T he antimicrobial activity of phagocytes depends in part on the cells' ability to reduce oxygen to reactive microbicidal oxidants by means of an NADPH oxidase. In resting phagocytes, the NADPH oxidase is in a dormant state, but exposure of the cell to any of a variety of stimuli can activate the enzyme, causing it to release large amounts of O 2 Ϫ by reducing oxygen at the expense of NADPH. The oxidase is activated by the phosphorylation of one of its cytosolic subunits, p47 PHOX , on particular serines (1). In whole cells, the stimulation of an appropriate receptor activates PKC and phosphatidylinositol 3-kinase (PI3-kinase) (2-4). Although NADPH oxidase activation by PKC has been well characterized (5, 6), the association between PI3-kinase and NADPH oxidase activation remains to be established. A possible connection is through the activation of Akt (7), whose role in oxidase activation is suggested by the finding that Akt is activated rapidly when neutrophils are treated with oxidase-activating agents (8) and by the finding that oxidase activation is inhibited by wortmannin (9). The experiments described here show that Akt is able to activate the oxidase by phosphorylating p47 PHOX on serines S304 and S328. Experimental ProceduresMaterials. Active Akt was obtained from Upstate Biotechnology (Lake Placid, NY) or from J.-H.L., whose recombinant material was 95% pure by SDS͞PAGE. [␥-32 P]ATP was purchased from New England Nuclear. Phosphorylated peptides were obtained from Sigma.Isolation and Fractionation of Neutrophils. Neutrophils were obtained from normal subjects by dextran sedimentation and Ficoll͞Hypaque fractionation of freshly drawn citrateanticoagulated blood (10). After treatment on ice for 10-20 min with 5 l of 0.54 M diisopropyl fluorophosphate, the neutrophils, suspended at 10 8 cells per ml in a modified relaxation buffer (0.1 M KCl͞3 mM NaCl͞3.5 mM MgCl 2 ͞10 mM Pipes buffer, pH 7.3), were subjected to nitrogen cavitation. Membranes and cytosol were separated by centrifugation through a Percoll gradient. Aliquots of membrane and cytosol were stored at Ϫ70°C until use.Recombinant Oxidase Components. Recombinant fusion proteins composed of an upstream GST linked to a downstream p47 PHOX , p67 PHOX , or Rac2 were isolated from Escherichia coli transformed with pGEX-6P3 plasmids containing cDNA inserts encoding the downstream proteins. The fusion proteins were isolated by the addition of a prewashed 50% slurry of glutathione-Sepharose 4B (Amersham Biosciences) in PBS (133 mM NaCl͞16 mM Na 2 HPO 4 ͞2.7 mM KCl͞1.5 mM KH 2 PO 4 , pH 7.3). The tube was rotated end-over-end for 1 h at 4°C and then microcentrifuged for 3 min at 200 ϫ g to sediment the glutathione-Sepharose beads. The beads were washed four times with 10 bead volumes of PBS, and the bound fusion proteins were eluted by incubating for 30 min at 4°C with three 1-ml washes of 50 mM Tris⅐HCl, pH 8.0͞20 mM glutathione͞0.2 M NaCl. Excess glutathione was removed from the purified recombinant protein by dialysis against relaxation buffer and conce...

We conducted a large-scale association study to identify genes that influence nonfamilial breast cancer risk using a collection of German cases and matched controls and >25,000 single nucleotide polymorphisms located within 16,000 genes. One of the candidate loci identified was located on chromosome 19p13.2 [odds ratio (OR) ‫؍‬ 1.5, P ‫؍‬ 0.001]. The effect was substantially stronger in the subset of cases with reported family history of breast cancer (OR ‫؍‬ 3.4, P ‫؍‬ 0.001). The finding was subsequently replicated in two independent collections (combined OR ‫؍‬ 1.4, P < 0.001) and was also associated with predisposition to prostate cancer in an independent sample set of prostate cancer cases and matched controls (OR ‫؍‬ 1.4, P ‫؍‬ 0.002). High-density single nucleotide polymorphism mapping showed that the extent of association spans 20 kb and includes the intercellular adhesion molecule genes ICAM1, ICAM4, and ICAM5. Although genetic variants in ICAM5 showed the strongest association with disease status, ICAM1 is expressed at highest levels in normal and tumor breast tissue. A variant in ICAM5 was also associated with disease progression and prognosis. Because ICAMs are suitable targets for antibodies and small molecules, these findings may not only provide diagnostic and prognostic markers but also new therapeutic opportunities in breast and prostate cancer.

A B S T R A C T PurposeWhole-genome scan association analysis was carried out to identify genetic variants predictive of lung cancer risk in smokers and to confirm the identified variants in an independent sample. Patients and MethodsA case-control study was performed using two pools consisting of DNA from 321 German smoking lung cancer patients and 273 healthy smoking controls, respectively. A replication study was carried out using 254 Italian lung adenocarcinoma (ADCA) patients and 235 healthy controls. ResultsPatients with genotypes GG or CG for the rs1862214 single nucleotide polymorphism, 5Ј upstream of the programmed cell death 5 (PDCD5) gene, compared with those with the common genotype CC showed an increased risk of lung cancer (odds ratio [OR], 1.6; 95% CI, 1.2 to 2.1) and a higher incidence of poor clinical stage disease (hazard ratio [HR], 1.9; 95% CI, 1.1 to 3.4; P ϭ .023), nodal involvement (HR, 1.9; 95% CI, 1.1 to 3.6; P ϭ .033), and short-term survivorship (HR, 1.8; 95% CI, 1.2 to 2.6, P ϭ .003). PDCD5 mRNA expression levels were ϳ2.4-fold lower in lung ADCA as compared to normal lung tissue. Human NCI-H520 cancer cells transfected with PDCD5 cDNA showed decreased colony-forming ability. ConclusionThese results suggest that the rs1862214 polymorphism in PDCD5 is predictive for lung cancer risk and prognosis, and that PDCD5 may represent a novel tumor suppressor gene influencing lung cancer.J Clin Oncol 24.

Genome-wide association studies using large numbers of bi-allelic single nucleotide polymorphisms (SNPs) have been proposed as a potentially powerful method for identifying genes involved in common diseases. To assemble a SNP collection appropriate for large-scale association, we designed assays for 226,099 publicly available SNPs located primarily within known and predicted gene regions. Allele frequencies were estimated in a sample of 92 CEPH Caucasians using chip-based MALDI-TOF mass spectrometry with pooled DNA. Of the 204,200 designed assays that were functional, 125,799 SNPs were determined to be polymorphic (minor allele frequency >0.02), of which 101,729 map uniquely to the human genome. Many of the commonly available RefSNP annotations were predictive of polymorphic status and could be used to improve the selection of SNPs from the public domain for genetic research. The set of uniquely mapping, polymorphic SNPs is located within 10 kb of 66% of known and predicted genes annotated in LocusLink, which could prove useful for large-scale disease association studies.[Supplemental material is available online at www.genome.org. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: E. Lai, and O. Osamu.]Single nucleotide polymorphisms (SNPs) are the most abundant genetic variations in the human genome. They occur, on average, once every 300 base pairs of sequence with a minor allele frequency (MAF) greater than 1% (Kruglyak and Nickerson 2001;Stephens et al. 2001;Reich et al. 2003). The high abundance of SNPs and the availability of increasingly high-throughput and cost effective methods of measuring them encourage their use in many kinds of human genetic studies (Marnellos 2003). SNPs are of particular value in whole-genome association studies for identifying the genes involved in complex trait variation (Lander 1996;Risch and Merikangas 1996). The expected number of SNPs required for successful population-based association studies depends on the distribution of linkage disequilibrium (LD) across the genome in the populations of interest. The lengths of genomic segments in strong LD varies tremendously throughout the genome, ranging from less than 1 kilobase pairs (kb) to over 500 kb, with most blocks estimated to be less than 20 kb in Caucasian populations (Daly et al. 2001;Reich et al. 2001;Dawson et al. 2002;Gabriel et al. 2002;Clark et al. 2003). The international haplotype mapping (HapMap) project was initiated to characterize the patterns of LD throughout the human genome to help identify the most informative set of SNP markers for LD mapping (Gibbs et al. 2003).To explore the potential of large-scale association studies, we set out to develop a suitable collection of approximately 100,000 SNPs. With the HapMap project far from completion, the SNPs could not be selected on the basis of LD patterns. Since a collection of 100,000 SNPs would be far too few to provide dense coverage throughout the genome, we primarily focused on SNPs located with...

The leukocyte NADPH oxidase catalyzes the 1-electron reduction of oxygen to O 2 ؊ at the expense of NADPH: 2 O 2 ؉ NADPH 3 2 O 2 ؊ ؉ NADP ؉ ؉ H ؉ . The oxidase is dormant in resting cells but acquires activity when the cells are stimulated with a suitable agent. Activation in whole cells is accompanied by extensive phosphorylation of p47 PHOX, an oxidase subunit located in the cytosol of resting cells that during oxidase activation migrates to the plasma membrane to complex with cytochrome b 558 , an oxidase-specific flavohemoprotein. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile as activating agent. We now report a cell-free system in which the oxidase can be activated in two stages using phosphorylated p47 PHOX . The first stage, which effects a change in the membrane, requires ATP and GTP and is blocked by the protein kinase inhibitor GF-109203X, suggesting a protein kinase requirement. The second stage requires phosphorylated p47 PHOX and GTP, but no ATP, and is unaffected by GF-109203X; assembly of the oxidase may take place during this stage. Activation is accomplished by p47 PHOX phosphorylated by protein kinase C but not protein kinase A or mitogen-activated protein kinase. We believe that activation by phosphorylated p47 PHOX is more physiological than activation by amphiphiles, because the mutant p47 PHOX S379A, which is inactive in whole cells, is also inactive in this system but works in systems activated by amphiphiles.