技术资料

BACKGROUND: Cancer stem cells (CSCs) are believed to be responsible for breast cancer formation and recurrence; therefore,therapeutic strategies targeting CSCs must be developed. One approach may be targeting signaling pathways,like Notch,that are involved in stem cell self-renewal and survival. MATERIALS AND METHODS: Breast cancer stem-like cells derived from cell lines and patient samples were examined for Notch expression and activation. The effect of Notch inhibition on sphere formation,proliferation,and colony formation was determined. RESULTS: Breast cancer stem-like cells consistently expressed elevated Notch activation compared with bulk tumor cells. Blockade of Notch signaling using pharmacologic and genomic approaches prevented sphere formation,proliferation,and/or colony formation in soft agar. Interestingly,a gamma-secretase inhibitor,MRK003,induced apoptosis in these cells. CONCLUSION: Our findings support a crucial role for Notch signaling in maintenance of breast cancer stem-like cells,and suggest Notch inhibition may have clinical benefits in targeting CSCs. View Publication

Mucopolysaccharidosis type I (MPS IH; Hurler syndrome) is a congenital deficiency of α-L-iduronidase,leading to lysosomal storage of glycosaminoglycans that is ultimately fatal following an insidious onset after birth. Hematopoietic cell transplantation (HCT) is a life-saving measure in MPS IH. However,because a suitable hematopoietic donor is not found for everyone,because HCT is associated with significant morbidity and mortality,and because there is no known benefit of immune reaction between the host and the donor cells in MPS IH,gene-corrected autologous stem cells may be the ideal graft for HCT. Thus,we generated induced pluripotent stem cells from 2 patients with MPS IH (MPS-iPS cells). We found that α-L-iduronidase was not required for stem cell renewal,and that MPS-iPS cells showed lysosomal storage characteristic of MPS IH and could be differentiated to both hematopoietic and nonhematopoietic cells. The specific epigenetic profile associated with de-differentiation of MPS IH fibroblasts into MPS-iPS cells was maintained when MPS-iPS cells are gene-corrected with virally delivered α-L-iduronidase. These data underscore the potential of MPS-iPS cells to generate autologous hematopoietic grafts devoid of immunologic complications of allogeneic transplantation,as well as generating nonhematopoietic cells with the potential to treat anatomical sites not fully corrected with HCT. View Publication

Human embryonic stem cells (hESCs) are pluripotent cells derived from the embryo at the blastocyst stage. Their embryonic origin confers upon them the capacity to proliferate indefinitely in vitro while maintaining the capacity to differentiate into a large variety of cell types. Based on these properties of self-renewal and pluripotency,hESCs represent a unique source to generate a large quantity of certain specialized cell types with clinical interest for transplantation-based therapy. However,hESCs are usually grown in culture conditions using fetal bovine serum and mouse embryonic fibroblasts,two components that are not compatible with clinical applications. Consequently,the possibility to expand hESCs in serum-free and in feeder-free culture conditions is becoming a major challenge to deliver the clinical promises of hESCs. Here,we describe the basic principles of growing hESCs in a chemically defined medium (CDM) devoid of serum and feeders. View Publication

Nuclear reprogramming of somatic tissue enables derivation of induced pluripotent stem (iPS) cells from an autologous,non-embryonic origin. The purpose of this study was to establish efficient protocols for lineage specification of human iPS cells into functional glucose-responsive,insulin-producing progeny. We generated human iPS cells,which were then guided with recombinant growth factors that mimic the essential signaling for pancreatic development. Reprogrammed with four stemness factors,human fibroblasts were here converted into authentic iPS cells. Under feeder-free conditions,fate specification was initiated with activin A and Wnt3a that triggered engagement into definitive endoderm,followed by priming with fibroblast growth factor 10 (FGF10) and KAAD-cyclopamine. Addition of retinoic acid,boosted by the pancreatic endoderm inducer indolactam V (ILV),yielded pancreatic progenitors expressing pancreatic and duodenal homeobox 1 (PDX1),neurogenin 3 (NGN3) and neurogenic differentiation 1 (NEUROD1) markers. Further guidance,under insulin-like growth factor 1 (IGF-1),hepatocyte growth factor (HGF) and N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT),was enhanced by glucagon-like peptide-1 (GLP-1) to generate islet-like cells that expressed pancreas-specific markers including insulin and glucagon. Derived progeny demonstrated sustained expression of PDX1,and functional responsiveness to glucose challenge secreting up to 230 pM of C-peptide. A pancreatogenic cocktail enriched with ILV/GLP-1 offers a proficient means to specify human iPS cells into glucose-responsive hormone-producing progeny,refining the development of a personalized platform for islet-like cell generation. View Publication

Induced pluripotent stem (iPS) cells have great potential for regenerative medicine and gene therapy. Thus far,iPS cells have typically been generated using integrating viral vectors expressing various reprogramming transcription factors; nonintegrating methods have been less effective and efficient. Because there is a significant risk of malignant transformation and cancer involved with the use of iPS cells,careful evaluation of transplanted iPS cells will be necessary in small and large animal studies before clinical application. Here,we have generated and characterized nonhuman primate iPS cells with the goal of evaluating iPS cell transplantation in a clinically relevant large animal model. We developed stable Phoenix-RD114-based packaging cell lines that produce OCT4,SOX2,c-MYC,and KLF4 (OSCK) expressing gammaretroviral vectors. Using these vectors in combination with small molecules,we were able to efficiently and reproducibly generate nonhuman primate iPS cells from pigtailed macaques (Macaca nemestrina). The established nonhuman primate iPS cells exhibited pluripotency and extensive self-renewal capacity. The facile and reproducible generation of nonhuman primate iPS cells using defined producer cells as a source of individual reprogramming factors should provide an important resource to optimize and evaluate iPS cell technology for studies involving stem cell biology and regenerative medicine. View Publication

Cancer stem cells (CSC) are rare drug-resistant cancer cell subsets proposed to be responsible for the maintenance and recurrence of cancer and metastasis. Telomerase is constitutively active in both bulk tumor cell and CSC populations but has only limited expression in normal tissues. Thus,inhibition of telomerase has been shown to be a viable approach in controlling cancer growth in nonclinical studies and is currently in phase II clinical trials. In this study,we investigated the effects of imetelstat (GRN163L),a potent telomerase inhibitor,on both the bulk cancer cells and putative CSCs. When breast and pancreatic cancer cell lines were treated with imetelstat in vitro,telomerase activity in the bulk tumor cells and CSC subpopulations were inhibited. Additionally,imetelstat treatment reduced the CSC fractions present in the breast and pancreatic cell lines. In vitro treatment with imetelstat,but not control oligonucleotides,also reduced the proliferation and self-renewal potential of MCF7 mammospheres and resulted in cell death after textless4 weeks of treatment. In vitro treatment of PANC1 cells showed reduced tumor engraftment in nude mice,concomitant with a reduction in the CSC levels. Differences between telomerase activity expression levels or telomere length of CSCs and bulk tumor cells in these cell lines did not correlate with the increased sensitivity of CSCs to imetelstat,suggesting a mechanism of action independent of telomere shortening for the effects of imetelstat on the CSC subpopulations. Our results suggest that imetelstat-mediated depletion of CSCs may offer an alternative mechanism by which telomerase inhibition may be exploited for cancer therapy. View Publication

We have studied the effects of purvalanol A on the cell cycle progression,proliferation and viability. In synchronized cells,purvalanol A induced a reversible arrest the progression in G1 and G2 phase of the cell cycle,but did not prevent the completion of DNA synthesis in S-phase cells. The specificity of action of the drug was supported by the selective inhibition of the phosphorylation of cyclin-dependent kinase (cdk) substrates such as Rb and cyclin E. The cell contents of cyclins D1 and E were lower in cells incubated with purvalanol A compared to controls,but the level of the cdk inhibitory protein p21(WAF1/CIP1) was increased,indicating that the drug did not cause a general inhibition of gene expression. Purvalanol A did not inhibit transcription under cell-free conditions. This compound,however,caused an inhibition of the estradiol-induced expression of an integrated luciferase gene,suggesting that cdk or related enzymes may participate in the regulation of the activity of certain promoters. When exponentially growing cells,both mouse fibroblasts and human cancer cell lines,were incubated with purvalanol A for prolonged periods of time (24 hr),a lasting inhibition of cell proliferation as well as cell death were observed. In contrast,a 24 hr incubation of quiescent (non-transformed) cells with purvalanol A did not prevent their resumption of cell cycle after removal of the drug. View Publication

The p38 MAP kinase plays a crucial role in regulating the production of proinflammatory cytokines,such as tumor necrosis factor and interleukin-1. Blocking this kinase may offer an effective therapy for treating many inflammatory diseases. Here we report a new allosteric binding site for a diaryl urea class of highly potent and selective inhibitors against human p38 MAP kinase. The formation of this binding site requires a large conformational change not observed previously for any of the protein Ser/Thr kinases. This change is in the highly conserved Asp-Phe-Gly motif within the active site of the kinase. Solution studies demonstrate that this class of compounds has slow binding kinetics,consistent with the requirement for conformational change. Improving interactions in this allosteric pocket,as well as establishing binding interactions in the ATP pocket,enhanced the affinity of the inhibitors by 12,000-fold. One of the most potent compounds in this series,BIRB 796,has picomolar affinity for the kinase and low nanomolar inhibitory activity in cell culture. View Publication

Telomere length must be tightly regulated in highly proliferative tissues,such as the lymphohematopoietic system. Under steady-state conditions,the levels and functionality of hematopoietic-committed or multipotent progenitors were not affected in late-generation telomerase-deficient mice (mTerc(-/-)) with critically short telomeres. Evaluation of self-renewal potential of mTerc(-/-) day-12 spleen colony-forming units demonstrated no alteration as compared with wildtype progenitors. However,the replating ability of mTerc(-/-) granulocyte-macrophage CFUs (CFU-GMs) was greatly reduced as compared with wildtype CFU-GMs,indicating a diminished capacity of late-generation mTerc(-/-) committed progenitors when forced to proliferate. Long-term bone marrow cultures of mTerc(-/-) bone marrow (BM) cells show a reduction in proliferative capacity; this defect can be mainly attributed to the hematopoietic,not to the stromal,mTerc(-/-) cells. In serial and competitive transplantations,mTerc(-/-) BM stem cells show reduced long-term repopulating capacity,concomitant with an increase in genetic instability compared with wildtype cells. Nevertheless,in competitive transplantations late-generation mTerc(-/-) precursors can occasionally overcome this proliferative impairment and reconstitute irradiated recipients. In summary,our results demonstrate that late-generation mTerc(-/-) BM cells with short telomeres,although exhibiting reduced proliferation ability and reduced long-term repopulating capacity,can still reconstitute myeloablated animals maintaining stem cell function. View Publication

Overexpression of the breast cancer resistance protein (BCRP) efflux pump in human cancer cell lines results in resistance to a variety of cytostatic agents. The aim of this study was to analyze BCRP protein expression and activity in acute myeloid leukemia (AML) samples and to determine whether it is up-regulated due to clonal selection at relapse/refractory disease. BCRP protein expression was measured flow cytometrically with the monoclonal antibodies BXP-34 and BXP-21 in 20 paired samples of de novo and relapsed/refractory AML. BXP-34/immunoglobulin G1 ratios were observed of 1.6 +/- 0.5 (mean +/- SD,range 0.8-2.7) and BXP-21/immunoglobulin G2a ratios of 4.9 +/- 3.0 (range 1.1-14.5) in the patient samples versus 9.8 +/- 6.8 and 6.5 +/- 2.4,respectively,in the MCF-7 cell line. BCRP activity was determined flow cytometrically by measuring mitoxantrone accumulation in absence and presence of the inhibitor fumitremorgin C. Mitoxantrone accumulation,expressed as mean fluorescence intensity (MFI),varied between 44 and 761 MFI (227 +/- 146 MFI) and correlated inversely with BCRP expression (r = -0.58,P textless.001). Addition of fumitremorgin C showed a small increase in mitoxantrone accumulation (11 +/- 29 MFI,n = 40) apart from the effect of PSC833 and MK-571. No consistent up-regulation of BCRP expression or activity was observed at relapse/refractory disease; some cases showed an increase and other cases a decrease at relapse. Relatively high BCRP expression correlated with immature immunophenotype,as determined by expression of the surface marker CD34 (r = 0.54,P =.001). In conclusion,this study shows that BCRP protein is expressed at low but variable levels in AML,especially in immature CD34(+) cells. BCRP was not consistently up-regulated in relapsed/refractory AML. View Publication

BACKGROUND AND OBJECTIVES: Leptin receptors can be expressed by acute myelogenous leukemia (AML) cells,but the functional effects of leptin on native AML blasts have not been characterized in detail. We investigated systemic leptin levels in AML patients and in vitro effects of leptin on cultured AML blasts. DESIGN AND METHODS: Serum leptin levels were compared for patients with untreated AML and healthy controls. Native AML blasts were derived from a large group of consecutive patients,and effects of leptin on proliferation (suspension cultures and colony formation),constitutive cytokine secretion,differentiation and apoptosis regulation were assayed in vitro. RESULTS: Systemic leptin levels were decreased in patients with untreated AML,and leptin levels in acute leukemia patients were not altered during severe chemotherapy-induced cytopenia and complicating febrile neutropenia. In vitro studies demonstrated that leptin increased AML blast release of interleukin (IL) 1beta,IL6,tumor necrosis factor (TNF) alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF). This enhancing effect showed no correlation with CD34 expression and was not dependent on the presence of serum,induction of differentiation or alteration of caspase 3 activity with decreased in vitro apoptosis. Leptin also increased spontaneous AML blast proliferation,whereas divergent effects on blast proliferation were observed in the presence of exogenous cytokines. The in vitro effects were usually observed at concentrations exceeding the systemic levels. INTERPRETATION AND CONCLUSIONS: Our results suggest that systemic leptin levels alone do not have a major influence on native AML blasts,but the systemic levels in combination with local leptin release in the bone marrow may affect the functional characteristics of these cells. View Publication

Small molecule inhibitors have proven extremely useful for investigating signal transduction pathways and have the potential for development into therapeutics for inhibiting signal transduction pathways whose activities contribute to human diseases. Transforming growth factor beta (TGF-beta) is a member of a large family of pleiotropic cytokines that are involved in many biological processes,including growth control,differentiation,migration,cell survival,adhesion,and specification of developmental fate,in both normal and diseased states. TGF-beta superfamily members signal through a receptor complex comprising a type II and type I receptor,both serine/threonine kinases. Here,we characterize a small molecule inhibitor (SB-431542) that was identified as an inhibitor of activin receptor-like kinase (ALK)5 (the TGF-beta type I receptor). We demonstrate that it inhibits ALK5 and also the activin type I receptor ALK4 and the nodal type I receptor ALK7,which are very highly related to ALK5 in their kinase domains. It has no effect on the other,more divergent ALK family members that recognize bone morphogenetic proteins (BMPs). Consistent with this,we demonstrate that SB-431542 is a selective inhibitor of endogenous activin and TGF-beta signaling but has no effect on BMP signaling. To demonstrate the specificity of SB-431542,we tested its effect on several other signal transduction pathways whose activities depend on the concerted activation of multiple kinases. SB-431542 has no effect on components of the ERK,JNK,or p38 MAP kinase pathways or on components of the signaling pathways activated in response to serum. View Publication