her with p75NTR. NRAGE has been shown to 2583244 be proapoptotic in various cell types and to be involved in the neuronal differentiation of pheochromocytoma cells. PC12 cells endogenously express the NRAGE activator p75NTR, which is known to mediate NGF-signalling in cell survival, differentiation and cell death. Praja1 binds to the necdin homology domain of NRAGE and less efficiently to necdin itself, leading to ubiquitination and proteasomal degradation of NRAGE and to a modulation of Msx2 and Dlx5dependent transcription. Control of NRAGE expression and activity through Praja1 may thus provide an important mechanism for controlling neuronal differentiation. We tested this hypothesis and investigated the role of Praja1 in NGF-induced differentiation of PC12 cells. Two validated transcript variants of mouse praja1 were used, that code for two isoforms, referred to as Praja1.1 and Praja1.2, with a predicted molecular weight of 64 kD and 44 kD, respectively. Our data demonstrate the induction of Praja1 during neuronal differentiation, its intracellular localization and co-localization with NRAGE, and the Praja1-mediated reduction of NRAGE expression levels and of neurite outgrowth. Materials and Methods mRNA expression analysis Isoform-specific gene expression was analysed with quantitative “real-time”polymerase chain reaction using FAM-labelled probes and a mouse-II-cDNA-panel. Primers for praja1.1 were tagged with the FAM-reporter 59-TTGGAGTCGCCACATTC-39, and for praja1.2 with the FAM-reporter 59CCCGCCACCTGGAATA-39. For detection of the housekeeping gene phospho-glycerate kinase, we used assay Mm00435617_m1. For amplification and real-time quantification, samples were uracil-N-glycosylated for 2 min at 50uC before being denatured for 10 min at 95uC and amplified with up to 50 cycles of 15 s at 95uC and 1 min at 60uC. Typical quantification was performed within a range of 25 to 35 cycles. However, detection of rare splice 10760364 forms occasionally required 40 cycles or more. For data analysis, mean cycle threshold values were determined for each triplicate assay and used for sample comparison, using PGK as an internal control. Individual dCT values were obtained by subtraction of the individual CT of the housekeeping gene PGK from the CT of the corresponding individual triplicate GW 5074 according to the ddCT method. For illustration, a relative quantification of the mean dCT values The growth rate of cancer is commonly estimated by measuring a cell proliferation-related protein, Ki-67, with Mib1 monoclonal antibody. The Mib1 score provides a particularly strong prognostic marker in human breast cancer. However, its relation to this cancer’s content of linoleic acid, arachidonic acid, and the oxygenase-derived metabolites of these two fatty acids is not clear. Application of LA or 
AA to breast cancer cell cultures stimulates proliferation apparently because these cells over-express 5-lipoxygenase, 12-LO, 15-LO-1, 15-LO-2, cyclooxygenase -1 and/or CO-2, and thereby over-produce LA and/or AA metabolites some of which feedback to up-regulate their parent cells’ proliferation. Since dietary excesses of these FA may promote human breast cancer, one or more of these autocoid loops may contribute to this disease. 5-LO and 12-LO are over-expressed in the breast cancer of patients who suffer poor survival. Two 5-LO products, 5hydroxy-eicosatetraenoate and 5-oxo-eicosatetraenoate, and a 12-LO product, 12-HETE, stimulate breast cancer cells in culture to proliferate and
