Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells - PubMed (original) (raw)
Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells
A R Karpf et al. Proc Natl Acad Sci U S A. 1999.
Abstract
Inhibitors of DNA methyltransferase, typified by 5-aza-2'-deoxycytidine (5-Aza-CdR), induce the expression of genes transcriptionally down-regulated by de novo methylation in tumor cells. We utilized gene expression microarrays to examine the effects of 5-Aza-CdR treatment in HT29 colon adenocarcinoma cells. This analysis revealed the induction of a set of genes that implicated IFN signaling in the HT29 cellular response to 5-Aza-CdR. Subsequent investigations revealed that the induction of this gene set correlates with the induction of signal transducer and activator of transcription (STAT) 1, 2, and 3 genes and their activation by endogenous IFN-alpha. These observations implicate the induction of the IFN-response pathway as a major cellular response to 5-Aza-CdR and suggests that the expression of STATs 1, 2, and 3 can be regulated by DNA methylation. Consistent with STAT's limiting cell responsiveness to IFN, we found that 5-Aza-CdR treatment sensitized HT29 cells to growth inhibition by exogenous IFN-alpha2a, indicating that 5-Aza-CdR should be investigated as a potentiator of IFN responsiveness in certain IFN-resistant tumors.
Figures
Figure 1
5-Aza-CdR inhibits HT29 cell proliferation and sequesters DNA MeTase I. (A) HT29 cells were treated with vehicle or 500 nM 5-Aza-CdR for 24 hr. After this treatment, the drug was removed and cell proliferation was measured by directly counting cells at the indicated time points (see Materials and Methods). Data are presented as mean count ± 1 SD, (n = 3). (B) HT29 cells were treated with the indicated concentrations of 5-Aza-CdR for 24 hr, and the presence of DNA MeTase I (200 kDa) was assessed in nuclear protein extracts by immunoblotting. Sequestration of DNA MeTase I by 500 nM 5-Aza-CdR continued for 4 days after treatment (data not shown) (C) The expression of p16 in HT29 cells at time points after treatment with 500 nM 5-Aza-CdR was measured by Northern blot analysis.
Figure 2
Microarray analysis of gene expression changes in HT29 cells after 5-Aza-CdR treatment. (A) A cDNA microarray containing 4,608 target genes was constructed from a set of minimally redundant expressed sequence tags (ESTs). The microarray was hybridized with cDNAs prepared from vehicle-treated [Cy3-dCTP-labeled (green)] and 500 nM 5-Aza-CdR-treated HT29 cells [Cy5-dCTP (red)] 9 days after treatment. Two representative 12 × 32 gene grids (of 12) are displayed. (B) The fluorescent signal from the hybridized microarray slide was detected, quantified, and plotted as a ratio (Cy-5 signal/Cy-3 signal) for each array element. The average expression ratio for all genes on the array was normalized to 1.0 and had a SD of 0.177. The black line indicates a trend line 2 SD above the mean expression ratio for all genes on the microarray. The small, blue diamonds are genes below this cutoff; the large, red diamonds are genes above the cutoff. (C) Microarray expression data were confirmed by Northern blot analysis. Induction of five representative transcripts (see Table 1) 5 and 9 days after 5-Aza-CdR treatment is shown, along with glyceraldehyde-3-phosphate dehydrogenase (gapdh), an RNA-loading control. 11.5 kD, IFN-inducible protein 27; 17.5 kD, IFN-induced 17-kDa protein; 56 kD, IFN-induced protein 56.
Figure 3
Microarray expression profiling of 5-Aza-CdR and IFN-treated HT29 cells. HT29 cells were treated with 500 nM 5-Aza-CdR, 1 × 105 units/ml IFN-α2a, or 5 × 102units/ml IFN-γ. RNA was harvested 9 days (5-Aza-CdR) or 4 days (96 hr) (IFN-α or -γ) after treatment and used to generate probes for microarray analysis. Shown in the figure is a representative section of the microarray after hybridization with Cy-5-labeled cDNAs from 5-Aza-CdR-, IFN-α-, or IFN-γ-treated cells and Cy-3-labeled cDNAs from control cells. Four genes up-regulated by 5-Aza-CdR treatment are on the displayed grid. They are IFN-α-inducible protein 6–16 (row 4, column 9), expressed sequence tag (EST) Hs.109309 (8, 7), EST Hs.165240 (9, 14), and human mRNA for Stac (9, 16).
Figure 4
5-Aza-CdR treatment activates STATs 1, 2, and 3 in HT29 cells. (A) The expression level of IFN-α in HT29 cells before and after 500 nM 5-Aza-CdR treatment was measured by reverse transcription–PCR along with gapdh to confirm equivalent cDNA input. (B) STAT transcription factor levels were measured by Western blotting. Cytoplasmic (C) and nuclear (N) cell extracts were prepared from HT29 cells after treatment with vehicle or 500 nM 5-Aza-CdR. A poly(vinylidene difluoride) membrane harboring the protein extracts was probed sequentially with mAbs specific to STATs 1, 2, and 3. In each case, the antibodies recognized proteins of the appropriate molecular weight for each STAT. Molecular mass markers are indicated. (C) The expression of STAT 1, 2, and 3 genes was measured by Northern blotting. RNA was isolated from HT29 cells after treatment with vehicle or 500 nM 5-Aza-CdR. The locations of molecular mass markers are indicated. Ethidium bromide staining confirmed equal RNA loading (28S, 18S rRNAs).
Figure 5
5-Aza-CdR treatment increases the responsiveness of HT29 cells to growth inhibition mediated by exogenous IFN-α2A. HT29 cells were treated with 500 nM 5-Aza-CdR or vehicle (PBS). Ten days after removal of the drug, triplicate wells were treated with a concentration curve of IFN-α2a. Four days later, cell proliferation was measured by using a Coulter counter. Percentage of control growth was calculated by dividing the mean cell count at each IFN concentration by the mean cell count of untreated control cells (either HT29 or 5-Aza-CdR-treated HT29 cells, respectively). Data are presented as mean ± 1 SD, (n = 3). Similar results were obtained in four independent experiments.
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