S-Adenosylmethionine decarboxylase belongs to a small class of amino acid decarboxylases that use a covalently bound pyruvate as a prosthetic group. It is an essential enzyme for polyamine biosynthesis and provides an important target for the design of anti-parasitic and cancer chemotherapeutic agents. We have determined the structures of S-adenosylmethionine decarboxylase complexed with the competitive inhibitors methylglyoxal bis(guanylhydrazone) and 4-amidinoindan-1-one-2'-amidinohydrazone as well as the irreversible inhibitors 5'-deoxy-5'-[N-methyl-N-[(2-aminooxy)ethyl]amino]adenosine, 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)amino]adenosine, and the methyl ester analogue of S-adenosylmethionine. These structures elucidate residues important for substrate binding and show how those residues interact with both covalently and noncovalently bound inhibitors. S-Adenosylmethionine decarboxylase has a four-layer alphabeta betaalpha sandwich fold with residues from both beta-sheets contributing to substrate and inhibitor binding. The side chains of conserved residues Phe7, Phe223, and Glu247 and the backbone carbonyl of Leu65 play important roles in binding and positioning the ligands. The catalytically important residues Cys82, Ser229, and His243 are positioned near the methionyl group of the substrate. One molecule of putrescine per monomer is observed between the two beta-sheets but far away from the active site. The activating effects of putrescine may be due to conformational changes in the enzyme, to electrostatic effects, or both. The adenosyl moiety of the bound ligand is observed in the unusual syn conformation. The five structures reported here provide a framework for interpretation of S-adenosylmethionine decarboxylase inhibition data and suggest strategies for the development of more potent and more specific inhibitors of S-adenosylmethionine decarboxylase.

Key words: adhesion; breast cancer; connexin; cadherin; gap junctional intercellular communication; metastasisGap junctions are intercellular channels that facilitate the movement of small (Ͻ1 kDa) ions and signaling molecules between cells. 1 A breakdown in GJIC has been correlated with tumorigenesis. 2 Previous studies suggested that the breakdown in homotypic communication may contribute to breast cancer metastatic potential. 3 The breast metastasis suppressor gene BRMS1 suppresses metastasis in human breast carcinomas. 4 Transfection of BRMS1 cDNA into the metastatic breast cancer cell line MDA-MB-435 (435) reduces their metastatic potential and restores homotypic GJIC, supporting the concept that metastasis-suppressed cells are capable of significant homotypic GJIC as well as the correlation between breakdown in homotypic GJIC and metastasis. 3 The purpose of the current study was 3-fold: (i) to determine whether breast carcinoma cells can form gap junctions with bone cells; (ii) to ascertain whether heterotypic GJIC might be relevant to cancer metastasis; and (iii) to explore whether BRMS1 expression might regulate heterotypic GJIC. Since breast carcinomas so commonly colonize bone, we explored heterotypic GJIC (i.e., between 435 cells and hFOB1.19 cells). We examined 435 cells because they metastasize to bone when injected intracardially in mice. 5 BRMS1-transfected 435 cells were used to study the effect of metastasis suppression on heterotypic GJIC with hFOB cells since BRMS1 transfectants are genotypically related to 435 cells. Osteoblasts were used for heterotypic studies because osteoblastic bone lining cells cover all bone surfaces 6 and tumor cells might have to adhere to and migrate through a layer of bone lining cells in addition to a layer of endothelial cells to reach the marrow cavity. 7 MATERIAL AND METHODS Cell linesHTERT-HME1 (HTERT) cells were maintained in specially formulated medium (both cells and medium from Clontech, Lexington, KY). 8 MCF-7 cells were purchased from the ATCC (Rockville, MD) 9 and maintained in EMEM supplemented with 10% FBS (Hyclone, Logan, UT) and 1 mg/ml bovine insulin. 435 cells 10 were maintained in DMEM/F-12 medium (GIBCO Life Technologies, Rockville, MD) supplemented with 5% FBS. 435/ BRMS1 cells (clones 3 and 6) are 435 cells transfected with BRMS1 cDNA 4 and were maintained in the 435 cell medium supplemented with geneticin (500 g/ml). The hFOB1.19 cell line 11 was maintained in DMEM/F-12 medium supplemented with 10% FBS and 1% penicillin-streptomycin. In previous studies, 435/BRMS1 cells were subcultured by an EDTA method while parental 435 cells were maintained by trypsinization. 12 Therefore, Abbreviations: BRMS1, breast metastasis suppressor gene; Cx32, connexin 32; DiI, 1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethyl indocarbocyanine perchlorate (DiIC18); E-cadherin, epithelial cadherin; ECL, enhanced chemiluminescence; GJIC, gap junctional intercellular communication; hFOB, human fetal osteoblast; HRP, horseradish peroxidase; MAb, monoclonal antibody; N-cadherin,...

No abstract

Breast cancer cells preferentially metastasize to bone, leading to the formation of primarily osteolytic lesions. Osteoprotegerin (OPG) plays multifactorial roles in the development of osteolytic bone metastases. An increase in the ratio of receptor activator of nuclear factor kappaB ligand (RANKL) to OPG increases osteoclastogenesis within the bone microenvironment. OPG also acts as a survival factor for cancer cells by protecting them from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apoptosis. This study compares OPG production in vitro in a number of breast cancer cell lines exhibiting both differences in metastatic capacity and in preferential metastasis to bone. Our studies demonstrated that OPG expression by MDA-231, MDA-MET, and MDA-231/K cancer cells was directly correlated with bone specific homing and colonization potential but not with metastasis of cancer cells to other organs; both in IL-1 beta stimulated and control cells. We also demonstrated expression of other bone-related markers including type I collagen, osteocalcin, osteopontin, and Runx2 in these cells. However, the generally lower expression of these markers in the bone selective cell line MDA-MET suggested that increased OPG expression in the bone specific variant was not merely a consequence of enhanced osteomimicry by these cells but that it has a significant role in the metastatic process. Co-culture of breast cancer cells with osteoblastic cells (hFOB 1.19) led to an overall downregulation in OPG production, which was not affected by the bone homing and colonization potential of the cell lines, suggesting that OPG alone is not indicative of osteolytic bone activity by breast cancer cells.