Background. The aim of this study is to measure global 5-methylcystosine (5MeC) methylation in paraffin embedded prostatic tissues and cell lines using flow cytometry. Methods. Cell/nuclei suspension from 10 cases of benign prostatic hyperplasia (BPH), 10 cases of prostatic adenocarcinoma, and two prostatic cell lines (PNT1A and LNCaP) were prepared using modified heat pretreatment technique. 5MeC global methylation was assessed by flow cytometry of cell/nuclei suspension and immunostaining of tissue sections. Results. Higher percentage of positively stained cells (PPSC) and mean channel fluorescence (MCF) were detected in PNT1A cell line and BPH cell/nuclei suspensions as compared to LNCaP cell lines and adenocarcinoma cell/nuclei suspensions. Lower scores of 5MeC immunostaining were observed in all prostate adenocarcinoma tissue sections as compared to BPH sections indicating global hypomethylation in prostate adenocarcinoma. Two distinctive populations of cells were detected in histograms generated from most of the BPH cell/nuclei suspensions. Conclusion. The study developed a novel technique that could measure 5MeC global methylation in paraffin embedded prostatic tissues. This represents a rapid and objective assessment of methylation and when combined with tissue micro-dissection and cell sorting, this technique could be applied to larger tissue samples such as post radical prostatectomy and transurethral resected specimens.
Prostate, Methylation, Flow cytometry, LNCaP
Holliday R, Pugh J. DNA modification mechanisms and gene activity during development. Science. 1975;187(4173):226-232.
Baylin SB, Makos M, Wu JJ, Yen RW, de Bustros A, Vertino P, Nelkin BD. Abnormal patterns of DNA methylation in human neoplasia: potential consequences for tumor progression. Cancer Cells. 1991; 3(10):383-90.
Lee WH, Morton RA, Epstein JI, Brooks JD, Campbell PA, Bova GS, Hsieh WS, Isaacs WB, Nelson WG. Cytidine methylation of regulatory sequences near the pi-class glutathione S-transferase gene accompanies human prostatic carcinogenesis. Proceedings of the National Academy of Sciences. 1994;91(24):11733-11737.
Herman JG, Latif F, Weng Y, Lerman MI, Zbar B, Liu S, Samid D, Duan DS, Gnarra JR, Linehan WM. Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proceedings of the National Academy of Sciences. 1994;91(21):9700-9704.
Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nature Genetics. 2003;33(3s):245-254.
Gama-Sosa M, Slagel V, Trewyn R, Oxenhandler R, Kuo K, Gehrke C et al. The 5-methylcytosine content of DNA from human tumors. Nucleic Acids Research. 1983;11(19):6883-6894.
Bedford MT and van Helden PD. Hypomethylation of DNA in pathological conditions of the human prostate. Cancer Research. 1987; 47(20):5274-6.
Sakai T, Toguchida J, Ohtani N, Yandell DW, Rapaport JM, and Dryja TP. Allele-specific hypermethylation of the retinoblastoma tumor-suppressor gene. The American Journal of Human Genetics. 1991; 48(5): 880-8.
Graff JR, Herman JG, Lapidus RG, Chopra H, Xu R, Jarrard DF, Isaacs WB, Pitha PM, Davidson NE, Baylin SB. E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas. Cancer Research. 1995; 55(22): 5195-9.
Reik W and Surani MA. Cancer genetics. Genomic imprinting and embryonal tumours. Nature. 1989; 338(6211):112-3.
Rainier S, Johnson L, Dobry C, Ping A, Grundy P, Feinberg A. Relaxation of imprinted genes in human cancer. Nature. 1993;362(6422):747-749.
Costello J, Frühwald M, Smiraglia D, Rush L, Robertson G, Gao X et al. Aberrant CpG-island methylation has non-random and tumour-type–specific patterns. Nature Genetics. 2000;24(2):132-138.
Esteller M, Corn PG, Baylin SB, Herman JG. A gene hypermethylation profile of human cancer. Cancer Research. 2001; 61(8): 3225-9.
Mohamed M, Greif P, Diamond J, Sharaf O, Maxwell P, Montironi R, Young RA, Hamilton PW. Epigenetic events, remodelling enzymes and their relationship to chromatin organization in prostatic intraepithelial neoplasia and prostatic adenocarcinoma. BJU International. 2007;99(4):908-915.
Leers M, Schutte B, Theunissen P, Ramaekers F, Nap M. Heat pretreatment increases resolution in DNA flow cytometry of paraffin-embedded tumor tissue. Cytometry. 1999;35(3):260-266.
Jordanova E, Corver W, Vonk M, Leers M, Riemersma S, Schuuring E, Kluin PM. Flow Cytometric Sorting of Paraffin-Embedded Tumor Tissues Considerably Improves Molecular Genetic Analysis. American Journal of Clinical Pathology. 2003;120(3):327-334.
Feinberg AP and Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature. 1983; 301(5895):89-92.
Jones PA. DNA methylation and cancer. Oncogene. 2002; 21(35): 5358-60.