SummaryCD14 is a 55-kD protein found both as a glycosylphosphatidyl inositol-linked protein on the surface of mononudear phagocytes and as a soluble protein in the blood. CD14 on the call membrane (mCD14) has been shown to serve as a receptor for complexes of lipopolysaccharide (LPS) with LPS binding protein, but a function for soluble CD14 (sCD14) has not been described. Here we show that sCD14 enables responses to LPS by cells that do not express CD14. We have examined induction of endothdial-leukocyte adhesion molecule 1 expression by human umbilical vein endothdial cells, interleukin 6 secretion by U373 astrocytoma cells, and cytotoxicity of bovine endothelial ceUs. None of these cell types express mCD14, yet all respond to LPS in a serumdependent fashion, and all responses are completely blocked by anti-CD14 antibodies. Immunodepletion of sCD14 from serum prevents responses to LPS, and the responses are restored by addition of sCD14. These studies suggest that a surface anchor is not needed for the function of CD14 and further imply that sCD14 must bind to additional proteins on the cell surface to associate with the cell and transduce a signal. They also indicate that sCD14 may have an important role in potentiating responses to LPS in cells lacking mCD14.

Human Toll like receptor (TLR) 2 has been implicated as a signaling receptor for LPS from Gram-negative bacteria and cell wall components from Gram-positive organisms. In this study, we investigated whether TLR2 can signal cell activation by the heat-killed group B streptococci type III (GBS) and Listeria monocytogenes (HKLM). HKLM, but not GBS, showed a time- and dose-dependent activation of Chinese hamster ovary cells transfected with human TLR2, as measured by translocation of NF-κB and induction of IL-6 production. A mAb recognizing a TLR2-associated epitope (TL2.1) was generated that inhibited IL-6 production from Chinese hamster ovary-TLR2 cells stimulated with HKLM or LPS. The TL2.1 mAb reduced HKLM-induced TNF production from human monocytes by 60%, whereas a CD14 mAb (3C10) reduced the TNF production by 30%. However, coadministrating TL2.1 and 3C10 inhibited the TNF response by 80%. In contrast to this, anti-CD14 blocked LPS-induced TNF production from monocytes, whereas anti-TLR2 showed no inhibition. Neither TL2.1 nor 3C10 affected GBS-induced TNF production. These results show that TLR2 can function as a signaling receptor for HKLM, possibly together with CD14, but that TLR2 is unlikely to be involved in cell activation by GBS. Furthermore, although LPS can activate transfected cell lines through TLR2, this receptor does not seem to be the main transducer of LPS activation of human monocytes. Thus, our data demonstrate the ability of TLR2 to distinguish between different pathogens.

Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA) is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM). Thus inhibiting PCNA’s protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells’ sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment.

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IntroductionMembers of the MYC family are important oncogenes involved in the development of malignant cells. 1 This may also be the case in multiple myeloma (MM), a malignancy of antibodyproducing plasma cells in bone marrow. The activity of c-MYC in MM increases with disease stage. 2,3 The mechanism by which c-MYC is activated in each case is unclear; however, multiple signaling pathways converge on c-MYC. Translocations involving MYC and immunoglobulin genes (IG) are relatively rare in MM and considered late progression events. 4 c-MYC regulates transcription of up to 15% of the genes in human cells by binding to its obligate partner MAX. Many cancer cells may develop a dependency on c-MYC activity; and by preventing this activity, the cells may stop dividing or even undergo apoptosis. In agreement with this, short-hairpin RNA targeting MYC was shown to be lethal to a number of human myeloma cell lines. 5 A small-molecule inhibitor, termed 10058-F4, has been identified that is proposed to specifically inhibit c-MYC-MAX heterodimerization, thereby preventing transactivation of c-MYC target genes. 6,7 The inhibitor has been shown to have growth inhibitory effects on lymphoma and acute myelogenous leukemia cells. 8,9 Methods CellsMyeloma cell lines used in this study were U266, INA-6, JJN-3, KMS-12-BM, IH-1, and KJON. Details on cell culture conditions are found in supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). CD138 ϩ patient cells obtained through the Norwegian Myeloma Biobank were purified using RoboSep automated cell separator and Human CD138 Positive Selection Kit (Stem Cell Technologies). Bone marrow stromal cells (BMSCs) from patients were obtained by plastic adherence and cultivated as stated in supplemental Methods. The project was approved by the Regional Ethics Committee, and patients gave informed consent in accordance with the Declaration of Helsinki. Patient characteristics are described in supplemental Table 1.Cell viability measurements and quantitative RT-PCR were performed as described previously. 10 PCR TaqMan assays used were as follows: MYC, Hs00153408_m1; MYCL1, Hs00420495_m1; and GAPDH, Hs99999905_m1 (Applied Biosystems).Description of other reagents, immunoblotting, and knockdown experiments is found in supplemental Methods. Results and discussionTo address the c-MYC dependency of myeloma cells, we decided to evaluate the effect of 10058-F4 in myeloma cell lines and primary cells by in vitro studies. First, both mRNA and protein expression of c-MYC and L-MYC was determined in 6 cell lines by quantitative RT-PCR and immunoblotting ( Figure 1A). Five of the cell lines expressed c-MYC, whereas U266 only had L-MYC, as previously reported. 11 L-MYC mRNA was also detected in KMS-12-BM and to a lesser extent in INA-6 and JJN-3, although the levels of L-MYC were negligible compared with c-MYC. The effect on cell viability was evaluated in myeloma cell lines treated with increasing concentrations of 10058-F4 for 48 hour...

BackgroundActivins are members of the TGF-β family of ligands that have multiple biological functions in embryonic stem cells as well as in differentiated tissue. Serum levels of activin A were found to be elevated in pathological conditions such as cachexia, osteoporosis and cancer. Signaling by activin A through canonical ALK4-ACVR2 receptor complexes activates the transcription factors SMAD2 and SMAD3. Activin A has a strong affinity to type 2 receptors, a feature that they share with some of the bone morphogenetic proteins (BMPs). Activin A is also elevated in myeloma patients with advanced disease and is involved in myeloma bone disease.ResultsIn this study we investigated effects of activin A binding to receptors that are shared with BMPs using myeloma cell lines with well-characterized BMP-receptor expression and responses. Activin A antagonized BMP-6 and BMP-9, but not BMP-2 and BMP-4. Activin A was able to counteract BMPs that signal through the type 2 receptors ACVR2A and ACVR2B in combination with ALK2, but not BMPs that signal through BMPR2 in combination with ALK3 and ALK6.ConclusionsWe propose that one important way that activin A regulates cell behavior is by antagonizing BMP-ACVR2A/ACVR2B/ALK2 signaling.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-015-0104-z) contains supplementary material, which is available to authorized users.

IntroductionBone is a dynamic tissue in which the synthesis of bone matrix by osteoblasts and bone resorption by osteoclasts are coupled processes. Osteoclasts differentiate from hematopoietic precursor cells under the control of humoral factors and cell-cell contact with osteoblasts or stromal cells. Key regulators of osteoclastogenesis are members of the tumor necrosis family of receptors and ligands: receptor activator of nuclear factor (NF)-B (RANK), receptor activator of NF-B ligand (RANKL), and osteoprotegerin (OPG). RANKL is expressed by activated T cells, stromal cells, and osteoblasts. [1][2][3] When RANKL binds to RANK on osteoclast precursors, maturation and differentiation of the osteoclasts are induced, leading to bone resorption. 4,5 OPG is a soluble decoy receptor that is secreted by osteoblasts and bone marrow (BM) stromal cells and that competes with RANK for binding to RANKL. Binding of OPG to RANKL inhibits the development of osteoclasts. 6,7 The importance of OPG as a negative regulator of osteoclastogenesis is evident from experiments with transgenic mice, where overexpression of OPG leads to severe osteopetrosis and reduced numbers of mature osteoclasts. 6 In contrast, OPG knockout mice are osteoporotic. 8 Multiple myeloma (MM) is a malignancy characterized by accumulation of plasma cells in the BM. Bone destruction is a common complication of the disease and is associated with severe morbidity. A number of osteoclast-activating factors are implicated in myeloma bone disease (for a review, see Callander and Roodman 9 ). However, accumulating data suggest that a disruption of the balance between RANKL and OPG is of major importance. Histologic examination of BM biopsies from patients with MM showed enhanced expression of RANKL in the BM, as well as reduced OPG expression. 10,11 Furthermore, we have recently shown that serum OPG levels are lower in myeloma patients than in healthy individuals and that myeloma patients with osteolytic lesions have reduced levels of OPG in serum compared to myeloma patients without clinical bone disease. 12 Osteoprotegerin has a highly basic heparin-binding domain, 13 making interactions with heparin and heparan sulfates possible. A feature of both normal and malignant plasma cells is the abundant expression of syndecan-1, 14,15 which is a transmembrane proteoglycan with heparan sulfate side chains. These side chains allow interactions with several macromolecules, including extracellular matrix proteins, growth factors, cytokines, and pathogens (for a review, see Tumova et al 16 ). In addition to modifying the action of its ligands, 17,18 syndecan-1 has been shown to mediate the catabolism of several proteins. 19 Patients, materials, and methods MM and control patientsBone marrow was aspirated from the iliac crest or sternum of 33 patients with MM for diagnostic purposes before treatment (median age, 65 years; 19 men, 14 women). Twenty-seven patients who underwent BM aspiration for diagnostic purposes, but who had normal BM morphology and subsequently were not ...

Interleukin-21 (IL-21