trichostatin-a and Astrocytoma

Many progesterone (P4) effects are mediated by its intracellular receptor (PR), which has two isoforms, PR-A and PR-B, each of them with different function and regulation. Differential PR expression in cancer cells has been associated to a PR isoform-specific promoter methylation. In astrocytomas, the most frequent and aggressive brain tumors, PR isoforms expression is directly correlated to the tumor's evolution grade. However, there is no evidence of the role of epigenetic regulation of PR expression in astrocytomas. We evaluated the effect of the demethylating agent 5-aza-2'-deoxycytidine (5AzadC) and the histone deacetylase inhibitor trichostatin A (TSA) on PR expression in human astrocytoma cell lines U373 (grade III) and D54 (grade IV) by RT-PCR and Western blot. Total PR expression increased with 5 μM 5AzadC treatment, whereas PR-B expression increased with 5 and 10 μM 5AzadC treatment in U373 cells, but not in D54 cells. In U373 cells, PR-A protein content augmented with 10 μM 5AzadC treatment, while PR-B content increased with 5 and 10 μM 5AzadC. PR-B expression was not modified by the TSA concentrations that were used, and the combination with 5AzadC did not change the effects of the latter. The study of 5AzadC effects on the number of astrocytoma cells showed that P4 treatment increased the number of U373 cells, whereas 5AzadC and the combined treatment with P4 reduced it. Our results suggest that PR-B expression is regulated by methylation and not by histone acetylation in U373 cells, and that DNA demethylation reduced the number of U373 cells.

Topics: Acetylation; Astrocytoma; Azacitidine; Cell Line, Tumor; Cell Proliferation; Cell Survival; Decitabine; DNA Methylation; Gene Expression Regulation, Neoplastic; Histones; Humans; Hydroxamic Acids; Neoplasm Grading; Protein Isoforms; Receptors, Progesterone

To identify novel genes involved in glioma progression we performed suppression subtractive hybridization combined with cDNA array analysis on 4 patients with primary low-grade gliomas of World Health Organization (WHO) grade II that recurred as secondary glioblastomas (WHO grade IV). Eight genes showing differential expression between primary and recurrent tumors in 3 of the 4 patients were selected for further analysis using real-time reverse transcription-PCR on a series of 10 pairs of primary low-grade and recurrent high-grade gliomas as well as 42 astrocytic gliomas of different WHO grades. These analyses revealed that 5 genes, i.e., AMOG (ATP1B2, 17p13.1), APOD (3q26.2-qter), DMXL1 (5q23.1) DRR1 (TU3A, 3p14.2) and PSD3 (KIAA09428/HCA67/EFA6R, 8p22), were expressed at significantly lower levels in secondary glioblastomas as compared to diffuse astrocytomas of WHO grade II. In addition, AMOG, DRR1 and PSD3 transcript levels were significantly lower in primary glioblastomas than in diffuse astrocytomas. Treatment of glioma cell lines with 5-aza-2'-deoxycytidine and trichostatin A resulted in increased expression of AMOG and APOD transcripts. Sequencing of sodium bisulfite-modified DNA demonstrated AMOG promoter hypermethylation in the glioma cell lines and 1 primary anaplastic astrocytoma with low AMOG expression. Taken together, we identified interesting novel candidate genes that likely contribute to glioma progression and provide first evidence for a role of epigenetic silencing of AMOG in malignant glioma cells.

Topics: Adenosine Triphosphatases; Antimetabolites, Antineoplastic; Apolipoproteins; Apolipoproteins D; Astrocytoma; Azacitidine; Biomarkers, Tumor; Brain Neoplasms; Cation Transport Proteins; Cell Adhesion Molecules, Neuronal; Decitabine; Disease Progression; DNA Methylation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Glycoproteins; Guanine Nucleotide Exchange Factors; Histone Deacetylases; Humans; Hydroxamic Acids; Membrane Transport Proteins; Nerve Tissue Proteins; Nuclear Proteins; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Protein Synthesis Inhibitors; Proteins; Reverse Transcriptase Polymerase Chain Reaction