The glitazones or thiazolidinediones are ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma). The glitazones are used in the treatment of diabetes, regulate adipogenesis, inflammation, cell proliferation, and induce apoptosis in several cancer cell types. High grade astrocytomas are rapidly growing tumors derived from astrocytes, for which new treatments are needed. We determined the effects of two glitazones, ciglitazone and the therapeutic rosiglitazone, on the survival of serum-deprived primary rat astrocytes and glioma cell lines C6 and U251, which were assessed by the methylthiazolyl tetrazolium assay and lactate dehydrogenase release. Rosiglitazone (5-20 microM) decreased survival of glioma cells without affecting primary astrocytes, whereas ciglitazone at 20 microM was toxic for both cell types. Ciglitazone at 10 microM was cytoprotective for primary astrocytes but toxic to glioma cells. Cell death induced by ciglitazone, but not rosiglitazone, presented apoptotic features (Hoechst staining and externalization of phosphatidylserine). Two mechanisms to explain cytotoxicity were investigated: activation of PPARgamma and production of reactive oxygen species (ROS). PPARgamma does not seem to be the main mechanism involved, because the order of efficacy for cytotoxicity, ciglitazone > rosiglitazone, was inverse of their reported affinities for activating PPARgamma. In addition, GW9662, an inhibitor of PPARgamma, only slightly attenuated cytotoxicity. However, the rapid increase in ROS production and the marked reduction of cell death with the antioxidants ebselen and N-acetylcysteine, indicate that ROS have a key role in glitazone cytotoxicity. Ciglitazone caused a dose-dependent and rapid loss (in minutes) of mitochondrial membrane potential in glioma cells. Therefore, mitochondria are a likely source of ROS and early targets of glitazone cytotoxicity. Our results highlight the potential of rosiglitazone and related compounds for the treatment of astrogliomas.

Neurons are highly dependent on astrocyte survival during brain damage. To identify genes involved in astrocyte function during ischemia, we performed mRNA differential display in astrocytes after oxygen and glucose deprivation (OGD). We detected a robust down-regulation of S6 kinase 1 (S6K1) mRNA that was accompanied by a sharp decrease in protein levels and activity. OGD-induced apoptosis was increased by the combined deletion of S6K1 and S6K2 genes, as well as by treatment with rapamycin that inhibits S6K1 activity by acting on the upstream regulator mTOR (mammalian target of rapamycin). Astrocytes lacking S6K1 and S6K2 (S6K1;S6K2-/-) displayed a defect in BAD phosphorylation and in the expression of the anti-apoptotic factors Bcl-2 and Bcl-xL. Furthermore reactive oxygen species were increased while translation recovery was impaired in S6K-deficient astrocytes following OGD. Rescue of either S6K1 or S6K2 expression by adenoviral infection revealed that protective functions were specifically mediated by S6K1, because this isoform selectively promoted resistance to OGD and reduction of ROS levels. Finally, "in vivo" effects of S6K suppression were analyzed in the permanent middle cerebral artery occlusion model of ischemia, in which absence of S6K expression increased mortality and infarct volume. In summary, this article uncovers a protective role for astrocyte S6K1 against brain ischemia, indicating a functional pathway that senses nutrient and oxygen levels and may be beneficial for neuronal survival.

Numerous eukaryotic mRNAs contain sequences complementary to segments of the 18S and 28S rRNAs. Previous studies raised the possibility that these complementarities might allow mRNA–rRNA interactions and affect rates of translation. In the present study, we investigated the mRNA encoding the mouse Gtx homeodomain protein. This mRNA contains within its 5′ untranslated region (UTR) a segment that is complementary to two regions of the 18S rRNA, located at nucleotides 701–741 and 1104–1136. A Gtx RNA probe containing this complementarity could be photochemically cross-linked to ribosomal subunits through a linkage to 18S rRNA but not to 28S rRNA. Oligonucleotide-directed RNase H digestion of the rRNA and a reverse transcription analysis localized the cross-linked probe to the complementary segment of 18S rRNA at nucleotides 1104–1136 but not at nucleotides 701–741. To determine whether complementarity in the Gtx mRNA affected translation, a mutational analysis was performed with a Gtx-luciferase fusion construct and four related constructs with altered complementarity to the 18S rRNA. These constructs were examined for their ability to be translated in cell-free lysates prepared from P19 embryonal carcinoma and C6 glioma cell lines and after cellular transfection into these same cell lines. In both cell-free translation and transfection studies, the rate of translation decreased more than 9-fold as the degree of complementarity to nucleotides 1104–1136 of the 18S rRNA increased. We hypothesize that segments complementary to rRNA, such as those contained within the Gtx mRNA, form a category of cis-acting regulatory elements in mRNAs that affect translation by base pairing to rRNA within ribosomes.