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

Abstract

Transcriptional silencing of antitumor genes via CpG island methylation could be a mechanism mediating prostate cancer (PCa) progression from an androgensensitive (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 LNCaP CS generated by maintaining LNCaP in medium with charcoal-stripped (CS) serum for over 30 passages. One such sequence identified was located on a 5 0 CpG island that was found to span part of the promoter, exon 1, and part of intron 1 of the peroxisomal membrane protein 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 0 CpG island of PMP24. PMP24 mRNA was absent in LNCaP CS 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 0 CpG island remained unmethylated. Treatment of LNCaP CS and PC-3 with the demethylating agent 5-aza-2 0 -deoxycytidine (5-AZAdC) reactivated PMP24 mRNA expression. Transient transfection of PMP24 into LNCaP CS 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.

Early effects of doxorubicin in perfused heart: transcriptional profiling reveals inhibition of cellular stress response genes

“…Among the notable upregulated genes with known antiproliferative action in tumor growth are PMP24 and quiescin Q6 (QSCN6). Silencing of PMP24, the most strongly DXR-induced transcript, is associated with cancer progression (67). Cytoprotective upregulation may be found in DNA synthesis/repair with polymerases and nibrin, in cardiac remodeling with tropomyosin 1␣ and osteoglycin, whose expression correlates with a cardiac hypertrophic response (44), as well as in immune response and mainly in energy metabolism.…”

Epigenetics, Environment, and Allergic Diseases

“…Perera et al studied Hispanic and African American nonsmoking mothers who were monitored regularly during pregnancy in an attempt to explore the epigenetic impact of transplacental exposures to PAHs. After isolation of T cells from umbilical cord, the group focused on the global methylation patterns through MSRF (for its acronym in Methylation Sensitive Restriction Fingerprint) [47,48]. The authors reported that changes in methylation of the promoter region of ASCL3 gene (acyl-CoA synthetase long-chain family member 3), were significantly associated with maternal exposure to PAHs, as well as appearance of asthma symptoms during the first 5 years of life.…”

Techniques used in studies of epigenome dysregulation due to aberrant DNA methylation: An emphasis on fetal-based adult diseases

“…One of the genes, phosphodiesterase 4D4, was further characterized for its functional role in modulating prostate cancer risk in adulthood [20]. In an earlier study [49], using a similar approach, we identified hypermethylation of peroxisome membrane protein 24 as a marker/modifier for the transition of prostate cancer cells from an androgen-dependent to an androgen-independent state. Others have used this method to identify genes whose promoters are aberrantly methylated in breast cancers [48], nasopharyngeal carcinoma [50], and hepatocellular carcinoma [51].…”

“…By increasing the number of arbitrary primers used in MSRF, one can increase the coverage of the genome and the number of candidate sequences identified. For example, in two of the experiments we have conducted [20,49], both using 4 primers and 6 permutations of primer pairs, we identified approximately 50 candidate sequences. However, if 9 primers were to be used, a total of 36 [n × (n-1)/2; n is the number of primers] permutations for arbitrary PCRs could be achieved, likely yielding over 1,800 candidates.…”