搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

An in vivo transplantation system has been used to evaluate the developmental capacities of specific mouse mammary epithelial cell populations. Specifically,mouse mammary epithelial cells with distinctly limited developmental potentials have been identified using this procedure. Two distinct epithelial cell progenitors have been identified by experiments designed to determine whether basal lobular and ductal phenotypes could develop independently under conditions imposed by a limiting dilution. The prediction that these separate epithelial progenitors must exist was based upon the results from transplantation experiments carried out in epithelium-divested mammary fat pads of syngeneic mice with mammary epithelium from two different transgenic mouse models. The results presented here demonstrate the following points: 1) lobular,i.e. secretory,progenitor cells are present as distinct entities among the mammary epithelial cells found in immature virgin female mice; 2) similarly,ductal epithelial progenitors are present within the same population; 3) lobular progenitors are present in greater numbers,although both cell populations are extremely small; 4) as expected,some inocula produce outgrowths with simultaneous development of both lobular and ductal phenotypes--it is not known whether this indicates cooperative interaction between the two epithelial progenitors or signals the presence of a third progenitor type capable of producing both ductular and lobular committed daughters; 5) these findings have important consequences in the design of experiments aimed at testing the effects of known and putative mammary oncogenes and tumor suppressor genes,using techniques which include cellular transformation in vitro followed by in vivo cultivation and evaluation. 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

The bioorthogonal keto group has attracted interest for the site-specific chemical conjugation of recombinant proteins under mild conditions,e.g. with aminooxy-functionalised fluorescent probes,radiometal chelates,toxins or polymers. However,the cotranslational incorporation of the corresponding non-canonical amino acid p-acetyl-L-phenylalanine (Apa) into proteins expressed in Escherichia coli by means of amber suppression using a previously described system with a mutated tRNA and an engineered tyrosyl-tRNA synthetase from Methanococcus jannaschii shows limited efficiency and considerable promiscuity towards endogenous amino acids. Employing a one-plasmid system that encodes all three components required for selection,i.e. the modified aminoacyl-tRNA synthetase (aaRS),the cognate amber suppressor tRNA and the enhanced green fluorescent protein equipped with an amber stop codon and serving as reporter,we have generated an Apa-specific aaRS&tRNA pair with considerably improved efficiency (17-fold increased expression) and also fidelity (6-fold). To this end,both the aaRS and the tRNA were subjected to doped random mutagenesis and selection in altogether four evolutionary cycles using fluorescence-activated bacterial cell sorting as well as automated screening of microcultures. The resulting aaRS&tRNA pair was applied to the functionalisation of an Anticalin with specificity towards oncofetal fibronectin by introducing a keto group at a permissible site for subsequent conjugation with a fluorescent dye,thus allowing visualisation of this tumour target under the microscope. View Publication

BACKGROUND: The mitomycins are natural products that contain a variety of functional groups,including aminobenzoquinone- and aziridine-ring systems. Mitomycin C (MC) was the first recognized bioreductive alkylating agent,and has been widely used clinically for antitumor therapy. Precursor-feeding studies showed that MC is derived from 3-amino-5-hydroxybenzoic acid (AHBA),D-glucosamine,L-methionine and carbamoyl phosphate. A genetically linked AHBA biosynthetic gene and MC resistance genes were identified previously in the MC producer Streptomyces lavendulae NRRL 2564. We set out to identify other genes involved in MC biosynthesis. RESULTS: A cluster of 47 genes spanning 55 kilobases of S. lavendulae DNA governs MC biosynthesis. Fourteen of 22 disruption mutants did not express or overexpressed MC. Seven gene products probably assemble the AHBA intermediate through a variant of the shikimate pathway. The gene encoding the first presumed enzyme in AHBA biosynthesis is not,however,linked within the MC cluster. Candidate genes for mitosane nucleus formation and functionalization were identified. A putative MC translocase was identified that comprises a novel drug-binding and export system,which confers cellular self-protection on S. lavendulae. Two regulatory genes were also identified. CONCLUSIONS: The overall architecture of the MC biosynthetic gene cluster in S. lavendulae has been determined. Targeted manipulation of a putative MC pathway regulator led to a substantial increase in drug production. The cloned genes should help elucidate the molecular basis for creation of the mitosane ring system,as well efforts to engineer the biosynthesis of novel natural products. View Publication

Human embryonic stem cells (hESCs) is a potential unlimited ex vivo source of ventricular (V) cardiomyocytes (CMs),but hESC-VCMs and their engineered tissues display immature traits. In adult VCMs,sarcolemmal (sarc) and mitochondrial (mito) ATP-sensitive potassium (KATP) channels play crucial roles in excitability and cardioprotection. In this study,we aim to investigate the biological roles and use of sarcKATP and mitoKATP in hESC-VCM. We showed that SarcIK,ATP in single hESC-VCMs was dormant under baseline conditions,but became markedly activated by cyanide (CN) or the known opener P1075 with a current density that was ˜8-fold smaller than adult; These effects were reversible upon washout or the addition of GLI or HMR1098. Interestingly,sarcIK,ATP displayed a ˜3-fold increase after treatment with hypoxia (5% O2). MitoIK,ATP was absent in hESC-VCMs. However,the thyroid hormone T3 up-regulated mitoIK,ATP,conferring diazoxide protective effect on T3-treated hESC-VCMs. When assessed using a multi-cellular engineered 3D ventricular cardiac micro-tissue (hvCMT) system,T3 substantially enhanced the developed tension by 3-folds. Diazoxide also attenuated the decrease in contractility induced by simulated ischemia (1% O2). We conclude that hypoxia and T3 enhance the functionality of hESC-VCMs and their engineered tissues by selectively acting on sarc and mitoIK,ATP. View Publication

PURPOSE: Despite their preclinical promise,previous MEK inhibitors have shown little benefit for patients. This likely reflects the narrow therapeutic window for MEK inhibitors due to the essential role of the P42/44 MAPK pathway in many nontumor tissues. GSK1120212 is a potent and selective allosteric inhibitor of the MEK1 and MEK2 (MEK1/2) enzymes with promising antitumor activity in a phase I clinical trial (ASCO 2010). Our studies characterize GSK1120212' enzymatic,cellular,and in vivo activities,describing its unusually long circulating half-life. EXPERIMENTAL DESIGN: Enzymatic studies were conducted to determine GSK1120212 inhibition of recombinant MEK,following or preceding RAF kinase activation. Cellular studies examined GSK1120212 inhibition of ERK1 and 2 phosphorylation (p-ERK1/2) as well as MEK1/2 phosphorylation and activation. Further studies explored the sensitivity of cancer cell lines,and drug pharmacokinetics and efficacy in multiple tumor xenograft models. RESULTS: In enzymatic and cellular studies,GSK1120212 inhibits MEK1/2 kinase activity and prevents Raf-dependent MEK phosphorylation (S217 for MEK1),producing prolonged p-ERK1/2 inhibition. Potent cell growth inhibition was evident in most tumor lines with mutant BRAF or Ras. In xenografted tumor models,GSK1120212 orally dosed once daily had a long circulating half-life and sustained suppression of p-ERK1/2 for more than 24 hours; GSK1120212 also reduced tumor Ki67,increased p27(Kip1/CDKN1B),and caused tumor growth inhibition in multiple tumor models. The largest antitumor effect was among tumors harboring mutant BRAF or Ras. CONCLUSIONS: GSK1120212 combines high potency,selectivity,and long circulating half-life,offering promise for successfully targeting the narrow therapeutic window anticipated for clinical MEK inhibitors. View Publication

Molluscum contagiosum poxvirus (MCV) type 1 and type 2 encode two chemokine-like proteins MC148R1 and MC148R2. It is believed that MC148R proteins function by blocking the inflammatory response. However,the mechanism of the proposed biological activities of MC148R proteins and the role of the additional C-terminal cysteines that do not exist in other chemokines are not understood. Here,we demonstrated in two different assay systems that His-tagged MC148R1 displaces the interaction between CXCL12α and CXCR4. The N-terminal cysteines but not the additional C-terminal cysteines modulate this displacement. His-tagged MC148R1 blocked both CXCL12α-mediated and MIP-1α-mediated chemotaxis. In contrast,MC148R2 blocked MIP-1α-mediated but not CXCL12α-mediated chemotaxis. Immunoprecipitation by antibodies to MC148R1 or CXCL12α followed by immunoblotting and detection by antibodies to the other protein demonstrated physical interaction of His-tagged CXCL12α and His-tagged MC148R1. Interaction with chemokines might mask the receptor interaction site resulting in decreased binding and impairment of the biological activities. View Publication

First developed for hematologic disorders,the concept of cancer stem cells (CSCs) was expanded to solid tumors,including colorectal cancer (CRC). The traditional model of colon carcinogenesis includes several steps that occur via mutational activation of oncogenes and inactivation of tumor suppressor genes. Intestinal epithelial cells exist for a shorter amount of time than that required to accumulate tumor-inducing genetic changes,so researchers have investigated the concept that CRC arises from the long-lived stem cells,rather than from the differentiated epithelial cells. Colon CSCs were originally identified through the expression of the CD133 glycoprotein using an antibody directed to its epitope AC133. It is not clear if CD133 is a marker of colon CSCs-other cell surface markers,such as epithelial-specific antigen,CD44,CD166,Musashi-1,CD29,CD24,leucine-rich repeat-containing G-protein-coupled receptor 5,and aldehyde dehydrogenase 1,have been proposed. In addition to initiating and sustaining tumor growth,CSCs are believed to mediate cancer relapse after chemotherapy. How can we identify and analyze colon CSCs and what agents are being designed to kill this chemotherapy-refractory population? View Publication

BACKGROUND Transplantation of endothelial progenitor cells (EPCs) improves neovascularization after ischemia,but patients with coronary artery disease (CAD) or diabetes mellitus show a reduced number of EPCs and impaired functional activity. Therefore,we investigated the effects of risk factors,such as glucose and TNF-alpha,on the number of EPCs in vitro to elucidate the underlying mechanisms. METHODS AND RESULTS EPCs of patients or healthy subjects were isolated from peripheral blood. Incubation with glucose or TNF-alpha dose-dependently reduced the number of EPCs (79.9+/-1.3% and 74.3+/-8.1% of control; Ptextless0.05,respectively). This reduction was not caused by apoptosis. TNF-alpha and glucose induced a dose- and time-dependent activation of the p38 MAP kinase,the downstream kinase mitogen- and stress-activated kinase 1,and the transcription factor cAMP-responsive element-binding protein (CREB),in EPCs. Moreover,EPCs from CAD patients had significantly higher basal p38-phosphorylation levels (1.83+/-0.2-fold increase; Ptextless0.05) compared with healthy subjects. The inhibition of the p38-kinase by SB203580 or infection with a dominant negative p38 kinase adenovirus significantly increased basal number of EPCs (136.7+/-6.3% and 142.9+/-18% versus control,respectively). Likewise,ex vivo cultivation of EPCs from patients with CAD with SB203580 significantly increased the number of EPCs and partially reversed the impaired capacity for neovascularization of EPCs in vivo (relative blood flow: 0.40+/-0.03 versus 0.64+/-0.08,Ptextless0.05). The increased numbers of EPCs by SB203580 were associated with an augmentation of EPC proliferation and a reduction of cells expressing the monocytic marker proteins CD14 and CD64,suggesting that p38 regulates proliferation and differentiation events. CONCLUSIONS These results demonstrate that p38 MAP kinase plays a pivotal role in the signal transduction pathways regulating the number of EPCs ex vivo. SB203580 can prevent the negative effects of TNF-alpha and glucose on the number of EPCs and may be useful to improve the number of EPCs for potential cell therapy. View Publication

Development of resistance to therapy continues to be a serious clinical problem in breast cancer management. Cancer stem/progenitor cells have been shown to play roles in resistance to chemo? and radiotherapy. Here,we examined their role in the development of resistance to the oestrogen receptor antagonist tamoxifen. Tamoxifen?resistant cells were enriched for stem/progenitors and expressed high levels of the stem cell marker Sox2. Silencing of the SOX2 gene reduced the size of the stem/progenitor cell population and restored sensitivity to tamoxifen. Conversely,ectopic expression of Sox2 reduced tamoxifen sensitivity in vitro and in vivo. Gene expression profiling revealed activation of the Wnt signalling pathway in Sox2?expressing cells,and inhibition of Wnt signalling sensitized resistant cells to tamoxifen. Examination of patient tumours indicated that Sox2 levels are higher in patients after endocrine therapy failure,and also in the primary tumours of these patients,compared to those of responders. Together,these results suggest that development of tamoxifen resistance is driven by Sox2?dependent activation of Wnt signalling in cancer stem/progenitor cells. View Publication