p73 Gene Promoter Methylation Patterns in Prostate Cancer Cell Lines
Authors: Schabort Johannes J. , Braganca Nicholas E. , Gandy Keith Y. , Park Jong Y. , Carastro L. Michael 
Published: June 2021

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 Information

This 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.