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PMP24, a gene identified by MSRF, undergoes DNA hypermethylation-associated gene silencing during cancer progression in an LNCaP model

Oncogene volume 23, pages 250–259 (2004)Cite this article

Abstract

Transcriptional silencing of antitumor genes via CpG island methylation could be a mechanism mediating prostate cancer (PCa) progression from an androgen-sensitive (AS) to an androgen-insensitive (AI) state. We have used the methylation-sensitive restriction fingerprinting (MSRF) technique to identify novel CpG-rich sequences that are differentially methylated between the genome of the AS PCa cell line LNCaP and that of an AI subline LNCaPCS generated by maintaining LNCaP in medium with charcoal-stripped (CS) serum for over 30 passages. One such sequence identified was located on a 5′ CpG island that was found to span part of the promoter, exon 1, and part of intron 1 of the p eroxisomal m embrane p rotein 24 kDa (PMP24) gene. Using semiquantitative RT–PCR and bisulfite genomic sequencing, we established an inverse relationship between mRNA expression and methylation of the 5′ CpG island of PMP24. PMP24 mRNA was absent in LNCaPCS and the androgen receptor-negative PC-3 cell line; both exhibited dense methylation in the said CpG island. In contrast, PMP24 mRNA was expressed in LNCaP and normal prostatic epithelial cells (NPrECs) whose PMP24 5′ CpG island remained unmethylated. Treatment of LNCaPCS and PC-3 with the demethylating agent 5-aza-2′-deoxycytidine (5-AZAdC) reactivated PMP24 mRNA expression. Transient transfection of PMP24 into LNCaPCS and PC-3 cells induced a significant reduction in cell growth and soft-agar colony formation potential, suggesting that PMP24 gene product has antitumor properties. These results demonstrate the utility of MSRF in the identification of novel, differentially methylated DNA sequences in the genome and suggest that hypermethylation-mediated silencing of PMP24 is an epigenetic event involved in PCa progression to androgen independence.

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Acknowledgements

The research is supported in part by NIH Grants CA62269, CA94221, and DK61084 awarded to SMH. We thank Dr Samuel C Mok of Brigham and Women's Hospital, Boston, Massachusetts for his generous gifts of HOSE and OVCA cell lines and Dr Brian Liu at the same institution for providing the LNCaPCS. Likewise, we thank Dr Jinquiang Chen of Johns Hopkins University for providing the MCF-10A cell line.

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Authors and Affiliations

  1. Division of Urology, Department of Surgery, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, 01655, MA, USA

    Mengchu Wu & Shuk-Mei Ho

  2. Interdisciplinary Graduate Program, Graduate School of Biomedical Sciences, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, 01655, MA, USA

    Mengchu Wu & Shuk-Mei Ho

  3. Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, 01655, MA, USA

    Shuk-Mei Ho

  4. Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, 01655, MA, USA

    Shuk-Mei Ho

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Wu, M., Ho, SM. PMP24, a gene identified by MSRF, undergoes DNA hypermethylation-associated gene silencing during cancer progression in an LNCaP model. Oncogene 23, 250–259 (2004). https://doi.org/10.1038/sj.onc.1207076

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