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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

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

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

Differentiation of totipotent mouse embryonic stem (ES) cells to various lymphohematopoietic cells is an in vitro model of the hematopoietic cell development during embryogenesis. To understand this process at cellular levels,differentiation intermediates were investigated. ES cells generated progeny expressing CD34,which was significantly enhanced by vascular endothelial growth factor (VEGF). The isolated CD34+ cells were enriched for myeloid colony-forming cells but not significantly for erythroid colony-forming cells. When cultured on OP9 stroma cells in the presence of interleukin-2 and interleukin-7,the CD34+ cells developed two types of B220+ CD34- lymphocytes: CD3- cytotoxic lymphocytes and CD19+ pre-B cells,and such lymphoid potential was highly enriched in the CD34+ population. Interestingly,the cytotoxic cells expressed the natural killer (NK) cell markers,such as NKR-P1,perforin,and granzymes,classified into two types,one of which showed target specificity of NK cells. Thus,ES cells have potential to generate NK-type cytotoxic lymphocytes in vitro in addition to erythro-myeloid cells and pre-B cells,and both myeloid and lymphoid cells seem to be derived from the CD34+ intermediate,on which VEGF may play an important role. 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

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

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