The discovery of translocations that involve one of the genes of the ETS family (ERG, ETV1, ETV4 and ETV5) has been a major advance in understanding the molecular basis of prostate cancer (PC). Each one of these translocations results in deregulated expression of one of the ETS proteins. Here, we focus on the mechanism whereby overexpression of the ETV4 gene mediates oncogenesis in the prostate. By siRNA technology, we show that ETV4 inhibition in the PC3 cancer cell line reduces not only cell mobility and anchorage-independent growth, but also cell proliferation, cell cycle progression and tumor growth in a xenograft model. Conversely, ETV4 overexpression in the nonmalignant human prostate cell line (RWPE) increases anchorage-independent growth, cell mobility and cell proliferation, which is probably mediated by downregulation of p21, producing accelerated progression through the cell cycle. ETV4 overexpression is associated with changes in the pattern of E-cadherin and N-cadherin expression; the cells also become spindle-shaped, and these changes are characteristic of the so-called epithelial to mesenchymal transition (EMT). In RWPE cells overexpressing ETV4 EMT results from a marked increase in EMT-specific transcription factors such as TWIST1, SLUG1, ZEB1 and ZEB2. Thus, whereas ETV4 shares with the other ETS proteins (ERG, ETV5 and ETV1) a major role in invasiveness and cell migration, it emerges as unique in that it increases at the same time also the rate of proliferation of PC cells. Considering the wide spectrum in the clinical course of patients with PC, it may be highly relevant that ETV4 is capable of inducing most and perhaps all of the features that make a tumor aggressive.

© F e r r a t a S t o r t i F o u n d a t i o nphic alleles of the C3 gene and of the complement receptor 1 (CR1) gene. Methods PatientsSeventy-two patients with hemolytic PNH who had received eculizumab treatment were analyzed after a median follow-up of 52 months (range, 11-98 months). Patients with evidence of bone marrow failure (reticulocytes ≤60x10 9 /L, platelets ≤50x10 9 /L, neutrophils ≤1x10 9 /L) were not included in this series because this condition may affect the clinical response to eculizumab regardless of how well hemolysis is controlled. Peripheral blood samples were collected for clinical testing, flow cytometry (GPI-molecules and C3-binding) 7 and genotyping after the patients had signed an informed consent form approved by the respective Institutional Review Board. As a criterion of response to eculizumab we used the one that is most stringent and objective: namely RBC transfusion at any time after the first 6 months on eculizumab treatment. Of the 72 patients 60 were transfusiondependent before eculizumab: of these 60, those who on eculizumab received no further transfusion during follow-up have been classified as good responders; those who on eculizumab received one or more RBC transfusion at any time during follow-up have been classified as suboptimal responders. Twelve patients started eculizumab before having received any RBC transfusions. Of these, the ten patients who on eculizumab remained transfusion-independent have been classified as good responders because their hemoglobin level increased, on average, by 2.2 g/dL; the remaining two patients required RBC transfusions on eculizumab and have been classified as sub-optimal responders. Only one patient, who had hemolytic PNH at the time of starting eculizumab and who had already been classified as a sub-optimal responder, became aplastic during follow-up. GenotypingThe polymorphism rs2230199 C>G of the C3 gene was investigated by a newly designed tetra-primer amplification refractory mutation system-polymerase chain reaction method.11 Three polymorphisms of the CR1 gene were genotyped by restriction fragment length (RFLP) analysis:12 HindIII RFLP (intron 27); His1208Arg (exon 22); Pro1827Arg (exon 33). Kinetics of C3 binding in vitroRBC and sera were promptly separated from peripheral blood freshly collected from PNH patients. The RBC were then incubated with sera containing eculizumab as previously described. 13,14 Briefly, a 2% suspension of RBC from PNH patients was incubated at 37°C with a pool of ABO-compatible sera from patients on eculizumab, and the complement alternative pathway was activated by mild acidification (HCl 0.016 M). At serial time points (15,30, 60, and 120 min) after complement activation the fraction of GPI-negative RBC with newly bound C3 fragments was measured by flow cytometry (AccuriC6, Becton Dickinson, NJ, USA) after staining with anti-CD59 Alexa647 (Mem43, Serotec, UK) and anti-C3d-neoantigen (A250, Quidel, CA, USA); secondary staining was performed with phycoerythrin-labeled polyclonal rabbit-an...

SummaryA family from the Southeast of Italy was found to have a novel b-globin mutant, b+45 G fi C, with the features of a silent b-thalassaemia mutation. It was asymptomatic in two heterozygotes, but its interaction with the severe thalassaemia mutation b-IVS-II-654 C fi T worsened the haematological and biosynthetic phenotype in two compound heterozygotes; moreover, another compound heterozygote, who was also heterozygote for the aaa anti3AE7 , suffered from thalassaemia intermedia. The mutation was found associated in cis with the IVS-II-754 T fi C substitution, which did not lead to abnormally spliced mRNA. Furthermore, the amount of b+45 mRNA was the same as the bA mRNA in the reticulocytes of the carriers. In vitro transcription/translation experiments demonstrated that the b+45 G fi C decreased the efficiency of translation of the b-globin chain by about 30%: this slight impairment was consistent with the observed clinical phenotype. The b+45 G fi C is the first mutation found in the Kozak sequence (GACACCATGG) of the b-globin gene and the first one at the position -6 upstream the ATG. The Kozak consensus sequence plays a major role in the initiation of translation process. The present finding supports the hypothesis that the G in position -6 is important in this process.