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Prostaglandin H synthase (PGHS) is the primary enzyme responsible for the biosynthesis of prostaglandins and thromboxanes. Of the two isoenzymes of PGHS,PGHS-1 is constitutively expressed and PGHS-2 is inducible by mitogens or other inflammatory stimuli. Constitutive expression of PGHS-2 in neoplastic tissues has been implicated in carcinogenesis. Resveratrol,a lignan,was recently shown to be an anticarcinogen that selectively inhibits PGHS-1. In vitro experiments to resolve these seemingly paradoxical observations revealed that resveratrol is not only an inhibitor of PGHS-1 but also is an activator of PGHS-2. Resveratrol non-competitively inhibited PGHS-1 with a K1 of 26 +/- 2 microM but enhanced the PGHS-2 activity nearly twofold. Additionally,resveratrol did not serve as a reducing co-substrate for the peroxidase activities of either enzyme despite being an easily oxidizable phenolic compound. Resveratrol inhibited the peroxidase activity of PGHS-1 (IC50 = 15 microM) better than that of PGHS-2 (IC50 = textgreater 200 microM). Inhibition of the perxidase activity but not the cyclooxygenase activity of PGHS-2 resulted in the production of PGG2 from arachidonic acid. A plausible relationship between these observation and the anticarcinogenic activity of resveratrol is discussed. View Publication

The histologic distinction between epithelial peritoneal mesothelioma and papillary serous carcinoma diffusely involving the peritoneum may be difficult. Although some investigators have indicated that immunohistochemistry can facilitate this differential diagnosis. only a few studies using a limited number of markers have been published. In this study,the immunoreactivity of keratin 5/6,vimentin,epithelial membrane antigen,thrombomodulin,calretinin,MOC-31,Ber-EP4,carcinoembryonic antigen,TAG-72 (B72.3),CD15 (Leu-M1),placental alkaline phosphatase,CA19-9,CA-125,HBME-1,44-3A6,and S-100 protein was investigated in 35 epithelial peritoneal mesotheliomas,and 45 papillary serous carcinomas [30 ovarian (10 primary and 20 metastatic to the peritoneum) and 15 papillary serous carcinomas of the peritoneum]. After analyzing the results,it is concluded that calretinin,thrombomodulin,and keratin 5/6 are the best positive markers for differentiating epithelial malignant mesotheliomas from papillary serous carcinomas diffusely involving the peritoneum. The best diagnostic discriminators among the antibodies considered to be negative markers for mesothelioma are MOC-31,B72.3,Ber-EP4,CA19-9,and Leu-M1. Immunostaining for carcinoembryonic antigen,placental alkaline phosphatase,epithelial membrane antigen,vimentin,HBME-1,44-3A6,CA-125,or S-100 have little or no diagnostic utility in establishing the differential diagnosis between these conditions. The results of this study also confirm previous observations indicating that both papillary serous carcinomas of the peritoneum and serous carcinomas of the ovary have a similar phenotype and,therefore,immunohistochemical studies are not useful in separating these entities. View Publication

Angiogenesis,the sprouting of new blood vessels from pre-existing ones,is an essential physiological process in development,yet also plays a major role in the progression of human diseases such as diabetic retinopathy,atherosclerosis and cancer. The effects of the most potent angiogenic factors,vascular endothelial growth factor (VEGF),angiopoietin and fibroblast growth factor (FGF) are mediated through cell surface receptors that possess intrinsic protein tyrosine kinase activity. In this report,we describe a synthetic compound of the pyrido[2,3-d]pyrimidine class,designated PD 173074,that selectively inhibits the tyrosine kinase activities of the FGF and VEGF receptors. We show that systemic administration of PD 173074 in mice can effectively block angiogenesis induced by either FGF or VEGF with no apparent toxicity. To elucidate the determinants of selectivity,we have determined the crystal structure of PD 173074 in complex with the tyrosine kinase domain of FGF receptor 1 at 2.5 A resolution. A high degree of surface complementarity between PD 173074 and the hydrophobic,ATP-binding pocket of FGF receptor 1 underlies the potency and selectivity of this inhibitor. PD 173074 is thus a promising candidate for a therapeutic angiogenesis inhibitor to be used in the treatment of cancer and other diseases whose progression is dependent upon new blood vessel formation. View Publication

Mitomycin C (MMC) is the prototype bioreductive DNA alkylating agent. To exploit its unique properties and maximize patient responses,different therapeutic approaches have been investigated. Recently,the focus has concentrated on monitoring the levels of the proteins metabolizing the drug and relating these to activity in a regimen referred to as enzyme-directed bioreductive drug development. To be successful,it is important to understand the enzymology of metabolic activation not only in cell lines but also in solid tumour models. A general mechanism of action for MMC has now emerged that is activated regardless of the source of reducing equivalents,comprising three competing pathways that give rise to unique reactive intermediates and different DNA adducts. Partitioning into the pathways is dictated by chemical considerations such as pH and drug concentration. DT-diaphorase stands out in this mechanism,since it is much less effective at metabolizing MMC at neutral pH. At least five different enzymes can catalyse MMC bioreduction in vitro,and as many activities may be present in solid tumours,including a series of novel mitochondrial reductases such as a cytochrome P450 reductase. Competition between reductases for MMC appears to be based solely on protein levels rather than enzyme kinetics. Consequentially,DT-diaphorase can occupy a central role in MMC metabolic activation since it is often highly overexpressed in cancer cells. Although a good correlation has been observed in cell lines between DT-diaphorase expression and aerobic cytotoxicity,this does not hold consistently in vivo for any single bioreductive enzyme,suggesting revision of the enzyme-directed hypothesis as originally formulated. View Publication

Phosphatidylinositol,a component of eukaryotic cell membranes,is unique among phospholipids in that its head group can be phosphorylated at multiple free hydroxyls. Several phosphorylated derivatives of phosphatidylinositol,collectively termed phosphoinositides,have been identified in eukaryotic cells from yeast to mammals. Phosphoinositides are involved in the regulation of diverse cellular processes,including proliferation,survival,cytoskeletal organization,vesicle trafficking,glucose transport,and platelet function. The enzymes that phosphorylate phosphatidylinositol and its derivatives are termed phosphoinositide kinases. Recent advances have challenged previous hypotheses about the substrate selectivity of different phosphoinositide kinase families. Here we re-examine the pathways of phosphoinositide synthesis and the enzymes involved. View Publication

We determined the inhibitory activities of gatifloxacin against Staphylococcus aureus topoisomerase IV,Escherichia coli DNA gyrase,and HeLa cell topoisomerase II and compared them with those of several quinolones. The inhibitory activities of quinolones against these type II topoisomerases significantly correlated with their antibacterial activities or cytotoxicities (correlation coefficient [r] = 0.926 for S. aureus,r = 0.972 for E. coli,and r = 0.648 for HeLa cells). Gatifloxacin possessed potent inhibitory activities against bacterial type II topoisomerases (50% inhibitory concentration [IC50] = 13.8 microg/ml for S. aureus topoisomerase IV; IC50 = 0.109 microg/ml for E. coli DNA gyrase) but the lowest activity against HeLa cell topoisomerase II (IC50 = 265 microg/ml) among the quinolones tested. There was also a significant correlation between the inhibitory activities of quinolones against S. aureus topoisomerase IV and those against E. coli DNA gyrase (r = 0.969). However,the inhibitory activity against HeLa cell topoisomerase II did not correlate with that against either bacterial enzyme. The IC50 of gatifloxacin for HeLa cell topoisomerase II was 19 and was more than 2,400 times higher than that for S. aureus topoisomerase IV and that for E. coli DNA gyrase. These ratios were higher than those for other quinolones,indicating that gatifloxacin possesses a higher selectivity for bacterial type II topoisomerases. View Publication

MUC1 mucin is expressed by normal and malignant epithelial cells and is thought to function through cell-cell interactions and transmembrane signal transduction events. Secreted cancer-associated MUC1 is immunosuppressive and inhibits human T-cell proliferation. We report here that newly synthesized MUC1 is expressed on the surface of mitogen-activated human T cells and is also found in soluble form in the supernatants from cultures of mitogen-activated human T cells. After removal of the mitogenic stimulus from the T-cell cultures,MUC1 expression is downregulated. The addition of anti-MUC1 monoclonal antibody to mitogen-activated cultures partially inhibits the T-cell proliferative response. These data suggest that MUC1 serves an immunodulatory function for human T lymphocytes. View Publication

The detection of primitive hematopoietic cells based on repopulation of immune-deficient mice is a powerful tool to characterize the human stem-cell compartment. Here,we identify a newly discovered human repopulating cell,distinct from previously identified repopulating cells,that initiates multilineage hematopoiesis in NOD/SCID mice. We call such cells CD34neg-SCID repopulating cells,or CD34neg-SRC. CD34neg-SRC are restricted to a Lin-CD34-CD38- population without detectable surface markers for multiple lineages and CD38 or those previously associated with stem cells (HLA-DR,Thy-1 and CD34). In contrast to CD34+ subfractions,Lin-CD34-CD38- cells have low clonogenicity in short-and long-term in vitro assays. The number of CD34neg-SRC increased in short-term suspension cultures in conditions that did not maintain SRC derived from CD34+ populations,providing independent biological evidence of their distinctiveness. The identification of this newly discovered cell demonstrates complexity of the organization of the human stem-cell compartment and has important implications for clinical applications involving stem-cell transplantation. View Publication

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Simultaneous ex vivo expansion of different progenitor cell types may be beneficial for cord blood (CB) transplantation,to overcome a potential limitation due to restricted cell numbers. Therefore,1.5 x 10(6) CD34+ cells isolated from fresh or thawed CB samples were inoculated in a large-scale stirred suspension bioreactor and cultured in the presence of Flt3-L,SCF and IL-3. At days 0,7,10,14,21 and 28,the spinner cultures were analyzed for viable cells,colony-forming cells (CFC),including erythroid burst-forming unit (BFU-E),granulocyte-macrophage colony-forming unit (CFU-GM) and granulocyte-erythrocyte-megakaryocyte-monocyte colony-forming unit (CFU-GEMM) as well as long-term culture-initiating cells (LTC-IC). Expansion of thawed CD34+ cells resulted in a substantial amplification of total cells (maximal at day 28: 154 +/- 132-fold),CFC (maximal at day 14: 45 +/- 36-fold),CFU-GM (maximal at day 14: 88 +/- 85-fold),CFU-GEMM (maximal at day 7: 4 +/- 2-fold) and of LTC-IC (maximal at day 10: 8 +/- 3-fold). There was no significant difference between fresh and thawed CD34+ cells. These results demonstrate that simultaneously committed progenitors as well as the more immature CFU-GEMM and LTC-IC can be substantially amplified from CD34+-enriched CB samples in large-scale stirred suspension cultures within 7-14 days without exhausting the proliferative potential and,thus,it may be possible to improve CB transplantation by ex vivo generated cells. View Publication

Selective protein kinase inhibitors were developed on the basis of the unexpected binding mode of 2,6,9-trisubstituted purines to the adenosine triphosphate-binding site of the human cyclin-dependent kinase 2 (CDK2). By iterating chemical library synthesis and biological screening,potent inhibitors of the human CDK2-cyclin A kinase complex and of Saccharomyces cerevisiae Cdc28p were identified. The structural basis for the binding affinity and selectivity was determined by analysis of a three-dimensional crystal structure of a CDK2-inhibitor complex. The cellular effects of these compounds were characterized in mammalian cells and yeast. In the latter case the effects were characterized on a genome-wide scale by monitoring changes in messenger RNA levels in treated cells with high-density oligonucleotide probe arrays. Purine libraries could provide useful tools for analyzing a variety of signaling and regulatory pathways and may lead to the development of new therapeutics. View Publication

6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN or CD437),originally identified as a retinoic acid receptor gamma-selective retinoid,was previously shown to induce growth inhibition and apoptosis in human breast cancer cells. In this study,we investigated the role of AHPN/CD437 and its mechanism of action in human lung cancer cell lines. Our results demonstrated that AHPN/CD437 effectively inhibited lung cancer cell growth by inducing G0/G1 arrest and apoptosis,a process that is accompanied by rapid induction of c-Jun,nur77,and p21(WAF1/CIP1). In addition,we found that expression of p53 and Bcl-2 was differentially regulated by AHPN/CD437 in different lung cancer cell lines and may play a role in regulating AHPN/CD437-induced apoptotic process. On constitutive expression of the c-JunAla(63,73) protein,a dominant-negative inhibitor of c-Jun,in A549 cells,nur77 expression and apoptosis induction by AHPN/CD437 were impaired,whereas p21(WAF1/CIP1) induction and G0/G1 arrest were not affected. Furthermore,overexpression of antisense nur77 RNA in A549 and H460 lung cancer cell lines largely inhibited AHPN/CD437-induced apoptosis. Thus,expression of c-Jun and nur77 plays a critical role in AHPN/CD437-induced apoptosis. Together,our results reveal a novel pathway for retinoid-induced apoptosis and suggest that AHPN/CD437 or analogs may have a better therapeutic efficacy against lung cancer. View Publication