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1.  RIT2, a neuron-specific small guanosine triphosphatase, is expressed in retinal neuronal cells and its promoter is modulated by the POU4 transcription factors 
Molecular Vision  2013;19:1371-1386.
Purpose
Ras-like without CAAX 2 (RIT2), a member of the Ras superfamily of small guanosine triphosphatases, is involved in regulating neuronal function. RIT2 is a unique member of the Ras family in that RIT2 is preferentially expressed in various neurons, including retinal neurons. The mechanisms that regulate RIT2 expression in neurons were studied.
Methods
Reverse transcription-quantitative PCR (RT-qPCR), immunohistochemistry, western blotting, bioinformatic prediction, electrophoretic mobility shift assay (EMSA), and cell transfection methods were used.
Results
With immunohistochemistry of the mouse retina, RIT2 protein was detected in the ganglion cell layer (GCL), inner plexiform layer, inner nuclear layer, and outer plexiform layer, with the strongest staining in the GCL and the inner plexiform layer. RT-qPCR combined with laser capture microdissection detected Rit2 messenger RNA in the GCL and the inner nuclear layer. Western blot analysis showed a large increase in the RIT2 protein in the retina during maturation from newborn to adult. Transient transfection identified the 1.3 kb upstream region of human RIT2 as capable of driving expression in neuronal cell lines. Based on the known expression pattern and biological activity, we hypothesized that POU4 family factors might modulate RIT2 expression in retinal ganglion cells (RGCs). Bioinformatic analyses predicted six POU4 factor-binding sites within the 1.3 kb human RIT2 promoter region. EMSA analyses showed binding of POU4 proteins to three of the six predicted sites. Cotransfection with expression vectors demonstrated that POU4 proteins can indeed modulate the human RIT2 promoter, and that ISL1, a LIM homeodomain factor, can further modulate the activity of the POU4 factors.
Conclusions
These studies confirm the expression of RIT2 in retinal neuronal cells, including RGCs, begin to reveal the mechanisms responsible for neuronal expression of RIT2, and suggest a role for the POU4 family factors in modulating RIT2 expression in RGCs.
PMCID: PMC3692409  PMID: 23805044
2.  Cryptococcus neoformans as a Model for Radioimmunotherapy of Infections 
There is an obvious and urgent need for novel approaches to treat infectious diseases. The use of monoclonal antibodies in therapy of infectious diseases is now experiencing renewed interest. During the last 5 years radioimmunotherapy (RIT), a modality previously developed only for cancer treatment, has been successfully adapted for the treatment of experimental fungal, bacterial, and viral infections. As our model organism for studying the efficacy, mechanisms, potential toxicity, and radioresistance to RIT, as well as for comparison of RIT with the existing antimicrobial therapies we have chosen the encapsulated yeast Cryptococcus neoformans (CN). The success of RIT approach in laboratory studies provides encouragement for feasibility of therapeutically targeting microbes with labeled antibodies. In addition, the creation of “panantibodies” for RIT which would recognize antigens shared by the whole class of pathogens such as fungi, for example, would facilitate the introduction of RIT into the clinic.
doi:10.1155/2011/830286
PMCID: PMC3124862  PMID: 21747848
3.  Continuous Requirement for the Clr4 Complex But Not RNAi for Centromeric Heterochromatin Assembly in Fission Yeast Harboring a Disrupted RITS Complex 
PLoS Genetics  2010;6(10):e1001174.
Formation of centromeric heterochromatin in fission yeast requires the combined action of chromatin modifying enzymes and small RNAs derived from centromeric transcripts. Positive feedback mechanisms that link the RNAi pathway and the Clr4/Suv39h1 histone H3K9 methyltransferase complex (Clr-C) result in requirements for H3K9 methylation for full siRNA production and for siRNA production to achieve full histone methylation. Nonetheless, it has been proposed that the Argonaute protein, Ago1, is the key initial trigger for heterochromatin assembly via its association with Dicer-independent “priRNAs.” The RITS complex physically links Ago1 and the H3-K9me binding protein Chp1. Here we exploit an assay for heterochromatin assembly in which loss of silencing by deletion of RNAi or Clr-C components can be reversed by re-introduction of the deleted gene. We showed previously that a mutant version of the RITS complex (Tas3WG) that biochemically separates Ago1 from Chp1 and Tas3 proteins permits maintenance of heterochromatin, but prevents its formation when Clr4 is removed and re-introduced. Here we show that the block occurs with mutants in Clr-C, but not mutants in the RNAi pathway. Thus, Clr-C components, but not RNAi factors, play a more critical role in assembly when the integrity of RITS is disrupted. Consistent with previous reports, cells lacking Clr-C components completely lack H3K9me2 on centromeric DNA repeats, whereas RNAi pathway mutants accumulate low levels of H3K9me2. Further supporting the existence of RNAi–independent mechanisms for establishment of centromeric heterochromatin, overexpression of clr4+ in clr4Δago1Δ cells results in some de novo H3K9me2 accumulation at centromeres. These findings and our observation that ago1Δ and dcr1Δ mutants display indistinguishable low levels of H3K9me2 (in contrast to a previous report) challenge the model that priRNAs trigger heterochromatin formation. Instead, our results indicate that RNAi cooperates with RNAi–independent factors in the assembly of heterochromatin.
Author Summary
Centromeres are the chromosomal regions that promote chromosome movement during cell division. They consist of repetitive DNA sequences that are packaged into heterochromatin. Disruption of centromeric heterochromatin leads to chromosome loss that can result in miscarriages and genetic disorders. We have sought to define the precise steps leading to heterochromatin assembly using fission yeast as the model system. To accomplish this we employed our novel Tas3WG mutant strain that can propagate preassembled heterochromatin but cannot support its de novo establishment. Current models suggest that small RNAs initiate heterochromatin assembly by targeting the RNAi machinery and subsequently the Clr-C chromatin-modifying complex to the centromere. Here, we demonstrate that transient depletion of components of the RNAi pathway that generate or bind small RNAs does not perturb heterochromatin assembly in our Tas3WG strain. Instead, transient depletion of the Clr-C complex blocks heterochromatin assembly, suggesting a critical role for continuous Clr-C activity during heterochromatin assembly in Tas3WG cells. We have directly tested whether Clr-C can target centromeres when expressed in cells deficient for RNAi and Clr-C. We find that RNAi–independent recruitment of Clr-C can occur and likely contributes to the critical initiating mechanisms of heterochromatin assembly.
doi:10.1371/journal.pgen.1001174
PMCID: PMC2965749  PMID: 21060862
4.  Radioimmunotherapy of Fungal Diseases: The Therapeutic Potential of Cytocidal Radiation Delivered by Antibody Targeting Fungal Cell Surface Antigens 
Radioimmunotherapy is the targeted delivery of cytocidal radiation to cells via specific antibody. Although mature for the treatment of cancer, RIT of infectious diseases is in pre-clinical development. However, as there is an obvious and urgent need for novel approaches to treat infectious diseases, RIT can provide us with a powerful approach to combat serious diseases, including invasive fungal infections. For example, RIT has proven more effective than standard amphotericin B for the treatment of experimental cryptococcosis. This review will discuss the concepts of RIT, its applications for infectious diseases, and the strides made to date to bring RIT of infectious diseases to fruition. Finally, we will discuss the potential of PAN-FUNGAL RIT, the targeting of conserved fungal cell surface antigens by RIT, as a treatment modality for fungi prior to the formal microbiological identification of the specific pathogen. In sum, RIT provides a mechanism for the targeted killing of drug susceptible or resistant fungi irrespective of the host immune status and may dramatically reduce the length of therapy currently required for many invasive fungal diseases.
doi:10.3389/fmicb.2011.00283
PMCID: PMC3257868  PMID: 22275913
Histoplasma capsulatum; Cryptococcus neoformans; Candida albicans; antibody; heat shock protein 60; beta-glucan; melanin
5.  Radioimmunotherapy of infectious diseases 
Seminars in nuclear medicine  2009;39(2):146-153.
The need for novel approaches to treat infectious diseases is obvious and urgent. This situation has renewed interest in using monoclonal antibodies (mAbs) in therapy of infectious diseases. During the last 5 years radioimmunotherapy (RIT), a modality developed for cancer treatment, has been successfully adapted for the treatment of experimental fungal (C. neoformans and H. capsulatum), bacterial (S. pneumoniae and B. anthracis) and viral (HIV-1) infections. RIT produced none or only transient hematological toxicity in experimental animals. Investigation of radiobiological mechanisms of RIT of infections showed that microbial cells are killed by both "direct hit" and "cross-fire" radiation. MAbs radiolabeled with either alpha- or beta-emitters stimulated apoptosis-like cell death, while only mAbs radiolabeled with alpha-emitter 213Bi also decreased the metabolic activity of microbial cells. The success of this approach in laboratory studies combined with earlier nuclear medicine experience on pre-clinical and clinical studies utilizing radiolabeled organism-specific antibodies for imaging of infections provides encouragement for feasibility of therapeutically targeting microbes with labeled antibodies. We envision that first the organism-specific mAbs will be radiolabeled with imaging radionuclides such as 99mTc or 111In to localize the sites of infection with SPECT followed by RIT with 188Re- or 90Y-labeled mAb, respectively. Also, immunoPET might be utilized for imaging of infection before treatment if such positron-emitting radionuclides as 86Y (matching pair for 90Y) or 124I (matching pair for 131I) are available. It might be possible to create a so-called “pan-antibody” which would recognize an antigen shared by a particular class of human pathogens such as fungi, for example. The availability of such antibodies would eliminate the necessity of having antibodies specific for each particular microorganism and would enormously enhance the development of RIT of infectious diseases.
doi:10.1053/j.semnuclmed.2008.10.007
PMCID: PMC2676918  PMID: 19187806
6.  A Novel Cyclic AMP-Dependent Epac-Rit Signaling Pathway Contributes to PACAP38-Mediated Neuronal Differentiation▿  
Molecular and Cellular Biology  2006;26(23):9136-9147.
Pituitary adenylate cyclase-activating polypeptide (PACAP38) stimulation results in the activation of Gsα protein-coupled receptors to regulate neuronal differentiation in a cyclic AMP (cAMP)-dependent manner. These pathways involve protein kinase A (PKA)-dependent processes, but a growing body of evidence indicates that cAMP also regulates cellular functions through PKA-independent signaling cascades. Here we show that the Rit small GTPase is regulated by PACAP38 in a cAMP-dependent but PKA-independent fashion. Rit activation results from stimulation of the cAMP-activated guanine nucleotide exchange factor Epac but does not appear to rely upon the activation of Rap GTPases, the accepted cellular Epac substrates. Although RNA interference studies demonstrated that Epac is required for PACAP38-mediated Rit activation, neither Epac1 nor Epac2 activates Rit directly, indicating that Epac signals to Rit through a novel mechanism in which Rap signaling is not essential. Loss-of-function analysis demonstrated that Rit makes an important contribution to PACAP38-mediated neuronal differentiation. Surprisingly, although Rit is required for sustained extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase signaling following nerve growth factor stimulation of pheochromocytoma 6 (PC6) cells, Rit silencing selectively suppressed PACAP38-elicited activation of p38, without obvious effects on ERK signaling in the same cells. Moreover, the ability of PACAP38 to stimulate CREB-dependent transcription and to promote neurite outgrowth was inhibited by Rit knockdown. Together, these studies identify an unsuspected connection between cAMP and Rit signaling pathways and imply that Rit can function downstream of Gsα/cAMP/Epac in a novel signal transduction pathway necessary for PACAP38-mediated neuronal differentiation and CREB signaling.
doi:10.1128/MCB.00332-06
PMCID: PMC1636835  PMID: 17000774
7.  Regulation of Iron Transport in Streptococcus pneumoniae by RitR, an Orphan Response Regulator 
Journal of Bacteriology  2004;186(23):8123-8136.
RitR (formerly RR489) is an orphan two-component signal transduction response regulator in Streptococcus pneumoniae that has been shown to be required for lung pathogenicity. In the present study, by using the rough strain R800, inactivation of the orphan response regulator gene ritR by allele replacement reduced pathogenicity in a cyclophosphamide-treated mouse lung model but not in a thigh model, suggesting a role for RitR in regulation of tissue-specific virulence factors. Analysis of changes in genome-wide transcript mRNA levels associated with the inactivation of ritR compared to wild-type cells was performed by the use of high-density DNA microarrays. Genes with a change in transcript abundance associated with inactivation of ritR included piuB, encoding an Fe permease subunit, and piuA, encoding an Fe carrier-binding protein. In addition, a dpr ortholog, encoding an H2O2 resistance protein that has been shown to reduce synthesis of reactive oxygen intermediates, was activated in the wild-type (ritR+) strain. Microarray experiments suggested that RitR represses Fe uptake in vitro by negatively regulating the Piu hemin-iron transport system. Footprinting experiments confirmed site-specific DNA-binding activity for RitR and identified three binding sites that partly overlap the +1 site for transcription initiation upstream of piuB. Transcripts belonging to other gene categories found to be differentially expressed in our array studies include those associated with (i) H2O2 resistance, (ii) repair of DNA damage, (iii) sugar transport and capsule biosynthesis, and (iv) two-component signal transduction elements. These observations suggest that RitR is an important response regulator whose primary role is to maintain iron homeostasis in S. pneumoniae. The name ritR (repressor of iron transport) for the orphan response regulator gene, rr489, is proposed.
doi:10.1128/JB.186.23.8123-8136.2004
PMCID: PMC529065  PMID: 15547286
8.  RNA Interference (RNAi)-Dependent and RNAi-Independent Association of the Chp1 Chromodomain Protein with Distinct Heterochromatic Loci in Fission Yeast 
Molecular and Cellular Biology  2005;25(6):2331-2346.
The establishment of centromeric heterochromatin in the fission yeast Schizosaccharomyces pombe is dependent on the RNA interference (RNAi) pathway. Dicer cleaves centromeric transcripts to produce short interfering RNAs (siRNAs) that actively recruit components of heterochromatin to centromeres. Both centromeric siRNAs and the heterochromatin component Chp1 are components of the RITS (RNA-induced initiation of transcriptional gene silencing) complex, and the association of RITS with centromeres is linked to Dicer activity. In turn, centromeric binding of RITS promotes Clr4-mediated methylation of histone H3 lysine 9 (K9), recruitment of Swi6, and formation of heterochromatin. Similar to centromeres, the mating type locus (Mat) is coated in K9-methylated histone H3 and is bound by Swi6. Here we report that Chp1 associates with the mating type locus and telomeres and that Chp1 localization to heterochromatin depends on its chromodomain and the C-terminal domain of the protein. Another protein component of the RITS complex, Tas3, also binds to Mat and telomeres. Tas3 interacts with Chp1 through the C-terminal domain of Chp1, and this interaction is necessary for Tas3 stability. Interestingly, in cells lacking the Argonaute (Ago1) protein component of the RITS complex, or lacking Dicer (and hence siRNAs), Chp1 and Tas3 can still bind to noncentromeric loci, although their association with centromeres is lost. Thus, Chp1 and Tas3 exist as an Ago1-independent subcomplex that associates with noncentromeric heterochromatin independently of the RNAi pathway.
doi:10.1128/MCB.25.6.2331-2346.2005
PMCID: PMC1061622  PMID: 15743828
9.  Antigen shedding may improve efficiencies for delivery of antibody-based anticancer agents in solid tumors 
Cancer research  2012;72(13):3143-3152.
Recombinant immunotoxins (RIT) are targeted anti-cancer agents that are composed of a targeting antibody fragment and a protein toxin fragment. SS1P is a RIT that targets mesothelin on the surface of cancer cells and is being evaluated in patients with mesothelioma. Mesothelin, like many other target antigens, is shed from the cell surface. However, whether antigen shedding positively or negatively affects the delivery of RIT remains unknown. In this study, we used experimental data with SS1P to develop a mathematical model that describes the relationship between tumor volume changes and the dose level of the administered RIT, while accounting for the potential effects of antigen shedding. We found that antigen shedding is a favorable biological process for targeted therapy of solid tumors. Shed antigens acted as a protective reservoir of RIT and buffered against the well-known binding site barrier effect, promoting a more uniform distribution of RIT in the tumor.
In addition, our model reproduced the decrease in tumor size upon RIT treatment in animal experiments. Our findings therefore can be used to study the delivery efficacy of RITs and also antibody drug conjugates currently in clinical trials.
doi:10.1158/0008-5472.CAN-11-3925
PMCID: PMC3408876  PMID: 22562466
mathematical model; antigen shedding; immunoconjugate delivery
10.  Rit Mutants Confirm Role of MEK/ERK Signaling in Neuronal Differentiation and Reveal Novel Par6 Interaction 
Biochimica et biophysica acta  2007;1773(12):1793-1800.
Rit is a novel member of the Ras superfamily of small GTP-binding proteins that regulates signaling pathways controlling cellular fate determination. Constitutively activated mutants of Rit induce terminal differentiation of pheochromocytoma (PC6) cells resulting in a sympathetic neuron-like phenotype characterized by the development of highly-branched neurites. Rit signaling has been found to activate several downstream pathways including MEK/ERK, p38 MAPK, Ral-specific guanine nucleotide exchange factors (GEFs), and Rit associates with the Par6 cell polarity machinery. In this study, a series of Rit effector loop mutants was generated to test the importance of these cellular targets to Rit-mediated neuronal differentiation. We find that Rit-mediated neuritogenesis is dependent upon MEK/ERK MAP kinase signaling but independent of RalGEF activation. In addition, in vivo binding studies identified a novel mechanism of Par6 interaction, suggesting that the cell polarity machinery may serve to spatially restrict Rit signaling.
doi:10.1016/j.bbamcr.2007.09.008
PMCID: PMC2175081  PMID: 17976838
Neuronal differentiation; PC12 cell; Rit; GTPase; Ras; ERK MAP kinase; Par6
11.  Radioimmunotherapy of infection with 213Bi-labeled antibodies 
Current radiopharmaceuticals  2008;1(3):234-239.
Bismuth-213 (213Bi) (physical half-life 46 min) is a beta-emitter (97%) and an alpha-emitter (3%) which decays to short lived alpha-emitter Polonium-213 and could therefore be used as an in vivo generator of alpha particles with the energy of around 8 MeV. 213Bi has been successfully used during the last decade in both clinical and pre-clinical work for radioimmunotherapy (RIT) of cancer with 213Bi-labeled monoclonal antibodies (mAbs). RIT has been proposed as a novel techonology for treatment of infectious diseases. 213Bi-labeled mAbs have been successfully used for treatment of experimental fungal, bacterial and viral infections with transient or none hematologic toxicity. The mechanisms of RIT of infection with 213Bi-labeled mAbs include “direct” killing of cells and induction of apoptosis. In vivo RIT results in decrease of inflammation in infected organs. Among the delivery vehicles for RIT of infection whole IgG1 mAbs seem to be the most suitable in terms of the highest uptake in the target organs and the lowest - in normal tissues. RIT with alpha-emitter 213Bi involves the application of established technology developed for the treatment of malignancies to infectious diseases. The development of RIT for infectious diseases is potentially easier than its application to tumor therapy given antigenic and tissue perfusion differences between sites of microbial infection and tumor infiltration. Nevertheless, considerable pre-clinical and clinical development work is likely to be required to learn how to use RIT for infection optimally.
doi:10.2174/1874471010801030234
PMCID: PMC2836599  PMID: 20228957
Bismuth-213; radioimmunotherapy; fungal infection; bacterial infection; viral infection; apoptosis; radiobiological mechanisms; inflammation
12.  Discrimination between initiation and elongation of protein biosynthesis in yeast: identity assured by a nucleotide modification in the initiator tRNA. 
Nucleic Acids Research  1993;21(24):5679-5683.
Cytoplasmic initiator tRNAs from plants and fungi possess an unique 2'-phosphoribosyl residue at position 64 of their sequence. In yeast tRNA(iMet), this modified nucleotide located in the T-stem of the tRNA is a 2'-1''-(beta-O-ribofuranosyl-5''-phosphoryl)-adenosine. The phosphoribosyl residue of this modified nucleoside was removed chemically by treatment involving periodate oxidation of tRNA(iMet) and regeneration of the 3'-terminal adenosine with ATP (CTP):tRNA nucleotidyl transferase. The role of phosphoribosylation at position 64 for interaction with elongation factor eEF-1 alpha and initiation factor 2 (eIF-2) was investigated in the homologous yeast system. Whereas the 5'-phosphoribosyl residue prevents the binding of Met-tRNA(iMet) to eEF-1 alpha, it does not influence the interaction with eIF-2. After removal of the ribosyl group, the demodified initiator tRNA showed binding to eEF-1 alpha, but no change was detected with respect to the interaction with the initiation factor eIF-2. This observation is interpreted to mean that a single modification of an eucaryotic initiator tRNA in yeast serves as a negative discriminant for eEF-1 alpha, thus preventing the initiator tRNA(iMet) from entering the elongation cycle of protein biosynthesis.
Images
PMCID: PMC310535  PMID: 8284215
13.  High-throughput decoding of anti-trypanosomal drug efficacy and resistance 
Nature  2012;482(7384):232-236.
Summary
The concept of specific chemotherapy was developed a century ago by Paul Ehrlich and others. Dyes and arsenical compounds that displayed selectivity against trypanosomes were central to this work 1,2, and the drugs that emerged remain in use for treating Human African Trypanosomiasis (HAT) 3. Ehrlich recognised the importance of understanding the mechanisms underlying selective drug action and resistance for the development of improved HAT therapies, but these mechanisms have remained largely mysterious. Here, we use all five current HAT drugs for genome-scale RNA interference (RNAi) target sequencing (RIT-seq) screens in Trypanosoma brucei, revealing the transporters, organelles, enzymes and metabolic pathways that function to facilitate anti-trypanosomal drug action. RIT-seq profiling identifies both known drug importers 4,5 and the only known pro-drug activator 6, and links more than fifty additional genes to drug action. A specific bloodstream stage invariant surface glycoprotein (ISG75) family mediates suramin uptake while the AP-1 adaptin complex, lysosomal proteases and major lysosomal transmembrane protein, as well as spermidine and N-acetylglucosamine biosynthesis all contribute to suramin action. Further screens link ubiquinone availability to nitro-drug action, plasma membrane P-type H+-ATPases to pentamidine action, and trypanothione and multiple putative kinases to melarsoprol action. We also demonstrate a major role for aquaglyceroporins in pentamidine and melarsoprol cross-resistance. These advances in our understanding of mechanisms of anti-trypanosomal drug efficacy and resistance will aid the rational design of new therapies and help to combat drug resistance, and provide unprecedented levels of molecular insight into the mode of action of anti-trypanosomal drugs.
doi:10.1038/nature10771
PMCID: PMC3303116  PMID: 22278056
DFMO; eflornithine; ISG75; nifurtimox; RNAi
14.  The Chp1-Tas3 core is a multifunctional platform critical for gene silencing by RITS 
Nature structural & molecular biology  2011;18(12):1351-1357.
RNA interference (RNAi) is critical for the assembly of heterochromatin at fission yeast centromeres. Central to this process is the RNA-induced Initiation of Transcriptional gene Silencing (RITS) complex, which physically anchors small non-coding RNAs to chromatin. RITS includes Ago1, the chromodomain protein Chp1, and Tas3, which bridges between Chp1 and Ago1. Chp1 is a large protein with, apart from its chromodomain, no recognizable domains. Here we describe how the structured C-terminal half of Chp1 binds the Tas3 N-terminal domain, revealing Chp1's tight embrace of Tas3. The structure also reveals a PIN domain at the C-terminal tip of Chp1 that controls subtelomeric transcripts through a post-transcriptional mechanism. We suggest that the Chp1-Tas3 complex provides a solid and versatile platform to recruit both RNAi-dependent and RNAi-independent gene-silencing pathways for locus-specific regulation of heterochromatin.
doi:10.1038/nsmb.2151
PMCID: PMC3230742  PMID: 22081013
15.  Rit Contributes to Nerve Growth Factor-Induced Neuronal Differentiation via Activation of B-Raf-Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase Cascades 
Molecular and Cellular Biology  2005;25(2):830-846.
Rit is one of the original members of a novel Ras GTPase subfamily that uses distinct effector pathways to transform NIH 3T3 cells and induce pheochromocytoma cell (PC6) differentiation. In this study, we find that stimulation of PC6 cells by growth factors, including nerve growth factor (NGF), results in rapid and prolonged Rit activation. Ectopic expression of active Rit promotes PC6 neurite outgrowth that is morphologically distinct from that promoted by oncogenic Ras (evidenced by increased neurite branching) and stimulates activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways. Furthermore, Rit-induced differentiation is dependent upon both MAP kinase cascades, since MEK inhibition blocked Rit-induced neurite outgrowth, while p38 blockade inhibited neurite elongation and branching but not neurite initiation. Surprisingly, while Rit was unable to stimulate ERK activity in NIH 3T3 cells, it potently activated ERK in PC6 cells. This cell type specificity is explained by the finding that Rit was unable to activate C-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf. Importantly, selective down-regulation of Rit gene expression in PC6 cells significantly altered NGF-dependent MAP kinase cascade responses, inhibiting both p38 and ERK kinase activation. Moreover, the ability of NGF to promote neuronal differentiation was attenuated by Rit knockdown. Thus, Rit is implicated in a novel pathway of neuronal development and regeneration by coupling specific trophic factor signals to sustained activation of the B-Raf/ERK and p38 MAP kinase cascades.
doi:10.1128/MCB.25.2.830-846.2005
PMCID: PMC543422  PMID: 15632082
16.  RNAi-Mediated Targeting of Heterochromatin by the RITS Complex 
Science (New York, N.Y.)  2004;303(5658):672-676.
RNA interference (RNAi) is a widespread silencing mechanism that acts at both the posttranscriptional and transcriptional levels. Here, we describe the purification of an RNAi effector complex termed RITS (RNA-induced initiation of transcriptional gene silencing) that is required for heterochromatin assembly in fission yeast. The RITS complex contains Ago1 (the fission yeast Argonaute homolog), Chp1 (a heterochromatin-associated chromodomain protein), and Tas3 (a novel protein). In addition, the complex contains small RNAs that require the Dicer ribonuclease for their production. These small RNAs are homologous to centromeric repeats and are required for the localization of RITS to heterochromatic domains. The results suggest a mechanism for the role of the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci.
doi:10.1126/science.1093686
PMCID: PMC3244756  PMID: 14704433
17.  Direct evidence of nuclear Argonaute distribution during transcriptional silencing links the actin cytoskeleton to nuclear RNAi machinery in human cells 
Nucleic Acids Research  2011;40(4):1579-1595.
Mammalian RNAi machinery facilitating transcriptional gene silencing (TGS) is the RNA-induced transcriptional gene silencing-like (RITS-like) complex, comprising of Argonaute (Ago) and small interfering RNA (siRNA) components. We have previously demonstrated promoter-targeted siRNA induce TGS in human immunodeficiency virus type-1 (HIV-1) and simian immunodeficiency virus (SIV), which profoundly suppresses retrovirus replication via heterochromatin formation and histone methylation. Here, we examine subcellular co-localization of Ago proteins with promoter-targeted siRNAs during TGS of SIV and HIV-1 infection. Analysis of retrovirus-infected cells revealed Ago1 co-localized with siRNA in the nucleus, while Ago2 co-localized with siRNA in the inner nuclear envelope. Mismatched and scrambled siRNAs were observed in the cytoplasm, indicating sequence specificity. This is the first report directly visualizing nuclear compartment distribution of Ago-associated siRNA and further reveals a novel nuclear trafficking mechanism for RITS-like components involving the actin cytoskeleton. These results establish a model for elucidating mammalian TGS and suggest a fundamental mechanism underlying nuclear delivery of RITS-like components.
doi:10.1093/nar/gkr891
PMCID: PMC3287199  PMID: 22064859
18.  A Comparative Analysis of Prognostic Factor Models for Follicular Lymphoma based on a Phase III Trial of CHOP-Rituximab vs CHOP + 131Iodine-Tositumomab 
Purpose
There is currently no consensus on optimal front-line therapy for patients with follicular lymphomas (FL). We analyzed a Phase III randomized intergroup trial comparing 6 cycles of CHOP-R with six cycles of CHOP followed by iodine I-131 tositumomab radioimmunotherapy (RIT) to assess whether any subsets benefitted more from one treatment or the other, and to compare three prognostic models.
Experimental Design
We conducted univariate and multivariate Cox regression analyses of 532 patients enrolled on this trial and compared the prognostic value of the FLIPI, FLIPI2, and LDH + β2M models.
Results
Outcomes were excellent, but not statistically different between the two study arms (5 year PFS of 60% with CHOP-R and 66% with CHOP-RIT [p =0.11]; 5-yr OS of 92% with CHOP-R and 86% with CHOP-RIT [p=0.08]; overall response rate of 84% for both arms). The only factor found to potentially predict the impact of treatment was serum β2 microglobulin (β2M); among patients with normal β2M, CHOP-RIT patients had better PFS compared to CHOP-R patients, whereas among patients with high serum β2M, PFS by arm was similar (interaction p-value=.02).
Conclusions
All three prognostic models (FLIPI, FLIPI2, LDH + β2M) predicted both PFS and OS well, though the LDH + β2M model is easiest to apply and identified an especially poor risk subset. In an exploratory analysis using the latter model, there was a statistically significant trend suggesting that low risk patients had superior observed PFS if treated with CHOP-RIT, whereas high risk patients had a better PFS with CHOP-R.
doi:10.1158/1078-0432.CCR-13-1120
PMCID: PMC3872052  PMID: 24130072
Follicular Lymphoma; Prognostic Factors; Subset Analysis; β2 microglobulin; Front-Line Therapy
19.  Pentostatin Plus Cyclophosphamide Safely and Effectively Prevents Immunotoxin Immunogenicity in Murine Hosts 
Purpose
The success of immunotoxin therapy of cancer is limited by host production of neutralizing antibodies, which are directed toward the Pseudomonas exotoxin A (PE) component. In this proof-of-principle study using a well-established murine model, we hypothesized that a newly developed immune depletion regimen consisting of pentostatin plus cyclophosphamide would abrogate anti-immunotoxin reactivity.
Experimental Design
BALB/c hosts were injected weekly with recombinant immunotoxin (RIT) SS1P, which is an anti-mesothelin Fv antibody fragment genetically fused to a 38 kDa portion of PE, and has been evaluated in clinical trials. Experimental cohorts received induction chemotherapy consisting of pentostatin (P) plus cyclophosphamide (C) prior to initial RIT exposure; some cohorts received further maintenance PC therapy of varying intensity just prior to each weekly RIT challenge. Cohorts were monitored for T, B, and myeloid cell depletion and for total anti-SS1P antibody (Ab) formation.
Results
Controls uniformly developed anti-SS1P Ab after the third RIT exposure. Induction PC therapy reduced the frequency of hosts with anti-SS1P Ab. Abrogation of antibody generation was improved by maintenance PC therapy: nearly 100% of recipients of intensive PC maintenance were free of anti-SS1P Ab after 9 weekly RIT doses. The most effective PC regimen yielded the greatest degree of host B cell depletion, moderate T cell depletion, and minimal myeloid cell depletion.
Conclusions
Induction and maintenance PC chemotherapy safely prevented anti-immunotoxin antibody formation with uniform efficacy. These data suggest that immunotoxin therapy might be used in combination with pentostatin plus cyclophosphamide chemotherapy to improve the targeted therapy of cancer.
doi:10.1158/1078-0432.CCR-11-0493
PMCID: PMC3107891  PMID: 21521777
adjuvant chemotherapy; combination chemotherapy; pentostatin; cyclophosphamide; immunotoxin
20.  Radioimmunotherapy based conditioning regimens for stem cell transplantation 
Seminars in hematology  2008;45(2):118-125.
Radioimmunotherapy (RIT) combines the mechanism of action and targeting capability of monoclonal antibodies with the tumoricidal effect of radiation and has shown promising results in the treatment of various hematologic malignancies. Based on RIT’s efficacy and safety profile, many investigators have evaluated its use in transplant conditioning regimens with the goal of improving long-term disease control with limited toxicity. In lymphoma, two basic transplant approaches targeting CD20 have emerged: 1. Myeloablative doses of RIT with or without chemotherapy, and 2. Standard non-myeloablative doses of RIT combined with high-dose chemotherapy. Myeloablative RIT has been shown to be feasible and efficacious using escalated doses of I-131-Tositumomab (Bexxar), Y-90-ibritumomab tiuxetan (Zevalin), and I-131-rituximab with or without chemotherapy followed by autologous stem cell transplant (ASCT). The second approach predominantly has used standard doses of Y-90-ibritumomab tiuxetan or I-131 Tositumomab plus BEAM chemotherapy followed ASCT. RIT targeting CD-45, CD-33 and CD-66 prior to allogeneic transplantation has also been evaluated for the treatment of acute leukemia. Overall RIT-based transplant conditioning for lymphoma and leukemia has been shown to be safe, effective, and feasible with ongoing randomized trials currently underway to definitively establish its place in the treatment of hematologic malignancies.
doi:10.1053/j.seminhematol.2008.02.002
PMCID: PMC2460561  PMID: 18381107
Radioimmunotherapy; stem cell transplantation; CD20; CD45; I-131; Y-90
21.  A Comparison Between Radioimmunotherapy and Hyperthermic Intraperitoneal Chemotherapy for the Treatment of Peritoneal Carcinomatosis of Colonic Origin in Rats 
Annals of Surgical Oncology  2007;14(11):3274-3282.
Background
Cytoreductive surgery (CS) followed by heated intraperitoneal chemotherapy (HIPEC) is considered the standard of care for the treatment of patients with peritoneal carcinomatosis (PC) of colorectal cancer (CRC). These surgical procedures result in a median survival of 2 years at the cost of considerable morbidity and mortality. In preclinical studies, radioimmunotherapy (RIT) improved survival after CS in a model of induced PC of colonic origin.
In the present studies we aimed to compare the efficacy and toxicity of CS followed by adjuvant RIT in experimental PC to the standard of care, HIPEC.
Methods
PC was induced by intraperitoneal inoculation of CC-531 colon carcinoma cells in three groups of Wag/Rij rats. Treatment comprised CS only, CS + RIT or CS + HIPEC, immediately after surgery. RIT consisted of intraperitoneal administration of 74 MBq Lutetium-177 labeled MG1. HIPEC was performed by a closed abdomen perfusion technique using mitomycin C (16 mg/L during 60 minutes). The primary endpoint was survival.
Results
CS only or combined with RIT was well tolerated. Rats receiving CS + HIPEC were lethargic, suffered from diarrhea, and lost significantly more weight in the first postoperative week. Median survival of rats treated with CS + RIT was significantly longer than after CS alone (97 and 57 days, respectively, P < .004), whereas survival after CS + HIPEC or CS alone were not significantly different (76 and 57 days, respectively, P = .17).
Conclusion
Survival after CS was significantly improved by RIT with Lutetium-177-MG1 in rats with PC of colorectal origin. Adjuvant HIPEC did not improve survival and was more toxic than adjuvant RIT.
doi:10.1245/s10434-007-9509-2
PMCID: PMC2039838  PMID: 17653591
Radioimmunotherapy; Cytoreductive surgery; Heated intraperitoneal chemotherapy; Peritoneal carcinomatosis; Colon cancer; Adjuvant
22.  An evolutionarily conserved Rit GTPase–p38 MAPK signaling pathway mediates oxidative stress resistance 
Molecular Biology of the Cell  2011;22(17):3231-3241.
Rit knockout mice and D-Ric null Drosophila were used to identify the Rit/RIC subfamily of Ras-related GTPases as regulators of an evolutionarily conserved, p38-dependent signaling cascade that functions as a survival mechanism for cells in response to reactive oxygen species exposure.
Ras-related small GTP-binding proteins control a wide range of cellular processes by regulating a variety of effector pathways, including prominent roles in the control of mitogen-activated protein kinase (MAPK) cascades. Although the regulatory role(s) for many Ras family GTPases are well established, the physiological function for the Rit/Rin subfamily has been lacking. Here, using both knockout mice and Drosophila models, we demonstrate an evolutionarily conserved role for Rit subfamily GTPases (mammalian Rit and Rin, and the Drosophila RIC homologue) in governing survival in response to oxidative stress. Primary embryonic fibroblasts derived from Rit knockout mice display increased apoptosis and selective disruption of MAPK signaling following reactive oxygen species (ROS) exposure but not in response to endoplasmic reticulum stress or DNA damage. These deficits include a reduction in ROS-mediated stimulation of a p38-MK2-HSP27 signaling cascade that controls Akt activation, directing Bad phosphorylation to promote cell survival. Furthermore, D-RIC null flies display increased susceptibility to environmental stresses and reduced stress-dependent p38 signaling, extending the Rit-p38 survival pathway to Drosophila. Together, our studies establish the Rit GTPases as critical regulators of an evolutionarily conserved, p38 MAPK–dependent signaling cascade that functions as an important survival mechanism for cells in response to oxidative stress.
doi:10.1091/mbc.E11-05-0400
PMCID: PMC3164468  PMID: 21737674
23.  RNIE: genome-wide prediction of bacterial intrinsic terminators 
Nucleic Acids Research  2011;39(14):5845-5852.
Bacterial Rho-independent terminators (RITs) are important genomic landmarks involved in gene regulation and terminating gene expression. In this investigation we present RNIE, a probabilistic approach for predicting RITs. The method is based upon covariance models which have been known for many years to be the most accurate computational tools for predicting homology in structural non-coding RNAs. We show that RNIE has superior performance in model species from a spectrum of bacterial phyla. Further analysis of species where a low number of RITs were predicted revealed a highly conserved structural sequence motif enriched near the genic termini of the pathogenic Actinobacteria, Mycobacterium tuberculosis. This motif, together with classical RITs, account for up to 90% of all the significantly structured regions from the termini of M. tuberculosis genic elements. The software, predictions and alignments described below are available from http://github.com/ppgardne/RNIE.
doi:10.1093/nar/gkr168
PMCID: PMC3152330  PMID: 21478170
24.  Chp1-Tas3 Interaction Is Required To Recruit RITS to Fission Yeast Centromeres and for Maintenance of Centromeric Heterochromatin▿ † 
Molecular and Cellular Biology  2008;28(7):2154-2166.
The maintenance of centromeric heterochromatin in fission yeast relies on the RNA interference-dependent complexes RITS (RNA-induced transcriptional silencing complex) and RDRC (RNA-directed RNA polymerase complex), which cooperate in a positive feedback loop to recruit high levels of histone H3 K9 methyltransferase activity to centromeres and to promote the assembly and maintenance of centromeric heterochromatin. However, it is unclear how these complexes are targeted to chromatin. RITS comprises Chp1, which binds K9-methylated histone H3; Ago1, which binds short interfering (siRNAs); the adaptor protein Tas3, which links Ago1 to Chp1; and centromeric siRNAs. We have generated mutants in RITS to determine the contribution of the two potential chromatin-targeting proteins Chp1 and Ago1 to the centromeric recruitment of RITS. Mutations in Tas3 that disrupt Ago1 binding are permissive for RITS recruitment and maintain centromeric heterochromatin, but the role of Tas3's interaction with Chp1 is unknown. Here, we define the Chp1 interaction domain of Tas3. A strain expressing a tas3 mutant that cannot bind Chp1 (Tas3Δ10-24) failed to maintain centromeric heterochromatin, with a loss of centromeric siRNAs, a failure to recruit RITS and RDRC to centromeres, and high levels of chromosome loss. These findings suggest a pivotal role for Chp1 and its association with Tas3 for the recruitment of RITS, RDRC, and histone H3 K9 methyltransferase activity to centromeres.
doi:10.1128/MCB.01637-07
PMCID: PMC2268443  PMID: 18212052
25.  Dosimetric evaluation and radioimmunotherapy of anti-tumour multivalent Fab́ fragments 
British Journal of Cancer  1999;81(6):972-980.
We have been investigating the use of cross-linked divalent (DFM) and trivalent (TFM) versions of the anti-carcinoembryonic antigen (CEA) monoclonal antibody A5B7 as possible alternatives to the parent forms (IgG and F(ab́)2) which have been used previously in clinical radioimmunotherapy (RIT) studies in colorectal carcinoma. Comparative biodistribution studies of similar sized DFM and F(ab́)2 and TFM and IgG, radiolabelled with both 131I and 90Y have been described previously using the human colorectal tumour LS174T nude mouse xenograft model (Casey et al (1996) Br J Cancer 74: 1397–1405). In this study quantitative estimates of radiation distribution and RIT in the xenograft model provided more insight into selecting the most suitable combination for future RIT. Radiation doses were significantly higher in all tissues when antibodies were labelled with 90Y. Major contributing organs were the kidneys, liver and spleen. The extremely high absorbed dose to the kidneys on injection of 90Y-labelled DFM and F(ab́)2 as a result of accumulation of the radiometal would result in extremely high toxicity. These combinations are clearly unsuitable for RIT. Cumulative dose of 90Y-TFM to the kidney was 3 times lower than the divalent forms but still twice as high as for 90Y-IgG. TFM clears faster from the blood than IgG, producing higher tumour to blood ratios. Therefore when considering only the tumour to blood ratios of the total absorbed dose, the data suggests that TFM would be the most suitable candidate. However, when corrected for equitoxic blood levels, doses to normal tissues for TFM were approximately twice the level of IgG, producing a two-fold increase in the overall tumour to normal tissue ratio. In addition RIT revealed that for a similar level of toxicity and half the administered activity, 90Y-IgG produced a greater therapeutic response. This suggests that the most promising A5B7 antibody form with the radionuclide 90Y may be IgG. Dosimetry analysis revealed that the tumour to normal tissue ratios were greater for all 131I-labelled antibodies. This suggests that 131I may be a more suitable radionuclide for RIT, in terms of lower toxicity to normal tissues. The highest tumour to blood dose and tumour to normal tissue ratio at equitoxic blood levels was 131I-labelled DFM, suggesting that 131I-DFM may be best combination of antibody and radionuclide for A5B7. The dosimetry estimates were in agreement with RIT results in that twice the activity of 131I-DFM must be administered to produce a similar therapeutic effect as 131I-TFM. The toxicity in this therapy experiment was minimal and further experiments at higher doses are required to observe if there would be any advantage of a higher initial dose rate for 131I-DFM. © 1999 Cancer Research Campaign
doi:10.1038/sj.bjc.6690795
PMCID: PMC2362943  PMID: 10576653
dosimetry; radioimmunotherapy; tumour targeting; divalent Fab́; tri-valent Fab́

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