技术资料

Adult-derived hippocampal progenitors generate neurons,astrocytes,and oligodendrocytes in vitro and following grafting into the adult brain. Although these progenitors have a considerable capacity for in vitro self renewal,it is not known if each lineage is generated by separate committed precursors or by multipotent stem cells. By genetic marking,we have followed individual cells through the process of proliferative expansion,commitment,and differentiation. All three lineages are generated by single marked cells and the relative proportions of each lineage can be strongly influenced by environmental cues. Differentiation is accompanied by a characteristic progression of lineage-specific markers and can be potentiated by retinoic acid,elevated cyclic AMP,or neurotrophic factors. The ability to genetically mark and clone normal diploid hippocampal progenitors provides the first definitive evidence that multipotent neural stem cells exist outside of the adult striatal subventricular zone and supports the hypothesis that FGF-2-responsive neural stem cells may be broadly distributed in the adult brain. View Publication

Ligand-dependent chimeric Cre recombinases are powerful tools to induce specific DNA rearrangements in cultured cells and in mice. We report here the construction and characterization of a series of chimeric recombinases,each consisting of Cre fused to a mutated human oestrogen receptor (ER) ligand-binding domain (LBD). Two new ligand-dependent recombinases which contain either the G400V/M543A/L544A or the G400V/L539A/L540A triple mutation of the human ER LBD are efficiently induced by the synthetic ER antagonists 4-hydroxytamoxifen (OHT) and ICI 182,780 (ICI),respectively,but are insensitive to 17 beta-oestradiol (E2). Both chimeric recombinases should be useful for efficient spatio-temporally controlled site-directed somatic mutagenesis. View Publication

Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. We have developed a producer cell line that yields high titers of amphotropic retroviral vectors carrying the enhanced green fluorescent protein (EGFP) gene,a codon humanized,red-shifted variant of the green fluorescent protein (GFP) gene,which can be used as a selectable marker. We have used a hybrid vector that has been shown to efficiently drive gene expression in hematopoietic cells. Virtually all murine and human cell lines and primary human hematopoietic cells tested were transduced with varying efficiency after incubation with vector-containing supernatants. Human CD34(+) cells obtained from cord blood or aphereses products were transduced using a protocol that involves daily addition of vector-containing supernatants for 6 consecutive days. At day 6,up to 16% of the cells expressed EGFP,as assessed by flow cytometry. Sorted EGFP-expressing cells were able to produce fluorescent hematopoietic colonies. EGFP's main advantages are its fast flow cytometry determination and the possibility of cell sorting and simultaneous evaluation of the transduction efficiency along with other phenotypic markers. View Publication

Mitomycin C,an important antitumor drug and antibiotic,has an extraordinary ability to crosslink DNA with high efficiency and absolute specificity for the sequence CpG. Recent results have shown how mitomycin C crosslinks DNA,and why the sequence specificity is so complete. This new understanding may allow the design of agents that mimic mitomycin C's economy of structure and can crosslink other sequences. View Publication

This is the first study to describe the association between expression of MUC1 and MUC2 mucins and prognosis in ovarian cancer. Paraffin sections of epithelial ovarian tumours (n = 182: 29 benign,21 low malignant potential,and 132 invasive tumours) were analysed immunohistochemically for expression of MUC1 and MUC2 mucin core proteins. Most benign,low malignant potential,and invasive tumours showed high MUC1 expression in the cytoplasm. Low cytoplasmic expression of MUC1 was a predictor for good prognosis,particularly within stage III tumours. A minority of benign epithelial tumours,but most low malignant potential and invasive non-mucinous tumours,showed high MUC1 expression on the cell membrane. High apical MUC1 reactivity was associated with non-mucinous tumours. Low expression of MUC1 in the apical membrane was associated with early stage and good outcome for invasive tumours. Most benign and low malignant potential tumours,but only a minority of invasive tumours,showed MUC2 expression. MUC2 was found in non-mucinous as well as in mucinous tumours. The presence of MUC2 was inversely associated with high tumour grade but was not associated with altered survival. These results support experimental evidence that MUC1 influences the metastatic ability of ovarian cancer. View Publication

The prototype of a new class of antiproliferative phospholipid analogs,hexadecylphosphocholine (HePC),has been shown to inhibit tumor growth and is currently used for the treatment of cutaneous metastases of mammary carcinomas. Although several cellular targets of HePC,e.g. protein kinase C and CTP:phosphocholine cytidylyltransferase,have been proposed,the mechanisms of HePC-induced anticancer activity are still unclear. Considering that the antiproliferative effect of HePC correlates with inhibition of phosphatidylcholine biosynthesis,which is tightly coupled to sphingomyelin biosynthesis,we tested the hypothesis that treatment of cells with the anticancer drug leads to increased cellular ceramide and subsequently to apoptotic cell death. In the present study,we showed that 25 micromol/liter HePC induced apoptosis. In further experiments,we demonstrated that HePC inhibited the incorporation of radiolabeled choline into phosphatidylcholine and at a later time point into sphingomyelin. This was confirmed by metabolic labeling of the lipid backbone using radiolabeled serine,and it was shown that HePC decreased the incorporation of serine into sphingomyelin by 35% and simultaneously increased the incorporation of serine into ceramide by 70%. Determination of the amount of ceramide revealed an increase of 53% in HePC-treated cells compared with controls. In accordance with the hypothesis that elevated ceramide levels may be the missing link between the metabolic effects of HePC and its proapoptotic properties,HePC-induced apoptosis was blocked by fumonisin B1,an inhibitor of ceramide synthesis. Furthermore,we found that membrane-permeable ceramides additively increased the apoptotic effect of HePC. View Publication

In the present study,we describe the cloning and sequence analysis of rat IL-3. Two different mRNA isoforms were isolated after transfection of COS cells with the cytokine genomic sequences. One of the isoforms has been predicted before by Cohen et al. (1986),and the other one is identical except that it encodes a protein with an insertion of three amino acids at position 56. As names for the two isoforms,we propose IL-3alpha for the predicted and IL-3beta for the novel molecule. IL-3beta mRNA was detected as the predominant isoform in rat lymphocytes in vivo. High levels of the cytokine were obtained after infection of human cells (A549) with a recombinant adenovirus harboring rIL-3beta cDNA (IG.Ad.CMV.IL-3beta). The biological properties of the IL-3beta protein were tested in a FDC-P1 proliferation assay and in a hematopoietic progenitor colony forming assay. To assess in-vivo bioactivity,lysed 293 cells containing IG.Ad.CMV.rIL-3beta virus were injected subcutaneously into F344 rats. Stimulation of hematopoiesis and leucocytosis were observed during the treatment. After subcutaneous injections of the lysed adeno-producer cells in mice,the only effect observed was a cellular infiltration at the site of injection,confirming the poor cross-reactivity between the two species. The biological properties in vitro and in vivo demonstrate that the cDNA sequences of IL-3beta presented here encode active rat IL-3 protein. View Publication

Three mitogen-activated protein kinase pathways are up-regulated during the activation of T lymphocytes,the extracellular signal-regulated kinase (ERK),Jun NH2-terminal kinase,and p38 mitogen-activated protein kinase pathways. To examine the effects of blocking the ERK pathway on T cell activation,we used the inhibitor U0126,which has been shown to specifically block mitogen-activated protein kinase/ERK kinase (MEK),the kinase upstream of ERK. This compound inhibited T cell proliferation in response to antigenic stimulation or cross-linked anti-CD3 plus anti-CD28 Abs,but had no effect on IL-2-induced proliferation. The block in T cell proliferation was mediated by down-regulating IL-2 mRNA levels. Blocking Ag-induced proliferation by inhibiting MEK did not induce anergy,unlike treatments that block entry into the cell cycle following antigenic stimulation. Surprisingly,induction of anergy in T cells exposed to TCR cross-linking in the absence of costimulation was also not affected by blocking MEK,unlike cyclosporin A treatment that blocks anergy induction. These results suggest that inhibition of MEK prevents T cell proliferation in the short term,but does not cause any long-term effects on either T cell activation or induction of anergy. These findings may help determine the viability of using mitogen-activated protein kinase inhibitors as immune suppressants. View Publication

The novel quinazoline derivative 4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154) exhibited significant cytotoxicity against U373 and U87 human glioblastoma cell lines,causing apoptotic cell death at micromolar concentrations. The in vitro antiglioblastoma activity of WHI-P154 was amplified textgreater 200-fold and rendered selective by conjugation to recombinant human epidermal growth factor (EGF). The EGF-P154 conjugate was able to bind to and enter target glioblastoma cells within 10-30 min via receptor (R)-mediated endocytosis by inducing internalization of the EGF-R molecules. In vitro treatment with EGF-P154 resulted in killing of glioblastoma cells at nanomolar concentrations with an IC50 of 813 +/- 139 nM,whereas no cytotoxicity against EGF-R-negative leukemia cells was observed,even at concentrations as high as 100 microM. The in vivo administration of EGF-P154 resulted in delayed tumor progression and improved tumor-free survival in a severe combined immunodeficient mouse glioblastoma xenograft model. Whereas none of the control mice remained alive tumor-free beyond 33 days (median tumor-free survival,19 days) and all control mice had tumors that rapidly progressed to reach an average size of textgreater 500 mm3 by 58 days,40% of mice treated for 10 consecutive days with 1 mg/kg/day EGF-P154 remained alive and free of detectable tumors for more than 58 days with a median tumor-free survival of 40 days. The tumors developing in the remaining 60% of the mice never reached a size textgreater 50 mm3. Thus,targeting WHI-P154 to the EGF-R may be useful in the treatment of glioblastoma multiforme. View Publication

A variety of cytokines mediate the activation of Janus protein tyrosine kinases (Jaks). The Jaks then phosphorylate cellular substrates,including members of the signal transducers and activators of transcription (Stat) family of transcription factors. Among the Stats,the two highly related proteins,Stat5a and Stat5b,are activated by a variety of cytokines. To assess the role of the Stat5 proteins,mutant mice were derived that have the genes deleted individually or together. The phenotypes of the mice demonstrate an essential,and often redundant,role for the two Stat5 proteins in a spectrum of physiological responses associated with growth hormone and prolactin. Conversely,the responses to a variety of cytokines that activate the Stat5 proteins,including erythropoietin,are largely unaffected. View Publication

The compound U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene) was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 was also shown to inhibit endogenous promoters containing AP-1 response elements but did not affect genes lacking an AP-1 response element in their promoters. These effects of U0126 result from direct inhibition of the mitogen-activated protein kinase kinase family members,MEK-1 and MEK-2. Inhibition is selective for MEK-1 and -2,as U0126 shows little,if any,effect on the kinase activities of protein kinase C,Abl,Raf,MEKK,ERK,JNK,MKK-3,MKK-4/SEK,MKK-6,Cdk2,or Cdk4. Comparative kinetic analysis of U0126 and the MEK inhibitor PD098059 (Dudley,D. T.,Pang,L.,Decker,S. J.,Bridges,A. J.,and Saltiel,A. R. (1995) Proc. Natl. Acad. Sci U. S. A. 92,7686-7689) demonstrates that U0126 and PD098059 are noncompetitive inhibitors with respect to both MEK substrates,ATP and ERK. We further demonstrate that the two compounds bind to deltaN3-S218E/S222D MEK in a mutually exclusive fashion,suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for deltaN3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly,we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the deltaN3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126,its selectivity for MEK over other kinases,and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction. 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