Prevalence of copy-number neutral LOH in glioblastomas revealed by genomewide analysis of laser-microdissected tissues

We have employed a laser-capture microdissection technique and single-nucleotide polymorphism arrays to characterize genomic alterations associated with the development of glioblastoma multiforme (GBM). Combined analysis of loss of heterozygosity (LOH) and copy number revealed that more than half (56.3%) of the 254 identified LOH loci showed no copy-number alteration, indicating the presence of copy-number neutral LOH (cnLOH). Furthermore, we found a GBM case that showed cnLOH in 18 of the 22 autosomes. These results were confirmed by quantitative real-time PCR, microsatellite analysis, and fluorescence in situ hybridization. The high rate of cnLOH suggests that epigenetic abnormalities of many genes are involved in the development and progression of GBMs

Mechanisms of action of rapamycin in gliomas1

Rapamycin has previously been shown to be efficacious against intracerebral glioma xenografts and to act in a cytostatic manner against gliomas. However, very little is known about the mechanism of action of rapamycin. The purpose of our study was to further investigate the in vitro and in vivo mechanisms of action of rapamycin, to elucidate molecular end points that may be applicable for investigation in a clinical trial, and to examine potential mechanisms of treatment failure. In the phosphatase and tensin homolog deleted from chromosome 10 (PTEN)-null glioma cell lines U-87 and D-54, but not the oligodendroglioma cell line HOG (PTEN null), doses of rapamycin at the IC50 resulted in accumulation of cells in G1, with a corresponding decrease in the fraction of cells traversing the S phase as early as 24 h after dosing. All glioma cell lines tested had markedly diminished production of vascular endothelial growth factor (VEGF) when cultured with rapamycin, even at doses below the IC50. After 48 h of exposure to rapamycin, the glioma cell lines (but not HOG cells) showed downregulation of the membrane type–1 matrix metalloproteinase (MMP) invasion molecule. In U-87 cells, MMP-2 was downregulated, and in D-54 cells, both MMP-2 and MMP-9 were downregulated after treatment with rapamycin. Treatment of established subcutaneous U-87 xenografts in vivo resulted in marked tumor regression (P < 0.05). Immunohistochemical studies of subcutaneous U-87 tumors demonstrated diminished production of VEGF in mice treated with rapamycin. Gelatin zymography showed marked reduction of MMP-2 in the mice with subcutaneous U-87 xenografts that were treated with rapamycin as compared with controls treated with phosphate-buffered saline. In contrast, treatment of established intracerebral U-87 xenografts did not result in increased median survival despite inhibition of the Akt pathway within the tumors. Also, in contrast with our findings for subcutaneous tumors, immunohistochemistry and quantitative Western blot analysis results for intracerebral U-87 xenografts indicated that there is not significant VEGF production, which suggests possible deferential regulation of the hypoxia-inducible factor 1α in the intracerebral compartment. These findings demonstrate that the complex operational mechanisms of rapamycin against gliomas include cytostasis, anti-VEGF, and anti-invasion activity, but these are dependent on the in vivo location of the tumor and have implications for the design of a clinical trial