In prior work we established that EC cells are exquisitely sensitive to low nanomolar doses of 5-aza and that this hypersensitivity is partially dependent on high levels of DNMT3B. The current study utilizes genome-wide transcriptional and promoter methylation analyses to discover that 5-aza not only exerts its biological effects through DNA demethylation and gene re-expression but also by an unexpected repression of pluripotency in NT2/D1 cells. Further, low dose 5-aza mediates DNA damage as assessed by accumulation of pH2AX that is associated with a robust induction of p53 target genes. Interestingly, the transcriptional reprogramming with 5-aza in NT2/D1-R1 cells appears largely dependent on high levels of DNMT3B while the accumulation of pH2AX and p53 does not. The relationship between DNA damage, p53 target gene activation, DNA demethylation, and repression of pluripotency in response to low-dose 5-aza in NT2/D1-R1 cells is depicted in .
Model of mechanisms of low-dose 5-aza hypersensitivity in EC cells.
The exact anticancer mechanism of 5-aza is controversial and most studies have used somatic cells at doses substantially higher than used in the current study. There is evidence that incorporation of 5-aza in DNA results in adduct formation with DNMTs leading to sequestration-mediated hypomethylation or a trigger for a DNA-damage response 
. Doses of 5-aza as low as 10 nM are sufficient to induce DNA damage and apoptosis in NT2/D1 cells as monitored by pH2AX () and this is associated with the induction of a classic p53 target gene signature coupled with transcriptional repression of core pluripotency genes (). Interestingly, the transcriptional effects commence even after only 1 day of 5-aza treatment suggesting that they are proximal to the actions of 5-aza on NT2/D1 cells (). The data also suggests that either one cell cycle is sufficient to meet threshold 5-aza incorporation for demethylation/DNA damage responses or that 5-aza can have additional effects on EC cells independent of DNA incorporation.
We and others have shown that EC cells undergo hyperactive activation of pro-apoptotic p53 target genes in response to cisplatin, suggesting that TGCTs responsiveness to DNA damaging agents may relate to a unique cellular context during p53 activation 
. Notably, recent evidence suggests that p53 is a barrier for induced pluripotency of somatic cells and has antistemness and prodifferentiation functions in ES and EC cells 
. It has also been suggested that p53 can directly repress pluripotency genes including NANOG, OCT4 and GDF3 after treatment with DNA damaging agents 
. However, we failed to see downregulation of pluripotency genes with cisplatin under the conditions employed here and 5-aza repression of pluripotency genes was not affected by shRNA knockdown of p53 (data not shown). Interestingly, 5-aza only induced a subset of the p53 target genes that were induced with cisplatin, further suggesting that 5-aza and cisplatin may activate p53 in distinct manners, which for 5-aza may include mechanisms apart from DNA damage.
Doses of 5-aza as low as 10 nM led to global hypomethylation of LINE-1 repetitive elements and a decrease in promoter methylation in NT2/D1 and NT2/D1-R1 cells (, , and ). The promoter hypomethylation was in both CpG islands and non CpG island promoters. The genes induced only by 5-aza (Cluster 1, ) may be induced by a demethylation mechanism and are likely important in mediating the acute hypersensitivity of 5-aza in NT2/D1 cells. There was also a substantial degree of overlap between genes with decreased promoter DNA methylation and induced expression with low-dose 5-aza, suggesting effective re-expression through DNA demethylation is occurring (). RIN1, TLR4 and SOX15 are novel candidate biomarkers and tumors suppressor genes in TGCTs (). In addition, GSEA analysis suggests that a subset of genes altered with high-dose 5-aza in other tumor types is also altered with low-dose 5-aza in NT2/D1 cells, further supporting demethylation as a participating mechanism for the hypersensitivity of NT2/D1 cells to 5-aza.
Recent analysis of the methylome of ES and induced pluripotent stem (iPS) cells supports a unique pattern of DNA methylation in ES cells that suggests potential additional mechanisms responsible for the hypersensitivity of EC cells to 5-aza compared to somatic cells 
. A potential mechanism for acute low-dose 5-aza toxicity in EC cells is increased genomic instability due to demethylation of centromeric and pericentromeric satellite repeats 
. Recently, pluripotent cells have been exclusively shown to possess high non-CpG methylation in gene bodies that correlates with the expression and specificity of DNMT3B 
. It would be of interest to investigate whether demethylation of non-CpGs plays a role in 5-aza response in EC and in a boarder sense whether the unique methylome of the pluripotent genome sensitizes cells to 5-aza.
The use of 5-aza in the treatment of myelodysplastic syndrome and recent trials in lung cancer suggest low-dose 5-aza treatment mediates delayed and long-term anticancer responses 
. A possible implication of these findings is that low-dose 5-aza may preferentially target cancer-initiating or stem-like cells and that the prolonged time to response in patients might involve progressive exhaustion of discrete cell populations. An elegant recent report from Tsai and colleagues used in vivo
and in vitro
models to demonstrate that 3 day 10 nM 5-aza treatments did not elicit DNA damage or acute toxicity in a variety of solid tumors but did mediate delayed toxicity associated with depletion of tumor initiating cells 
. It is tempting to speculate that rare malignant stem-like cells in somatic solid tumors may undergo acute toxicity similar to EC cells, and by the mechanisms outlined above (), to account for the delayed responses to 5-aza in bulk somatic solid tumors.
Other reports have also seen that knockdown of DNMT expression in ES cells results in decreased sensitivity to 5-aza 
. Surprisingly, a similar level of DNA damage with 5-aza, as monitored by pH2AX and p53 induction, was seen in our control and DNMT3B knockdown cells (). However, there is a dramatic decrease in 5-aza mediated repression of pluripotency genes and 5-aza induction of p53 target genes with DNMT3B knockdown (). These results support a cause-and-effect relationship between 5-aza gene expression alterations in NT2/D1-R1 cells and acute toxicity and suggest that the role of DNMT3B in 5-aza toxicity is at a level downstream of induction of DNA damage. One explanation for the apparent paradoxical effect of DNMT3B knockdown on 5-aza mediated gene expression and survival of NT2/D1-R1 cells is that in the absence of DNMT3B DNA adducts formed with 5-aza may be qualitatively or quantitatively altered in a manner that is insensitive to discrimination by pH2AX staining. An example would be differential recruitment of chromatin modifying proteins. Perhaps there is also redistribution of DNA-adducts and DNA damage in DNMT3B knockdown cells that is not detected by total pH2AX staining that originates from the unique localization and high levels of DNMT3B in EC cells 
. There is extensive evidence that different DNMTs are differentially recruited to DNA and participate in DNMT isoform-specific protein interactions to exert scaffolding functions for chromatin dynamics and regulation 
. DNMT3B- and pH2AX-specific chromatin immunoprecipitation and co-immunoprecipitation assays may help resolve the apparent paradoxical role of DNMT3B in the 5-aza response of NT2/D1 cells.
Interestingly, although few genes were changed in expression by DNMT3B knockdown alone (), there was substantial decreases in gene promoter methylation with DNMT3B knockdown in NT2/D1-R1 cells () and these promoter demethylations had a large degree of overlap with demethylation induced with 5-aza (). This data suggests that DNA demethylation with low-dose 5-aza in NT2/D1-R1 cells does not fully explain its acute toxicity and effects on gene expression.
TGCT-derived pluripotent EC cells, even those resistant to cisplatin, are hypersensitive to low-dose 5-aza compared to solid somatic tumors cells 
. We have shown in NT2/D1 and NT2/D1-R1 cells that this acute, low-dose sensitivity to 5-aza is likely mediated through a multifactorial mechanism involving the combined activation of p53 targets, repression of pluripotency genes, and activation of genes repressed by DNA methylation. Low-dose 5-aza therapy may be a general strategy to treat those tumors that are sustained by cells with embryonic stem-like properties. Studies on pluripotent EC cells may have important biological and clinical relevance based on the growing appreciation that cancer stem or initiating cells share gene expression programs with pluripotent cells and the recent findings that low-dose 5-aza may target tumor initiating cells in solid tumors.