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

Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays. Relative to unmutated (native) Bcr-Abl,the ATP binding loop mutants Y253F and E255K exhibited increased transformation potency,M351T and H396P were less potent,and the performance of T315I was assay dependent. The transformation potency of Y253F and M351T correlated with intrinsic Bcr-Abl kinase activity,whereas the kinase activity of E255K,H396P,and T315I did not correlate with transforming capabilities,suggesting that additional factors influence transformation potency. Analysis of the phosphotyrosine proteome by mass spectroscopy showed differential phosphorylation among the mutants,a finding consistent with altered substrate specificity and pathway activation. Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion,leading to gain- or loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors. View Publication

Overcoming imatinib mesylate (IM) resistance and disease persistence in patients with chronic myeloid leukemia (CML) is of considerable importance to the issue of potential cure. Here we asked whether autocrine signaling contributes to survival of BCR/ABL+ cells in the presence of IM and nilotinib (NI; AMN107),a novel,more selective Abl inhibitor. Conditioned media (CM) of IM-resistant LAMA84 cell clones (R-CM) was found to substantially protect IM-naive LAMA cells and primary CML progenitors from IM- or NI-induced cell death. This was due to an increased secretion of the granulocyte-macrophage colony-stimulating factor (GM-CSF),which was identified as the causative factor mediating IM resistance in R-CM. GM-CSF elicited IM and NI drug resistance via a BCR/ABL-independent activation of the janus kinases 2 (JAK-2)/signal transducer and activator of transcription 5 (STAT-5) signaling pathway in GM-CSF receptor alpha receptor (CD116)-expressing cells,including primary CD34+/CD116+ GM progenitors (GMPs). Elevated mRNA and protein levels of GM-CSF were detected in IM-resistant patient samples,suggesting a contribution of GM-CSF secretion for IM and NI resistance in vivo. Importantly,inhibition of JAK-2 with AG490 abrogated GM-CSF-mediated STAT-5 phosphorylation and NI resistance in vitro. Together,adaptive autocrine secretion of GM-CSF mediates BCR/ABL-independent IM and NI resistance via activation of the antiapoptotic JAK-2/STAT-5 pathway. Inhibition of JAK-2 overcomes GM-CSF-induced IM and NI progenitor cell resistance,providing a rationale for the application of JAK-2 inhibitors to eradicate residual disease in CML. View Publication

Multiple myeloma is characterized by increased osteoclast activity that results in bone destruction and lytic lesions. With the prolonged overall patient survival achieved by new treatment modalities,additional drugs are required to inhibit bone destruction. We focused on a novel and more potent structural analog of the nonsteroidal anti-inflammatory drug etodolac,known as SDX-308,and its effects on osteoclastogenesis and multiple myeloma cells. SDX-101 is another structural analog of etodolac that is already used in clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Compared with SDX-101,a 10-fold lower concentration of SDX-308 induced potent (60%-80%) inhibition of osteoclast formation,and a 10- to 100-fold lower concentration inhibited multiple myeloma cell proliferation. Bone resorption was completely inhibited by SDX-308,as determined in dentin-based bone resorption assays. SDX-308 decreased constitutive and RANKL-stimulated NF-kappaB activation and osteoclast formation in an osteoclast cellular model,RAW 264.7. SDX-308 effectively suppressed TNF-alpha-induced IKK-gamma and IkappaB-alpha phosphorylation and degradation and subsequent NF-kappaB activation in human multiple myeloma cells. These results indicate that SDX-308 effectively inhibits multiple myeloma cell proliferation and osteoclast activity,potentially by controlling NF-kappaB activation signaling. We propose that SDX-308 is a promising therapeutic candidate to inhibit multiple myeloma growth and osteoclast activity and that it should receive attention for further study. View Publication

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

Maintenance of genomic integrity is an essential cellular function. We previously reported that the transcription factor and tumor suppressor CCAAT/enhancer binding protein δ (C/EBPδ,CEBPD; also known as NFIL-6β") promotes genomic stability. However� View Publication

Bone marrow (BM) hematopoietic cells are selectively sensitive to polycyclic aromatic hydrocarbons (PAH) in vivo. 7,12-Dimethylbenz(a)anthracene (DMBA),but not benzo(a)pyrene (BP),depletes BM hematopoietic cells in C57BL/6 mice. This difference is due to a BP-selective aryl hydrocarbon receptor (AhR)-mediated recovery. Colony-forming unit assays show suppression of lymphoid progenitors by each PAH within 6 h but a subsequent recovery,exclusively after BP treatment. Suppression of myeloid progenitors (6 h) occurs only for DMBA. Each progenitor responded equally to DMBA and BP in congenic mice expressing the PAH-resistant AhR (AhR(d)). AhR,therefore,mediates this BP recovery in each progenitor type. These PAH suppressions depend on Cyp1b1-mediated metabolism. Paradoxically,few genes responded to DMBA,whereas 12 times more responded to BP. Progenitor suppression by DMBA,therefore,occurs with minimal effects on the general BM population. Standard AhR-mediated stimulations (Cyp1a1,Cyp1b1,Ahrr) were similar for each PAH and for the specific agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin but were absent in AhR(d) mice. A group of 12 such AhR responses was sustained from 6 to 24 h. A second,larger set of BP responses (chemokines,cytokines,cyclooxygenase 2) differed in two respects; DMBA responses were low and BP responses declined extensively from 6 to 24 h. A third cluster exhibited BP-induced increases in protective genes (Nqo1,GST-mu) that appeared only after 12 h. Conversion of BP to quinones contributes oxidative signaling not seen with DMBA. We propose that genes in this second cluster,which share oxidative signaling and AhR activation,provide the AhR-dependent protection of hematopoietic progenitors seen for BP. View Publication

The mammalian embryonic zeta-globin genes,including that of humans,are expressed at the early embryonic stage and then switched off during erythroid development. This autonomous silencing of the zeta-globin gene transcription is probably regulated by the cooperative work of various protein-DNA and protein-protein complexes formed at the zeta-globin promoter and its upstream enhancer (HS-40). We present data here indicating that a protein-binding motif,ZF2,contributes to the repression of the HS-40-regulated human zeta-promoter activity in erythroid cell lines and in transgenic mice. Combined site-directed mutagenesis and EMSA suggest that repression of the human zeta-globin promoter is mediated through binding of the zinc finger factor RREB1 to ZF2. This model is further supported by the observation that human zeta-globin gene transcription is elevated in the human erythroid K562 cell line or the primary erythroid culture upon RNA interference (RNAi)(2) knockdown of RREB1 expression. These data together suggest that RREB1 is a putative repressor for the silencing of the mammalian zeta-globin genes during erythroid development. Because zeta-globin is a powerful inhibitor of HbS polymerization,our experiments have provided a foundation for therapeutic up-regulation of zeta-globin gene expression in patients with severe hemoglobinopathies. View Publication

Aldehyde dehydrogenase isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. However,our knowledge of the expression and activity of ALDH1 in common epithelial cancers and their corresponding normal tissues is still largely absent. Therefore,we characterized ALDH1 expression in 24 types of normal tissues and a large collection of epithelial tumor specimens (six cancer types,n = 792) by immunohistochemical staining. Using the ALDEFUOR assay,ALDH1 activity was also examined in 16 primary tumor specimens and 43 established epithelial cancer cell lines. In addition,an ovarian cancer transgenic mouse model and 7 murine ovarian cancer cell lines were analyzed. We found that the expression levels and patterns of ALDH1 in epithelial cancers are remarkably distinct,and they correlate with their corresponding normal tissues. ALDH1 protein expression levels are positively correlated with ALDH1 enzymatic activity measured by ALDEFLUOR assay. Long-term in vitro culture doesn't significantly affect ALDH1 activity in epithelial tumor cells. Consistent with research on other cancers,we found that high ALDH1 expression is significantly associated with poor clinical outcomes in serous ovarian cancer patients (n = 439,p = 0.0036). Finally,ALDH(br) tumor cells exhibit cancer stem cell properties and are resistant to chemotherapy. As a novel cancer stem cell marker,ALDH1 can be used for tumors whose corresponding normal tissues express ALDH1 in relatively restricted or limited levels such as breast,lung,ovarian or colon cancer. View Publication

Because video data are complex and are comprised of many images,mining information from video material is difficult to do without the aid of computer software. Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article,we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments,hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT. To determine the rate of growth of these colonies,recipes were developed using CL-Quant software which enables users to extract various types of data from video images. To accurately evaluate colony growth,three recipes were created. The first segmented the image into the colony and background,the second enhanced the image to define colonies throughout the video sequence accurately,and the third measured the number of pixels in the colony over time. The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes,the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared,results were virtually identical,indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition,other video bioinformatics recipes can be developed in the future for other cell processes such as migration,apoptosis,and cell adhesion. View Publication

Osteoclasts are resident cells of the bone that are primarily involved in the physiological and pathological remodeling of this tissue. Mature osteoclasts are multinucleated giant cells that are generated from the fusion of circulating precursors originating from the monocyte/macrophage lineage. During inflammatory bone conditions in vivo,de novo osteoclastogenesis is observed but it is currently unknown whether,besides increased osteoclast differentiation from undifferentiated precursors,other cell types can generate a multinucleated giant cell phenotype with bone resorbing activity. In this study,an animal model of calvaria-induced aseptic osteolysis was used to analyze possible bone resorption capabilities of dendritic cells (DCs). We determined by FACS analysis and confocal microscopy that injected GFP-labeled immature DCs were readily recruited to the site of osteolysis. Upon recruitment,the cathepsin K-positive DCs were observed in bone-resorbing pits. Additionally,chromosomal painting identified nuclei from female DCs,previously injected into a male recipient,among the nuclei of giant cells at sites of osteolysis. Finally,osteolysis was also observed upon recruitment of CD11c-GFP conventional DCs in Csf1r(-/-) mice,which exhibit a severe depletion of resident osteoclasts and tissue macrophages. Altogether,our analysis indicates that DCs may have an important role in bone resorption associated with various inflammatory diseases. View Publication

Human embryonic stem (hES) cells have a potential use for the repair and regeneration of injured tissues. However,teratoma formation can be a major obstacle for hES-mediated cell therapy. Therefore,tracking the fate and function of transplanted hES cells with noninvasive imaging could be valuable for a better understanding of the biology and physiology of teratoma formation. In this study,hES cells were stably transduced with a double fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein. Following bioluminescence imaging and histology,we demonstrated that engraftment of hES cells was followed by dramatically increasing signaling and led to teratoma formation confirmed by histology. Studies of the angiogenic processes within teratomas revealed that their vasculatures were derived from both differentiated hES cells and host. Moreover,FACS analysis showed that teratoma cells derived from hES cells expressed high levels of CD56 and SSEA-4,and the subcultured SSEA-4(+) cells showed a similar cell surface marker expression pattern when compared to undifferentiated hES cells. We report here for the first time that SSEA-4(+) cells derived from teratoma exhibited multipotency,retained their differentiation ability in vivo as confirmed by their differentiation into representative three germ layers. View Publication