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 (). 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 (). 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 (). 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 (). 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 (). 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 [5
]. The transcript is present in the T24 cell line (). These results suggest that MIM
may be related to cancer progression or tumor metastasis in a variety of organ sites.
Figure 1 The 316-bp cDNA is not expressed in TccSup (Lane 6) by PCR. Lane 1: 100-bp DNA marker. Lane 2: RT4 (bladder papilloma cell line). Lane 3: 5637 (bladder superficial transitional cell line). Lane 4: HT 1376 (bladder invasive TCC cell line). Lane 5: T24 (more ...)
Northern blot analysis of cell lines and tissues utilizing the C11 300 cDNA as a probe. A. Bladder cancer cell lines. B. Normal tissues. C. Breast cancer cell lines. D. Prostate cancer cell lines.
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 ().
The protein sequence for MIM. The predicted protein sequence is 356 bp. The proline-rich region stretches from bp 209 to 284 (bold italicized). The WH2 domain encompasses the bp region 328 to 345 (bold underlined).
A protein homology search demonstrates that MIM
has a proline-rich region and a W Wiskott-Aldrich syndrome protein (WASp) Homology 2 (WH2) motif () [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 [6
]. Their activity is regulated by the Rho-family GTPases and these interactions are thought to provide a final common signaling pathway for actin polymerization [6
The mammalian WASp/Scar family currently consists of five members: WASp, N-WASP, and three Scar (suppressor of cAMP receptor) isoforms [7
]. 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 [7
]. 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.