Methylation of the androgen receptor promoter CpG island is associated with loss of androgen receptor expression in prostate cancer cells.

Androgen-independent metastatic prostate cancer is characterized by a heterogeneous loss of androgen receptor (AR) expression among tumor cells. In this study, we evaluate DNA hypermethylation as a potential transcriptional regulatory mechanism in AR-negative prostate cancer cell lines. Nucleotide sequence analysis demonstrates an approximately 15-kb CpG island in the AR gene that encompasses the transcription start site and exon 1. Using Southern blotting with methylation-sensitive restriction enzymes and methylation-specific PCR, we find aberrant methylation in the AR expression-negative cell lines Du145, DuPro, TSU-PR1, and PPC1. Incomplete methylation in the AR CpG island is also seen in normal female breast and ovarian tissues consistent with the inactivation of one X chromosome by hypermethylation. In contrast, prostate cancer cell lines LNCaP and PC3 express AR and are unmethylated. Normal prostate epithelial cell strains demonstrate no methylation. Exposure of AR-negative prostate cancer cell lines to 5-aza-2' deoxycytidine, a demethylating agent, induces the reexpression of AR RNA in DuPro and TSU-PR1. This reexpression is associated with a demethylation of this region. Prostate-specific antigen, an androgen-responsive gene, is also specifically induced in these lines after AR reexpression. Therefore, in vitro DNA methylation of the 5' CpG AR island may be associated with the loss of AR expression. Furthermore, our results demonstrate that treatment with demethylating agents may engender the reexpression and function of the androgen receptor in AR-negative cell lines.

Methylation of the androgen receptor minimal promoter silences transcription in human prostate cancer.

Advanced hormone-independent prostate cancer is characterized by a significant loss of androgen receptor (AR) expression in 20-30% of the tumors. The transcriptional block underlying this phenomenon is not known, but we have proposed that methylation of CpG sites in the AR promoter may reversibly inactivate transcription of the AR (D. F. Jarrard et al, Cancer Res., 58: 5310-5314, 1998). In this study, detailed methylation analysis using bisulfite sequencing was performed on a series of AR expression-positive and -negative prostate cancer cells. We found that methylation of several consensus sequences in the AR promoter (from -131 to -121 and +44 to +54) are tightly linked to the loss of AR expression in metastatic hormone-independent prostate cancer cell lines. These consensus sites of methylation correlate with the minimal promoter region critical for AR transcription. In human tissues, no methylation was demonstrated in normal or primary prostate cancers that express the AR. Four of 15 tumors obtained from men who had died from hormone-independent prostate cancer demonstrated a significant loss of AR expression immunohistochemically and two (50%) of these AR-negative tumors contained AR methylation. We conclude that the AR promoter contains specific CpG methylation hot spots that are markers for gene silencing. Furthermore, AR methylation may represent a phenotype important in the development of hormone independence in a subset of advanced prostate cancer in which AR expression is lost. The finding of AR methylation also represents the first report of aberrant methylation on an X-linked gene associated with a somatic male cancer.

Screening hypermethylated regions by methylation-sensitive single-strand conformational polymorphism.

DNA methylation is an important epigenetic modification that alters transcription in those genes containing CpG islands. In this report we demonstrate that the familiar technique single-strand conformation polymorphism (SSCP) can be easily applied to bisulfite-treated DNA to detect methylation differences over large ( approximately 250 bp) CG-enriched regions. In the methylation-sensitive SSCP (Ms-SSCP) technique, sodium bisulfite modification of DNA converts unmethylated cytosine to uracil under conditions in which 5-methylcytosine remains unaltered. Amplification of these regions to be tested is then performed using primers specific for bisulfite-treated DNA. The resulting products are then subjected to nondenaturing gel analysis. Based on differential band mobility, Ms-SSCP is able to sensitively detect alterations in methylation density and determine if unmethylated alleles are present in a sample. Ms-SSCP is a rapid and simple technique for screening multiple samples for methylation changes. It has several advantages over existing techniques, including the utilization of nanogram amounts of DNA, the avoidance of difficulties in sequencing CG-enriched regions, and the screening of multiple sites for methylation across large regions of DNA.

Abrupt onset of severe pain at term. A case report.

This case report involves an adolescent primigravida at term who was admitted with urinary complaints to the labor and delivery unit of a medical center. Within an hour, she suddenly began screaming and complaining of severe pain running from her anterior pelvis through her vagina and up her spine. Three days of very challenging co-management of the patient, with several recurrences of acute pain, followed. Differential diagnoses that could explain this patient's symptoms are reviewed and discussed. Difficult management issues, including the stress of clinical management in the face of unidentified disease processes, are addressed. Lacking a certain diagnosis even retrospectively, the authors request comments from readers.