Daniel Meruelo, Ph.D.

Activation of cytotoxic and regulatory functions of NK cells by sindbis viral vectors
Granot, Tomer; Venticinque, Lisa; Tseng, Jen-Chieh; Meruelo, Daniel
2011 ;6(6):e20598-e20598, PLoS ONE
Oncolytic viruses (OVs) represent a relatively novel anti-cancer modality. Like other new cancer treatments, effective OV therapy will likely require combination with conventional treatments. In order to design combinatorial treatments that work well together, a greater scrutiny of the mechanisms behind the individual treatments is needed. Sindbis virus (SV) based vectors have previously been shown to target and kill tumors in xenograft, syngeneic, and spontaneous mouse models. However, the effect of SV treatment on the immune system has not yet been studied. Here we used a variety of methods, including FACS analysis, cytotoxicity assays, cell depletion, imaging of tumor growth, cytokine blockade, and survival experiments, to study how SV therapy affects Natural Killer (NK) cell function in SCID mice bearing human ovarian carcinoma tumors. Surprisingly, we found that SV anti-cancer efficacy is largely NK cell-dependent. Furthermore, the enhanced therapeutic effect previously observed from Sin/IL12 vectors, which carry the gene for interleukin 12, is also NK cell dependent, but works through a separate IFNgamma-dependent mechanism, which also induces the activation of peritoneal macrophages. These results demonstrate the multimodular nature of SV therapy, and open up new possibilities for potential synergistic or additive combinatorial therapies with other treatments
— id: 134464, year: 2011, vol: 6, page: e20598, stat: Journal Article,

Efficient in vivo microRNA targeting of liver metastasis
Huynh, C; Segura, M F; Gaziel-Sovran, A; Menendez, S; Darvishian, F; Chiriboga, L; Levin, B; Meruelo, D; Osman, I; Zavadil, J; Marcusson, E G; Hernando, E
2011 Mar 24;30(12):1481-1488, Oncogene
Targeting oncogenic microRNAs (miRNAs) is emerging as a promising strategy for cancer therapy. In this study, we provide proof of principle for the safety and efficacy of miRNA targeting against metastatic tumors. We tested the impact of targeting miR-182, a pro-metastatic miRNA frequently overexpressed in melanoma, the in vitro silencing of which represses invasion and induces apoptosis. Specifically, we assessed the effect of anti-miR-182 oligonucleotides synthesized with 2' sugar modifications and a phosphorothioate backbone in a mouse model of melanoma liver metastasis. Luciferase imaging showed that mice treated with anti-miR-182 had a lower burden of liver metastases compared with control. We confirmed that miR-182 levels were effectively downregulated in the tumors of anti-miR-treated mice compared with tumors of control-treated mice, both in the liver and in the spleen. This effect was accompanied by an upregulation of multiple miR-182 direct targets. Transcriptional profiling of tumors treated with anti-miR-182 or with control oligonucleotides revealed an enrichment of genes controlling survival, adhesion and migration modulated in response to anti-miR-182 treatment. These data indicate that in vivo administration of anti-miRs allows for efficient miRNA targeting and concomitant upregulation of miRNA-controlled genes. Our results demonstrate that the use of anti-miR-182 is a promising therapeutic strategy for metastatic melanoma and provide a solid basis for testing similar strategies in human metastatic tumors
— id: 138159, year: 2011, vol: 30, page: 1481, stat: Journal Article,

Structure-guided identification of a laminin binding site on the laminin receptor precursor
Jamieson, Kelly V; Hubbard, Stevan R; Meruelo, Daniel
2011 Jan 7;405(1):24-32, Journal of molecular biology
The 37/ 67-kDa human laminin receptor (LamR) is a cell surface receptor for laminin, prion protein, and a variety of viruses. Because of its wide range of ligands, LamR plays a role in numerous pathologies. LamR overexpression correlates with a highly invasive cell phenotype and increased metastatic ability, mediated by interactions between LamR and laminin. In addition, the specific targeting of LamR with small interfering RNAs, blocking antibodies, and Sindbis viral vectors confers anti-tumor effects. We adopted a structure-based approach to map a laminin binding site on human LamR by comparing the sequences and crystal structures of LamR and Archaeoglobus fulgidus S2p, a non-laminin-binding ortholog. Here, we identify a laminin binding site on LamR, comprising residues Phe32, Glu35, and Arg155, which are conserved among mammalian species. Mutation of these residues results in a significant loss of laminin binding. Further, recombinant wild-type LamR is able to act as a soluble decoy to inhibit cellular migration towards laminin. Mutation of this laminin binding site results in loss of migration inhibition, which demonstrates the physiological role of Phe32, Glu35, and Arg155 for laminin binding activity. Mapping of the LamR binding site should contribute to the development of therapeutics that inhibit LamR interactions with laminin and may aid in the prevention of tumor growth and metastasis
— id: 116207, year: 2011, vol: 405, page: 24, stat: Journal Article,

Interactions between laminin receptor and the cytoskeleton during translation and cell motility
Venticinque, Lisa; Jamieson, Kelly V; Meruelo, Daniel
2011 ;6(1):e15895-e15895, PLoS ONE
Human laminin receptor acts as both a component of the 40S ribosomal subunit to mediate cellular translation and as a cell surface receptor that interacts with components of the extracellular matrix. Due to its role as the cell surface receptor for several viruses and its overexpression in several types of cancer, laminin receptor is a pathologically significant protein. Previous studies have determined that ribosomes are associated with components of the cytoskeleton, however the specific ribosomal component(s) responsible has not been determined. Our studies show that laminin receptor binds directly to tubulin. Through the use of siRNA and cytoskeletal inhibitors we demonstrate that laminin receptor acts as a tethering protein, holding the ribosome to tubulin, which is integral to cellular translation. Our studies also show that laminin receptor is capable of binding directly to actin. Through the use of siRNA and cytoskeletal inhibitors we have shown that this laminin receptor-actin interaction is critical for cell migration. These data indicate that interactions between laminin receptor and the cytoskeleton are vital in mediating two processes that are intimately linked to cancer, cellular translation and migration
— id: 120657, year: 2011, vol: 6, page: e15895, stat: Journal Article,

Extraribosomal functions associated with the C terminus of the 37/67 kDa laminin receptor are required for maintaining cell viability
Scheiman, J; Jamieson, K V; Ziello, J; Tseng, J-C; Meruelo, D
2010 ;1:e42-e42, Cell death & disease
The 37/67 kDa laminin receptor (LAMR) is a multifunctional protein, acting as an extracellular receptor, localizing to the nucleus, and playing roles in rRNA processing and ribosome assembly. LAMR is important for cell viability; however, it is unclear which of its functions are essential. We developed a silent mutant LAMR construct, resistant to siRNA, to rescue the phenotypic effects of knocking down endogenous LAMR, which include inhibition of protein synthesis, cell cycle arrest, and apoptosis. In addition, we generated a C-terminal-truncated silent mutant LAMR construct structurally homologous to the Archaeoglobus fulgidus S2 ribosomal protein and missing the C-terminal 75 residues of LAMR, which displays more sequence divergence. We found that HT1080 cells stably expressing either silent mutant LAMR construct still undergo arrest in the G1 phase of the cell cycle when treated with siRNA. However, the expression of full-length silent mutant LAMR rescues cell viability, whereas the expression of the C-terminal-truncated LAMR does not. Interestingly, we also found that both silent mutant constructs restore protein translation and localize to the nucleus. Our findings indicate that the ability of LAMR to regulate viability is associated with its C-terminal 75 residues. Furthermore, this function is distinct from its role in cell proliferation, independent of its ribosomal functions, and may be regulated by a nonnuclear localization
— id: 138316, year: 2010, vol: 1, page: e42, stat: Journal Article,

Multiple functions of the 37/67-kd laminin receptor make it a suitable target for novel cancer gene therapy
Scheiman, Jonathan; Tseng, Jen-Chieh; Zheng, Yun; Meruelo, Daniel
2010 Jan;18(1):63-74, Molecular therapy
The 37/67-kd laminin receptor, LAMR, is a multifunctional protein that associates with the 40S ribosomal subunit and also localizes to the cell membrane to interact with the extracellular matrix. LAMR is overexpressed in many types of cancer, playing important roles in tumor-cell migration and invasion. Here, we show that LAMR is also vital for tumor-cell proliferation, survival, and protein translation. Small-interfering RNA (siRNA)-mediated reduction in expression of LAMR leads to G1 phase cell-cycle arrest in vitro by altering cyclins A2/B1, cyclin-dependent kinases (CDKs) 1/2, Survivin, and p21 expression levels. In vivo, reduction in LAMR expression results in inhibition of HT1080 cells to develop tumors. We also found that LAMR's ribosomal functions are critical for translation as reduction in LAMR expression leads to a dramatic decrease in newly synthesized proteins. Further, cells with lower expression of LAMR have fewer 40S subunits and 80S monosomes, causing an increase in free 60S ribosomal subunits. These results indicate that LAMR is able to regulate tumor development in many ways; further enhancing its potential as a target for gene therapy. To test this, we developed a novel Sindbis/Lenti pseudotype vector carrying short-hairpin RNA (shRNA) designed against lamr. This pseudotype vector effectively reduces LAMR expression and specifically targets tumors in vivo. Treatment of tumor-bearing severe combine immunodeficient (SCID) mice with this pseudotype vector significantly inhibits tumor growth. Thus, we show that LAMR can be used as a target in novel therapy for tumor reduction and elimination
— id: 106089, year: 2010, vol: 18, page: 63, stat: Journal Article,

Enhanced specific delivery and targeting of oncolytic Sindbis viral vectors by modulating vascular leakiness in tumor
Tseng, J-C; Granot, T; DiGiacomo, V; Levin, B; Meruelo, D
2010 Apr;17(4):244-255, Cancer gene therapy
Genetic instability of cancer cells generates resistance after initial responses to chemotherapeutic agents. Several oncolytic viruses have been designed to exploit specific signatures of cancer cells, such as important surface markers or pivotal signaling pathways for selective replication. It is less likely for cancer cells to develop resistance given that mutations in these cancer signatures would negatively impact tumor growth and survival. However, as oncolytic viral vectors are large particles, they suffer from inefficient extravasation from tumor blood vessels. Their ability to reach cancer cells is an important consideration in achieving specific oncolytic targeting and potential vector replication. Our previous studies indicated that the Sindbis viral vectors target tumor cells by the laminin receptor. Here, we present evidence that modulating tumor vascular leakiness, using VEGF and/or metronomic chemotherapy regimens, significantly enhances tumor vascular permeability and directly enhances oncolytic Sindbis vector targeting in tumor models. Because host-derived vascular endothelium cells are genetically stable and less likely to develop resistance to chemotherapeutics, a combined metronomic chemotherapeutics and oncolytic vector regimen should provide a new approach for cancer therapy. This mechanism could explain the synergistic treatment outcomes observed in clinical trials of combined therapies
— id: 108426, year: 2010, vol: 17, page: 244, stat: Journal Article,

Sindbis viral vector induced apoptosis requires translational inhibition and signaling through Mcl-1 and Bak
Venticinque, Lisa; Meruelo, Daniel
2010 ;9:37-37, Molecular cancer
ABSTRACT: BACKGROUND: Sindbis viral vectors are able to efficiently target and kill tumor cells in vivo, as shown using pancreatic and ovarian cancer models. Infection results in apoptosis both in vitro and in vivo. Sindbis vector uptake is mediated by the LAMR, which is upregulated on a number of different tumor types, thus conferring specificity of the vector to a wide range of cancers. In this study we elucidate the mechanism of apoptosis in two tumor cell lines, MOSEC, derived from the ovarian epithelium and Pan02, derived from a pancreatic adenocarcinoma. A comprehensive understanding of the mechanism of apoptosis would facilitate the design of more effective vectors for cancer therapy. RESULTS: The initial phase of Sindbis vector induced apoptosis in MOSEC and Pan02 models reconfirms that viral infection is sensed by PKR due to double-stranded RNA intermediates associated with genomic replication. PKR activation results in translation inhibition through eIF2alpha phosphorylation and initiation of the stress response. Our studies indicate that the roles of two proteins, Mcl-1 and JNK, intimately link Sindbis induced translational arrest and cellular stress. Translational arrest inhibits the synthesis of anti-apoptotic Bcl-2 protein, Mcl-1. JNK activation triggers the release of Bad from 14-3-3, which ultimately results in apoptosis. These signals from translational arrest and cellular stress are propagated to the mitochondria where Bad and Bik bind to Bcl-xl and Mcl-1 respectively. Formation of these heterodimers displaces Bak, which results in caspase 9 cleavage and signaling through the mitochondrial pathway of apoptosis. CONCLUSION: The host cell response to Sindbis is triggered through PKR activation. Our studies demonstrate that PKR activation and subsequent translational arrest is linked to both cellular stress and apoptosis. We have also found the linkage point between translational arrest and apoptosis to be Mcl-1, a protein whose constant translation is required for inhibition of apoptosis. With this information vectors can be designed, which express or repress proteins implicated in this study, to enhance their therapeutic potential
— id: 108791, year: 2010, vol: 9, page: 37, stat: Journal Article,

CCR7 signalling as an essential regulator of CNS infiltration in T-cell leukaemia
Buonamici, Silvia; Trimarchi, Thomas; Ruocco, Maria Grazia; Reavie, Linsey; Cathelin, Severine; Mar, Brenton G; Klinakis, Apostolos; Lukyanov, Yevgeniy; Tseng, Jen-Chieh; Sen, Filiz; Gehrie, Eric; Li, Mengling; Newcomb, Elizabeth; Zavadil, Jiri; Meruelo, Daniel; Lipp, Martin; Ibrahim, Sherif; Efstratiadis, Argiris; Zagzag, David; Bromberg, Jonathan S; Dustin, Michael L; Aifantis, Iannis
2009 Jun 18;459(7249):1000-1004, Nature
T-cell acute lymphoblastic leukaemia (T-ALL) is a blood malignancy afflicting mainly children and adolescents. T-ALL patients present at diagnosis with increased white cell counts and hepatosplenomegaly, and are at an increased risk of central nervous system (CNS) relapse. For that reason, T-ALL patients usually receive cranial irradiation in addition to intensified intrathecal chemotherapy. The marked increase in survival is thought to be worth the considerable side-effects associated with this therapy. Such complications include secondary tumours, neurocognitive deficits, endocrine disorders and growth impairment. Little is known about the mechanism of leukaemic cell infiltration of the CNS, despite its clinical importance. Here we show, using T-ALL animal modelling and gene-expression profiling, that the chemokine receptor CCR7 (ref. 5) is the essential adhesion signal required for the targeting of leukaemic T-cells into the CNS. Ccr7 gene expression is controlled by the activity of the T-ALL oncogene Notch1 and is expressed in human tumours carrying Notch1-activating mutations. Silencing of either CCR7 or its chemokine ligand CCL19 (ref. 6) in an animal model of T-ALL specifically inhibits CNS infiltration. Furthermore, murine CNS-targeting by human T-ALL cells depends on their ability to express CCR7. These studies identify a single chemokine-receptor interaction as a CNS 'entry' signal, and open the way for future pharmacological targeting. Targeted inhibition of CNS involvement in T-ALL could potentially decrease the intensity of CNS-targeted therapy, thus reducing its associated short- and long-term complications
— id: 105354, year: 2009, vol: 459, page: 1000, stat: Journal Article,

Controlled propagation of replication-competent Sindbis viral vector using suicide gene strategy
Tseng, J-C; Daniels, G; Meruelo, D
2009 Feb;16(2):291-296, Gene therapy
A major concern of using viral gene therapy is the potential for uncontrolled vector propagation and infection that might result in serious deleterious effects. To enhance the safety, several viral vectors, including vectors based on Sindbis virus, were engineered to lose their capability to replicate and spread after transduction of target cells. Such designs, however, could dramatically reduce the therapeutic potency of the viral vectors, resulting in the need for multiple dosages to achieve treatment goals. Earlier, we showed that a replication-defective (RD) Sindbis vector achieved specific tumor targeting without any adverse effects in vivo. Here, we present a replication-competent Sindbis viral vector that has an hsvtk suicide gene incorporated into ns3, an indispensable non-structural gene for viral survival. The capability of viral propagation significantly increases tumor-specific infection and enhances growth suppression of tumor compared with the conventional RD vectors. Furthermore, in the presence of the prodrug ganciclovir, the hsvtk suicide gene serves as a safety mechanism to prevent uncontrolled vector propagation. In addition to suppressing vector propagation, toxic metabolites, generated by prodrug activation, could spread to neighboring uninfected tumor cells to further enhance tumor killing
— id: 105914, year: 2009, vol: 16, page: 291, stat: Journal Article,

Crystal structure of the human laminin receptor precursor
Jamieson, Kelly V; Wu, Jinhua; Hubbard, Stevan R; Meruelo, Daniel
2008 Feb 8;283(6):3002-3005, Journal of biological chemistry
The human laminin receptor (LamR) interacts with many ligands, including laminin, prions, Sindbis virus, and the polyphenol (-)-epigallocatechin-3-gallate (EGCG), and has been implicated in a number of diseases. LamR is overexpressed on tumor cells, and targeting LamR elicits anti-cancer effects. Here, we report the crystal structure of human LamR, which provides insights into its function and should facilitate the design of novel therapeutics targeting LamR
— id: 76763, year: 2008, vol: 283, page: 3002, stat: Journal Article,

Restricted tissue tropism and acquired resistance to Sindbis viral vector expression in the absence of innate and adaptive immunity
Tseng, J-C; Zheng, Y; Yee, H; Levy, D E; Meruelo, D
2007 Aug;14(15):1166-1174, Gene therapy
Our previous studies suggest that replication-defective Sindbis vectors might be promising agents for specific tumor targeting and detection. However, the effects of innate and/or adaptive anti-viral immunity, in particular, the IFN-I/STAT1 signaling pathway, may impact their therapeutic potential. Using a bioluminescent imaging system, we demonstrate that although most normal cells are not permissively transduced by replication-defective Sindbis vector, transduction of liver non-sinusoidal endothelial occurs the first time IFN-I/STAT1 signaling deficient mice are inoculated with the vector. Transduction of some cells is not surprising since STAT1 knockout animals show significant delay in IFN responses such as the production of IFN-alpha/beta and transcriptional activation of several anti-viral genes (IRF7, RIG-I, PKR, TLR3, USP18, ISG15). However, beyond the initial vector transduction, which resolves rapidly, secondary inoculums of Sindbis vectors do not transduce any liver cells, suggesting that an alternative antiviral pathway may protect against further transduction. Other known signaling pathways were examined using mice lacking functional TLR3, tumor necrosis factoralphaR or nuclear factor-kappa B (p50). Surprisingly, none of those pathways seem to play a significant role in anti-Sindbis responses. Thus it appears that in vivo, in contrast to the ready transduction of tumor cells, transduction of normal cells by replication-defective Sindbis vector is limited, possibly by a novel mechanism
— id: 74661, year: 2007, vol: 14, page: 1166, stat: Journal Article,

Gene therapy that safely targets and kills tumor cells throughout the body
Hurtado, Alicia; Tseng, Jen-Chieh; Meruelo, Daniel
2006 Spring;9(1):36-44, Rejuvenation research
The authors studied the therapeutic value of Sindbis vectors for advanced metastatic cancer by using a variety of clinically accurate mouse models and demonstrated through imaging, histological, and molecular data that Sindbis vectors systemically and specifically infect/detect and kill metastasized tumors in vivo, leading to significant suppression of tumor growth and enhanced survival. Use of two different bioluminescent genetic markers for the IVIS Imaging System (Xenogen Corp., Alameda, CA) permitted demonstration of an excellent correlation between vector delivery and metastatic locations in vivo. Sindbis tumor specificity is not attributable to a species difference between human tumor and mouse normal cells. Sindbis virus is known to infect mammalian cells using the Mr 67,000 laminin receptor, which is elevated in tumor versus normal cells, and downregulated expression of laminin receptor with small interfering RNA significantly reduces the infectivity of Sindbis vectors. Tumor overexpression of the laminin receptor may explain the specificity and efficacy that Sindbis vectors demonstrate for tumor cells in vivo. Laser capture microdissection of mouse tumor implants showed equivalent laminin receptor expression levels in the different tumor metastases in the peritoneal cavity. Incorporation of antitumor cytokine genes such as interleukin-12 and interleukin-15 genes enhances the efficacy of the vector. These results suggest that Sindbis viral vectors may be promising agents for both specific detection and growth suppression of metastatic ovarian cancer
— id: 64391, year: 2006, vol: 9, page: 36, stat: Journal Article,

Tumor-specific in vivo transfection with HSV-1 thymidine kinase gene using a Sindbis viral vector as a basis for prodrug ganciclovir activation and PET
Tseng, Jen-Chieh; Zanzonico, Pat B; Levin, Brandi; Finn, Ronald; Larson, Steven M; Meruelo, Daniel
2006 Jul;47(7):1136-1143, Journal of nuclear medicine
One type of gene therapy of tumors, gene-directed enzyme-prodrug therapy (GDEPT), holds considerable promise, although practical considerations limit its clinical applicability. These include the lack of acceptable noninvasive methods that are adaptable to humans for selective tumor targeting of the therapeutic genetic material. Sindbis virus is an oncolytic, alpha-virus that selectively targets tumors through the 67-kDa laminin receptor (LAMR). In this report we describe a novel approach that permits tumor-selective tumor targeting and quantitative in vivo monitoring using PET of a commonly applied GDEPT, based on herpes simplex virus thymidine kinase type 1 (HSVtk) and ganciclovir (GCV). METHODS: Sindbis/tk vectors were harvested from the supernatant of in vitro cultures of a packaging cell produced by electroporation of both replicon RNA (SinRep5/tk) and helper RNA (DH-BB) into baby hamster kidney (BHK) cells. The therapeutic effect of GCV was determined by incubation of transfected tumor cells with increasing concentrations of GCV. BHK tumors growing as xenografts in severe combined immunodeficiency disease (SCID) mice were transfected by parenteral administration of the vector. Imaging was performed using small-animal PET at 2 h after injection of 18F fluoro-ethyl-arabinosyluridine (18F-FEAU) and 24 h after the final parenteral injection of Sindbis/tk viral vector. RESULTS: The vector efficiently expresses the HSVtk enzyme in infected tumor cells, both in vitro and in vivo. High levels of HSVtk expression ensure sufficient prodrug GCV conversion and activation for bystander effects that kill the surrounding untransduced tumor cells. Tumor localization of intravenously administered 18F-FEAU after 2 and 3 parenteral vector treatments of Sindbis/tk demonstrated uptake of 1.7 and 3.1 %ID/g (percentage injected dose per gram), respectively. CONCLUSION: The vector efficiently targets the HSVtk enzyme gene into Sindbis-infected tumor cells. High levels of HSVtk expression ensure sufficient prodrug GCV conversion and activation for bystander effects that killed many surrounding untransduced tumor cells. In addition, the HSVtk activities in tumors can be noninvasively monitored using PET after systemic Sindbis/tk treatments as a basis for determining the levels and tissue distribution of vector, noninvasively in living animals, and for optimizing in vivo transfection rates of tumor
— id: 67390, year: 2006, vol: 47, page: 1136, stat: Journal Article,

Identification of amino acids of Sindbis virus E2 protein involved in targeting tumor metastases in vivo
Hurtado, Alicia; Tseng, Jen-Chieh; Boivin, Christopher; Levin, Brandi; Yee, Herman; Pampeno, Christine; Meruelo, Daniel
2005 Nov;12(5):813-823, Molecular therapy
Previous studies conducted in our laboratory with Sindbis viral vectors in animal models demonstrated excellent in vivo targeting of tumor cells and significant reduction of metastatic implant size. To explore the influence of Sindbis strain on these factors, we constructed new plasmids from the wild-type Ar-339 Sindbis virus strain and compared their sequences. We found differences in the replicase and envelope proteins between JT, HRSP, and Ar-339 sequences. We made chimeras combining both strains and studied their efficiency in SCID mice bearing tumor xenograft using IVIS in vivo imaging techniques. We found that JT envelope proteins targeted tumors more efficiently than those of Ar-339, while the Ar-339 replicase showed increased efficacy in tumor reduction. To determine which residues are responsible for tumor targeting, we made mutants of Ar-339 E2 envelope protein and tested them by IVIS imaging in ES-2 tumor-bearing and tumor-free mice. The change of only one amino acid from E70 to K70 in Ar-339 E2 suppressed the ability to target metastatic tumor implants in mice. A K70 and V251 double E2 mutant did not reverse the loss of targeting capability. Only the mutant with JT E2 and Ar-339 helper targeted tumor, though with less intensity
— id: 61365, year: 2005, vol: 12, page: 813, stat: Journal Article,

Anti-angiogenic activities of hypericin in vivo: potential for ophthalmologic applications
Lavie, Gad; Mandel, Mathilda; Hazan, Sadick; Barliya, Tilda; Blank, Michael; Grunbaum, Aaron; Meruelo, Daniel; Solomon, Arieh
2005 ;8(1):35-42, Angiogenesis
Hypericin, a perihydroxylated dianthraquinone is shown here to be a highly potent inhibitor of angiogenesis in several ocular models examined in rat eyes. Extensive angiogenesis induced in the cornea and iris by intra-ocular administration of FGF-2 was effectively inhibited by a minimum of four dose regimens of hypericin (2 mg/kg) administered via the intraperitoneal route at 48 h intervals. Maximal inhibition was achieved when animal treatment with hypericin was initiated 48 h prior to inoculation of FGF-2. The molecular basis for the hypericin-mediated inhibition of angiogenesis in the anterior eye compartment appears to involve several sites in the cascade leading to angiogenesis. We show that the activating phosphorylation of extracellular signal-regulated MAP kinases (ERK1/2) is inhibited by hypericin in human retinal pigment epithelial cells and in EA.hy926 cells, an endothelial hybridoma expressing endothelial cell properties. ERK1/2 activity is required for the transactivation of hypoxia-inducible factor 1 alpha (HIF-1alpha) and in VEGF-induced blood vessel sprouting. MT1-MMP activity in human microvascular endothelial cells was also inhibited. The findings identify hypericin as a potentially useful agent in the treatment of ophthalmic neovascularization pathogeneses
— id: 64515, year: 2005, vol: 8, page: 35, stat: Journal Article,

The effect of sindbis viral vectors on the N-nitroso-bis(2-oxopropyl)amine(BOP) induced hamster pancreatic ductal adenocarcinoma and cholangiocarcinoma
Lin, Q; West, AB; Merali, S; Levin, B; Meruelo, D; Pellicer, A
2005 ;85(Suppl 1):283A-284A, Laboratory investigation
— id: 50475, year: 2005, vol: 85, page: 283A, stat: Journal Article,

The effect of Sindbis viral vectors on the N-nitroso-bis(2-oxopropyl)amine(BOP) induced hamster pancreatic ductal adenocarcinoma and cholangiocarcinoma
Liu, Q; West, AB; Merali, S; Levin, B; Meruelo, D; Pellicer, A
2005 ;18(Suppl 1):283A-284A, Modern pathology
— id: 50445, year: 2005, vol: 18, page: 283A, stat: Journal Article,

Antimetastatic activity of the photodynamic agent hypericin in the dark
Blank, Michael; Lavie, Gad; Mandel, Mathilda; Hazan, Sadick; Orenstein, Arie; Meruelo, Daniel; Keisari, Yona
2004 Sep 10;111(4):596-603, International journal of cancer
A unique property of the photodynamic signal transduction inhibitor hypericin (HY) is high functionality in the dark, which has been shown to result in portfolio of anticancer activities both in vitro and in vivo. Here we show that treatment with HY significantly reduces growth rate of metastases in 2 murine models: breast adenocarcinoma (DA3) and squamous cell carcinoma (SQ2). Focus on metastases was achieved by resection of primary tumors at stages in which micrometastases exist in lungs. Long-term animal survival in DA3 tumor-excised groups increased from 15.6% in controls to 34.5% following supplementary treatment with HY. In mice bearing SQ2 tumor metastases, therapy with HY increased animal survival from 17.7% in controls to 46.1%. Using Laser-induced fluorescence and multipixel spectral image analyses, we demonstrate that HY has a high tendency to accumulate in primary and metastatic tumors; HY content in lungs bearing metastases was approximately 2-fold higher than in the lungs of healthy animals. The tendency of HY to preferentially concentrate in lung metastases, combined with its potent antiproliferative activities, may render HY as a useful supplementary modality in the treatment of metastatic cancer irrespective of photoactivation
— id: 44810, year: 2004, vol: 111, page: 596, stat: Journal Article,

Systemic gene therapy by Sindbis vectors: A potentially safe and effective targeted therapy for identifying and killing tumor cells in vivo
Meruelo, Daniel
2004 Feb;4(20):54-57, Discovery medicine
Extract: A major obstacle to the development of gene therapy for cancer has been the inability to specifically and systemically deliver gene therapy vectors throughout the body to primary and/or metastasized tumor cells. Although intratumor injections of gene therapy vectors have sometimes been possible, no viral vector has been available that could be administered systemically and would selectively and efficiently target tumors without infection of normal tissues. Furthermore, even when locally injected, many viral vectors end up at high concentrations in the liver, because many cells of the body have low receptor numbers for some of the vectors in current use, whereas the liver has high numbers of such receptors. A number of ingenious approaches have been tried but none so far have fully resolved the problem. For example, tumor-specific promoters have been incorporated into the vectors so that gene expression and/or replication can occur in tumor cells but not normal cells. Unfortunately, only a small fraction of these vectors are typically taken up by the target tumor and expressed. In such cases, tumor cell death is generally insufficient to eradicate or significantly slow tumor growth
— id: 111829, year: 2004, vol: 4, page: 54, stat: Journal Article,

Using sindbis viral vectors for specific detection and suppression of advanced ovarian cancer in animal models
Tseng, Jen-Chieh; Hurtado, Alicia; Yee, Herman; Levin, Brandi; Boivin, Christopher; Benet, Marta; Blank, Stephanie V; Pellicer, Angel; Meruelo, Daniel
2004 Sep 15;64(18):6684-6692, Cancer research
We studied the therapeutic value of Sindbis vectors for advanced metastatic ovarian cancer by using two highly reproducible and clinically accurate mouse models: a SCID xenograft model, established by i.p. inoculation of human ES-2 ovarian cancer cells, and a syngenic C57BL/6 model, established by i.p. inoculation of mouse MOSEC ovarian cancer cells. We demonstrate through imaging, histologic, and molecular data that Sindbis vectors systemically and specifically infect/detect and kill metastasized tumors in the peritoneal cavity, leading to significant suppression of the carcinomatosis in both animal models. Use of two different bioluminescent genetic markers for the IVIS Imaging System permitted demonstration, for the first time, of an excellent correlation between vector delivery and metastatic locations in vivo. Sindbis vector infection and growth suppression of murine MOSEC tumor cells indicate that Sindbis tumor specificity is not attributable to a species difference between human tumor and mouse normal cells. Sindbis virus is known to infect mammalian cells using the M(r) 67,000 laminin receptor. Immunohistochemical staining of tumor cells indicates that laminin receptor is elevated in tumor versus normal cells. Down-regulated expression of laminin receptor with small interfering RNA significantly reduces the infectivity of Sindbis vectors. Tumor overexpression of the laminin receptor may explain the specificity and efficacy that Sindbis vectors demonstrate for tumor cells in vivo. We show that incorporation of antitumor cytokine genes such as interleukin-12 and interleukin-15 genes enhances the efficacy of the vector. These results suggest that Sindbis viral vectors may be promising agents for both specific detection and growth suppression of metastatic ovarian cancer
— id: 44950, year: 2004, vol: 64, page: 6684, stat: Journal Article,

Systemic tumor targeting and killing by Sindbis viral vectors
Tseng, Jen-Chieh; Levin, Brandi; Hurtado, Alicia; Yee, Herman; Perez de Castro, Ignacio; Jimenez, Maria; Shamamian, Peter; Jin, Ruzhong; Novick, Richard P; Pellicer, Angel; Meruelo, Daniel
2004 Jan;22(1):70-77, Nature biotechnology
Successful cancer gene therapy requires a vector that systemically and specifically targets tumor cells throughout the body. Although several vectors have been developed to express cytotoxic genes via tumor-specific promoters or to selectively replicate in tumor cells, most are taken up and expressed by just a few targeted tumor cells. By contrast, we show here that blood-borne Sindbis viral vectors systemically and specifically infect tumor cells. A single intraperitoneal treatment allows the vectors to target most tumor cells, as demonstrated by immunohistochemistry, without infecting normal cells. Further, Sindbis infection is sufficient to induce complete tumor regression. We demonstrate systemic vector targeting of tumors growing subcutaneously, intrapancreatically, intraperitoneally and in the lungs. The vectors can also target syngeneic and spontaneous tumors in immune-competent mice. We document the anti-tumor specificity of a vector that systemically targets and eradicates tumor cells throughout the body without adverse effects
— id: 44812, year: 2004, vol: 22, page: 70, stat: Journal Article,

Enhanced ubiquitinylation of heat shock protein 90 as a potential mechanism for mitotic cell death in cancer cells induced with hypericin
Blank, Michael; Mandel, Mathilda; Keisari, Yona; Meruelo, Daniel; Lavie, Gad
2003 Dec 1;63(23):8241-8247, Cancer research
A unique property of the photodynamic signal transduction inhibitor hypericin is functionality in the dark. We show in tumor cells that hypericin targets the heat shock protein (Hsp) 90 chaperone but not Hsp70 (Hsc70) to enhanced ubiquitinylation. As a consequence Hsp90 chaperone functionality is abrogated and the client proteins, mutant p53, Cdk4, Raf-1, and Plk, are displaced from complexes with Hsp90, destabilized, and degraded via a proteasome-independent pathway. Decline in Raf-1 prevents downstream activation of extracellular signal-regulated kinase 1/2 kinases, the Ras/Raf pathway is inhibited, and tumor cell proliferation is arrested. The cells exhibit multiple aberrations including retardation at G(2)-M, increased cell volume, and multinucleation, all of which are hallmarks of mitotic cell death. The studies demonstrate that ubiquitinylation of Hsp90 inactivates the chaperone, destabilizes the plethora of client proteins, and creates deficiencies in multiple unrelated cellular functions. This combination constitutes a mechanism by which hypericin generates mitotic cell death in cancer cells
— id: 44811, year: 2003, vol: 63, page: 8241, stat: Journal Article,

Transduction of dominant negative ATF-1 suppresses the pX gene expression in joint fibroblastic cells derived from HTLV-I transgenic rats
Ishizu, Akihiro; Tsuji, Takahiro; Abe, Asami; Saito, Saori; Takahashi, Toshiyuki; Ikeda, Hitoshi; Meruelo, Daniel; Yoshiki, Takashi
2003 Jun;74(3):309-313, Experimental & molecular pathology
Tax (p40Tax) encoded by the env-pX gene of human T-cell leukemia virus type I (HTLV-I) interacts with cyclic adenosine-3',5'-monophosphate response element binding protein/activation transcription factor 1 (CREB/ATF-1) transcription factors of host cells and activates the viral long terminal repeat (LTR) promoter. This molecular interaction induces augmentation of viral gene expression and may result in development of HTLV-I-associated diseases, including adult T-cell leukemia, HTLV-I associated myelopathy/tropical spastic paraparesis, and HTLV-I uveitis. To inhibit this pathway, a dominant negative molecule of ATF-1, ATF-1DN, was used. We transduced ATF-1DN into joint fibroblastic cells derived from transgenic rats carrying the LTR-env-pX-LTR gene of HTLV-I, using the Sindbis virus-based vectors. Expression of the pX gene in cells transduced with ATF-1DN was lower than that in cells with control transfection. A possible application of ATF-1DN to suppress viral gene expression in HTLV-I infected cells can be considered
— id: 44813, year: 2003, vol: 74, page: 309, stat: Journal Article,

"Competitive quenching" between photosensitizers. A novel concept in protecting cells from verteporfin-induced phototoxicity using hypericin
Ron, YD; Weinberger, D; Blank, M; Mandel, M; Livnat, T; Lusky, M; Barliya, T; Orenstein, A; Meruelo, D; Lavie, G
2003 MAY ;44(3):U385-U385, Investigative ophthalmology & visual science. IOVS
— id: 55419, year: 2003, vol: 44, page: U385, stat: Journal Article,

Characterization of the transcriptional expression of Notch-1 signaling pathway members, Deltex and HES-1, in developing mouse thymocytes
Choi, Jung W; Pampeno, Christine; Vukmanovic, Stanislav; Meruelo, Daniel
2002 Jul;26(6):575-588, Developmental & comparative immunology
The Notch transmembrane protein is involved in a broad range of different developmental pathways in vertebrates and invertebrates. Targeted thymocyte expression of the Notch-1 intracellular domain has been shown to affect lineage commitment decisions such as those involving T cell vs. B cell, thymocyte alpha beta vs. gamma delta TCR, as well as CD4 vs. CD8 thymocyte commitment. In this paper, we quantitatively characterize thymocyte RNA expression of two purported transcriptional markers of Notch-1 signaling activity, Deltex and HES-1. Using a semiquantitative RTPCR approach, we show that both Deltex and HES-1 transcriptional levels are developmentally regulated as thymocytes mature from the earliest CD4/CD8 double negative thymocyte stage, through the intermediate CD4/CD8 double positive stage, and finally to the mature CD4 or CD8 single positive stage. Deltex and HES-1, despite both being transcriptional markers of Notch-1 activity, express different patterns of transcriptional activity among the thymocyte subsets. Neither treatment with combined (alpha CD3)/(alpha CD28) antibodies nor the combination of the phorbol ester PMA and calcium ionophore ionomycin affects expression of Deltex in immature thymocytes; however, PMA/ionomycin treatment does downregulate expression of HES-1, an affect mostly mediated by ionomycin. Finally, a difference in HES-1 expression is seen between CD4/CD8 double positive thymocytes isolated from wild-type vs. MHC class I/II deficient mice, suggesting that Notch-1 activity is modulated during in vivo TCR/MHC-ligand selection events
— id: 32466, year: 2002, vol: 26, page: 575, stat: Journal Article,

In vivo antitumor activity of Sindbis viral vectors
Tseng, Jen-Chieh; Levin, Brandi; Hirano, Tadamichi; Yee, Herman; Pampeno, Christine; Meruelo, Daniel
2002 Dec 4;94(23):1790-1802, Journal of the National Cancer Institute
BACKGROUND: Sindbis virus, a blood-borne virus transmitted by mosquitoes, has been used as a vector to efficiently express exogenous genes in vitro and in vivo and to induce apoptosis. Because Sindbis virus infects mammalian cells by interacting with the high-affinity laminin receptors, which are expressed at higher levels in several human cancers than in normal cells, we determined whether a Sindbis viral vector could be used to target cancers in vivo. METHODS: C.B-17-SCID mice with established xenografts were given daily intraperitoneal injections of the Sindbis viral vector SinRep/LacZ containing the bacterial beta-galactosidase gene. Control mice were untreated or received injections with phosphate-buffered saline. Tumor size was measured daily. Expression of beta-galactosidase and Factor VIII (a marker for endothelial cells) was determined by immunohistochemical staining of tumor sections. Apoptosis was analyzed by TUNEL (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labeling) staining. C.B-17-SCID beige mice, which lack natural killer (NK) cells, were used to assess the importance of NK cells in antitumor efficacy of Sindbis viral vectors. RESULTS: Tumors from mice treated with SinRep/LacZ were statistically significantly smaller than tumors from control mice. This effect was observed for tumor xenografts derived from BHK (kidney, hamster), LS174T (colon, human), HT29 (colon, human), and CFPAC (pancreas, human) cells. Expression of beta-galactosidase co-localized with that of Factor VIII in tumor sections. Tumors from SinRep/LacZ-treated mice contained more apoptotic cells than tumors from control mice. Complete tumor regression was observed in three of five C.B-17-SCID mice but in none of five C.B-17-SCID beige mice treated with SinRep/LacZ. CONCLUSION: Sindbis viral vectors efficiently targeted tumors in vivo, were apparently delivered through the circulation, and were more effective in the presence of NK cells
— id: 39358, year: 2002, vol: 94, page: 1790, stat: Journal Article,

Genomic analysis and localization of murine Deltex, a modulator of notch activity, to mouse chromosome 5 and its human homolog to chromosome 12
Pampeno CL; Vallerie AM; Choi J; Meruelo NC; Meruelo D
2001 Mar;20(3):141-148, DNA & cell biology
Deltex is a component of the Notch signaling network, which mediates cellular differentiation, proliferation, and apoptosis during development. Murine Deltex was initially isolated as a cDNA transcript that displayed increased expression in T-cell tumors induced by gamma irradiation. The in vivo function of Deltex is unknown; however, the emerging role of Notch signaling in T-cell development and lymphomagenesis indirectly supports a role for Deltex in these processes. To investigate the regulation of Deltex expression in both normal and transformed tissue, we have begun analyzing the Deltex genomic locus. Here, we report the exon-intron organization of Deltex and map the locus to the middistal region of mouse chromosome 5, tightly linked to the Adam1a, Lnk, Tbx5, and Nos1 loci. The human homolog of Deltex has been localized to chromosome 12
— id: 20725, year: 2001, vol: 20, page: 141, stat: Journal Article,

Transduction of a murine dominant negative activation transcription factor 1 increases cell surface expression of the class I MHC on a human epidermoid tumor cell line
Ishizu A; Sawai K; Ikeda H; Hirano T; Ishiguro N; Meruelo D
2000 Feb;12(2):161-168, International immunology
The transcription of the MHC class I genes is regulated by interaction of cis-elements, located in the 5' genomic flanking regions, with sequence-specific trans-factors. We have identified a cis-regulatory element, 5'-TGACGCG-3', of the H-2D(d) gene. This cyclic adenosine-3',5'-monophosphate regulatory element (CRE)-like sequence, named H-2 binding factor 1 (H-2 BF1) binding motif, is highly conserved among species. In addition, we found that homo- and heterodimers of activation transcription factor 1 (ATF-1) and CRE binding protein (CREB) associate with the H-2 BF1 binding motif and activate transcription of the H-2D(d) gene. Here we demonstrate that a homologue of ATF-1, originally isolated and designated ATF-1DN, acts as a dominant repressor, blocking the ability of wild-type ATF-1 and CREB to bind to the H-2 BF1 probe in electrophoretic mobility shift assays (EMSA). We have utilized this molecule to analyze the participation of the H-2 BF1 complexes, consisting of the H-2 BF1 binding motif and ATF-1/CREB trans-factors, in the physiological regulation of MHC class I expression in tissue culture cells. A human epidermoid carcinoma cell line, A431, was transfected with ATF-1DN and clones expressing the gene transcripts were selected. When analyzed in the EMSA, nuclear proteins prepared from these clones exhibited a decreased shift of the H-2 BF1 probe corresponding to the levels of the ATF-1DN gene expression. Additionally, MHC class I expression of cells with reduced H-2 BF1 activity was significantly higher than in control cells lacking ATF-1DN. These findings indicate that in these carcinoma cells, the H-2 BF1 complexes negatively regulate the constitutive expression of MHC class I
— id: 8544, year: 2000, vol: 12, page: 161, stat: Journal Article,

Inhibition of the CD8+ T cell-mediated cytotoxicity reaction by hypericin: potential for treatment of T cell-mediated diseases
Lavie G; Meruelo D; Aroyo K; Mandel M
2000 Apr;12(4):479-486, International immunology
The cytotoxicity reaction of murine CD8 T lymphocytes has been found to be strongly inhibited by nanomolar concentrations of hypericin, a lipophilic dianthraquinone with photodynamic properties. Cytotoxic T lymphocyte (CTL)-induced target cell apoptosis, as well as exocytosis of cytolytic granules from these cells, were ablated by hypericin, administered at the onset of the reaction, without affecting CTL viability. The inhibition of cytolysis occurred without the light irradiation which is essential for photosensitization. The findings suggest that the action of hypericin targets the effector CTL; however, apoptosis induced in murine L-cells with recombinant tumor necrosis factor (TNF)-alpha was also prevented by hypericin. Since hypericin is a known inhibitor of protein kinase C, MAP kinase and at least one other tyrosine kinase, this inhibitory activity could play a role in the down-modulation of CTL-induced cytotoxicity. Furthermore, our studies show that the action of hypericin induces rapid dephosphorylation of phospholipids associated with low-density membranes in CTL, but not with membranes of the cytotoxic granules. The ability of hypericin to interfere with cytotoxicity may render it useful in the treatment of T cell-mediated diseases
— id: 15240, year: 2000, vol: 12, page: 479, stat: Journal Article,

Strategies for evaluation of enveloped virus inactivation in red cell concentrates using hypericin
Prince AM; Pascual D; Meruelo D; Liebes L; Mazur Y; Dubovi E; Mandel M; Lavie G
2000 Feb;71(2):188-195, Photochemistry & photobiology
Photodynamically induced virus inactivation appears promising in preventing transmission of enveloped virus infections in transfusible blood products. The potential for utilizing hypericin as a photosensitizer to inactivate key enveloped viruses in packed red cell concentrates (PRC) was evaluated. In addition to inactivating effectively > or = 10(6) TCID50 of human immunodeficiency virus (HIV), inactivation of bovine viral diarrhea virus (BVDV) in PRC was used as a model for hepatitis C virus to overcome the deficiency in reliable experimental systems for hepatitis C virus (HCV) inactivation. BVDV was two orders of magnitude more sensitive to inactivation by hypericin than HIV. As part of the virucidal efficacy analyses, the effects of photosensitization on hemopoietic cell lines carrying quiescent integrated HIV provirus were studied as models for evaluating virus inactivation in latently infected cells. Phorbol ester-induced virus production by these cells was effectively prevented by photosensitization with hypericin. A refinement of the illumination conditions, incorporating a monochromatic sodium light source with an emission spectrum coinciding with the absorption peak of hypericin, was highly virucidal, however, caused unacceptable levels of hemolysis. Red blood cells could be protected from phototoxic cellular damage by complexing hypericin with human serum albumin (albumin-hypericin), but the decrease in hemolysis was at the expense of virucidal efficacy. Thus, excitation of hypericin with a fluorescent source appears to be useful potentially for virus inactivation in PRC
— id: 57560, year: 2000, vol: 71, page: 188, stat: Journal Article,

Specific cell targeting for delivery of toxins into small-cell lung cancer using a streptavidin fusion protein complex
Yu A; Choi J; Ohno K; Levin B; Rom WN; Meruelo D
2000 Jul;19(7):383-388, DNA & cell biology
New modalities of treatment for small-cell lung cancer (SCLC) are needed, because the majority of patients continue to die of disseminated disease despite an initial response to conventional chemotherapy. Abnormal surface expression of the neural-cell adhesion molecule (NCAM) has been noted to be highly associated with SCLC. We examined the ability and efficiency of a streptavidin-Protein A (ST-PA) fusion protein complexed with an anti-NCAM monoclonal antibody (Mab) to transfer biotinylated beta-galactosidase into human SCLC cell lines NCI-H69, NCI-H526, and NCI-H446. When the surface molecule NCAM was targeted with this system, more than 99% of the targeted cells internalized and exhibited beta-galactosidase activity. In addition, we evaluated cytotoxic activity against SCLC lines NCI-H69 and NCI-H526 by efficient delivery of biotinylated glucose oxidase using the same ST-PA/anti-NCAM Mab complex. Cytotoxicity of the transduced cells (SCLC) was 10-fold and 100-fold greater, respectively, than the glucose oxidase control. This system could be widely applied for specific therapy of cancer cells by targeting unique surface molecules (antigens) using the corresponding Mab/ST-PA complex to transfer a variety of effector molecules; e.g., immunotoxic compounds, into target cells with a high degree of efficiency and specificity
— id: 11544, year: 2000, vol: 19, page: 383, stat: Journal Article,

Targeted gene transfer system using a streptavidin-transforming growth factor-alpha chimeric protein
Garcia-Espana A; Biria S; Malumbres M; Levin B; Meruelo D; Pellicer A
1999 Oct;18(10):743-749, DNA & cell biology
The previously reported streptavidin-TGFalpha chimeric protein-based delivery system (Ohno and Meruelo, DNA Cell Biol. 15:401-406, 1996) could efficiently transfer protein molecules into A431 cells via the epidermal growth factor (EGF) receptor. We have modified this delivery system for the transfer of DNA. For this purpose, we have linked the chimeric protein ST-TGFalpha to DNA through biotinylated polylysine molecules. We show with this system, in the presence of the endosome-destabilizing reagent chloroquine, an average of 50-fold increase in reporter gene expression in comparison with polylysine DNA complexes alone. This gene expression is specific for EGF receptor-expressing cells and is blocked by EGF-binding molecules. These results suggest that the ST-TGFalpha biotinylated polylysine system could be used to deliver DNA to targeted cells
— id: 11938, year: 1999, vol: 18, page: 743, stat: Journal Article,

Cell-specific targeting of a thymidine kinase/ganciclovir gene therapy system using a recombinant Sindbis virus vector
Iijima Y; Ohno K; Ikeda H; Sawai K; Levin B; Meruelo D
1999 Jan 5;80(1):110-118, International journal of cancer
Transfer of the herpes simplex virus type I thymidine kinase (HSV-TK) gene into tumor cells using virus-based vectors in conjunction with ganciclovir (GCV) exposure provides a potential gene therapy strategy for the treatment of cancer. The possibility of using a novel targetable Sindbis virus expression vector containing the HSV-TK gene was examined. Baby hamster kidney (BHK) cells and several human tumor cells infected with a Sindbis virus containing the HSV-TK gene showed strong expression of HSV-TK protein. Cells transduced with the HSV-TK gene exhibited increased TK activity, ranging from 3- to 20-fold over an average baseline level. The human HeLa-CD4+ cells infected with recombinant Sindbis virus containing the HSV-TK gene were sensitive to low concentrations of GCV (0.1-1 microg/ml) and the 50% growth inhibitory concentration (IC50) was 0.6 microg/ml. We also demonstrated applications of cell type-specific Sindbis virus-mediated antigen-antibody targeting of the HSV-TK/GCV system in vitro. Sindbis virus containing the HSV-TK gene packaged in a helper virus displaying the IgG-binding domain of protein A on its envelope could infect various tumor cell lines in the presence of specific antibodies that recognize antigens on their surfaces. HSV-TK-transduced tumor cell lines exhibited sensitivity to GCV. Our data suggest the potential for targeted gene therapy of the HSV-TK/GCV system using a cell type-specific recombinant Sindbis virus vector-antibody system
— id: 57170, year: 1999, vol: 80, page: 110, stat: Journal Article,

Suicide gene therapy for human uterine adenocarcinoma cells using herpes simplex virus thymidine kinase
Kunishige I; Samejima Y; Shiki Y; Moriyama A; Meruelo D; Saji F; Murata Y
1999 Jan;72(1):16-25, Gynecologic oncology
In gene therapy, the herpes simplex virus thymidine kinase (HSV-tk) gene is widely used as a suicide agent. Tumor cells expressing HSV-tk are sensitive to nucleoside analogs such as ganciclovir (GCV). An advantage of this system is the bystander killing effect whereby HSV-tk-positive cells exposed to GCV are lethal to surrounding HSV-tk-negative cells. We transfected the HSV-tk gene into a human cervical adenocarcinoma cell line, BU25TK-, and a human endometrial adenocarcinoma cell line, HHUA, by the Lipofectine method. The sensitivity of HSV-tk-positive cells to GCV and bystander killing effect on HSV-tk-negative cells were examined in vitro. HSV-tk-positive cells were sensitive to GCV at concentrations of 1 to 100 microg/ml in a dose- and time-dependent manner. The growth of HSV-tk-negative cells was inhibited when the population of cultured cells contained more than about 3% HSV-tk-positive cells. Moreover, for BU25TK- cells, HSV-tk-positive cells were injected into SCID mice subcutaneously and the effects of GCV therapy and bystander killing at a daily concentration of 25 mg/kg for 14 days were examined. HSV-tk-positive tumors transduced into SCID mice almost disappeared upon GCV treatment. Furthermore, tumor reduction was observed when mixtures of HSV-tk-negative cells containing more than 20% HSV-tk-positive cells were injected into SCID mice. In conclusion, the HSV-tk/GCV system might be applied to both cervical and endometrial adenocarcinoma.
— id: 15242, year: 1999, vol: 72, page: 16, stat: Journal Article,

A photodynamic pathway to apoptosis and necrosis induced by dimethyl tetrahydroxyhelianthrone and hypericin in leukaemic cells: possible relevance to photodynamic therapy
Lavie G; Kaplinsky C; Toren A; Aizman I; Meruelo D; Mazur Y; Mandel M
1999 Feb;79(3-4):423-432, British journal of cancer
The mechanism of cell death induction by dimethyl tetrahydroxyhelianthrone (DTHe), a new second-generation photodynamic sensitizer, is analysed in human leukaemic cell lines in comparison with the structurally related hypericin. DTHe has a broad range of light spectrum absorption that enables effective utilization of polychromatic light. Photosensitization of HL-60 cells with low doses of DTHe (0.65 microM DTHe and 7.2 J cm(-2) light energy) induced rapid apoptosis of > or =90% of the cells. At doses > or =2 microM, dying cells assumed morphological necrosis with perinucleolar condensation of chromatin in HL-60 and K-562 cell lines. Although nuclear fragmentation that is characteristic to apoptosis was prevented, DNA digestion to oligonucleosomes proceeded unhindered. Such incomplete apoptosis was more prevalent with the related analogue hypericin throughout most doses of photosensitization. Despite hypericin being a stronger photosensitizer, DTHe exhibited advantageous phototoxic properties to tumour cells, initiating apoptosis at concentrations about threefold lower than hypericin. Photosensitization of the cells induced dissociation of the nuclear envelope, releasing lamins into the cytosol. DTHe also differed from hypericin in effects exerted on the nuclear lamina, causing release of an 86-kDa lamin protein into the cytosol that was unique to DTHe. Within the nucleus, nuclear envelope lamin B underwent covalent polymerization, which did not affect apoptotic nuclear fragmentation at low doses of DTHe. At higher doses, polymerization may have been extensive enough to prevent nuclear collapse. Hut-78, CD4+ cells were resistant to the photodynamically activated apoptotic pathway. Beyond the tolerated levels of photodynamic damage, these cells died exclusively via necrosis. Hut-78 cells overexpress Bcl-X(L) as well as a truncated Bcl-X(L)tr isoform that could contribute to the observed resistance to apoptosis
— id: 15241, year: 1999, vol: 79, page: 423, stat: Journal Article,

Reducing cytotoxicity induced by Sindbis viral vectors
Sawai K; Ikeda H; Ishizu A; Meruelo D
1999 May;67(1):36-42, Molecular genetics & metabolism
Sindbis virus has been recognized as a potentially useful virus vector for gene therapy. In an effort to improve its utility and provide cell-targeting capability to gene therapy vectors, we recently developed Sindbis virus vectors possessing chimeric envelopes with cell-specific targeting ability [K. Ohno et al. Nature Biotechnol 15:763-767, 1997; K. Sawai et al. Biochem Biophys Res Commun 248:315-323, 1998]. However, a residual problem associated with Sindbis virus vectors is the apoptotic effect of this virus on infected cells. To address this issue, we have studied the possible role of bcl-2 expression. Bcl-2 expression has been postulated to facilitate the establishment of persistent Sindbis viral infection by blocking virus-induced apoptosis. In this study we produced a Sindbis virus vector capable of expressing human bcl-2 and the reporter gene, lacZ. This chimeric virus (SinRep/lacZ/bcl-2/DH-BB) showed a marked reduction in induced apoptosis in infected cells. For example, after infection with this vector, cell proliferation of BHK cells was 55% of that of uninfected cells 2 days after infection and 40% 3 days after infection. While this reflected a significant degree of apoptosis, the effect was much less pronounced than that seen with wild-type Sindbis virus. Cell proliferation was reduced to 26% 2 days after wild-type virus infection of BHK cells and to only 7% 3 days after infection. Although additional work will be required to eliminate apoptosis induced by Sindbis virus vectors, the studies reported here suggest that such a goal may be achievable after additional modification of the vectors
— id: 56437, year: 1999, vol: 67, page: 36, stat: Journal Article,

The regulation of murine H-2Dd expression by activation transcription factor 1 and cAMP response element binding protein
Ishiguro N; Brown GD; Ishizu A; Meruelo D
1998 Jun 15;160(12):5907-5914, Journal of immunology
Resistance to radiation leukemia virus (RadLV)-induced leukemia is correlated with an increase in H-2Dd expression on the thymocyte surface. It has been shown that elevated H-2Dd expression on infected thymocytes is a result of elevated mRNA transcription and that the transcriptional increase is correlated with elevated levels of a DNA binding activity, H-2 binding factor 1 (H-2 BF1), which recognizes the 5'-flanking sequence (5'-TGACGCG-3') of the H-2Dd gene. Recently, it has been shown that the activation transcription factor 1 (ATF-1) homodimer is one form of the H-2 BF1 complex. Here we demonstrate that the cAMP response element binding protein (CREB) homodimer and the heterodimer of CREB/ATF-1 also recognize the cis regulatory motif and are two additional forms of the H-2 BF1 complex. The levels of mRNA encoding ATF-1 and CREB were both increased in RadLV-infected thymocytes that showed increased levels of H-2 mRNA. Also, all three H-2 BF1 binding activities, ATF-1 homodimer, CREB homodimer, and ATF-1/CREB heterodimer, were increased in RadLV-infected thymocytes that expressed high levels of H-2Dd Ag on the cell surface. Transfection experiments demonstrated that ATF-1 and CREB activated a reporter plasmid containing the H-2 BF1 motif. These observations strongly suggest that both ATF-1 and CREB are involved in the regulation of H-2 gene expression following RadLV infection of mouse thymocytes
— id: 7616, year: 1998, vol: 160, page: 5907, stat: Journal Article,

Cell-specific transfection of choriocarcinoma cells by using Sindbis virus hCG expressing chimeric vector
Sawai K; Meruelo D
1998 Jul 20;248(2):315-323, Biochemical & biophysical research communications
The development of Sindbis virus vectors that can target specific cell types would provide an important gene therapy strategy. We explored the possibility of designing a Sindbis virus vector that can target human choriocarcinoma cells via ligand-receptor interaction. The Sindbis virus envelope gene was modified by insertion of the alpha- and beta-hCG genes. The chimeric helper RNA was then transfected into BHK cells along with a virus-based expression vector, allowing the production of virus particles containing hCG-envelope chimeras. The hCG-envelope chimeric virus vector has minimal infectivities against BHK cells and human cancer cells which do not contain LH/CG receptors on their surface. This vector can, however, infect and transfer a reporter gene to choriocarcinoma cells as well as other cells bearing LH/CG receptors. This chimeric Sindbis virus vector may provide a novel approach for gene therapy of gestational trophoblast disease and placental dysfunction.
— id: 7779, year: 1998, vol: 248, page: 315, stat: Journal Article,

A novel method of cell-specific mRNA transfection
Sawai K; Ohno K; Iijima Y; Levin B; Meruelo D
1998 May;64(1):44-51, Molecular genetics & metabolism
In this study, we developed a cell-specific mRNA transfection system using streptavidin-protein A (ST-PA) fusion protein and monoclonal antibodies (mAbs). We previously reported that ST-PA fusion protein and mAb complexes can transfer certain biotinylated proteins into specific cell types. At this time, we combined an in vitro transcribed biotinylated and self-replicating Sindbis virus genomic RNA with ST-PA fusion protein and mAbs. In the presence of cationic liposomes, to prevent RNA degradation, this complex is able to transfect a reporter gene to specific cancer cells in a mAb does-dependent manner. Even in the absence of cationic liposomes, biotinylated mRNA, ST-PA fusion, and mAb complexes can transfer some types of cancer cell suspension cultures. This cell-specific transfection system is a novel method of introducing various mRNAs into cells that results in high levels of transient protein expression
— id: 57247, year: 1998, vol: 64, page: 44, stat: Journal Article,

Conservation of the H-2 BF1 binding motif 5' of the H-2Ds, Ks and Dq genes
Brown GD; Morris DR; Meruelo D
1997 Aug;24(4):241-257, European journal of immunogenetics
The biological consequences of radiation leukaemia virus (RadLV) infection include the stimulation of H-2 antigen expression soon after injection of the virus. Early studies demonstrated that resistance to RadLV-induced leukaemia in certain mouse strains is mediated by genes in the H-2D region of the major histocompatibility complex (MHC). Recent studies have shown that elevated H-2Dd expression on the thymocyte cell surface of resistance mouse strains results from increased mRNA transcription and is correlated with elevated levels of a DNA-binding activity that recognizes a short DNA sequence 5' of the start of transcription for the H-2Dd gene. This binding activity has been termed H-2 binding factor 1 (H-2 BF1) and is found exclusively in the thymus. In an effort to examine the H-2 genes of RadLV-susceptible mice for the presence of the H-2 BF1 binding target, we have cloned class I genes from the highly susceptible B10.S mouse strain and have identified both the Ds and the Ks genes. The entire genomic sequence for the Ds gene has been determined and is reported here. In addition, the 5' regulatory region of the previously cloned Dq gene has been sequenced; mice of the Dq haplotype are also susceptible to RadLV-induced leukaemia. In this report, we show that the H-2 BF1 DNA binding sequence is present 5' of each of these three class I genes
— id: 7123, year: 1997, vol: 24, page: 241, stat: Journal Article,

Activation transcription factor 1 involvement in the regulation of murine H-2Dd expression
Ishiguro N; Brown GD; Meruelo D
1997 Jun 20;272(25):15993-16001, Journal of biological chemistry
Resistance to radiation leukemia virus-induced leukemia is correlated with an increase in H-2D expression on the thymocyte surface. Recently, it has been shown that elevated H-2Dd expression on the infected thymocyte is a result of elevated mRNA transcription and that the transcriptional increase is correlated with elevated levels of a DNA binding activity, H-2 binding factor 1 (H-2 BF1), which recognizes the 5'-flanking sequences (5'-TGACGCG-3') of the H-2Dd gene. This target for transcription factor binding has been found to be identical in the 5'-regulatory region of 12 rodent class I genes, nine of which have been shown to be functional genes. Furthermore, this cis-element is found 5' of 20 primate class I genes (15 human genes), seven of which are known to be functional. Here, we demonstrate that activation transcription factor 1 (ATF-1) is one component of H-2 BF1. In addition, the levels of ATF-1 mRNA in uninfected and radiation leukemia virus-infected thymocytes parallel those of H-2Dd mRNA, and therefore, it is suggested that ATF-1 up-regulates the transcription of the H-2Dd gene after radiation leukemia virus infection of thymocytes. Transfection experiments also demonstrate that ATF-1 activates a reporter plasmid that contains the H-2 BF1 motif, but not a reporter lacking this motif. This is the first demonstration of the interaction of ATF-1 with 5'-regulatory sequences of major histocompatibility complex class I genes
— id: 7170, year: 1997, vol: 272, page: 15993, stat: Journal Article,

Retrovirus vectors displaying the IgG-binding domain of protein A
Ohno K; Meruelo D
1997 Oct;62(1):123-127, Biochemical & molecular medicine
We have designed and constructed retrovirus particles displaying the IgG-binding domain of protein A. We fused the gene for the synthetic antibody-binding portion of protein A with the envelope gene of ecotropic Moloney murine leukemia virus. The fusion gene was coexpressed in ecotropic retroviral packaging cells, and retrovirus particles with IgG-binding activities were recovered. In principle, the protein A-envelope chimeric retrovirus complexed with specific monoclonal antibody could be used for cell-targeted gene delivery.
— id: 12166, year: 1997, vol: 62, page: 123, stat: Journal Article,

Cell-specific targeting of Sindbis virus vectors displaying IgG-binding domains of protein A
Ohno K; Sawai K; Iijima Y; Levin B; Meruelo D
1997 Aug;15(8):763-767, Nature biotechnology
Sindbis virus can infect a broad range of insect and vertebrate cell types due to the widespread distribution of the cellular receptor for the virus. The development of Sindbis virus vectors that target specific cell types could have important implications for the design of gene therapy strategies. To achieve this goal we have designed and constructed Sindbis virus particles displaying the IgG-binding domain of protein A. The protein A-envelope chimeric Sindbis virus vector has minimal infectivities against baby hamster kidney and human cell lines. When used in conjunction with monoclonal antibodies that react with cell-surface antigens, however, the protein A-envelope chimeric virus was able to infect human cell lines with high efficiency. Infection rates were 90% or higher for human lymphoblastoid cells. A variety of cells could be targeted by changing the monoclonal antibody without generating a new recombinant virus
— id: 7955, year: 1997, vol: 15, page: 763, stat: Journal Article,

Cell-specific, multidrug delivery system using streptavidin-protein A fusion protein
Ohno K; Levin B; Meruelo D
1996 Aug;58(2):227-233, Biochemical & molecular medicine
Tissue-specific delivery of variety of molecules has been a valuable technique for biological and medical research and for the diagnosis and therapy of cancer. We have therefore examined the ability of streptavidin-protein A (ST-PA) fusion protein complexed with monoclonal antibodies (mAbs) to transfer biotinylated proteins into specific type of cells. ST-PA/mAbs complexes could efficiently deliver biotinylated beta-galactosidase into a variety of cancer cell lines through molecules expressed on their surface. In addition, ST-PA/mAb complexed with either biotinylated glucose oxidase or biotinylated ribonuclease A could be transferred to specific cell types and made to display cytotoxic activity against the transduced cell. The flexibility of the system was enhanced by the fact that the cell-targeting specificity could be altered by just changing the mAb used and the 'payload' molecule could be replaced by substituting one biotinylated protein or enzyme with another. This flexibility was achieved without the need to generate a covalent chemical link or engineering new recombinant molecules. Results obtained to date suggest that the ST-PA fusion protein may be used as a nearly 'universal carrier' to transfer a variety of effector molecules into target cells with a high degree of specificity. Essentially, the ST-PA fusion protein effectively serves as a high-efficiency, modular 'molecular bridge' for the transfer into cells of a wide variety of effector molecules
— id: 56913, year: 1996, vol: 58, page: 227, stat: Journal Article,

Multi-drug delivery system using streptavidin-transforming growth factor-alpha chimeric protein
Ohno K; Meruelo D
1996 May;15(5):401-406, DNA & cell biology
Tissue-specific delivery of a variety of molecules has been a valuable technique for biological and medical research. Therefore, we have constructed a recombinant plasmid containing the coding regions for streptavidin core and mature human transforming growth factor-alpha (TGF-alpha). The recombinant plasmid has been expressed in Escherichia coli to produce a chimeric protein with both streptavidin and TGF-alpha activity. The streptavidin-TGF-alpha chimeric protein (ST-TGF-alpha) could efficiently transfer biotinylated beta-galactosidase into A431 cells via the epidermal growth factor receptor. More than 99% of the cells contained the enzyme transferred. Furthermore, ST-TGF-alpha complexed with biotinylated-glucose oxidase had a significant cytotoxic effect when incubated with A431 cells. These findings suggest that the ST-TGF-alpha chimeric protein could be used to deliver proteins of interest into target cells without the need for chemical linkage or genetic construction. Essentially, ST-TGF-alpha serves as a high-modular 'molecular bridge' for the passage of a wide variety of effector molecules into target cells
— id: 7085, year: 1996, vol: 15, page: 401, stat: Journal Article,

A novel cDNA transcript expressed in fractionated X-irradiation-induced murine thymomas
Pampeno CL; Meruelo D
1996 Aug;7(8):1113-1123, Cell growth & differentiation
Elucidation of the leukemogenic process induced by fractionated X-irradiation (FX) requires the identification of molecules that mediate the differentiation and regeneration of T cells. To isolate cDNA transcripts associated with FX-induced leukemia in C57BL/6 mice, a cDNA library was constructed from FX-induced thymoma mRNA and differentially screened with cDNA probes. A novel cDNA transcript, FX-induced transcript 1 (FXI-T1), showed strong differential mRNA expression in all C57BL/6 FX-induced thymomas examined when compared with normal thymus tissue. FXI-T1 was not universally expressed in proliferative or other neoplastic cells. Expression of FXI-T1 mRNA in untreated mouse organs was not restricted to the thymus; highest expression was observed in brain and skeletal muscle tissue. The translated FXI-T1 sequence encodes a basic, prolinerich protein that contains a RING-H2-finger motif. The COOH-terminal region of the putative FXI-T1 protein has sequence similarity with the COOH-terminal domain of the Drosophila deltex protein, a component of a signal pathway that functions during cell differentiation. The described observations suggest an association of FXI-T1 with FX-induced leukemogenesis. The study of FXI-T1 should contribute to an understanding of the processes of T-cell differentiation and regeneration in addition to leukemogenesis
— id: 12569, year: 1996, vol: 7, page: 1113, stat: Journal Article,

Infection of human cells by murine ecotropic viruses: retroviral vectors carrying the hygromycin resistance-encoding gene
Suzuki H; Brown GD; Ohno K; Meruelo D
1996 May 8;170(2):255-259, Gene
The construction of a new retroviral vector, pSKV, is described. This vector carries two unique cloning sites, located between two Moloney leukemia virus-derived LTR, into which genes of interest may be introduced. The gene encoding hygromycin resistance (HyR) was subsequently introduced into one of the two sites, producing a second vector (pSKV/HyR) containing a unique SfiI site for the introduction of cDNA clones under the control of the cytomegalovirus (CMV) promoter (P-CMV). The cDNA (mH13), encoding a protein that has been shown to serve as a murine ecotropic retroviral receptor in transient assays, was cloned into the SfiI site (pSKV/HyR/mH13). Both constructs can be packaged into retroviral particles following transfection into an appropriate packaging cell line. Stable transfectants of the human glioblastoma cell line (U118MG) carrying each of these two constructs were generated by transfection and subsequent Hy selection. Clones expressing both the selectable marker and the mH13 gene, but not those expressing only the selectable marker, are shown to be susceptible to infection with murine ecotropic retroviral particles. These cells (HyR and mH13 positive) were then exposed to CRE/Xtk culture supernatant, a packaging cell line producing ecotropic retroviral particles carrying the HSV-TK (Herpes simplex virus-thymidine kinase) and neoR (neomycin-resistance) genes. Selection was in the presence of G418. In vitro growth of the U118MG/HyR/mH13/TK cells, but not that of the U118MG/HyR/mH13 cells, was inhibited by ganciclovir (GCV), indicating the successful transfer of HSV-TK by infection of human cells with murine retroviruses via the mH13 product
— id: 7051, year: 1996, vol: 170, page: 255, stat: Journal Article,

The chemical and biological properties of hypericin--a compound with a broad spectrum of biological activities [published erratum appears in Med Res Rev 1995 May;15(3):259]
Lavie G; Mazur Y; Lavie D; Meruelo D
1995 Mar;15(2):111-119, Medicinal research reviews
— id: 6925, year: 1995, vol: 15, page: 111, stat: Journal Article,

Hypericin as an inactivator of infectious viruses in blood components
Lavie G; Mazur Y; Lavie D; Prince AM; Pascual D; Liebes L; Levin B; Meruelo D
1995 May;35(5):392-400, Transfusion
BACKGROUND: Hypericin is a potent virucidal agent with activity against a broad range of enveloped viruses and retroviruses. The effective virucidal activity emanates from a combination of photodynamic and lipophilic properties. Hypericin binds cell membranes (and, by inference, virus membranes) and crosslinks virus capsid proteins. This action results in a loss of infectivity and an inability to retrieve the reverse transcriptase enzymatic activity from the virion. STUDY DESIGN AND METHODS: Since hypericin is devoid of adverse action in most blood components and blood analyses, it is investigated as an additive with potential to inactivate infective viruses in blood components intended for transfusion. RESULTS: Complete inactivation of 10(6) tissue culture-infective doses of human immunodeficiency virus was obtained in whole blood and in diluted packed red cells after illumination with fluorescent light for 1 hour. Loss of viral infectivity to cultured CEM cells has been monitored by use of a detection assay for human immunodeficiency virus p55 in enzyme-linked immunosorbent assay and cytopathic assays. In physiologic media, hypericin interacts with albumin and lipoproteins, retaining the virucidal activity in bound form. The molecule is negatively charged and forms organic and inorganic monobasic salts (ion pairs) in physiologic pH. Various ion pairs differ in virucidal efficacy. CONCLUSION: The apparent transfusibility of hypericin, taken together with the efficacy of the virucidal activity, the broad range of enveloped viruses affected, and the absence of adverse effects on stored red cells, may render hypericin useful for inactivation of infectious viruses in red cells
— id: 56669, year: 1995, vol: 35, page: 392, stat: Journal Article,

Cell targeting for gene delivery: use of fusion protein containing the modified human receptor for ecotropic murine leukemia virus
Ohno K; Brown GD; Meruelo D
1995 Dec;56(2):172-175, Biochemical & molecular medicine
We have previously cloned a human gene (H13) homologous to the murine ecotropic retrovirus (E-MuLV) receptor, which, however, does not confer susceptibility to E-MuLV infection. The extracellular domain 3 (ECD3) of H13 contains amino acid residues critical for E-MuLV binding in that the modified H13 gene (mH13), substituted with amino acids from the actual receptor, has the ability to bind E-MuLV. Here we have expressed a fusion protein consisting of mH13/ECD3 and transforming growth factor-alpha in Escherichia coli and demonstrated its binding activity to both ecotropic AKR virus and the epidermal growth factor receptor expressed on the cell surface. Fusion proteins of mH13/ECD3 and ligands to cell surface molecules might be useful for specific cell targeting in E-MuLV-based gene delivery systems
— id: 56836, year: 1995, vol: 56, page: 172, stat: Journal Article,

'Bystander killing' induces apoptosis and is inhibited by forskolin
Samejima Y; Meruelo D
1995 Jan;2(1):50-58, Gene therapy
'Bystander killing' is a term used to describe the broad cell death associated with the transduction of the herpes simplex virus thymidine kinase gene (HSV1-tk) and administration of nucleoside analogs and which extends the killing effect to adjacent cells not transduced with HSV1-tk ('bystander cells'). HSV1-tk negative cells can be killed by co-culture with HSV1-tk positive cells at a ratio as small as one HSV1-tk positive to 32 HSV1-tk negative cells (1:32). In this report, several aspects of bystander killing are characterized. First, the sensitivity to bystander killing is shown to differ among cell lines. Second, cell-to-cell contact, or at least proximity between cells, is demonstrated to be necessary for bystander killing. Third, forskolin is shown to inhibit bystander killing. We also show that bystander killing is not species specific. Finally, it is demonstrated that cell death induced by bystander killing is mediated via apoptosis
— id: 6741, year: 1995, vol: 2, page: 50, stat: Journal Article,

ACIDIC PROPERTIES OF HYPERICIN AND ITS OCTAHYDROXY ANALOG IN THE GROUND AND EXCITED-STATES
FREEMAN, D; FROLOW, F; KAPINUS, E; LAVIE, D; LAVIE, G; MERUELO, D; MAZUR, Y
1994 APR 7 ;33(7):891-892, Journal of the Chemical Society. Chemical communications
pH dependent absorption and fluorescence spectra of hypericin 1 and its octahydroxy analogue 3 demonstrate their acidic properties, their deprotonation occurring in the ground and excited states from OH in the bay region and in the peri position to the carbonyl, respectively; the presence of the anion of 1 in the crystalline state is established by X-ray diffraction
— id: 52428, year: 1994, vol: 33, page: 891, stat: Journal Article,

PHOTOSENSITIZATION OF THE ANTIVIRALLY ACTIVE HYPERICIN COMPLEXES WITH ALBUMIN
FREEMAN, D; KAPINUS, E; LAVIE, D; LAVIE, G; MERUELO, D; MAZUR, Y
1994 JUL ;68(7):1435-1436, Polish journal of chemistry
— id: 52398, year: 1994, vol: 68, page: 1435, stat: Journal Article,

Photodynamic inactivation of radiation leukemia virus produced from hypericin-treated cells
Degar S; Lavie G; Meruelo D
1993 Dec;197(2):796-800, Virology
Hypericin is a polycyclic, aromatic, naphthodianthrone which has been shown to possess in vivo and in vitro antiretroviral activity. To gain further insight into the mechanism(s) by which hypericin exerts its antiretroviral effects, we have studied Radiation Leukemia virus (RadLV) produced from cells pulse-treated with hypericin. Hypericin-treatment did not inhibit retroviral production or the proteolytic cleavage of the gag-encoded precursor proteins. Rather, hypericin was found to be associated with RadLV particles, the retrovirions showed an increased density in sucrose, and the RadLV protein banding patterns were altered. RadLV produced from hypericin-treated cells was rendered noninfectious upon exposure to visible light. Our results suggest that RadLV produced from hypericin-treated cells is inactivated by a hypericin-mediated photodynamic process
— id: 6355, year: 1993, vol: 197, page: 796, stat: Journal Article,

THE POTENTIAL USE OF HYPERICIN AS AN INACTIVATOR OF RETROVIRUSES AND OTHER VIRUSES IN BLOOD PRODUCTS
MERUELO, D; PRINCE, AM; PASCUAL, D; GILCHER, R; LAVIE, D; MAZUR, Y; LAVIE, G
1993 NOV 15 ;82(10):A205-A205, Blood
— id: 52142, year: 1993, vol: 82, page: A205, stat: Journal Article,

Linkage of superantigen-like stimulation of syngeneic T cells in a mouse model of follicular center B cell lymphoma to transcription of endogenous mammary tumor virus
Tsiagbe VK; Yoshimoto T; Asakawa J; Cho SY; Meruelo D; Thorbecke GJ
1993 Jun;12(6):2313-2320, EMBO journal
The MHC class II I-A(s) positive B cell lymphomas reticulum cell sarcoma (RCS) that arise in > 90% of SJL mice by the age of 12 months have superantigen-like stimulating properties. In the present study, therefore, RCS cell lines were examined for abnormal expression of endogenous mouse mammary tumor virus (MMTV) proviruses. Extraordinarily high expression of a 1.8 kb mRNA hybridizing with the long terminal repeat (LTR) of MMTV was found in both primary lymphomas and in vitro RCS lines, but not in an SJL B cell lymphoma, NJ101, that does not stimulate syngeneic T cells, or in LPS activated SJL B cells. A cDNA was cloned from cRCS-2 and sequenced. A 31mer oligonucleotide probe, prepared based on the unique C-terminal sequence of this RCS-Mtv LTR, detected the 1.8 kb mRNA in all RCS lymphomas, while a similar probe for the C-terminal sequence of Mtv-8 LTR hybridized with the larger mRNA present in normal B cells and in NJ101. Preincubation with 19mer antisense S-oligonucleotides, prepared based on the sequences of the first two potential translation initiation sites common to both Mtv-8 and the RCS-Mtv LTR, significantly reduced the ability of RCS cells to stimulate syngeneic T cells. Moreover, transfection of NJ101 cells with the cloned RCS-MMTV cDNA conferred V beta 16 T cell stimulating properties on to these cells. It is concluded that expression of the product of this MMTV-LTR mRNA provides RCS with the strong T cell stimulating properties that it needs for its growth. These results thus identify a novel oncogenic property of MMTV-LTR
— id: 8456, year: 1993, vol: 12, page: 2313, stat: Journal Article,

MTV encoded superantigen expression in B lymphoma cells in SJL mice as a stimulus for "reversed immunological surveillance"
Tsiagbe, V.K.; Asakawa, J.; Yoshimoto, T.; Cho, S.Y.; Meruelo, D.; Thorbecke, G.J
Superantigens : a pathogen's view of the immune system Plainview, N.Y. : Cold Spring Harbor Laboratory Press, 1993 ,
— id: 2519, year: 1993, vol: , page: 93, stat: Chapter,

MMTV-ENCODED SUPERANTIGEN ON LYMPHOMA-CELLS MEDIATES REVERSED IMMUNOLOGICAL SURVEILLANCE IN SJL MICE
TSIAGBE, VK; YOSHIMOTO, T; ASAKAWA, J; CHO, SY; MERUELO, D; THORBECKE, GJ
1993 APR 15 ;150(8):A19-A19, Journal of immunology
— id: 54224, year: 1993, vol: 150, page: A19, stat: Journal Article,

Identification of amino acid residues critical for infection with ecotropic murine leukemia retrovirus
Yoshimoto T; Yoshimoto E; Meruelo D
1993 Mar;67(3):1310-1314, Journal of virology
The murine cationic amino acid transporter is also the receptor for murine ecotropic leukemia retrovirus (MuLV-E). Recently, we have cloned a human gene (H13) homologous to the murine ecotropic retroviral receptor (ERR). Although the human homolog is very similar to murine ERR in sequence (87.6% amino acid identity) and structure (14 transmembrane-spanning domains), the human protein fails to function as a receptor for MuLV-E. To identify amino acid residues critical for MuLV-E infection, we took advantage of this species difference and substituted human H13 and murine ERR amino acid residues. Mouse-human chimeric receptor molecules were generated by taking advantage of using common restriction sites. These studies demonstrated that extracellular domains 3 and/or 4 contain the critical amino acid residues. Oligonucleotide-directed mutagenesis was then used to create 13 individual ERR mutants containing one or two amino acids substitutions or insertions within these two extracellular domains. Substitution of as few as one amino acid residue (Tyr) at position 235 in ERR with the corresponding H13 amino acid residue Pro abrogates the ability to function as a receptor for MuLV-E infection. Conversely, substitution of just two amino acid residues at positions 240 and 242 or 242 and 244 in H13 with the corresponding amino acid residues in ERR endows H13 with the ability to function as the receptor. This observation can be utilized to significantly improve the safety of retrovirus-mediated gene therapy in humans
— id: 57546, year: 1993, vol: 67, page: 1310, stat: Journal Article,

Inactivation of the human immunodeficiency virus by hypericin: evidence for photochemical alterations of p24 and a block in uncoating
Degar S; Prince AM; Pascual D; Lavie G; Levin B; Mazur Y; Lavie D; Ehrlich LS; Carter C; Meruelo D
1992 Nov;8(11):1929-1936, AIDS research & human retroviruses
Following attachment and entry of human immunodeficiency virus (HIV) into a host cell, the HIV genomic RNA is reverse transcribed to cDNA. This step may be inhibited by hypericin, a compound that induces alterations of the retroviral capsid. Incubation of HIV with hypericin rendered the virus noninfectious. The replication of HIV was blocked early; HIV cDNA could not be detected in cells challenged with hypericin-treated HIV. Hypericin did not inhibit the binding of recombinant gp120 to CD4+ cells, nor did hypericin inhibit syncytium formation. However, reverse transcriptase activity could not be released from hypericin-treated virions. Western blot analysis revealed altered mobility of the HIV major capsid protein (p24) following hypericin treatment. Hypericin-treated recombinant HIV p24 exhibited similar altered mobility. The inactivation of HIV infectivity and the alterations in p24 mobility required hypericin incubations in the presence of visible light. Collectively, these data suggest that photochemical alterations of the HIV capsid may contribute to the hypericin-mediated inactivation of HIV. Such alterations may inhibit the release of RT activity from treated HIV, and prevent uncoating and subsequent reverse transcription of the HIV genome within a target cell
— id: 13391, year: 1992, vol: 8, page: 1929, stat: Journal Article,

Long-range physical map of the Ly-6 complex: mapping the Ly-6 multigene family by field-inversion and two-dimensional gel electrophoresis
Kamiura S; Nolan CM; Meruelo D
1992 Jan;12(1):89-105, Genomics
The Ly-6 proteins are encoded by a recently identified multigene family. Much attention has been focused on these proteins because they may be involved in lymphocyte activation, and expression of some of them occurs at critical times in the differentiation of lymphocytes. These features make it important to investigate and to characterize further this family of molecules and the genes that encode them. To aid our investigation of these issues, we have constructed a physical map of the entire Ly-6 complex in the C57BL/6 murine genome using the combined techniques of field-inversion gel electrophoresis (FIGE), phage and cosmid genomic library screening, and two-dimensional DNA electrophoresis. This map spans approximately 1600 kb, and comparison of the FIGE map and cosmids indicates that most of the Ly-6 complex has been isolated in the cosmid clones
— id: 13717, year: 1992, vol: 12, page: 89, stat: Journal Article,

MODULATION OF MAJOR HISTOCOMPATIBILITY COMPLEX ANTIGENS ACCOMPANIES REVERSION OF MALIGNANT-MELANOMA PHENOTYPE BY CONTACT INHIBITORY FACTOR
LIPKIN, G; MERUELO, D
1992 APR ;40(2):A528-A528, Clinical research
— id: 52018, year: 1992, vol: 40, page: A528, stat: Journal Article,

MODULATION OF MAJOR HISTOCOMPATIBILITY COMPLEX ANTIGENS ACCOMPANIES REVERSION OF MALIGNANT-MELANOMA PHENOTYPE BY CONTACT INHIBITORY FACTOR
LIPKIN, G; MERUELO, D
1992 APR ;98(4):643-643, Journal of investigative dermatology
— id: 52034, year: 1992, vol: 98, page: 643, stat: Journal Article,

A novel DNA binding activity is elevated in thymocytes expressing high levels of H-2Dd after radiation leukemia virus infection
Nobunaga T; Brown GD; Morris DR; Meruelo D
1992 Aug 1;149(3):871-879, Journal of immunology
Resistance to radiation leukemia virus-induced leukemia is mediated by gene(s) in the H-2D region of the MHC; a clear correlation exists between disease resistance and increased H-2Dd expression on the thymocyte surface. We have investigated the molecular basis for this stimulation of H-2Dd class I expression. Elevated H-2 mRNA and H-2 transcription are demonstrated in the infected thymocytes as compared to normal thymocytes indicating that the elevation of H-2 surface expression is the result of transcriptional activation. Gel mobility assays performed with nuclear extracts of normal and infected thymocytes and sequences 5' of the H-2Dd gene show that specific binding occurs with both extracts; the binding differs both quantitatively and qualitatively, however. DNase I protection analysis detects a protein binding site that is protected only by extracts from infected cells. The protected region contains a sequence similar to the AP-1 consensus sequence. Gel shift competition assays and UV photo-cross-linking to an oligonucleotide containing this sequence demonstrate that specific binding of an H-2 binding factor 1 occurs and that this factor is not the AP-1 binding complex. This novel binding factor, activated in vivo, might also be involved in the normal regulation of H-2 gene expression by recognizing the highly conserved binding sequence (TGACGCG) found in the 5' flanking region of many MHC class I genes. This is the first demonstration of the parallel stimulation of a DNA binding activity and increased transcription occurring in thymocytes after infection with a leukemogenic retrovirus
— id: 13483, year: 1992, vol: 149, page: 871, stat: Journal Article,

Enhanced gene expression of the murine ecotropic retroviral receptor and its human homolog in proliferating cells
Yoshimoto T; Yoshimoto E; Meruelo D
1992 Jul;66(7):4377-4381, Journal of virology
The receptor for gp70 envelope glycoprotein of murine ecotropic leukemia virus is essential for virus entry into the host cell and has been recently demonstrated to function as a cationic amino acid transporter. In the experiments reported herein, we compared the gene expression of the murine ecotropic retroviral receptor (ERR) and its human homolog (H13) in rapidly proliferating cells versus resting cells using four different systems. (i) The expression of ERR gene is enhanced during activation of T and B lymphocytes by concanavalin A and lipopolysaccharide, respectively. Similar enhancement is observed by adding phorbol 12-myristate 13-acetate (PMA) or calcium ionophore (A23187). These phenomena appear to involve protein kinase C; two PMA analogs, 4 alpha-phorbol and 4 alpha-PMA, lacking the ability to activate protein kinase C fail to induce elevated levels of gene expression, and the protein kinase C inhibitor, H7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride[, inhibits the enhancement induced by PMA. (ii) Friend murine leukemia virus induces rapid splenomegaly, and acute erythroleukemia in sensitive mice. Concomitantly with splenomegaly, ERR gene expression in spleen cells increases dramatically. (iii) The level of expression of the ERR or H13 gene in a variety of tumor cells is highly elevated compared with the level in noncancerous cells. (iv) H13 gene expression decreases upon terminal differentiation of the human promyelocytic leukemia cell line HL-60 into granulocytes or macrophages by dimethyl sulfoxide or PMA, respectively. These results suggest that ERR and H13 genes play an important role in cellular proliferation
— id: 13555, year: 1992, vol: 66, page: 4377, stat: Journal Article,

In vivo stimulation of H-2Dd expression following RadLV infection of thymocytes: increased transcription and DNA-binding activity to sequences 5' of the Dd gene
Brown GD; Nobunaga T; Morris DR; Meruelo D
1991 Jun-Aug;142(5-6):431-440, Research in immunology
Early studies showed that resistance to RadLV-induced leukaemia is mediated by gene(s) in the H-2D region of the MHC. Furthermore, these experiments correlated disease resistance with changes in H-2 expression occurring very early after virus inoculation. In the present study, we have begun to study at the molecular level this stimulation of H-2Dd class I expression in thymocytes of resistant mouse strains following infection by RadLV. The resistant strain of B10.T(6R) mice is used in these studies. When these infected thymocytes are assayed by fluorescence-activated cell sorting analysis, we can detect increased levels of H-2Dd expression on the surface of the thymocytes as early as 12 days following intrathymic injection of RadLV. RNA was prepared and examined by Northern blot analysis; H-2 mRNA levels are shown to be elevated on the order of four-fold. Nuclei were prepared from normal and infected thymocytes and the run-off transcripts were analysed by slot-blot hybridization. The rate of H-2 mRNA transcription is shown to be two- to three-fold higher in RadLV-infected thymocytes at 14 days post-infection when compared to that of normal thymocytes. These data demonstrate that elevation of H-2 surface expression following RadLV infection is the result of transcriptional activation. Extracts have been prepared from both normal and infected B10.T(6R) thymocytes and have been used in gel mobility assays in order to detect the interaction of potential trans-acting regulatory factors with sequences 5' of the H-2Dd gene. Specific binding occurs in both extracts, but the assay shows that the extracts differ both quantitatively and qualitatively; the extracts from infected thymocytes bind to additional sequences and to a higher degree than that from normal thymocytes. DNase I protection analysis locates a number of protein-binding sites, some of which are protected by extracts of either origin and some of which are only protected by extracts from infected cells. Two of these sequences are similar to the previously recognized consensus recognition sequences for the binding of AP-1 and NF-chi B. Oligonucleotides have been synthesized for both the genomic sequences being protected from DNase I digestion as well the published consensus sequences. While the DNA-binding activity in infected thymocytes for both AP-1 and NF-chi B-binding sites is increased, the binding to the genomic 'AP-1 like' binding site is activated to a considerably greater level.(ABSTRACT TRUNCATED AT 400 WORDS)
— id: 14012, year: 1991, vol: 142, page: 431, stat: Journal Article,

Isolation of virus-like (VL30) elements from the Q10 and D regions of the major histocompatibility complex
Choi YC; Meruelo D
1991 Feb;29(1-2):91-101, Biochemical genetics
Previous studies from our laboratory have described two endogenous provirus-like sequences in a series of cosmids spanning the TL region of the major histocompatibility complex (MHC) of normal C57BL/10 mice. At least one of these viruses shares similarities with VL30 elements. To determine if additional VL30-like retroviral elements are integrated in the MHC, we constructed a cosmid library using DNA from a radiation leukemia virus (RadLV)-transformed cell line derived from C57BL/6 mice. The library was first screened using the H-2III (5') probe, which detects Class I genes of the H-2 complex. In the primary screening 163 H-2III positives were isolated. The H-2III-positive isolates were then hybridized with an AKR-derived virus probe, EcoB/S, which contains sequences from both the pol and the env genes of the virus. Nine virus-positive isolates were detected. Localization of these cosmid isolates containing viral sequences within the H-2 complex was done utilizing low-copy probes and confirmed using previously mapped cosmid isolates from other laboratories. We report here the isolation and characterization of VL30-like elements from the Qa and D regions of the MHC of several inbred mouse strains
— id: 14153, year: 1991, vol: 29, page: 91, stat: Journal Article,

INHIBITION OF HIV INFECTIVITY BY HYPERICIN - EVIDENCE FOR A BLOCK IN CAPSID UNCOATING
DEGAR, S; LAVIE, G; LEVIN, B; MAZUR, Y; LAVIE, D; PASCUAL, D; PRINCE, A; MERUELO, D
1991 FEB 14 ;4(3):352-353, Journal of acquired immune deficiency syndromes & human retrovirology
— id: 51728, year: 1991, vol: 4, page: 352, stat: Journal Article,

RETROVIRAL PARTICLE INACTIVATION BY ORGANIC POLYCYCLIC QUINONES - A NOVEL MECHANISM OF VIRUCIDAL ACTIVITY CHARACTERIZED BY DIMINUTION OF VIRUS PARTICLE DERIVED REVERSE-TRANSCRIPTASE ENZYMATIC-ACTIVITY
LAVIE, G; MERUELO, D; DAUB, M; DEGAR, S; LEVIN, B; LAVIE, D; MAZUR, Y; NASR, M
1991 FEB 14 ;4(3):356-357, Journal of acquired immune deficiency syndromes & human retrovirology
— id: 51729, year: 1991, vol: 4, page: 356, stat: Journal Article,

A method for the quantitation of hypericin, an antiviral agent, in biological fluids by high-performance liquid chromatography
Liebes L; Mazur Y; Freeman D; Lavie D; Lavie G; Kudler N; Mendoza S; Levin B; Hochster H; Meruelo D
1991 May 15;195(1):77-85, Analytical biochemistry
Hypericin, a polycyclic aromatic dianthroquinone, is a natural plant product with antiviral properties. We report here the development of a methodology for the extraction and quantitation of hypericin from plasma and biological fluids and the adaptation of a sensitive and selective method for detection of the compound by high-performance liquid chromatography. The methodology offers a rapid and specific means of monitoring drug blood levels in clinical and pharmacokinetic studies. The chromatographic procedure utilizes the substantial retentive properties of hypericin on reverse-phase media and detection by the strong visible absorbance maximum at 590 nm. Verification by the fluorescence spectral properties of hypericin in organic media can also be utilized. The assay is linear over a 3 log concentration range and hypericin is consistently recovered from murine, simian, and human plasma. The methodology was applied to assess the pharmacokinetic properties of hypericin in mice receiving a single bolus injection of 350 micrograms. A distribution half-life of 2.0 h and an elimination half-life of 38.5 h were calculated. We also discuss the limitations of direct analysis of hypericin by absorbance or fluorescence measurements
— id: 14022, year: 1991, vol: 195, page: 77, stat: Journal Article,

A HYPOTHESIS FOR THE MODE OF ACTION OF HYPERICIN AS AN ANTIRETROVIRAL AGENT
MERUELO, D; DEGAR, S; LEVIN, B; LAVIE, D; MAZUR, Y; LAVIE, G
1991 AUG 25 ;202(3):188-AGFD, Abstracts of papers (American Chemical Society)
— id: 52074, year: 1991, vol: 202, page: 188, stat: Journal Article,

INACTIVATION OF RETROVIRAL PARTICLES BY HYPERICIN - POSSIBLE ROLE OF OXIDATIVE REACTIONS IN THE ANTIRETROVIRAL ACTIVITY
MERUELO, D; DEGAR, S; LEVIN, B; LAVIE, D; MAZUR, Y; LAVIE, G
1991 FEB 14 ;4(3):357-358, Journal of acquired immune deficiency syndromes & human retrovirology
— id: 51730, year: 1991, vol: 4, page: 357, stat: Journal Article,

Molecular cloning and characterization of a novel human gene homologous to the murine ecotropic retroviral receptor
Yoshimoto T; Yoshimoto E; Meruelo D
1991 Nov;185(1):10-17, Virology
A novel human cDNA homologous to the murine ecotropic retroviral receptor was cloned from a cDNA library derived from a human T-cell line. The human cDNA is highly homologous to the murine counterpart (87.6% amino acid identity), and its sequence predicts a protein with 629 amino acids (approximately 68 kDa), which is 7 amino acids more than the murine counterpart (622 amino acids). The predicted protein is highly hydrophobic and contains 14 potential transmembrane-spanning domains. No other gene and protein with significant homology to the cloned human gene and the predicted protein were identified by a computer-based search of sequence data banks other than the murine T-cell early activation gene (52.5% amino acid identity) and the murine ecotropic retroviral receptor gene. The human gene is ubiquitously expressed in human tissues and conserved among mammalian species. The genomic gene was also isolated from a cosmid library derived from human lymphocytes, and its organization was elucidated. The gene mapped to human chromosome 13
— id: 13860, year: 1991, vol: 185, page: 10, stat: Journal Article,

Effects of fractionated x-irradiation on the Ly-6--Ril-1--Pol-5 region
Amari NM; Kamiura S; Meruelo D
1990 ;32(4):252-262, Immunogenetics
Our laboratory has focused on defining, localizing, and understanding the mode of action of genes involved in fractionated x-irradiation (FXI) leukemia in susceptible and restraint mouse strains. We have described the genetic and molecular evidence suggesting the existence of multiple independent loci involved in FXI-induced leukemogenesis. These studies indicated that one of these, Ril-1, a locus on the distal portion of chromosome 15, is the major locus influencing susceptibility to the disease. Our data unequivocally place Ril-1 in the gene complex Ly-6--Ril-1--Sis--H-30--Pol-5. Ril-1 appears to be closest to Ly-6 and Sis. We report that in FXI-induced leukemias there are hypomethylation changes in the Ly-6 region as compared to normal thymocytes. In contrast, Sis was found to be hypermethylated and not expressed. In addition, we have noted DNA rearrangements in the Ly-6--Pol-5 region in the majority of tumors examined using the Ly-6 and spleen focus-forming virus (SFFLV) molecular probes. Increased expression of Ly-6 and other surface markers encoded in this region has been noted in FXI-induced thymomas
— id: 15244, year: 1990, vol: 32, page: 252, stat: Journal Article,

Increased H-2Dd expression following infection by a molecularly cloned ecotropic MuLV
Brown GD; Egan G; Dowling T; Meruelo D
1990 ;31(2):94-103, Immunogenetics
The biological consequences of radiation leukemia virus (RadLV) infection include the stimulation of H-2Dd antigen expression in resistant mouse strains and thymoma induction in susceptible strains. In an effort to understand the genetic basis of these phenomena, the integrated ecotropic RadLV genome has been examined in a number of primary RadLV-induced tumors, as well as thymomas adapted to in vitro passage; considerable heterogeneity was observed. Examination of these polymorphic viral sequences should help define the viral gene(s) involved in the biological effects of RadLV infection; toward this end, integrated RadLV genomes were molecularly cloned and examined. The genomes and their flanking sequence were characterized by restriction enzyme analysis. Three unique viral genomes were obtained which represent four integration sites. The three RadLV genomes are shown to carry polymorphisms of the original tumor. Following DNA transfection, one of the three genomes replicated in and reinfected both mouse thymocytes and fibroblasts, but not mink fibroblasts in vitro. Virus encoded by the other two DNA genomes could not be recovered following transfection into any of the three cell types. One of these two apparently defective retroviruses encodes a truncated p15E molecule, while the other has elongated long terminal repeats (LTRs). The non-defective ecotropic isolate was collected from in vitro tissue culture supernatants, concentrated, and used to infect mice. Thymocytes of infected, resistant mice were shown to express elevated levels of H-2Dd antigen as early as 12 days post infection, a hallmark of RadLV infection
— id: 15243, year: 1990, vol: 31, page: 94, stat: Journal Article,

REVISED NOMENCLATURE OF MOUSE H-2 GENES
Klein, J; Benoist, C; David, CS; Demant, P; Lindahl, KF; Flaherty, L; Flavell, RA; Hammerling, U; Hood, LE; Hunt, SW; Jones, PP; Kourilsky, P; Mcdevitt, HO; Meruelo, D; Murphy, DB; Nathenson, SG; Sachs, DH; Steinmetz, M; Tonegawa, S; Wakeland, EK; Weiss, EH
1990 Sep;32(3):147-149, Immunogenetics
— id: 31836, year: 1990, vol: 32, page: 147, stat: Journal Article,

HYPERICIN AS AN ANTIRETROVIRAL AGENT - MODE OF ACTION AND RELATED ANALOGS
Lavie, G; Mazur, Y; Lavie, D; Levin, B; Ittah, Y; Meruelo, D
1990 Dec 26;616(3):556-562, Annals of the New York Academy of Sciences
— id: 32177, year: 1990, vol: 616, page: 556, stat: Journal Article,

Radiation leukemia virus and its effect on H-2 gene expression
Brown GD; Meruelo D
1989 Aug-Oct;16(4-5):351-361, Journal of immunogenetics
In this report we demonstrate that lowered expression of the H-2 antigens on RadLV-induced tumour cells is a result of depressed levels of stable mRNA in these cells. Whether this observation is a result of lowered transcription or of mRNA instability is under investigation. In an effort to determine which viral sequences are essential for mediating both the H-2 regulatory function and the transforming function of RadLV, we have begun to assemble newly integrated proviral genomes from tumours. The restriction enzyme cleavage sites of four isolates are presented; these isolates differ substantially from RadLV genomes previously presented. One of these molecular clones is shown to encode a non-defective B-tropic, ecotropic virus which when reinjected into resistant mouse strains can mediate the up-regulation of H-2Dd antigen expression. Finally, possible mechanisms of H-2 regulation are discussed
— id: 10540, year: 1989, vol: 16, page: 351, stat: Journal Article,

A TCR gamma delta cell recognizing a novel TL-encoded gene product
Houlden BA; Matis LA; Cron RQ; Widacki SM; Brown GD; Pampeno C; Meruelo D; Bluestone JA
1989 ;54 Pt 1:45-55, Cold Spring Harbor symposia on quantitative biology
— id: 15245, year: 1989, vol: 54 Pt 1, page: 45, stat: Journal Article,

Studies of the mechanisms of action of the antiretroviral agents hypericin and pseudohypericin
Lavie G; Valentine F; Levin B; Mazur Y; Gallo G; Lavie D; Weiner D; Meruelo D
1989 Aug;86(15):5963-5967, Proceedings of the National Academy of Sciences of the United States of America
Administration of the aromatic polycyclic dione compounds hypericin or pseudohypericin to experimental animals provides protection from disease induced by retroviruses that give rise to acute, as well as slowly progressive, diseases. For example, survival from Friend virus-induced leukemia is significantly prolonged by both compounds, with hypericin showing the greater potency. Viremia induced by LP-BM5 murine immunodeficiency virus is markedly suppressed after infrequent dosage of either substance. These compounds affect the retroviral infection and replication cycle at least at two different points: (i) Assembly or processing of intact virions from infected cells was shown to be affected by hypericin. Electron microscopy of hypericin-treated, virus-producing cells revealed the production of particles containing immature or abnormally assembled cores, suggesting the compounds may interfere with processing of gag-encoded precursor polyproteins. The released virions contain no detectable activity of reverse transcriptase. (ii) Hypericin and pseudohypericin also directly inactivate mature and properly assembled retroviruses as determined by assays for reverse transcriptase and infectivity. Accumulating data from our laboratories suggest that these compounds inhibit retroviruses by unconventional mechanisms and that the potential therapeutic value of hypericin and pseudohypericin should be explored in diseases such as AIDS
— id: 10542, year: 1989, vol: 86, page: 5963, stat: Journal Article,

Reverse genetics approaches for cloning RIL-1, a major locus involved in susceptibility to leukemia
Amari NM; Scandalis S; Zhang D; Pampeno CL; Arant S; Meruelo D
1988 ;137:256-263, Current topics in microbiology & immunology
— id: 15246, year: 1988, vol: 137, page: 256, stat: Journal Article,

Extension of the H-2 TLb molecular map. Isolation and characterization of T13, T14, and T15 from the C57BL/6 mouse
Brown GD; Choi Y; Egan G; Meruelo D
1988 ;27(4):239-251, Immunogenetics
A region of the TLb locus encompassing T11 to T13 contains retroviral sequences TLev1 and TLev2. As part of a study to determine whether the retroviral elements are involved in the expression of TL genes, the genomic organization of this region was reexamined in greater detail. A result of these investigations is the extension of the H-2 TLb molecular map. Two additional TL genes have been isolated from C57BL/6 mice, T14 and T15. The genomic organization of T9 through T15 is presented. The nucleotide sequence has been determined for exons 4, 5, and 6 of T13. As a result of a C to T conversion, a termination codon is introduced into exon 4, indicating that T13 either encodes a secreted protein or is a pseudogene. T13 was found to be more homologous to the H-2 genes outside the TL region. T14 has been physically disrupted by the integration of TLev1, and the H-2 sequences appear to have diverged greatly. The relationship of the TL regions of the b and c haplotypes has been investigated using numerous low copy probes. The genome of BALB/c (TLc) is shown to lack a counterpart of the T13-T15b region. Homologous regions exist in the two haplotypes; yet considerable polymorphism is observed. TLb mice do not express TLa on the cell surface of normal thymocytes while TLc mice do; TLa expression is activated in many TLb leukemias. The diversity seen in the T13-T15 region may provide insights into the phenotypic expression or regulatory mechanisms of TL expression in these two haplotypes
— id: 11286, year: 1988, vol: 27, page: 239, stat: Journal Article,

Regulation of H-2 class I gene expression in virally transformed and infected cells
Brown GD; Choi Y; Pampeno C; Meruelo D
1988 ;8(3):175-215, Critical reviews in immunology
Early studies of the resistance and susceptibility of mouse strains to radiation-induced leukemia virus have demonstrated the important role of altered histocompatibility (H-2) antigen expression in the effectiveness of the immune response of the host to virus-infected and transformed cells. Changes in H-2 gene expression have now been correlated with disease resistance in a variety of viral systems. The experiments discussed indicate that viruses may directly or indirectly affect H-2 antigen expression at various levels of gene expression. These investigations generate a framework for approaching a molecular understanding of viral-induced changes in H-2 gene expression
— id: 11230, year: 1988, vol: 8, page: 175, stat: Journal Article,

Therapeutic agents with dramatic antiretroviral activity and little toxicity at effective doses: aromatic polycyclic diones hypericin and pseudohypericin
Meruelo D; Lavie G; Lavie D
1988 Jul;85(14):5230-5234, Proceedings of the National Academy of Sciences of the United States of America
Two aromatic polycyclic diones hypericin and pseudohypericin have potent antiretroviral activity; these substances occur in plants of the Hypericum family. Both compounds are highly effective in preventing viral-induced manifestations that follow infections with a variety of retroviruses in vivo and in vitro. Pseudohypericin and hypericin probably interfere with viral infection and/or spread by direct inactivation of the virus or by preventing virus shedding, budding, or assembly at the cell membrane. These compounds have no apparent activity against the transcription, translation, or transport of viral proteins to the cell membrane and also no direct effect on the polymerase. This property distinguishes their mode of action from that of the major antiretro-virus group of nucleoside analogues. Hypericin and pseudohypericin have low in vitro cytotoxic activity at concentrations sufficient to produce dramatic antiviral effects in murine tissue culture model systems that use radiation leukemia and Friend viruses. Administration of these compounds to mice at the low doses sufficient to prevent retroviral-induced disease appears devoid of undesirable side effects. This lack of toxicity at therapeutic doses extends to humans, as these compounds have been tested in patients as antidepressants with apparent salutary effects. Our observations to date suggest that pseudohypericin and hypericin could become therapeutic tools against retroviral-induced diseases such as acquired immunodeficiency syndrome (AIDS)
— id: 11059, year: 1988, vol: 85, page: 5230, stat: Journal Article,

Genomic organization of the mouse Tla locus: study of an endogenous retroviruslike locus reveals polymorphisms related to different Tla haplotypes
Pampeno C; Meruelo D
1988 ;28(4):247-254, Immunogenetics
A retrovirus element (TLev1) is located within the Thymus leukemia antigen (Tla) locus of the C57BL/10 mouse major histocompatibility complex. Low-copy probes have been isolated from sequences flanking the TLev1 integration site to examine the distribution of TLev1 among inbred mouse strains having genotypically determined variations in TL-antigen expression. It was found that the low-copy probes cross-hybridize to regions within the Tla locus in a genotype-specific manner. Although a strong association was found between TL mouse strains and TLev1, the presence or absence of the TLev1 locus did not exclusively correlate with expression or nonexpression of TL antigens. Analysis of different Mus subspecies indicates that TLev1 integrated into a common ancestor of the species Mus musculus. It is suggested that the loss of the TLev1 locus from certain mouse genomes reflects evolutionary rearrangements in the TL region; the resulting diversity may relate to the differential expression of TL antigens among mouse strains. The probes described here provide a useful tool for examining the genomic expansions and contractions which have occurred during the evolution of the Tla locus
— id: 11274, year: 1988, vol: 28, page: 247, stat: Journal Article,

Murine thymomas induced by fractionated-X-irradiation have specific T-cell receptor rearrangements and characteristics associated with day-15 to -16 fetal thymocytes
Amari NM; Meruelo D
1987 Dec;7(12):4159-4168, Molecular & cellular biology
We report here that specific T-cell receptor rearrangements were observed in fractionated-X-irradiation-induced murine leukemias. Consistent gamma-chain rearrangements, limited beta-chain rearrangements, and no detectable alpha-chain rearrangements were observed. Gene expression studies revealed that, in comparison with normal thymus tissue, expression of alpha T-cell receptor genes was lower in the thymomas, beta expression was much higher but approximately equal to that of normal thymocytes, and gamma expression was significantly increased. After coupling these data with those from analyses using reagents against other surface markers, such as Lyt-2, L3T4, H-2, IL-2R and MEL-14, we concluded that the target T cells for fractionated-X-irradiation-induced transformation resemble fetal thymocytes from days 15 and 16 of gestation
— id: 11313, year: 1987, vol: 7, page: 4159, stat: Journal Article,

Assignment of the Ly-6--Ril-1--Sis--H-30--Pol-5/Xmmv-72--Ins-3--Krt-1--Int-1 --Gdc-1 region to mouse chromosome 15
Meruelo D; Rossomando A; Scandalis S; D'Eustachio P; Fournier RE; Roop DR; Saxe D; Blatt C; Nesbitt MN
1987 ;25(6):361-372, Immunogenetics
Previous work has demonstrated linkage between Ly-6, H-30, and a locus, Ril-1, that affects susceptibility to radiation-induced leukemia. Results or preliminary linkage analyses suggested further that the cluster might be linked to Ly-11 on the proximal portion of mouse chromosome 2. Using molecular probes to examine somatic cell lines and recombinant inbred and congenic strains of mice, we have re-evaluated these linkage relationships. A cloned genomic DNA fragment derived from a retroviral site has been used to define a novel locus, Pol-5, that is tightly linked to both H-30 and Ril-1 as shown by analysis of the B6.C-H-30c congenic mouse strain. Following the segregation of the Pol-5 mouse-specific DNA fragment in a series of somatic cell hybrids carrying various combinations of mouse chromosomes on a rat or Chinese hamster background mapped Pol-5 to mouse chromosome 15. During the course of these studies, restriction fragment length polymorphisms were defined associated with several loci, including Pol-5, Ly-6, Sis, Ins-3, Krt-1, Int-1, and Gdc-1. Three of these loci, Sis, Int-1, and Gdc-1, have been previously mapped to chromosome 15 by others using somatic cell hybrids or isoenzyme analyses. Following the inheritance of these eight loci in recombinant inbred strains of mice allowed the definition of a linkage group on the chromosome with the order Ly-6--Ril-1--Sis--H-30--Pol-5--Ins-3--Krt-1--Int-1--Gdc-1. Analyses of alleles inherited as passengers in B6.C-H-30c, C3H.B-Ly-6b, and C57BL/6By-Eh/+ congenic mouse strains and in situ hybridization experiments support the above gene order and indicate further that the cluster is located on distal chromosome 15, with Ly-6 and Sis near Eh
— id: 15248, year: 1987, vol: 25, page: 361, stat: Journal Article,

Phosphoproteins recognized by an H-2-linked immune response gene and their association with cell proliferation
Zalman MA; Meruelo D
1987 Jan 1;47(1):193-200, Cancer research
An H-2-associated immune response gene which maps to the I-A subregion of the H-2 complex governs the ability of H-2 congenic mice to mount an antibody response to five phosphoproteins with molecular weights of 33,000, 29,000, 23,000, 17,000, and 16,000 when inoculated with BW5147, a spontaneous AKR T-cell leukemic cell line. The phosphoproteins are present in all tumor cell lines tested, including those of murine and human origins. The phosphoproteins are associated with the proliferative state of the cell as studied in many systems including growth stimulation of normal lymphoid cells with mitogens, interleukin 2 dependency for growth of a cloned T-cell line, cessation of proliferation by serum starvation of Swiss 3T3 fibroblasts, retention of the proliferative capacity of SV40-transformed 3T3 fibroblasts, and the differentiation and inhibition of proliferation of human promyelocytic leukemic cells. Phosphoproteins with molecular weights of 33,000, 29,000, 23,000, 17,000, and 16,000 are therefore not specific to a particular inducible cellular pathway but are associated with cell proliferation in general
— id: 15247, year: 1987, vol: 47, page: 193, stat: Journal Article,

Lack of class I H-2 antigens in cells transformed by radiation leukemia virus is associated with methylation and rearrangement of H-2 DNA
Meruelo D; Kornreich R; Rossomando A; Pampeno C; Boral A; Silver JL; Buxbaum J; Weiss EH; Devlin JJ; Mellor AL; et al
1986 Jun;83(12):4504-4508, Proceedings of the National Academy of Sciences of the United States of America
Transformation of murine thymocytes by radiation leukemia virus is associated with reduced expression of the class I antigens encoded in the major histocompatibility complex (MHC) and increased methylation and altered restriction enzyme patterns of MHC DNA. These changes may play a role in host susceptibility to virus-induced leukemogenesis and accord with the notion that viral genomes play a regulatory function when they integrate adjacent to histocompatibility genes
— id: 15249, year: 1986, vol: 83, page: 4504, stat: Journal Article,

A molecular and genetic approach to understanding the mechanisms by which fractionated X-irradiation induces leukemia in mice
Meruelo D; Rossomando A
1986 ;10(7):819-832, Leukemia research
Our laboratory's approach to try to shed light on the question of a viral etiology for radiation-induced leukemia has focused on defining, localizing and understanding the mode of action of genes involved in susceptibility to FXI-induced disease. These studies have indicated that multiple genes control the process of leukemogenesis. In addition not every mouse strain which shows some susceptibility to FXI-induced leukemia carries the susceptible gene at each of the multiple loci involved in the disease process. Thus, it is plausible to conclude that more than one mechanism of leukemogenesis can be triggered by FXI. Our studies have focused on the mode of action of one such locus Ril-1. Several reagents have been developed to help us clone and characterize this locus. Currently chromosomal 'walking' and 'hopping' techniques are being used in conjunction with an RFLP molecular probe which is adjacent to Ril-1. In addition a cDNA library has been prepared from a radiation-induced thymoma and subtraction hybridization analysis is being used in the search for Ril-1
— id: 15251, year: 1986, vol: 10, page: 819, stat: Journal Article,

Isolation of a retroviruslike sequence from the TL locus of the C57BL/10 murine major histocompatibility complex
Pampeno CL; Meruelo D
1986 May;58(2):296-306, Journal of virology
Two retroviruslike sequences have been isolated from the TL locus of the major histocompatibility complex of C57BL/10 mice. One sequence (TLev2) hybridizes only with probes derived from the pol region of the murine leukemia provirus AKR; the other sequence (TLev1) hybridizes with gag, pol, and env AKR region probes. This 9-kilobase endogenous, TL region-associated virus (TLev1) has been further characterized. The TLev1 genome has been shown to contain murine leukemia virus-related sequences bounded by retroviruslike, VL30 long terminal repeats. Hybridization of TLev1-derived probes to mouse genomic digests reveals multiple copies which show distinct patterns compared with those observed with murine leukemia virus probes. The study of TLev1 may prove significant with respect to the interaction of retroviral sequences within the genome, expression of genes within the TL locus, and polymorphisms within the major histocompatibility complex
— id: 15250, year: 1986, vol: 58, page: 296, stat: Journal Article,

Viral sequences are associated with many histocompatibility genes
Rossomando A; Meruelo D
1986 ;23(4):233-245, Immunogenetics
A C57BL/6By 5.5 kb Pvu II polymorphic restriction fragment which hybridizes with a spleen focus-forming env probe and maps in the H-30 region has been cloned, and a 358 bp subfragment subcloned. Hybridization and sequencing studies show that the 358 bp fragment is encoded by the region of the pol gene of murine retrovirus which codes for an endonuclease critical for viral integration. Hybridizations of digested murine genomic DNAs with the 358 bp probe generate 31 restriction fragment length polymorphisms (RFLPs); 16 of these can be placed near the following 15 minor histocompatability (H) loci: H-3, H-4, H-7, H-13, H-15, H-16, H-17, H-19, H-22, H-24, H-27, H-30, H-34, H-36, and H-38. We suggest that the proximity of viral sequences to H loci is probably evolutionarily and functionally significant and that the closeness of viral sequences and minor H loci can probably be utilized to facilitate the cloning of minor H genes. During the course of these studies, it has become possible to tentatively assign H-17, H-34, and H-38 to chromosome 12. In addition, it was observed that several H-2 congenic strains retain portions of chromosome 12 from the parental donor strains used in their derivation
— id: 15253, year: 1986, vol: 23, page: 233, stat: Journal Article,

Analysis of H-2-linked immune responses involved in resistance to AKR tumor growth
Zalman MA; Meruelo D
1986 ;24(1):51-62, Immunogenetics
H-2-associated immune response gene(s) govern resistance to growth of a spontaneous AKR lymphoma, BW5147. The antigenic specificities recognized by the anti-BW5147 humoral response have been characterized and include: Thy-1, a T-cell differentiation antigen; gp70, a retroviral envelope protein; and several previously uncharacterized proteins, including a 78 000 molecular mass protein, p78, which is restricted to expression on BW5147 cells and five phosphoproteins with molecular masses of 33 000, 29 000, 23 000, 17 000, and 16 000. Only mice which are able to respond to Thy-1, p78, and the phosphoproteins can survive an inoculation of BW5147. Thus, resistance to BW5147 is complex and involves multiple antigens with possible roles in tumor rejection
— id: 15252, year: 1986, vol: 24, page: 51, stat: Journal Article,

Heterogeneity of p15(E)-related polypeptides expressed by MuLV-infected cells
Bach RG; Meruelo D
1985 Aug;145(1):165-170, Virology
The p15(E)-related polypeptides of radiation leukemia virus (RadLV)-derived viruses and of cells infected with prototype MuLV were analyzed by immunoprecipitation, SDS-PAGE, and immunofluorescence analyses. It was found that the p15(E)-related molecules of ecotropic and xenotropic viruses derived from RadLV lymphoma cell lines were distinguishable by reactivity with monoclonal anti-p15(E) antibodies and by SDS-PAGE profile. Ecotropic MuLV of RadLV origin encoded the p15(E)a antigen and produced a Pr15(E) of 20K MW. In contrast, xenotropic virus derived from RadLV did not express the p15(E)a antigen and by SDS-PAGE its Pr15(E) migrated at 21K. A previously undescribed, p15(E)-related molecule of 18.5K MW was associated with xenotropic RadLV. These differences were also reproduced by the prototype ecotropic, xenotropic, and dualtropic viruses
— id: 15254, year: 1985, vol: 145, page: 165, stat: Journal Article,

Relationship between leukemia virus genomes and histocompatibility genes
Meruelo D
1985 ;:163-170, Year in immunology
— id: 15255, year: 1985, vol: , page: 163, stat: Journal Article,

Identification of a 36,000-molecular weight, gag-related phosphoprotein in lymphoma cells transformed by radiation leukemia virus
Bach RG; Meruelo D
1984 Jul 1;160(1):270-285, Journal of experimental medicine
Radiation leukemia virus (RadLV) causes thymic lymphoma in 90% of susceptible mice after a latent period of several months. The virally encoded polypeptides produced by RadLV-induced lymphoma cells were analyzed by immunoprecipitation and NaDodSO4/polyacrylamide gel electrophoresis. Along with the expected precursor and mature forms of gag and env gene products, a polypeptide of 36,000 molecular weight (p36) was precipitated by anti-gag antisera. It was not precipitable by normal sera or anti-env antibodies. Like the gag-associated fusion proteins of some acute leukemia viruses, p36 was found to be phosphorylated in vivo, although it lacked detectable ATP-specific protein kinase activity in vitro. By kinetics during pulse-chase labeling experiments and by comparison of two-dimensional tryptic peptide maps, this protein is not an intermediate in gag precursor processing. One lymphoma cell line is described that resembles a nonproducer RadLV-transformant, synthesizing relatively large amounts of p36 in the absence of Pr66gag or p30 production. Several RadLV-induced lymphoma cell lines also produce p36, while it was not detectable in the radiation-induced lines tested. In addition, p36 was not produced by mouse or mink fibroblasts or cultured thymocyte cell lines infected with virus passaged from the RadLV-induced lymphomas. We conclude that p36 may represent a previously unrecognized transformation-related protein induced directly or indirectly by infection with RadLV
— id: 15256, year: 1984, vol: 160, page: 270, stat: Journal Article,

Murine leukemia virus sequences are encoded in the murine major histocompatibility complex
Meruelo D; Kornreich R; Rossomando A; Pampeno C; Mellor AL; Weiss EH; Flavell RA; Pellicer A
1984 Mar;81(6):1804-1808, Proceedings of the National Academy of Sciences of the United States of America
The studies reported here localize murine leukemia viral sequences to the TL region of the major histocompatibility complex, H-2. We examined a battery of 38 cosmids, isolated from two large genomic libraries constructed from C57BL/10 spleen DNA, that define 25 class I gene sequences. The viral probes used hybridized with only four cosmids, containing overlapping mouse sequences, that define four class I gene-related sequences in a region of 90 kilobases of DNA. The data show that two distinct viral envelope sequences are contained in the cluster. One of these sequences is situated with its 3' end next to the 3' end of a class I sequence. The other sequence, which does not contain the entire viral envelope, is proximal to the 3' end of a different class I sequence. Hybridization of the viral probes with the H-2 cosmid clones does not appear to be due to homology between viral and H-2 sequences. Rather, the viral sequences detected appear to be linked to or inserted amid class I genes. These findings may be significant in understanding molecular mechanisms involved in the generation of H-2 class I gene diversity
— id: 15257, year: 1984, vol: 81, page: 1804, stat: Journal Article,

Genetics of resistance to virus-induced leukemias
Meruelo D; Bach R
1983 ;40:107-188, Advances in cancer research
— id: 15260, year: 1983, vol: 40, page: 107, stat: Journal Article,

A new lymphocyte cell surface antigen, Ly-22.2, controlled by a locus on chromosome 4 and a second unlinked locus
Meruelo D; Offer M; Flieger N
1983 Feb;130(2):946-950, Journal of immunology
A serum raised by immunizing (B10.A(4R) X B10.HTT)F1 mice against A.AL lymphocytes detects a new antigenic determinant designated Ly-22.2. Ly-22.2 expression is under the control of two independently segregating genes, one of which maps to chromosome 4 adjacent to a locus affecting XenCSA expression. Ly-22.2 is present in varying amounts in all lymphoid organs, but appears to be expressed primarily on T lymphocytes. Ly-22.2 is not detectable in brain, kidney, lung, liver, or erythrocytes. Strain distribution studies show Ly-22.2 is present in all strains examined except B10-derived congenic strains. It is of interest that C57BL/6 and C57BL/10 mice differ in the expression of this antigen
— id: 15259, year: 1983, vol: 130, page: 946, stat: Journal Article,

Induction of leukemia by both fractionated x-irradiation and radiation leukemia virus involves loci in the chromosome 2 segment H-30-A
Meruelo D; Offer M; Rossomando A
1983 Jan;80(2):462-466, Proceedings of the National Academy of Sciences of the United States of America
A common link between the induction of leukemia by (i) fractionated doses of x-irradiation and (ii) radiation leukemia virus in mice may be established by the observation that the segment of chromosome 2 between the loci for the minor histocompatibility antigen H-30 and color coat agouti (H-30-A) includes distinct loci involved in susceptibility to leukemogenesis induced by factors i and ii
— id: 15261, year: 1983, vol: 80, page: 462, stat: Journal Article,

Association of endogenous viral loci with genes encoding murine histocompatibility and lymphocyte differentiation antigens
Meruelo D; Rossomando A; Offer M; Buxbaum J; Pellicer A
1983 Aug;80(16):5032-5036, Proceedings of the National Academy of Sciences of the United States of America
Several polymorphic DNA restriction endonuclease fragments hybridizing with xenotropic and ecotropic envelope virus probes map adjacent to minor histocompatibility and lymphocyte (H/Ly) antigen-encoding loci. Viral DNA restriction fragments are associated with Ly-17 on chromosome 1, H-30, H-3, and H-13 on chromosome 2, Ly-21 on chromosome 7, H-28 on chromosome 3, and H-38 (chromosomal location as yet undetermined). In each case no recombinant can be found between the H/Ly locus in question and the virus-related restriction fragment, suggesting that linkage is very tight. Although some viral loci map to locations where no H/Ly has yet been mapped, the frequency and tightness of linkage in the seven instances described, coupled with the large number of as yet unmapped H/Ly loci, suggests that the associations found are significant
— id: 15258, year: 1983, vol: 80, page: 5032, stat: Journal Article,

A new murine lymphocyte alloantigen, Ly-21.2, mapping to the seventh chromosome
Kennard J; Meruelo D
1982 Mar;15(3):239-250, Immunogenetics
Using a monoclonal antibody raised by fusing spleen cells from A/J mice, immunized with B10.A splenocytes and lymph-node cells, with a BALB/c myeloma, we have described a new surface alloantigen, Ly-21.2, Ly-21.2 is present in varying amounts in all lymphoid tissues, is not detectable in the brain, kidney, lung or erythrocytes, and is found in only trace amounts in the liver. Strain distribution studies showed that Ly-21.2 is present in all strains examined, including B10, except the A strain and segregation analysis of (A/J x B10) F2 mice showed that Ly-21.2 expression (1) is encoded by one gene and (2) is linked to albinism on chromosome 7. Studies performed on mice developing T-cell leukemia showed that, regardless of the etiologic agent, Ly-21.2 expression increases dramatically in mice with overt leukemia. In addition, preliminary studies suggest that expression of Ly-21.2 is linked to increased susceptibility of mice to Friend-virus-induced erythroleukemia
— id: 15264, year: 1982, vol: 15, page: 239, stat: Journal Article,

Precise mapping of the gene for Ly-21.2 on the 7th chromosome
Kennard J; Meruelo D
1982 Dec;9(6):389-395, Journal of immunogenetics
We have recently described a murine lymphocyte alloantigen, Ly-21.2, and showed that the gene which controls Ly-21.2 expression is linked to albinism on the 7th chromosome. In order to map the gene more precisely, we have studied the linkage of Ly-21.2 with two other genetic markers on the 7th chromosome, the electrophoretic variant of the beta chain of haemoglobin and glucose phosphate isomerase, in (A/J x C57BL/10)F2 mice. The results of these 3-point cross studies show that the gene encoding Ly-21.2 maps 27 units distal to albinism
— id: 15262, year: 1982, vol: 9, page: 389, stat: Journal Article,

Evidence for a major cluster of lymphocyte differentiation antigens on murine chromosome 2
Meruelo D; Offer M; Rossomando A
1982 Dec;79(23):7460-7464, Proceedings of the National Academy of Sciences of the United States of America
The region of chromosome 2 between H-13 and H-3 has been shown to contain loci coding for a variety of other alloantigens, including Ly-4 and the locus coding for beta 2-microglobulin. Herein we show that Ly-6 and Ly-11 are coded for by genes in a segment of chromosome 2 adjacent to the H-3-H-13 region and that this segment of chromosome also contains the tightly linked loci coding for antigens Ala-1, DAG, H9/25, H-30, Ly-8, and ThB. In addition, at least one locus (and probably more) affecting susceptibility to leukemia induction is found within this gene cluster
— id: 15263, year: 1982, vol: 79, page: 7460, stat: Journal Article,

H-2D CONTROL OF RADIATION LEUKEMIA-VIRUS INDUCED NEOPLASIA - EVIDENCE FOR INTERACTION OF VIRAL AND H-2 GENOMIC INFORMATION
Meruelo, D; Kramer, J; Rossomando, A; Kornreich, R
1982 ;163(2-4):311-311, Immunobiology
— id: 30342, year: 1982, vol: 163, page: 311, stat: Journal Article,

EVIDENCE FOR A MAJOR CLUSTER OF LYMPHOCYTE DIFFERENTIATION ANTIGENS ON MURINE CHROMOSOME .2. (MAPPING OF LY-11/LY-6/VIRAL INTEGRATION RADIATION LEUKEMIA)
Meruelo, D; Offer, M; Rossomando, A
1982 ;163(2-4):230-231, Immunobiology
— id: 30341, year: 1982, vol: 163, page: 230, stat: Journal Article,

IDENTIFICATION OF A UNIQUE PROTEIN INVOLVED IN H-2 LINKED RESISTANCE TO SPONTANEOUS AKR MURINE LEUKEMIA
Zalman, MA; Meruelo, D
1982 ;41(3):428-428, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 30481, year: 1982, vol: 41, page: 428, stat: Journal Article,

CELLULAR ANTIGENS OF VIRAL AND X-IRRADIATION INDUCED MURINE LYMPHOMAS RECOGNIZED BY MONOCLONAL-ANTIBODIES
BACH, RG; FLIEGER, N; MERUELO, D
1981 ;40(3):822-822, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 40252, year: 1981, vol: 40, page: 822, stat: Journal Article,

"120-A NEW THYMOCYTE CELL-SURFACE MARKER, MAPPING NEAR A MINOR HISTOCOMPATIBILITY LOCUS, CRITICALLY INVOLVED IN LEUKEMOGENESIS"
KENNARD, J; MERUELO, D
1981 ;40(3):998-998, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 40255, year: 1981, vol: 40, page: 998, stat: Journal Article,

H-2D control of radiation leukemia virus induced neoplasia: evidence for interaction of viral and H-2 genomic information
Meruelo D; Kramer J
1981 Dec;13(4):1858-1862, Transplantation proceedings
— id: 15265, year: 1981, vol: 13, page: 1858, stat: Journal Article,

Genetics of susceptibility for radiation-induced leukemia. Mapping of genes involved to chromosomes 1, 2, and 4, and implications for a viral etiology in the disease
Meruelo D; Offer M; Flieger N
1981 Oct 1;154(4):1201-1211, Journal of experimental medicine
Susceptibility to radiation-induced leukemia in (A/J x B10)F2 mice is encoded for by genes in chromosomes 1, 2, and 4. The loci involved in chromosomes 1 and 4 are close to or similar to xenotropic virus inducibility locus on chromosome 1 and a locus-affecting expression of xenotropic MuLV envelope-related cell surface antigens. Radiation-induced leukemia-1 (Ril-1) on chromosome 2 plays an overriding influence in susceptibility to the disease. This locus might encode ecotropic viral-associated genetic information or might contain cellular sequences with oncogenic potential. These findings are of interest in view of the importance of recombinant viruses to leukemogenesis. Furthermore, it is intriguing that Ril-1 is located in a chromosomal site rich in thymus differentiation-specific loci. An explanation for tissue-specific activation of endogenous viruses is that activation of the virus in question is dependent on differentiation-specific steps
— id: 15266, year: 1981, vol: 154, page: 1201, stat: Journal Article,

Ly 11.2: a cell surface antigen that may identify murine lymphocytes susceptible to transformation by oncogenic agents
Meruelo D; Paolino A; Flieger N; Offer M; Dworkin J
1981 Oct;40(12):2717-2720, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 15267, year: 1981, vol: 40, page: 2717, stat: Journal Article,

Effect of type I and type II interferons on murine thymocyte surface antigen expression: induction or selection?
Sonnenfeld G; Meruelo D; McDevitt HO; Merigan TC
1981 Jan 15;57(2):427-439, Cellular immunology
— id: 15268, year: 1981, vol: 57, page: 427, stat: Journal Article,

H-2D control of leukaemia susceptibility: mechanism and implications
Meruelo D
1980 Feb;7(1):81-90, Journal of immunogenetics
Genes in the D region of the murine major histocompatibility complex, H-2, confer resistance to radiation-induced leukaemia virus. H-2D gene control appears to ensue at a step subsequent to virus infection, since elimination of virus infected cells does not become apparent until 3--5 weeks after virus infection. Nonetheless, almost immediately after virus infection, expression of H-2D-coded antigens is markedly elevated on the surface of thymocytes from resistant (H-2Dd) but not susceptible mice (H-2Ds or H-2Dq). This increased H-2D antigen expression triggers a vigorous cell-mediated immune response which probably plays a key role in resistance to leukaemia via elimination of virus-infected cells. A hypothesis is put forth to explain the induction of increased sythesis and expression of H-2D antigens. This hypothesis postulates that the oncogenic segment of RadLV bears a close resemblance to H-2.4, the private specificity for H-2Dd, allowing it to integrate at or near the H-2Dd murine gene. Subsequent to integration, the rates of transcription and translation are altered with a resulting increase in cell surface antigen expression. Other possibilities are also discussed
— id: 15272, year: 1980, vol: 7, page: 81, stat: Journal Article,

The biological function of the major histocompatibility complex: hypotheses
Meruelo D; Edidin M
1980 ;9:231-253, Contemporary topics in immunobiology
— id: 15273, year: 1980, vol: 9, page: 231, stat: Journal Article,

In vivo or in vitro treatments with anti-I-J alloantisera abolish immunity to AKR leukemia
Meruelo D; Flieger N; Smith D; McDevitt HO
1980 Apr;77(4):2178-2182, Proceedings of the National Academy of Sciences of the United States of America
This paper provides evidence for the involvement of immune mechanisms in conferring resistance to a spontaneous AKR leukemia. It is shown that genes in the B, J, or E subregions of the H-2 complex confer resistance to a spontaneously arisen, tissue culture-adapted AKR thymoma, BW5147. A direct correlation is demonstrated between survival to injected BW5147 cells and humoral responsiveness in various hybrids obtained from crosses of AKR mice and C57BL/10 or C3H/DiSn derived congeneic strains differing at H-2. Cellular immunity appears to play no role in resistance to the proliferation of tumor cells. It is further established that development of effective humoral immunity depends on B cells and Ly-1+, 2-, 3- helper T-cells bearing the I-Jk phenotype. These findings seem directly applicable to the spontaneous disease, and results of studies using transformed cells from an overtly leukemic AKR mouse parallel those obtained using BW1547 cells
— id: 15271, year: 1980, vol: 77, page: 2178, stat: Journal Article,

Functional properties of Ly 11.2 lymphocytes: a role for these cells in leukemia?
Meruelo D; Paolino A; Flieger N; Dworkin J; Offer M; Hirayama N; Ovary Z
1980 Dec;125(6):2719-2726, Journal of immunology
Functional studies of lymphocyte subpopulations reveal that Ly 11.2, a newly defined T cell surface antigen, is present on prothymocytes and natural killer cells, but not on suppressor T cells for antigen-specific IgE antibody responses, Ly 1+, 2-, 3- helper T cells nor on tumor-specific cytotoxic effector cells. Changes in the expression of Ly 11.2 regularly accompany leukemogenesis and are quite distinct from changes of other cell surface antigens thus far observed. After intrathymic inoculation of radiation leukemia virus (RadLV), many more Ly 11.2-positive cells are found expressing viral antigens than cells expressing other cell surface phenotypes. In addition, after RadLV inoculation, significantly more Ly 11.2-positive cells can be found in the thymus of susceptible mice than in the thymus of resistant mice. The greater availability of permissive (Ly 11.2-positive) cells in susceptible vs resistant hosts at the time when infectious virus is present may account for the shorter latency period and high leukemia incidence of susceptible vs resistant mice
— id: 15270, year: 1980, vol: 125, page: 2719, stat: Journal Article,

Definition of a new T lymphocyte cell surface antigen, Ly 11.2
Meruelo D; Paolino A; Flieger N; Offer M
1980 Dec;125(6):2713-2718, Journal of immunology
The present communication defines a new cell surface antigen, Ly 11.2, which appears from its strain and tissue distribution to be distinct from all other previously defined normal or virally coded antigens or traits of the mouse. Lymphocytes of the T cell lineage, but not B cells or nonlymphoid cells, bear this marker. Although the locus coding for this antigen appears to be closely linked to the minor histocompatibility locus H-30 and to the locus coding for Ly 6.2, a chromosome assignment has not yet been made for my of these loci
— id: 15269, year: 1980, vol: 125, page: 2713, stat: Journal Article,

DEFINITION OF A NEW LYMPHOCYTE-T CELL-SURFACE ANTIGEN LY 10.2
Meruelo, D; Paolino, A; Flieger, N; Dworkin, J
1980 ;39(3):800-800, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 28038, year: 1980, vol: 39, page: 800, stat: Journal Article,

DEFINITION OF A NEW LYMPHOCYTE-T CELL-SURFACE ANTIGEN, LY-11.2
Meruelo, D; Paolino, A; Flieger, N; Offer, M
1980 ;125(6):2708-2713, Journal of immunology
— id: 27886, year: 1980, vol: 125, page: 2708, stat: Journal Article,

GENETIC-CONTROL OF LIVER CAMP LEVELS IN MICE
Lafuse, W; Meruelo, D; Edidin, M
1979 ;9(1):57-65, Immunogenetics
— id: 29718, year: 1979, vol: 9, page: 57, stat: Journal Article,

A role for elevated H-2 antigen expression in resistance to neoplasia caused by radiation-induced leukemia virus. Enhancement of effective tumor surveillance by killer lymphocytes
Meruelo D
1979 Apr 1;149(4):898-909, Journal of experimental medicine
Resistance to neoplasia caused by radiation-induced leukemia virus (RadLV) is mediated by gene(s) in the H-2D region of the major histocompatibility complex. The previous observation that rapid increases in cellular synthesis and cell-surface expression of H-2 antigens are detectable immediately after virus inoculation has suggested that altered expression of H-2 antigens may play a significant role in the mechanism(s) of host defense to virus infection. This concept is supported by the following observations. First, cell-mediated immunity against RadLV transformed or infected cells can be detected with ease when H-2-positive target cells are used in the cell-mediated lympholysis (CML) assay. (Although RadLV transformed cells obtained from overtly leukemic animals and maintained in tissue culture are H-2 negative, these cells can regain their H-2 phenotype by in vivo passage in normal animals. The H-2-negative cells are poor targets in a CML assay.) Second, resistant mice develop greater numbers of effectors when infected with RadLV than do susceptible mice. Third, injection of normal (uninfected) thymocytes into syngeneic recipients of resistant or susceptible H-2 type does not stimulate a CML response. However, injection of RadLV infected thymocytes from resistant mice produces a vigorous CMI response, and such thymocytes elicit the strongest response at a time when both H-2 and viral antigen expression is elevated. By contrast, injection of infected thymocytes from susceptible mice, which express viral antigens, but low levels of H-2 antigens, does not stimulate a CML reaction. These findings may explain the easier induction of leukemia found by many investigators when virus is inoculated into neonatal mice and the preferential thymus tropism of some oncogenic type-C RNA virus. Cells expressing very low levels of H-2, such as thymocytes, may serve as permissive targets for virus infection because they lack an important component (H-2 antigens) of the dual or altered recognition signal required to trigger a defensive host immune response
— id: 15274, year: 1979, vol: 149, page: 898, stat: Journal Article,

H-2 LINKED RESISTANCE TO SPONTANEOUS AKR LEUKEMIA - MECHANISM
Meruelo, D; Smith, D; Flieger, N; Mcdevitt, HO
1979 ;43(1):320-320, Journal of supramolecular structure
— id: 30137, year: 1979, vol: 43, page: 320, stat: Journal Article,

Expression of a single major histocompatibility complex locus controls the immune response to poly-L-(tyrosine, glutamic acid)-poly-DL-alanine-poly-L-lysine
Deak BD; Meruelo D; McDevitt HO
1978 Feb 1;147(2):599-604, Journal of experimental medicine
— id: 15276, year: 1978, vol: 147, page: 599, stat: Journal Article,

Recent studies on the role of the immune response in resistance to virus-induced leukemias and lymphomas
Meruelo D; McDevitt HO
1978 Oct;15(4):399-419, Seminars in hematology
— id: 15275, year: 1978, vol: 15, page: 399, stat: Journal Article,

Increased synthesis and expression of H-2 antigens on thymocytes as a result of radiation leukemia virus infection: a possible mechanism for H-2 linked control of virus-induced neoplasia
Meruelo D; Nimelstein SH; Jones PP; Lieberman M; McDevitt HO
1978 Feb 1;147(2):470-487, Journal of experimental medicine
Previous studies from this laboratory have mapped resistance and/or susceptibility to radiation-induced leukemia virus (RadLV)-induced neoplasia to the H-2D region. H-2 linked effects on virus replication can be detected subsequent to the initial virus infection, and clear-cut differences in numbers of virus infected thymus cells can be detected as early as 5 wk after RadLV inoculation. Rapid increases in cellular synthesis and cell surface expression of H-2 antigens are detectable immediately after virus inoculation. These changes have been studied by immunofluorescence, absorption, cell surface iodination followed by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis, and two dimensional gel electrophoretic analysis of internally labeled lymphocyte proteins. Expression of H-2K molecules is significantly increased in cells of susceptible and resistant animals. However, significant increases in expression of H-2D antigens occurs only on thymus cells from resistant strains (H-2Dd). Transformed cells of resistant and susceptible H-2 haplotypes adapted to tissue culture lack detectable H-2 antigens as determined by serological absorption studies. It is argued that altered expression of H-2 antigens plays a very significant role in the mechanism of host defense to virus infection
— id: 15277, year: 1978, vol: 147, page: 470, stat: Journal Article,

CHANGES IN LEVELS OF H-2 ANTIGENS AFTER RADLV INFECTION AFFECT CELLULAR IMMUNE-RESPONSES INVOLVED IN LEUKEMIA RESISTANCE
Meruelo, D
1978 ;37(6):1569-1569, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 29803, year: 1978, vol: 37, page: 1569, stat: Journal Article,

Genetic control of cell-mediated responsiveness to an AKR tumor-associated antigen: mapping of the locus involved to the I region of the H-2 complex
Meruelo D; Deak B; McDevitt HO
1977 Nov 1;146(5):1367-1379, Journal of experimental medicine
The role of H-2-linked genes in controlling resistance to murine leukemia viruses has been studied by measuring the cell-mediated immune response of F1 hybrid mice (between AKR and various C3H and C57BL/10 derived, H-2 congenic strains) to an AKR tumor cell line, BW5147. The studies have shown that the ability to generate a primary or secondary cell-mediated response to an AKR tumor cell antigenic determinant is under H-2 linked control. The locus determining CML responsiveness maps in the I-J subregion. Nonresponsiveness is associated with the H-2q/k and H-2b/k hybrid genotypes, whereas responsiveness is associated with the H-2k/k homozygous genotype. Nonresponsiveness may result from (a) dominant suppression; (b) recessive responsiveness; or (c) an alternate mechanism not yet understood. This type of control may be one of several H-2-associated mechanisms of defense against virus-induced neoplasms
— id: 15278, year: 1977, vol: 146, page: 1367, stat: Journal Article,

Genetic control of radiation leukemia virus-induced tumorigenesis. I. Role of the major murine histocompatibility complex, H-2
Meruelo D; Leiberman M; Ginzton N; Deak B; McDevitt HO
1977 Oct 1;146(4):1079-1087, Journal of experimental medicine
Resistance to radiation leukemia virus-induced leukemogenesis is associated with the H-2D region of the H-2 complex, or with closely linked loci. The H-2Dd allele confers resistance ot the disease, while the H-2D-Q and H-2Ds alleles are associated with susceptibility. It is not clear whether Ir genes, or an alternative mechanism are responsible for the observed H-2-linked resistance to the disease
— id: 15280, year: 1977, vol: 146, page: 1079, stat: Journal Article,

Genetic control of radiation leukemia virus-induced tumorigenesis II. Influence of Srlv-1, a locus not linked to H-2
Meruelo D; Lieberman M; Deak B; McDevitt HO
1977 Oct 1;146(4):1088-1095, Journal of experimental medicine
A single locus, tentatively denoted Srlv-1 (susceptibility to radiation leukemia virus [RadLV]-1), confers dominant susceptibility to RadLV-induced leukemogenesis. Srlv-1 is not linked to H-2, and appears to be distinct from Fv-1 and Fv-2. Preliminary data suggest that Srlv-1 affected virus proliferation. A striking feature of this system is that Srlv-1 overrides the protection afforded by the H2D-associated dominant resistance to RadLV-induced neoplasia
— id: 15279, year: 1977, vol: 146, page: 1088, stat: Journal Article,

An assay for adenyl cyclase based on a combination of sodium borate electrophoresis and paper chromatography
Meruelo D; Bromberg FG; Edidin M
1975 Jul;67(1):30-43, Analytical biochemistry
— id: 15282, year: 1975, vol: 67, page: 30, stat: Journal Article,

Association of mouse liver adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels with histocompatibility-2 genotype
Meruelo D; Edidin M
1975 Jul;72(7):2644-2648, Proceedings of the National Academy of Sciences of the United States of America
When the content of cyclic AMP (cAMP) was compared in livers of a series of congenic mouse strains differing at the H-2 locus, significant variation in concentration of cAMP per unit wet weight was found among strains, and also for animals of a given strain with increasing age. For a given age, from 8 to 22 weeks, cAMP levels in liver of H-2a and H-2b genotype animals were significantly higher than that in liver of H-2k type animals. This difference was seen whether the H-2 gene was on the genetic background of strain C57BL/10, C3H, or A. Levels of cAMP in livers of H-2d animals were between those of H-2a and H-2k animals
— id: 15281, year: 1975, vol: 72, page: 2644, stat: Journal Article,