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Phosphatidylinositol (PtdIns) 3-kinase is an enzyme implicated in growth factor signal transduction by associating with receptor and nonreceptor tyrosine kinases,including the platelet-derived growth factor receptor. Inhibitors of PtdIns 3-kinase could potentially give a better understanding of the function and regulatory mechanisms of the enzyme. Quercetin,a naturally occurring bioflavinoid,was previously shown to inhibit PtdIns 3-kinase with an IC50 of 1.3 microgram/ml (3.8 microM); inhibition appeared to be directed at the ATP-binding site of the kinase. Analogs of quercetin were investigated as PtdIns 3-kinase inhibitors,with the most potent ones exhibiting IC50 values in the range of 1.7-8.4 micrograms/ml. In contrast,genistein,a potent tyrosine kinase inhibitor of the isoflavone class,did not inhibit PtdIns 3-kinase significantly (IC50 textgreater 30 micrograms/ml). Since quercetin has also been shown to inhibit other PtdIns and protein kinases,other chromones were evaluated as inhibitors of PtdIns 3-kinase without affecting PtdIns 4-kinase or selected protein kinases. One such compound,2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (also known as 2-(4-morpholinyl)-8-phenylchromone,LY294002),completely and specifically abolished PtdIns 3-kinase activity (IC50 = 0.43 microgram/ml; 1.40 microM) but did not inhibit PtdIns 4-kinase or tested protein and lipid kinases. Analogs of LY294002 demonstrated a very selective structure-activity relationship,with slight changes in structure causing marked decreases in inhibition. LY294002 was shown to completely abolish PtdIns 3-kinase activity in fMet-Leu-Phe-stimulated human neutrophils,as well as inhibit proliferation of smooth muscle cells in cultured rabbit aortic segments. Since PtdIns 3-kinase appears to be centrally involved with growth factor signal transduction,the development of specific inhibitors against the kinase may be beneficial in the treatment of proliferative diseases as well as in elucidating the biological role of the kinase in cellular proliferation and growth factor response. View Publication

Significant activity of phosphatidylinositol 3-kinase (PI 3-kinase) was detected in the membrane fractions,or in the immunoprecipitates prepared with anti-phosphotyrosine antibodies,from rat adipocytes that had been incubated with insulin for 20 min. The PI 3-kinase activity in these preparations as well as in the whole cell lysates of adipocytes not treated with insulin was inhibited by the addition of wortmannin,a fungal metabolite,to the enzyme assay mixture. The inhibition was dependent on the inhibitor concentration with IC50 being less than 10 nM and perfect inhibition at 100 nM. The effect of insulin to induce membrane PI 3-kinase activity was mostly abolished,but its effects to tyrosine-phosphorylate the beta-subunit of the insulin receptor or other cellular substrate proteins including insulin-receptor-substrate-1 were not at all antagonized,by wortmannin added to the cell incubation medium. Insulin stimulation of cellular 2-deoxyglucose uptake and inhibition of isoproterenol-induced lipolysis observable in adipocytes under the same conditions were also antagonized by wortmannin added in the same concentration range as used for the inhibition of insulin-susceptible PI 3-kinase. It is concluded,therefore,that activation of wortmannin-sensitive PI 3-kinase plays a pivotal role in the intracellular signaling pathways arising from the insulin receptor autophosphorylation and leading to certain metabolic responses. View Publication

The effects of nicotinamide (NIC) on human fetal and adult endocrine pancreatic cells were studied in tissue culture. Treatment of the fetal cells with 10 mM NIC resulted in a twofold increase in DNA content and a threefold increase in insulin content. This was associated with the development of beta cell outgrowths from undifferentiated epithelial cell clusters and an increase in the expression of the insulin,glucagon,and somatostatin genes. DNA synthesis was stimulated only in the undifferentiated cells. Half-maximal doses for the insulinotropic and mitogenic effects of NIC were 5-10 and 1-2 mM,respectively. Islet-like cell clusters cultured with NIC responded to glucose stimulation with a biphasic increase in insulin release (fourfold peak),whereas control cells were unresponsive to glucose. Both control and NIC-treated cells developed into functional islet tissue after transplantation into athymic nude mice. As compared with adult islets,the insulinotropic action of NIC could only be demonstrated in the fetal cells. Our results indicate that NIC induces differentiation and maturation of human fetal pancreatic islet cells. This model should be useful for the study of molecular mechanisms involved in beta cell development. View Publication

We demonstrate an essential role for the proteasome complex in two proteolytic processes required for activation of the transcription factor NF-kappa B. The p105 precursor of the p50 subunit of NF-kappa B is processed in vitro by an ATP-dependent process that requires proteasomes and ubiquitin conjugation. The C-terminal region of p105 is rapidly degraded,leaving the N-terminal p50 domain. p105 processing can be blocked in intact cells with inhibitors of the proteasome or in yeast with proteasome mutants. These inhibitors also block the activation of NF-kappa B and the rapid degradation of I kappa B alpha induced by tumor necrosis factor alpha. Thus,the ubiquitin-proteasome pathway functions not only in the complete degradation of polypeptides,but also in the regulated processing of precursors into active proteins. View Publication

An efficient system was developed that induced the differentiation of embryonic stem (ES) cells into blood cells of erythroid,myeloid,and B cell lineages by coculture with the stromal cell line OP9. This cell line does not express functional macrophage colony-stimulating factor (M-CSF). The presence of M-CSF had inhibitory effects on the differentiation of ES cells to blood cells other than macrophages. Embryoid body formation or addition of exogenous growth factors was not required,and differentiation was highly reproducible even after the selection of ES cells with the antibiotic G418. Combined with the ability to genetically manipulate ES cells,this system will facilitate the study of molecular mechanisms involved in development and differentiation of hematopoietic cells. View Publication

Stroma-dependent long-term bone marrow cultures (LTBMC) assay the ability of primitive haematopoietic stem cells (HSC) for long-term production of clonable progenitors. We have developed a limiting dilution type LTBMC assay allowing frequency analysis of transiently repopulating HSC and long-term culture initiating cells (LTC-IC) without the necessity to replate large numbers of wells. Normal or 5-FU-treated Ficoll bone marrow cells (BMC),or BMCs sorted on CD34 or HLA-DR expression,or Rh123 retention,(input range 40-70,000 CFU-GM/BFU-E/10(5) cells) were plated at limiting dilution on unirradiated adherent layers formed by a novel murine preadipose cell line (FBMD-1). The percentage of wells with at least one phase-dark haematopoietic clone (cobblestone area,CA) beneath the stromal layer was weekly determined for at least 8 weeks,and CA-forming cell (CAFC) frequencies were calculated using Poisson statistics. Parallel LTBMCs of the same samples were weekly assessed for supernate CFU-GM/BFU-E production. Weekly addition of rhIL-3 with rhG-CSF supported a high average clonogenic output per CA and dramatically increased CA size,but did not significantly alter the apparent CAFC frequency. The generation of CFU-GM per CA was constant over a period of 6 weeks with weekly means of eight normal BM samples,ranging between 5-16. At week 6 the mean CAFC frequency was 29 (1 SEM,8.8)/10(5). Early appearing CAFC were highly sensitive to 5-FU,and were contained over the full Rh123 and HLA-DR fluorescence profile of CD34pos cells,whereas CAFC week 5-8 were predominantly contained in the CD34pos Rh123dull HLA-DRlow fraction in agreement with previously reported LTC-IC characteristics. In conclusion,the CAFC assay enumerates LTC-IC using a direct visual endpoint and allows study of LTC-IC heterogeneity with respect to progenitor cell generation per stem cell clone in various haematologic diseases. View Publication

In fura-2-loaded ECV304 cells ionomycin elicited a saturable biphasic change in intracellular Ca2+ concentration ([Ca2+]i),where the initial phase represented mobilization of intracellular stores and the sustained component represented Ca2+ influx. To examine whether ionomycin could stimulate influx via a store-dependent mechanism. Mn2+ entry was monitored by the quenching of fura-2 fluorescence: influx was enhanced even after ionomycin wash-out,provided that internal stores were not refilled with Ca2+. Moreover,the maximal rate of histamine-stimulated Mn2+ entry was unaffected by ionomycin,suggesting a common route of entry. The Ca(2+)-entry blocker SK&F 96365 inhibited both the ionomycin-induced Mn2+ entry and the sustained [Ca2+]i response to the ionophore (leaving the initial peak [Ca2+]i response unaffected). In other experiments,although addition of ionomycin further increased the plateau phase induced by 100 microM histamine,the increase was completely abolished by pretreatment with the store Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA). Furthermore,in store-depleted cells,re-addition of 1 mM extracellular Ca2+ (in the presence of CPA plus histamine) led to a rapid rise in [Ca2+]i,dependent on Ca2+ influx,with kinetics that were not enhanced by ionomycin. These data suggest that ionomycin acts primarily at the level of the internal Ca2+ stores,so that,at the concentrations used here (textless or = 1 microM),it increases Ca2+ (and Mn2+) influx via activation of endogenous entry pathways and not by plasmalemmal translocation. View Publication

We have cloned the FP receptor from rat corpus luteum and human uterus cDNA libraries,respectively. The coding DNA sequence in the rat cDNA is 1101 bp and is similar to the mouse cDNA coding for a receptor protein of 366 amino acids. The human sequence shows a 5 bp deficiency in the 3' region,truncating the coding sequence to 359 amino acids. Northern blot analysis indicates highest expression in the ovary. Cell lines have been established giving stable expression of the FP receptor. Activation of the cloned FP receptor gave an increase in intracellular calcium,indicating signaling via phospholipase C-mediated phosphoinositide turnover. Using [3H]PGF2 alpha,binding of PGs showed the rank order of fluprostenol textgreater PhXA70 textgreater PGF2 alpha textgreater or = PhXA85 textgreater PGD2 textgreater PGE2. View Publication

We examined the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3],25-hydroxyvitamin D3 (25OHD3),and vitamin D3 on human keratinocyte proliferation and differentiation in a serum-free or defined culture system. Concentrations greater than 10(-8) M 1,25-(OH)2D3 or 10(-7) M 25(OH)2D3 caused marked inhibition of cell growth. Growth inhibition with high doses of 1,25-(OH)2D3 was not stringent,but was mainly exerted in the G1 phase of the cell cycle. Early release from the cell cycle block restored the proliferation of human keratinocytes. The calcium concentration in the medium had no significant effect on the antiproliferative action of 1,25-(OH)2D3,25OHD3,and vitamin D3. We also show that human keratinocyte proliferation is enhanced at doses of 1,25-(OH)2D3 and 25OH2D3 of 10(-9) M or less. Enhanced proliferation of human keratinocytes with physiological concentrations of 1,25-(OH)2D3 could only be shown in fully defined medium that contained no vitamin D3,related sterols,or bovine pituitary extract. Human keratinocyte differentiation was enhanced with higher doses of 1,25-(OH)2D3 when cells were grown in the presence of high calcium concentrations. These studies demonstrate that the lower,physiological concentrations of vitamin D3 metabolites are capable of stimulating the proliferation of epidermal keratinocytes grown under selected conditions that eliminate confounding or unidentified medium culture factors. Vitamin D3 metabolites are shown to exert mitogenic trophic effects in cultured human epithelial cells similar to their established activities in vivo. View Publication

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