Matrix metalloproteinase (MMP)-9, the 92-kDa type IV collagenase, contributes to tumor invasion and metastases, and strategies to down-regulate its expression could ultimately be of clinical utility. A pyrrole-imidazole (PI) polyamide that targets the activator protein-1 (AP-1)-binding site of the MMP-9 promoter was designed and synthesized as a gene-silencing agent for tumor metastases. The synthesized product showed selective DNA binding ability. The MMP-9 PI polyamide significantly inhibited MMP-9's mRNA expression, protein level, and enzymatic activity in human breast adenocarcinoma cells (MDA-MB-231). Furthermore, the MMP-9 PI polyamide inhibited migration and invasion by in vitro wound-healing and matrigel-invasion assay. The FITC-labeled PI polyamide was localized in nuclei in 45 min of incubation with an MDA-MB-231 cell and remained in the nuclei for up to 96 h after incubation in vitro. It was also quickly localized in the mouse cellular nuclei of many tissues, including liver, kidney, and spleen, after intravenous injection without using any drug-delivery system. Moreover, the polyamide treatment significantly decreased metastasis in a mouse model of liver metastasis. Our results suggest that this PI polyamide, which targets the MMP-9 gene promoter, can be a novel MMP-9 down-regulating molecule for antimetastasis.

Background-Epigenetic changes such as aberrant DNA methylation and histone modification have been shown to play an important role in the tumorigenesis of malignant melanoma.Objective-To identify novel tumor-specific differentially methylated regions (DMRs) in human malignant melanoma.Methods-The aberrant methylation at 14 candidate human genomic regions identified through a mouse model study with quantitative DNA methylation analysis using the Sequenom MassARRAY system was performed.Results-The CpG island Exon 1 region of the Zygote arrest 1 (ZAR1) gene, which is responsible for oocyte-to-embryo transition, showed frequent aberrant methylation of 28 out of 30 (93%) melanoma surgical specimens, 16 of 17 (94%) melanoma cell lines, 0% of 4 normal human epidermal melanocyte (NHEM) cell lines, 0% of 10 melanocytic nevi and 100% of 51 various cancer cell lines. According to the real-time RT-PCR, the ZAR1 gene was overexpressed in part of Correspondence author: Hiroki Nagase, Division of Cancer Genetics, Department of Advanced Medical Research Center, Nihon, University School of Medicine, Tokyo, 173-8610, Japan. Phone: +81-3-3972-8337, FAX: +81-3-3972-8337, nagase.hiroki@med.nihon-u.ac.jp. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

5 Pichler WJ. Delayed drug hypersensitivity reactions. Ann Intern Med 2003; 139:683-93. 6 Mashiah J, Brenner S. A systemic reaction to patch testing for the evaluation of acute generalized exanthematous pustulosis. Arch Dermatol 2003; 139:1181-83. 7 Schmid S, Kuechler PC, Britschgi M et al. Acute generalized exanthematous pustulosis: role of cytotoxic T cells in pustule formation. Am J Pathol 2002; 161:2079-86. 8 Sidoroff A, Halevy S, Bavinck JAB et al. Acute generalized exanthematous pustulosis (AGEP)-a clinical reaction pattern. Conflicts of interest: none declared.(a) Fig 2. (a) Abdominal contrast computed tomography showing a lowdensity 6 cm mass in segment 5 of the liver and a high-density 2 cm mass in the neck of the gallbladder. (b) The tumour cells and the surrounding matrix showed positive immunohistochemical staining for granulocyte colony-stimulating factor (original magnification ·25).

Tissue-specific differentially methylated regions (tDMRs) have been identified and implicated for their indispensable involvement in mammalian development and tissue differentiation. In this report, a quantitative DNA methylation analysis was performed for 13 human orthologous regions of recently confirmed mouse tDMRs by using Sequenom Mass Array, by which bisulfite-treated fragments are quantitatively detected using time of flight masspectroscopy analysis. Eight regions were shown as tDMRs in various tissues from three independent individuals. Testis DNA samples from eight individuals were also analyzed for methylation. Interestingly, there is evidence that the DNA methylation level is divergent among individuals. DNA methylation levels of five testisspecific DMRs were significantly inversely correlated with the number of spermatocytes. However, a positive correlation was seen at tDMRs located near the TRIM38 and CASZ1 genes. Our results indicate that tDMRs are conserved between mouse and human and may have an important role in regulating tissue function, differentiation and aging.