参考文献

DNA-dependent protein kinase (DNA-PK),which is involved in DNA double-stranded break repair and V(D)J recombination,comprises a DNA-targeting component called Ku and an approximately 460 kDa catalytic subunit,DNA-PKcs. Here,we describe the cloning of the DNA-PKcs cDNA and show that DNA-PKcs falls into the phosphatidylinositol (PI) 3-kinase family. Biochemical assays,however,indicate that DNA-PK phosphorylates proteins but has no detectable activity toward lipids. Strikingly,DNA-PKcs is most similar to PI kinase family members involved in cell cycle control,DNA repair,and DNA damage responses. These include the FKBP12-rapamycin-binding proteins Tor1p,Tor2p,and FRAP,S. pombe rad3,and the product of the ataxia telangiectasia gene,mutations in which lead to genomic instability and predisposition to cancer. The relationship of these proteins to DNA-PKcs provides important clues to their mechanisms of action. View Publication

The microbial product wortmannin has previously been shown to be a potent inhibitor of phosphatidylinositol-3-kinase. In view of the potential role of this enzyme in transduction of mitogenic signals,we determined the cytotoxic activity of wortmannin against several human tumor cell lines in vitro. The most sensitive lines included GC3 colon carcinoma,IGROV1 ovarian carcinoma,and CCRF-CEM leukemia (IC-50s ranging from 0.7-2.1 microM). The cytotoxicity of wortmannin was decreased approximately 10-fold by serum-free conditions. Wortmannin was generally less active in low passage human breast cancer cell lines that overexpress either epidermal growth factor receptor or Her2/neu. Wortmannin was also tested for in vivo antitumor activity against seven murine tumor and ten human tumor xenograft models. Activity (textgreater 60% inhibition of tumor growth) was observed in only the C3H mammary carcinoma and the human BxPC-3 pancreatic carcinoma xenograft. In vivo antitumor activity did not correlate with in vitro sensitivity to wortmannin cytotoxicity. View Publication

Treatment of cells with a variety of growth factors triggers a phosphorylation cascade that leads to activation of mitogen-activated protein kinases (MAPKs,also called extracellular signal-regulated kinases,or ERKs). We have identified a synthetic inhibitor of the MAPK pathway. PD 098059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one] selectively inhibited the MAPK-activating enzyme,MAPK/ERK kinase (MEK),without significant inhibitory activity of MAPK itself. Inhibition of MEK by PD 098059 prevented activation of MAPK and subsequent phosphorylation of MAPK substrates both in vitro and in intact cells. Moreover,PD 098059 inhibited stimulation of cell growth and reversed the phenotype of ras-transformed BALB 3T3 mouse fibroblasts and rat kidney cells. These results indicate that the MAPK pathway is essential for growth and maintenance of the ras-transformed phenotype. Further,PD 098059 is an invaluable tool that will help elucidate the role of the MAPK cascade in a variety of biological settings. View Publication

Retinoids elicit biological responses by activating a series of nuclear receptors. Six retinoid receptors belonging to two families are currently known: retinoic acid receptors (RAR alpha,beta,and gamma) and retinoid X receptors (RXR alpha,beta,and gamma). Stilbene retinoid analogs of retinoic acid (RA),such as (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)prope n-1- yl]benzoic acid (TTNPB,1) and (E)-4-[2-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)pro pen-1- yl]benzoic acid (3-methyl-TTNPB,2),display differential RAR and RXR activities,depending on the substituent at C3 of the naphthalene ring. We report here structural modifications of the benzoate moiety of 2 that result in analogs with greater RXR selectivity as well as those with pan-agonist (activate both RAR and RXR receptors) activities,analyze the structural features that impart receptor selectivity,and describe a stereoselective method for the synthesis of these analogs. The biological activities associated with the RAR and RXR receptors were examined by testing representative examples with different receptor activation profiles for their ability to induce tissue transglutaminase (Tgase) activity in a human promyelocytic leukemia cell line (HL-60 cdm-1) and to inhibit tumor-promoter-induced ornithine decarboxylase (ODC) activity in hairless mouse skin. These results suggest that RAR agonists and RXR agonists may have different therapeutic applications. Finally,we show that RXR agonists are significantly reduced in teratogenic potency relative to RAR agonists and may therefore have significant advantages in clinical practice. View Publication

In this report,we describe a modification of the assay for long-term culture-initiating cells (LTC-IC) that allows a subset of murine LTC-IC (designated as LTC-ICML) to express both their myeloid (M) and lymphoid (L) differentiative potentials in vitro. The modified assay involves culturing test cells at limiting dilutions on irradiated mouse marrow feeder layers for an initial 4 weeks under conditions that support myelopoiesis and then for an additional week under conditions permissive for B-lymphopoiesis. All of the clonogenic pre-B progenitors (colony-forming unit [CFU] pre-B) detected in such postswitch LTC appear to be the progeny of uncommitted cells present in the original cell suspension because exposure of lymphoid-restricted progenitors to myeloid LTC conditions for textgreater or = 7 days was found to irreversibly terminate CFU-pre-B production and,in cultures initiated with limiting numbers of input cells (no progenitors of any type detected in textgreater 70% of cultures 1 week after the switch),the presence of CFU-pre-B was tightly associated with the presence of myeloid clonogenic cells,regardless of the purity of the input population. Limiting dilution analysis of the proportion of negative cultures measured for different numbers of input cells showed the frequency of LTC-ICML in normal adult mouse marrow to be 1 per 5 x 10(5) cells with an enrichment of approximately 500-fold in the Sca-1+ Lin-WGA+ fraction,as was also found for competitive in vivo repopulating units (CRU) and conventionally defined LTC-IC. LTC-ICML also exhibited the same resistance to treatment in vivo with 5-fluorouracil (5-FU) as CRU and LTC-IC,thereby distinguishing these three populations from the great majority of both in vitro clonogenic cells and day 12 CFU-S. The ability to quantitate cells with dual lymphoid and myeloid differentiation potentials in vitro,without the need for their prior purification,should facilitate studies of totipotent hematopoietic stem cell regulation. View Publication

A method for quantitating the proportion of cycling long-term culture-initiating cells (LTC-IC) in heterogeneous populations of human hematopoietic cells is described. This procedure involves incubating the cells of interest for 16 to 24 hours in a serum-free medium containing 100 ng/mL Steel factor (SF),20 ng/mL interleukin-3 (IL-3),and 20 ng/mL granulocyte-colony-stimulating factor (G-CSF),with or without 20 microCi/mL of high specific activity 3H-thymidine (3H-Tdr) before plating the recovered cells in standard LTC-IC assays. The details of this procedure are based in part on the finding that the number of LTC-IC (regardless of their cycling status) remains constant for at least 24 hours under these culture conditions,as long as 3H-Tdr is not present. In addition,we have determined that a 16-hour period of exposure to the 3H-Tdr is sufficient to maximize the discrimination of cycling LTC-IC but not long enough to allow a detectable redistribution of LTC-IC between noncycling and cycling compartments. Finally,any isotope reutilization that may occur is not sufficient to affect the LTC-IC 3H-Tdr suicide values measured. Application of this methodology to normally circulating LTC-IC showed these to be a primarily quiescent population. However,within 72 hours of incubation in a serum-free medium containing SF,IL-3,and G-CSF,most had entered S-phase,although there was no net change in their numbers. This suggests that,under certain conditions in vitro,self-renewal divisions of LTC-IC can occur and,at least initially,balance any losses of these cells due to their differentiation or death. In contrast,many of the LTC-IC in freshly aspirated samples of normal marrow were found to be proliferating,although those that were initially quiescent could also be recruited into S-phase within 72 hours in vitro when incubated under the same conditions used to stimulate circulating LTC-IC. This modified 3H-Tdr suicide procedure should facilitate further investigation of the mechanisms regulating the turnover of the most primitive compartments of human hematopoietic cells and how these may be altered in disease states or exploited for a variety of therapeutic applications. View Publication

The requirement of complex sphingolipid biosynthesis for growth of neurons was examined in developing rat cerebellar Purkinje neurons using a dissociated culture system. Purkinje cells developed well-differentiated dendrites and axons after 2 weeks in a serum-free nutrient condition. Addition of 2 microM fumonisin B1,a fungal inhibitor of mammalian ceramide synthase,inhibited incorporation of [3H]galactose/glucosamine and [14C]-serine into complex sphingolipids of cultured cerebellar neurons. Under this condition,the expression of Purkinje cell-enriched sphingolipids,including GD1 alpha,9-O-acetylated LD1 and GD3,and sphingomyelin,was significantly decreased. After 2 weeks' exposure to fumonisin B1,dose-dependent measurable decreases in the survival and visually discernible differences in the morphology were seen in fumonisin-treated Purkinje cells. The Purkinje cell dendrites exhibited two types of anomalies; one population of cells developed elongated but less-branched dendrites after a slight time lag,but their branches began to degenerate. In some cells,formation of elongated dendrite trees was severely impaired. However,treatment with fumonisin B1 also led to the formation of spinelike protrusions on the dendrites of Purkinje cells as in control cultures. In contrast to the alterations observed in Purkinje cells,morphology of other cell types including granule neurons appeared to be almost normal after treatment with fumonisin B1. These observations indicated strongly that membrane sphingolipids participate in growth and maintenance of dendrites and in the survival of cerebellar Purkinje cells. Indeed,these effects of fumonisin B1 were reversed,but not completely,by the addition of 6-[[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino dcaproyl]sphingosine (C6-NBD-ceramide),a synthetic derivative of ceramide. Thus,we conclude that deprivation of membrane sphingolipids in a culture environment is responsible for aberrant growth of Purkinje cells. View Publication

Taxol stabilizes microtubules,prevents tubulin depolymerization,and promotes tubulin bundling and is one of the most effective drugs for the treatment of metastatic breast and ovarian cancer. Although its interaction with tubulin has been well characterized,the mechanism by which taxol induces growth arrest and cytotoxicity is not well understood. Herein,we show that taxol induced dose- and time-dependent accumulation of the cyclin inhibitor p21WAF1 in both p53 wild-type and p53-null cells,although the degree of induction was greater in cells expressing wild-type p53. In MCF7 cells,wild-type p53 protein was also induced after taxol treatment,and this induction was mediated primarily by increased protein stability. Taxol induced both p21WAF1 and wild-type p53 optimally in MCF7 cells after 20-24-h exposure with an EC50(3) of 5 nM. In p53-null PC3M cells,p21WAF1 was similarly induced after 24-h exposure to taxol. Coincident with these biochemical effects,taxol altered the electrophoretic mobility of c-raf-1 and stimulated mitogen activated protein kinase. Previous depletion of c-raf-1 inhibited both the p21WAF1- and p53-inducing properties of taxol,as well as the activation of MAP kinase. These data suggest that induction of p21WAF1 by taxol requires c-raf-1 activity,but that it is not strictly dependent on wild-type p53. Furthermore,the ability of taxol to both induce wild-type p53 in MCF7 cells and activate MAP kinase is also dependent on c-raf-1 expression. View Publication

Hypoxia-inducible factor 1 (HIF-1) is found in mammalian cells cultured under reduced O2 tension and is necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. We show that both HIF-1 subunits are basic-helix-loop-helix proteins containing a PAS domain,defined by its presence in the Drosophila Per and Sim proteins and in the mammalian ARNT and AHR proteins. HIF-1 alpha is most closely related to Sim. HIF-1 beta is a series of ARNT gene products,which can thus heterodimerize with either HIF-1 alpha or AHR. HIF-1 alpha and HIF-1 beta (ARNT) RNA and protein levels were induced in cells exposed to 1% O2 and decayed rapidly upon return of the cells to 20% O2,consistent with the role of HIF-1 as a mediator of transcriptional responses to hypoxia. View Publication

We have investigated the potential of stirred suspension cultures to support hematopoiesis from starting innocula of normal human bone marrow cells. Initial studies showed that the short-term maintenance of both colony-forming cell (CFC) numbers and their precursors,detected as long-term culture-initiating cells (LTC-IC),could be achieved as well in stirred suspension cultures as in static cultures. Neither of these progenitor cell populations was affected in either type of culture when porous microcarriers were added to provide an increased surface for adherent cell attachment. Supplementation of the medium with 10 ng/ml of Steel factor (SF) and 2 ng/ml of interleukin-3 (IL-3) resulted in a significant expansion of LTC-IC,CFC and total cell numbers in stirred cultures. Both the duration and ultimate magnitude of these expansions were correlated with the initial cell density and after 4 weeks the number of LTC-IC and CFC present in stirred cultures initiated with the highest starting cell concentration tested reflected average increases of 7- and 22-fold,respectively,above input values. Stirred suspension cultures offer the combined advantages of homogeneity and lack of dependence on the formation and maintenance of an adherent cell layer. Our results suggest their applicability to the development of scaled-up bioreactor systems for clinical procedures requiring the production of primitive hematopoietic cell populations. In addition,stirred suspension cultures may offer a new tool for the analysis of hematopoietic regulatory mechanisms. View Publication

Normal murine bone marrow (BM) cells were sorted on the basis of low forward and orthogonal light scatter properties,Sca-1 expression (Sca-1+),lack of staining with a cocktail of mature hematopoietic lineage markers (Lin-),and binding of wheat germ agglutinin (WGA+). This approach allowed the reproducible isolation of a very small subpopulation (0.037% +/- 0.023% of all nucleated BM cells) that was approximately 400-fold enriched in cells capable of reconstituting both lymphoid and myeloid lineages in lethally irradiated recipients. Transplantation of 30 or 10 of these Sca-1+Lin-WGA+ cells resulted in textgreater or = to 20% donor-derived nucleated peripheral blood cells 3 months posttransplantation in 100% and 22% of the recipients,respectively. When Sca-1+Lin-WGA+ cells were cultured in serum-free medium supplemented with Steel factor,interleukin-6 (IL-6),and erythropoietin (with or without IL-3),a large increase in total cell number,including cells with an Sca-1+Lin-WGA+ phenotype was observed. Single cell cultures showed that 90% to 95% of the input cells underwent at least one division during the first 2 weeks and the remainder died. Interestingly,this proliferative response was not accompanied by a parallel increase in the number of cells with both lymphoid and myeloid repopulating potential in vivo,as quantitation of these by limiting dilution analysis showed they had decreased slightly (1.3-fold) but not significantly below the number initially present. These results demonstrate that Sca-1+Lin-WGA+ cells with long-term repopulating potential can be maintained for 2 weeks in a serum- and stroma cell-free culture,providing a simple in vitro system to study their behavior under well-defined conditions. The observed expansion of Sca-1+Lin-WGA+ cells in vitro without a concomitant increase in reconstituting cells also shows that extensive functional heterogeneity exists within populations of cells with this surface phenotype. View Publication

PD 098059 has been shown previously to inhibit the dephosphorylated form of mitogen-activated protein kinase kinase-1 (MAPKK1) and a mutant MAPKK1(S217E,S221E),which has low levels of constitutive activity (Dudley,D. T.,Pang,L.,Decker,S. J.,Bridges,A. J.,and Saltiel,A. R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92,7686-7689). Here we report that PD 098059 does not inhibit Raf-activated MAPKK1 but that it prevents the activation of MAPKK1 by Raf or MEK kinase in vitro at concentrations (IC50 = 2-7 microM) similar to those concentrations that inhibit dephosphorylated MAPKK1 or MAPKK1(S217E,S221E). PD 098059 inhibited the activation of MAPKK2 by Raf with a much higher IC50 value (50 microM) and did not inhibit the phosphorylation of other Raf or MEK kinase substrates,indicating that it exerts its effect by binding to the inactive form of MAPKK1. PD 098059 also acts as a specific inhibitor of the activation of MAPKK in Swiss 3T3 cells,suppressing by 80-90% its activation by a variety of agonists. The high degree of specificity of PD 098059 in vitro and in vivo is indicated by its failure to inhibit 18 protein Ser/Thr kinases (including two other MAPKK homologues) in vitro by its failure to inhibit the in vivo activation of MAPKK and MAP kinase homologues that participate in stress and interleukin-1-stimulated kinase cascades in KB and PC12 cells,and by lack of inhibition of the activation of p70 S6 kinase by insulin or epidermal growth factor in Swiss 3T3 cells. PD 098059 (50 microM) inhibited the activation of p42MAPK and isoforms of MAP kinase-activated protein kinase-1 in Swiss 3T3 cells,but the extent of inhibition depended on how potently c-Raf and MAPKK were activated by any particular agonist and demonstrated the enormous amplification potential of this kinase cascade. PD 098059 not only failed to inhibit the activation of Raf by platelet-derived growth factor,serum,insulin,and phorbol esters in Swiss 3T3 cells but actually enhanced Raf activity. The rate of activation of Raf by platelet-derived growth factor was increased 3-fold,and the subsequent inactivation that occurred after 10 min was prevented. These results indicate that the activation of Raf is suppressed and that its inactivation is accelerated by a downstream component(s) of the MAP kinase pathway. View Publication