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In herpesvirus replicating cells, host cell gene transcription is frequently down-regulated because important transcriptional apparatuses are appropriated by viral transcription factors. Here, we show a small peptide derived from the Kaposi’s sarcoma-associated herpesvirus transactivator (K-Rta) sequence, which attenuates cellular MYC expression, reduces cell proliferation, and selectively kills cancer cell lines in both tissue culture and a xenograft tumor mouse model. Mechanistically, the peptide functions as a decoy to block the recruitment of coactivator complexes consisting of Nuclear receptor coactivator 2 (NCOA2), p300, and SWI/SNF proteins to the MYC promoter in primary effusion lymphoma cells. Thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM seq) with target-transcriptional analyses further confirm that the viral peptide directly attenuates MYC and MYC-target gene expression. This study thus provides a unique tool to control MYC activation, which may be used as a therapeutic payload to treat MYC-dependent diseases such as cancers and autoimmune diseases., Shimoda et al. present evidence that a small functional peptide derived from the KSHV transactivator could be used to target MYC in cancer. The authors find that the peptide attenuates MYC expression, reduces cell proliferation, and selectively kills cancer cells in tissue culture and a xenograft tumor mouse model, by blocking recruitment of coactivator complexes (Nuclear receptor coactivator 2, p300, and SWI/SNF proteins) to the MYC promoter.

In herpesvirus replicating cells, host cell gene transcription is frequently down-regulated because important transcriptional apparatuses are appropriated by viral transcription factors. Here, we identified a small peptide derived from the Kaposi's sarcoma-associated herpesvirus transactivator (K-Rta) sequence, which attenuates cellular c-MYC expression, reduces cell proliferation, and selectively kills cancer cell lines in both tissue culture and a xenograft tumor mouse model. Mechanistically, the peptide functions as a decoy to block the recruitment of coactivator complexes consisting of Nuclear receptor coactivator 2 (NCOA2), p300, and SWI/SNF proteins to the MYC promoter in primary effusion lymphoma cells. Thiol(SH)-linked alkylation for the metabolic sequencing of RNA (​SLAM seq) with target-transcriptional analyses further confirmed that the viral peptide directly attenuates MYC and MYC-target gene expression. This study thus provides a unique tool to control MYC activation, which may have significant potential as a therapeutic payload to treat MYC-dependent diseases such as cancers and autoimmune diseases.

Background: While many humanized monoclonal antibodies utilize complement-dependent cytotoxicity, the complement depleting effects of these antibodies and the impact of complement replacement on treatment response are not well-described. Methods: We conducted a phase 2 trial involving patients with relapsed/refractory chronic lymphocytic leukemia (CLL). Patients were treated with ofatumumab with fresh frozen plasma (FFP) used as a source of complement replacement. The primary endpoint was objective response rate. Correlative endpoints included complement levels (C3 and C4) and complement activity (CH50) which was drawn at baseline and after ofatumumab with FFP administration. Results: Among 12 enrolled patients, overall response rate was 83% with two patients (17%) achieving a complete response. While only two (17%) patients had low complement activity at baseline, eight (67%) developed low levels of complement activity after ofatumumab treatment with FFP replacement. The magnitude of complement depletion did not correlate with response. Adverse events were minimal. The combination of ofatumumab and FFP demonstrated tolerability and surprising activity in high-risk CLL patients. Conclusions: The combination of ofatumumab and FFP demonstrated tolerability and surprising activity in high-risk CLL patients. Complement replacement should be studied further as a minimally toxic approach to improve efficacy of monoclonal antibody-based regimens. J Hematol. 2020;9(3):79-83 doi: https://doi.org/10.14740/jh721

BackgroundNeuroblastoma is the second most common extracranial cancer in children. Current therapies for neuroblastoma, which use a combination of chemotherapy drugs, have limitations for high-risk subtypes and can cause significant long-term adverse effects in young patients. Therefore, a new therapy is needed. In this study, we investigated the transcription factor MXD3 as a potential therapeutic target in neuroblastoma.MethodsMXD3 expression was analyzed in five neuroblastoma cell lines by immunocytochemistry and quantitative real-time reverse transcription PCR, and in 18 primary patient tumor samples by immunohistochemistry. We developed nanocomplexes using siRNA and superparamagnetic iron oxide nanoparticles to target MXD3 in neuroblastoma cell lines in vitro as a single-agent therapeutic and in combination with doxorubicin, vincristine, cisplatin, or maphosphamide - common drugs used in current neuroblastoma treatment.ResultsMXD3 was highly expressed in neuroblastoma cell lines and in patient tumors that had high-risk features. Neuroblastoma cells treated in vitro with the MXD3 siRNA nanocomplexes showed MXD3 protein knockdown and resulted in cell apoptosis. Furthermore, on combining MXD3 siRNA nanocomplexes with each of the four drugs, all showed additive efficacy.ConclusionThese results indicate that MXD3 is a potential new target and that the use of MXD3 siRNA nanocomplexes is a novel therapeutic approach for neuroblastoma.

In this study, the HB22.7 anti-CD22 mAb, was used for specific, targeted delivery of the potent anti-cancer agent, monomethyl auristatin E (MMAE) to non-Hodgkin lymphoma (NHL). MMAE was covalently coupled to HB22.7 through a valine-citrulline peptide linker (vc). Maleimide-functionalized vcMMAE (mal-vcMMAE) was reacted with thiols of the partially reduced mAb. Approximately 4 molecules of MMAE were conjugated to HB22.7 as determined by residual thiol measurement and hydrophobic interaction chromatography-HPLC (HIC-HPLC). HB22.7-vcMMAE antibody drug conjugate (ADC) retained its binding to Ramos NHL cells and also exhibited potent and specific in vitro cytotoxicity on a panel of B cell NHL cell lines with IC(50)s of 20 - 284 ng/ml. HB22.7-vcMMAE also showed potent efficacy in vivo against established NHL xenografts using the DoHH2 and Granta 519 cell lines. One dose of the ADC induced complete and persistent response in all DoHH2 xenografts and 90% of Granta xenografts. Minimal toxicity was observed. In summary, HB22.7-vcMMAE is an effective ADC that should be evaluated for clinical translation.

Immunotherapy has revolutionized cancer therapy. Two of the most recently FDA-approved immunotherapies for B-cell malignancies target CD19, in the form of a Bispecific T-Cell Engager (BiTE) antibody construct or CAR-T cells. Blinatumomab, and FDA-approved BiTE, binds to CD19 on B cells and to CD3 on T cells, mediating killer-target cell contact and T cell activation that results in effective elimination of B cells. Although CD19 is expressed by essentially all cases of B cell malignancies at clinical presentation, relapses with loss or reduction of CD19 surface expression are increasingly recognized as a cause of treatment failure. We have developed a novel BiTE consisting of humanized anti-CD22 and anti-CD3 single chain variable fragments, expressed as a single molecule and purified from tissue culture supernatant. Binding of the anti-CD22 and anti-CD3 moieties to B and T cells, respectively, was confirmed by flow cytometry. CD22-BiTE promoted cell-mediated cytotoxicity in a dose and E:T-dependent fashion, with a maximum NHL and ALL-specific killing activity of 90% in vitro. Besides canonical expression in B cells, CD22 has been reported in non-hematologic malignancies. Our analysis of TCGA data suggests CD22 is expressed in a variety of solid tumors, including sarcoma. We verified expression of CD22 message and protein in solid tumor cell lines. Accordingly, our CD22-BiTE mediated killing of HT1080 sarcoma cells in vitro. In conclusion, we report here the development of a new BiTE with killing activity against CD22+ malignancies which could represent a therapeutic option for B-cell malignancies and potentially solid tumors.

MXD3 is a transcription factor that plays an important role in proliferation of human DAOY medulloblastoma cells. Here, we demonstrate that MXD3 is highly enriched in human precursor B acute lymphoblastic leukemia (preB ALL) samples compared to mobilized peripheral blood mononuclear cells, bone marrow, or hematopoietic stem cells from healthy donors. MXD3 knock-down in the preB ALL cell line Reh resulted in decreased cell numbers with no change in G0/G1, S or G2/M populations but increased apoptosis compared to control cells. Our results suggest that MXD3 is important for survival of Reh preB ALL cells, possibly as an anti-apoptotic factor.

Non-Hodgkin lymphoma (NHL) affects over 400,000 people in the United States; its incidence increases with age. Treatment options are numerous and expanding, yet efficacy is often limited by toxicity, particularly in the elderly. Nearly 70% patients eventually die of the disease. Many patients explore less toxic alternative therapeutics proposed to boost anti-tumor immunity, despite a paucity of rigorous scientific data. Here we evaluate the lymphomacidal and immunomodulatory activities of a protein fraction isolated from fermented wheat germ. Fermented wheat germ extract was produced by fermenting wheat germ with Saccharomyces cerevisiae. A protein fraction was tested for lymphomacidal activity in vitro using NHL cell lines and in vivo using mouse xenografts. Mechanisms of action were explored in vitro by evaluating apoptosis and cell cycle and in vivo by immunophenotyping and measurement of NK cell activity. Potent lymphomacidal activity was observed in a panel of NHL cell lines and mice bearing NHL xenografts. This activity was not dependent on wheat germ agglutinin or benzoquinones. Fermented wheat germ proteins induced apoptosis in NHL cells, and augmented immune effector mechanisms, as measured by NK cell killing activity, degranulation and production of IFNγ. Fermented wheat germ extract can be easily produced and is efficacious in a human lymphoma xenograft model. The protein fraction is quantifiable and more potent, shows direct pro-apoptotic properties, and enhances immune-mediated tumor eradication. The results presented herein support the novel concept that proteins in fermented wheat germ have direct pro-apoptotic activity on lymphoma cells and augment host immune effector mechanisms.

Checkpoint kinase inhibition has been studied as a way of enhancing the effectiveness of DNA-damaging agents. More recently, histone deacetylase inhibitors have shown efficacy in several cancers, including non-Hodgkin lymphoma. To evaluate the effectiveness of this combination for the treatment of lymphoma, we examined the combination of AR42, a histone deacetylase inhibitor, and checkpoint kinase 2 (CHEK2) inhibitor II in vitro and in vivo. The combination resulted in up to 10-fold increase in potency in five Burkitt lymphoma cell lines when compared with either drug alone. Both drugs inhibited tumor progression in xenograft models, but the combination was more effective than either agent alone, resulting in regression of established tumors. No toxicity was observed. These results suggest that the combination of histone deacetylase inhibition and checkpoint kinase inhibition represent an effective and nontoxic treatment option that should be further explored in preclinical and clinical studies.

Objective Hyperamylinemia, a common pancreatic disorder in obese and insulin-resistant patients, is known to cause amylin oligomerization and cytotoxicity in pancreatic islets, leading to β-cell mass depletion and development of type 2 diabetes. Recent data has revealed that hyperamylinemia also affects the vascular system, heart, and kidneys. We therefore hypothesized that oligomerized amylin might accumulate in the cerebrovascular system and brain parenchyma of diabetic patients. Methods Amylin accumulation in the brain of diabetic patients with vascular dementia or Alzheimer disease (AD), nondiabetic patients with AD, and age-matched healthy controls was assessed by quantitative real time polymerase chain reaction, immunohistochemistry, Western blot, and enzyme-linked immunosorbent assay. Results Amylin oligomers and plaques were identified in the temporal lobe gray matter from diabetic patients, but not controls. In addition, extensive amylin deposition was found in blood vessels and perivascular spaces. Intriguingly, amylin deposition was also detected in blood vessels and brain parenchyma of patients with late onset AD without clinically apparent diabetes. Mixed amylin and amyloid β (Aβ) deposits were occasionally observed. However, amylin accumulation leads to amyloid formation independent of Aβ deposition. Tissues infiltrated by amylin showed increased interstitial space, vacuolation, spongiform change, and capillaries bent at amylin accumulation sites. Unlike the pancreas, there was no evidence of amylin synthesis in the brain. Interpretation Metabolic disorders and aging promote accumulation of amylin amyloid in the cerebrovascular system and gray matter, altering microvasculature and tissue structure. Amylin amyloid formation in the wall of cerebral blood vessels may also induce failure of elimination of Aβ from the brain, thus contributing to the etiology of AD. Ann Neurol 2013;74:517–526

Summary During development of the central nervous system, precise synaptic connections between presynaptic and postsynaptic neurons are formed. While significant progress has been made in our understanding of AMPA receptor trafficking during synaptic plasticity, less is known about the molecules that recruit AMPA receptors to nascent synapses during synaptogenesis. Here we identify a type II transmembrane protein (SynDIG1) that regulates AMPA receptor content at developing synapses in dissociated rat hippocampal neurons. SynDIG1 colocalizes with AMPA receptors at synapses and at extrasynaptic sites and associates with AMPA receptors in heterologous cells and brain. Altered levels of SynDIG1 in cultured neurons result in striking changes in excitatory synapse number and function. SynDIG1-mediated synapse development is dependent on association with AMPA receptors via its extracellular C terminus. Intriguingly, SynDIG1 content in dendritic spines is regulated by neuronal activity. Altogether, we define SynDIG1 as an activity-regulated transmembrane protein that regulates excitatory synapse development.

Vitelline envelopes (VEs) of Bufo arenarum were isolated in order to study their composition and their role in fertilization. VEs are composed of four glycoproteins, with molecular masses of 120, 75, 41, and 38 kDa. To characterize its biological properties, we quantitatively determined sperm-VE binding and the induction of the acrosome reaction. Heterologous binding of B. arenarum sperm to Xenopus laevis VE components was observed with about one-third the efficiency of homologous binding. Equivalent binding of X. laevis sperm to the B. arenarum VE was observed. When B. arenarum sperm were incubated with fluorescein isothiocyanate-labeled VE, the labeled glycoproteins bound to the anterior end of the sperm head, showing a lateral distribution. Induction of the acrosome reaction was evaluated by incubating sperm in hypotonic saline media with VE glycoproteins. VEs induced the acrosome reaction in a time- and concentration-dependent manner. The acrosome reaction was maximal after 10 min. The half-m...

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals(1). In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)(2). When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci(1) that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

Conventional chemotherapy for precursor B-cell (preB) acute lymphoblastic leukaemia (ALL) has limitations that could be overcome by targeted therapy. Previously, we discovered a potential therapeutic molecular target, MDX3 (MAX dimerization protein 3), in preB ALL. In this study, we hypothesize that an effective siRNA therapy for preB ALL can be developed using antiCD22 antibody (αCD22 Ab) and nanoparticles. We composed nanocomplexes with super paramagnetic iron oxide nanoparticles (SPIO NPs), αCD22 Abs and MXD3 siRNA molecules based on physical interactions between the molecules. We demonstrated that the MXD3 siRNA-αCD22 Ab-SPIO NP complexes entered leukaemia cells and knocked down MXD3, leading the cells to undergo apoptosis and resulting in decreased live cell counts in the cell line Reh and in primary preB ALL samples in vitro. Furthermore, the cytotoxic effects of the MXD3 siRNA-αCD22 Ab-SPIO NP complexes were significantly enhanced by addition of the chemotherapy drugs vincristine or doxorubicin. We also ruled out potential cytotoxic effects of the MXD3 siRNA-αCD22 Ab-SPIO NP complexes on normal primary haematopoietic cells. Normal B cells were affected while CD34-positive haematopoietic stem cells and non-B cells were not. These data suggest that MXD3 siRNA-αCD22 Ab-SPIO NP complexes have the potential to be a new targeted therapy for preB ALL.

Background MXD3 is a basic-helix-loop-helix-leucine-zipper transcription factor involved in cellular proliferation. In previous studies we demonstrated that knock-down of MXD3 in the human medulloblastoma cell line DAOY resulted in decreased proliferation. Surprisingly, overexpression of MXD3 in DAOY cells also decreased proliferation and increased cell death, suggesting that persistent expression of MXD3 triggers an apoptotic response, perhaps as a fail-safe mechanism. To investigate this apparent paradox in detail we developed a tamoxifen inducible system to analyze the temporal effects of MXD3 in the proliferation and transcriptional response of DAOY cells upon acute induction compared with long-term expression of MXD3. Results We find that acute induction of MXD3 initially promotes cell cycle progression as assessed by a transient increase in bromodeoxyuridine incorporation. However, persistent induction of MXD3 ultimately results in decreased proliferation based on cell counts. Finally, with microarray expression profiling and gene ontology analysis we identify several major pathways enriched in response to acute (immune response, apoptosis, cell cycle) versus persistent (cell adhesion) MXD3 activation. Conclusions In this study, we demonstrate that acute MXD3 activation results in a transient increase in cell proliferation while persistent activation of MXD3 eventually results in an overall decrease in cell number, suggesting that the time course of MXD3 expression dictates the cellular outcome. Microarray expression profiling and gene ontology analysis indicate that MXD3 regulates distinct genes and pathways upon acute induction compared with persistent expression, suggesting that the cellular outcome is specified by changes in MXD3 transcriptional program in a time-dependent manner.

HB22.7, an anti-CD22 monoclonal antibody has shown consistent preclinical activity against non-Hodgkin lymphoma (NHL). Histone deacetylase inhibitors (HDACi) have demonstrated efficacy in lymphoma and can modulate cell surface receptor expression. To augment the lymphomacidal activity of HB22.7 we examined the combination of AR42 (an HDACi) and HB22.7 in vitro and in vivo. The combination resulted in 10-fold increased potency in 6 NHL cell lines when compared to either drug alone. Both drugs reduced tumor progression in xenografts, but the combination was significantly more efficacious and resulted in regression of established tumors, without toxicity. AR42 inhibited HB22.7-mediated CD22 internalization, suggesting that increased efficacy could be due to higher availability of CD22. Overall, the synergistic effects of HB22.7 and AR42 on in vitro cytotoxicity and in vivo anti-tumor activity make this combination an attractive option for further pre-clinical and clinical evaluation.

SummaryGalectins are a group of soluble animal lectins that exhibit specificity for β-galactosides and conserve sequence homology in the carbohydrate-recognition domain. The galectin fromBufo arenarumovary showed a strong cross-reaction with the lectin of 14.5 kDa purified from embryos at early blastula stage. In this paper, we studied the immunohistochemical localisation of the galectin of 14.5 kDa from ovary of the toadB. arenarumin adult ovary sections. We also analysed the immunohistochemical localisation of the embryonic lectin during early development using the antiserum anti-ovary galectin. In the ovary, oocytes in the previtellogenic stage showed strong reactivity in the nucleus and the cortex but not in the cytoplasm. Oocytes in the stage of primary vitellogenesis exhibited a similar pattern in the nuclear and cortical areas but showed immunostaining in the cytoplasm. Intense nuclear staining was detected in oocytes in the stage of late vitellogenesis and in mature oocytes, which also presented strong reactions in the yolk platelets that completely covered the cytoplasm. In blastula embryos the staining was found in the blastomeres, the yolk platelets and the blastocoele. Each lectin localisation is discussed in relation to potential biological roles in the corresponding tissues.

Hyperamylinemia, a common pancreatic disorder in obese and insulin-resistant patients, is known to cause amylin oligomerization and cytotoxicity in pancreatic islets, leading to β-cell mass depletion and development of type 2 diabetes. Recent data has revealed that hyperamylinemia also affects the vascular system, heart, and kidneys. We therefore hypothesized that oligomerized amylin might accumulate in the cerebrovascular system and brain parenchyma of diabetic patients.Amylin accumulation in the brain of diabetic patients with vascular dementia or Alzheimer disease (AD), nondiabetic patients with AD, and age-matched healthy controls was assessed by quantitative real time polymerase chain reaction, immunohistochemistry, Western blot, and enzyme-linked immunosorbent assay.Amylin oligomers and plaques were identified in the temporal lobe gray matter from diabetic patients, but not controls. In addition, extensive amylin deposition was found in blood vessels and perivascular spaces. Intriguingly, amylin deposition was also detected in blood vessels and brain parenchyma of patients with late onset AD without clinically apparent diabetes. Mixed amylin and amyloid β (Aβ) deposits were occasionally observed. However, amylin accumulation leads to amyloid formation independent of Aβ deposition. Tissues infiltrated by amylin showed increased interstitial space, vacuolation, spongiform change, and capillaries bent at amylin accumulation sites. Unlike the pancreas, there was no evidence of amylin synthesis in the brain.Metabolic disorders and aging promote accumulation of amylin amyloid in the cerebrovascular system and gray matter, altering microvasculature and tissue structure. Amylin amyloid formation in the wall of cerebral blood vessels may also induce failure of elimination of Aβ from the brain, thus contributing to the etiology of AD.

The goal of our project is to develop a new therap y for childhood malignancies using nanoformulated siRNA targeting Mxd3, a molecule in the Sonic Hedgehog signali ng pathway, which we belie ve is important for cell survival. We plan to use cancer-s pecific ligands and superparamagnetic iron oxide nanoparticles (SPIO NPs) to carry siRNA. This delivery system will be tested in mouse xenograft models that we developed with primary cancer tissues. Our current focus is acute lymphoblastic leukemia (ALL), the most common cancer in children. We report our progress to date. Keywords: siRNA, nanoparticles, acute ly mphoblastic leukemia, leukemia-speci fic ligand, targeted therapy 1. INTRODUCTION 1.1 Childhood acute lymphoblastic leukemia and current treatment Leukemia is the most common cancer in children, with B-cell type ALL being the most common type (Figure 1 ) [1,2]. Despite the progress in medicine, certain types of ALL continue to have poor outcomes, with survival rates as low as 30% [2]. Current standard treatments take 2 ½ to 3 years and include systemic chemotherapy or radiation therapy. These treatments ar e highly toxic to growing ch ildren, as they inevitably damage healthy cells along with the leukemia cells. These toxicities are not limited to short-term side effects, but also result in long-term irreversible side effects th at decrease the quality of life of children who survive. These long-term effects involve multi-organ systems leading to poor growth, osteoporosis, infertility, heart failure, and secondary cancers [3]. To overcome the limitations and significant side effects of current treatments, we need new approaches that specifically target leukemia cells. A few promising targeted therapies have been clinically tested in different le ukemias, such as a BCR-ABL ty rosine kinase inhibitor in chronic myeloid leukemia (and in BCR-ABL positive ALL) and all-trans retinoic acid in acute myeloid leukemia [7]. However, no targeted therapy h as been applied exclusively for childhood ALL.

Background Consistent asymmetry of the left-right (LR) axis is a crucial aspect of vertebrate embryogenesis. Asymmetric gene expression of the TGFβ superfamily member Nodal related 1 (Nr1) in the left lateral mesoderm plate is a highly conserved step regulating the situs of the heart and viscera. In Xenopus, movement of maternal serotonin (5HT) through gap-junctional paths at cleavage stages dictates asymmetry upstream of Nr1. However, the mechanisms linking earlier biophysical asymmetries with this transcriptional control point are not known. Results To understand how an early physiological gradient is transduced into a late, stable pattern of Nr1 expression we investigated epigenetic regulation during LR patterning. Embryos injected with mRNA encoding a dominant-negative of Histone Deacetylase (HDAC) lacked Nr1 expression and exhibited randomized sidedness of the heart and viscera (heterotaxia) at stage 45. Timing analysis using pharmacological blockade of HDACs implicated cleavage stages as the active period. Inhibition during these early stages was correlated with an absence of Nr1 expression at stage 21, high levels of heterotaxia at stage 45, and the deposition of the epigenetic marker H3K4me2 on the Nr1 gene. To link the epigenetic machinery to the 5HT signaling pathway, we performed a high-throughput proteomic screen for novel cytoplasmic 5HT partners associated with the epigenetic machinery. The data identified the known HDAC partner protein Mad3 as a 5HT-binding regulator. While Mad3 overexpression led to an absence of Nr1 transcription and randomized the LR axis, a mutant form of Mad3 lacking 5HT binding sites was not able to induce heterotaxia, showing that Mad3's biological activity is dependent on 5HT binding. Conclusion HDAC activity is a new LR determinant controlling the epigenetic state of Nr1 from early developmental stages. The HDAC binding partner Mad3 may be a new serotonin-dependent regulator of asymmetry linking early physiological asymmetries to stable changes in gene expression during organogenesis.

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

During development, Sonic hedgehog (Shh) regulates the proliferation of cerebellar granule neuron precursors (GNPs) in part via expression of Nmyc. Mutations in the Shh signaling pathway lead to brain tumors in mice and humans. We have recently identified a novel role for the Mad family member Mad3 in GNP proliferation and Nmyc expression. Interestingly, Mad3 expression is upregulated in mouse models of medulloblastoma, the most common brain tumor in children. These results are surprising because current models suggest that Mad proteins should antagonize Myc proteins by competition for direct DNA binding via Max heterodimerization to inhibit cellular proliferation and potentially tumor progression. Here, we discuss our recent work in the context of candidate Mad3-interacting proteins and Mad3 expression in human brain tumors that together suggest interesting insights into the role of Mad3 in cellular proliferation and tumorigenesis.

The vitelline envelope (VE) parti- cipates in sperm-egg interactions during the first steps of fertilization. In Bufo arenarum, this envelope is composed of at least four glycoproteins, with molecular masses of 120, 75, 41, and 38 kDa and molar ratio of 1:1.3:7.4:4.8, respectively. These components were isolated and covalently coupled to silanized glass slides in order to study their sperm-binding capacity. When considering the molar ratio of the glycoproteins in the egg-envelope and assuming that each protein is monovalent for sperm, the assay showed that gp41 and gp38 possess 55 and 25% of total sperm-binding activity. We obtained a full-length cDNA of gp41 (ZPC), comprising a sequence for 486 amino acids, with 43.3% homology with Xenopus laevis ZPC. As in the case of mammalian ZP3 and Xenopus ZPC, Bufo ZPC presented a furin-like (convertase) and a C- terminal transmembrane domain (TMD) reflecting common biosynthetic and secretory pathways. As it was reported for some fishes, we obtained evidence that suggests the presence of more than one zpc gene in Bufo genome, based on different partial cDNA sequences of zpc, Southern blots and two-dimensional SDS-PAGE of deglycosylated egg-envelope compo- nents. As far as we are aware, this is the first observation of the presence of different zpc genes in an Amphibian species. Mol. Reprod. Dev. 74: 629- 640, 2007. 2006 Wiley-Liss, Inc.

Vitelline envelopes (VEs) of Bufo arenarum were isolated in order to study their composition and their role in fertilization. VEs are composed of four glycoproteins, with molecular masses of 120, 75, 41, and 38 kDa. To characterize its biological properties, we quantitatively determined sperm-VE binding and the induction of the acrosome reaction. Heterologous binding of B. arenarum sperm to Xenopus laevis VE components was observed with about one-third the efficiency of homologous binding. Equivalent binding of X. laevis sperm to the B. arenarum VE was observed. When B. arenarum sperm were incubated with fluorescein isothiocyanate-labeled VE, the labeled glycoproteins bound to the anterior end of the sperm head, showing a lateral distribution. Induction of the acrosome reaction was evaluated by incubating sperm in hypotonic saline media with VE glycoproteins. VEs induced the acrosome reaction in a time- and concentration-dependent manner. The acrosome reaction was maximal after 10 min. The half-maximal effect was obtained at a glycoprotein concentration of 1 microg/ml. Specificity was determined using fertilization envelope glycoproteins, which failed to induce the acrosome reaction. The B. arenarum VE is biochemically similar to other egg envelopes. It also seems that its biological properties are similar to other species in regard to sperm binding and induction of the acrosome reaction. However, as far as we are aware, this is the first observation of the VE inducing the sperm acrosome reaction in amphibians. The relatively small differences observed in heterologous sperm-VE binding in X. laevis and B. arenarum are inconsistent with the current paradigm that species specificity in fertilization is regulated at the sperm-VE binding step.

Neuroblastoma is a cancer of the sympathetic nervous system and the most common extracranial solid tumor in children. Almost half of neuroblastoma patients have a high-risk phenotype at diagnosis: metastatic disease. Prognosis of these patients is very poor with only 30% survival despite the most intensive therapies currently available. In addition, patients who respond successfully to treatment suffer from side effects from chemo and radiation therapies, leading to life-long irreversible complications. Therefore, there is a desperate need for a neuroblastoma-targeted therapy that is more effective and has fewer side effects than conventional therapies. Our goal is to develop a molecular-targeted therapy that can be used in tandem with current treatments. In our previous study, we identified the transcription factor MXD3 as a novel molecular target for neuroblastoma (presented in the 2013 AACR annual meeting). In the current study, we demonstrated in vitro efficacy of an MXD3 targeted therapeutic using siRNA and nanoparticles. We have developed MXD3 targeting methods using MXD3 siRNA and superparamagnetic iron oxide nanoparticles (NPs). We assembled siRNA on the surface of NPs and introduced them to SK-N-DZ cells (neuroblastoma cell line with high MXD3 expression) in vitro. Upon a single treatment with the siRNA-NP-complexes, the siRNAs were successfully delivered into the cells and MXD3 knockdown was confirmed at the protein level as early as 4 hours post treatment. The cells treated with the MXD3 siRNA-NP-complexes showed significantly decreased proliferation compared to those treated with control siRNA-NP-complexes or untreated cells (p In conclusion, we have demonstrated novel in vitro delivery of the MXD3 siRNA-NP complexes into neuroblastoma cells. We have also shown that MXD3 knockdown induced apoptosis and inhibited cell proliferation. Furthermore, we showed additive effects of the MXD3 siRNA-NP complexes with doxorubicin and vincristine. Further work is ongoing to determine the therapeutic effects in other neuroblastoma cell lines with different MXD3 expression and side effects in normal blood cells. The future plan is to optimize our nanocomplexes for efficient in vivo delivery, which is the major hurdle in the development of siRNA-based therapeutics. Citation Format: Connie Duong, Cathy Chen, Sakiko Yoshida, Gustavo Barisone, Jan Nolta, Elva Diaz, Nitin Nitin, Noriko Satake. Novel targeted therapy for neuroblastoma: Silencing the MXD3 gene using siRNA. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5423. doi:10.1158/1538-7445.AM2014-5423

Prothymosin alpha (PTA) was detected by immunocytological and biochemical methods in oocytes at different stages of oogenesis, and in early embryos of the amphibian Bufo arenarum. In all cases PTA was detected in the nucleus and was absent from the cytoplasm. This indicates that this protein could act at the level of regulating transcription. Western blots were carried out using polyclonal antibodies with extracts of embryos at different stages of development from early fertilisation up to neural tube. With this method PTA was detected in all the samples under study.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Up to 45% of the cases are high risk at diagnosis, representing very aggressive, usually metastasized tumors that are refractory or recurrent after all available therapies. Prognosis for these patients is very poor with only 30% survival. MXD3 is a transcription factor previously shown in our lab to be a novel member of the Hedgehog (Hh) pathway (Yun, Rust, Ishimaru, Diaz, Mol Cell Biol, 2007) in granule neuron precursors. In these cells, overexpression of MXD3 results in higher MYCN expression, suggesting a functional interaction between the two transcription factors. MXD3 phenocopies MYCN expression in mouse models of medulloblastoma; we have also shown that it is expressed in human medulloblastoma and that its knockdown reduces proliferation of human medulloblastoma cell lines (Barisone et al., PLoS One, 2012). More than 20% of NBs have MYCN amplification, which is considered a marker of poor prognosis. In the current study, we investigated a possible role for MXD3 in NB cell proliferation. Using qRT-PCR we observed high levels of MXD3 mRNA expression in 5 NB cell lines: SK-N-DZ, IMR-32, SH-SY5Y, SK-N-BE and SK-N-SH. Immunoblot analysis with anti-MXD3 monoclonal antibodies confirmed that the protein was present in these cells. Furthermore, immunohistochemistry analysis showed strong immunoreactivity in a cohort of 16 primary NB tissue specimens. We investigated the role of MXD3 in NB cell proliferation by silencing MXD3 in the SK-N-DZ cell line. We used lentiviral delivery to knockdown MXD3 using an RNA interference approach. Upon transduction by the viral vector, MXD3 knockdown was confirmed at both the RNA and protein level. Within 48 hours, MXD3 protein levels were reduced >90% in cells infected with the shMXD3 virus but not with the control virus. MXD3 knockdown resulted in decreased proliferation in NB cells, supporting our hypothesis that it may be involved in the maintenance of NB. We analyzed cell cycle progression after knockdown using flow cytometry. We observed no significant differences in the G0/G1, S or G2/M populations between experimental and control samples. Our results suggest that MXD3 is important for NB cell proliferation, possibly by acting as an anti-apoptotic factor. Therefore, MXD3 is a potential candidate for targeted therapies against neuroblastoma. Citation Format: Gustavo Barisone, Noriko Satake, Carly Lewis, Connie Duong, Cathy Chan, Kit Lam, Jan Nolta, Elva Diaz. Mxd3 is required for proliferation of human neuroblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3128. doi:10.1158/1538-7445.AM2013-3128

Abstract 3523 Disregulation of the Hedgehog (Hh) signaling pathway is known to drive proliferation in several cancers, including hematopoietic malignancies. The role of the Hh pathway in precursor B cell (preB) acute lymphoblastic leukemia (ALL), the most common leukemia in children, is unclear; however, recent reports suggest that Hh signaling is involved in the development of B cell precursors as well as the self-renewal and survival of B cell malignancies. MXD3 is a transcription factor previously shown in our lab to be a novel member of the Hh pathway (Yun, Rust, Ishimaru, Diaz, Mol Bio Cell, 2007). We have previously shown that MXD3 is expressed in medulloblastoma (the most common brain tumor in children) and that its knockdown reduces proliferation of human medulloblastoma cell lines (Barisone et al., PLoS, 2012). In the current study, we investigated a possible role for MXD3 in preB ALL cell proliferation. Using quantitative real-time reverse-transcription PCR (qRT-PCR) we observed high levels of MXD3 mRNA expression in 8 primary preB ALL samples as well as in the preB ALL cell lines Reh and JM1. However, MXD3 levels were very low in mobilized peripheral blood mononuclear cells from healthy donors, mouse bone marrow and spleen. Indeed, MXD3 levels in the primary ALL cells and cell lines ranged between 13 and 35 fold increase when compared to the normal cells. Immunoblot analysis with anti-MXD3 monoclonal antibodies confirmed that the protein was present in the ALL samples but not in normal cells. We investigated the role of MXD3 in cell proliferation and survival, by silencing MXD3 in the Reh cell line. We used lentiviral delivery to knockdown MXD3 using an RNA interference approach. Briefly, lentiviruses were produced carrying a short hairpin RNA sequence specific for MXD3 (shMXD3) or a negative control sequence (shCTRL). Upon transduction by the viral vector, MXD3 knockdown was confirmed at both the RNA and protein level. Within 48 hours after transduction, MXD3 protein levels were reduced >90% in cells infected with the shMXD3 virus but not with the control virus. Interestingly, MXD3 knockdown resulted in decreased proliferation in Reh cells, supporting our hypothesis that it may be involved in the maintenance of this malignancy. As a first step toward understanding the mechanisms by which MXD3 exerts its function in leukemia cells, we analyzed cell cycle progression and apoptosis levels after knockdown using flow cytometry. We observed no significant differences in the G0/G1, S or G2/M populations between experimental and control samples. However, samples where MXD3 had been knocked down showed higher levels of apoptosis when compared to controls. Taken together, our results suggest that MXD3 is important for preB ALL cell proliferation, possibly by acting as an anti-apoptotic factor. Therefore, MXD3 may represent a suitable candidate for future efforts in developing targeted therapies against preB ALL. Disclosures: No relevant conflicts of interest to declare.

A subset of medulloblastomas, the most common brain tumor in children, is hypothesized to originate from granule neuron precursors (GNPs) in which the sonic hedgehog (SHH) pathway is over-activated. MXD3, a basic helix-look-helix zipper transcription factor of the MAD family, has been reported to be upregulated during postnatal cerebellar development and to promote GNP proliferation and MYCN expression. Mxd3 is upregulated in mouse models of medulloblastoma as well as in human medulloblastomas. Therefore, we hypothesize that MXD3 plays a role in the cellular events that lead to medulloblastoma biogenesis. In agreement with its proliferative role in GNPs, MXD3 knock-down in DAOY cells resulted in decreased proliferation. Sustained overexpression of MXD3 resulted in decreased cell numbers due to increased apoptosis and cell cycle arrest. Structure-function analysis revealed that the Sin3 interacting domain, the basic domain, and binding to E-boxes are essential for this activity. Microarray-based expression analysis indicated up-regulation of 84 genes and down-regulation of 47 genes. Potential direct MXD3 target genes were identified by ChIP-chip. Our results suggest that MXD3 is necessary for DAOY medulloblastoma cell proliferation. However, increased level and/or duration of MXD3 expression ultimately reduces cell numbers via increased cell death and cell cycle arrest.

A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1) when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE), 2) after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE), and 3) after oocyte activation (surrounded by the fertilization envelope, (FE). The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE.