The prognosis for patients with malignant glioma has not significantly changed in two decades, despite advances in surgery, radiation, and chemotherapy, emphasizing the growing need for novel approaches to glioma therapy. Perillyl alcohol (POH) is a naturally occurring monoterpene that has been shown to possess chemotherapeutic as well as chemopreventive activity in animal tumor models and is currently in Phase I and Phase II clinical trials. In the present study, we have demonstrated that POH is an effective radiosensitizer at clinically relevant doses of radiation using established glioma cell lines. POH caused a transient arrest in the G 2 /M phase of the cell cycle and induced apoptosis in glioma cells. POH treatment sensitized glioma cells to Fas-mediated apoptosis, which was further augmented in the presence of ionizing radiation and abrogated in the presence of antagonistic antibody. POH-induced radiosensitization was partially inhibited in glioma cells expressing dominant negative Fas-associated death domain and completely inhibited in glioma cells overexpressing the cytokine response modifier A. In addition, POH treatment resulted in a dose-dependent sensitization to cisplatin and doxorubicin induced cytotoxicity in glioma cells, highlighting its usefulness as a potent radio/chemosensitizer in the treatment of malignant glioma.Over the past 2 decades, minimal survival gains for malignant glioma have been recorded, and the disease remains almost universally fatal (1-3). To date, radiotherapy has proven to be the most effective treatment for malignant glioma, extending median survival to 8 -9 months, and hence the development of an effective radiosensitizer should be particularly relevant in the management of malignant glioma, since 90% of these patients will ultimately develop recurrences immediately within the treatment field (4, 5).Monoterpenes are naturally occurring nonnutritive dietary components with significant antitumor activity, and their mechanism of action is not similar to classic cytotoxic chemotherapeutic agents. Perillyl alcohol (POH), 1 also called p-metha, 1,7-diene-6-ol, or 4-isopropenylcyclohexenecarbinol, is a monoterpene, isolated from the essential oils of lavendin, peppermint, spearmint, and several other plants and synthesized by the mevalonate pathway (6). It has chemotherapeutic activity in pancreatic, mammary, and prostatic model tumor systems and also has chemopreventive activity in rodent mammary, skin, liver, lung, and forestomach cancers (6 -8 (14,15) studied thymidine incorporation of POH-treated malignant and nonmalignant hamster pancreatic ductal epithelial cells and concluded that the inhibitory effect of POH was due to stimulation of apoptosis and an increase in the proapoptotic protein Bak. The malignant cells treated with 100, 300, and 500 M POH exhibited a 2.6, 8.8, and 18-fold higher rate of apoptosis than the untreated malignant cells. As little as 2000 ppm of POH in the diet inhibited azoxymethane-induced adenoma and adenocarcinoma development in rat colons (6, ...
PrrA is a global trancription regulator activated upon phosphorylation by its cognate kinase PrrB in response to low oxygen conditions in Rhodobacter sphaeroides. Here we show by gel filtration, analytical ultracentrifugation and NMR diffusion measurements that treatment of PrrA by a phosphate analogue, BeF 3 -, results in dimerization of the protein, producing a protein that binds DNA. No dimeric species was observed in the absence of BeF 3 -. On addition of BeF 3 -, the inhibitory activity of the N-terminal domain on the C-terminal DNA-binding domain is relieved, after which PrrA becomes capable of binding DNA as a dimer. The interaction surface of the DNA-binding domain with the regulatory domain of PrrA is identified by NMR as being a well conserved region centered on helix α6, which is on the opposite face to the DNA recognition helix. This suggests that there is no direct blockage of DNA binding in the inactive state, but rather that PrrA dimerization promotes a correct arrangement of two adjacent DNA binding domains to recognise specific DNA binding sequences. KeywordsTwo-Component System; PrrA; RegA; dimerization; phosphorylation; DNA recognitionThe purple, non-sulfur bacterium Rhodobacter sphaeroides has very versatile metabolic activities including aerobic and anaerobic respiration, photosynthesis, and carbon and nitrogen assimilation. The R. sphaeroides global regulator PrrA coordinately controls a large number of genes involved in the complex switch between aerobic and anaerobic lifestyles and the optimum use of reducing power. It regulates genes necessary for the synthesis of the photosynthetic apparatus, electron transport, nitrogen and carbon fixation, anaerobic respiration, [NiFe] hydrogenase and aerotaxis, as well as the expression of the Prr gene cluster itself (1-6). † This work was supported by an equipment grant from the Wellcome Trust and by grants from the NIH (GM37509) and DOE (ER63232-1018220-0007203 The Prr system is a bacterial two-component signal transduction system (TCS) (7), consisting of the two proteins PrrB and PrrA. Two-component systems homologous to Prr have been found in other proteobacteria including other photosynthetic species, like the very similar and well-studied Reg system from R. capsulatus, but also in non-photosynthetic bacteria, and suggest a very conserved transduction mechanism, despite different in vivo functions (8,9). PrrB is a membrane-bound histidine kinase and is activated under low oxygen conditions, probably via a third member of the pathway, PrrC, through formation of an intermolecular disulfide bond using a conserved cysteine (10,11). It then autophosphorylates on a conserved histidine and the phosphate is transferred to the response regulator (RR) PrrA on a conserved aspartate residue.PrrA is a two-domain protein. The N-terminal receiver domain (residues 1-130) is a CheY-like domain common to all bacterial TCS response regulators, and there are a number of structures of these domains including CheY (12), FixJ (13) and NtrC (14). The phosphory...
The Rhodobacter sphaeroides response regulator PrrA directly activates transcription of genes necessary for energy conservation at low O2 tensions and under anaerobic conditions. It is proposed that PrrA homologues contain a C-terminal DNA-binding domain (PrrA-CTD) that lacks significant amino acid sequence similarity to those found in other response regulators. To test this hypothesis, single amino acid substitutions were created at 12 residues in the PrrA-CTD. These mutant PrrA proteins were purified and tested for the ability to be phosphorylated by the low-molecular-mass phosphate donor acetyl phosphate, to activate transcription and to bind promoter DNA. Each mutant PrrA protein accepted phosphate from 32P-labelled acetyl phosphate. At micromolar concentrations of acetyl phosphate-treated wild-type PrrA, a single 20 bp region in the PrrA-dependent cycA P2 promoter was protected from DNase I digestion. Of the mutant PrrA proteins tested, only acetyl phosphate-treated PrrA-N168A and PrrA-I177A protected cycA P2 from DNase I digestion at similar protein concentrations compared to wild-type PrrA. The use of in vitro transcription assays with the PrrA-dependent cycA P2 and puc promoters showed that acetyl phosphate-treated PrrA-N168A produced transcript levels similar to that of wild-type PrrA at comparable protein concentrations. Using concentrations of acetyl phosphate-treated PrrA that are saturating for the wild-type protein, PrrA-H170A and PrrA-I177A produced <45 % as much transcript as wild-type PrrA. Under identical conditions, the remaining mutant PrrA proteins produced little or no detectable transcripts from either promoter in vitro. Explanations are presented for why these amino acid side chains in the PrrA-CTD are important for its ability to activate transcription.
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