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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

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

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

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

We have recently shown that more than 90% of long-term culture initiating cells (LTC-IC) mobilized in the peripheral blood (PB) of normal individuals express HLA-DR and CD38 antigens and can sustain hematopoiesis for only 5 weeks. However,10% of LTC-IC in mobilized PB are CD34+ HLA-DR- and CD34+ CD38- and can sustain hematopoiesis for at least 8 weeks. We now examine the ex vivo expansion potential of CD34+ HLA-DR+ cells (rich in mature LTC-IC) and CD34+ HLA-DR- cells (rich in primitive LTC-IC) in granulocyte colony-stimulating factor (G-CSF) mobilized PB progenitor cells (PBPC). Cells were cultured in contact with M2-10B4 cells (contact) or in transwells above M2-10B4 (noncontact) without and with interleukin-3 (IL-3) and macrophage inflammatory protein (MIP-1alpha) for 2 and 5 weeks. Progeny were evaluated for the presence of colony-forming cells (CFC) and LTC-IC. When CD34+ HLA-DR+ PB cells were cultured in contact cultures without cytokines,a threefold expansion of CFC was seen at 2 weeks,but an 80% decrease in CFC was seen at week 5. Further,the recovery of LTC-IC at week 2 was only 17% and 1% at week 5. This confirms our previous observation that although CD34+ HLA-DR+ mobilized PB cells can initiate long-term cultures,they are relatively mature and cannot sustain long-term hematopoiesis. In contrast,when CD34+ HLA-DR- mobilized PB cells were cultured in contact cultures without cytokines,CFC expansion persisted until week 5 and 49% and 11% of LTC-IC were recovered at week 2 and 5,respectively. As we have shown for steady state bone marrow (BM) progenitors,recovery of LTC-IC was threefold higher when CD34+ HLA-DR- PBPC were cultured in noncontact rather than contact cultures,and improved further when IL-3 and MIP-1alpha were added to noncontact cultures (96 +/- 2% maintained at week 5). We conclude that although G-CSF mobilizes a large population of mature" CD34+ HLA-DR+ LTC-IC with a limited proliferative capacity� View Publication

We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay,the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 x 10(5) cells. This was significantly higher than the frequency of 1 SRC in 3.0 x 10(6) adult BM cells or 1 in 6.0 x 10(6) mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus,the findings reported here will have important clinical as well as biologic implications. View Publication

Prostaglandin F2alpha inhibits adipose differentiation of primary culture of adipocyte precursors and of the adipogenic cell line 1246 in defined medium. In the present paper,we investigated the effect of FP receptor agonists cloprostenol and fluprostenol on the differentiation of newborn rat adipocyte precursors in primary culture. The results show that cloprostenol and fluprostenol are very potent inhibitors of adipose differentiation. Dose response studies indicate that both agonists are more potent than PGF2alpha in inhibiting adipocyte precursors differentiation. 50% inhibition of adipose differentiation was observed at a concentration of 3 x 10(-12) M for cloprostenol and 3 to 10 x 10(-11) M for fluprostenol respectively whereas the PGF2alpha concentration required to elicit the same effect was 10(-8) M. In contrast compounds structurally related to PGE2 such as 17-phenyl trinor PGE2 had no effect on adipose differentiation except when added at a 10,000-fold higher concentration. View Publication

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

We have identified three non-cross-reacting anti-human CD44 monoclonal antibodies that have significant positive or negative (or no) effects on normal human haemopoiesis in the long-term culture (LTC) system. These effects manifested as increases or decreases in the number of LTC-initiating cells (LTC-IC),and the number of colony-forming cells (CFC) recovered from cultures in which either unseparated or highly purified CD34+ CD38- normal marrow cells were placed on pre-established normal marrow feeder layers in the presence or absence of each antibody. The effects seen were rapid and sustained,and dependent on the presence of a preformed feeder layer. Interestingly,the same anti-CD44 antibodies had no effect on the maintenance of leukaemic (Ph+) progenitors (from patients with chronic myeloid leukaemia) when these cells were cultured on preformed feeder layers established from normal marrow. CD44 appears to be part of a mechanism by which stromal elements can regulate primitive normal haemopoietic cells but not their leukaemic (Ph+) counterparts. View Publication