技术资料

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

Butein,a plant polyphenol,was shown to be a specific protein tyrosine kinase inhibitor. This compound inhibited not only the epidermal growth factor (EGF)-stimulated auto-phosphotyrosine level of EGF receptor in HepG2 cells but also tyrosine-specific protein kinase activities of EGF receptor (IC50 = 65 microM) and p60c-src (IC50 = 65 microM) in vitro. The inhibition was competitive to ATP and non-competitive to the phosphate acceptor,poly (Glu,Ala,Tyr) 6:3:1 for EGF receptor tyrosine kinase. In contrast,butein non-significantly inhibited the activities of serine- and threonine-specific protein kinases,such as protein kinase C (PKC) and cAMP-dependent protein kinase (PKA). View Publication

Recent studies have shown efficient gene transfer to primitive progenitors in human cord blood (CB) when the cells are incubated in retrovirus-containing supernatants on fibronectin-coated dishes. We have now used this approach to achieve efficient gene transfer to human CB cells with the capacity to regenerate lymphoid and myeloid progeny in nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. CD34(+) cell-enriched populations were first cultured for 3 days in serum-free medium containing interleukin-3 (IL-3),IL-6,granulocyte colony-stimulating factor,Flt3-ligand,and Steel factor followed by two 24-hour incubations with a MSCV-NEO virus-containing medium obtained under either serum-free or serum-replete conditions. The presence of serum during the latter 2 days made no consistent difference to the total number of cells,colony-forming cells (CFC),or long-term culture-initiating cells (LTC-IC) recovered at the end of the 5-day culture period,and the cells infected under either condition regenerated similar numbers of human CD34(+) (myeloid) CFC and human CD19(+) (B lymphoid) cells for up to 20 weeks in NOD/SCID recipients. However,the presence of serum increased the viral titer in the producer cell-conditioned medium and this was correlated with a twofold to threefold higher efficiency of gene transfer to all progenitor types. With the higher titer viral supernatant,17% +/- 3% and 17% +/- 8%,G418-resistant in vivo repopulating cells and LTC-IC were obtained. As expected,the proportion of NEO + repopulating cells determined by polymerase chain reaction analysis of in vivo generated CFC was even higher (32% +/- 10%). There was no correlation between the frequency of gene transfer to LTC-IC and colony-forming unit-granulocyte-macrophage (CFU-GM),or to NOD/SCID repopulating cells and CFU-GM (r2 = 0.16 and 0.17,respectively),whereas values for LTC-IC and NOD/SCID repopulating cells were highly and significantly correlated (r2 = 0.85). These findings provide further evidence of a close relationship between human LTC-IC and NOD/SCID repopulating cells (assessed using a textgreater/= 6-week CFC output endpoint) and indicate the predictive value of gene transfer measurements to such LTC-IC for the design of clinical gene therapy protocols. 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

Any epithelial portion of a normal mouse mammary gland can reproduce an entire functional gland when transplanted into an epithelium-free mammary fat pad. Mouse mammary hyperplasias and tumors are clonal dominant populations and probably represent the progeny of a single transformed cell. Our study provides evidence that single multipotent stem cells positioned throughout the mature fully developed mammary gland have the capacity to produce sufficient differentiated progeny to recapitulate an entire functional gland. Our evidence also demonstrates that these stem cells are self-renewing and are found with undiminished capacities in the newly regenerated gland. We have taken advantage of an experimental model where mouse mammary tumor virus infects mammary epithelial cells and inserts a deoxyribonucleic acid copy(ies) of its genome during replication. The insertions occur randomly within the somatic genome. CzechII mice have no endogenous nucleic acid sequence homology with mouse mammary tumor virus; therefore all viral insertions may be detected by Southern analysis provided a sufficient number of cells contain a specific insertional event. Transplantation of random fragments of infected CzechII mammary gland produced clonal-dominant epithelial populations in epithelium-free mammary fat pads. Serial transplantation of pieces of the clonally derived outgrowths produced second generation glands possessing the same viral insertion sites providing evidence for self-renewal of the original stem cell. Limiting dilution studies with cell cultures derived from third generation clonal outgrowths demonstrated that three multipotent but distinct mammary epithelial progenitors were present in clonally derived mammary epithelial populations. Estimation of the potential number of multipotent epithelial cells that may be evolved from an individual mammary-specific stem cell by self-renewal is in the order of 10(12)-10(13). Therefore,one stem cell might easily account for the renewal of mammary epithelium over several transplant generations. View Publication

A series of N-heteroaryl hydrazones derived from aryl N-heteroaryl or bis-N-heteroaryl methanones was prepared in search for potential novel antitumor agents. The stereochemistry of these compounds was established by means of NMR spectroscopy. Antiproliferative activity was determined in a panel of human tumor cell lines (CCRF-CEM,Burkitt's lymphoma,HeLa,ZR-75-1,HT-29,and MEXF 276L) in vitro. Generally,the new compounds were found to be more potent (IC50 = 0.011-0.436 microM) than the ribonucleotide reductase inhibitor hydroxyurea (IC50 = 140 microM). Most of the compounds exhibited the highest activity against Burkitt's lymphoma with an IC50 of 0.011-0.035 microM. [14C]Cytidine incorporation into DNA was quantitated for selected hydrazones (Z-A,E-1,Z-3,Z-4,E-5,Z-5,E-13,E-18,Z-19,Z-24,and E-26) as a measure of the inhibition of ribonucleotide reductase in Burkitt's lymphoma cells. The E-configurated compounds were found to inhibit [14C]cytidine incorporation to a greater extent (IC50 = 0.67-5.05 microM) than the Z-isomers (IC50 = 7.20 to textgreater 10 microM). Principal component analysis of the IC50 values obtained for inhibition of cell proliferation revealed that the cell lines tested can be grouped into three main families showing different sensitivities toward the compounds in our series [(i) CCRF-CEM,Burkitt's lymphoma,and Hela; (ii) HT-29; and (iii) MEXF 276 L]. View Publication

In human HL-60 promyelocytic leukemia cells,diazepinylbenzoic acid derivatives can exhibit either antagonistic or synergistic effects on the differentiation-inducing activities of natural or synthetic retinoids,the activity depending largely on the nature of the substituents on the diazepine ring. Thus,a benzolog of the retinoid antagonist LE135 (6),4-(13H-10,11,12,13-tetrahydro-10,10,13,13,15-pentamethyldinaphtho[2,3-b][1,2-e]diazepin-7-yl) benzoic acid (LE540,17),exhibits a 1 order of magnitude higher antagonistic potential than the parental LE135 (6). In contrast,4-[5H-2,3-(2,5-dimethyl-2,5-hexano)-5-methyldibenzo[b,e] [1,4]diazepin-11-yl]-benzoic acid (HX600,7),a structural isomer of the antagonistic LE135 (6),enhanced HL-60 cell differentiation induced by RAR agonists,such as Am80 (2). This synergistic effect was further increased for a thiazepine,HX630 (29),and an azepine derivative,HX640 (30); both synergized with Am80 (2) more potently than HX600 (7). Notably,the negative and positive effects of the azepine derivatives on retinoidal actions can be related to their RAR-antagonistic and RXR-agonistic properties,respectively,in the context of the RAR-RXR heterodimer. View Publication

Elucidation of mechanisms that regulate hematopoietic stem cell self-renewal and differentiation would be facilitated by the identification of defined culture conditions that allow these cells to be amplified. We now demonstrate a significant net increase (3-fold,P textless 0.001) in vitro of cells that are individually able to permanently and competitively reconstitute the lymphoid and myeloid systems of syngeneic recipient mice when Sca-1(+)lin- adult marrow cells are incubated for 10 days in serum-free medium with interleukin 11,flt3-ligand,and Steel factor. Moreover,the culture-derived repopulating cells continued to expand their numbers in the primary hosts at the same rate seen in recipients of noncultured stem cells. In the expansion cultures,long-term culture-initiating cells increased 7- +/- 2-fold,myeloid colony-forming cells increased 140- +/- 36-fold,and total nucleated cells increased 230- +/- 62-fold. Twenty-seven of 100 cultures initiated with 15 Sca-1(+)lin- marrow cells were found to contain transplantable stem cells 10 days later. This frequency of positive cultures is the same as the frequency of transplantable stem cells in the original input suspension,suggesting that most had undergone at least one self-renewal division in vitro. No expansion of stem cells was seen when Sca-1+TER119- CD34+ day 14.5 fetal liver cells were cultured under the same conditions. These findings set the stage for further investigations of the mechanisms by which cytokine stimulation may elicit different outcomes in mitotically activated hematopoietic stem cells during ontogeny and in the adult. View Publication

CGP 57148 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the ABL and the platelet-derived growth factor receptor (PDGFR) protein tyrosine kinases. We previously showed that CGP 57148 selectively kills p210BCR-ABL-expressing cells. To extend these observations,we evaluated the ability of CGP 57148 to inhibit other activated ABL tyrosine kinases,including p185BCR-ABL and TEL-ABL. In cell-based assays of ABL tyrosine phosphorylation,inhibition of ABL kinase activity was observed at concentrations similar to that reported for p210BCR-ABL. Consistent with the in vitro profile of this compound,the growth of cells expressing activated ABL protein tyrosine kinases was inhibited in the absence of exogenous growth factor. Growth inhibition was also observed with a p185BCR-ABL-positive acute lymphocytic leukemia (ALL) cell line generated from a Philadelphia chromosome-positive ALL patient. As CGP 57148 inhibits the PDGFR kinase,we also showed that cells expressing an activated PDGFR tyrosine kinase,TEL-PDGFR,are sensitive to this compound. Thus,this compound may be useful for the treatment of a variety of BCR-ABL-positive leukemias and for treatment of the subset of chronic myelomonocytic leukemia patients with a TEL-PDGFR fusion protein. 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

Peripheral blood progenitor cells (PBPCs) are increasingly used instead of bone marrow for autologous or allogeneic transplantation. In this study PBPCs mobilized in cancer patients by chemotherapy and granulocyte-colony stimulating factor were collected by apheresis and first enriched by immunoaffinity removal of lineage positive cells. When these cells were exposed to both cyclophosphamide and taxol or cultured for 7 days in the presence of 5-fluorouracil,stem cell factor,and interleukin-3,88% to 93% of the enriched PBPCs were killed and short-term clonogenic capacity in methylcellulose assays was lost,but week-5 cobblestone area-forming cell (CAFC) enrichment was higher than 10-fold in comparison to enriched PBPCs and higher than 700-fold in comparison to unmanipulated apheresis cells. After drug exposure,most of the progenitors displayed a CD34+,CD38-,multidrug-resistance (MDR+),Rhodamine 123 low,Hoechst 33342 low phenotype,and as few as 180 of these drug-resistant cells were able to generate a stable multilineage human hematopoiesis in sublethally irradiated immunodeficient mice. In these animals,the level of human hematopoietic engraftment was significantly increased by cotransplantation of irradiated cells from the human L87/4 stromal cell line. These observations are consistent with the functional isolation of a population of very early hematopoietic progenitors and might help to design new protocols for the removal of neoplastic cells from autografts. View Publication

We compared glucocorticoid receptor binding characteristics and glucocorticoid responsiveness of human mononuclear leukocytes (HML) from hypertensive patients and matched normotensive volunteers. We also considered associations of these variables with plasma renin activity,aldosterone,cortisol,corticotropin,and electrolyte concentrations. We calculated binding affinity (Kd; nmol/L) and capacity (Bmax; sites/cell) for dexamethasone and cortisol from homologous and heterologous competition curves for specific [3H]dexamethasone binding sites on HML isolated from the blood of normotensive volunteers and subjects with essential hypertension. Glucocorticoid responsiveness of HML was evaluated as IC50 values (nmol/L) for dexamethasone and cortisol for the inhibition of lysozyme release. We measured plasma hormones by radioimmunoassay. Kd values (mean+/-SE) for cortisol in HML of hypertensive patients were higher than in control subjects (24.6+/-2.4 versus 17.5+/-1.7 nmol/L,Ptextless.04). Binding capacity (4978+/-391 versus 4131+/-321 sites/cell),Kd values for dexamethasone (6.7+/-0.5 versus 5.7+/-0.3 nmol/L),and IC50 values for dexamethasone (3.4+/-0.3 versus 3.1+/-0.2 nmol/L) and cortisol (12.2+/-1.6 versus 9.5+/-0.3 nmol/L) were not significantly different. Patients with renin values less than 0.13 ng angiotensin I/L per second were markedly less sensitive to cortisol than those with higher values. Both Kd (30.3+/-2.5 versus 19.2+/-2.4 nmol/L) and IC50 values (15.5+/-1.8 versus 8.9+/-1.2 nmol/L) for cortisol were significantly higher in patients with lower renin values (Ptextless.03). Other variables,including plasma hormone and electrolyte values and binding characteristics for dexamethasone,were not different. These data suggest that cortisol binding to glucocorticoid receptor is slightly impaired in patients with essential hypertension. In vivo,this could lead to inappropriate binding of cortisol to mineralocorticoid receptors. Hence,decreased sensitivity to cortisol is associated with renin suppression. This hypothesis is supported by evidence of hypertension and low renin activity,which others have described in patients with primary glucocorticoid resistance due to mutations of the glucocorticoid receptor. View Publication