DNA methylation regulates constitutive expression of Stat6 regulatory genes SOCS-1 and SHP-1 in colon cancer cells

Abstract

Purpose: Stat6 signaling is active in cancer cells and IL-4-induced Stat6 activities or Stat6 activational phenotypes vary among cancer cells. This study aimed at investigating possible mechanism(s) involved in the formation of varying Stat6 activities/phenotypes.

Methods: Stat6 regulatory genes, SOCS-1 and SHP-1, were examined for mRNA expression using RT-PCR, and their promoter DNA methylation was assayed by methylation-specific PCR in Stat6-phenotyped colon cancer cell lines. DNA methylation was then verified by sequencing. RT-PCR assay and Western blotting were used to detect the expression of SOCS-1 and SHP-1 after demethylation using 5-aza-2'-deoxycytidine.

Results: Compared with Stat6(null) Caco-2 cells, Stat6(high) HT-29 cells showed decreased constitutive expression of SOCS-1 and SHP-1, which correlated with DNA hypermethylation in these genes' promoters. Interestingly, demethylation in HT-29 cells recovered the constitutive expression of SOCS-1 and SHP-1.

Conclusions: These findings suggest that DNA methylation controls the constitutive expression of negative Stat6 regulatory genes, which may affect Stat6 activities.

Fig. 1

Colon cancer cell lines HT-29 and Caco-2 show distinct IL-4/Stat6 EMSA profiles corresponding to differential Stat6 phenotypes. The criteria of assigning Stat6 phenotypes were established previously (Zhang et al. 2003). The phenotyping of these two cell lines was reported by us in a previous report (reproduced from Li et al. with permission)

Fig. 2

Active Stat6^high HT-29 cells express less constitutive mRNA of Stat6 negative regulators SOCS-1 and SHP-1 than inactive Stat6^null Caco-2 cells. a Gel images of RT-PCR products of SOCS-1, SHP-1, and SHP-2. The OD readings of individual target gene products were compared with those of GAPDH products within the same lane and the results were expressed as a ratio of target gene/GAPDH. b Summary presentation of OD ratios from at least three independent culture/RT-PCR tests for each gene. **P<0.01 by statistical analyses

Fig. 3

Active Stat6^high HT-29 cells exhibit higher DNA methylation in the promoter of SOCS-1 than that in inactive Stat6^null Caco-2 cells. a MSP detection of SOCS-1 methylation in HT-29 and Caco-2 cells. Relative methylation levels were measured for SOCS-1 whose OD readings over methylated product (M) were compared with that of a methylation-positive control (MPC, indicated by a vertical arrow), and the results were shown as a ratio of M/MPC. U and UPC indicate unmethylated product and unmethylation-positive control, respectively. As can be seen under U, both HT-29 and Caco-2 carry unmethylated products suggesting incomplete methylation of SOCS-1. b Diagrammatic comparison of methylation levels (OD ratios) obtained from three independent tests for HT-29 and Caco-2. c Confirmation of methylation by sequencing. Wild-type sequence of SOCS-1 shown at top and the bottom sequence is methylation-positive sequence. Caco-2 was sequenced reversely and therefore, the middle sequence represents the complementary sequence of the sequenced DNA. After bisulfite treatment, unmethylated C is converted to “T” whereas methylated C remains as C (underlined). **P < 0.01 by statistical analysis

Fig. 4

Active Stat6^high HT-29 cells exhibit higher DNA methylation in the promoter of SHP-1 than that in inactive Stat6^null Caco-2 cells. a MSP detection of SHP-1 methylation in HT-29 and Caco-2 cells. Relative methylation levels were measured and presented for SHP-1 in the same way as shown in Fig. 3. Note that SHP-1 is completely methylated in both HT-29 and Caco-2 with varying magnitude. U and UPC indicate unmethylated product and unmethylation-positive control, respectively. b Diagrammatic comparison of methylation levels (OD ratios) obtained from three independent tests for HT-29 and Caco-2. c Confirmation of methylation by sequencing. The presentation of methylated and unmethylated sequences is in the same way as in Fig. 3 (c). **P<0.01 by statistical analysis

Fig. 5

Expression of mRNA and protein is recovered after demethylation in HT-29 cells. a Gel images of RT-PCR detection of SOCS-1 and SHP-1 mRNA after demethylation. The relative expression of mRNA was measured as described in Fig. 2. b Diagrammatic presentation of mRNA expression levels (OD ratios) from at least three independent tests. c Gel images of Western blots for SHP-1 and β-actin. The OD readings of SHP-1 were compared with those of β-actin and the results were expressed as a ratio of SHP-1/β-actin. d Summary presentation of OD ratios obtained from three independent tests. ***P<0.001 and **P<0.01 by statistical analyses