|Home | About | Journals | Submit | Contact Us | Français|
Using a modified version of the mRNA differential display technique, five human bladder cancer cell lines from low grade to metastatic were analyzed to identify differences in gene expression. A 316-bp cDNA (C11-300) was isolated that was not expressed in the metastatic cell line TccSuP. Sequence analysis revealed that this gene was identical to KIAA 0429, has a 5.3-kb transcript that mapped to 8q24.1. The protein is predicted to be 356 amino acids in size and has an actin-binding WH2 domain. Northern blot revealed expression in multiple normal tissues, but none in a metastatic breast cancer cell line (SKBR3) or in metastatic prostatic cancer cell lines (LNCaP, PC3). We have named this gene Missing in Metastasis (MIM) and our data suggest that it may be involved in cytoskeletal organization.
Bladder cancer is the second most common cancer of the genitourinary tract in the United States. Approximately 30% to 70% of superficial bladder tumors will recur after initial treatment and 10% to 30% will progress to invasive and/or metastatic disease . It is likely that this progression is the result of genetic changes that transform a superficial cancer to one with metastatic potential, including activation of oncogenes or loss of tumor suppressor genes . The objective of this study was to identify differentially expressed genes related to the progression and metastasis of transitional cell bladder cancer utilizing a modified, nonradioactive differential display technique .
Total RNA was isolated using TRIzol reagent (Life Technologies, Grand Island, NY), and purified by digestion with RNAse-free DNAse I from the following cell lines: RT4, a bladder papilloma cell line; 5637, a bladder superficial transitional cell cancer cell line; HT1376, a bladder invasive transitional cell cancer cell line; T24, a bladder invasive transitional cell cancer cell line; TccSuP, a metastatic bladder transitional cell cancer cell line; MCF7, a human breast cancer cell line; MCF10A, a benign breast fibroadenoma cell line; SKBr3, a metastatic breast cancer cell line; PrEC-1, a human prostate cell line; and metastatic prostate cancer cell lines PC-3, TSU-PR1, DU145; and LNCaP. All cell lines were obtained from the American Type Culture Collection (Manassas, VA) except PrEC-1 cells that were obtained from Clonetics (East Rutherford, NJ). mRNA was isolated from total RNA using PolyA-Ttract mRNA Isolation Systems (Promega, Madison, WI) according to the manufacturer's recommendations.
DEPC H2O was added to 2 to 5 µg of poly A+ RNA per sample to bring the total volume to 22 µl. The RNA was heated at 65°C for 5 minutes and then placed on ice. Master mix containing 8 µl of 5x reverse transcription buffer, 4 µl of 0.1 M DTT, 2 µl of 10 mM dNTP, 2 µl of random hexamers (50 ng/µl) and 2 µl of MML-V reverse transcriptase (200 U/µl was added per tube containing denatured RNA, then incubated as follows: 22°C for 10 minutes, 37°C for 50 minutes, 70°C for 15 minutes and then 4°C.
Reverse-transcribed RNA (cDNA) was amplified in separate polymerase chain reactions (PCR) using 40 random 10-mers at 5 µM per reaction (Operon, Alameda, CA). Master mix containing 3 µl of 10xPCR buffer, 0.3 µl of 10 mM dNTPs, 0.9 µl of 50 mM MgCl2, 0.2 µl Taq DNA polymerase (5U/µl) and 20.6 µl of sterile dH2O was added into 2 µl of cDNA and 3 µl of random primer. The cycling parameters using Perkin-Elmer 9600 thermocycler (Perkin-Elmer, Shelton, CT) were as follows: 94°C, 2 minutes at first cycle; 94°C, 45 seconds; 38°C 2 minutes; 72°C, 1 minute 15 seconds for 40 cycles followed by 72°C 15 minutes and then 4°C soaked. The PCR products were separated on 2% agarose (DNA typing agarose, Gibco BRL, Grand Island, NY) and visualized with 0.3 µg/ml ethidium bromide staining under UV light. Differentially expressed bands were excised from the agarose gel using a scalpel and extracted using a Qiaex gel extraction kit (Qiagen, Valencia, CA) [3,6].
cDNAs were purified utilizing the High Pure PCR product purification kit (Boehringer Mannheim, Indianapolis, IN), cloned into a plasmid TA cloning vector, pGEM-T easy (Promega) and sequenced using the T7 and/or the SP6 primer. Sequences were blasted against Genbank.
Two micrograms of poly A+ RNA was denatured and electrophoresed through a 1.2% denatured agarose/formaldehyde gel and transferred to nylon membrane (Scleicher & Schuell, Keene, NH). The membrane was cross-linked by a UV Stratalinker and dried under vacuum for 2 hours, then prehybridized with 20 ml of the hybridization solution (50% formamide, 5x SSC, 1x PE, and 125 µg/ml of denatured sheared salmon sperm DNA) for 2 to 8 hours. The human multiple tissue Northern blot membrane was purchased from Clontech (Palo Alto, CA). All probes were random labeled with α32PdCTP using an Oligolabeling kit (Pharmacia Biotech, Piscataway, NJ). Hybridization was performed for 16 hours at 42°C, then the membrane was washed under stringent conditions: twice for 10 minutes in 2x SSC plus 0.1% sodium pyrophosphate plus 0.1% SDS at 65°C, once for 10 minutes and 30 minutes in 0.2x SSC plus 0.1% sodium pyrophosphate plus 0.1% SDS at 65°C, respectively. Autoradiography was performed using Kodak XAR film (Kodak, New Haven, CT) at -80°C. After removal of cDNA probes by washing in 100°C H2O for 10 minutes, the same blot was rehybridized with human tubulin or -actin as controls.
Chromosomal mapping was obtained using the BLAST algorithm with the C11-300 sequence against the Human Genome sequence database through the National Center for Biotechnology website as accessed through the Internet (http://www.ncbi.nlm.nih.gov/).
The predicted 356-residue MIM protein sequence was examined for the presence of functional motifs through a ProfileScan search of the protein profile entries in PROSITE, Pfam and MultAlin accessed through the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB) (http://www.expasy.ch).
We have modified the original differential display technique developed by Liang and Pardee by utilizing a 5′ random decamer and 2% DNA typing agarose with visualization after ethidium bromide staining [3,4]. Using this technique, a 300-bp amplicon, C11-300, was amplified from RNA using the random primer 5′ AAAGCTGCGG 3′ in the RT4, 5637, HT1376 T24, but not the TccSuP cells TccSup (Figure 1). Because this gene was not expressed in metastatic bladder cells we have termed it Missing in Metastasis (MIM). The amplicon was cloned and used as a probe in Northern blot analysis. The differential expression pattern of MIM was confirmed for RT4, 5637, HT1376, T24 and TccSuP cells by Northern blot analysis (Figure 2A). TccSuP cells were derived from an anaplastic transitional cell carcinoma from a patient with metastases to the bone. A human normal multiple tissue Northern blot demonstrated that MIM was expressed in many tissues including spleen, thymus, prostate, testis, uterus, colon, and peripheral blood (Figure 2B). In addition to the 5.3-kb major transcript, testis expresses a 2.0-kb transcript. Northern blot analysis in breast cell lines showed that MIM was not expressed in the SkBr3, a metastatic breast cancer cell line, but was expressed in MCF10A, a benign breast fibroadenoma cell line, and MCF7, a breast cancer cell line derived from pleural fluid (Figure 2C). Northern blot analysis in prostate cell lines demonstrated MIM was not expressed in PC-3 and LNCaP, metastatic prostate cancer cell lines, but was minimally expressed in PrEC-1, a prostate epithelial cell line, and DU145, a metastatic prostate cancer cell line (Figure 2D). The transcript was expressed in TSU-PR1, a presumably metastatic prostate cancer cell line. Interestingly, a recent report suggests that TSU-PR1 may be a transitional cell cancer cell line derived from the bladder tumor cell line T24 . The transcript is present in the T24 cell line (Figure 2A). These results suggest that MIM may be related to cancer progression or tumor metastasis in a variety of organ sites.
The amplicon was cloned and sequenced and found to be 100% homologous to the 5645-bp KIAA0429 gene transcript (GenBank accession # NM_014751). BLAST analysis of the C11-300 amplicon sequence against the Human Genome sequence database showed that it localized to contig NT_023726 and maps to chromosomal region 8q24.1. The NM-014751 nucleotide sequence contains a 1068 nt open-reading frame that encodes a 38-kDa protein 356 amino acid residues in length (Figure 3).
A protein homology search demonstrates that MIM has a proline-rich region and a W Wiskott-Aldrich syndrome protein (WASp) Homology 2 (WH2) motif (Figure 3) [6,7]. The WH-2 motif participates in actin monomer binding. The WASp family of proteins is thought to help initiate the growth of a new actin filament on the side of an existing one . Their activity is regulated by the Rho-family GTPases and these interactions are thought to provide a final common signaling pathway for actin polymerization .
The mammalian WASp/Scar family currently consists of five members: WASp, N-WASP, and three Scar (suppressor of cAMP receptor) isoforms . The gene encoding WASp is mutated in Wiskott-Aldrich syndrome, an X-linked human disease with selective defects in platelet development and lymphocytes. WASp has a binding motif for activated Cdc42 and Rac, suggesting that WASp might regulate actin, because these Rho family GTPases influence actin dynamics, and because transfection of WASp rearranges actin filaments in cultured cells . N-WASP, originally described in neural cells, is expressed more widely in vertebrate cells than WASp and causes filopodial formation. Scar was discovered in Dictyostelium and deletion causes cytoskeletal defects. G-protein-coupled receptors trigger the reorganization of the actin cytoskeleton in many cell types. Scar cells have reduced levels of F-actin staining, and abnormal cell morphology and actin distribution during chemotaxis.
Our data suggest that MIM may be lost in certain cells that express a metastatic phenotype. Actin filament assembly is associated with cytoskeletal structure organization and many forms of cell motility. Alteration of actin assembly/disassembly dynamics may have serious consequences on the ability of cells to metastasize. MIM appears to be an exciting new gene product that may be involved in invasion and metastasis, most likely through an interaction with the actin cytoskeleton. Work is underway to characterize its exact role in regulation of the cytoskeleton and to further describe its distribution in normal tissue as well as primary and metastatic tumors.
This work was supported by National Institutes of Health (NIH) SPORE P50 CA 69568 (K.J.P.), NIH R21 CA 80660 (K.J.P.), NIH R01 CA 60948 (J.A.M.).