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

Retinoic acid (RA) is used to treat leukemia and other cancers through its ability to promote cancer cell differentiation. Strategies to enhance the anticancer effects of RA could deepen and broaden its beneficial therapeutic applications. In this study,we describe a receptor cross-talk system that addresses this issue. RA effects are mediated by RAR/RXR receptors that we show are modified by interactions with the aryl hydrocarbon receptor (AhR),a protein functioning both as a transcription factor and a ligand-dependent adaptor in an ubiquitin ligase complex. RAR/RXR and AhR pathways cross-talk at the levels of ligand-receptor and also receptor-promoter interactions. Here,we assessed the role of AhR during RA-induced differentiation and a hypothesized convergence at Oct4,a transcription factor believed to maintain stem cell characteristics. RA upregulated AhR and downregulated Oct4 during differentiation of HL-60 promyelocytic leukemia cells. AhR overexpression in stable transfectants downregulated Oct4 and also decreased ALDH1 activity,another stem cell-associated factor,enhancing RA-induced differentiation as indicated by cell differentiation markers associated with early (CD38 and CD11b) and late (neutrophilic respiratory burst) responses. AhR overexpression also increased levels of activated Raf1,which is known to help propel RA-induced differentiation. RNA interference-mediated knockdown of Oct4 enhanced RA-induced differentiation and G(0) cell-cycle arrest relative to parental cells. Consistent with the hypothesized importance of Oct4 downregulation for differentiation,parental cells rendered resistant to RA by biweekly high RA exposure displayed elevated Oct4 levels that failed to be downregulated. Together,our results suggested that therapeutic effects of RA-induced leukemia differentiation depend on AhR and its ability to downregulate the stem cell factor Oct4. View Publication

BACKGROUND: Cancer stem cells (CSCs) are purported to be epithelial tumour cells expressing CD44(+)CD24(lo) that exhibit aldehyde dehydrogenase activity (Aldefluor(+)). We hypothesised that if CSCs are responsible for tumour dissemination,disseminated cells in the bone marrow (BM) would be positive for putative breast CSC markers. Therefore,we assessed the presence of Aldefluor(+) epithelial (CD326(+)CD45(dim)) cells for the presence of the CD44(+)CD24(lo) phenotype in BM of patients with primary breast cancer (PBC). METHODS: BM aspirates were collected at the time of surgery from 66 patients with PBC. Thirty patients received neoadjuvant chemotherapy (NACT) prior to aspiration. BM was analysed for Aldefluor(+) epithelial cells with or without CD44(+)CD24(lo) expression by flow cytometry. BM aspirates from three healthy donors (HD) were subjected to identical processing and analyses and served as controls. RESULTS: Patients with triple-receptor-negative (TN) tumours had a significantly higher median percentage of CD44(+)CD24(lo) CSC within Aldefluor(+) epithelial cell population than patients with other immunohistochemical subtypes (P=0.018). Patients with TN tumours or with pN2 or higher pathologic nodal status were more likely to have a proportion of CD44(+)CD24(lo) CSC within Aldefluor(+) epithelial cell population above the highest level of HD. Furthermore,patients who received NACT were more likely to have percentages of Aldefluor(+) epithelial cells than the highest level of HD (P=0.004). CONCLUSION: The percentage of CD44(+)CD24(lo) CSC in the BM is higher in PBC patients with high risk tumour features. The selection or enrichment of Aldefluor(+) epithelial cells by NACT may represent an opportunity to target these cells with novel therapies. View Publication

Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab,the mechanisms mediating this resistance remain elusive. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrate that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of the cancer stem cell (CSC) population. Long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies demonstrate that trastuzumab resistance may be mediated by an IL6 inflammatory loop and suggest that blocking this loop may provide alternative strategy to overcome trastuzumab resistance. 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

BACKGROUND Interleukin (IL-6) and transforming growth factor (TGF)-beta have been shown to play a role in skin development and maintenance. OBJECTIVES A link between these two cytokines has yet to be identified and therefore in this study we investigated the modulation of TGF-beta1 and TGF-beta type 2 receptor (TGF-betaR2) by IL-6 in skin. METHODS An IL-6 knockout (IL-6KO) fibroblast-populated lattice model and intradermal injections of IL-6 into unwounded IL-6KO mice were used to investigate the direct effects of IL-6 treatment on TGF-beta and TGF-betaR2 expression and to determine the signalling mechanism. In addition,IL-6KO and C57BL/6 control mice were wounded by a 4-mm punch biopsy to monitor expression of TGF-beta1 and TGF-betaR2 within a wound over time. The expression of TGF-beta1 and TGF-betaR2 was assessed by real-time quantitative polymerase chain reaction,enzyme-linked immunosorbent assay and immunohistology. RESULTS Recombinant IL-6 treatment of IL-6KO lattices and intradermal injections of IL-6 showed a significant induction of TGF-beta1 mRNA and protein,with TGF-beta1 expression localized in the dermis,while TGF-betaR2 expression was primarily in the epidermis in IL-6KO mice. During healing,the expression of TGF-beta1 and TGF-betaR2 mRNA was significantly greater in unwounded and 7-day-old wounds from wild-type mice; however,protein expression did not differ. Treatment with signal transduction inhibitors indicated that IL-6 modulates TGF-beta through a mitogen-activated protein kinase/extracellular signal-regulated kinase (Mapk/Erk)-dependent mechanism. CONCLUSION These studies indicate that IL-6 has the ability to modulate the expression of TGF-beta and TGF-betaR2 to varying degrees in the skin,which may provide a possible mechanism for defining the role of IL-6 in skin maintenance and a new association of IL-6 with TGF-beta in pathologies associated with fibrosis. View Publication

The expression of an enzyme,GnT-V,that catalyzes a specific posttranslational modification of a family of glycoproteins,namely a branched N-glycan,is transcriptionally up-regulated during breast carcinoma oncogenesis. To determine the molecular basis of how early events in breast carcinoma formation are regulated by GnT-V,we studied both the early stages of mammary tumor formation by using 3D cell culture and a her-2 transgenic mouse mammary tumor model. Overexpression of GnT-V in MCF-10A mammary epithelial cells in 3D culture disrupted acinar morphogenesis with impaired hollow lumen formation,an early characteristic of mammary neoplastic transformation. The disrupted acinar morphogenesis of mammary tumor cells in 3D culture caused by her-2 expression was reversed in tumors that lacked GnT-V expression. Moreover,her-2-induced mammary tumor onset was significantly delayed in the GnT-V null tumors,evidence that the lack of the posttranslational modification catalyzed by GnT-V attenuated tumor formation. Inhibited activation of both PKB and ERK signaling pathways was observed in GnT-V null tumor cells. The proportion of tumor-initiating cells (TICs) in the mammary tumors from GnT-V null mice was significantly reduced compared with controls,and GnT-V null TICs displayed a reduced ability to form secondary tumors in NOD/SCID mice. These results demonstrate that GnT-V expression and its branched glycan products effectively modulate her-2-mediated signaling pathways that,in turn,regulate the relative proportion of tumor initiating cells and the latency of her-2-driven tumor onset. View Publication

BACKGROUND & AIMS The etiology of abdominal pain in postinfectious,diarrhea-predominant irritable bowel syndrome (PI-IBS-D) is unknown,and few treatment options exist. Catechol-O-methyltransferase (COMT),an enzyme that inactivates and degrades biologically active catecholamines,plays an important role in numerous physiologic processes,including modulation of pain perception. Our objective was to determine the mechanism(s) of how decreased colonic COMT in PI-IBS-D patients contributes to the chronic abdominal pain phenotype after enteric infections. METHODS Colon neurons,epithelial cells,and macrophages were procured with laser capture microdissection from PI-IBS-D patients to evaluate cell-specific colonic COMT,microRNA-155 (miR-155),and tumor necrosis factor (TNF) ? expression levels compared to recovered patients (infection cleared: did not develop PI-IBS-D) and control individuals. COMT-/-,colon-specific COMT-/-,and miR-155-/- mice and human colonoids were used to model phenotypic expression of COMT in PI-IBS-D patients and to investigate signaling pathways linking abdominal pain. Citrobacter rodentium and trinitrobenzene sulfonic acid animal models were used to model postinflammatory changes seen in PI-IBS-D patients. RESULTS Colonic COMT levels were significantly decreased and correlated with increased visual analog scale abdominal pain ratings in PI-IBS-D patients compared to recovered patients and control individuals. Colonic miR-155 and TNF-? were increased in PI-IBS-D patients with diminished colonic COMT. COMT-/- mice had significantly increased expression of miR-155 and TNF-? in both colon tissues and dorsal root ganglia. Introduction of cV1q antibody (anti-TNF-?) into mice reversed visceral hypersensitivity after C rodentium and trinitrobenzene sulfonic acid. CONCLUSIONS Decreased colonic COMT in PI-IBS-D patients drives abdominal pain phenotypes via the COMT/miR-155/TNF-? axis. These important findings will allow new treatment paradigms and more targeted and personalized medicine approaches for gastrointestinal disorders after enteric infections. View Publication

In vitro models of human bronchial epithelium are useful for toxicological testing because of their resemblance to in vivo tissue. We constructed a model of human bronchial tissue which has a fibroblast layer embedded in a collagen matrix directly below a fully-differentiated epithelial cell layer. The model was applied to whole cigarette smoke (CS) exposure repeatedly from an air-liquid interface culture while bronchial epithelial cells were differentiating. The effects of CS exposure on differentiation were determined by histological and gene expression analyses on culture day 21. We found a decrease in ciliated cells and perturbation of goblet cell differentiation. We also analyzed the effects of CS exposure on the inflammatory response,and observed a significant increase in secretion of IL-8,GRO-α,IL-1β,and GM-CSF. Interestingly,secretion of these mediators was augmented with repetition of whole CS exposure. Our data demonstrate the usefulness of our bronchial tissue model for in vitro testing and the importance of exposure repetition in perturbing the differentiation and inflammation processes. View Publication

OBJECTIVE To evaluate the effects of MUC18 on IL-13-mediated airway inflammatory responses in human airway epithelial cells and in mice. MATERIALS Primary normal human tracheobronchial epithelial (HTBE) cells,wild-type (WT) and Muc18 knockout (KO) mice,and mouse tracheal epithelial cells (mTECs) were utilized. TREATMENT Cultured HTBE cells treated with MUC18 siRNA or MUC18 expressing lentivirus were incubated with IL-13 (10 ng/mL) for 24 h. Mice were intranasally instilled with 500 ng of IL-13 for 3 days. mTECs were treated with IL-13 (10 ng/mL) for 3 days. METHODS PCR was used to measure mRNA expression. Western Blot and ELISAs were used to quantify protein expression. Cytospins of bronchoalveolar lavage (BAL) cells were used to obtain leukocyte differentials. RESULTS MUC18 siRNA reduced IL-13-mediated eotaxin-3 (183 ± 44 vs. 380 ± 59 pg/mL,p < 0.05),while MUC18 overexpression increased IL-13-mediated eotaxin-3 (95 ± 3 vs. 58 ± 3 pg/mL,p < 0.05) in HTBE cells. IL-13-treated Muc18 KO mice had a lower percentage of neutrophils in BAL than WT mice (25 ± 3 vs. 35 ± 3%,p = 0.0565). CONCLUSIONS These results implicate MUC18 as a potential enhancer of airway inflammation in a type 2 cytokine (e.g.,IL-13) milieu. View Publication