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The immunosuppressant,rapamycin,inhibits cell growth by interfering with the function of a novel kinase,termed mammalian target of rapamycin (mTOR). The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases. This study demonstrates that mTOR is a component of a cytokine-triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor-4E (eIF-4E) binding protein,PHAS-1,in activated T lymphocytes. This event promotes G1 phase progression by stimulating eIF-4E-dependent translation initiation. A mutant YAC-1 T lymphoma cell line,which was selected for resistance to the growth-inhibitory action of rapamycin,was correspondingly resistant to the suppressive effect of this drug on PHAS-1 phosphorylation. In contrast,the PI 3-kinase inhibitor,wortmannin,reduced the phosphorylation of PHAS-1 in both rapamycin-sensitive and -resistant T cells. At similar drug concentrations (0.1-1 microM),wortmannin irreversibly inhibited the serine-specific autokinase activity of mTOR. The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3-kinase inhibitor,LY294002,at concentrations (1-30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85-p110 heterodimer. These studies indicate that the signaling functions of mTOR,and potentially those of other high molecular weight PI 3-kinase homologs,are directly affected by cellular treatment with wortmannin or LY294002. View Publication

Natural and synthetic retinoids have proved to be effective in the treatment and prevention of various human cancers. In the present study,we investigated the effect of retinoids on Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines (LCLs),since these cells closely resemble those that give rise to EBV-related lymphoproliferative disorders in the immunosuppressed host. All six compounds tested inhibited LCL proliferation with no significant direct cytotoxicity,but 9-cis-retinoic acid (RA),13-cis-RA,and all-trans-RA (ATRA) were markedly more efficacious than Ro40-8757,Ro13-6298,and etretinate. The antiproliferative action of the three most effective compounds was confirmed in a large panel of LCLs,thus appearing as a generalized phenomenon in these cells. LCL growth was irreversibly inhibited even after 2 days of treatment at drug concentrations corresponding to therapeutically achievable plasma levels. Retinoid-treated cells showed a marked downregulation of CD71 and a decreased S-phase compartment with a parallel accumulation in Gzero/ G1 phases. These cell cycle perturbations were associated with the upregulation of p27 Kip1,a nuclear protein that controls entrance and progression through the cell cycle by inhibiting several cyclin/cyclin-dependent kinase complexes. Unlike what is observed in other systems,the antiproliferative effect exerted by retinoids on LCLs was not due to the acquisition of a terminally differentiated status. In fact,retinoid-induced modifications of cell morphology,phenotype (downregulation of CD19,HLA-DR,and s-Ig,and increased expression of CD38 and c-Ig),and IgM production were late events,highly heterogeneous,and often slightly relevant,being therefore only partially indicative of a drug-related differentiative process. Moreover,EBV-encoded EBV nuclear antigen-2 and latent membrane protein-1 proteins were inconstantly downregulated by retinoids,indicating that their growth-inhibitory effect is not mediated by a direct modulation of viral latent antigen expression. The strong antiproliferative activity exerted by retinoids in our experimental model indicates that these compounds may represent a useful tool in the medical management of EBV-related lymphoproliferative disorders of immunosuppressed patients. View Publication

beta 1 Integrins are known to regulate terminal differentiation and morphogenesis in the adult epidermis. We have investigated their role in the embryonic development of keratinocytes by comparing the differentiation of wild-type and beta 1-null mouse embryonal stem (ES) cells. By 12-15 days in culture,differentiation of embryonic or simple epithelial cells occurred in both ES cell populations,as detected by expression of keratins 8,18,and 19. From 21 days,expression of keratins 10 and 14 and of the cornified envelope precursor involucrin indicated that some of the wild-type cells had differentiated into keratinocytes. In contrast,keratinocyte markers were not expressed in beta 1-null cultures. The beta 1-null cells failed to express the alpha 2 and alpha 3 integrin subunits on the cell surface,consistent with the association of these a subunits with beta 1. Furthermore,alpha 6 and beta 4 expression was reduced in the beta 1-null cultures. Although beta 1-null ES cells failed to undergo differentiation into keratinocytes in vitro,they did form keratinocyte cysts expressing alpha 6 beta 4,keratins 1 and 14,and involucrin when allowed to form teratomas by subcutaneous injection in mice; furthermore,beta 1-null keratinocytes were found in the epidermis of a wild-type/beta 1-null chimeric mouse. As judged by immunofluorescence microscopy,extracellular matrix assembly was severely impaired in beta 1-null ES cell cultures,but not in the teratomas or chimeric mouse skin. We therefore speculate that the failure of beta 1-null cells to differentiate into keratinocytes in vitro may reflect an inability to assemble a basement membrane. View Publication

The inhibitory and bactericidal activities of anacardic acid and totarol,alone and in combination with methicillin,were investigated against methicillin-resistant Staphylococcus aureus (MRSA). The growth of two MRSA strains was inhibited by 6 x 25 microg ml-1 of anacardic acid and 0 x 78 microg ml-1 of totarol. The time-kill curve study showed that these two compounds were bactericidal against MRSA. Anacardic acid killed MRSA cells more rapidly than totarol,and no viable cells were detected after being exposed to 6 x 25 microg ml-1 of anacardic acid for 6 h. Anacardic acid showed bactericidal activity against MRSA at any stage of growth,and also even when cell division was inhibited by chloramphenicol. In the combination studies,the minimal inhibitory concentration (MIC) of methicillin was lowered from 800 to 1 x 56 microg ml-1 for MRSA ATCC 33591,and from 800 to 6 x 25 microg ml-1 for MRSA ATCC 33592,by combining with 1/2 x MIC of anacardic acid. The time-kill curves demonstrated synergistic bactericidal activities for these combinations. View Publication

Elevated numbers of primitive Philadelphia chromosome-positive (Ph+) progenitors,including long-term culture-initiating cells (LTC-IC) as well as colony-forming cells (CFC),have been previously described in the blood of patients with chronic myeloid leukemia (CML) in chronic phase with high white blood cell counts. In the present study,which focused primarily on an analysis of circulating progenitors present in such patients at diagnosis,we discovered the frequent and occasionally exclusive presence of circulating normal (Ph-) LTC-IC,often at levels above those seen for LTC-IC in the blood of normal individuals. The presence of detectable numbers of circulating Ph- LTC-IC was independent of the fact that the same peripheral blood samples also contained elevated numbers of predominantly or exclusively Ph+ CFC. Interestingly,both the Ph+ and Ph- LTC-IC in these samples were CD34+CD71- and variably CD38- and Thy-1+,as previously documented for LTC-IC in normal marrow. Thus,neither CD38 nor Thy-1 expression was useful for discriminating between Ph+ and Ph- LTC-IC in mixed populations. Nevertheless,an association of these phenotypes with LTC-IC function did allow highly enriched (textgreater 5% pure) suspensions of either Ph+ or Ph- LTC-IC to be obtained from selected samples of CML blood in which the initial LTC-IC population was either predominantly Ph+ or Ph-,respectively. These findings suggest that the mechanisms causing mobilization of leukemic stem cells in untreated CML patients may affect their normal counterparts. They also indicate a possible new source of autologous cells for the support of intensive therapy of CML patients. Finally,they provide a method for obtaining the most highly purified populations of Ph+ LTC-IC described to date. This method should be useful for further analyses of the molecular activities of these very primitive neoplastic cells. View Publication

1. We have investigated the inhibitory effects of RP 73401 (piclamilast) and rolipram against human monocyte cyclic AMP-specific phosphodiesterase (PDE4) in relation to their effects on prostaglandin (PG)E2-induced cyclic AMP accumulation and lipopolysaccharide (LPS)-induced TNF alpha production and TNF alpha mRNA expression. 2. PDE4 was found to be the predominant PDE isoenzyme in the cytosolic fraction of human monocytes. Cyclic GMP-inhibited PDE (PDE3) was also detected in the cytosolic and particulate fractions. Reverse transcription polymerase chain reaction (RT-PCR) of human monocyte poly (A+) mRNA revealed amplified products corresponding to PDE4 subtypes A and B of which the former was most highly expressed. A faint band corresponding in size to PDE4D was also observed. 3. RP 73401 was a potent inhibitor of cytosolic PDE4 (IC50: 1.5 +/- 0.6 nM,n = 3). (+/-)-Rolipram (IC50: 313 +/- 6.7 nM,n = 3) was at least 200 fold less potent than RP 73401. R-(-)-rolipram was approximately 3 fold more potent than S-(+)-rolipram against cytosolic PDE4. 4. RP 73401 (IC50: 9.2 +/- 2.1 nM,n = 6) was over 50 fold more potent than (+/-)-rolipram (IC50: 503 +/- 134 nM,n = 6) ) in potentiating PGE2-induced cyclic AMP accumulation. R-(-)-rolipram (IC50: 289 +/- 121 nM,n = 5) was 4.7 fold more potent than its S-(+)-enantiomer (IC50: 1356 +/- 314 nM,n = 5). A strong and highly-significant,linear correlation (r = 0.95,P textless 0.01,n = 13) was observed between the inhibitory potencies of a range of structurally distinct PDE4 inhibitors against monocyte PDE4 and their ED50 values in enhancing monocyte cyclic AMP accumulation. A poorer,though still significant,linear correlation (r = 0.67,P textless 0.01,n = 13) was observed between the potencies of the same compounds in potentiating PGE2-induced monocyte cyclic AMP accumulation and their abilities to displace [3H]-rolipram binding to brain membranes. 5. RP 73401 (IC50: 6.9 +/- 3.3 nM,n = 5) was 71 fold more potent than (+/-)-rolipram (IC50: 490 +/- 260 nM,n = 4) in inhibiting LPS-induced TNF alpha release from monocytes. R-(-)-rolipram (IC50: 397 +/- 178 nM,n = 3) was 5.2-fold more potent than its S-(+)- enantiomer (IC50: 2067 +/- 659 nM,n = 3). As with cyclic AMP,accumulation a closer,linear correlation existed between the potency of structurally distinct compounds in suppressing TNF alpha with PDE4 inhibition (r = 0.93,P textless 0.01,n = 13) than with displacement of [3H]-rolipram binding (r = 0.65,P textless 0.01,n = 13). 6. RP 73401 (IC50: 2 nM) was 180 fold more potent than rolipram (IC50: 360 nM) in suppressing LPS (10 ng ml-1)-induced TNF alpha mRNA. 7. The results demonstrate that RP 73401 is a very potent inhibitor of TNF alpha release from human monocytes suggesting that it may have therapeutic potential in the many pathological conditions associated with over-production of this pro-inflammatory cytokine. Furthermore,PDE inhibitor actions on functional responses are better correlated with inhibition of PDE4 catalytic activity than displacement of [3H]-rolipram from its high-affinity binding site,suggesting that the native PDE4 in human monocytes exists predominantly in a 'low-affinity' state. View Publication

We used reverse transcription-polymerase chain reaction (RT-PCR) to clone a rat complementary DNA that encoded the PVG rat granulocyte-macrophage colony-stimulating factor (GM-CSF). PCR products were cloned into a eukaryotic expression vector and transfected into the mouse myeloma cell line Sp2/0-Ag14. Cell culture supernatants of two of these transfectants supported proliferation of the growth factor-dependent cell line,DA-3,and promoted myeloid colony formation in rat and mouse bone marrow cell (BMC) cultures. The GM-CSF activity in these supernatants was neutralized by a polyclonal antibody to mouse GM-CSF. The cloning and expression of rat GM-CSF provides a valuable reagent for the study of the biology and clinical applications of the GM-CSFs. View Publication

An in vivo transplantation system has been used to evaluate the developmental capacities of specific mouse mammary epithelial cell populations. Specifically,mouse mammary epithelial cells with distinctly limited developmental potentials have been identified using this procedure. Two distinct epithelial cell progenitors have been identified by experiments designed to determine whether basal lobular and ductal phenotypes could develop independently under conditions imposed by a limiting dilution. The prediction that these separate epithelial progenitors must exist was based upon the results from transplantation experiments carried out in epithelium-divested mammary fat pads of syngeneic mice with mammary epithelium from two different transgenic mouse models. The results presented here demonstrate the following points: 1) lobular,i.e. secretory,progenitor cells are present as distinct entities among the mammary epithelial cells found in immature virgin female mice; 2) similarly,ductal epithelial progenitors are present within the same population; 3) lobular progenitors are present in greater numbers,although both cell populations are extremely small; 4) as expected,some inocula produce outgrowths with simultaneous development of both lobular and ductal phenotypes--it is not known whether this indicates cooperative interaction between the two epithelial progenitors or signals the presence of a third progenitor type capable of producing both ductular and lobular committed daughters; 5) these findings have important consequences in the design of experiments aimed at testing the effects of known and putative mammary oncogenes and tumor suppressor genes,using techniques which include cellular transformation in vitro followed by in vivo cultivation and evaluation. View Publication

The PDE2,cyclic GMP-stimulated,and the PDE4,cyclic AMP-specific enzymes provide the major,detectable cyclic AMP phosphodiesterase activities in murine thymocytes. In the absence of the cyclic GMP,PDE4 activity predominated (approximately 80% total) but in the presence of low (10 microM) cyclic GMP concentrations,PDE2 activity constituted the major PDE activity in thymocytes (approximately 80% total). The PDE4 selective inhibitor rolipram dose-dependently inhibited thymocyte PDE4 activity (IC50 approximately 65 nM). PDE2 was dose-dependently activated (EC50 approximately 1 microM) by cyclic GMP and inhibited by erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) (IC50 approximately 4 microM). EHNA was shown to serve as a selective inhibitor of PDE-2 activity as assessed from studies using separated PDE1,PDE2,PDE3 and PDE4 species from hepatocytes as well as human PDE2 and PDE4 enzymes. EHNA completely ablated the ability of cyclic GMP to activate PDE2 activity,whilst having a much smaller inhibitory effect on the unstimulated PDE2 activity. EHNA exhibited normal Michaelian kinetics of inhibition for the cyclic GMP-stimulated PDE2 activity with Hill plots near unity. Apparent negative co-operative effect were seen in the absence of cyclic GMP with Hill coefficients of approximately 0.3 for inhibition of PDE2 activity. Within 5 min of challenge of thymocytes with the lectin phytohaemagglutinin (PHA) there was a transient decrease (approximately 83%) in PDE-4 activity and in PDE2 activity (approximately 40%). Both anti-TCR antibodies also caused an initial reduction in the PDE4 activity which was followed by a sustained and profound increase in activity. In contrast to that observed with PHA,anti-TCR/CD3 antisera had little effect on PDE2 activity. It is suggested that,dependent upon the intracellular concentrations of cyclic GMP,thymocyte cyclic AMP metabolism can be expected to switch from being under the predominant control of PDE4 activity to that determined predominantly by PDE2 activity. These activities may be rapidly and differentially regulated following ligation of different cell surface receptors. View Publication

Three series of analogs were regioselectively prepared from a protected forskolin precursor to afford 7-carbamoyl-7-desacetylforskolins (series 1),6-carbamoyl-7-desacetylforskolins (series 2),and 6-carbamoylforskolins (series 3). The analogs were pharmacologically evaluated for binding (IC50) to and activation (EC50) of type I adenylyl cyclase in membranes from stably transfected Sf9 cell lines expressing a single adenylate cyclase subtype. The following ranges were determined for the IC50's and EC50's of each individual series: series 1,IC50 = 43-1600 nM,EC50 = 0.5-9.6 microM; series 2,IC50 = 65-680 nM,EC50 = 0.63-6.5 microM; series 3,IC50 = 21-271 nM,EC50 = 0.5-8.1 microM (forskolin IC50 = 41 nM and EC50 = 0.5 microM). Activation paralleled binding; however,some analogs exhibited poor binding and good activation whereas others demonstrated good binding but poor activation. Steric bulk tended to diminish binding and activation when at the 6- or 7-position,although bulk was accommodated at the 6-position if the 7-site was reacetylated. Acylation of the 7-position by the carbamoyl linker or acetyl was important for obtaining good binding and activation; however,the effect was more pronounced with binding. For both binding and activation,small,linear,lipophilic substituents (propyl,allyl,isopropyl) are well tolerated at the 7-position but less so in the 6-position,even when the 7-site is reacetylated. Planar aromatic moieties (phenyl and 2-pyridinyl) demonstrated moderate to good potency for binding and activation when located at either the 6- or 7-positions. There is an overall trend toward increasing potency for both binding and activation with polar substituents. View Publication

Retinoids exert pleiotropic effects on the development of vertebrates through the action of retinoic acid receptors (RAR) and retinoid X receptors (RXR). We have investigated the effect of synthetic retinoids selective for RXR and RAR on the development of Xenopus and zebrafish embryos. In Xenopus,both ligands selective for RAR and RXR caused striking malformations along the anterior-posterior axis,whereas in zebrafish only ligands specific for RAR caused embryonic malformations. In Xenopus,RAR- and RXR-selective ligands regulated the expression of the Xlim-1,gsc,and HoxA1 genes similarly as all-trans-retinoic acid. Nevertheless,RXR-selective ligands activated only an RXR responsive reporter but not an RAR responsive reporter introduced by microinjection into the Xenopus embryo,consistent with our failure to detect conversion of an RXR-selective ligand to different derivatives in the embryo. These results suggest that Xenopus embryos possess a unique response pathway in which liganded RXR can control gene expression. Our observations further illustrate the divergence in retinoid responsiveness between different vertebrate species. View Publication

A high proportion of the CD34+CD38- cells in normal human marrow are defined as long-term culture-initiating cells (LTC-IC) because they can proliferate and differentiate when co-cultured with cytokine-producing stromal feeder layers. In contrast,very few CD34+CD38- cells will divide in cytokine-containing methylcellulose and thus are not classifiable as direct colony-forming cells (CFC),although most can proliferate in serum-free liquid cultures containing certain soluble cytokines. Analysis of the effects of 16 cytokines on CD34+CD38- cells in the latter type of culture showed that Flt3-ligand (FL),Steel factor (SF),and interleukin (IL)-3 were both necessary and sufficient to obtain an approximately 30-fold amplification of the input LTC-IC population within 10 d. As single factors,only FL and thrombopoietin (TPO) stimulated a net increase in LTC-IC within 10 d. Interestingly,a significantly increased proportion of the CFC produced from the TPO-amplified LTC-IC were erythroid. Increases in the number of directly detectable CFC of textgreater 500-fold were also obtainable within 10 d in serum-free cultures of CD34+CD38- cells. However,this required the presence of IL-6 and/or granulocyte/colony-stimulating factor and/or nerve growth factor beta in addition to FL,SF,and IL-3. Also,for this response,the most potent single-acting factor tested was IL-3,not FL. Identification of cytokine combinations that differentially stimulate primitive human hematopoietic cell self-renewal and lineage determination should facilitate analysis of the intracellular pathways that regulate these decisions as well as the development of improved ex vivo expansion and gene transfer protocols. View Publication