•Versican- and hyaluronan-deficient mouse mutants fail to form yolk sac vasculature and blood.•Mouse embryonic stem cells with CRISPR-Cas9 Vcan mutation have impaired angiogenesis and ...hematopoiesis.•The versican-hyaluronan complex is strongly associated with emerging Flk1+ hematoendothelial precursors.•Versican and hyaluronan are each destabilized by the absence of the other.•The versican chondroitin sulfate chains sequester vasculogenic factors VEGF and Indian Hedgehog.
Little is known about extracellular matrix (ECM) contributions to formation of the earliest cell lineages in the embryo. Here, we show that the proteoglycan versican and glycosaminoglycan hyaluronan are associated with emerging Flk1+ hematoendothelial progenitors at gastrulation. The mouse versican mutant Vcanhdf lacks yolk sac vasculature, with attenuated yolk sac hematopoiesis. CRISPR/Cas9-mediated Vcan inactivation in mouse embryonic stem cells reduced vascular endothelial and hematopoietic differentiation within embryoid bodies, which generated fewer blood colonies, and had an impaired angiogenic response to VEGF165. Hyaluronan was severely depleted in Vcanhdf embryos, with corresponding upregulation of the hyaluronan-depolymerase TMEM2. Conversely, hyaluronan-deficient mouse embryos also had vasculogenic suppression but with increased versican proteolysis. VEGF165 and Indian hedgehog, crucial vasculogenic factors, utilized the versican-hyaluronan matrix, specifically versican chondroitin sulfate chains, for binding. Versican-hyaluronan ECM is thus an obligate requirement for vasculogenesis and primitive hematopoiesis, providing a vasculogenic factor-enriching microniche for Flk1+ progenitors from their origin at gastrulation.
SHARPIN, an adaptor for the linear ubiquitin chain assembly complex (LUBAC), plays important roles in NF-κB signaling and inflammation. Here, we have demonstrated a LUBAC-independent role for SHARPIN ...in regulating melanoma growth. We observed that SHARPIN interacted with PRMT5, a type II protein arginine methyltransferase, and increased its multiprotein complex and methyltransferase activity. Activated PRMT5 controlled the expression of the transcription factors SOX10 and MITF by SHARPIN-dependent arginine dimethylation and inhibition of the transcriptional corepressor SKI. Activation of PRMT5 by SHARPIN counteracted PRMT5 inhibition by methylthioadenosine, a substrate of methylthioadenosine phosphorylase, which is codeleted with cyclin-dependent kinase inhibitor 2A (CDKN2A) in approximately 15% of human cancers. Collectively, we identified a LUBAC-independent role for SHARPIN in enhancing PRMT5 activity that contributes to melanomagenesis through the SKI/SOX10 regulatory axis.
The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae ...also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity.
(Cancer Sci 2010; 101: 594–600)
WT1 (Wilms’ tumor gene 1) protein is a potent pan‐tumor‐associated antigen (TAA) and WT1‐specific cytotoxic T lymphocytes (WT1 tetramer+ CD8+ T cells) are ...spontaneously induced in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). We conducted a single‐cell level comparative analysis of T‐cell receptor β‐chain variable region (TCR‐BV) gene families of a total of 1242 spontaneously induced WT1 tetramer+ CD8+ T cells in HLA‐A*2402+ patients with AML or MDS and those in healthy donors (HDs). This is the first report of direct usage analysis of TCR‐BV gene families of individual TAA‐specific CD8+ T cells at single‐cell level. Usage analysis using single‐cell RT‐PCR of TCR‐BV gene families of individual FACS‐sorted WT1 tetramer+ CD8+ T cells showed for the first time (i) that BVs 5, 6, 20, and 27 were commonly biased in both HDs and patients; (ii) that BV4 was commonly biased in HDs and MDS patients; (iii) that BV19 was commonly biased in the patients; and (iv) that BVs 7 and 28, BVs 9 and 15, and BVs 12 and 29 were specifically biased in HDs, AML, and MDS patients, respectively. However, statistical analysis of similarity among HD, AML, and MDS of individual usage frequencies of 24 kinds of TCR‐BV gene families indicated that the usage frequencies of TCR‐BV gene families in AML and MDS patients reflect those in HDs. These findings represent a novel insight for a better understanding of WT1‐specific immune response.
In tumor‐bearing patients, tumor‐associated antigen (TAA)‐specific CTLs are spontaneously induced as a result of immune response to TAAs and play an important role in anti‐tumor immunity. Wilms’ ...tumor gene 1 (WT1) is overexpressed in various types of tumor and WT1 protein is a promising pan‐TAA because of its high immunogenicity. In this study, to clarify the immune response to the WT1 antigen, WT1‐specific CD8+ T cells that were spontaneously induced in patients with solid tumor were comparatively analyzed in both bone marrow (BM) and peripheral blood (PB). WT1‐specific CD8+ T cells more frequently existed in BM than in PB, whereas frequencies of naïve (CCR7+ CD45RA+), central memory (CCR7+ CD45RA−), effector‐memory (CCR7− CD45RA−), and effector (CCR7− CD45RA+) subsets were not significantly different between BM and PB. However, analysis of these subsets for the expression of CD57 and CD28, which were associated with differentiation, revealed that effector‐memory and effector subsets of the WT1‐specific CD8+ T cells in BM had less differentiated phenotypes and more proliferative potential than those in PB. Furthermore, CD107a/b functional assay for WT1 peptide‐specific cytotoxic potential and carboxyfluorescein diacetate succinimidyl ester dilution assay for WT1 peptide‐specific proliferation also showed that WT1‐specific CD8+ T cells in BM were less cytotoxic and more proliferative in response to WT1 peptide than those in PB. These results implied that BM played an important role as a secondary lymphoid organ in tumor‐bearing patients. Preferential residence of WT1‐specific CD8+ T cells in BM could be, at least in part, explained by higher expression of chemokine receptor CCR5, whose ligand was expressed on BM fibroblasts on the WT1‐specific CD8+ T cells in BM, compared to those in PB. These results should provide us with an insight into WT1‐specific immune response in tumor‐bearing patients and give us an idea of enhancement of clinical response in WT1 protein‐targeted immunotherapy.
(Cancer Sci 2010; 101: 848–854)
The extracellular matrix (ECM) plays an important role in maintaining tissue homeostasis and poses a significant physical barrier to in vivo cell migration. Accordingly, as a means of enhancing ...tissue invasion, tumor cells use matrix metalloproteinases to degrade ECM proteins. However, the in vivo ECM is comprised not only of proteins but also of a variety of nonprotein components. Hyaluronan (HA), one of the most abundant nonprotein components of the interstitial ECM, forms a gel-like antiadhesive barrier that is impenetrable to particulate matter and cells. Mechanisms by which tumor cells penetrate the HA barrier have not been addressed. Here, we demonstrate that transmembrane protein 2 (TMEM2), the only known transmembrane hyaluronidase, is the predominant mediator of contact-dependent HA degradation and subsequent integrin-mediated cell–substrate adhesion. We show that a variety of tumor cells are able to eliminate substrate-bound HA in a tightly localized pattern corresponding to the distribution of focal adhesions (FAs) and stress fibers. This FA-targeted HA degradation is mediated by TMEM2, which itself is localized at site of FAs. TMEM2 depletion inhibits the ability of tumor cells to attach and migrate in an HA-rich environment. Importantly, TMEM2 directly binds at least two integrins via interaction between extracellular domains. Our findings demonstrate a critical role for TMEM2-mediated HA degradation in the adhesion and migration of cells on HA-rich ECM substrates and provide novel insight into the early phase of FA formation.
How to treat CML patients who are resistant to inhibitors of BCR‐ABL tyrosine kinase such as Imatinib is a very important and urgent issue in clinical hematology. Here, we report a case of ...Imatinib‐treated CML in which intradermally administered WT1 peptide vaccine elicited WT1‐specific immune responses and the resultant reduction in the persistent residual disease in co‐administration of Imatinib. BCR‐ABL mRNA levels were being maintained under the detection limit for 8 months since week 77 of vaccination. No adverse effects except local erythema at the injection sites were observed. The tetramer assay revealed that the decrease in BCR‐ABL mRNA levels was associated with the increase in frequency of WT1‐specific cytotoxic T lymphocytes, notably effector‐memory type of that, in the patient’s peripheral blood. The case presented here indicates that WT1 peptide vaccine may become a safe and cure‐oriented therapy for CML patients who have residual disease regardless of the treatment with Imatinib.
Immunotherapy using a Wilms tumor (WT1) peptide has been undergoing clinical trials for adulthood leukemia and solid cancer with promising results. In this study, the authors used WT1 peptide ...vaccination to treat a 6-year-old girl with metastatic alveolar rhabdomyosarcoma. She received weekly intradermal injection with HLA-A*2404-restricted, 9-mer WT1 peptide against residual bone disease. After 3 months her bone disease disappeared, concurrent with an increase in the frequency of WT1-specific cytotoxic T lymphocytes (CTLs). A high proportion of WT1-specific CTLs with effector or effector memory phenotype were detected in peripheral blood of this patient. She is currently still on continued WT1 peptide immunotherapy in a disease-free condition for 22 months. WT1 peptide-based immunotherapy should be a promising option for high-risk rhabdomyosarcoma in childhood.
Synergistic and supportive interactions among genes can be incorporated in engineering biology to enhance and stabilize the performance of biological systems, but combinatorial numerical explosion ...challenges the analysis of multigene interactions. The incorporation of DNA barcodes to mark genes coupled with next-generation sequencing offers a solution to this challenge. We describe improvements for a key method in this space, CombiGEM, to broaden its application to assembling typical gene-sized DNA fragments and to reduce the cost of sequencing for prevalent small-scale projects. The expanded reach of the method beyond currently targeted small RNA genes promotes the discovery and incorporation of gene synergy in natural and engineered processes such as biocontainment, the production of desired compounds, and previously uncharacterized fundamental biological mechanisms.
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