SW480, RKO and LIM1215, the cells exhibiting the highest methylation indices atAREG CpG p150 and CpG p220, were tested for methylation of these CpGs after treatment with the DNA methyltransferase inhibitor DAC (see figure 15). SW480 cells became fully unmethylated in both CpGs after 72 h of treatment. Demethylation remained up to 144 h of treatment. In LIM1215 and RKO cells, treatment with DAC also led to a decrease of methylation in both CpGs, however, methylation did not disappear completely in all timepoints as seen for SW480. Nevertheless, the methylation indices of both CpGs dropped in RKO cells from high (0.8<MI<1) to low (0<MI<0.3) and in a similar way in LIM1215 from high (0.5< MI <0.8) to low (0.1 <MI <0.3). Since it was shown earlier thatAREG mRNA expression correlated with methylation inAREG CpG p150 and CpG p220, an explanation of theAREG mRNA expression change after DAC treatment might be due to the demethylating effect of DAC on these CpGs.
Figure 15:
SW480:
RKO:
LIM1215:
Figure 15: AREG intragenic methylation after DAC treatment: Three cancer cell lines were tested for their intragenic methylation atAREG CpGs p150 and p220 after treatment with 2.5 µM DAC at four different timepoints. The methylation index (MI) shows the content of methy-lation indicating products within the primer extension products in msSNuPE-experiments and is an indicator for DNA methylation at the given CpG. A methylation index of 1 means fully methylated whereas a methylation index of 0 means fully unmethylated.
Figure 16:
SW480:
RKO:
LIM1215:
CaCO2:
Figure 16: EREG intragenic methylation after DAC treatment: Four cancer cell lines were tested for their intragenic methylation atEREG CpGs p143 and p297 after treatment with 2.5 µM DAC at four different timepoints. The methylation index (MI) shows the content of methy-lation indicating products within the primer extension products in msSNuPE-experiments and is an indicator for DNA methylation at the given CpG. A methylation index of 1 means fully methylated whereas a methylation index of 0 means fully unmethylated.
The intragenic EREG CpGs p143 and p297 were methylated in the four cell lines SW480, RKO, LIM1215 and CaCO2. Therefore, it was also tested if methylation changed after DAC treatment (see figure 16). In contrast to what was observed for AREG, methylation of the EREG CpGs did not decrease in SW480 and LIM1215, although SW480, for example, had a methylation index similar to theAREG CpGs. In RKO and CaCO2 methylation of the CpGs decreased. The strongest effect was observed in CaCO2.
However, EREG mRNA expression did not change in CaCO2 after DAC treatment (see figure 4). Unfortunately, the samples RKO 72 h and 144 h as well as LIM1215 96 h -144 h did not give a result at the msSNuPE experiment.
Figure 17:
SW480:
LIM1215:
Figure 17: AREG intragenic methylation after TSA treatment: SW480 and LIM1215 were tested for their intragenic methylation at AREG CpG p150 and p220 after treatment with 25 ng/ml TSA at six different timepoints. The methylation index (MI) shows the content of methy-lation indicating products within the primer extension products in msSNuPE-experiments and is an indicator for DNA methylation at the given CpG. A methylation index of 1 means fully methylated whereas a methylation index of 0 means fully unmethylated.
In addition to investigating the effect of DAC treatment it was also tested, whether HDACis have an effect on the intragenic methylation of theAREG gene, since treatment with HDACis mainly led to an increase of AREG expression, too (see section 3.2.2). The effect of TSA onto the methylation ofAREG CpGs p150 and p220 is shown for the cells SW480 and LIM1215 in figure 17. Both cells differ in their initial methylation index, but in contrast to DAC treatment, methylation of the tested CpGs was changed upon
TSA treatment in none of the cells. However, in LIM1215 a reduction of CpG p150 methylation was observed from MI = 0.8 to MI = 0.6. A tendency was also seen for CpG p220 but to a lower extend.
Besides the effect of TSA, the effect of Valproat on the methylation of the intragenic AREG CpGs (see figure 18) and EREG CpGs (see figure 19) was tested, too. Neither AREG nor EREG methylation was influenced by Valproat in the tested cells.
Figure 18:
SW480:
RKO:
LIM1215:
Figure 18: AREG intragenic methylation after Valproat treatment: SW480, RKO and LIM1215 were tested for their intragenic methylation at AREG CpG p150 and p220 after treatment with 1 mM or 2 mM Valproat (Valproat 1 and 2) in four different timepoints. The methylation index shows the content of methylation indicating products within the primer ex-tension products in msSNuPE-experiments and is an indicator for DNA methylation at the given CpG. A methylation index of 1 means fully methylated whereas a methylation index of 0 means fully unmethylated.
Figure 19:
SW480:
RKO:
LIM1215:
CaCO2:
Figure 19: EREG intragenic methylation after Valproat treatment: Four different cell lines were tested for their intragenic methylation at EREG CpG p143 and p297 after treatment with 1 mM or 2 mM Valproat (Valproat 1 and 2) in four different timepoints. The Methylation index was calculated after msSNuPE experiment. A methylation index of 1 means fully methylated whereas a methylation index of 0 means fully unmethylated.
To summarize, DAC treatment led to a decrease of methylation at the intragenic AREG CpGs. Contrary, DAC had a lower effect on the methylation of the intragenic CpGs of theEREG gene. The HDACis TSA and Valproat did not influence methylation.