• Development and application of RT-PCR systems to determine HERV expression in astrocytoma cell lines

      Nelson, Paul N.; Smith, R.; Conde, Gillian; Roden, Denise A.; Darling, John L. (Society for Neuro-Oncology and Duke University Press, 2005)
      Human endogenous retroviruses (HERVs) belong to the family of transposable elements that make up 8% of the human genome. Unlike exogenous retrovirus (e.g., HIV and HTLV), HERVs are inherited in a Mendelian manner. More than 22 families of HERVs have been identified over the past two decades. Importantly, some HERVs have been found to possess large open reading frames and produce viral like particles. More latterly, these viruses have been linked with certain autoimmune diseases and cancers. Indeed, HERVs may contribute toward carcinogenesis through retrotransposition, promoter insertion, immunomodulation, disruption of normal HERV-related functions, recombination, or by the production of fusion proteins. Of importance, HERV-K, HERV-W, and HERV-H have the potential to be transcriptionally active in the brain. We have developed robust RT-PCR systems using primers/probes specific to HERV-K and HERV-W to assess mRNA expression in conjunction with the house keeping gene, histidyl tRNA synthetase. In employing a gel-documentation system, we are able to provide semiquantitative levels of HERV expression in cell lines. Pilot data shows markedly enhanced expression of HERV-K in the cell line U251-MG (derived from a glioblastoma multiforme; WHO grade IV astrocytoma) as compared to a control cell line SW480 (colon adenocarcinoma): RT-PCR values; 1.0 and 0.42, respectively. This observation raises an intriguing possibility that HERV-K expression may be elevated in malignant brain tumors. In addition, this approach provides a useful approach to optimize primers and probes prior to using real-time quantitative PCR.
    • Regulation of uncoupling protein-2 (UCP-2) expression in human glioma cells by peroxisome proliferators-activated receptor (PPAR) agonists

      Brown, James E. P.; Darling, John L.; Dunmore, Simon J.; Bassey, S. (Society for Neuro-Oncology and Duke University Press, 2005)
      Recent studies have suggested that the glitazones, a group of PPAR agonists commonly prescribed as therapy in type 2 diabetes, could have a role in regulation of cell viability in astrocytomas and glioma cell lines, possibly due to a modulation of reactive oxygen species (ROS) production. PPAR agonists are also known to regulate expression of the mitochondrial protein UCP-2, and UCP-2 has a purported role in ROS regulation amongst others. This study investigated the expression of UCP-2 in U251MG glioma cells and its regulation by PPAR agonists. U251MG glioma cells were cultured according to standard methods and treated with PPAR alpha, delta, and gamma agonists (10 μM WY14643, 10 nM PGI2, 10 μM rosiglitazone and 10 nM PGJ2, respectively) for 24 h. Total RNA was subsequently extracted and semiquantitative RT-PCR used to evaluate UCP-2 mRNA expression. Results showed that UCP-2 was expressed in all control and treatment samples and that its expression was regulated differentially by the various PPAR receptor subtype agonists tested. This novel finding that UCP-2 is expressed in glioma cells, and that its expression is regulated by PPAR agonists, suggests a potential mechanism for the cytotoxic effects of glitazones that have been previously reported, and describes a mechanism which could possibly be manipulated as a potential therapeutic avenue in the future.