title: Shortening of microsatellite deoxy(CA) repeats involved in GL331-induced down-regulation of matrix metalloproteinase-9 gene expression.
abstract: Matrix metalloproteinase-9 (MMP-9) associates with cancer cell invasion and metastasis. CL1-5 cells, a human lung adenocarcinoma cell line, expressed an elevated level of MMP-9 and exhibited a highly invasive and metastatic ability. By Matrigel assay and gelatinase zymography, the topoisomerase II poison GL331 was found to dose-dependently inhibit the invasiveness and the level of secreted MMP-9 of CL1-5 cells. Northern blot analysis indicated that cellular MMP-9 mRNA level was decreased after GL331 treatment. Furthermore, GL331-induced down-regulation of mmp-9 gene promoter was demonstrated by using a luciferase reporter gene driven by the -216 to -13 region of the mmp-9 gene promoter cloned from CL1-5 cells. By PCR amplification and gel electrophoresis, we found that GL331 caused shortening of the -216 to -13 region of the mmp-9 promoter. Direct sequencing analysis revealed that the number of d(CA) was reduced from 24 to 18 at the microsatellite d(CA) repeat region of the mmp-9 promoter. The CL1-5 cells transfected with the luciferase reporter containing 18 d(CA)s expressed only 53% of those when the reporter contained 24 d(CA)s. The promoter region of mmp-9 gene contains other positive regulatory elements, such as TRE and kappaB. We found that GL331 did not significantly influence the luciferase activity driven by TRE or kappaB. Taken together, these data suggested that GL331 inhibited MMP-9 mRNA expression at least partly through the selective induction of shortening of microsatellite d(CA) repeats. This is the first report that an anti-cancer agent can inhibit mmp-9 gene expression by inducing microsatellite DNA shortening.
authors.affiliation: Division of Cancer Research, National Health Research Institutes, Taipei, Taiwan, ROC. tshuang@nhri.org.tw
authors.forename: Tze-Sing
authors.initials: TS
authors.lastname: Huang
authors.forename: Chun-Chung
authors.initials: CC
authors.lastname: Lee
authors.forename: Ai-Chi
authors.initials: AC
authors.lastname: Chang
authors.forename: Shankung
authors.initials: S
authors.lastname: Lin
authors.forename: Chuan-Chuan
authors.initials: CC
authors.lastname: Chao
authors.forename: Yuh-Shan
authors.initials: YS
authors.lastname: Jou
authors.forename: Yi-Wen
authors.initials: YW
authors.lastname: Chu
authors.forename: Cheng-Wen
authors.initials: CW
authors.lastname: Wu
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Whang-Peng
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000031:analogs & derivatives* / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000235:genetics* / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D018895:Microsatellite Repeats / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D061965:Matrix Metalloproteinase Inhibitors; D011993:Recombinant Fusion Proteins; C121391:GL 331; D005047:Etoposide; D020780:Matrix Metalloproteinase 9