DNA methylation-mediated silencing of nonsteroidal anti-inflammatory drug-activated gene (NAG-1/GDF15) in glioma cell lines

Abstract

Nonsteroidal anti-inflammatory drug-activated gene, NAG-1, a transforming growth factor-β member, is involved in tumor progression and development. The association between NAG-1 expression and development and progression of glioma has not been well defined. Glioblastoma cell lines have lower basal expression of NAG-1 than other gliomas and normal astrocytes. Most primary human gliomas have very low levels of NAG-1 expression. NAG-1 basal expression appeared to inversely correlate with tumor grade in glioma. Aberrant promoter hypermethylation is a common mechanism for silencing of tumor suppressor genes in cancer cells. In glioblastoma cell lines, NAG-1 expression was increased by the demethylating agent, 5-aza-2'-deoxycytidine. To investigate whether the NAG-1 gene was silenced by hypermethylation in glioblastoma, we examined DNA methylation status using genomic bisulfite sequencing. The NAG-1 promoter was densely methylated in several glioblastoma cell lines as well as in primary oligodendroglioma tumor samples, which have low basal expression of NAG-1. DNA methylation at two specific sites (-53 and +55 CpG sites) in the NAG-1 promoter was strongly associated with low NAG-1 expression. The methylation of the NAG-1 promoter at the -53 site blocks Egr-1 binding and thereby suppresses Nag-1 induction. Treatment of cells with low basal NAG-1 expression with NAG-1 inducer also did not increase NAG-1. Incubation with a demethylation chemical increased Nag-1 basal expression and subsequent incubation with a NAG-1 inducer increased NAG-1 expression. We concluded from these data that methylation of specific promoter sequences causes transcriptional silencing of the NAG-1 locus in glioma and may ultimately contribute to tumor progression.

FIGURE 1. NAG-1 basal expression in glioma cells, NHA cells, and primary tumor samples

A, Western blot analysis depicts the expression of β-actin and NAG-1 protein basal levels. B, ELISA analysis depicts secret NAG-1 protein concentration in growth media of cell culture. Secret NAG-1 concentration was normalized by total protein concentration. C, quantitative real-time RT-PCR analysis shows the basal level of NAG-1 mRNA reported as copy number. The values shown in the graph are the mean±S.D. of three independent experiments. The values shown in the graph are the mean±S.D. of three independent experiments.

FIGURE 2. DNA demethylation agent, 5-AZA-dC induces NAG-1 expression in glioblastoma cells

A, NAG-1 protein levels as determined by Western blot analysis. B, NAG-1 mRNA levels as determined by quantitative real-time RT-PCR. U87, U118, U138, U373 cells were treated with vehicle (white bar) or 1µM 5-AZA-dC (black bar) in serum containing media for 48 h. β-actin is a control for the amount of protein loaded. The values shown in the graph are the mean±S.D. of three independent experiments.

FIGURE 3. Hypermethylation prevents Nag-1 expression in glioblastoma cells

A, Western blot analysis depicts the expression of β-Actin and NAG-1 protein. β-Actin is a control for the amount of protein loaded. B, ELISA analysis depicts secret NAG-1 protein concentration in growth media of cell culture at different time points as indicated. Secret NAG-1 concentration was normalized by total protein concentration. T98G and U118 cells were treated with vehicle or 30µM SS in serum-free media. C.ELISA analysis depicts secret NAG-1 protein concentration in growth media of cell culture. Secret NAG-1 concentration was normalized by total protein concentration. T98G and U118 cells were pretreated with or without 5-Aza-dC in serum contain media for 5 days, then treated with vehicle, 30µM SS or 500ng/ml TSA in serum-free media for 24 h. The values shown in the graph are the mean±S.E. of three independent experiments. p values derived from Student’s t test is (*) p < 0.01, (**) p < 0.05.

FIGURE 4. Hypermethylation blocks Egr-1 binding to Nag-1 promoter

A, Egr-1 mRNA levels as determined by quantitative real-time RT-PCR analysis. T98G and U118 cells were treated with vehicle or 30µM SS at different time points as indicated. The values shown in the graph are the mean±S.E. of three independent experiments. B, The Egr-1 binding region amplified by the specific PCR primers in Chip assays is indicated. T98G and U118 cells were treated with vehicle or 30µM SS for 4 h and the Egr-1 protein and target DNA complex was examined by Chip and quantitative real-time PCR. C, Cells were pre-treated with 5-Aza for 5 days, passaged, starved overnight, treated with 30µM SS for 4 h and then examined by the Chip assay. The values shown in the graph are the mean±S.E. of three independent experiments. p values derived from Student’s t test is (*) p < 0.01.

FIGURE 5. NAG-1 over-expression increased apoptosis in glioblastoma cells

A, NAG-1 protein levels in cell lysates (A) and secret NAG-1 protein levels in growth media (B) after transfection as determined by Western blot analysis and ELISA. U87, U118, U138, U373 cells were transfected with pcDNA3 (white bar) or pcDNA3-NAG-1 (black bar) for 24 hr, then measured. β-Actin is a control for the amount of protein loaded. Secret NAG-1 concentration was normalized by total protein concentration. The values shown in the graph are the mean±S.D. of three independent transfections. C. the population of apoptotic cells by flow cytometry. After transfection for 24 hr, cells were incubated with serum free media for 5 days. The values shown in the graph are the mean±S.D. of three independent transfections. p values derived from Student’s t test is (**) p < 0.01, 0.01 < (*) p < 0.05. D, Alteration of NAG-1 expression with NAG-1 overexpression plasmid affects soft agar growth. U87 cells were transiently transfected with pcDNA3.1 or pcDNA3-NAG-1 for 24 h, then re-suspended and grown for 2 weeks in soft agar. The bar graphs represent mean ± S.D. of colony number in 5 randomly chosen microscope fields. **p < 0.01, significant differences.