Anti-PrP antibodies block PrPSc replication in prion-infected cell cultures by accelerating PrPC degradation

The use of anti-PrP antibodies represents one of the most promising strategies for the treatment of prion diseases. In the present study, we screened various anti-PrP antibodies with the aim of identifying those that would block PrP(Sc) replication in prion-infected cell culture. Two antibodies, SAF34 recognizing the flexible octarepeats region on HuPrP protein, and SAF61 directed against PrP amino acid residues (144-152), not only inhibited PrP(Sc) formation in prion-infected neuroblastoma cells but also decreased the PrP(C) levels in non-infected N2a cells. In addition, treatment with both SAF34 and SAF61 antibodies decreased PrP(C) and PrP(Sc) levels in the cells synergistically. In the presence of both antibodies, our results showed that the mode of action which leads to the disappearance of PrP(Sc) in cells is directly coupled to PrP(C) degradation by reducing the half-life of the PrP(C) protein.     

Only Pyrimidinoceptors Are Functionally Expressed in Mouse Neuroblastoma Cell Lines

The ability of UTP, UDP, ATP, and ADP to influence inositol phospholipid hydrolysis in neuroblastoma origin cell lines was assessed. The mouse neuroblastoma lines N1E 115, Neuro 2a, and NB4 1A3 and the rat glioma/mouse neuroblastoma hybrid line NG108-15 gave robust responses to both UTP and UDP, which were essentially equipotent. Thus a range of cell lines of mouse neuroblastoma origin express a pyrimidine-selective P2Y receptor. The NG108-15 cells were the only cell type tested at which ATP and ADP displayed activity with EC₅₀ values of greater than 100 μM, compared with values of 0.58 and 1.25 μM for UTP and UDP, respectively. In contrast to the cell lines derived from mouse neuroblastoma, the human neuroblastoma lines SH-SY5Y and SK-N-SH did not respond to any nucleotides, although both responded well to carbachol.     

Comparison of the Side Populations in Pretreatment and Postrelapse Neuroblastoma Cell Lines

Cancer stem-like cells have been identified in both primary tumors and in cell lines and seem to have a high degree of inherent resistance to traditional chemotherapeutic agents. Relapsed cancers including neuroblastoma are generally chemotherapy-resistant and carry a very poor prognosis. We investigated the side populations of three pairs of neuroblastoma cell lines derived from single patients at the time of their initial presentation and then at relapse after multimodality therapy. We found that the size of the side populations in the relapsed cell lines was significantly increased compared with its paired pretreatment cell line. In addition, these side population cells showed increased proliferation and were significantly more efficient at forming colonies in soft agar than their prerelapse pair. Gene expression analysis of the stem cell genes NANOG and POU5F1 (Oct3/4) showed increased expression in the unsorted relapsed cell lines compared with pretreatment lines as well as in the side populations of the relapsed versus prerelapse cell line pairs. The increased size, proliferative ability, and colony-forming efficiency of the side populations of the postrelapse cell lines demonstrated in this study suggest that a population of stemlike cells is not being efficiently targeted by conventional therapy and implies that strategies to specifically target the stem cell fraction of neuroblastomas are needed to improve outcomes in this devastating childhood disease.     

Replication of Nosema Algerae In Three Insect Cell Lines

SYNOPSIS Nosema algerae , a microsporidan parasite of anopheline mosquitoes, was successfully replicated in 3 insect cell culture lines: Trichoplusia ni (TN-368); Heliothis zea (IPLB-1075); and Mamestra brassicae (IZD-Mb-0503). Infectious spores were produced in vitro. Spores were observed at 48 h postinfection, and some cells were filled with sproes by 72 h.
The number of parasites per cell increased with time. At 72 h postinfection, the infection rates for the 3 cell lines ranged from 23 to 32%. Infected cell lines were subcultured, and by the 6th passage spore production had ceased.     

Thyroid Hormone Binding and Regulation of Adrenergic Enzymes in Two Neuroblastoma Cell Lines

Two neuroblastoma cell lines were cultured in control (euthyroid) and hypothyroid media and examined for protein, RNA and DNA content, activity of the catecholaminergic enzymes tyrosine hydroxylase (TH, EC 1.14.16.2) and monoamine oxidase-A (MAO-A, EC 1.4.3.4), and for L-triiodothyronine (T3) nuclear receptors. In the hypothyroid condition, the rate of cell division and the levels of RNA and protein as well as the activities of TH and MAO were lower than in the euthyroid condition, the reduction being more marked in the E than in the A2(1) cell line. T3 nuclear receptors, unaltered in affinity, were increased in number in the hypothyroid medium, possibly as a regulatory response to hormonal deficiency. Examination of a possible relationship between T3 occupancy and TH activity in the E cells, most sensitive to thyroid hormone deficiency, revealed that induction of TH activity by T3 is dose-dependent and correlates with the number of nuclear sites occupied by the hormone. When neuroblastoma cells were induced to differentiate by the addition of sodium butyrate to the medium, parameters of cell growth (protein, RNA) and enzyme activity (TH and MAO-A) increased in both cell lines irrespective of the presence of thyroid hormones. These data indicate that thyroid hormones, through their nuclear receptors, directly affect the activity of catecholaminergic enzymes in cultured, immature (undifferentiated) neurons.     

Identification of Proteins Sensitive to Thermal Stress in Human Neuroblastoma and Glioma Cell Lines

Heat-shock is an acute insult to the mammalian proteome. The sudden elevation in temperature has far-reaching effects on protein metabolism, leads to a rapid inhibition of most protein synthesis, and the induction of protein chaperones. Using heat-shock in cells of neuronal (SH-SY5Y) and glial (CCF-STTG1) lineage, in conjunction with detergent extraction and sedimentation followed by LC-MS/MS proteomic approaches, we sought to identify human proteins that lose solubility upon heat-shock. The two cell lines showed largely overlapping profiles of proteins detected by LC-MS/MS. We identified 58 proteins in detergent insoluble fractions as losing solubility in after heat shock; 10 were common between the 2 cell lines. A subset of the proteins identified by LC-MS/MS was validated by immunoblotting of similarly prepared fractions. Ultimately, we were able to definitively identify 3 proteins as putatively metastable neural proteins; FEN1, CDK1, and TDP-43. We also determined that after heat-shock these cells accumulate insoluble polyubiquitin chains largely linked via lysine 48 (K-48) residues. Collectively, this study identifies human neural proteins that lose solubility upon heat-shock. These proteins may represent components of the human proteome that are vulnerable to misfolding in settings of proteostasis stress.     

Mesenchymal Stromal Cell Secretome Up-Regulates 47 kDa CXCR4 Expression,and Induce Invasiveness in Neuroblastoma Cell Lines

Neuroblastoma accounts for 15% of childhood cancer deaths and presents with metastatic disease of the bone and the bone marrow at diagnosis in 70% of the cases. Previous studies have shown that the Mesenchymal Stromal Cell (MSC) secretome, triggers metastases in several cancer types such as breast and prostate cancer, but the specific role of the MSC factors in neuroblastoma metastasis is unclear. To better understand the effect of MSC secretome on chemokine receptors in neuroblastoma, and its role in metastasis, we studied a panel of 20 neuroblastoma cell lines, and compared their invasive potential towards MSC-conditioned-RPMI (mRPMI) and their cytokine receptor expression profiles. Western blot analysis revealed the expression of multiple CXCR4 isoforms in neuroblastoma cells. Among the five major isoforms, the expression of the 47 kDa isoform showed significant correlation with high invasiveness. Pretreatment with mRPMI up-regulated the expression of the 47 kDa CXCR4 isoform and also increased MMP-9 secretion, expression of integrin α3 and integrin β1, and the invasive potential of the cell; while blocking CXCR4 either with AMD 3100, a CXCR4 antagonist, or with an anti-47 kDa CXCR4 neutralizing antibody decreased the secretion of MMP-9, the expression of integrin α3 and integrin β1, and the invasive potential of the cell. Pretreatment with mRPMI also protected the 47 kDa CXCR4 isoform from ubiquitination and subsequent degradation. Our data suggest a modulatory role of the MSC secretome on the expression of the 47 kDa CXCR4 isoform and invasion potential of the neuroblastoma cells to the bone marrow.     

A Cell Internalizing Antibody Targeting Capsid Protein (p24) Inhibits the Replication of HIV-1 in T Cells Lines and PBMCs: A Proof of Concept Study

There remains a need for newer therapeutic approaches to combat HIV/AIDS. Viral capsid protein p24 plays important roles in HIV pathogenesis. Peptides and small molecule inhibitors targeting p24 have shown to inhibit virus replication in treated cell. High specificity and biological stability of monoclonal antibodies (mAbs) make them an attractive contender for in vivo treatments. However, mAbs do not enter into cells, thus are restricted to target surface molecules. This also makes targeting intracellular HIV-1 p24 a challenge. A mAb specific to p24 that can internalize into the HIV-infected cells is hypothesized to inhibit the virus replication. We selected a mAb that has previously shown to inhibit p24 polymerization in an in vitro assay and chemically conjugated it with cell penetrating peptides (CPP) to generate cell internalizing anti-p24 mAbs. Out of 8 CPPs tested, κFGF-MTS -conjugated mAbs internalized T cells most efficiently. At nontoxic concentration, the κFGF-MTS-anti-p24-mAbs reduced the HIV-1 replication up to 73 and 49% in T-lymphocyte and PBMCs respectively. Marked inhibition of HIV-1 replication in relevant cells by κFGF-MTS-anti-p24-mAbs represents a viable strategy to target HIV proteins present inside the cells.     

Magnetosome Expression of Functional Camelid Antibody Fragments (Nanobodies) in Magnetospirillum gryphiswaldense

Numerous applications of conventional and biogenic magnetic nanoparticles (MNPs), such as in diagnostics, immunomagnetic separations, and magnetic cell labeling, require the immobilization of antibodies. This is usually accomplished by chemical conjugation, which, however, has several disadvantages, such as poor efficiency and the need for coupling chemistry. Here, we describe a novel strategy to display a functional camelid antibody fragment (nanobody) from an alpaca (Lama pacos) on the surface of bacterial biogenic magnetic nanoparticles (magnetosomes). Magnetosome-specific expression of a red fluorescent protein (RFP)-binding nanobody (RBP) in vivo was accomplished by genetic fusion of RBP to the magnetosome protein MamC in the magnetite-synthesizing bacterium Magnetospirillum gryphiswaldense. We demonstrate that isolated magnetosomes expressing MamC-RBP efficiently recognize and bind their antigen in vitro and can be used for immunoprecipitation of RFP-tagged proteins and their interaction partners from cell extracts. In addition, we show that coexpression of monomeric RFP (mRFP or its variant mCherry) and MamC-RBP results in intracellular recognition and magnetosome recruitment of RFP within living bacteria. The intracellular expression of a functional nanobody targeted to a specific bacterial compartment opens new possibilities for in vivo synthesis of MNP-immobilized nanobodies. Moreover, intracellular nanotraps can be generated to manipulate bacterial structures in live cells.     

A PCNA-Derived Cell Permeable Peptide Selectively Inhibits Neuroblastoma Cell Growth

Proliferating cell nuclear antigen (PCNA), through its interaction with various proteins involved in DNA synthesis, cell cycle regulation, and DNA repair, plays a central role in maintaining genome stability. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was significantly expressed in a broad range of cancer cells and tumor tissues, but not in non-malignant cells. We found that the caPCNA-specific antigenic site lies between L126 and Y133, a region within the interconnector domain of PCNA that is known to be a major binding site for many of PCNA''s interacting proteins. We hypothesized that therapeutic agents targeting protein-protein interactions mediated through this region may confer differential toxicity to normal and malignant cells. To test this hypothesis, we designed a cell permeable peptide containing the PCNA L126-Y133 sequence. Here, we report that this peptide selectively kills human neuroblastoma cells, especially those with MYCN gene amplification, with much less toxicity to non-malignant human cells. Mechanistically, the peptide is able to block PCNA interactions in cancer cells. It interferes with DNA synthesis and homologous recombination-mediated double-stranded DNA break repair, resulting in S-phase arrest, accumulation of DNA damage, and enhanced sensitivity to cisplatin. These results demonstrate conceptually the utility of this peptide for treating neuroblastomas, particularly, the unfavorable MYCN-amplified tumors.     

Comparison of the Anti-Prion Mechanism of Four Different Anti-Prion Compounds,Anti-PrP Monoclonal Antibody 44B1, Pentosan Polysulfate,Chlorpromazine, and U18666A,in Prion-Infected Mouse Neuroblastoma Cells

Molecules that inhibit the formation of an abnormal isoform of prion protein (PrP^Sc) in prion-infected cells are candidate therapeutic agents for prion diseases. Understanding how these molecules inhibit PrP^Sc formation provides logical basis for proper evaluation of their therapeutic potential. In this study, we extensively analyzed the effects of the anti-PrP monoclonal antibody (mAb) 44B1, pentosan polysulfate (PPS), chlorpromazine (CPZ) and U18666A on the intracellular dynamics of a cellular isoform of prion protein (PrP^C) and PrP^Sc in prion-infected mouse neuroblastoma cells to re-evaluate the effects of those agents. MAb 44B1 and PPS rapidly reduced PrP^Sc levels without altering intracellular distribution of PrP^Sc. PPS did not change the distribution and levels of PrP^C, whereas mAb 44B1 appeared to inhibit the trafficking of cell surface PrP^C to organelles in the endocytic-recycling pathway that are thought to be one of the sites for PrP^Sc formation. In contrast, CPZ and U18666A initiated the redistribution of PrP^Sc from organelles in the endocytic-recycling pathway to late endosomes/lysosomes without apparent changes in the distribution of PrP^C. The inhibition of lysosomal function by monensin or bafilomycin A1 after the occurrence of PrP^Sc redistribution by CPZ or U18666A partly antagonized PrP^Sc degradation, suggesting that the transfer of PrP^Sc to late endosomes/lysosomes, possibly via alteration of the membrane trafficking machinery of cells, leads to PrP^Sc degradation. This study revealed that precise analysis of the intracellular dynamics of PrP^C and PrP^Sc provides important information for understanding the mechanism of anti-prion agents.     

Comparative Replication of Lymantria dispar Nuclear Polyhedrosis Virus Strains in Three Continuous-Culture Cell Lines

We compared the replication of the gypsy moth (Lymantria dispar) nuclear polyhedrosis virus in two new cell lines, from embryos and fat body of L. dispar, and in a previously available ovarian cell line. Three virus isolates (the Hamden strain [LDP-67] used commercially as GYPCHEK, a plaque-purified clone of Hamden [5-7d], and an isolate from Abington, Mass. [Ab]) were each tested on the three cell lines. The fat-body-derived cell line proved best in terms of occlusion body production for all three virus strains, with the highest yield produced by the Abington strain. On the basis of these results, we conclude that a more efficient in vitro production of gypsy moth virus can be obtained by using the fat body cell line in conjunction with the Abington strain of the virus.     

Expression of Endothelin-Converting Enzyme in Both Neuroblastoma and Glial Cell Lines and Its Localization in Rat Hippocampus

Abstract: Endothelin-converting enzyme is a phosphoramidon-sensitive metalloprotease that cleaves big endothelin to the potent vasoconstrictor peptide, endothelin. The converting enzyme is expressed in endothelial cells in a variety of tissues and in some secretory cells. In the present study, phosphoramidon-sensitive endothelin-converting enzyme activity has been demonstrated by radioimmunoassay in the neuroblastoma cell line, SH-SY5Y, and in Bu17 and C6 glioma lines. The identity of the activity was confirmed by immunoblotting, revealing a polypeptide of ∼120 kDa in each of these lines, in D384 glioma cells, and in primary astrocytes. Immunofluorescence revealed the cell-surface location of endothelin-converting enzyme in the neuronal and glial cell lines and in primary astrocytes. Pretreatment of SH-SY5Y and Bu17 cells with phosphoramidon resulted in an apparent concentration of the enzyme protein in an intracellular compartment. Immunoperoxidase-staining of rat brain sections located this metalloprotease to the pyramidal cells of the hippocampus. Endothelin-converting enzyme-1 was revealed by in situ hybridisation in the neuronal and glial cell lines.     

Close Interactions between Mesenchymal Stem Cells and Neuroblastoma Cell Lines Lead to Tumor Growth Inhibition

Mesenchymal stem cells (MSCs) have attracted much interest in oncology since they exhibit marked tropism for the tumor microenvironment and support or suppress malignant cell growth depending on the tumor model tested. The aim of this study was to investigate the role of MSCs in the control of the growth of neuroblastoma (NB), which is the second most common solid tumor in children. In vivo experiments showed that systemically administered MSCs, under our experimental conditions, did not home to tumor sites and did not affect tumor growth or survival. However, MSCs injected intratumorally in an established subcutaneous NB model reduced tumor growth through inhibition of proliferation and induction of apoptosis of NB cells and prolonged the survival of hMSC-treated mice. The need for contact between MSCs and NB cells was further supported by in vitro experiments. In particular, MSCs were found to be attracted by NB cells, and to affect NB cell proliferation with different results depending on the cell line tested. Moreover, NB cells, after pre-incubation with hMSCs, acquired a more invasive behavior towards CXCL12 and the bone marrow, i.e., the primary site of NB metastases. In conclusion, this study demonstrates that functional cross-talk between MSCs and NB cell lines used in our experiments can occur only within short range interaction. Thus, this report does not support the clinical use of MSCs as vehicles for selective delivery of antitumor drugs at the NB site unless chemotherapy and/or radiotherapy create suitable local conditions for MSCs recruitment.     

抗GD2 抗体疗法用于高危神经母细胞瘤治疗的研究进展

高危神经母细胞瘤是幼儿及青少年中高发型恶性疾病,为胚胎性肿瘤,其表面高度表达神经节苷脂GD2,故GD2 成为此类恶疾的治疗靶标。在过去几十年中,抗GD2 抗体疗法用于神经母细胞瘤的治疗研究取得了巨大进展,其安全性和有效性得到了充分验证。简介GD2 的生物合成、结构与功能,综述用于神经母细胞瘤的抗GD2 抗体疗法及其有效性的提高策略。     

Neuroblastoma Cell Lines Contain Pluripotent Tumor Initiating Cells That Are Susceptible to a Targeted Oncolytic Virus

Background

Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers.

Methodology/Principal Findings

Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice.

Conclusions/Significance

These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.