Reactive oxygen species-linked regulation of the multidrug resistance transporter P-glycoprotein in Nox-1 overexpressing prostate tumor spheroids

AbstractExpression of the multidrug resistance (MDR) transporter P-glycoprotein (P-gp) has been demonstrated to be regulated by hypoxia-inducible factor-1α (HIF-1α) and inhibited by intracellular reactive oxygen species (ROS). Herein, P-gp and HIF-1α expression were investigated in multicellular prostate tumor spheroids overexpressing the ROS-generating enzyme Nox-1 in comparison to the mother cell line DU-145. In Nox-1-overexpressing tumor spheroids (DU-145Nox1) generation of ROS as well as expression of Nox-1 was significantly increased as compared to DU-145 tumor spheroids. ROS generation was significantly inhibited in the presence of the NADPH-oxidase antagonists diphenylen-iodonium chloride (DPI) and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). Albeit growth kinetic of DU-145Nox1 tumor spheroids was decreased as compared to DU-145 spheroids, elevated expression of Ki-67 was observed indicating increased cell cycle activity. In DU-145Nox1 tumor spheroids, expression of HIF-1α as well as P-gp was significantly decreased as compared to DU-145 spheroids, which resulted in an increased retention of the anticancer agent doxorubicin. Pretreatment with the free radical scavengers vitamin E and vitamin C increased the expression of P-gp as well as HIF-1α in Nox-1-overexpressing cells, whereas no effect of free radical scavengers was observed on mdr-1 mRNA expression. In summary, the data of the present study demonstrate that the development of P-gp-mediated MDR is abolished under conditions of elevated ROS levels, suggesting that the MDR phenotype can be circumvented by modest increase of intracellular ROS generation

The BCR-ABL1 Kinase Bypasses Selection for the Expression of a Pre–B Cell Receptor in Pre–B Acute Lymphoblastic Leukemia Cells

The BCR-ABL1 kinase expressed in acute lymphoblastic leukemia (ALL) drives malignant transformation of human pre–B cells. Comparing genome-wide gene expression profiles of BCR-ABL1+ pre–B ALL and normal bone marrow pre–B cells by serial analysis of gene expression, many genes involved in pre–B cell receptor signaling are silenced in the leukemia cells. Although normal pre–B cells are selected for the expression of a functional pre–B cell receptor, BCR-ABL1+ ALL cells mostly do not harbor a productively rearranged IGH allele. In these cases, we identified traces of secondary VH gene rearrangements, which may have rendered an initially productive VH region gene nonfunctional. Even BCR-ABL1+ ALL cells harboring a functional VH region gene are unresponsive to pre–B cell receptor engagement and exhibit autonomous oscillatory Ca2+ signaling activity. Conversely, leukemia subclones surviving inhibition of BCR-ABL1 by STI571 restore responsiveness to antigen receptor engagement and differentiate into immature B cells expressing immunoglobulin light chains. BCR-ABL1 kinase activity is linked to defective pre–B cell receptor signaling and the expression of a truncated isoform of the pre–B cell receptor–associated linker molecule SLP65. Also in primary leukemia cells, truncated SLP65 is expressed before but not after treatment of the patients with STI571. We conclude that inhibition of BCR-ABL1 reconstitutes selection for leukemia cells expressing a functional (pre–) B cell receptor

14-3-3 Proteins and Other Candidates form Protein-Protein Interactions with the Cytosolic C-terminal End of SOS1 Affecting Its Transport Activity

The plasma membrane transporter SOS1 (SALT-OVERLY SENSITIVE1) is vital for plant survival under salt stress. SOS1 activity is tightly regulated, but little is known about the underlying mechanism. SOS1 contains a cytosolic, autoinhibitory C-terminal tail (abbreviated as SOS1 C-term), which is targeted by the protein kinase SOS2 to trigger its transport activity. Here, to identify additional binding proteins that regulate SOS1 activity, we synthesized the SOS1 C-term domain and used it as bait to probe Arabidopsis thaliana cell extracts. Several 14-3-3 proteins, which function in plant salt tolerance, specifically bound to and interacted with the SOS1 C-term. Compared to wild-type plants, when exposed to salt stress, Arabidopsis plants overexpressing SOS1 C-term showed improved salt tolerance, significantly reduced Na+ accumulation in leaves, reduced induction of the salt-responsive gene WRKY25, decreased soluble sugar, starch, and proline levels, less impaired inflorescence formation and increased biomass. It appears that overexpressing SOS1 C-term leads to the sequestration of inhibitory 14-3-3 proteins, allowing SOS1 to be more readily activated and leading to increased salt tolerance. We propose that the SOS1 C-term binds to previously unknown proteins such as 14-3-3 isoforms, thereby regulating salt tolerance. This finding uncovers another regulatory layer of the plant salt tolerance program

ECCO Essential Requirements for Quality Cancer Care : Soft Tissue Sarcoma in Adults and Bone Sarcoma. A critical review

Background: ECCO essential requirements for quality cancer care (ERQCC) are checklists and explanations of organisation and actions that are necessary to give high-quality care to patients who have a specific tumour type. They are written by European experts representing all disciplines involved in cancer care. ERQCC papers give oncology teams, patients, policymakers and managers an overview of the elements needed in any healthcare system to provide high quality of care throughout the patient journey. References are made to clinical guidelines and other resources where appropriate, and the focus is on care in Europe. Sarcoma: essential requirements for quality care Sarcomas - which can be classified into soft tissue and bone sarcomas - are rare, but all rare cancers make up more than 20% of cancers in Europe, and there are substantial inequalities in access to high-quality care. Sarcomas, of which there are many subtypes, comprise a particularly complex and demanding challenge for healthcare systems and providers. This paper presents essential requirements for quality cancer care of soft tissue sarcomas in adults and bone sarcomas. High-quality care must only be carried out in specialised sarcoma centres (including paediatric cancer centres) which have both a core multidisciplinary team and an extended team of allied professionals, and which are subject to quality and audit procedures. Access to such units is far from universal in all European countries. It is essential that, to meet European aspirations for high-quality comprehensive cancer control, healthcare organisations implement the requirements in this paper, paying particular attention to multidisciplinarity and patient-centred pathways from diagnosis and follow-up, to treatment, to improve survival and quality of life for patients. Conclusion: Taken together, the information presented in this paper provides a comprehensive description of the essential requirements for establishing a high-quality service for soft tissue sarcomas in adults and bone sarcomas. The ECCO expert group is aware that it is not possible to propose a 'one size fits all' system for all countries, but urges that access to multidisciplinary teams is guaranteed to all patients with sarcoma. (C) 2016 The Authors. Published by Elsevier Ireland Ltd.Peer reviewe

Mimicry of a constitutively active pre–B cell receptor in acute lymphoblastic leukemia cells

Pre–B cells undergo apoptosis unless they are rescued by pre–B cell receptor–dependent survival signals. We previously showed that the BCR-ABL1 kinase that is expressed in pre–B lymphoblastic leukemia bypasses selection for pre–B cell receptor–dependent survival signals. Investigating possible interference of BCR-ABL1 with pre–B cell receptor signaling, we found that neither SYK nor SLP65 can be phosphorylated in response to pre–B cell receptor engagement. Instead, Bruton's tyrosine kinase (BTK) is constitutively phosphorylated by BCR-ABL1. Activated BTK is essential for survival signals that otherwise would arise from the pre–B cell receptor, including activation of PLCγ1, autonomous Ca(2+) signaling, STAT5-phosphorylation, and up-regulation of BCLX (L). Inhibition of BTK activity specifically induces apoptosis in BCR-ABL1 (+) leukemia cells to a similar extent as inhibition of BCR-ABL1 kinase activity itself. However, BCR-ABL1 cannot directly bind to full-length BTK. Instead, BCR-ABL1 induces the expression of a truncated splice variant of BTK that acts as a linker between the two kinases. As opposed to full-length BTK, truncated BTK lacks kinase activity yet can bind to BCR-ABL1 through its SRC-homology domain 3. Acting as a linker, truncated BTK enables BCR-ABL1–dependent activation of full-length BTK, which initiates downstream survival signals and mimics a constitutively active pre–B cell receptor

Aspf2 From Aspergillus fumigatus Recruits Human Immune Regulators for Immune Evasion and Cell Damage

The opportunistic fungal pathogen Aspergillus fumigatus can cause life-threatening infections, particularly in immunocompromised patients. Most pathogenic microbes control host innate immune responses at the earliest time, already before infiltrating host immune cells arrive at the site of infection. Here, we identify Aspf2 as the first A. fumigatus Factor H-binding protein. Aspf2 recruits several human plasma regulators, Factor H, factor-H-like protein 1 (FHL-1), FHR1, and plasminogen. Factor H contacts Aspf2 via two regions located in SCRs6–7 and SCR20. FHL-1 binds via SCRs6–7, and FHR1 via SCRs3–5. Factor H and FHL-1 attached to Aspf2-maintained cofactor activity and assisted in C3b inactivation. A Δaspf2 knockout strain was generated which bound Factor H with 28% and FHL-1 with 42% lower intensity. In agreement with less immune regulator acquisition, when challenged with complement-active normal human serum, Δaspf2 conidia had substantially more C3b (>57%) deposited on their surface. Consequently, Δaspf2 conidia were more efficiently phagocytosed (>20%) and killed (44%) by human neutrophils as wild-type conidia. Furthermore, Aspf2 recruited human plasminogen and, when activated by tissue-type plasminogen activator, newly generated plasmin cleaved the chromogenic substrate S2251 and degraded fibrinogen. Furthermore, plasmin attached to conidia damaged human lung epithelial cells, induced cell retraction, and caused matrix exposure. Thus, Aspf2 is a central immune evasion protein and plasminogen ligand of A. fumigatus. By blocking host innate immune attack and by disrupting human lung epithelial cell layers, Aspf2 assists in early steps of fungal infection and likely allows tissue penetration