技术资料

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

The effect of 9-cis-retinoic acid (9-cis-RA) on the growth of eight gastric cancer cell lines was related to their transcription levels of mRNAs for retinoid receptors. Northern blot analysis showed that seven (TMK-1,MKN-1,-28,-45,-74,HSC-39,KATO-III) out of eight gastric cancer cell lines synthesized mRNAs for retinoic acid receptors (RARs) and retinoid X receptor-alpha (RXR-alpha). MKN-7 cells did not transcribe either RARs or RXR-alpha at the mRNA level although they appeared to have no alterations at the gene level. The growth of all of the cell lines except for MKN-7 cells was inhibited by 1 x 10(-6) M 9-cis-RA. Cell cycle distribution analysis revealed that G0-G1 arrest was not induced by exposure to 9-cis-RA in the sensitive TMK-1 and KATO-III cells or the resistant MKN-7 cells. Interestingly,9-cis-RA temporarily increased the amount of the cyclin dependent kinase (cdk) inhibitor,Waf1/Cip1/Sdi1/p21 protein,and also reduced the amount of cdk-7,epidermal growth factor receptor (EGFR) and cyclin D1 proteins,followed by reduction in phosphorylation of the product of the retinoblastoma tumor suppressor gene (Rb) in the sensitive TMK-1 cells,but not in the resistant MKN-7 cells. These results suggest that 9-cis-RA has a cytostatic effect on gastric cancer cells that synthesize the receptor molecules through cell cycle regulatory machinery. View Publication

Analysis of the mitogenic activity of interleukin-3 (IL-3),Steel factor (SF),and flt-3 ligand (FL) on acute myelogenous leukemia (AML) blasts using the short-term endpoints of proliferation in 3H-thymidine (3H-Tdr) incorporation assays or methylcellulose cultures (colony assays) showed that greater than 90% of samples contained cells that were responsive to one or more of these cytokines. With this information,culture conditions that were known to support normal long-term culture-initiating cells (LTC-IC) were tested,with or without supplements of one or more of these three growth factors,for their ability to support primitive progenitors from 10 cell samples from patients with AML. In all cases cytogenetically abnormal colony forming cells (CFC) were detected after 5 weeks when AML peripheral blood or marrow cells were cocultured on preestablished,normal human marrow feeders (HMF) and/or SI/SI mouse fibroblast feeders and the number of CFC detected in these 5-week-old LTC maintained a linear relationship to the number of input AML cells. Limiting dilution analysis,performed on 6 of the 10 samples,showed the frequency of AML cells initiating LTC (AML LTC-IC) to be 5- to 300-fold lower than the frequency of AML-CFC in the same cell sample,whereas the average number of CFC produced per LTC-IC varied from 1 to 13. Surprisingly,in each case the concentration of cytogenetically normal LTC-IC detected in AML patient blood was at least 10-fold higher than that previously observed in the blood of normal individuals. Mixed" mouse fibroblast feeders engineered to produce human G-CSF 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

Human hematopoiesis originates in a population of stem cells with transplantable lympho-myeloid reconstituting potential,but a method for quantitating such cells has not been available. We now describe a simple assay that meets this need. It is based on the ability of sublethally irradiated immunodeficient nonobese diabetic-scid/scid (NOD/SCID) mice to be engrafted by intravenously injected human hematopoietic cells and uses limiting dilution analysis to measure the frequency of human cells that produce both CD34(-)CD19(+) (B-lymphoid) and CD34(+) (myeloid) colony-forming cell progeny in the marrow of such recipients 6 to 8 weeks post-transplant. Human cord blood (CB) contains approximately 5 of these competitive repopulating units (CRU) per ml that have a similar distribution between the CD38(-) and CD38(+) subsets of CD34(+) CB cells as long-term culture-initiating cells (LTC-IC) (4:1 vs. 2:1). Incubation of purified CD34(+)CD38(-) human CB cells in serum-free medium containing flt-3 ligand,Steel factor,interleukin 3,interleukin 6,and granulocyte colony-stimulating factor for 5-8 days resulted in a 100-fold expansion of colony-forming cells,a 4-fold expansion of LTC-IC,and a 2-fold (but significant,P textless 0.02) increase in CRU. The culture-derived CRU,like the original CB CRU,generated pluripotent,erythroid,granulopoietic,megakaryopoietic,and pre-B cell progeny upon transplantation into NOD/SCID mice. These findings demonstrate an equivalent phenotypic heterogeneity amongst human CB cells detectable as CRU and LTC-IC. In addition,their similarly modest response to stimulation by a combination of cytokines that extensively amplify LTC-IC from normal adult marrow underscores the importance of ontogeny-dependent changes in human hematopoietic stem cell proliferation and self-renewal. View Publication

Retinoid induction of epidermal hyperplasia was investigated in hairless mice with synthetic ligands for the retinoic acid (RAR) and retinoid X (RXR) nuclear receptors. Induction of hyperplasia by all-trans retinoic acid and the RAR-specific retinoids TTNPB,tazarotene and AGN 190121 varied over a wide range (ED50 = 0.2-100 nmol/animal in three daily applications). Potency of induction was not directly correlated to receptor-binding affinity,but specificity of action could be demonstrated by inhibition with the high-affinity antagonist of the RARs,AGN 193109. Although RAR is functionally complexed with RXR in vivo,RXR-selective compounds have only weak potency in induction of hyperplasia. The ED50 value of the RXR-selective AGN 191701 was 600 nmol/animal compared with an ED50 value of 0.2 nmol for the structurally similar RAR-selective TTNPB. SR11237 and SR11217,also RXR-selective,each have an ED50 value of textgreater1000 nmol. Unlike RAR-specific retinoids,RXR-selective retinoids cause only very mild skin flaking at high doses. Relative potencies for cumulative topical irritation (flaking and abrasion) of both RAR and RXR ligands were well correlated with epidermal hyperplasia. These data are consistent with RXR as a silent partner in the RAR-RXR heterodimer in skin. View Publication

A variety of factors produced by stromal fibroblasts,including Flt3-ligand (FL),interleukin-11 (IL-11),Steel factor (SF),and IL-7,have been implicated in stimulating the production of pre-B cells and myeloid cells from primitive hematopoietic precursors. To investigate their relative roles in this process,either as single-acting or synergistic agents,we compared the yield and types of cells produced after 2 weeks from small numbers of Sca-1+ Lin- (i.e.,B220-,Ly-1-,Gr-1-,and Ter-119-) day 14.5 murine fetal liver cells placed in stromal cell-free cultures containing all possible combinations of FL,SF,IL-7,and IL-11. None of these factors alone supported the production (or survival) of any cells beyond 1 week: only pairs of factors consisting of either FL or SF plus either IL-11 or IL-7 were effective in this regard,with FL plus IL-11 being the most potent pair (approximately 7 x 10(4) cells obtained per 100 Sca-1+ Lin- input cells). The maximum numbers of cells were produced in the presence of FL,IL-11,and IL-7: these included both B220+ and Mac-1+/Gr-1+ cells (approximately 10(6) and approximately 2 x 10(5),respectively,per 100 Sca-1+ Lin- input cells). Both of these lineages were also obtained with each of the other possible three-factor combinations,albeit with variable effectiveness. Omission of either FL or IL-7 caused the greatest reduction in the yield of B220+ cells (approximately 130-fold and approximately 80-fold,respectively). Omission of IL-11 and,to a lesser extent,FL caused the greatest reduction in the yield of Mac-1+/Gr-1+ cells (approximately 90-fold and approximately 3-fold,respectively). When fetal calf serum was replaced with a defined serum substitute,the out put of B220+ cells remained the same but myelopoiesis was consistently enhanced (approximately 5- to 20-fold). These findings support a model involving factor redundancy in the extracellular signals required to stimulate the production and amplification of both lymphoid and myeloid cells from early Sca-1+ Lin- cells. They also reveal quantitative differences in the abilities of different competent factor combinations to promote this process,which may be further modulated by the presence of undefined serum components. View Publication

Ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications. Although current culture conditions have been optimized using quantitative in vitro progenitor assays,knowledge of the conditions that permit maintenance of primitive human repopulating cells is lacking. We report that primitive human cells capable of repopulating nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (SCID-repopulating cells; SRC) can be maintained and/or modestly increased after culture of CD34+CD38- cord blood cells in serum-free conditions. Quantitative analysis demonstrated a 4- and 10-fold increase in the number of CD34+CD38- cells and colony-forming cells,respectively,as well as a 2- to 4-fold increase in SRC after 4 d of culture. However,after 9 d of culture,all SRC were lost,despite further increases in total cells,CFC content,and CD34+ cells. These studies indicate that caution must be exercised in extending the duration of ex vivo cultures used for transplantation,and demonstrate the importance of the SRC assay in the development of culture conditions that support primitive cells. View Publication

Ceramide has emerged as an important lipid messenger for many cellular processes triggered via surface receptors. In the present study,inflammatory activation of P388D1 macrophages with bacterial lipopolysaccharide (LPS) and platelet-activating factor (PAF) stimulated a transient accumulation of ceramide. Moreover,cell-permeable ceramide mimicked LPS/PAF in triggering arachidonate mobilization in these cells. LPS/PAF-induced ceramide synthesis did not result from sphingomyelinase activation but from increased de novo synthesis. Participation of this pathway in arachidonate signaling was detected since fumonisin B1,an inhibitor of de novo ceramide synthesis,was able to inhibit the LPS/PAF-induced response. These studies have uncovered a new role for sphingolipid metabolism in cellular signaling and constitute evidence that products of the sphingomyelin biosynthetic pathway may serve a specific role in signal transduction by influencing the activity of the novel Group V secretory phospholipase A2. View Publication

Pluripotent embryonic stem (ES) cells spontaneously differentiate via embryo-like aggregates into cardiomyocytes of pacemaker-,atrium- and ventricle-like type,which can be distinguished by their specific patterns of action potentials. It has been shown that retinoic acid (RA) treatment during ES cell differentiation increases the number of cardiomyocytes in a time- and concentration-dependent manner. In order to test the effect of RA on cardiomyocyte differentiation and specialization into ventricle-like cardiomyocytes,we studied gene expression of beta-galactosidase driven by the ventricular myosin light chain-2 (MLC-2v) promoter as an indicator for ventricular differentiation. Clones containing the stably integrated expression vector pGNA/MLC-2.1 were selected,which revealed an increase of beta-galactosidase activity in cardiomyocytes of embryoid bodies at day 7 + 16. RA,both,in the all-trans and in the 9-cis configuration resulted in a significant acceleration of cardiomyocyte differentiation and a transient increase of beta-galactosidase activity. To test whether this acceleration of cardiac differentiation and RA-induced increase of the MLC-2v promotor/beta-galactosidase activity reflects an increase of cardiac- and ventricle-specific gene expression,a semi-quantitative RT-PCR analysis was performed for alpha-cardiac myosin heavy chain (alpha-MHC) and MLC-2v genes. It was shown that both 10(-8) M and 10(-9) M RA resulted in an increased level of alpha-cardiac MHC and MLC-2v mRNA in embryoid bodies in early,but not in terminal developmental stages. This led us to the conclusion that the RA-induced accelerated expression of cardiac-specific genes results in an enhanced development of ventricular cardiomyocytes. An increased number of ventricle-like cells after RA treatment was also found by patch-clamp analysis. The number of cardiomyocytes with Purkinje- and ventricle-like properties was shown to be increased by RA,whereas the number of pacemaker- and atrium-like cells was reduced and early pacemaker cells were not quantitatively affected. View Publication

A single entity,the AMP-activated protein kinase (AMPK),phosphorylates and regulates in vivo hydroxymethylglutaryl-CoA reductase and acetyl-CoA carboxylase (key regulatory enzymes of sterol synthesis and fatty acid synthesis,respectively),and probably many additional targets. The kinase is activated by high AMP and low ATP via a complex mechanism,which involves allosteric regulation,promotion of phosphorylation by an upstream protein kinase (AMPK kinase),and inhibition of dephosphorylation. This protein-kinase cascade represents a sensitive system,which is activated by cellular stresses that deplete ATP,and thus acts like a cellular fuel gauge. Our central hypothesis is that,when it detects a 'low-fuel' situation,it protects the cell by switching off ATP-consuming pathways (e.g. fatty acid synthesis and sterol synthesis) and switching on alternative pathways for ATP generation (e.g. fatty acid oxidation). Native AMP-activated protein kinase is a heterotrimer consisting of a catalytic alpha subunit,and beta and gamma subunits,which are also essential for activity. All three subunits have homologues in budding yeast,which are components of the SNF1 protein-kinase complex. SNF1 is activated by glucose starvation (which in yeast leads to ATP depletion) and genetic studies have shown that it is involved in derepression of glucose-repressed genes. This raises the intriguing possibility that AMPK may regulate gene expression in mammals. AMPK/SNF1 homologues are found in higher plants,and this protein-kinase cascade appears to be an ancient system which evolved to protect cells against the effects of nutritional or environmental stress. View Publication

Embryonic stem cells,derived from the inner cell mass of murine blastocysts,can be maintained in a totipotent state in vitro. In appropriate conditions embryonic stem cells have been shown to differentiate in vitro into various derivatives of all three primary germ layers. We describe in this paper conditions to induce differentiation of embryonic stem cells reliably and at high efficiency into adipocytes. A prerequisite is to treat early developing embryonic stem cell-derived embryoid bodies with retinoic acid for a precise period of time. Retinoic acid could not be substituted by adipogenic hormones nor by potent activators of peroxisome proliferator-activated receptors. Treatment with retinoic acid resulted in the subsequent appearance of large clusters of mature adipocytes in embryoid body outgrowths. Lipogenic and lipolytic activities as well as high level expression of adipocyte specific genes could be detected in these cultures. Analysis of expression of potential adipogenic genes,such as peroxisome proliferator-activated receptors gamma and delta and CCAAT/enhancer binding protein beta,during differentiation of retinoic acid-treated embryoid bodies has been performed. The temporal pattern of expression of genes encoding these nuclear factors resembled that found during mouse embryogenesis. The differentiation of embryonic stem cells into adipocytes will provide an invaluable model for the characterisation of the role of genes expressed during the adipocyte development programme and for the identification of new adipogenic regulatory genes. View Publication