Acute Hypersensitivity of Pluripotent Testicular Cancer-Derived Embryonal Carcinoma to Low-Dose 5-Aza Deoxycytidine Is Associated with Global DNA Damage-Associated p53 Activation, Anti-Pluripotency and DNA Demethylation

Human embryonal carcinoma (EC) cells are the stem cells of nonseminoma testicular germ cells tumors (TGCTs) and share remarkable similarities to human embryonic stem (ES) cells. In prior work we found that EC cells are hypersensitive to low nanomolar doses of 5-aza deoxycytidine (5-aza) and that this hypersensitivity partially depended on unusually high levels of the DNA methyltransferase, DNMT3B. We show here that low-dose 5-aza treatment results in DNA damage and induction of p53 in NT2/D1 cells. In addition, low-dose 5-aza results in global and gene specific promoter DNA hypomethylation. Low- dose 5-aza induces a p53 transcriptional signature distinct from that induced with cisplatin in NT2/D1 cells and also uniquely downregulates genes associated with pluripotency including NANOG, SOX2, GDF3 and Myc target genes. Changes in the p53 and pluripotency signatures with 5-aza were to a large extent dependent on high levels of DNMT3B. In contrast to the majority of p53 target genes upregulated by 5-aza that did not show DNA hypomethylation, several other genes induced with 5-aza had corresponding decreases in promoter methylation. These genes include RIN1, SOX15, GPER, and TLR4 and are novel candidate tumors suppressors in TGCTs. Our studies suggest that the hypersensitivity of NT2/D1 cells to low-dose 5-aza is multifactorial and involves 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. GEO number for the microarray data: GSE42647

Identification of G1-Regulated Genes in Normally Cycling Human Cells

BACKGROUND: Obtaining synchronous cell populations is essential for cell-cycle studies. Methods such as serum withdrawal or use of drugs which block cells at specific points in the cell cycle alter cellular events upon re-entry into the cell cycle. Regulatory events occurring in early G1 phase of a new cell cycle could have been overlooked. METHODOLOGY AND FINDINGS: We used a robotic mitotic shake-off apparatus to select cells in late mitosis for genome-wide gene expression studies. Two separate microarray experiments were conducted, one which involved isolation of RNA hourly for several hours from synchronous cell populations, and one experiment which examined gene activity every 15 minutes from late telophase of mitosis into G1 phase. To verify synchrony of the cell populations under study, we utilized methods including BrdU uptake, FACS, and microarray analyses of histone gene activity. We also examined stress response gene activity. Our analysis enabled identification of 200 early G1-regulated genes, many of which currently have unknown functions. We also confirmed the expression of a set of genes candidates (fos, atf3 and tceb) by qPCR to further validate the newly identified genes. CONCLUSION AND SIGNIFICANCE: Genome-scale expression analyses of the first two hours of G1 in naturally cycling cells enabled the discovery of a unique set of G1-regulated genes, many of which currently have unknown functions, in cells progressing normally through the cell division cycle. This group of genes may contain future targets for drug development and treatment of human disease

Caspase-Dependent Regulation and Subcellular Redistribution of the Transcriptional Modulator YY1 during Apoptosis

The transcriptional regulator Yin Yang 1 (YY1) controls many aspects of cell behavior and is essential for development. We analyzed the fate of YY1 during apoptosis and studied the functional consequences. We observed that this factor is rapidly translocated into the cell nucleus in response to various apoptotic stimuli, including activation of Fas, stimulation by tumor necrosis factor, and staurosporine and etoposide treatment. Furthermore, YY1 is cleaved by caspases in vitro and in vivo at two distinct sites, IATD(12)G and DDSD(119)G, resulting in the deletion of the first 119 amino acids early in the apoptotic process. This activity generates an N-terminally truncated YY1 fragment (YY1Δ119) that has lost its transactivation domain but retains its DNA binding domain. Indeed, YY1Δ119 is no longer able to stimulate gene transcription but interacts with DNA. YY1Δ119 but not the wild-type protein or the caspase-resistant mutant YY1D12A/D119A enhances Fas-induced apoptosis, suggesting that YY1 is involved in a positive feedback loop during apoptosis. Our findings provide evidence for a new mode of regulation of YY1 and define a novel aspect of the involvement of YY1 in the apoptotic process

Low-dose 5-aza and DNMT3B knockdown alters genome-wide promoter demethylation in NT2/D1-R1 cells.

<p><b>A</b>, Summary of results from Illumina 27K beadchip DNA promoter methylation analysis of NT2/D1-R1 sh-control cells (sh-ctrl) and NT2/D1-R1 sh-DNMT3B cells (sh3B) untreated or treated for 3 days with 10 nM 5-aza. The number of promoter methylation alterations was based on the average of 3 replicates with 1.3-fold or greater change and a p value<0.05. <b>B</b>, Venn diagrams depicting degree of overlap in genes altered in expression or DNA promoter methylation in NT2/D1-R1 cells due to low dose 5-aza or DNMT3B knockdown. <b>Top</b>, High degree of overlap in genes in NT2/D1-R1 cells demethylated with low dose 5-aza and demethylated with DNMT3B knockdown (left). Little overlap in genes in NT2/D1-R1 cells undergoing increased DNA methylation with low dose 5-aza and increased DNA methylation with DNMT3B knockdown (right). The numbers represent methylation changes of 1.3-fold or greater with p<0.05. <b>Middle</b>, A moderate degree of overlap in genes in NT2/D1-R1 cells that underwent decreased DNA methylation and increased gene expression with 5-aza (left) and little overlap in genes in NT2/D1-R1 cells that underwent increased DNA methylation and increased gene expression with 5-aza. The numbers represent methylation changes of 1.3-fold or greater with p<0.05 and expression changes of 1.5-fold or greater with p<0.01. <b>Bottom</b>, A large degree of overlap in genes demethylated after 5-aza treatment of NT2/D1 and NT2/D1-R1 cells. The six overlapping genes are shown. The numbers represent methylation changes of 1.3-fold or greater with p<0.05. <b>C</b>, Unsupervised hierarchical clustering of the promoter DNA methylation of the 4 treatment arm values among just the 388 genes changed 1.3-fold or greater, p value<0.05 with 5-aza in the control cells depicting the large degree of overlap in genes undergoing demethylation with 5-aza and DNMT3B knockdown. Increased methylation = red, decreased methylation = green.</p

DNMT3B knockdown inhibits low-dose 5-aza mediated genome-wide activation of p53 target genes and repression of pluripotency genes.

<p><b>A</b>, Summary of results from expression microarray data of NT2/D1-R1 sh-control cells (sh-ctrl) and NT2/D1-R1 sh-DNMT3B cells (sh3B) untreated or treated for 3 days with 10 nM 5-aza. Knockdown of DNMT3B greatly reduced the number of genes altered with 5-aza treatment while DNMT3B knockdown alone results in minimal expression changes. The number of genes altered was based on the average of 3 biological replicates with 1.5-fold or greater change and a p value<0.01. <b>B</b>, Unsupervised hierarchical cluster analysis of the expression data in (A) was performed on the 541 genes altered more than 1.8-fold with p value<0.01 ANOVA BH corrected, between NT2/D1-R1 sh-control cells (sh-ctrl) and NT2/D1-R1 sh-DNMT3B cells (sh3B) untreated or treated for 3 days with 10 nM 5-aza. Upregulated gene are red, downregulated genes are green. C, Partitioning around mediods (PAM) analysis of 1169 genes changed 1.5-fold or greater with BH corrected p-value of <0.01. Cluster 1 and Cluster 2 of 6 total clusters are shown. All clusters are provided in supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053003#pone.0053003.s004" target="_blank">Fig S4</a>. The number of genes in the clusters and the mean silhouette width (MSW) value is indicated. Expression intensity values for representative genes in Cluster 1 (pluripotent genes) and Cluster 2 (p53 target genes) is provided on the right and additional prominent members are provided below each cluster. Error bars are S.E.M. * = p<0.05 compared to untreated control.</p

Low-dose 5-aza induces an early, robust, and unique reprogramming of gene expression in NT2/D1 cells.

<p><b>A</b>, Scatter plot of microarray gene expression differences in NT2/D1 cells. Each point represents the average of 3 biological replicates. Points above the diagonal line represent genes upregulated and points below the diagonal line repressed with 1 day 5-aza treatment (top), 3 day 5-aza treatment (middle), and cisplatin treatment (bottom), compared to control. The number of altered genes above the indicated thresholds were changed with p<0.05. <b>B</b>, Robust induction of gene expression occurs with low-dose 5-aza. Box-whisker plot of expression levels of genes (1296) changed among groups >1.2-fold and p<0.02 ANOVA, Benjamini Hochberg (BH) corrected indicated a upward shift in gene expression with 5-aza. <b>C</b>, Low dose 5-aza induced an early and unique program of gene expression compared to cisplatin. Unsupervised hierarchical cluster analysis of the expression profile of 5-aza and cisplatin altered genes was performed on the 898 genes (rows) altered between the samples (columns) more than 1.5-fold with p value<0.01 ANOVA, BH corrected. Upregulated gene are red, downregulated genes are green. Large overlap in 1 day and 3 day 5-aza treatments and distinct regulation compared to cisplatin is evident. Genes regulated in a similar manner by 5-aza and cisplatin are in brackets.</p

Confirmation of promoter DNA demethylation with low-dose 5-aza in NT2/D1 cells.

<p><b>A</b>, Representative bisulfite pyrosequencing tracing from NT2/D1 cells treated with vehicle control or 10 nM 5-aza for 3 days in the promoter region of SOX15. Shaded areas are CpG sites. <b>B</b>, Decrease in DNA methylation of the RIN1, SOX15 and TLR4 promoter with 3 day 10 nM 5-aza treatment of NT2/D1 cells as determined by bisulfite pyrosequencing. Average of biological triplicate determinations. Error bars are standard deviation. ** = p<0.005. SOX15 values represent the average methylation value across two CpG sites. RIN1 and TRL4 values represent the average methylation across three CpG sites. <b>C</b>, Increased gene expression of RIN1, SOX15 and TLR4 with 3 day 10 nM 5-aza treatment of NT2/D1 cells as determined by real-time PCR. Average of biological triplicate determinations. Error bars are standard deviation. * = p<0.05; ** = p<0.005.</p

Low-dose 5-aza induces distinct genome-wide activation of p53 target genes and unique repression of pluripotency genes in NT2/D1 cells.

<p><b>A</b>, Venn diagrams of expression microarray data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053003#pone-0053003-g002" target="_blank">Figure 2</a> indicating a large overlap in the genes upregulated (left) and down regulated (middle) in NT2/D1 cells with 1 day 5-aza treatment (fold change >1.3) compared to 3 day 5-aza treatment (fold change >1.5). A Venn diagram (right) of microarray data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053003#pone-0053003-g002" target="_blank">Figure 2</a> showing a large degree of overlap in genes upregulated 1.5-fold or greater by both 3 day 5-aza and cisplatin treatments. Genes listed in each Venn diagram are altered with p<0.05. Of the overlap genes, 13 are known p53 target genes in NT2/D1 cells. <b>B</b>, Partitioning around mediods (PAM) analysis of 5-aza and cisplatin regulated genes in NT2/D1 cells. The 1130 genes changed 1.5-fold or greater with BH corrected p-value of <0.02 were subjected to PAM analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053003#s4" target="_blank">Methods</a>. The number of genes in each of the five clusters and the mean silhouette width (MSW) value for each cluster is indicated. Expression intensity values for representative genes in Cluster 2, Cluster 3, and Cluster 4 is provided on the left. Error bars are S.E.M. * = p<0.05; ** = p<0.005 compared to untreated controls.</p