SummaryStreptomyces coelicolor GlnR is a global regulator that controls genes involved in nitrogen metabolism. By genomic screening 10 new GlnR targets were identified, including enzymes for ammonium assimilation (glnII, gdhA), nitrite reduction (nirB), urea cleavage (ureA) and a number of biochemically uncharacterized proteins (SCO0255, SCO0888, SCO2195, SCO2400, SCO2404, SCO7155). For the GlnR regulon, a GlnR binding site which comprises the sequence gTnAc-n6-GaAAc-n6-GtnAC-n6-GAAAc-n6 has been found. Reverse transcription analysis of S. coelicolor and the S. coelicolor glnR mutant revealed that GlnR activates or represses the expression of its target genes. Furthermore, glnR expression itself was shown to be nitrogen-dependent. Physiological studies of S. coelicolor and the S. coelicolor glnR mutant with ammonium and nitrate as the sole nitrogen source revealed that GlnR is not only involved in ammonium assimilation but also in ammonium supply. BLAST analysis demonstrated that GlnRhomologous proteins are present in different actinomycetes containing the glnA gene with the conserved GlnR binding site. By DNA binding studies, it was furthermore demonstrated that S. coelicolor GlnR is able to interact with these glnA upstream regions. We therefore suggest that GlnR-mediated regulation is not restricted to Streptomyces but constitutes a regulon conserved in many actinomycetes.

BACKGROUNDGlioblastoma commonly is characterized by hypoxia and acidosis and the histologic features of tissue necrosis and neovascularization. Current approaches of adjuvant radiochemotherapy for patients with glioblastoma have only a modest impact on the natural course of this disease.METHODSThe authors examined the effects of acidosis on growth and response to irradiation and chemotherapy in cultured human malignant glioma cells.RESULTSThe authors found that mild acidosis (pH 7.0) inhibited the growth of cell lines that retained wild type p53 activity but did not inhibit the growth of cell lines that were devoid of p53 function. Transfer of a dominant‐negative p53 gene into p53 wild type cells failed to override the acidosis‐conferred growth arrest, suggesting that loss of p53 activity per se does not mediate escape from acidosis‐induced growth inhibition. Moderate acidosis (pH 6.6) inhibited the growth of all cell lines. Acidosis‐mediated growth arrest was not associated with a specific type of cell cycle arrest, e.g., in G0/G1 or G2/M phase. Acidosis did not result in consistent changes in radiosensitivity; however, it enhanced the cytotoxic effects of lomustine but conferred protection from topotecan, vincristine, teniposide, and cisplatin cytotoxicity. Lomustine exhibited enhanced stability at low pH, providing a putative mechanism for the enhanced cytotoxic effects of lomustine in acidotic conditions. Decreased sensitivity to the other drugs did not result from altered multidrug resistance drug transport activity.CONCLUSIONSTaken together, the current results suggest that tissue acidosis may be an important determinant of glioma cell responses to adjuvant radiochemotherapy. The superior activity of nitrosoureas, such as lomustine, compared with other agents in patients with glioblastoma may result in part from prolonged drug stability in an acidotic microenvironment. Cancer 2002;95:1113–9. © 2002 American Cancer Society.DOI 10.1002/cncr.10767

The death ligands CD95L and Apo2L/TRAIL are promising investigational agents for the treatment of malignant glioma. EGFR is overexpressed in a significant proportion of malignant gliomas in vivo. Here, we report that CD95L-induced cell death is enhanced by EGFR inhibition using tyrphostine AG1478 in 7 of 12 human malignant glioma cell lines. Conversely, CD95-mediated and Apo2L-induced cell death are both inhibited by overexpression of EGFR in LN-229 cells. CD95L-induced cell death augmented by AG1478 is accompanied by enhanced processing of caspase 8. LN-229 cells overexpressing the viral caspase inhibitor, crm-A, are not sensitized to CD95L-induced cell death by AG1478, indicating that EGFR exerts its antiapoptotic properties through a caspase 8-dependent pathway. These data define a modulatory effect of EGFR-activity on death ligand-induced apoptosis and indicate that EGFR inhibition is likely to improve the efficacy of death ligand-based cancer therapies. Furthermore, it is tempting to speculate that EGFR amplification protects tumor cells from death ligand-mediated host immune responses in vivo and that EGFR's effects on death receptor-mediated apoptosis may explain the anti-tumor effects of non-cytotoxic, unarmed anti-EGFR family antibodies.