技术资料

Protein geranylgeranyltransferase type I (GGTase-I) is responsible for the posttranslational lipidation of CAAX proteins such as RHOA,RAC1,and cell division cycle 42 (CDC42). Inhibition of GGTase-I has been suggested as a strategy to treat cancer and a host of other diseases. Although several GGTase-I inhibitors (GGTIs) have been synthesized,they have very different properties,and the effects of GGTIs and GGTase-I deficiency are unclear. One concern is that inhibiting GGTase-I might lead to severe toxicity. In this study,we determined the effects of GGTase-I deficiency on cell viability and K-RAS-induced cancer development in mice. Inactivating the gene for the critical beta subunit of GGTase-I eliminated GGTase-I activity,disrupted the actin cytoskeleton,reduced cell migration,and blocked the proliferation of fibroblasts expressing oncogenic K-RAS. Moreover,the absence of GGTase-I activity reduced lung tumor formation,eliminated myeloproliferative phenotypes,and increased survival of mice in which expression of oncogenic K-RAS was switched on in lung cells and myeloid cells. Interestingly,several cell types remained viable in the absence of GGTase-I,and myelopoiesis appeared to function normally. These findings suggest that inhibiting GGTase-I may be a useful strategy to treat K-RAS-induced malignancies. View Publication

Aldehyde dehydrogenase (ALDH) activity is used to define normal hematopoietic stem cell (HSC),but its link to leukemic stem cells (LSC) in acute myeloid leukemia (AML) is currently unknown. We hypothesize that ALDH activity in AML might be correlated with the presence of LSC. Fifty-eight bone marrow (BM) samples were collected from AML (n=43),acute lymphoblastic leukemia (ALL) (n=8) and normal cases (n=7). In 14 AML cases,a high SSC(lo)ALDH(br) cell population was identified (ALDH(+)AML) (median: 14.89%,range: 5.65-48.01%),with the majority of the SSC(lo)ALDH(br) cells coexpressing CD34(+). In another 29 cases,there was undetectable (n=23) or rare (textless or =5%) (n=6) SSC(lo)ALDH(br) population (ALDH(-)AML). Among other clinicopathologic variables,ALDH(+)AML was significantly associated with adverse cytogenetic abnormalities. CD34(+) BM cells from ALDH(+)AML engrafted significantly better in NOD/SCID mice (ALDH(+)AML: injected bone 21.11+/-9.07%; uninjected bone 1.52+/-0.75% vs ALDH(-)AML: injected bone 1.77+/-1.66% (P=0.05); uninjected bone 0.23+/-0.23% (P=0.03)) with the engrafting cells showing molecular and cytogenetic aberrations identical to the original clones. Normal BM contained a small SSC(lo)ALDH(br) population (median: 2.92%,range: 0.92-5.79%),but none of the ALL cases showed this fraction. In conclusion,SSC(lo)ALDH(br) cells in ALDH(+)AML might denote primitive LSC and confer an inferior prognosis in patients. View Publication

In order to investigate the biologic processes underlying and resulting from the megakaryocytic hyperplasia that characterizes idiopathic myelofibrosis (IMF),peripheral blood CD34+ cells isolated from patients with IMF,polycythemia vera (PV),and G-CSF-mobilized healthy volunteers were cultured in the presence of stem cell factor and thrombopoietin. IMF CD34+ cells generated 24-fold greater numbers of megakaryocytes (MKs) than normal CD34+ cells. IMF MKs were also shown to have a delayed pattern of apoptosis and to overexpress the antiapoptotic protein bcl-xL. MK hyperplasia in IMF is,therefore,likely a consequence of both the increased ability of IMF progenitor cells to generate MKs and a decreased rate of MK apoptosis. Media conditioned (CM) by CD61+ cells generated in vitro from CD34+ cells were then assayed for the levels of growth factors and proteases. Higher levels of transforming growth factor-beta (TGF-beta) and active matrix metalloproteinase-9 (MMP9) were observed in media conditioned with IMF CD61+ cells than normal or PV CD61+ cells. Both normal and IMF CD61+ cells produced similar levels of VEGF. MK-derived TGF-B and MMP-9,therefore,likely contribute to the development of many pathological epiphenomena associated with IMF. View Publication

UNLABELLED The differentiation capacity of human embryonic stem cells (hESCs) holds great promise for therapeutic applications. We report a novel three-stage method to efficiently direct the differentiation of human embryonic stem cells into hepatic cells in serum-free medium. Human ESCs were first differentiated into definitive endoderm cells by 3 days of Activin A treatment. Next,the presence of fibroblast growth factor-4 and bone morphogenetic protein-2 in the culture medium for 5 days induced efficient hepatic differentiation from definitive endoderm cells. Approximately 70% of the cells expressed the hepatic marker albumin. After 10 days of further in vitro maturation,these cells expressed the adult liver cell markers tyrosine aminotransferase,tryptophan oxygenase 2,phosphoenolpyruvate carboxykinase (PEPCK),Cyp7A1,Cyp3A4 and Cyp2B6. Furthermore,these cells exhibited functions associated with mature hepatocytes including albumin secretion,glycogen storage,indocyanine green,and low-density lipoprotein uptake,and inducible cytochrome P450 activity. When transplanted into CCl4 injured severe combined immunodeficiency mice,these cells integrated into the mouse liver and expressed human alpha-1 antitrypsin for at least 2 months. In addition,we found that the hESC-derived hepatic cells were readily infected by human immunodeficiency virus-hepatitis C virus pseudotype viruses. CONCLUSION We have developed an efficient way to direct the differentiation of human embryonic stem cells into cells that exhibit characteristics of mature hepatocytes. Our studies should facilitate searching the molecular mechanisms underlying human liver development,and form the basis for hepatocyte transplantation and drug tests. View Publication

Disruption of pathways leading to programmed cell death plays a major role in most malignancies,including multiple myeloma (MM). ABT-737 is a BH3 mimetic small-molecule inhibitor that binds with high affinity to Bcl-2 and Bcl-xL,preventing the sequestration of proapoptotic molecules and shifting the cell survival/apoptosis balance toward apoptosis induction. In this study,we show that ABT-737 is cytotoxic to MM cell lines,including those resistant to conventional therapies,and primary tumor cells. Flow cytometric analysis of intracellular levels of Bcl-2 family proteins demonstrates a clear inversion of the Bax/Bcl-2 ratio leading to induction of apoptosis. Activation of the mitochondrial apoptosis pathway was indicated by mitochondrial membrane depolarization and caspase cleavage. Additionally,several signaling pathways known to be important for MM cell survival are disrupted following treatment with ABT-737. The impact of ABT-737 on survival could not be overcome by the addition of interleukin-6,vascular endothelial growth factor or insulin-like growth factor,suggesting that ABT-737 may be effective in preventing the growth and survival signals provided by the microenvironment. These data indicate that therapies targeting apoptotic pathways may be effective in MM treatment and warrant clinical evaluation of ABT-737 and similar drugs alone or in combination with other agents in the setting of MM. View Publication

Ubiquitously acting chromatin opening elements (UCOEs) consist of methylation-free CpG islands encompassing dual divergently transcribed promoters of housekeeping genes that have been shown to confer resistance to transcriptional silencing and to produce consistent and stable transgene expression in tissue culture systems. To develop improved strategies for hematopoietic cell gene therapy,we have assessed the potential of the novel human HNRPA2B1-CBX3 UCOE (A2UCOE) within the context of a self-inactivating (SIN) lentiviral vector. Unlike viral promoters,the enhancer-less A2UCOE gave rise to populations of cells that expressed a reporter transgene at a highly reproducible level. The efficiency of expression per vector genome was also markedly increased in vivo compared with vectors incorporating either spleen focus-forming virus (SFFV) or cytomegalovirus (CMV) promoters,suggesting a relative resistance to silencing. Furthermore,an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease. These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector,largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression. These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile. View Publication

The present study reports for the first time a dual antiglioma effect of the well-known antidiabetic drug metformin. In low-density cultures of the C6 rat glioma cell line,metformin blocked the cell cycle progression in G(0)/G(1) phase without inducing significant cell death. In confluent C6 cultures,on the other hand,metformin caused massive induction of caspase-dependent apoptosis associated with c-Jun N-terminal kinase (JNK) activation,mitochondrial depolarization and oxidative stress. Metformin-triggered apoptosis was completely prevented by agents that block mitochondrial permeability transition (cyclosporin A) and oxygen radical production (N-acetylcisteine),while the inhibitors of JNK activation (SP600125) or glycolysis (sodium fluoride,iodoacetate) provided partial protection. The antiglioma effect of metformin was reduced by compound C,an inhibitor of AMP-activated protein kinase (AMPK),and was mimicked by the AMPK agonist AICAR. Similar effects were observed in the human glioma cell line U251,while rat primary astrocytes were completely resistant to the antiproliferative and proapoptotic action of metformin. View Publication

B cell-activating factor belonging to the TNF family (BAFF) plays a critical role in B cell maturation,yet its precise role in B cell differentiation into Ig-secreting cells (ISCs) remains unclear. In this study,we find that upon isolation human naive and memory B (MB) cells have prebound BAFF on their surface,whereas germinal center (GC) B cells lack detectable levels of prebound BAFF. We attribute their lack of prebound BAFF to cell activation,because we demonstrate that stimulation of naive and MB cells results in the loss of prebound BAFF. Furthermore,the absence of prebound BAFF on GC B cells is not related to a lack of BAFF-binding receptors or an inability to bind exogenous BAFF. Instead,our data suggest that accessibility to soluble BAFF is limited within GCs,perhaps to prevent skewing of the conventional B cell differentiation program. In support of this concept,whereas BAFF significantly enhances ISC differentiation in response to T cell-dependent activation,we report for the first time the ability of BAFF to considerably attenuate ISC differentiation of MB cells in response to CpG stimulation,a form of T cell-independent activation. Our data suggest that BAFF may be providing regulatory signals during specific T cell-independent events,which protect the balance between MB cells and ISCs outside GCs. Taken together,these data define a complex role for BAFF in humoral immune responses and show for the first time that BAFF can also play an inhibitory role in B cell differentiation. View Publication

BACKGROUND The epidermal growth factor receptor (EGFR) and Notch signaling pathways have been implicated in self-renewal of normal breast stem cells. We investigated the involvement of these signaling pathways in ductal carcinoma in situ (DCIS) of the breast. METHODS Samples of normal breast tissue (n = 15),pure DCIS tissue of varying grades (n = 35),and DCIS tissue surrounding an invasive cancer (n = 7) were used for nonadherent (i.e.,mammosphere) culture. Mammosphere cultures were treated at day 0 with gefitinib (an EGFR inhibitor),DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester) (a gamma-secretase inhibitor),or Notch 4-neutralizing antibody. Mammosphere-forming efficiency (MFE) was calculated by dividing the number of mammospheres of 60 microm or more formed by the number of single cells seeded and is expressed as a percentage. The Notch 1 intracellular domain (NICD) was detected immunohistochemically in paraffin-embedded DCIS tissue from 50 patients with at least 60 months of follow-up. All statistical tests were two-sided. RESULTS DCIS had a greater MFE than normal breast tissue (1.5% versus 0.5%,difference = 1%,95% confidence interval [CI] = 0.62% to 1.25%,Ptextless.001). High-grade DCIS had a greater MFE than low-grade DCIS (1.6% versus 1.09%,difference = 0.51%,95% CI = 0.07% to 0.94%,P = .01). The MFE of high-grade DCIS treated with gefitinib in the absence of exogenous EGF was lower than that of high-grade DCIS treated with mammosphere medium lacking gefitinib and exogenous EGF (0.56% versus 1.36%,difference 0.8%,95% CI = 0.33% to 1.4%,P = .004). Increased Notch signaling as detected by NICD staining was associated with recurrence at 5 years (P = .012). DCIS MFE was reduced when Notch signaling was inhibited using either DAPT (0.89% versus 0.51%,difference = 0.38%,95% CI = 0.2% to 0.6%,Ptextless.001) or a Notch 4-neutralizing antibody (0.97% versus 0.2%,difference = 0.77%,95% CI = 0.52% to 1.0%,Ptextless.001). CONCLUSION We describe a novel primary culture technique for DCIS. Inhibition of the EGFR or Notch signaling pathways reduced DCIS MFE. View Publication

Although butein (3,4,2',4'-tetrahydroxychalcone) is known to exhibit anti-inflammatory,anti-cancer,and anti-fibrogenic activities,very little is known about its mechanism of action. Because numerous effects modulated by butein can be linked to interference with the NF-kappaB pathway,we investigated in detail the effect of this chalcone on NF-kappaB activity. As examined by DNA binding,we found that butein suppressed tumor necrosis factor (TNF)-induced NF-kappaB activation in a dose- and time-dependent manner; suppressed the NF-kappaB activation induced by various inflammatory agents and carcinogens; and inhibited the NF-kappaB reporter activity induced by TNFR1,TRADD,TRAF2,NIK,TAK1/TAB1,and IKK-beta. We also found that butein blocked the phosphorylation and degradation of IkappaBalpha by inhibiting IkappaBalpha kinase (IKK) activation. We found the inactivation of IKK by butein was direct and involved cysteine residue 179. This correlated with the suppression of phosphorylation and the nuclear translocation of p65. In this study,butein also inhibited the expression of the NF-kappaB-regulated gene products involved in anti-apoptosis (IAP2,Bcl-2,and Bcl-xL),proliferation (cyclin D1 and c-Myc),and invasion (COX-2 and MMP-9). Suppression of these gene products correlated with enhancement of the apoptosis induced by TNF and chemotherapeutic agents; and inhibition of cytokine-induced cellular invasion. Overall,our results indicated that antitumor and anti-inflammatory activities previously assigned to butein may be mediated in part through the direct inhibition of IKK,leading to the suppression of the NF-kappaB activation pathway. View Publication

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain holds promise for the treatment of neurological diseases and has yielded new insight into brain cancer. However,the complete repertoire of signaling pathways that governs the proliferation and self-renewal of NSCs,which we refer to as the 'ground state',remains largely uncharacterized. Although the candidate gene approach has uncovered vital pathways in NSC biology,so far only a few highly studied pathways have been investigated. Based on the intimate relationship between NSC self-renewal and neurosphere proliferation,we undertook a chemical genetic screen for inhibitors of neurosphere proliferation in order to probe the operational circuitry of the NSC. The screen recovered small molecules known to affect neurotransmission pathways previously thought to operate primarily in the mature central nervous system; these compounds also had potent inhibitory effects on cultures enriched for brain cancer stem cells. These results suggest that clinically approved neuromodulators may remodel the mature central nervous system and find application in the treatment of brain cancer. View Publication

Ewing sarcoma gene EWS encodes a putative RNA-binding protein with proposed roles in transcription and splicing,but its physiological role in vivo remains undefined. Here,we have generated Ews-deficient mice and demonstrated that EWS is required for the completion of B cell development and meiosis. Analysis of Ews(-/-) lymphocytes revealed a cell-autonomous defect in precursor B lymphocyte (pre-B lymphocyte) development. During meiosis,Ews-null spermatocytes were deficient in XY bivalent formation and showed reduced meiotic recombination,resulting in massive apoptosis and complete arrest in gamete maturation. Inactivation of Ews in mouse embryonic fibroblasts resulted in premature cellular senescence,and the mutant animals showed hypersensitivity to ionizing radiation. Finally,we showed that EWS interacts with lamin A/C and that loss of EWS results in a reduced lamin A/C expression. Our findings reveal essential functions for EWS in pre-B cell development and meiosis,with proposed roles in DNA pairing and recombination/repair mechanisms. Furthermore,we demonstrate a novel role of EWS in cellular senescence,possibly through its interaction and modulation of lamin A/C. View Publication