Background: Wnt-3a is an intercellular signalling molecule that is involved in a variety of morphogenetic events. However, the molecular mechanisms underlying Wnt-3a signalling are poorly understood. We have sought to establish in vitro systems to assay the activity of this protein and investigate its biological roles.
When Axin, a negative regulator of the Wnt signaling pathway, was expressed in COS cells, it coeluted with glycogen synthase kinase-3b (GSK-3b), b-catenin, and adenomatous polyposis coli protein (APC) in a high molecular weight fraction on gel ®ltration column chromatography. In this fraction, GSK-3b, b-catenin, and APC were co-precipitated with Axin. Although bcatenin was detected in the high molecular weight fraction in L cells on gel ®ltration column chromatography, addition of conditioned medium expressing Wnt3a to the cells increased b-catenin in the low molecular weight fraction. However, Wnt-3a-dependent accumulation of b-catenin was greatly inhibited in L cells stably expressing Axin. Axin also suppressed Wnt-3a-dependent activation of Tcf-4 which binds to b-catenin and acts as a transcription factor. These results suggest that Axin forms a complex with GSK-3b, b-catenin, and APC, resulting in the stimulation of the degradation of bcatenin and that Wnt-3a induces the dissociation of bcatenin from the Axin complex and accumulates bcatenin.
Since membrane type-1 matrix metalloproteinase (MT1-MMP) plays pivotal roles in tumor progression and metastasis and holds great promise as an early biomarker for malignant tumors, a method of evaluating MT1-MMP expression levels would be valuable for molecular biological and clinical studies. Although we have previously developed a 99m Tc-labeled anti-MT1-MMP monoclonal IgG ( 99m Tc-MT1-mAb) as an MT1-MMP imaging probe by nuclear medical techniques for this purpose, slow pharmacokinetics were a problem due to its large molecular size. Thus, in this study, our aim was to develop miniaturized antibodies, a single chain antibody fragment (MT1-scFv) and a dimer of two molecules of scFv (MT1-diabody), as the basic structures of MT1-MMP imaging probes followed by in vitro and in vivo evaluation with an 111 In radiolabel. Phage display screening successfully provided MT1-scFv and MT1-diabody, which had sufficiently high affinity for MT1-MMP (K D = 29.8 and 17.1 nM). Both 111 In labeled miniaturized antibodies showed higher uptake in MT1-MMP expressing HT1080 cells than in non-expressing MCF7 cells. An in vivo biodistribution study showed rapid pharmacokinetics for both probes, which exhibited >20-fold higher tumor to blood radioactivity ratios (T ⁄ B ratio), an index for in vivo imaging, than 99m Tc-MT1-mAb 6 h post-administration, and significantly higher tumor accumulation in HT1080 than MCF7 cells. SPECT images showed heterogeneous distribution and ex vivo autoradiographic analysis revealed that the radioactivity distribution profiles in tumors corresponded to MT1-MMP-positive areas. These findings suggest that the newly developed miniaturized antibodies are promising probes for detection of MT1-MMP in cancer cells. (Cancer Sci 2013; 104: 495-501) T umor metastasis is the most frequent cause of death for cancer patients. In order to metastasize, tumor cells must acquire the ability to break through the basement membrane and invade dense networks of interstitial ECM proteins.(1) Matrix metalloproteinases (MMPs) are a family of enzymes responsible for degrading the various ECM components. While most MMPs are secreted as soluble zymogens, members of the subfamily of membrane-type MMPs (MT-MMPs) anchored to the cell membrane are suited for pericellular proteolysis. MT1-MMP is the major pericellular protease involved in processing triple helical collagen type I.(4) In addition, MT1-MMP activates MMP zymogens such as proMMP-2 and proMMP-13 that have significant involvement in tumor cell invasion and metastasis.(5,6) As MT1-MMP has a close relationship with tumor malignancy and holds great promise as an early biomarker of malignant tumors, (7,8) in vivo monitoring and ⁄ or quantitation of MT1-MMP expression could be valuable tools for molecular biological and clinical studies.Recently, in the course of focusing on nuclear medical techniques for noninvasive quantitative evaluation of biological molecules deep within the body, we developed a 99m Tc-labeled anti-MT1-MMP monoclonal IgG ( 99m Tc-MT1-mAb) as a radiolabeled...
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