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The long-term culture-initiating cell (LTC-IC) assay is a physiological approach to the quantitation of primitive human hematopoietic cells. The readout using identification of cobblestone area-forming cells (CAFC) has gained popularity over the LTC-IC readout where cells are subcultured in a colony-forming cell assay. However,comparing the two assays,cord blood (CB) mononuclear cell (MNC) samples were found to contain a higher frequency of CAFC than LTC-IC (126 +/- 83 versus 40 +/- 31 per 10(5) cells,p = 0.0001). Overall,60% of week-5 cobblestones produced by CB MNC were not functional LTC-IC and were classified as false." Separation of CB MNC using immunomagnetic columns showed that false cobblestones were CD34(-)/lineage(+). Purified CD34(+) cells� View Publication

We studied the actions of geldanamycin (GA) and herbimycin A (HMA),inhibitors of the chaperone proteins Hsp90 and GRP94,on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses,T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA,but not HMA,showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase,a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL. View Publication

We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells,we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma,particularly by monocytes and stromal cells,but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration,HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines,through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti-IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether,these data identify ErbB receptors as putative therapeutic targets in multiple myeloma. View Publication

In a patient with refractory anemia with excess blasts (RAEB),a somatic mutation of mitochondrial transfer RNA(Leu(UUR)) was detected in bone marrow cells. Heteroduplex analysis indicated that 40% to 50% of mitochondrial DNA (mtDNA) molecules in the bone marrow (BM) carried the novel G3242A mutation. The proportion of mutant mtDNA was higher in CD34(+) cells than in the unfractionated sample. Surprisingly,the mutation was not detectable by heteroduplex analysis in the peripheral blood (PB). However,PB CD34(+) cells selected by immunomagnetic beads harbored the mutation with a proportion of approximately 50%. In hematopoietic colony assays,CD34(+) cells from BM and PB yielded only colonies with wild-type mtDNA. These results indicate that the mtDNA mutation in CD34(+) cells was associated with a maturation defect. Mitochondrial tRNA mutations impair mitochondrial protein synthesis,thereby causing dysfunction of the mitochondrial respiratory chain. We propose that this effect contributed to ineffective hematopoiesis in our patient. View Publication

SU9516 is a 3-substituted indolinone compound with demonstrated potent and selective inhibition toward cyclin dependent kinases (cdks). Here,we describe the kinetic characterization of this inhibition with respect to cdk2,1,and 4,along with the crystal structure in complex with cdk2. The molecule is competitive with respect to ATP for cdk2/cyclin A,with a K(i) value of 0.031 microM. Similarly,SU9516 inhibits cdk2/cyclin E and cdk1/cyclin B1 in an ATP-competitive manner,although at a 2- to 8-fold reduced potency. In contrast,the compound exhibited non-competitive inhibition with respect to ATP toward cdk4/cyclin D1,with a 45-fold reduced potency. The X-ray crystal structure of SU9516 bound to cdk2 revealed interactions between the molecule and Leu83 and Glu81 of the kinase. This study should aid in the development of more potent and selective cdk inhibitors for potential therapeutic agents. View Publication

In mammals,the continuous production of hematopoietic cells (HCs) is sustained by a small number of hematopoietic stem cells (HSCs) residing in the bone marrow. Early HSC activity arises in the aorta-gonad mesonephros region,within cells localized to the ventral floor of the major blood vessels,suggesting that the first HSCs may be derived from cells capable of giving rise to the hematopoietic system and to the endothelial cells of the vasculature. TIE1 (TIE) and TIE2 (TEK) are related receptor tyrosine kinases with an embryonic expression pattern in endothelial cells,their precursors,and HCs,suggestive of a role in the divergence and function of both lineages. Indeed,gene targeting approaches have shown that TIE1,TIE2,and ligands for TIE2,the angiopoietins,are essential for vascular development and maintenance. To explore possible roles for these receptors in HCs,we have examined the ability of embryonic cells lacking both TIE1 and TIE2 to contribute to developmental and adult hematopoiesis by generating chimeric animals between normal embryonic cells and cells lacking these receptors. We show here that TIE receptors are not required for differentiation and proliferation of definitive hematopoietic lineages in the embryo and fetus; surprisingly,however,these receptors are specifically required during postnatal bone marrow hematopoiesis. View Publication

Vertebrate body axis extension involves progressive generation and subsequent differentiation of new cells derived from a caudal stem zone; however,molecular mechanisms that preserve caudal progenitors and coordinate differentiation are poorly understood. FGF maintains caudal progenitors and its attenuation is required for neuronal and mesodermal differentiation and to position segment boundaries. Furthermore,somitic mesoderm promotes neuronal differentiation in part by downregulating Fgf8. Here we identify retinoic acid (RA) as this somitic signal and show that retinoid and FGF pathways have opposing actions. FGF is a general repressor of differentiation,including ventral neural patterning,while RA attenuates Fgf8 in neuroepithelium and paraxial mesoderm,where it controls somite boundary position. RA is further required for neuronal differentiation and expression of key ventral neural patterning genes. Our data demonstrate that FGF and RA pathways are mutually inhibitory and suggest that their opposing actions provide a global mechanism that controls differentiation during axis extension. View Publication

Embryonic stem (ES) cells are permanent pluripotent stem cell lines established from pre-implantation mouse embryos. There is currently great interest in the potential therapeutic applications of analogous cells derived from human embryos. The isolation of ES cells is commonly presented as a straightforward transfer of cells in the early embryo into culture. In reality,however,continuous expansion of pluripotent cells does not occur in vivo,and in vitro is the exception rather than the norm. Both genetic and epigenetic factors influence the ability to derive ES cells. We have tracked the expression of a key marker and determinant of pluripotency,the transcription factor Oct-4,in primary cultures of mouse epiblasts and used this to assay the effect of experimental manipulations on the maintenance of a pluripotent cell compartment. We find that expression of Oct-4 is often lost prior to overt cytodifferentiation of the epiblast. The rate and extent of Oct-4 extinction varies with genetic background. We report that treatment with the MAP kinase/ERK kinase inhibitor PD98059,which suppresses activation of the mitogen-activated protein kinases Erk1 and Erk2,results in increased persistence of Oct-4-expressing cells. Oct-4 expression is also relatively sustained in cultures of diapause embryos and of isolated inner cell masses. Combination of all three conditions allowed the derivation of germline-competent ES cells from the normally refractory CBA mouse strain. These findings suggest that the genesis of an ES cell is a relatively complex process requiring epigenetic modulation of key gene expression over a brief time-window. Procedures that extend this time-window and/or directly regulate the critical genes should increase the efficiency of ES cell derivation. View Publication

Exposure of murine bone marrow (BM) cells to ionizing radiation (IR; 4 Gy) resulted in textgreater95% inhibition of the frequency of various day types of cobblestone area-forming cells in association with the induction of apoptosis in hematopoietic stem cell alike cells (Lin(-) ScaI(+) c-kit(+) cells; IR: 64.8 +/- 0.4% versus control: 20.4 +/- 0.5%; P textless 0.001) and progenitors (Lin(-) ScaI(-) c-kit(+) cells; IR: 46.2 +/- 1.4% versus control: 7.8 +/- 0.5%; P textless 0.001). Incubation of murine BM cells with busulfan (BU; 30 micro M) for 6 h also inhibited the cobblestone area-forming cell frequency but failed to cause a significant increase in apoptosis in these two types of hematopoietic cells. After 5 weeks of long-term BM cell culture,33% and 72% of hematopoietic cells survived IR- and BU-induced damage,respectively,as compared with control cells,but they could not form colony forming units-granulocyte macrophages. Moreover,these surviving cells expressed an increased level of senescence-associated beta-galactosidase,p16(Ink4a),and p19(Arf). These findings suggest that IR inhibits the function of hematopoietic stem cell alike cells and progenitors primarily by inducing apoptosis,whereas BU does so mainly by inducing premature senescence. In addition,induction of premature senescence in BM hematopoietic cells also contributes to IR-induced inhibition of their hematopoietic function. Interestingly,the induction of hematopoietic cell senescence by IR,but not by BU,was associated with an elevation in p53 and p21(Cip1/Waf1) expression. This suggests that IR induces hematopoietic cell senescence in a p53-p21(Cip1/Waf1)-dependent manner,whereas the induction of senescence by BU bypasses the p53-p21(Cip1/Waf1) pathway. View Publication

A wide variety of membrane transformations important in intracellular transport are inhibited by the fungal metabolite brefeldin A (refs 1-4),implying that the target for this drug is central to the formation and maintenance of subcellular compartments. Brefeldin A added to cells causes the rapid and reversible dissociation of a Golgi-associated peripheral membrane protein (M(r) 110,000) which was found to be identical to one of the subunits of the coat of Golgi-derived (non-clathrin) coated vesicles,beta-COP,implying that brefeldin A prevents transport by blocking the assembly of coats and thus the budding of enclosed vesicles. In addition to the coatomer (a cytosol-derived complex of seven polypeptide chains,one of which is beta-COP),the non-clathrin (COP) coat of Golgi-derived vesicles contains stoichiometric amounts of a small (M(r) approximately 20,000) GTP-binding protein,the ADP-ribosylation factor (ARF). Binding of ARF to Golgi membranes is necessary before coatomer/beta-COP can bind these membranes (ref. 12; and D. J. Palmer et al.,manuscript submitted),so the primary effect of brefeldin A seems to be on the reaction responsible for ARF binding. Indeed,like beta-COP,ARF is dissociated from the Golgi complex by treatment with brefeldin A and brefeldin A prevents ARF from associating in vitro,but the mechanism of this action by brefeldin A has been unclear. Here we report the discovery of an enzyme in a Golgi-enriched fraction that catalyses guanine nucleotide (GDP-GTP) exchange on ARF-1 protein,and which is inhibited by brefeldin A. We suggest that activation of ARF proteins for membrane localization by compartmentalized exchange enzymes is in general the first committed step in membrane transformation pathways. View Publication

To directly study the biological properties of purified hematopoietic colony-forming cell precursors,cells with a CD34+ CD45RAlo CD71lo phenotype were purified from human bone marrow using density separation and fluorescence-activated cell sorting,and were cultured in serum-free culture medium supplemented with various cytokines. In the presence of interleukin 3 (IL-3),IL-6,erythropoietin,and mast cell growth factor (a c-kit ligand),cell numbers increased approximately 10(6)-fold over a period of 4 wk,and the percentage of cells that expressed transferrin receptors (CD71) increased from less than 0.1% at day 0 to greater than 99% at day 14. Interestingly,the absolute number of CD34+ CD71lo cells did not change during culture. When CD34+ CD71lo cells were sorted from expanded cultures and recultured,extensive cell production was repeated,again without significant changes in the absolute number of cells with the CD34+ CD71lo phenotype that were used to initiate the (sub)cultures. These results document that primitive hematopoietic cells can generate progeny without an apparent decrease in the size of a precursor cell pool. View Publication

Deamination of the nucleoside analogues ARA-C and 5-AZA-CdR by CR deaminase results in a loss of antileukemic activity. To prevent the inactivation of these analogues,inhibitors of CR deaminase may prove to be useful agents. In the present study we investigated the effects of the deaminase inhibitors Zebularine,5-F-Zebularine,and diazepinone riboside on the deamination of CR,ARA-C,and 5-AZA-CdR using highly purified human CR deaminase (EC 3.5.4.5). These inhibitors produced a competitive type of inhibition with each substrate,the potency of which followed the patterns diazepinone riboside greater than 5-F-Zebularine and THU greater than Zebularine. 5-AZA-CdR was more sensitive than ARA-C to the inhibition produced by these deaminase inhibitors. The inhibition constants for diazepinone riboside lay in the range of 5-15 nM,suggesting that this inhibitor could be an excellent candidate for use in combination chemotherapy with either ARA-C or 5-AZA-CdR in patients with leukemia. View Publication