Chronic myelogenous leukemia (CML) in IntroductionThe management of chronic myelogenous leukemia (CML) has been revolutionized by kinase inhibitors that were developed in response to cues from biologic studies of the BCR-ABL1 oncogene. However two challenging problems persist: the progression of the disease to blast crisis and resistance to kinase inhibition. 1 Continued investigation of BCR-ABL1 kinase signaling will provide insight into these problems. Members of the Src kinase family, which regulate proliferation, differentiation, and motility, 2 are known downstream targets of BCR-ABL1. In myeloid cell lines, BCR-ABL1 activates Lyn and Hck. 3,4 Several reports link growth, survival, and imatinib resistance of Philadelphia chromosome-positive (Ph ϩ ) leukemias to Lyn kinase expression and activation. 5,6 However, reports examining Fyn, a ubiquitously expressed Src family member, are sparse. Of note, phase-specific gene expression in CML using microarray analyses revealed that Fyn gene expression was linked to imatinib relapse. 7 In addition, a separate study using combined systems biology and gene expression approaches in Ph ϩ acute lymphoblastic leukemia (ALL) specimens identified Fyn as a hub for signaling. 8 Here we show that Fyn protein expression is increased in patients with blast-crisis CML compared with chronic-phase disease. By examining effects of silencing Fyn using shRNA, we find that Fyn transduces a mitogenic signal. Collectively, our results identify a novel effect of BCR-ABL1-up-regulation of Fyn-and delineate consequences of the observed up-regulation. MethodsPatient specimens were used for this study and were collected after informed consent was obtained in accordance with the Declaration of Antibodies, chemicals, and cell linesAntibodies were purchased from sources outlined in Document S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). Imatinib was kindly provided by Dr Elisabeth Buchdunger at Novartis Pharmaceuticals (Basel, Switzerland). Murine growth factor-dependent pro-B lymphoid BaF3 cell lines transformed with vector, wild-type BCR-ABL1, or imatinib-resistant mutant BCR-ABL1 were kindly provided by Dr Charles Sawyers 9 and were cultured as previously described. 9 K562 cells, TonB210 cells stably expressing a tetracyclineinducible BCR-ABL1 expression vector (kindly provided by Dr George Daley, Children's Hospital Boston, Harvard Medical School, MA), 10 and mouse 32D and 32Dp210 cells were maintained in RPMI1640 medium with 10% FBS supplemented. Mouse 32D cells were supplemented with 10% WEHI-cultured conditioned medium as a source of interleukin-3 (IL-3) in addition to 10% FBS. Design of shRNA to FynK562 cells were transfected with Fyn shRNA and control vectors (TranSilent human shRNA from Panomics, Redwood, CA) using the Nucleofector system kit V and transfection program T-16 (Amaxa Biosystems, Cologne, Germany). Lentiviral knockdown of Fyn and rescue design is detailed in Document S1. To generate the rescue construct, 4 nucleo...
Cells are the most basic structural units that play vital roles in the functioning of living organisms.
Recent knowledge of the cellular and molecular mechanisms underlying cutaneous wound healing has advanced the development of medical products. However, patients still suffer from the failure of current treatments, due to the complexity of healing process and thus novel therapeutic approaches are urgently needed. Previously, our laboratories produced a range of low molecular weight hyaluronic acid (LMW-HA) fragments, where a proportion of the glucosamine moieties were chemically N-acyl substituted. Specifically, N-butyrylation results in anti-inflammatory properties in a macrophage system, and we demonstrate the importance of N-acyl substituents in modulating the inflammatory response of LMW-HA. We have set up an inter-institutional collaborative program to examine the biomedical applications of the N-butyrylated LMW-HA (BHA). In this study, the potentials of BHA for dermal healing are assessed in vitro and in vivo. Consequently, BHA significantly promotes dermal healing relative to a commercial wound care product. By contrast, the “parent” partially de-acetylated LMW-HA (DHA) and the re-acetylated DHA (AHA) significantly delays wound closure, demonstrating the specificity of this N-acylation of LMW-HA in wound healing. Mechanistic studies reveal that the BHA-mediated therapeutic effect is achieved by targeting three phases of wound healing (i.e., inflammation, proliferation and maturation), demonstrating the significant potential of BHA for clinical translation in cutaneous wound healing.
The Drosophila ovarian germline stem cells (GSCs) constantly experience self-renewal and differentiation, ensuring the female fertility throughout life. The balance between GSC self-renewal and differentiation is exquisitely regulated by the stem cell niche, the stem cells themselves and systemic factors. Increasing evidence has shown that the GSC regulation also involves epigenetic mechanisms including chromatin remodeling and histone modification. Here, we find that dBre1, an E3 ubiquitin ligase, functions in controlling GSC self-renewal and germ cell differentiation via distinct mechanisms. Removal or knock down of dBre1 function in the germline or somatic niche cell lineage leads to a gradual GSC loss and disruption of H3K4 trimethylation in the Drosophila ovary. Further studies suggest that the defective GSC maintenance is attributable to compromised BMP signaling emitted from the stem cell niche and impaired adhesion of GSCs to their niche. On the other hand, dBre1-RNAi expression in escort cells causes a loss of H3K4 trimethylation and accumulation of spectrosome-containing single germ cells in the germarium. Reducing dpp or dally levels suppresses the germ cell differentiation defects, indicating that dBre1 limits BMP signaling activities for the differentiation control. Strikingly, all phenotypes observed in dBre1 mutant ovaries can be mimicked by RNAi-based reduced expression of dSet1, a Drosophila H3K4 trimethylase. Moreover, genetic studies favor that dBre1 interacts with dSet1 in controlling GSC maintenance and germ cell differentiation. Taken together, we identify a dBre1/dSet1-dependent pathway for the H3K4 methylation involved in the cell fate regulation in the Drosophila ovary.
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