Cells require optimum protein synthetic activity in order to support cell proliferation, maintain homeostatic and metabolic integrity, and repair damage. Since growth depends on protein synthesis through ribosome biogenesis, the control of biosynthesis of ribosomes is necessarily a key element for control of growth. Nucleolin is a major nucleolar protein of exponentially growing eukaryotic cells, which is directly involved in the regulation of ribosome biogenesis and maturation. The highly conserved nucleolin contains three major domains through which it controls the organization of nucleolar chromatin, packaging of pre-RNA, rDNA transcription, and ribosome assembly. Numerous reports have implicated the involvement of nucleolin either directly or indirectly in the regulation of cell proliferation and growth, cytokinesis, replication, embryogenesis, and nucleogenesis. Nucleolin, an RNA binding protein, is also an autoantigen, a transcriptional repressor, and a switch region targeting factor. In addition, nucleolin exhibits autodegradation, DNA and RNA helicase activities, and DNA-dependent ATPase activity. An interesting aspect of nucleolin action is that it is a target for regulation by proteolysis, methylation, ADP-ribosylation, and phosphorylation by CKII, cdc2, PKC-xi, cyclic AMP-dependent protein kinase, and ecto-protein kinase. For these and other reasons, nucleolin is fundamental to the survival and proliferation of cells. Considerable progress has been made in recent years with the identification of new nucleolin binding proteins that may mediate these many nucleolin-dependent functions. Nucleolin also functions as a cell surface receptor, where it acts as a shuttling protein between cytoplasm and nucleus, and thus can even provide a mechanism for extracellular regulation of nuclear events. Exploration of the regulation of this multifaceted protein in a remarkable number of diverse functions is challenging.

Cystic Fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung arising from profound expression of inflammatory genes, including interleukin-8 (IL-8). We have previously reported that IL-8 mRNA is stabilized in CF lung epithelial cells, resulting in concomitant hyperexpression of IL-8 protein. However, the mechanistic link between mutations in CFTR and acquisition of the proinflammatory phenotype in the CF airway has remained elusive. We hypothesized that specific microRNAs (miRNAs) might mediate this linkage. To identify the potential link, we screened an miRNA library for differential expression in ⌬F508-CFTR and wild type CFTR lung epithelial cell lines. Of 22 differentially and significantly expressed miRNAs, we found that expression of miR-155 was more than 5-fold elevated in CF IB3-1 lung epithelial cells in culture, compared with control IB3-1/S9 cells. Clinically, miR-155 was also highly expressed in CF lung epithelial cells and circulating CF neutrophils biopsied from CF patients. We report here that high levels of miR-155 specifically reduced levels of SHIP1, thereby promoting PI3K/Akt activation. However, overexpressing SHIP1 or inhibition of PI3K in CF cells suppressed IL-8 expression. Finally, we found that phospho-Akt levels were elevated in CF lung epithelial cells and were specifically lowered by either antagomir-155 or elevated expression of SHIP1. We therefore suggest that elevated miR-155 contributes to the proinflammatory expression of IL-8 in CF lung epithelial cells by lowering SHIP1 expression and thereby activating the PI3K/Akt signaling pathway. These data suggest that miR-155 may play an important role in the activation of IL-8-dependent inflammation in CF.

The mammalian anx7 gene codes for a Ca 2؉ -activated GTPase, which supports Ca 2؉ ͞GTP-dependent secretion events and Ca 2؉ channel activities in vitro and in vivo. To test whether anx7 might be involved in Ca 2؉ signaling in secreting pancreatic ␤ cells, we knocked out the anx7 gene in the mouse and tested the insulinsecretory properties of the ␤ cells. The nullizygous anx7 (؊͞؊) phenotype is lethal at embryonic day 10 because of cerebral hemorrhage. However, the heterozygous anx7 (؉͞؊) mouse, although expressing only low levels of ANX7 protein, is viable and fertile. The anx7 (؉͞؊) phenotype is associated with a substantial defect in insulin secretion, although the insulin content of the islets, is 8-to 10-fold higher in the mutants than in the normal littermate control. We infer from electrophysiological studies that both glucose-stimulated secretion and voltage-dependent Ca 2؉ channel functions are normal. However, electrooptical recordings indicate that the (؉͞؊) mutation has caused a change in the ability of inositol 1,4,5-trisphosphate (IP3)-generating agonists to release intracellular calcium. The principle molecular consequence of lower anx7 expression is a profound reduction in IP3 receptor expression and function in pancreatic islets. The profound increase in islets, ␤ cell number, and size may be a means of compensating for less efficient insulin secretion by individual defective pancreatic ␤ cells. This is a direct demonstration of a connection between glucoseactivated insulin secretion and Ca 2؉ signaling through IP3-sensitive Ca 2؉ stores.

The ANX7 gene is located on human chromosome 10q21, a site long hypothesized to harbor a tumor suppressor gene(s) (TSG) associated with prostate and other cancers. To test whether ANX7 might be a candidate TSG, we examined the ANX7-dependent suppression of human tumor cell growth, stage-specific ANX7 expression in 301 prostate specimens on a prostate tissue microarray, and loss of heterozygosity (LOH) of microsatellite markers at or near the ANX7 locus. Here we report that human tumor cell proliferation and colony formation are markedly reduced when the wild-type ANX7 gene is transfected into two prostate tumor cell lines, LNCaP and DU145. Consistently, analysis of ANX7 protein expression in human prostate tumor microarrays reveals a significantly higher rate of loss of ANX7 expression in metastatic and local recurrences of hormone refractory prostate cancer as compared with primary tumors (P ‫؍‬ 0.0001). Using four microsatellite markers at or near the ANX7 locus, and laser capture microdissected tumor cells, 35% of the 20 primary prostate tumors show LOH. The microsatellite marker closest to the ANX7 locus showed the highest rate of LOH, including one homozygous deletion. We conclude that the ANX7 gene exhibits many biological and genetic properties expected of a TSG and may play a role in prostate cancer progression.cancer genetics ͉ chromosome 10q21 ͉ loss of heterozygosity T he gene for annexin 7 (ANX7 ‡ ‡ , synexin; refs. 1-6) is located on human chromosome 10q21, where potential tumor suppressor genes (TSGs) have been hypothesized to exist for prostate and other cancers (5, 7-15). However, the specific relevance of the ANX7 gene for cancer only became apparent after we created a knockout for this gene in the mouse (16). Although the homozygous Anx7(Ϫ͞Ϫ) deletion is embryonically lethal, the phenotype of the Anx7(ϩ͞Ϫ) heterozygote includes calcium signaling deficits and growth defects such as gigantism, and selective organomegaly. As these mice aged, we also began to observe a profoundly increased frequency of disparate spontaneous tumors in both male and female Anx7(ϩ͞Ϫ) mutants (17).Because of these observations, and the chromosomal location of the gene, we hypothesized that ANX7 might be a candidate TSG associated with 10q21 locus. Commonly, TSGs can suppress growth of tumor cells, in vitro, and are frequently inactivated by mutations, deletions, or loss of expression in tumors, in vivo. In addition, loss of heterozygosity (LOH) often is observed for these genes in clinical tumor specimens. Therefore, to test this hypothesis for the ANX7 gene, we analyzed the action of the ANX7 gene on colony formation by human tumor cell lines. We also examined the expression of the ANX7 protein in hundreds of prostate cancers by using tumor tissue microarray technology. Finally, we tested a panel of primary and metastatic prostate cancers for evidence of LOH.In this paper we show that the ANX7 gene suppresses the growth of the prostate tumor cell lines DU145 and LNCaP. Consistently, in a prostate tissue microarray, we...

A cDNA containing the entire coding region for human nucleolin has been isolated from a 1 gtl0 human retinal library using a bovine cDNA probe. The cDNA hybridized to a transcript of 3000 bases from fast-dividing cells, as well as terminally differentiated tissues of several species. Translation of the nucleotide sequence revealed a long open reading frame which predicts a 707 amino acid protein with several distinct domains. These include repeating elements, four conserved RNA-binding regions, a glycine-rich carboxy-terminal domain and sites for phosphorylation, glycosylation and dibasic cleavage. Human and bovine nucleolin exhibited more additions and/or substitutions of aspartate, glutamate and serine residues in the chromatin-binding domains by comparison with the hamster and mouse nucleolins. These differences may be related to species-specific functions in transcription.

Wild-type CFTR and CPX both suppress proinflammatory IL-8 secretion from CF epithelial cells. The mechanism, as defined by pharmacogenomic analysis, involves identified genes from the TNF-alphaR/NFkappaB pathway. The close relationship between IL-8 secretion and genes from the TNF-alphaR/NFkappaB pathway suggests that molecular or pharmaceutical targeting of these novel genes may have strategic use in the development of new therapies for CF. From the perspective of global gene expression, both gene and drug therapy have similar genomic consequences. This is the first example showing equivalence of gene and drug therapy in CF, and suggests that a gene therapy-defined endpoint may prove to be a powerful paradigm for CF drug discovery. Finally, because the GENESAVER algorithm is capable of isolating disease-relevant genes in a hypothesis-driven manner without recourse to any a priori knowledge about the system, this new algorithm may also prove useful in applications to other genetic diseases.

Exocytotic membrane fusion and secretion are promoted by the concerted action of GTP and Ca2 , although the precise site(s) of action in the process are not presently known. However, the calcium-dependent membrane fusion reaction driven by synexin (annexin VII) is an in vitro model for this process, which we have now found to be further activated by GTP. The mechanism of fusion activation depends on the unique ability of synexin to bind and hydrolyze GTP in a calcium-dependent manner, both in vitro and in vivo in streptolysin 0-permeabilized chromaffin cells. The required [Ca2+] for GTP binding by synexin is in the range of 50-200 ,AM, which is known to occur at exocytotic sites in chromaffin cells, neurons, and other cell types. Previous immunolocalization studies place synexin at exocytotic sites in chromaffin cells, and we conclude that synexin is an atypical G protein that may be responsible for both detecting and mediating the Ca2+/GTP signal for exocytotic membrane fusion.GTP and its nonhydrolyzable analogue guanosine 5'- [y-thio]triphosphate (GTP[,yS]) are known to promote Ca2+-dependent exocytotic secretion from many cell types by a mechanism thought to involve as yet unknown proteins in the GTPase superfamily (1-4). Specific members of this superfamily have been considered as mediators of these GTP effects on exocytosis, including heterotrimeric G proteins (5-9) and low molecular weight ras-like proteins such as Rab (10-15) and ARF (16). The current "fusion machine" hypothesis (17-19) envisions a core complex formed between plasma membrane syntaxin and SNAP-25 and the synaptic vesicle protein synaptobrevin/VAMP (20), with vesicular synaptotagmin putatively identified as a low-affinity calcium sensor that interacts with regulatory syntaxin 1 (21,22). However, none of the proteins presently identified in the hypothetical fusion machine have actually been shown to be activated by GTP or, indeed, even to fuse membranes (17,(23)(24)(25). Therefore, it has been suggested that other GTP-binding proteins, as yet unidentified, might control the activity of the fusion complex (19,25).The site of GTP action in exocytosis has been hypothesized to be closely associated with the site of calcium action in a common pathway (2,5,26,27). The most recent consensus on the nature of this calcium binding site is that it is involved in docking and fusion and that the affinity of the site for Ca2+ may be in the range of 50-200 ,tM (28-36

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