Background Environmental factors may influence the development of tetralogy of Fallot (TOF), and DNA methylation patterns may reveal specific chemical signatures of perturbations during cardiac development. We investigated whether blood and buccal cells could be viable surrogates for myocardium. Methods We measured epigenome‐wide DNA methylation at 866,895 5’‐cytosine‐phosphate‐guanine‐3’ (CpG) sites in blood (n=3), buccal cells (n=3), and right ventricular myocardium (n=4) collected from infants with TOF and compared the percent of differentially methylated CpG sites across tissue types. Gene‐specific DNA methylation profiles were also analyzed for ten representative genes associated with heart development. Welch's ANOVAs compared general methylation between tissue types. Results Comparison of DNA methylation profiles across blood, buccal, and myocardium suggested myocardium and buccal samples were most similar, differing in DNA methylation at only 1.3% (11,386) of CpG sites whereas myocardium and blood were most dissimilar, having 146,857 statistically dissimilar methylated CpG sites (~17% dissimilarity; adjusted p < 0.01 for each site). Buccal swabs were significantly more variable (p < .001) than either blood or myocardial samples. In gene‐specific analyses, SCO2, GATA4, NOTCH4, WNT7A, and DKK2 showed conserved DNA methylation profiles across tissue types, while HAND1, JAG1, NKX2‐5, TBX5 and TBX20 showed more distinctive tissue‐specific patterns of DNA methylation. Conclusions Compared with blood, buccal tissue more closely mirrors the myocardial methylome, with >10‐fold similarity. Nevertheless, both buccal and blood tissue capture highly conserved DNA methylation patterns at specific genetic loci related to cardiac development. Buccal cheek swabs may be a useful surrogate tissue type for future investigations of TOF‐specific epigenetic profiles.

Prostate cancer (PCa) is the most common malignancy among men in the USA. Polyphenon E (PolyE) is a standardized blend of polyphenols found in green tea extract, which has been shown to have chemoprevention value in PCa models, but the molecular mechanism(s) have not been elucidated. Polyphenols from green tea are known to alter the expression levels of some genes. Our experimental goal was to identify genes with expression level changes (up- or down-regulated by 2-fold or more) in normal, primary prostate cells as well as two PCa cell lines, DU145 and PC-3. Ultimately, we sought to identify genes whose expression level was differentially altered by PolyE treatment in normal versus PCa cells. We hypothesized that exposure to PolyE induces gene expression changes, which could provide rationales for the molecular mechanism(s) and provide potential targets for therapeutics and detection. A non-cancerous, human primary prostate cell line as well as two PCa cell lines (DU145 and PC-3) were cultured and treated them with varied concentrations of PolyE for 24 hours. Cellular RNA was isolated and subjected to microarray analyses (MA) at the Molecular Genomic Core facility of the Moffitt Cancer Center. A plethora of genes were identified by MA as having two-fold or higher gene expression level changes after PolyE exposure. Of these genes, fourteen with were selected for further analyses by TaqMan qRT-PCR. Cellular RNAs were converted into cDNA used in validated TaqMan qRT-PCR assays (StepOneTM RT-PCR System). RT-PCR data was analyzed using StepOneTM software and the ΔΔCt method. ACTB served as the endogenous control gene for all TaqMan qRT-PCR assays. Our qRT-PCR data from PolyE-treated DU145, PC-3, and primary prostate cells were consistent in the lack of expression changes greater than 2-fold for ATM, CASP8, HDAC4 and RB1. For RGCC, MXD1, CCSER2/FAM190B, CBLB and BCL2L11, gene expression levels increased by more than 2-fold in all three cell lines. For CCNB1 and SEC62, primary cells experienced more than a 2-fold change in gene expression, while the DU145 and PC-3 cell lines did not. Interestingly, ATM gene expression levels decreased by more than 10-fold in primary prostate cells, but in DU145 and PC-3 had changed less than 3-fold. Most notably, RGS4 gene expression levels were differentially altered in primary prostate cells compared to the PCa lines. In primary prostate cells, RGS4 decreased by more than 7-fold, but experienced a greater than 10-fold increases in DU145 and PC-3. In summary, we have identified RGS4 as a gene whose expression levels are differentially affected in primary prostate cells versus the PCa cell lines DU145 and PC-3. Citation Format: L. Michael Carastro, Ricardo A. Cordova, Daniel A. Barboto, Ricardo A. Declet-Bauzo, Irena Gushterova, Nicholas K. Lago, Nicholas E. Braganca, Joanna C. Burr, Dara E. Hoffman, Jong Y. Park. Gene expression changes in Polyphenon E treated prostate cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4211. doi:10.1158/1538-7445.AM2017-4211

Transcription is regulated through a variety of different mechanisms. One facet of transcriptional regulation is epigenetics, which is the regulation of gene expression without altering the nucleotide sequence of the genome, but rather reversible modifications, e.g. CpG methylation. The CpG islands in tumor suppressor gene promoters are often hypermethylated, thus down regulating their expression. The p73 gene is a member of the p53 tumor suppressor family of proteins, and like p53, has a bifurcated promoter. The p73 P1 promoter transcribes a full-length mRNA, which then translates into the transcriptionally active p73. The p73 P2 promoter transcribes a truncated mRNA, which translated into transcriptionally inactive ΔNp73. The ΔNp73 product lacks the N-terminal trans-activation domain that TAp73 possesses. This study sought to analyze the extent of DNA methylation in the CpG islands in the p73 P1 promoter of three prostate cancer (PCa) cell lines (LNCaP, PC-3, and DU145), and primary prostate cells. We hypothesized that the p73 P1 promoter would be hypermethylated in prostate cancer cell lines and hypomethylated in primary prostate cells. The methylation status of the p73 P1 promoter in all four prostate cell lines was analyzed by using bisulfite sequencing PCR (BSP). Our analyses revealed that a CpG island in the p73 P1 was hypermethylated in LNCaP, PC-3, and DU145 cells compared to primary prostate cells. Furthermore, among the three PCa cell lines evaluated, the p73 P1 promoter was the most methylated in DU145 cells, and the least methylated in LNCaP cells. The P1 region sequenced contained 28 CpG islands, and among them, only five were methylated in the primary prostate cells (18% methylated), 23 were methylated in LNCaP cells (82% methylated), 26 in PC-3 cells (93% methylated), and 28 in DU145 cells (100% methylated). Furthermore, of the 28 CpG sites, 14 of them were differentially methylated in the primary prostate cell line versus the three PCa cell lines evaluated in this study. These data are consistent with the PCa tumor-derived cell lines (LNCaP, PC-3, and DU145) having hypermethylated CpG sites in the p73 P1 promoter compared to the primary prostate cell line. Citation Format: Nicholas E. Braganca, L. Michael Carastro, Johannes J. Schabort, Jong Y. Park. Bisulfite DNA sequencing analyses to detect methylation patterns in the p73 gene promoter in prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4363. doi:10.1158/1538-7445.AM2017-4363

One facet of epigenetic regulation is methylation of CpG islands within the promoter region of genes. When CpG islands are hypermethylated, this often down regulates transcription of the gene as transcription factors are hindered from binding. This sometimes occurs in the promoter regions of tumor suppressor genes, such as in cancerous cell lines, e.g., p73. The p73 gene is a member of the p53 tumor suppressor family. The p73 gene promoter is bifurcated, with transcription of a full‐length, wild‐type mRNA, TAp73, from the P1 promoter, while the P2 promoter transcribes a truncated, dominant negative version of the p73 mRNA, deltaNp73. Previously, we reported bisulfite sequencing PCR (BSP) data which identified methylated CpG positions in the p73 gene P1 promoter in primary prostate cells, as well as three prostate cancer (PCa) cell lines: LNCaP, PC‐3, and DU 145. Upon analyzing this BSP methylation data for the P1 promoter region in all four cell lines studied, we reported the three PCa cell lines contained more methylated CpG positions compared to the primary prostate cell line which contained the fewest methylated CpG positions. Furthermore, within the three PCa cell lines, the number of methylated CpG positions was higher in more transformed cell lines, DU145 and PC‐3, compared to the less transformed cell lines, LNCaP. The purpose of this work was to combine our unreported BSP data with previously reported data and determine the overall degree of CpG methylation in the p73 gene P1 promoter region. We hypothesized the overall number of methylation CpG methylation observed in the P1 promoter of the p73 gene would be greater in the three PCa cell lines compared to primary prostate cells. Within the 635‐bp region of the P1 promoter analyzed, there were 230 potential CpG positions which could have been methylated. Of these 230 CpG position, 58 were found to be hypermethylated (>50% frequency) in one or more of these cell lines. Among these 58 confirmed CpG methylation positions, 13 (5.7%) were hypermethylated in primary prostate cells, 40 (17.4%) in LNCaP cells, 52 (22.6%) in PC‐3 cells, and 57 (24.8%) in DU145 cells. This analysis of our data is consistent with the stated hypothesis, in that, within the p73 P1 promoter region analyzed, a higher degree of CpG methylation was detected in the three PCa lines as compared to primary prostate cells. Currently, we are quantifying the extent of methylation, in all four cell lines, at all 58 detected CpG positions. Methylation quantification is being accomplished by subcloning p73 gene P1 promoter BSP amplimers into pET41a vectors, transforming ligation products into DH5‐alpha cells (NEB) from which plasmids are isolated. At least 20 clones from each amplimer ligation reaction are being analyzed by Sanger sequencing and the extent of CpG methylation at each position is being determined quantitatively.Support or Funding InformationThis work was financially supported by funding from the Department of Chemistry, Biochemistry & Physics at the University of Tampa to support student research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.