Sesquiterpene lactones (SLs) have potent antiinflammatory properties. We have shown previously that they exert this effect in part by inhibiting activation of the transcription factor NF-B, a central regulator of the immune response. We have proposed a molecular mechanism for this inhibition based on computer molecular modeling data. In this model, SLs directly alkylate the p65 subunit of NF-B, thereby inhibiting DNA binding. Nevertheless, an experimental evidence for the proposed mechanism was lacking. Moreover, based on experiments using the SL parthenolide, an alternative mode of action has been proposed by other authors in which SLs inhibit IB-␣ degradation. Here we report the construction of p65/ NF-B point mutants that lack the cysteine residues alkylated by SLs in our model. In contrast to wild type p65, DNA-binding of the Cys 38 3 Ser and Cys 38,120 3 Ser mutants is no longer inhibited by SLs. In addition, we provide evidence that parthenolide uses a similar mechanism to other SLs in inhibiting NF-B. Contrary to previous reports, we show that parthenolide, like other SLs, inhibits NF-B most probably by alkylating p65 at Cys 38 . Although a slight inhibition of IB degradation was detected for all SLs, the amount of remaining IB was too low to explain the observed NF-B inhibition.The transcription factor NF-B promotes the expression of over 150 target genes in response to inflammation, viral and bacterial infections, and other stressful situations (1, 2). Both the nature of the NF-B inducers and the function of its target genes highlight its role, as a central mediator of the human immune response.NF-B is most frequently composed of a p50 and a p65 subunit retained in the cytoplasm in an inactive form by binding to IB, an inhibitory subunit. Inducers of NF-B, such as bacterial lipopolysaccharides (LPS) 1 or inflammatory cytokines, activate the IB kinase complex (IKC), which phosphorylates IB on serines 32 and 36. Phosphorylation causes IB ubiquitinylation and its subsequent degradation by the 26-S proteasome. Degradation of the inhibitor allows NF-B to translocate to the nucleus, where it stimulates transcription of its target genes.2 Genes that are regulated by NF-B include, for example, proinflammatory and inflammatory cytokines such as interleukin-1, -2, -4, and -6 or TNF-␣, as well as genes encoding immunoreceptors, cell adhesion molecules, acute phase proteins, and enzymes such as cyclooxygenase-II. Because of its central role in regulating inflammatory responses, a pharmacological inhibition of NF-B activation could be beneficial in the treatment of inflammation (4). Using helenalin as a model, we have shown that SLs inhibit neither IB degradation nor NF-B nuclear translocation. SLs interact directly with NF-B. DNA binding of NF-B is prevented by selectively alkylating cysteine sulfhydryl groups in its p65 subunit (5, 6). There are strong indications that this is a general mechanism for SLs, which possess ␣,␤-or ␣,␤,␥-unsaturated carbonyl structures such as ␣-methylene-␥-lactones or ␣,␤-unsubstituted...

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Polycythemia vera (PV) is a clonal stem cell disorder characterized by hyperproliferation of the erythroid, myeloid, and megakaryocytic lineages. Although it has been shown that progenitor cells of patients with PV are hypersensitive to several growth factors, the molecular pathogenesis of this disease remains unknown. To investigate the molecular defects underlying PV, we used subtractive hybridization to isolate complementary DNAs (cDNAs) differentially expressed in patients with PV versus normal controls. We isolated a novel gene, subsequently named PRV-1, which is highly expressed in granulocytes from patients with PV (n = 19), but not detectable in normal control granulocytes (n = 21). Moreover, PRV-1 is not expressed in mononuclear cells from patients with chronic myelogenous leukemia (n = 4) or acute myelogenous leukemia (n = 5) or in granulocytes from patients with essential thrombocythemia (n = 4) or secondary erythrocytosis (n = 4). Northern blot analysis showed that PRV-1 is highly expressed in normal human bone marrow and to a much lesser degree in fetal liver. It is not expressed in a variety of other tissues tested. Although PRV-1 is not expressed in resting granulocytes from normal controls, stimulation of these cells with granulocyte colony-stimulating factor induces PRV-1 expression. The PRV-1 cDNA encodes an open reading frame of 437 amino acids, which contains a signal peptide at the N-terminus and a hydrophobic segment at the C-terminus. In addition, PRV-1 contains 2 cysteine-rich domains homologous to those found in the uPAR/Ly6/CD59/snake toxin-receptor superfamily. We therefore propose that PRV-1 represents a novel hematopoietic receptor.

Essential thrombocythemia (ET) is a heterogeneous disorder. For example, the growth of erythropoietin-independent erythroid colonies, termed "endogenous erythroid colonies (EECs)", has previously been observed in only 50% of ET patients. We have recently described the overexpression of a hematopoietic receptor, PRV-1 (polycythemia rubra vera-1), in patients with polycythemia vera (PV). Here, we compare PRV-1 expression and EEC formation in a cohort of 30 patients with ET; 50% of the ET patients in our cohort displayed EEC growth. Likewise, 50% of the ET patients overexpressed PRV-1. Remarkably, only the 15 ET patients displaying EEC growth showed elevated PRV-1 expression, while the 15 EEC-negative ET patients expressed normal PRV-1 levels. It has previously been reported that EEC-positive ET patients develop PV during long-term follow-up. Here, we show that 40% of the PRV-1-positive patients develop symptoms of PV during the course of their disease. In contrast, none of the 15 PRV-1-negative patients displayed such symptoms (p=0.017). Moreover, PRV-1-positive patients had a significantly higher number of thromboembolic or microcirculatory events (p=0.003). We propose that PRV-1-positive ET comprise a pathophysiologically distinct subgroup of patients, one that is at risk for the development of complications and for the emergence of PV.