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Occult metastases are a major cause of cancer mortality, even among patients undergoing curative resection. Therefore, practical strategies to target the growth and persistence of already established metastases would provide an important... more
Occult metastases are a major cause of cancer mortality, even among patients undergoing curative resection. Therefore, practical strategies to target the growth and persistence of already established metastases would provide an important advance in cancer treatment. Here, we assessed the potential of protein therapy using a cell permeable NM23-H1 metastasis suppressor protein. Hydrophobic transduction domains developed from a screen of 1,500 signaling peptide sequences enhanced the uptake of the NM23 protein by cultured cells and systemic delivery to animal tissues. The cell-permeable (CP)-NM23 inhibited metastasis-associated phenotypes in tumor cell lines, blocked the establishment of lung metastases, and cleared already established pulmonary metastases, significantly prolonging the survival of tumor-bearing animals. Therefore, these results establish the potential use of cell-permeable metastasis suppressors as adjuvant therapy against disseminated cancers. Cancer Res; 71(23); 7216–25. ©2011 AACR.
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Research Interests: Molecular Biology, Kinetics, Biology, Medicine, Gene expression, and 13 moreHumans, Animals, Actin, Rats, Transfection, Angiotensin II, Recombinant Proteins, Aorta, Biochemistry and cell biology, Gene Expression Regulation, Enzyme Induction, Vascular Smooth Muscle, and Medical biochemistry and metabolomics
Purpose: Gastric cancer is a leading cause of cancer death worldwide. Limited therapeutic options highlight the need to understand themolecular changes responsible for the disease and to develop therapies based on this understanding. The... more
Purpose: Gastric cancer is a leading cause of cancer death worldwide. Limited therapeutic options highlight the need to understand themolecular changes responsible for the disease and to develop therapies based on this understanding. The goal of this studywas to develop cell-permeable (CP-) forms of the RUNTrelated transcription factor 3, RUNX3–a candidate tumor suppressor implicated in gastric and other epithelial cancers–to study the therapeutic potential of RUNX3 in the treatment of gastric cancer. Experimental Design: We developed novel macromolecule transduction domains (MTD) which were tested for the ability to promote protein uptake by mammalian cells and tissues and used to deliver of biologically active RUNX3 into human gastric cancer cells. The therapeutic potential CP-RUNX3 was tested in the NCI-N87 human tumor xenograft animal model. Results:RUNX3 fusion proteins, HM57R andHM85R, containing hydrophobicMTDs enter gastric cancer cells and suppress cell phenotypes (e.g., ce...
Earl RuleyLlanes, Qing Lin, Kristen L. Hoek, Wasif N. Khan and H. Shang Cao, Gianluca Carlesso, Anna B. Osipovich, Joanhttp://www.jimmunol.org/content/181/1/476J Immunol€2008; 181:476-484;... more
Earl RuleyLlanes, Qing Lin, Kristen L. Hoek, Wasif N. Khan and H. Shang Cao, Gianluca Carlesso, Anna B. Osipovich, Joanhttp://www.jimmunol.org/content/181/1/476J Immunol€2008; 181:476-484; ;Referenceshttp://www.jimmunol.org/content/181/1/476.full#ref-list-1This article cites 50 articles, 14 of which you can access for free at: Subscriptionshttp://jimmunol.org/subscriptionsInformation about subscribing to The Journal of Immunology is online at: Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright permission requests at: Email Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts when new articles cite this article. Sign up at:
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Research article Gene trap mutagenesis of hnRNP A2/B1: a cryptic 3 ' splice site in the neomycin resistance gene allows continued expression of the disrupted cellular gene
Protein arginine N-methyltransferases have been implicated in a variety of processes, including cell proliferation, signal transduction, and protein trafficking. In this study, we have characterized essentially a null mutation induced by... more
Protein arginine N-methyltransferases have been implicated in a variety of processes, including cell proliferation, signal transduction, and protein trafficking. In this study, we have characterized essentially a null mutation induced by insertion of the U3 Geo gene trap retrovirus into the ...
Research Interests: Cellular Biology, Biological Sciences, Pregnancy, Mutation, Mice, and 15 moreMethylation, Female, Animals, Central Nervous System, Blastocyst, Cell Viability, Enzyme, Embryonic Development, Amino Acid Sequence, Base Sequence, Cell Survival, Fetal death, ES cell, Molecular Sequence Data, and Medical and Health Sciences
Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by mitochondrial dysfunction, Lewy body formation, and loss of dopaminergic neurons. Parkin, an E3 ubiquitin ligase, is thought to inhibit PD progression... more
Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by mitochondrial dysfunction, Lewy body formation, and loss of dopaminergic neurons. Parkin, an E3 ubiquitin ligase, is thought to inhibit PD progression by removing damaged mitochondria and suppressing the accumulation of α-synuclein and other protein aggregates. The present study describes a protein-based therapy for PD enabled by the development of a cell-permeable Parkin protein (iCP-Parkin) with enhanced solubility and optimized intracellular delivery. iCP-Parkin recovered damaged mitochondria by promoting mitophagy and mitochondrial biogenesis and suppressed toxic accumulations of α-synuclein in cells and animals. Last, iCP-Parkin prevented and reversed declines in tyrosine hydroxylase and dopamine expression concomitant with improved motor function induced by mitochondrial poisons or enforced α-synuclein expression. These results point to common, therapeutically tractable features in PD pathoph...
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PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) by neuronal differentiation and partial growth arrest. Mouse c-myc and adenovirus E1A genes were introduced into PC12 cells to study the influence of these nuclear... more
PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) by neuronal differentiation and partial growth arrest. Mouse c-myc and adenovirus E1A genes were introduced into PC12 cells to study the influence of these nuclear oncogenes on neuronal differentiation. Expression of myc or E1A blocked morphological differentiation and caused NGF to stimulate rather than inhibit cell proliferation. NGF binding to cell surface receptors and ornithine decarboxylase induction were similar in myc- and E1A-expressing clones compared with wild-type PC12 cells, suggesting that changes in the cellular response to NGF were at a post-receptor level. These results illustrate that NGF can promote either growth or differentiation of PC12 cells, and that myc or E1A alter the phenotypic responses to growth factors and hormones.
Research Interests: Life Sciences, DNA replication, Cell Division, Cell Differentiation, Oncogene, and 12 moreHealth Science, Pheochromocytoma, Enzyme, Clinical Sciences, Neuronal Differentiation, Oncogenes, Cell Proliferation, Nerve Growth Factor, Growth Factor, Cell Surface Markers, Enzyme Induction, and Ornithine decarboxylase
The specificity of gene expression in embryonic stem (ES) cells was analyzed both under in vitro culture conditions and during early embryogenesis. ES cells were infected with U3 beta geo, a U3 gene trap retrovirus that contains coding... more
The specificity of gene expression in embryonic stem (ES) cells was analyzed both under in vitro culture conditions and during early embryogenesis. ES cells were infected with U3 beta geo, a U3 gene trap retrovirus that contains coding sequences for a beta-galactosidase-...
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Cell-permeant Cre DNA site-specific recombinases provide an easily controlled means to regulate gene structure and function in living cells. Since recombination provides a stable and unambiguous record of protein uptake, the enzyme may... more
Cell-permeant Cre DNA site-specific recombinases provide an easily controlled means to regulate gene structure and function in living cells. Since recombination provides a stable and unambiguous record of protein uptake, the enzyme may also be used for quantitative studies of cis- and trans-acting factors that influence the delivery of proteins into cells. In the present study, 11 recombinant fusion proteins were analyzed to characterize sequences and conditions that affect protein uptake and/or activity and to develop more active cell-permeant enzymes. We report that the native enzyme has a low, but intrinsic ability to enter cells. The most active Cre proteins tested contained either an N-terminal 6xHis tag and a nuclear localization sequence from SV40 large T antigen (HNC) or the HIV Tat transduction sequence and a C-terminal 6xHis tag (TCH6). The NLS and 6xHis elements separately enhanced the delivery of the HNC protein into cells; moreover, transduction sequences from fibroblas...
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Tagged sequence mutagenesis is a process for constructing libraries of sequenced insertion mutations in embryonic stem cells that can be transmitted into the mouse germline. To better predict the functional consequences of gene entrapment... more
Tagged sequence mutagenesis is a process for constructing libraries of sequenced insertion mutations in embryonic stem cells that can be transmitted into the mouse germline. To better predict the functional consequences of gene entrapment on cellular gene expression, the present study characterized the effects of a U3Neo gene trap retrovirus inserted into an intron of the hnRNP A2/B1 gene. The mutation was selected for analysis because it occurred in a highly expressed gene and yet did not produce obvious phenotypes following germline transmission. Sequences flanking the integrated gene trap vector in 1B4 cells were used to isolate a full-length cDNA whose predicted amino acid sequence is identical to the human A2 protein at all but one of 341 amino acid residues. hnRNP A2/B1 transcripts extending into the provirus utilize a cryptic 3' splice site located 28 nucleotides downstream of the neomycin phosphotransferase start codon. The inserted Neo sequence and proviral poly(A) site...
Research Interests: Polyadenylation, Gene expression, Biological Sciences, Humans, Mutagenesis, and 14 moreMice, Animals, Embryonic Stem Cell, Microbial genetic and drug resistance, Molecular cloning, Amino Acid Sequence, Base Sequence, Full Length Movies, Nucleotides, Gene Expression Regulation, Molecular Sequence Data, virus integration, Medical and Health Sciences, and Neoplastic Stem Cells
Studies of mammalian gene function are hampered by temporal limitations in which phenotypes occurring at one stage of development interfere with analysis at later stages. Moreover, phenotypes resulting from altered gene activity include... more
Studies of mammalian gene function are hampered by temporal limitations in which phenotypes occurring at one stage of development interfere with analysis at later stages. Moreover, phenotypes resulting from altered gene activity include both direct and indirect effects that may be difficult to distinguish. In the present study, recombinant fusion proteins bearing the 12 amino acid membrane translocation sequence (MTS) from the Kaposi fibroblast growth factor (FGF-4) were used to transduce enzymatically active Cre proteins directly into mammalian cells. High levels of recombination were observed in a variety of cultured cell types and in all tissues examined in mice following intraperitoneal administration. This represents the first use of protein transduction to induce the enzymatic conversion of a substrate in living cells and animals and provides a rapid and efficient means to manipulate mammalian gene structure and function.
Research Interests: Multidisciplinary, Nature, Cell Culture, Transgenic Mice, Cell line, and 13 moreCell Differentiation, Mice, Animals, Fibroblast Growth Factor, Transduction, Gene Structure, Gene Function, Amino Acid Sequence, Recombination, Enzymatic Activity, Fusion Protein, Protein Transport, and Cell membrane permeability
Viruses are obligate intracellular parasites and rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding... more
Viruses are obligate intracellular parasites and rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies. A gene entrapment approach was used to identify candidate cellular genes that affect reovirus infection or virus induced cell lysis. Four of the 111 genes disrupted in clones selected for resistance to infection by reovirus type 1 involved the insulin growth factor-2 (IGF-II) pathway, including: the mannose-6-phosphate/IGF2 receptor (Igf2r), a protease associated with insulin growth factor binding protein 5 (Prss11), and the CTCF transcriptional regulator (Ctcf). The disruption of Ctcf, which encodes a repressor of Igf2, was associated with enhanced Igf2 gene expression. Plasmids expressing either the IGF-II pro-hormone or IGF-II without the carboxy terminal extension ...
Research Interests: Transcription Regulation, Gene expression, Signal Transduction, Cell line, Mutagenesis, and 15 moreMutation, Animals, Life Cycle, Rats, Gene Targeting, Cell Proliferation, Amino Acid Sequence, Base Sequence, Gene Disruption, Growth Factor, DNA binding proteins, Biochemistry and cell biology, Gene Expression Regulation, Molecular Sequence Data, and Antiviral Therapy
Viruses are obligate intracellular parasites that rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for... more
Viruses are obligate intracellular parasites that rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies. Candidate genes required for lytic reovirus infection were identified by tagged sequence mutagenesis, a process that permits rapid identification of genes disrupted by gene entrapment. One hundred fifty-one reovirus resistant clones were selected from cell libraries containing 2 x 105 independently disrupted genes, of which 111 contained mutations in previously characterized genes and functionally anonymous transcription units. Collectively, the genes associated with reovirus resistance differed from genes targeted by random gene entrapment in that known mutational hot spots were under represented, and a number of mutations appeared to cluster around specific cellular...
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nuclear riboprotein complexes4,5. FUS resembles a transcription factor in that it binds DNA, contributes a transcriptional activa- tion domain to the FUS-ERG oncoprotein and interacts with sev- eral transcription factors in vitro 6-8 . To... more
nuclear riboprotein complexes4,5. FUS resembles a transcription factor in that it binds DNA, contributes a transcriptional activa- tion domain to the FUS-ERG oncoprotein and interacts with sev- eral transcription factors in vitro 6-8 . To better understand FUS function in vivo, we examined the consequences of disrupting Fus in mice. Our results indicate that Fus is essential for viability of
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Most mammalian genes will soon be characterized as cDNA sequences with little information about their function. To utilize this sequence information for large-scale functional studies, a gene trap retrovirus shuttle vector has been... more
Most mammalian genes will soon be characterized as cDNA sequences with little information about their function. To utilize this sequence information for large-scale functional studies, a gene trap retrovirus shuttle vector has been developed to disrupt genes expressed in murine embryonic stem (ES) cells. A library of mutant clones was isolated, and regions of genomic DNA adjacent to 400 independent provirus inserts were cloned and sequenced. The flanking sequences, designated 'promoter-proximal sequence tags', or PSTs, identified 63 specific genes and anonymous cDNAs disrupted as a result of virus integration. The efficiency of tagged sequence mutagenesis suggests that many of the 10,000-20,000 genes expressed in ES cells can be targeted, providing defined mutations for the analysis of gene functions in vivo. In addition, PSTs provide the first expressed sequence tags derived from genomic DNA, and define gene features such as exon boundaries and promoters that are missing from cDNA sequences.