed with phosphate buffered saline 2% paraformaldehyde/ PBS for 15 min, and permeabilized with 0.1% Triton X100/PBS for 10 min. Slides were blocked with 5% bovine serum albumin (BSA) and then incubated with primary antibody (polyclonal anti-LIMD1 antibody, monoclonal anti-vinculin antibody [Calbiochem]) for 1 h at room temperature. Non-specific mouse and rabbit IgGs were used as controls. The cells were then incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse/rabbit (Alexa Fluor-488, Alexa Fluor-586; Molecular Probes) or anti-rabbit IgG secondary antibodies for 30 min. Cells were washed in PBS, stained with 40 -6-diamidino-2-phenylindole (DAPI) (Calbiochem) for 30 min, and washed. The stained cells were mounted on slides with mounting

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media (Calbiochem). Immunofluorescence was recorded using an Olympic 1X-70 inverted microscope, equipped with fluorescent optics and Deltavision Deconvolution Microscopy software (Applied Precision). 2.6. Methylation status of putative LIMD1 promoter Genomic DNA (1 lg) from HeLa and MDA–MB435 cell lines was isolated (Qiagen) and digested with MspI and HpaII restriction endonucleases (New England BioLabs). Reaction mixtures contained no enzyme, 25 U of MspI, or 25 U of HpaII, for 2 h at 37 °C. Two microliters of DNA from each digest was amplified by PCR using the LIMD1-F2/R2 primer set (LIMD1-F2, 50 -CAGTGCTAGAGCGTGG-30 ; LIMD1-R1, 50 -GAAACTCACAGCAAGGC-30 ) designed to amplify 934 to 422 of the LIMD1 locus, generating a 520 bp product. PCR was performed for one cycle of 95 °C for 4 min followed by 40 cycles of 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 1 min, followed by one cycle of 72 °C for 5 min using Plat Taq Polymerase (Invitrogen) using conditions for PCR Enhancer System (Invitrogen). The resulting amplification products were then analyzed by 1.5% agarose gel electrophoresis with ethidium bromide and examined under UV light. 2.7. Tissue samples Sporadic breast cancer tumors were obtained as part of a prospective study of axillary node-negative disease [10]. Subsequent to the diagnosis of invasive cancer, tumor specimens were catalogued by a pathologist, snap frozen, and stored in liquid nitrogen. 2.8. Real-time PCRs cDNA was reverse transcribed from cellular mRNA using MMLV (Invitrogen) according to the manufacturer’s instructions. cDNA was amplified using the Applied Biosystems Taqman Universal PCR Master Mix, no UNG (cat#4324018), 8 lL of diluted cDNA (1 lL cDNA + 7 lL of ddH20), 1lL of 20 Assay on Demand gene expression assay mix (Hypoxanthine phosphoribosyltransferase 1 [HPRT1] control primer/probe mix) and 1 lL of 20 Assay on Demand gene expression mix (test primer/probe mix-catalogue numbers listed below). PCR was performed as follows: samples were held at 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s, using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). Standard curves of serial dilutions ranging from 3 to 0.005 of a pool of cDNA were used for quantification. A reference pool of cDNA derived from 13 cell lines was generated from different tissue types. HPRT1 mRNA was used as an internal control and has been determined by our laboratory and others to have little variation

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between breast tumors [11,12]. Test probes were conjugated to the FAM fluor (Applied Biosystems), and the HPRT1 control probe was conjugated to the VIC fluor (Applied Biosystems). The test and control probes were analyzed either individually or multiplexed. ABI SDS2.1 software was used to analyze results.

2.9. Sodium bisulfite modification of DNA

TM

TM

Genomic DNA isolated from breast tumors and cell lines was isolated and subjected to sodium bisulfite modification according to instructions using EpiTect Bisulfite kit (Qiagen).

Fig. 1. Generation of polyclonal anti-LIMD1 antibodies and expression of LIMD1 in cancer cell lines. (a) Coomassie stained SDS– PAGE gel of LIMD1#5 (Lane 1; 26 kDa) and LIMD1#6 (Lane 2; 24 kDa) peptide antigens (arrows denote purified product). (b) Equalized 293 null (Lane 1), transfected empty vector (2B) (Lane 2) or LIMD1–FLAG (Lane 3) lysates were separated by SDS–PAGE and blotted with FLAG (b-i; control), pre-immune (b-i), or immune rabbit serum (#2023; LIMD1 a.a. 200–400) (b-ii). Arrows indicate 75 kDa, the theoretical size of LIMD1. The larger band in Lane 3 of (b-i) is the slower migrating form of the overexpressed LIMD1– FLAG protein.

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Fig. 2. LIMD1 is phosphorylated during mitosis. HeLa cells were synchronized to Go using a double-thymidine block. Cultures were released into S-phase and collected over 12 h. (a) Slower migrating forms of LIMD1 are seen around the 8, 9, and 10 h post-thymidine release, around mitosis. Monitoring of cyclin B1 expression was included as a guide for the G2/M boundary. (b) HeLa cells were subjected to 100 ng/mL nocodazole block for 18 h and mitotic cells collected. A slower migration form of LIMD1 is clearly evident (Lane 3) when compared to asynchronous (Lane 1) or Go synchronized cells (Lane 2). (c) Using nocodazole-treated HeLa cells, LIMD1 was immunoprecipitated and treated with or without CIAP. The loss of the higher LIMD1 migration band in the CIAP treated lane (Lane 1) demonstrates that LIMD1 is phosphorylated during mitosis. Asterisk indicates heavy chain.

3. Results 3.1. Generation of anti-LIMD1 antibodies To generate LIMD1 antibodies two regions of this protein were expressed as GST fusion protein, purified from bacteria and injected into rabbits. The two regions were referred to as LIMD1#5 (a.a. 1–200; 26 kDa) and LIMD1#6 (a.a. 200–400; 24 kDa) (Fig. 1a). Rabbit polyclonal antibodies raised against these two LIMD1 regions (a.a. 1–200; Rabbits #2046 and #2027 and a.a. 200–400; Rabbits #2023 and #2026) were used to detect endogenous LIMD1. Lysates from 293 cells, or cultures transfected with either empty FLAG vector (2B) or LIMD1–FLAG (Fig. 1b-i) were used to evaluate specificity of generated antibodies. Two hundred and ninety-three lysates were blotted with either pre-immune (P.I.) serum c-ii) or serum isolated from the second bleed (Rabbit #2023) (Fig. 1b-iii). The serum recognizes a band at approxi-

mately 75 kDa, the theoretical size of endogenous LIMD1. Overexpression of LIMD1–FLAG results in a slightly slower migration band compared to endogenous LIMD1 (Fig. 1b-iii). The remaining three antibodies all gave similar results (data not shown). 3.2. Cell cycle regulation and phosphorylation of LIMD1 To investigate the endogenous regulation of LIMD1, HeLa cells were synchronized in Go using a double-thymidine block and harvested every hour for 12 h thereafter (Fig. 2a). The amount of LIMD1 remained equal throughout the cell cycle, however, during the time prior to and during mitosis, slower migrating LIMD1 were visualized, indicating post-translational modification. Cyclin B1 is maximally expressed at the G2/M boundary of the cell cycle was included as a guide to HeLa cell synchronization (the slower migrating band in Lane 11 of the anti-LIMD1 blot in double-thymidine synchronization in Fig. 2a likely represents incomplete synchroniza-

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tion with this protocol). HeLa cells were then incubated with nocodazole, a drug that interferes with the structure

and function of microtubules in interphase and mitotic cells. Comparison of mitotic (Fig. 2b; Lane 3) to asyn-

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Fig. 4. Expression of LIMD1 mRNA (a) and protein (b) in cancer cell lines. (a) RT-PCR was performed on mRNA isolated from cell lines. LIMD1 expression was compared to control gene asparagine synthetase (AS). (b) Cell lysates were run on a 10% SDS–PAGE gel and probed with anti-LIMD1 with anti-b-actin antibody as loading control.

chronous (Fig. 2 b; Lane 1) or Go (Fig. 2b; Lane 2) lysates clearly showed a slower migrating band relative to the other two conditions. In our hands, treatment with nocodazole (Fig. 2b) was more efficient than the doublethymidine block at restricting cells to particular cell cycle phases, as evidenced by the complete slow migration form of LIMD1 and greater contrast in cyclin B1 expression. This has also been previously observed with ZYXIN [3]. To determine whether phosphorylation was involved in the shift in LIMD1, HeLa cells were incubated in nocodazole and mitotic cells collected. Lysates were immunoprecipitated with aLIMD1 and the immunoprecipitated LIMD1 was incubated with calf intestinal

phosphatase (CIAP). The slower migrating band was lost, indicating that LIMD1 is phosphorylated during mitosis (Fig. 2c). 3.3. Cellular localization of LIMD1 and colocalization with vinculin To determine the endogenous cellular localization of LIMD1, anti-LIMD1 antibodies were incubated with fixed HeLa cells. Antibodies raised to different LIMD1 antigens had similar patterns of localization. Generally, there was diffuse staining throughout the cytoplasm with a concentration at the outer edges of the cells with nuclear and peri-

3 Fig. 3. LIMD1 expression and colocalization with vinculin in HeLa cells. (i) HeLa cells were incubated with polyclonal aLIMD1 (#2027) (b) or aLIMD1 (#2023) (e). Fluorescein-secondary conjugated anti-rabbit antibodies marked anti-LIMD1 antibodies as green. Nucleus is visualized as blue using DAPI (a and d). (c and f) A merger of the two signals. Immunofluorescence of LIMD1 (#2027) also showed distinct cytoplasmic non-nuclear (h) in HeLa cells. (ii) HeLa and MDA–MB435 cells were incubated with either primary aLIMD1 (#2027) (a and d) or vinculin (b and e) and fluorescein-secondary conjugated anti-rabbit and anti-mouse antibodies. (c and f) Merged images. Arrows indicate localization of LIMD1 to focal adhesions as well as colocalization with vinculin, a marker of focal adhesions.

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nuclear staining as well (Fig. 3i-b and -e). Both antibodies exhibited nuclear staining at a frequency of 30%, around double that previously observed in overexpressed LIMD1 in U2OS cells [4]. In addition, using serum #2023, LIMD1 was clearly excluded from nucleoli (Fig. 3i-e). Focal adhesions represent sites of cellular attachments to the substratum and are characterized by the presence of structural proteins, such as actins, which help maintain cellular shape and polarity [13,14]. Since others have reported the localization of other LIM family members to focal adhesions, we investigated the localization of LIMD1 in HeLa cells by co-staining with aLIMD1 and avinculin, a marker of focal adhesion. As a negative control, MDA–MB435 cells, which lack LIMD1 expression, were also co-stained with aLIMD1 and avinculin. LIMD1 and vinculin colocalized in HeLa cells at sites of focal adhesions (Fig. 3ii. c) whereas in MDA–MB435 cells, LIMD1 was not visualized while vinculin was clearly seen (Fig. 3ii-d–f). 3.4. Expression of LIMD1 in cancer cell lines The LIMD1 gene is located in a chromosomal locus that has been reported to undergo LOH in a number of cancers, including breast tumors. To determine whether the gene is aberrantly expressed in cancer, the rabbit polyclonal antibodies generated against human LIMD1 peptides (as described in Section 2) were used to detect LIMD1 protein expression in human cancer cell lines. LIMD1 mRNA (Fig. 4a) and protein (Fig. 4b) were detected in all cell lines except MDA–MB435, a cell line previously reported to lack the expression of LIMD1 [4]. 3.5. Expression of the LIMD1 gene in breast tumors To investigate whether the expression of LIMD1 was altered in primary breast cancers, we determined the relative levels of LIMD1 mRNA in sporadic breast tumors (Table 1). Although most tumors expressed levels similar to a pool of cell line RNAs, several tumors (e.g. tumors 1096, 1728, 2160, 2253) exhibited low levels of LIMD1 relative to the pooled RNA. 3.6. Methylation of the LIMD1 gene in MDA–MB435 cell line A common mechanism by which gene expression is reduced, especially in tumors and cancer cell lines, is via methylation of CpG islands within their promoters leading to downregulation of transcription [15–17]. To study the mechanism of the reduced expression of LIMD1 in the MDA–MB435 cell line and primary breast tumors, we examined the potential role of DNA methylation. Since the LIMD1 promoter had not been previously described, we used 10 kb of

Table 1 Expression of LIMD1 in breast tumors relative to pooled cell line cDNA Tumor

LIMD1 expression levela

693 1035 1096 1131 1234 1728 2005 2063 2080 2081 2160 2253 2285 2290 2489 2498 2549 2592 2722 2850

1.27 1.01 0.54 2.32 0.93 0.58 2.67 2.23 0.99 0.88 0.33 0.43 1.03 1.06 0.9 0.9 1.64 2.53 0.89 0.91

a Normalized LIMD1/HPRT1 ratio as measured by quantitative real-time PCR.

upstream genomic DNA relative to the LIMD1 ATG start codon, under default settings, and inserted the sequence into the European Bioinformatics Institute CpG Island finder [18] (Fig. 5a). A 1.2 kb stretch of DNA was tagged as containing three CpG islands in close proximity, ending 450 bp upstream of the ATG start site (Fig. 5b). The methylation status of 11 CCGG restriction sites within the LIMD1 gene promoter was studied using PCR of this region, before and after DNA digestion with the isoschizomers MspI and HpaII. PCR of the putative promoter region containing CpG islands after DNA digestion with MspI failed to amplify this region in both cell lines (Fig. 5c-i); however, the highly methylated CCGG sites were resistant to HpaII digestion in MDA–MB435 (Fig. 5c.ii). Therefore, the HpaII-resistant putative LIMD1 promoter DNA was found to be hypermethylated. 3.7. LIMD1 expression in primary breast tumors is not related to methylation DNA from tumors was sodium bisulfite treated in order to determine methylation status of the putative LIMD1 promoter. We conducted methylation-specific PCR (MSP) on a dense 300 bp region of the putative LIMD1 promoter in a subset of breast cancers; two from low expressing tumors (<0.45) and two from high expressing tumors (>2.5). Sodium bisulfite treated HeLa and MDA–MB435 cell lines were chosen as positive and neg-

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Fig. 5. Putative LIMD1 promoter is methylated in MDA–MB435. (a) Bioinformatic analysis of DNA upstream of the LIMD1 ATG start codon indicated a stretch of CpG islands. (b) Primers were designed to amplify a 520 bp product harboring 11 of 17 CCGG sites within the hypothetical 1.1 kb island. (c) PCR amplification after MspI/HpaII restriction assay produces a 520 bp product in the MDA–MB435 sample, indicating methylation.

ative controls respectively to determine the specificity of the PCR reactions. The predicted promoter was not methylated in either the low expressing or high expressing tumors as shown by amplification using unmethylated primers (Fig. 6).

4. Discussion LIMD1, a member of the ZYXIN family of genes, has been considered a potential tumor suppressor gene [1]. Members of the ZYXIN family have been reported to be involved in maintaining cell structure, cell fate, and differentiation [19]. We found that

LIMD1 is expressed constantly and at similar levels during the cell cycle. Furthermore, we demonstrated that LIMD1 is phosphorylated during mitosis, similar to AJUBA [2] and ZYXIN [3]. Endogenous LIMD1 was striking in its colocalization with vinculin at focal adhesions in HeLa cells. Our results are in agreement with previous observations regarding the ZYXIN family of proteins. AJUBA and ZYXIN proteins are specifically phosphorylated during mitosis. Phosphorylation of AJUBA is required for the timely completion of the cell cycle via Aurora-A kinase and ZYXIN phosphorylation is thought to regulate its interac-

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Fig. 6. Methylation status of the predicted LIMD1 promoter region. Unmethylated-specific (M) and methylated-specific (U) primers were used in the analysis. Controls included sodium bisulfite modified HeLa (LIMD1 expression positive) and MDA–MB435 (LIMD1 expression negative) cell lines. Sodium bisulfite treated DNA from breast tumors with lower (<0.5) and higher (>2.5) expression of relative LIMD1 was evaluated. Negative is PCR control.

tion with WARTS/LATS1, as well as localize to the mitotic apparatus. The consequence of LIMD1 hyper-phosphorylation during mitosis is presently unclear. It may affect the localization of LIMD1 and/or its ability to interact with protein partners. Immunofluorescence using HeLa and MDA– MB435 cells demonstrated that LIMD1 is localized to the cytoplasm and the nucleus. A diffuse pattern of expression within the cytoplasm was usually seen, but additionally, it was shown that LIMD1 colocalized with vinculin at focal adhesions. Petit et al. previously reported that a GFP-tagged version of LIMD1 expressed in monkey CV-1 cells [20] colocalized with vinculin. We have extended this initial observation in the human HeLa cell line under endogenous conditions. Focal adhesion and cell-to-cell contacts, although different, serve a similar purpose by anchoring and maintaining cell shape and structure. ZYXIN family proteins are often found at sites of focal adhesions, where multiple protein interactions occur between integrin clusters and anchor actin filaments. Unlike most ZYXIN family proteins, LIMD1 lacks recognizable LD or SH3 protein interaction domains, often identified in other family members. LIMD1 could conceivably harbor a non-classical SH3 binding consen-

sus site given the prevalence of prolines within the Pre-LIM domain, but that has yet to be determined. LIMD1 has previously been associated with control of cell growth in vitro and in vivo and maps to a chromosomal region that undergoes frequent LOH in multiple cancers. Previously, we analyzed breast tumors for mutations and identified new variants; but we did not find any bona-fide tumor-associated mutation [9]. We therefore investigated the relative expression of LIMD1 in primary breast tumors since reduced expression could lead to inactivation. We detected a number of primary breast tumors with low levels of RNA expression, but we found that this was not due to promoter methylation. Other mechanisms which downregulate the expression of LIMD1 are likely to be involved in these tumors. In contrast, we found that LIMD1 was not expressed in MDA–MB435 cells, likely due to DNA methylation. Interestingly, there appears to be increasing evidence that the LIMD1 expression negative MDA–MB435 cell line is, in fact, a melanoma cell line and not a metastatic breast cancer. Recent work has suggested there may have been mix-ups in cell line handling as early as 1982 [21,22]. We found that the MDA–MB435 cell line is distinct from MDA–MB435‘‘S”, in that the ‘‘S”

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line expresses the LIMD1 transcript (Fig. 4a). In addition, the MspI/HpaII restriction assays in this study demonstrated that the putative LIMD1 promoter of MDA–MB435‘‘S” cells is not methylated (data not shown). According to ATCC, the MDA–MB435‘‘S” line is a ‘‘spindle shaped strain” which had evolved from the parental MDA– MB435 line. This may be an indication of phenotypic drift from the original MDA–MB435 line in which re-expression of LIMD1 has occurred, possibly through the loss of methylation. We have demonstrated the localization of endogenous LIMD1 to both the nucleus and cytoplasm. Furthermore, we show that endogenous LIMD1 colocalized with vinculin at focal adhesions, which are dynamic structures that serve to help maintain cell shape and structure. Additionally, we determined that LIMD1 is hyper-phosphorylated during mitosis. These observations are similar to that of the other ZYXIN members and suggest that LIMD1 shares characteristics of other members of the ZYXIN family of proteins. Alterations in the expression of LIMD1 may yet play a role in breast cancer but this requires further study.

[5]

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Acknowledgements We gratefully acknowledge the support of the National Cancer Institute of Canada with funds from the Terry Fox Run (I.L.A.) and a predoctoral fellowship from the United States Army Medical Research and Materiel Command (DAMD017-02-1-0497) (C.J.H.). The authors would also like to thank Lucie Bosnoyan-Collins for her technical assistance.

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