Shortening of microsatellite deoxy(CA) repeats involved in GL331-induced down-regulation of matrix metalloproteinase-9 gene expression.
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.
Huang
Tze-Sing
TS
Division of Cancer Research, National Health Research Institutes, Taipei, Taiwan, ROC. tshuang@nhri.org.tw
Lee
Chun-Chung
CC
Chang
Ai-Chi
AC
Lin
Shankung
S
Chao
Chuan-Chuan
CC
Jou
Yuh-Shan
YS
Chu
Yi-Wen
YW
Wu
Cheng-Wen
CW
Whang-Peng
Jacqueline
J
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
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D061965:Matrix Metalloproteinase Inhibitors; D011993:Recombinant Fusion Proteins; C121391:GL 331; D005047:Etoposide; D020780:Matrix Metalloproteinase 9