The present invention relates to methods of modulating inflammation by altering the expression and/or activity of OLD-35, a protein originally identified by its association with senescence and terminal cell differentiation. The invention is based on the discovery that OLD-35, at least in part through the generation of reactive oxygen species, induces a number of inflammatory cytokines and promotes nuclear translocation and binding of the transcriptional activator NF-κB. The present invention further provides for active portions of OLD-35 which may be used therapeutically.
Human polynucleotide phosphorylase (hPNPaseold-35, referred to as “OLD-35” herein) was identified as a previously unknown gene, old-35, by an overlapping pathway screening (OPS) approach due to its upregulation during cellular differentiation and senescence (1). Old-35, a 3′, 5′ exoribonuclease, is a predominantly type I interferon-inducible gene highly evolutionary conserved in plants, prokaryotes and eukaryotes having similar domain structures and functional properties in all species (1-3). Its expression is also augmented in senescent progeroid fibroblasts in comparison to young fibroblasts (1). Overexpression of old-35 via a replication incompetent adenovirus (Ad.hPNPase) in HO-1 human melanoma cells and in normal human melanocytes (NHuMel) produced a senescent phenotype characterized by growth arrest in the G1 phase, increased Senescence Associated β-galactosidase activity, decreased telomerase activity and defined senescence-associated gene expression changes (4). These profound alterations induced by old-35 suggest an essential role in controlling senescence and differentiation through its property as an exoribonuclease by targeting selective RNA degradation. This hypothesis is supported by the observations that old-35 induces specific degradation of c-myc mRNA and that overexpression of c-myc partially protects HO-1 cells from old-35-induced growth arrest (4).
Oxidative stress is a potential mediator of in vitro replicative and premature senescence and in vivo aging (5). The free radical theory of aging, as proposed by Harman, states that endogenous reactive oxygen species (“ROS”) are generated in cells resulting in a pattern of cumulative damage (6). Oxidative damage can be measured by formation of 8-oxo-2′-deoxyguanosine (oxo8dG) in DNA or free 8-oxoguanine base (oxo8Gua) release by cells (7). Replicative senescent cells contain approximately 30% more oxo8dG in their DNA and produce four times more free oxo8Gua bases (8). Tissues from aged individuals or aged experimental animals accumulate oxidative damage in their DNA, protein and lipids (9). Moreover, repeated subcytotoxic oxidative damage can induce premature senescence in multiple cell types such as fibroblasts, keratinocytes, melanocytes or umbilical vascular endothelial cells (10) and treatment with a cell-permeable anti-oxidant or culturing cells in a reduced ambient oxygen content can reverse growth arrest of fibroblasts induced by expression of activated Ras (11). ROS comprise a variety of diverse chemical species including superoxide anions, hydroxyl radicals and hydrogen peroxide (5). Although cytosolic enzymes such as NADPH oxidases contribute to the generation of ROS, the majority of intracellular ROS production generates from mitochondria (5). Additionally, aged animals contain defective mitochondria and can produce higher levels of ROS than their young counterparts (12).
A prominent mechanism by which ROS modulates diverse intracellular molecular processes is by regulating the activity of transcription factors, most notably nuclear factor (NF)-κB (13). As a corollary to increased ROS generation during the aging process, increased NF-κB DNA binding activity has been documented in multiple tissues of aged animals compared with young animals (14-21). In resting cells, NF-κB resides in the cytoplasm in an inactive form bound to an inhibitory protein known as IκB (22). Upon receiving a stimulus, such as ROS, IκB kinase (IKK) is activated which in turn phosphorylates IκB proteins making them susceptible to ubiquitin-proteosome-mediated degradation (23, 24). The destruction of IκB unmasks the nuclear localization signal of NF-κB, leading to nuclear translocation and regulation of gene transcription by binding to the decameric motif, “GGGRNNYYCC” in the promoters of target genes (25). Presently, five mammalian NF-κB family members, NF-κB1 (p50/p105), NF-κB2 (p52/p100), p65 (RelA), RelB and c-Rel, have been identified and cloned (26). The most abundant activated form of NF-κB is a heterodimer composed of a p50 and a p65 subunit that functions predominantly as a transcriptional activator.
The present invention relates to the discovery that OLD-35, at least in part through the generation of reactive oxygen species, induces a number of inflammatory cytokines and promotes nuclear translocation and binding of the transcriptional activator NF-κB. Accordingly, the present invention provides for assay systems (which either utilize the old-35 promoter or the old-35 gene) that may be used to identify new anti-inflammatory agents; model systems of inflammation based on over-expression of the old-35 gene in cells and tissues (including specific model systems for arthritis, atherosclerosis and Alzlieimer's disease); methods and kits for diagnosing old-35 associated inflammatory conditions, and methods of treatment and anti-inflammatory compositions that utilize agents that antagonize OLD-35 activity. The present invention further relates to portions of OLD-35 which exhibit PNPase activity. Such portions may be used therapeutically to inhibit cell proliferation (for example, in the context of malignancy) or to act as “vaccines” to inhibit inflammation
For clarity and not by way of limitation, the detailed description of the invention is divided into the following subsections:
Note that the old-35 gene and corresponding nucleic acids (genomic DNA, cDNA, mRNA, antisense RNA, RNA-i, etc.) is designated by all lower case letters, the protein is designated by all capital letters (i.e., OLD-35) and the gene and protein are collectively designated by a capitalized first letter only (i.e., Old-35).
The term “antibody,” as used herein, refers to a complete imnunoglobulin molecule as well as fragments and derivatives thereof, single-chain antibodies, and any other functional equivalents. The antibody may be polyclonal or monoclonal, chimeric, and biologically or chemically produced.
5.1. Assay Systems
The present invention provides for assay methods and systems that may be used to identify agents that modulate the transcription of old-35, its translation into protein and/or its biological activity. The agents may be molecules that occur in nature (e.g., a protein that binds to the old-35 promoter, an enzyme that specifically inhibits OLD-35 activity), or, alternatively, synthetic molecules such as (but not limited to) small molecules generated in a combinatorial chemical library. In sections 5.1.1 and 5.1.2, below, the assays identify agents that antagonize Old-35, but the skilled artisan would be able, given the instant disclosure, to use methods that look for an opposite result (e.g., an increase in promoter activity, an increase in reactive oxygen species, increased NF-κB binding or translocation into the nucleus, or increased cytokine levels) to identify agents that enhance rather than decrease the inflammatory activity of Old-35.
5.1.1 Assay Systems that Use the old-35 Promoter
The present invention provides for a method for identifying an agent that inhibits inflammation, comprising exposing a test agent to a system comprising an old-35 promoter element operatively linked to a reporter gene and determining whether the exposure to the test agent increases transcription of the reporter gene, wherein a decrease in transcription of the reporter gene indicates that the test agent inhibits inflammation.
As set forth above, the test agent may be a naturally occurring molecule or substance or may be synthetic.
An old-35 promoter element is a promoter element that is found in nature operatively linked to an old-35 gene. The human old-35 promoter element has been cloned and characterized and is set forth in
In preferred embodiments of the invention, the promoter element used is the human old-35 promoter (SEQ ID NO:1) or a variant thereof. The term “variant” includes fragments, deletion mutants, insertional mutants, point mutants, substitution mutants, nucleic acid molecules comprising one or more modified nucleic acid, etc. The wild-type old-35 promoter and variants thereof are collectively referred to as “old-35 promoters.” Preferably variants are at least 85 percent, preferably at least 90 percent homologous to a nucleic acid molecule having a sequence set forth in SEQ ID NO:1 (
Deletion mutants of the old-35 promoter preferably hybridize to a nucleic acid molecule having a sequence as set forth in SEQ ID NO:1 under stringent conditions.
In one non-limiting embodiment of the invention, a human old-35 promoter variant is contained in p2000, as depicted in
The present invention further provides for isolated nucleic acid molecules comprising subregions of an old-35 promoter, including but not limited to an old-35 Interferon-Stimulated Response Element (“ISRE”) having a sequence as set forth in SEQ ID NO:4 and depicted in
Data demonstrating that IFN-β was more effective in upregulating p400 than the p2000 construct indicate that one or more repressor element(s) is present in the p2000 construct. It may be desirable to omit this one or more repressor element from constructs intended to optimize promoter activity. One non-limiting example of an old-35 promoter variant lacking the repressor is the p400 variant.
The present invention provides for an old-35 promoter operatively linked to a gene of interest which, when introduced into a suitable host cell, results in the transcription of the gene of interest and preferably in the expression of a protein encoded by the gene of interest. The gene of interest may be an old-35 gene or may be another gene (a “heterologous”) gene. Examples of non-old-35 genes of interest include but are not limited to reporter genes, such as the genes encoding green fluorescent protein (or another naturally occurring fluorescent protein or engineered variant thereof), β-glucuronidase, β-galactosidase, luciferase, and dihydrofolate reductase.
The transcriptional activity of the old-35 promoter/reporter gene construct may be evaluated in vitro (e.g., nuclear run-off assays) or in vivo. The construct may be introduced into a cell using standard techniques, including transformation, transduction, transfection, electroporation, etc. The construct, for propagation purposes or for introduction into a cell, may be incorporated into a suitable vector molecule, such as a plasmid, a phage, a phagemid or a virus. Where the vector is an expression vector, suitable expression vectors include virus-based vectors and non-virus based DNA or RNA delivery systems. Examples of appropriate virus-based gene transfer vectors include, but are not limited to, those derived from retroviruses, for example Moloney murine leukemia-virus based vectors such as LX, LNSX, LNCX or LXSN (Miller and Rosman, 1989, Biotechniques 7:980-989); lentiviruses, for example human immunodeficiency virus (“HIV”), feline leukemia virus (“FIV”) or equine infectious anemia virus (“EIAV”)-based vectors (Case et al., 1999, Proc. Natl. Acad. Sci. U.S.A. 96: 22988-2993; Curran et al., 2000, Molecular Ther. 1:31-38; Olsen, 1998, Gene Ther. 5:1481-1487; U.S. Pat. Nos. 6,255,071 and 6,025,192); adenoviruses (Zhang, 1999, Cancer Gene Ther. 6(2):113-138; Connelly, 1999, Curr. Opin. Mol. Ther. 1(5):565-572; Stratford-Perricaudet, 1990, Human Gene Ther. 1:241-256; Rosenfeld, 1991, Science 252:431-434; Wang et al., 1991, Adv. Exp. Med. Biol. 309:61-66; Jaffe et al., 1992, Nat. Gen. 1:372-378; Quantin et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:2581-2584; Rosenfeld et al., 1992, Cell 68:143-155; Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; Ragot et al., 1993, Nature 361:647-650; Hayaski et al., 1994, J. Biol. Chem. 269:23872-23875; Bett et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:8802-8806), for example Ad5/CMV-based E1-deleted vectors (Li et al., 1993, Human Gene Ther. 4:403-409); adeno-associated viruses, for example pSub201-based AAV2-derived vectors (Walsh et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:7257-7261); herpes simplex viruses, for example vectors based on HSV-1 (Geller and Freese, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1149-1153); baculoviruses, for example AcMNPV-based vectors (Boyce and Bucher, 1996, Proc. Natl. Acad. Sci. U.S.A. 93:2348-2352); SV40, for example SVluc (Strayer and Milano, 1996, Gene Ther. 3:581-587); Epstein-Barr viruses, for example EBV-based replicon vectors (Hambor et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:4010-4014); alphaviruses, for example Semliki Forest virus- or Sindbis virus-based vectors (Polo et al., 1999, Proc. Natl. Acad. Sci. U.S.A. 96:4598-4603); vaccinia viruses, for example modified vaccinia virus (MVA)-based vectors (Sutter and Moss, 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10847-10851) or any other class of viruses that can efficiently transduce human tumor cells and that can accommodate the nucleic acid sequences required for therapeutic efficacy.
A decrease in transcription of the reporter gene may be detected by measuring reporter gene mRNA, the protein product of the reporter gene, or a property of the reporter gene product (e.g., enzyme activity, fluorescence, etc.).
The decrease in transcription is preferably measured relative to a control in which the old-35 promoter/reporter gene construct is not exposed to test agent.
Preferably, the decrease in transcription is at least about 10, 30, 50, 70 or 90 percent.
5.1.2 Assay Systems that Use the Old-35 Gene
In a first set of non-limiting embodiments, the present invention provides for a method for identifying an agent that inhibits inflammation, comprising administering a test agent (that is a putative anti-inflammatory agent) to a cell comprising an old-35 gene operatively linked to a promoter element, wherein the old-35 gene is transcribed and expressed as OLD-35 protein, and determining whether the exposure to the test agent decreases the amount of reactive oxygen species in the cell. In preferred, non-limiting related embodiments of the invention, the cell is a test cell into which an old-35 gene operatively linked to a promoter element has been introduced, and the amount of reactive oxygen species in the test cell exposed to the test agent is decreased relative to the amount of reactive oxygen species in a first control cell into which the old-35 gene operatively linked to the promoter has been introduced, but which is not exposed to the test agent. Further, the amount of reactive oxygen species in the first control cell is greater than that in a second control cell which does not contain the old-35 gene operatively linked to the promoter element and which is not senescent or terminally differentiated.
Reactive oxygen species that may be measured include, but are not limited to, superoxide anions, hydroxyl radicals and hydrogen peroxide. Reactive oxygen species may be detected by methods known in the art, for example, but not by way of limitation, by staining with hydroethidine and dichlorofluorescein diacetate, followed by flow cytometry, as set forth in example section 6, below.
In a second set of non-limiting embodiments, the present invention provides for a method for identifying an agent that inhibits inflammation, comprising administering a test agent to a cell comprising an old-35 gene operatively linked to a promoter element, wherein the old-35 gene is transcribed and expressed as OLD-35 protein, and determining whether the exposure to the test agent decreases the amount of binding between NF-κB and its target sequence in the cell. In preferred non-limiting related embodiments, the cell is a test cell into which an old-35 gene operatively linked to a promoter element has been introduced, and the amount of binding between NF-κB and its target sequence in the test cell exposed to the test agent is decreased relative to the amount of binding between NF-κB and its target sequence in a first control cell into which the old-35 gene operatively linked to the promoter has been introduced, but which is not exposed to the test agent. Further; the amount of binding between NF-κB and its target sequence in the first control cell is greater than that in a second control cell which does not contain the old-35 gene operatively linked to the promoter element and which is not senescent or terminally differentiated.
Binding of NF-κB to its target sequence may be demonstrated by any method known in the art. For example, a cell, test cell or control cell may contain a transgene comprising a reporter gene, such as luciferase, operably linked to a promoter containing one or more NF-κB binding site, and the amount of reporter gene produced in the presence and absence of the test agent may be monitored. Alternatively, electrophoretic mobility shift assays may be performed using nuclear extracts. See, for example, section 6 below.
In a third set of non-limiting embodiments, the present invention provides for a method for identifying an agent that inhibits inflammation, comprising administering a test agent to a cell comprising an old-35 gene operatively linked to a promoter element, wherein the old-35 gene is transcribed and expressed as OLD-35 protein, and determining whether the exposure to the test agent decreases the amount of translocation of a NF-κB protein from the cytoplasm into the nucleus of the cell. In preferred non-limiting related embodiments, the cell is a test cell into which an old-35 gene operatively linked to a promoter element has been introduced, and the amount of translocation of a NF-κB protein from the cytoplasm into the nucleus in the test cell exposed to the test agent is decreased relative to the amount of translocation of a NF-κB protein from the cytoplasm into the nucleus in a first control cell into which the old-35 gene operatively linked to the promoter has been introduced, but which is not exposed to the test agent. Further, the amount of translocation of a NF-κB protein from the cytoplasm into the nucleus in the first control cell is greater than that in a second control cell which does not contain the old-35 gene operatively linked to the promoter element and which is not senescent or terminally differentiated.
The translocation of a NF-κB protein from the cytoplasm into the nucleus may be detected and measured using standard laboratory techniques, for example, by cell fractionation into nuclear and cytoplasmic fractions followed by Western blot analysis using NF-κB specific antibodies (see example section 6, below).
In a fourth set of non-limiting embodiments, the present invention provides for a method for identifying an agent that inhibits inflammation, comprising administering a test agent to a cell comprising an old-35 gene operatively linked to a promoter element, wherein the old-35 gene is transcribed and expressed as OLD-35 protein, and determining whether the exposure to the test agent decreases the amount of a cytokine selected from the group consisting of interleukin-6, interleukin-8, TNFR1, RANTES and MMP-3 in the cell. In preferred related, non-limiting embodiments, the cell is a test cell into which an old-35 gene operatively linked to a promoter element has been introduced, and the amount of a cytokine selected from the group consisting of interleukin-6, interleukin-8, TNFR1, RANTES and MMP-3 in the test cell exposed to the test agent is decreased relative to the amount of a cytokine selected from the group consisting of interleukin-6, interleukin-8, TNFR1, RANTES and MMP-3 in a first control cell into which the old-35 gene operatively linked to the promoter has been introduced, but which is not exposed to the test agent. Further; the amount of a cytokine selected from the group consisting of interleukin-6, interleukin-8, TNFR1, RANTES and MMP-3 in the first control cell is greater than that in a second control cell which does not contain the old-35 gene operatively linked to the promoter element and which is not senescent or terminally differentiated. Cytokines may be measured using methods known in the art, including but not limited to ELISA analysis, Western blot, or antibody array using anti-cytokine antibodies (see example section 6, below) or Northern analysis of cellular RNA using cytokine specific nucleic acid probes.
In the foregoing four sets of embodiments, where a promoter/old-35 construct is not introduced, the cell may naturally express the old-35 gene, and may be identified as expressing the old-35 gene at a level that is at least equal to the level expressed in neonatal human fibroblasts. Examples of such cells include senescent cells, including cells of progeria patients, and terminally differentiated cells, such as melanoma cells exposed to interferon beta and mezerein (see United States Patent Application Publication No. 20030099660). The cell or test cell may be any cell that permits expression of OLD-35. Where a promoter/old-35 construct is introduced into the test cell, the test cell absent the construct preferably (but not necessarily) produces undetectable or low levels of OLD-35, to minimize background.
Any promoter that is active or inducible in the cell, test cell and/or first control cell may be used. Non-limiting examples of such promoters include the cytomegalovirus immediate early promoter, the Rous sarcoma virus long terminal repeat promoter, the human elongation factor lea promoter, the human ubiquitin c promoter, etc. Non-limiting examples of inducible promoters include the murine mammary tumor virus promoter (inducible with dexamethasone); commercially available tetracycline-responsive or ecdysone-inducible promoters, etc.
Old-35 genes which may be used according to the invention include the human old-35 gene, having a nucleic acid sequence as deposited in GenBank Accession No. AY027528 SEQ ID NO:5, or a nucleic acid that hybridizes thereto under stringent conditions (see section 5.1.1, above). In addition, a nucleic acid comprising an old-35 gene may encode human OLD-35 protein having a sequence as set forth in
In addition, non-human forms of old-35 may also be used according to the invention. For example, where murine assay systems are used, murine old-35 homolog, GenBank Acc. No. AF465249, may optionally be used.
The promoter/old-35 construct may be introduced into a cell using standard techniques, including transformation, transfection, transduction, electroporation, etc. For propagation or for introduction into a host cell, the construct may be comprised in a vector molecule, such as a plasmid, phage, phagemid, or virus (see section 5.1.1, above). In a preferred embodiment, the construct may be comprised in a recombinant adenovirus vector wherein transcription of the old-35 cDNA is driven by the cytomegalovirus immediate early (CMV) promoter.
5.2 Model Systems of Inflammation
The present invention provides for cells and animals engineered to overexpress (in the case of animals, in at least some cells and tissues) an old-35 gene, and thereby serve as model systems for the study of inflammation and for the evaluation of agents that modify inflammation (for example, for the discovery of novel anti-inflammatory compounds).
Suitable transgenic animals include, but are not limited to, mice, rats, goats, sheep, pigs, cows, etc.
Old-35 may be over expressed in some or all cells of said animals. Overexpression may be limited to certain tissues to provide model systems of particular inflammatory conditions. A number of non-limiting examples of such systems follow.
In one set of non-limiting embodiments, the present invention provides for a model system of arthritis, comprising a non-human animal carrying a transgene comprising an old-35 gene operatively linked to a promoter element that is selectively active in cells comprised in a joint of the animal. Examples of such promoters include but are not limited to the chondromodulin-1 (ChM-I) gene promoter (Aoyama et al., J Biol Chem. 2004, 279:28789-28797), chicken collagen X regulatory sequences (Campbell et al., Am J Pathol. 2004, 164:487-499, connective tissue growth factor (CTGF/Hcs24) gene promoter (Kubota et al., Bone. 2003; 33:694-702), the 4-kb murine Col10a1 promoter of the alpha1 (X) collagen gene (Zheng et al., J. Cell Biol. 2003; 162:833-842), cartilage oligomeric matrix protein gene (COMP) promoter (Issack et al., J Orthop Res. 2004, 22:751-758).
In another set of non-limiting embodiments, the present invention provides for a model system for atherosclerosis, comprising a non-human animal carrying a transgene comprising an old-35 gene operatively linked to a promoter element that is selectively active in cells of the vascular system. Examples of such promoters include but are not limited to the Angiopoietin-2 (Ang-2) gene promoter (Hegen et al., Arterioscler Thromb Vasc Biol. 2004 Jul. 29 [Epub ahead of print]), endothelial nitric-oxide synthase (eNOS) gene promoter (Chan et al., J Biol Chem. 2004, 279:35087-35100), Cysteine-rich protein (CRP)2 gene promoter (Chang et al., Am J Physiol Heart Circ Physiol. 2003 285:H1675-1683). intercellular adhesion molecule 2 (ICAM-2), platelet endothelial cell adhesion molecule 1 (PECAM-1) and endoglin. gene promoters (Cowan et al., Xenotransplantation. 2003; 10:223-231).
In yet another set of non-limiting embodiments, the present invention provides for a model system for Alzheimer's disease, comprising a non-human animal carrying a transgene comprising an old-35 gene operatively linked to a promoter element that is selectively active in cells of the central nervous system. Examples of such promoters include the neuron-specific enolase gene promoter (Tanaka et al., 2001, Anticancer Res. 21:291-294) and the excitatory amino acid transporter-2 promoter, as set forth in GenBank Acc. No. AF510107.
To study or monitor inflammation in said cells or animals, the present invention provides for a method for evaluating inflammation in a transgenic non-human animal carrying a transgene comprising an old-35 gene operatively linked to a promoter element, comprising determining, in a cell, tissue, or fluid of the animal, whether the amount of reactive oxygen species is increased, whether the amount of binding of a NF-κB protein to its target sequence is increased, whether the amount of a NF-κB protein translocated into the nucleus is increased, or whether the amount of a cytokine selected from the group consisting of interleukin-6, interleukin-8, TNFR1, RANTES and MMP-3 is increased. Such determinations may be made using methods known in the art (see section 5.1.2 above and example section 6 below).
The present invention also provides for administering a test agent to a transgenic animal carrying a transgene comprising an old-35 gene, and then determining whether inflammation is increased or decreased in said animal. Such transgenic animals may be used to discover new anti-inflammatory agents or to demonstrate anti-inflammatory activity of a putative or known anti-inflammatory agent.
Such transgenic animals may provide the advantage of creating a model system of inflammation as it occurs with aging.
5.3 Diagnostic Methods and Kits
The present invention provides for a method of detecting inflammation in a subject, comprising determining whether there is an increase in the expression of an old-35 gene in a cell of the subject relative to a control cell. Such an increase may be determined by showing an increase in old-35 mRNA (for example by Northern blot or dot-blot analysis) using a suitable old-35 nucleic acid probe, or by showing an increase in OLD-35 protein using, for example, an antibody directed toward OLD-35. The method may be practiced in vivo or preferably in vitro.
Inflammation may be detected so as to demonstrate acute or chronic inflammation. Detection of increased expression of Old-35 (nucleic acid and/or protein) may support a diagnosis of a chronic inflammatory condition such as, but not limited to, arthritis, atherosclerosis, periodontal disease, Alzheimer's disease or chronic obstructive pulmonary disease.
In related embodiments, the present invention provides for a kit for detecting inflammation in a subject, comprising a probe that binds to an old-35 gene product selected from the group consisting of old-35 mRNA (in which case the probe is a nucleic acid) and OLD-35 protein (in which case the probe is an antibody) and an antibody that binds to a cytokine selected from the group consisting of interleukin-6, interleukin-8, TNFR1, RANTES and MMP-3. Alternatively, the kit may comprise a nucleic acid probe that may be used to detect cytokine mRNA.
5.4 Methods of Treatment and Anti-Inflammatory Compositions
The present invention provides for a method of inhibiting inflammation in a subject in need of such treatment, comprising administering, to the subject, an effective amount of an agent that antagonizes the expression and/or activity of OLD-35, including, but not limited to, an effective amount of an antibody that binds to OLD-35 protein, an old-35 RNA-I, or an antisense old-35 nucleic acid.
As one non-limiting example, one of the foregoing Old-35 antagonist agents may be administered into the synovial fluid of an inflamed joint.
In non-limiting embodiments, the amount of antagonist may be administered to achieve a concentration of, for an OLD-35 antibody (10 μg-1 mg/ml), for an old-35 RNA-i or antisense RNA (25-100 nM).
The present invention further provides for a method of inhibiting inflammation in a subject in need of such treatment, comprising introducing, into cells of the subject, a nucleic acid comprising an old-35 promoter element operatively linked to a gene that inhibits inflammation. Non-limiting examples of such genes include antisense NFkB p65 subunit, dominant negative versions of IkB or NFkB p65 subunit etc.
The present invention still further provides for an anti-inflammatory composition, comprising an agent that antagonizes old-35 activity selected from the group consisting of an old-35 RNA-i, an old-35 antisense RNA, and an antibody directed toward OLD-35, and a second anti-inflammatory agent. Non-limiting examples of second anti-inflammatory agents include a steroid compound or a non-steroidal anti-inflammatory agent such as aspirin, ibuprofen, naprosyn, celecoxib, valdecoxib, diclofenac, and anti-inflammatory antibodies (or their fragments) such as etanercept, infliximab or anakinra.
5.5 Active Subregions of OLD-35
5.5.1 Domains of OLD-35
The present invention provides for a group of proteins collectively referred to as “OLD-35 variants” comprising active subregions of the OLD-35 protein. “Active” refers to anti-proliferative activity, inflammatory activity, PNPase activity, RNA degradation activity, cell-cycle arresting/slowing activity, and/or senescence inducing activity. Old-35 belongs to an RNA processing enzyme family comprising the polynucleotide phosphorylases (PNPases). PNPases typically contain RNAse PH (RPH) domains and RNA binding domains. Native full length OLD-35 protein contains 783 amino acid residues (SEQ ID NO:6,
In one embodiment of the invention an OLD-35 variant is a protein that is not the amino acid sequence set forth as SEQ ID NO:6, which encodes the full length OLD-35 protein consisting of 783 amino acids, but that comprises a RPH domain as set forth in SEQ ID NOS: 15-19 (
As set forth in SEQ ID NOS: 17-19, an Old-35 variant may comprise of either one or both RPH domains and any additional domain such as the mitochondrial localization signal (SEQ ID NO:20), the PNPase domain (SEQ ID NO:21), the KH domain (SEQ ID NO:23) or the S1 domain (SEQ ID NO:24) or that is at least about 85, at least about 90, or at least about 95 percent homologous thereto. In an additional non-limiting embodiment, an Old-35 variant need not have a contiguous arrangement of various its domains in the same linear order as present in the native OLD-35 protein.
In particular, non-limiting embodiments, the present invention provides for an OLD-35 variant protein comprising one, but not two, RPH domain and having an activity selected from the group consisting of anti-proliferative activity, PNPase activity, RNA degradation activity, cell-cycle arrest/slowing activity, senescence-inducing activity and a combination thereof. In an alternative non-limiting embodiment, an OLD-35 variant protein may comprise one, but not two, RPH domain which is immunogenic in a mammal. The invention also provides for an OLD-35 variant possessing anti-proliferative activity, PNPase activity, RNA degradation activity, cell-cycle slowing activity, senescence-inducing activity, a combination thereof and which is immunogenic in mammals, comprising amino acid residues 52-183 (SEQUENCE ID NO:15) or amino acid residues 366-501 (SEQUENCE ID NO:16) of native OLD-35 protein, or a sequence that is at least 90 percent, preferably at least 95 percent homologous to residues 52-183 or residues 366-501 respectively. The invention also provides for an OLD-35 variant possessing anti-proliferative activity, PNPase activity, RNA degradation activity, cell-cycle slowing activity, senescence-inducing activity and a combination thereof and which is immunogenic in mammals, comprising amino acid residues 289-363 (SEQUENCE ID NO:21) of native OLD-35 protein, or a sequence that is at least 90 percent, preferably at least 95 percent homologous to residues 289-363.
Accordingly, in specific, nonlimiting embodiments, the present invention provides for Old-35 variants as follows, where the residues identified are as set forth in SEQ ID NOS:
(i) lacking residues 676-750, and comprising residues 52-183 and residues 299-363, operably joined, optionally by a linker sequence, and having PNPase activity;
(ii) lacking residues 676-750, comprising residues 366-501 and residues 289-363, operably joined, optionally by a linker sequence, and having PNPase activity;
(iii) lacking residues 1-45, and comprising residues 52-183 and having anti-proliferative activity;
(iv) lacking residues 1-45, and comprising residues 366-501, and having antiproliferative activity;
(v) not the complete native OLD-35 sequence, comprising residues 289-363, and immunogenic in a mammal;
(vi) not the complete native OLD-35 sequence, comprising residues 366-501, and immunogenic in a mammal.
(vii) not the complete native OLD-35 sequence, comprising residues 607-667, and immunogenic in a mammal;
(viii) not the complete native OLD-35 sequence, comprising residues 676-750, and immunogenic in a mammal;
5.5.2 Expression Systems
The Old-35 variants of the invention may be produced by any method known in the art. Such methods include but are not limited to chemical synthesis and recombinant DNA techniques.
With regard to production of Old-35 variants using recombinant DNA techniques, the present invention provides for nucleic acids encoding said variants. Such nucleic acids may either be nucleic acid fragments of the aforelisted Old-35 nucleic acids derived from SEQ ID NO:5- and include SEQ ID NOS:24-28, encoding the variants, or may be nucleic acids designed, using the genetic code, to encode such variants, wherein an alternate or optimized codon usage provides the basis for conservative codon or amino acid substitutions.
A nucleic acid encoding a Old-35 variant of the invention may be comprised in a suitable vector molecule, and may optionally be operatively linked to a suitable promoter element, for example, but not limited to, the cytomegalovirus immediate early promoter, the Rous sarcoma virus long terminal repeat promoter, the human elongation factor 1α promoter, the human ubiquitin c promoter, etc. It may be desirable, in certain embodiments of the invention, to use an inducible promoter. Non-limiting examples of inducible promoters include the murine mammary tumor virus promoter (inducible with dexamethasone); commercially available tetracycline-responsive or ecdysone-inducible promoters, etc. In specific non-limiting embodiments of the invention, the promoter may be selectively active in cancer cells; one example of such a promoter is the PEG-3 promoter, as described in International Patent Application No. PCT/US99/07199, Publication No. WO 99/49898 by Fisher et al., published on Oct. 7, 1999; other non-limiting examples include the prostate specific antigen gene promoter (O'Keefe et al., 2000, Prostate 45:149-157), the kallikrein 2 gene promoter (Xie et al., 2001, Human Gene Ther. 12:549-561), the human alpha-fetoprotein gene promoter (Ido et al., 1995, Cancer Res. 55:3105-3109), the c-erbB-2 gene promoter (Takakuwa et al., 1997, Jpn. J. Cancer Res. 88:166-175), the human carcinoembryonic antigen gene promoter (Lan et al., 1996,Gastroenterol. 111:1241-1251), the gastrin-releasing peptide gene promoter (Inase et al., 2000, Int. J. Cancer 85:716-719). the human telomerase reverse transcriptase gene promoter (Pan and Koenman, 1999, Med. Hypotheses 53:130-135), the hexokinase II gene promoter (Katabi et al., 1999, Human Gene Ther. 10:155-164), the L-plastin gene promoter (Peng et al., 2001, Cancer Res. 61:4405-4413), the neuron-specific enolase gene promoter (Tanaka et al., 2001, Anticancer Res. 21:291-294), the midkine gene promoter (Adachi et al., 2000, Cancer Res. 60:4305-4310), the human mucin gene MUC1 promoter (Stackhouse et al., 1999, Cancer Gene Ther. 6:209-219), and the human mucin gene MUC4 promoter (Genbank Accession No. AF241535), which is particularly active in pancreatic cancer cells (Perrais et al., J Biol Chem. 2001, 276:30923-30933).
Suitable expression vectors include virus-based vectors and non-virus based DNA or RNA delivery systems. Examples of appropriate virus-based gene transfer vectors include, but are not limited to, pCEP4 and pREP4 vectors from Invitrogen, and, more generally, those derived from retroviruses, for example Moloney murine leukemia-virus based vectors such as LX, LNSX, LNCX or LXSN (Miller and Rosman, 1989, Biotechniques 7:980-989); lentiviruses, for example human immunodeficiency virus (“HIV”), feline leukemia virus (“FIV”) or equine infectious anemia virus (“EIAV”)-based vectors (Case et al., 1999, Proc. Natl. Acad. Sci. U.S.A. 96: 22988-2993; Curran et al., 2000, Molecular Ther. 1:31-38; Olsen, 1998, Gene Ther. 5:1481-1487; U.S. Pat. Nos. 6,255,071 and 6,025,192); adenoviruses (Zhang, 1999, Cancer Gene Ther. 6:113-138; Connelly, 1999, Curr. Opin. Mol. Ther. 1:565-572; Stratford-Perricaudet, 1990, Human Gene Ther. 1:241-256; Rosenfeld, 1991, Science 252:431-434; Wang et al., 1991, Adv. Exp. Med. Biol. 309:61-66; Jaffe et al., 1992, Nat. Gen. 1:372-378; Quantin et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:2581-2584; Rosenfeld et al., 1992, Cell 68:143-155; Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; Ragot et al., 1993, Nature 361:647-650; Hayaski et al., 1994, J. Biol. Chem. 269:23872-23875; Bett et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:8802-8806), for example Ad5/CMV-based E1-deleted vectors (Li et al., 1993, Human Gene Ther. 4:403-409); adeno-associated viruses, for example pSub201-based AAV2-derived vectors (Walsh et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:7257-7261); herpes simplex viruses, for example vectors based on HSV-1 (Geller and Freese, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1149-1153); baculoviruses, for example AcMNPV-based vectors (Boyce and Bucher, 1996, Proc. Natl. Acad. Sci. U.S.A. 93:2348-2352); SV40, for example SVluc (Strayer and Milano, 1996,Gene Ther. 3:581-587); Epstein-Barr viruses, for example EBV-based replicon vectors (Hambor et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:4010-4014); alphaviruses, for example Semliki Forest virus- or Sindbis virus-based vectors (Polo et al., 1999, Proc. Natl. Acad. Sci. U.S.A. 96:4598-4603); vaccinia viruses, for example modified vaccinia virus (MVA)-based vectors (Sutter and Moss, 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10847-10851) or any other class of viruses that can efficiently transduce human tumor cells and that can accommodate the nucleic acid sequences required for therapeutic efficacy.
Non-limiting examples of non-virus-based delivery systems which may be used according to the invention include, but are not limited to, so-called naked nucleic acids (Wolff et al., 1990, Science 247:1465-1468), nucleic acids encapsulated in liposomes (Nicolau et al., 1987, Methods in Enzymology 198:157-176), nucleic acid/lipid complexes (Legendre and Szoka, 1992, Pharmaceutical Research 9:1235-1242), and nucleic acid/protein complexes (Wu and Wu, 1991, Biother. 3:87-95).
Old-35 variant protein may also be produced by yeast or bacterial expression systems. For example, bacterial expression may be achieved using plasmids such as pGEX expression system (Amersham Biosciences, Piscataway, N.J.), pQE His-tagged expression system (Qiagen, Valencia, Calif.), pET His-tagged expression system (EMD Biosciences, Inc., La Jolla, Calif.), or IMPACT expression system (New England Biolabs, Beverly, Mass.).
In a specific, non-limiting embodiment of the invention, a nucleic acid encoding an Old-35 variant, in expressible form, may be in vitro translated to produce OLD-35 variant protein. In vitro translation may be performed using an appropriate system such as the TNT® coupled Reticulocyte Lysate Systems (Promega) or TNT® Coupled Wheat Germ Extract Systems. The expressed protein may include an affinity tag so that an Old-35 variant protein may be purified and recovered from the extracts for further use.
5.5.3 Delivery Systems
Depending on the expression system used, nucleic acid may be introduced by any standard technique, including transfection, transduction, electroporation, bioballistics, microinjection, etc.
In preferred, non-limiting embodiments of the invention, the expression vector is an E1-deleted human adenovirus vector of serotype 5. To prepare such a vector, an expression cassette comprising a transcriptional promoter element operatively linked to a Old-35 variant coding region and a polyadenylation signal sequence may be inserted into the multiple cloning region of an adenovirus vector shuttle plasmid, for example pXCJL.1 (Berkner, 1988, Biotechniques 6:616-624). In the context of this plasmid, the expression cassette may be inserted into the DNA sequence homologous to the 5′ end of the genome of the human serotype 5 adenovirus, disrupting the adenovirus E1 gene region. Transfection of this shuttle plasmid into the E1-transcomplementing 293 cell line (Graham et al., 1977, J. General Virology 36:59-74), or another suitable cell line known in the art, in combination with either an adenovirus vector helper plasmid such as pJM17 (Berkner, 1988, Biotechniques 6:616-624; McGrory et al., 1988, Virology 163:614-617) or pBHG10 (Bett et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91: 8802-8806) or a ClaI-digested fragment isolated from the adenovirus 5 genome (Berkner, 1988, Biotechniques 6:616-624), allows recombination to occur between homologous adenovirus sequences contained in the adenovirus shuttle plasmid and either the helper plasmid or the adenovirus genomic fragment. This recombination event gives rise to a recombinant adenovirus genome in which the cassette for the expression of the foreign gene has been inserted in place of a functional E1 gene. When transcomplemented by the protein products of the human adenovirus type 5 E1 gene (for example, as expressed in 293 cells), these recombinant adenovirus vector genomes can replicate and be packaged into fully-infectious adenovirus particles. The recombinant vector can then be isolated from contaminating virus particles by one or more rounds of plaque purification (Berkner, 1988, Biotechniques 6:616-624), and the vector can be further purified and concentrated by density ultracentrifugation.
In a specific, non-limiting embodiment of the invention, a nucleic acid encoding an Old-35 variant, in expressible form, may be inserted into the modified Ad expression vector pAd.CMV (Falck-Pedersen et al., 1994, Mol. Pharmacol. 45:684-689).
This vector contains, in order, the first 355 base pairs from the left end of the adenoviris genome, the cytomegalovirus immediate early promoter, DNA encoding splice donor and acceptor sites, a cloning site for the Old-35 variant gene, DNA encoding a polyadenylation signal sequence from the globin gene, and approximately three kilobase pairs of adenovirus sequence extending from within the EIB coding region. This construct may then be introduced into 293 cells (Graham et al., 1977, J. Gen. Virol. 36:59-72) together with plasmid JM17 (above), such that, as explained above, homologous recombination can generate a replication defective adenovirus containing Old-35 variant encoding nucleic acid.
5.6 Use of OLD-35 Variants
5.6.1 Use of OLD-35 Variants as Antiproliferative Agents
Old-35 variants are as effective as full length OLD-35 protein in inducing senescence or differentiation (
In preferred, non-limiting embodiments, the nucleic acid encoding the OLD-35 variant may be contained in a viral vector, operably linked to a promoter element that is inducible or constitutively active in the target cell. In preferred, non-limiting embodiments, the viral vector is a replication-defective adenovirus (as described in section 5.5.3 above).
In a specific, non-limiting embodiment of the invention, a viral vector containing a nucleic acid encoding a OLD-35 variant, such as a single RPH or two RPH domain containing proteins operably linked to a suitable promoter element, may be administered to a population of target cells at a multiplicity of infection (MOI) ranging from 10-100 MOI.
In another specific, non-limiting embodiment, the amount of a viral vector administered to a subject may be 1×109 pfu to 1×1012 pfu.
In specific, non-limiting embodiments, a nucleic acid encoding an OLD-35 variant comprised in a vector or otherwise, may be introduced into a cell ex vivo and then the cell may be introduced into a subject. For example, a nucleic acid encoding a OLD-35 variant may be introduced into a cell of a subject (for example; an irradiated tumor cell, glial cell or fibroblast) ex vivo and then the cell containing the nucleic acid may be optionally propagated and then (with its progeny) introduced into the subject.
5.6.2 Use of OLD-35 Variants as an Anti-Inflammatory Vaccine
The present invention also provides for administering an OLD-35 variant in protein or peptide form in a subject in need of treatment for an acute or chronic inflammatory condition. Enhanced levels and activity of full length OLD-35 protein is associated with enhancing the levels of reactive oxygen species, inducing activation of the NF-κB pathway and enhancing the level of proinflammatory cytokines (
As such, the OLD-35 variant of the invention may be prepared by chemical synthesis or recombinant DNA techniques, purified by methods known in the art, and then administered to a subject in need of such treatment. An OLD-35 variant may be comprised, for example, in solution, in suspension, and/or in a carrier particle such as microparticles, liposomes, or other protein-stabilizing formulations known in the art In a non-limiting specific example, formulations of OLD-35 variant peptides may stabilized by addition of zinc and/or protamine stabilizers as in the case of certain types of insulin formulations. Alternatively, in specific non-limiting embodiments, an OLD-35 variant may be linked covalently or non-covalently, to a carrier protein which is preferably non-immunogenic.
In preferred, non-limiting embodiments, an OLD-35 variant protein/peptide is administered in an amount which achieves a local concentration in the range of 18 to 50 ng per microliter. For example, a subject may be administered a range of 50-100 mg per kilogram. For a human subject, the dose range may be between 1000-2500 mg/day.
6.1 Materials and Methods
Cell Lines, Reagents and Virus Infection protocol. The human cervical carcinoma cell line HeLa was cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, penicillin (50 units/ml) and streptomycin (50 μg/ml) at 37° C. in 5% CO2 and 100% relative humidity. N-acetyl-L-cysteine and Tiron were obtained from Sigma (St. Louis, Mo.). The recombinant replication-incompetent adenovirus expressing old-35 (Ad.hPNPase) was created in two steps as described previously and plaque purified by standard procedures (1, 28). Cells were infected with a multiplicity of infection (m.o.i.) of 1 to 50 plaque forming units (pfu)/cell of Ad.vec (control replication-incompetent adenovirus) or Ad.hPNPase as described (29).
Transient Transfection and Luciferase Assay. Cells (5×103/well in 12-well plates) were either uninfected or infected with either Ad.vec or Ad. hPNPase at an m.o.i. of 50 p.f.u./cell. Transient transfection was conducted 12 hr post-infection using Lipofectamine-2000 transfection reagent (Invitrogen, Carlsbad, Calif.) and 1.2 μg of plasmid DNA per well that included 1 μg of pGL3Basic, 3 KB-Luc or 3 κBmut-Luc plasmids (30) and 0.2 μg of β-galactosidase-expression plasmid (pSV-β-gal; Promega, Madison, Wis.). For inhibition experiments, the cells were pre-treated with different inhibitors for 2 hr before transfection. Luciferase assays were performed 48 hr post-transfection using a Luciferase Reporter Gene Assay kit (Promega) according to the manufacturer's protocol. The β-galactosidase activity was determined using the Galacto-Light Plus kit (Tropix). Luciferase activity was normalized by β-galactosidase activity and the data from triplicate determinations were expressed as mean ± S.D.
Generation of Anti-OLD-35 Antibody. A C-terminal His-tagged old-35 protein was produced in a baculovirus expression system (Pharmingen, San Diego, Calif.) according to the manufacturer's instructions. The protein was purified by Ni-NTA agarose column and subsequently ion exchange chromatography. The purified protein was used to immunize chickens to generate anti-OLD-35 antibody (Genetel Laboratories, Madison, Wis.).
Cell Fractionation. Cells were harvested and the cytoplasm and nucleus were fractionated by the modified Schreiber's method as described (31). Briefly, the cells were washed with PBS and lysed for 10 min in Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Nonidet P-40) containing protease inhibitor cocktail (Roche, Mannheim, Germany). After centrifugation, the supernatant was saved as the cytoplasmic fraction and the pellet containing nuclei was lysed in Buffer B (20 mM HEPES pH 7.9, 400 mM KCl, 20% glycerol, 1 mM EDTA, 1 mM DTT) containing protease inhibitor cocktail (Roche) with one freeze-thaw cycle. After centrifugation the supernatant was collected as the nuclear fraction. Protein concentration was determined by Bio-Rad protein assay kit (Bio-Rad laboratories, Hercules, Calif.).
Electrophoretic Mobility Shift Assay (EMSA). EMSA was performed as described (31). The sequences of the consensus and mutated NF-κB probes are 5′-AGTTGAGGGGACTTTCCCAGGC-3′ (SEQ ID NO:7) and 5′-AGTTGAGGCGACTTTCCCAGGC-3′ (SEQ ID NO:8), respectively (Santa Cruz Biotechnology, Santa Cruz, Calif.). The probes were labeled using T4 polynucleotide kinase (Promega) and [γ32P]ATP. Ten (10) μg of nuclear extract was incubated in a final volume of 10 μl containing gel shift binding buffer (Promega) for 10 min at room temperature and 20,000 cpm of labeled probed was added and incubated for another 20 min at room temperature. For competition studies, unlabeled probes were added 15 min before the labeled probe. For supershift analysis, the reaction mixture was incubated for 1 hr with 1 μl of antibody before the addition of the radiolabeled probe. The antibodies used were anti-p50, anti-p65, anti-p52 and anti-cRel (Santa Cruz; rabbit polyclonal). Free and bound DNA was separated on a 4% non-denaturating polyacrylamide gel in 0.5% Tris-borate-EDTA at a constant voltage of 165v. The gel was dried on filter paper and autoradiographed.
Preparation of Whole Cell Lysates and Western Blot Analysis. Whole cell lysates were prepared and Western blotting was performed as described (4). Briefly, cells were harvested in RIPA buffer containing protease inhibitor cocktail (Roche, Mannheim, Germany), 1 mM Na3VO4 and 50 mM NaF and centrifuged at 12,000 rpm for 10 min at 4° C. The supernatant was used as total cell lysate. Thirty μg of total cell lysate were used for SDS-PAGE and transferred to a nitrocellulose membrane. The primary antibodies used were anti-p65, anti-p50, IκB-α (Santa Cruz; rabbit polyclonal; 1:250), anti-EF1α (Upstate Biotechnology; mouse monoclonal; 1:1000), and anti-OLD-355 (chicken; 1:10,000).
RNA Extraction and RT-PCR. Total RNA was extracted from the cells using Qiagen RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Two μg of total RNA was used for RT-PCR according to standard methods. The primers used were: IL-6 sense: 5′ CCACACAGACAGCCACTCACC 3′; (SEQ ID NO:9) IL-6 antisense: 5′ TGGCATTTGTGGTTGGGTCAG 3′; (SEQ ID NO:10) IL-8 sense: 5′ GGTGCAGAGGGTTGTGGAGAA 3′; (SEQ ID NO:11) IL-8 antisense: 5′ GCAGACTAGGGTTGCCAGATT 3′; (SEQ ID NO:12) GAPDH sense: 5′ ATGGGGAAGGTGAAGGTCGGAGTC 3′; (SEQ ID NO:13) GAPDH antisense; 5′ GCTGATGATCTTGAGGCTGTTGTC 3′ (SEQ ID NO:14).
Staining for Mitochondria and OLD-35. Live cells were loaded with 200 nM MitoTracker (Molecular Probes, Eugene, Oreg.) for 30 min according to the manufacturer's protocol, fixed in 3.7% formaldehyde in PBS for 15 min and permeabilized in 0.1% Triton X-100 in PBS. Immunocytochemistry was then performed by standard methods using chicken anti-old-35 antibody (1:1000) and FITC-conjugated anti-chicken secondary anibody (Genetel). The cells were visualized and the images were analyzed using a Zeiss confocal laser scanning microscope (LSM510) with a ×100 objective.
Monitoring ROS Production. Cells were stained with 2.5 μM hydroethidine (HE) and 5 μM 5,6-carboxy-2′,7′-dichlorofluorescein diacetate (DCFH-DA) in PBS for 30 min in the dark (32). Immediately after staining the cells were analyzed by flow cytometry (FACScan; Becton Dickinson, Mountain View, Calif.) and data were analyzed using CellQuest software, version 3.1 (Becton Dickinson). The cells were gated to exclude cell debris. For inhibition experiments, NAC was added 2 hr post-infection.
Human IL-6 and IL-8 ELISA. The levels of IL-6 and IL-8 were quantified by ELISA in culture supernatants using human IL-6 ELISA kit (Pierce Biotechnology, Rockford, Ill.) and human IL-8 ELISA kit (Pierce), respectively, according to the manufacturer's protocol.
Human Cytokine Arrays. The expression levels of 36 cytokines following Ad.vec and Ad. hPNPaseold-35 infection were analyzed in culture supernatants using the TranSignal™ human cytokine antibody array (Panomics, Redwood city, CA) according to the manufacturer's protocol.
Statistical analysis. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Fisher's protected least significant difference analysis. A p value of <0.05 was considered as significant.
6.2 Results
Ad.hPNPaseold-35 Infection Generates OLD-35 Protein that Localizes to Mitochondria. The expression of OLD-35 in HeLa cells following Ad.hPNPaseold-35 infection was analyzed two days post-infection by Western blot analysis. As shown in
To confirm the subcellular localization of Ad.hPNPaseold-35-generated protein, cells were loaded with the mitochondria-specific dye MitoTracker and immunostained for OLD-35. Image analysis revealed that merging the signals generated from OLD-35 (green) and mitochondria (red) resulted in intense yellow color indicating overlapping distribution of the two signals (
Infection with Ad.hPNPaseold-35 Induces ROS. The levels of intracellular ROS following Ad.hPNPaseold-35 infection were determined using two dyes, DCFH-DA and HE. Nonfluorescent DCFH-DA diffuses into cells where it is deacetylated to DCF that fluoresces on reaction with hydrogen peroxide or nitrous oxide (32). HE enters the cells and can be oxidized by superoxide or free hydroxyl radicals to yield fluorescent ethidium (32). Infection with Ad.hPNPaseold-35 induced both DCFH-DA and HE fluorescence. Flow cytometry analysis of cellular fluorescence revealed a time-dependent increase in ROS production following Ad.hPNPaseold-35 infection in comparison to Ad.vec-infected cells (
ROS Mediates Activation of the NF-κB Pathway following Ad.hPNPaseold-35 Infection. To test whether Ad.hPNPaseold-35 infection activates the NF-κB pathway a luciferase-based reporter gene assay was employed. HeLa cells were either uninfected or infected with Ad.vec or Ad.hPNPaseold-35 and then transfected with either empty vector (pGL3Basic), 3κB-Luc containing 3 tandem NF-κB binding sites upstream of the luciferase gene or 3κBmut-Luc containing mutated NF-κB binding sites. As shown in
The activation of NF-κB upon Ad.hPNPaseold-35 infection was further analyzed by EMSA using radiolabeled consensus NF-κB binding site as a probe and nuclear extracts from HeLa cells. As shown in
To characterize the shifted bands, competition assays using excess amounts of cold wild type and mutated probes and supershift assays using antibodies against different subunits of NF-κB were employed. As shown in
To confirm that the induction of NF-κB by Ad.hPNPaseold-35 is specific and not mediated by non-specific events such as protein overload or additional adenoviral proteins, that might activate NF-κB, HeLa cells were infected with three additional replication incompetent adenovirus constructs expressing functionally different genes and NF-κB binding was analyzed by EMSA. The constructs included Ad.mda-5 that expresses mda-5, an interferon-inducible putative RNA helicase (33), Ad.mda-7 that expresses the apoptosis-inducing cytokine mda-7/IL-24 (34) and Ad.PEG-3, that express PEG-3 which is involved in tumor progression (35). As shown in
The involvement of ROS in mediating increased NF-κB DNA binding by Ad.hPNPaseold-35 was also evaluated by EMSA. As shown in
The levels of p50 and p65 subunits of NF-κB and its inhibitor IκBcc were analyzed in cytoplasmic and nuclear extracts following Ad.hPNPaseold-35 infection. As shown in
IL-6 and IL-8 are Induced by Ad.hPNPaseold-35 Infection. Expressions of mRNAs of IL-6 and IL-8, two NF-κB target genes, were analyzed by RT-PCR following Ad.hPNPaseold-35 infection. A time-dependent increase in the expressions of both IL-6 and IL-8 mRNAs occurred from two days post-infection with Ad.hPNPaseold-35, but not with Ad.vec infection (
ELISA assays quantified secretion of IL-6 and IL-8 protein following Ad.hPNPaseold-35 infection (
Analysis of Cytokine Expression Profiles following Ad.hPNPaseold-35 Infection. Since Ad.hPNPaseold-35 infection induced two potently pro-inflammatory cytokines, IL-6 and IL-8, the induction of other cytokines in culture supernatants two days post-infection were also tested using a human cytokine antibody array that analyzes the expression levels of 36 cytokines. Ad.hPNPaseold-35 infection had a very specific cytokine induction profile resulting in marked upregulation of IL-8, moderate elevation of IL-6 and TNFR1 and upregulation of RANTES and MMP-3 to a lesser extent (
6.3 Discussion
The foregoing demonstrates that OLD-35 activates the NF-κB pathway via the generation of ROS in HeLa cells. ROS is involved in the induction of a senescent phenotype characterized by irreversible growth arrest (10). Overexpression of Old-35 induces a senescence-like growth arrest and also generates ROS. Moreover, inhibition of ROS impedes the induction of NF-κB-responsive genes.
Contrasting results have been obtained regarding NF-κB binding activity during aging. No difference was observed in NF-κB binding between senescent and pre-senescent human fibroblasts as a function of in vitro replicative senescence (36). A reduced NF-κB activation in T cells from aged humans and mice has been reported (37). On the other hand, an increase in constitutive NF-κB DNA binding in older animals over young animals has been demonstrated in multiple studies (14-21). A gradual rise in ROS was evident in kidneys from Fischer rats from 6 to 24 months of age and this increase correlated with an age-dependent augmentation in binding of p50/p65 NF-κB, IκBot degradation, p65 nuclear translocation and elevated expression of cyclogenase-2 (COX-2), an NF-κB-responsive enzyme involved in pro-inflaimmatory prostanoid synthesis (16). Vascular smooth muscle cells from 18-month old rats showed considerably higher p50/p65 NF-κB DNA binding than that from new-born rats which correlated with increased expression of inducible nitric oxide synthase and intracellular adhesion molecule-1, two pro-inflammatory molecules, in old smooth muscle cells upon inflaimmatory stimulation (17). A similar age-dependent elevation in NF-κB DNA binding has been reported in mouse and rat liver and heart and in rat brain (14, 19). In these tissues an age-dependent rise in the levels of NF-κB subunits, such as p50, p52 and p65, could be observed. However, no change in the level of IκBr was detected. From these studies it might be inferred that tissue-specific regulatory mechanisms may be involved in NF-κB activation during senescence. Although NF-κB activation requires degradation of IκBα, IκBα itself is an NF-κB-responsive gene (22, 38). During acute activation of NF-κB by TNFα or related stimuli, there is an initial decrease in the cytoplasmic IκBα level followed by gradual restoration because of NF-κB-mediated transcription (38). Infection with Ad.hPNPaseold-35 resulted in a persistent decrease in the cytoplasmic IκBcc level, indicating that even though NF-κB is activated by OLD-35 there might be an additional regulatory mechanism of IκBc transcription during senescence as compared to acute stimuli. A recent study has shown the lack of involvement of ROS in NF-κB activation by acute stimuli such as TNFα. (39). It is possible that in a state of chronic oxidative stress, such as senescence, ROS plays a role in activating NF-κB. Another intriguing observation is the selective induction of NF-κB-responsive genes by OLD-35 (
What is the significance of induction of NF-κB-responsive genes by OLD-35 in the context of senescence? Ad.hPNPaseold-35 infection results in the upregulation of pro-inflammatory cytokines via activation of NF-κB. By turning on pro-inflammatory cytokines, NF-κB functions as a central transcription factor for the development of chronic inflammatory diseases (40). Gene expression analysis by microarray in human hepatic stellate cells confirms that replicative senescence in these cells is associated with a pronounced inflammatory phenotype characterized by upregulation of pro-inflammatory cytokines, including IL-6 and IL-8 (41). An aging-induced pro-inflammatory shift in cytokine expression profile has been observed in rat coronary arteries (42). Several studies have documented increased blood level of pro-inflammatory cytokines such as IL-1, IL-6, TNFα and IL-8 in aged individuals as compared to young individuals (43). The onset and course of a spectrum of age-associated diseases, such as cardiovascular disease, osteoporosis, arthritis, type 2 diabetes, Alzheimer's disease, certain cancers, periodontal disease, frailty and functional decline, might be associated with the production of pro-inflammatory cytokines (44, 45). Multiple studies have established an association between elevated levels of IL-6 and diseases of old age. IL-6 induces the production of C-reactive protein (CRP), an important risk factor for myocardial infarction (45). High concentrations of CRP predict the risk of future cardiovascular disease in apparently healthy men (45). IL-8 plays a crucial role in initiating atherosclerosis by recruiting monocytes/macrophages to the vessel wall, which promotes atherosclerotic lesions and plaque vulnerability (46). Elevated levels of IL-6 and CRP predict the development of type 2 diabetes in healthy women (47). In another study, elevated serum IL-6 levels predicted future disability in older adults especially by inducing muscle atrophy (48). IL-6 and CRP also play a pathogenic role in several diseases such as osteoporosis, arthritis and congestive heart failure all of which have increasing incidence with age (48). Moreover, increased serum levels of IL-6 and IL-8 have been detected in patients with chronic obstructive pulmonary diseases and chemokines such as IL-8 and RANTES play important roles in the pathogenesis of these diseases (49, 50). Various inflammatory mediators, such as IL-1, TNF-α, IL-6, IL-8, RANTES, MMP-3 are responsible for chronic inflammatory rheumatoid diseases, such as osteoarthritis and rheumatoid arthritis both of which occur during aging (51). The observation that the senescence-associated molecule OLD-35 induces pro-inflammatory cytokines which are intimately involved in the development of aging-associated diseases are consistent with involvement of OLD-35 in these pathological processes.
7.1 Materials and Methods
Cell lines and culture conditions: The human metastatic melanoma cell line HO-1 and the human embryonic kidney cell line HEK-293 were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 50 units/ml penicillin G and 50 μg/ml streptomycin at 37° C. in 5% CO2 and 100% relative humidity. HO-1 cells were cultured with IFN-β at a dose of 1000 OLD-35 units/ml for 2 days to detect hPNPase protein.
Plasmid Construction: All plasmids were constructed in a backbone of pcDNA3.1+(Hygro) (Invitrogen, Carlsbad, Calif.) into the NheI and BamHI sites and all the constructs contained a COOH-terminal Hemagglutinin (HA)-tag. phPNPaseold-35, expressing the full-length hPNPaseOLD-35 protein, was amplified by PCR using the cloned hPNPaseold-35 cDNA as a template (75) and primers sense (1) 5′-GCTAGCATGGCGGCCTGCAGGTAC-3′ (SEQ ID NO:33) and anti-sense (1) 5′-GGATCCTCAAGCGTAATCTGGAACATCGTATGGGTACTGAGAATTAGATG ATGA-3′ (SEQ ID NO:34). pΔRPH1 was created by PCR using primers sense (2) 5′-GCTAGCATGCCTTGGAATGGACCTGTTGGG-3′ (SEQ ID NO:35) and antisense (1). pΔRPH2 was cloned in two steps. First, a 3′ PCR fragment was amplified by PCR using primers sense (3) 5′-GTTAACATGGATTCAGGGGTTCCAATT-3′ (SEQ ID NO:36) and antisense (1) and ligated to pGEMT-easy vector (Promega, Madison, Wis.) by TA-cloning. This fragment was digested with HpaI and BamHI and ligated into HpaI and BamHI-digested phPNPaseold-35. pΔRPH1+2 was generated by PCR using primer sense (4) 5′-GCTAGCATGGATTCAGGGGTTCCAATT-3′ (SEQ ID NO:37) and antisense (1). pΔC-term was generated by PCR using primers sense (1) and antisense (2) 5′-GGATCCTCAAGCGTAATCTGGAACATCGTATGGGTACTGCAACAGCAGAT GAAATTGG-3′ (SEQ ID NO:38). The authenticity of all the constructs was confirmed by sequencing. The PCR fragments were first cloned into the vector pGEMT-easy by TA-cloning and then transferred to pcDNA3.1+(Hygro). The c-myc expression plasmid [p290-myc(2,3)] was provided by Dr. Riccardo Dalla-Favera (Columbia University Medical Center, N.Y.).
Virus Construction and Infection Protocol: The construction of hPNPaseold-35 expressing replication-defective Ad.hPNPaseold-35 was performed by cloning the transgene into a shuttle vector (p0TgCMV) and then performing homologous recombination of the shuttle vector with E1 and E3 region deleted parental adenoviral vector in E. coli as described previously (69, 75). A similar method was employed to generate Ad.ΔRPH1, Ad.ΔRPH2, Ad.ΔRPH1+2 and Ad.ΔC-term. The transgene was digested from the pGEMT-easy vector with NotI and ligated into the NotI site of p0TgCMV. The direction of the cloning was confirmed by restriction enzyme digestion and sequencing. The empty adenoviral vector (Ad.vec) was used as a control. The Ad was propagated in HEK293 cells (69), purified by BD AdenoX™ Virus Purification Kit (BD Biosciences, Palo Alto, Calif.) and viral titer was determined by measuring O.D. at 260 nm and using BD AdenoX™ rapid titer kit (BD Biosciences). Ad infection was performed 24 h after cell plating in one-fifth the volume of the original culture medium in a serum-free condition for 2 h with rocking the plates several times (69).
Colony formation assays: HO-1 cells were plated at a density of 3×105 cells per 6-cm dish and 24 h later were infected with different Ad at an m.o.i. of 10, 20 and 50 pfu/cell. For colony formation assays with c-myc overexpression, HO-1 cells were plated at a density of 3×105 cells per 6-cm dish and 24 h later were transfected with 5 μg of either empty vector or p290-myc(2,3) using Superfect® (Qiagen, Hilden, Germany) transfection reagent according to the manufacturer's protocol. After 36 h, the cells were infected with different Ad at an m.o.i. of 50 pfu/cell. Six h after infection, the cells were trypsinized, counted and 1 cells were plated in 6-cm dishes. Colonies>50 cells were counted after 2 weeks.
Western Blot Analysis. Western blotting was performed as previously described (84). Briefly, cells were harvested in RIPA buffer (1×PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitor cocktail (Roche, Mannheim, Germany), 1 mM Na3VO4 and 50 mM NaF and centrifuged at 12,000 rpm for 10 min at 4° C. The supernatant was used as total cell lysate. Thirty μg of total cell lysate were used for SDS-PAGE and transferred to a nitrocellulose membrane. The primary antibodies included: from Santa Cruz Biotechnology, Santa Cruz, Calif.: Myc (1:200; mouse monoclonal), Max (1:200; rabbit polyclonal), Mad1 (1:200; rabbit polyclonal), p21 (1:200; rabbit polyclonal), p27 (1:200; rabbit polyclonal), CDK2 (1:250; rabbit polyclonal); from BD Biosciences: p16 (1:500; mouse monoclonal), Rb (1:500, mouse monoclonal), actin (1:1000, mouse monoclonal), cytochrome c (1:1000, mouse monoclonal), cyclin B1 (1:5000; mouse monoclonal); anti-hPNPaseOLD-35 (1:10,000; chicken polyclonal) (82); anti-HA (1:1000; mouse monoclonal; Covance Research Products, Inc, Berkeley, Calif.); and EF1α(1:1000; mouse monoclonal; Upstate Biotechnology, Waltham, Mass.).
Immunofluorescence analysis: HO-1 cells were plated on chamber slides (Falcon 4102; Becton Dickinson; Franklin Lakes, N.J.) and infected with different Ad. After 36 h, the cells were loaded with 250 mM MitoTracker (Molecular Probes, Eugene, Oreg.) for 30 min at 37° C., fixed with 3.7% formaldehyde for 15 min at 37° C. and permeabilized with 0.1% Triton-X-100 in PBS for 5 min at room temperature (RT). The cells were blocked with PBS containing 10% normal rabbit serum for 2 h at RT and incubated in the blocking solution containing anti-HA antibody (1:200) overnight at 4° C. After washing in PBS, the cells were incubated in the blocking solution containing anti-mouse-FITC (1:200) for 2 hi at RT, washed again in PBS, mounted and visualized using a Zeiss confocal laser scanning microscope (LSM510) and a 40× objective. In case of double immunofluorescence studies for hPNPaseOLD-35 and p27KIP-1, HO-1 cells were plated on chamber slides, infected with Ad.hPNPaseold-35 and 48 h later they were fixed, permeabilized and blocked with PBS containing 5% normal goat serum. The cells were incubated first with anti-p27 antibody and Alexa Fluor 594 (red) goat anti-rabbit IgG (Molecular probes) followed by anti-hPNPaseOLD-35 antibody and rabbit anti-chicken-FITC (Genetel Laboratories, Madison, Wis.). Image analysis was performed using a Zeiss confocal laser scanning microscope (LSM510) and a 40× objective.
Cell Fractionation: 2×107 cells were harvested by trypsinization and mitochondrial and cytoplasmic fractions were separated using a Mitochondria Isolation kit (Pierce, Rockford, Ill.) according to the manufacturer's protocol.
Assay for Senescence Associated β-galactosidase (SA-β-gal) activity: SA-β-gal activity was assayed 4 days after infection with different Ad at an m.o.i. of 50 pfu/cell (61). The cells were fixed with 2% formaldehyde+0.2% glutaraldehyde and then stained with X-gal (1 mg/ml) in 40 mM citric acid/Na phosphate buffer (pH 6.0) containing 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl and 2 mM MgCl2. After the development of color the cells were washed with PBS and methanol, air-dried and micro-photographed. Quantification of SA-β-gal positive cells was determined by counting at least 1,000 cells for each group.
Cell cycle analysis: Cell cycle was analyzed at 1, 2 and 3 days post-infection. Cells were harvested, washed in PBS and fixed overnight at −20° C. in 70% ethanol. Cells were treated with RNase A (1 mg/ml) at 37° C. for 30 min and then with propidium iodide (50 μg/ml). Cell cycle was analyzed using a FACS Calibur flow cytometer, and data was analyzed using CellQuest software (Becton Dickinson, San Jose, Calif.).
Cell sorting analysis: HO-1 cells were transfected with either empty vector or c-myc expression plasmid and infected the next day with either Ad.vec or Ad.hPNPaseold-35 at an m.o.i. of 50 pfu/cell. Two days after infection, live cells were incubated with 5 μg/ml of Hoechst 33342 (Molecular Probes) for 1 h in the dark. After trypsinization, cells were resuspended at a concentration of 1×107 cells/ml for sorting. Cells were sorted based on the amount of DNA by defining three regions for sorting: one for G1, one for S and one for G2+M using BDFACSAria (BD Biosciences) equipped with a UV laser required for Hoechst 33342 excitation. The separated cells (at least 1×106 cells from each sorted population) were collected, protein was extracted and Western blot analysis was performed.
RNA Isolation and Northern Blot Analysis: Total RNA was extracted from the cells using Qiagen RNeasy mini kit (Qiagen) according to the manufacturer's protocol and Northern blotting was performed as described (84). The cDNA probes used were a 500-bp fragment from human c-myc, full-length human GADD34 and full-length human GAPDH.
In vitro Translation and in vitro mRNA Degradation Assays: In vitro translation was performed using the TNT coupled Reticulocyte Lysate Systems (Promega) using the plasmids pcDNA3.1+(Hygro) as a control, GADD153 expression plasmid, phPNPaseold-35, pΔRPH1, pΔRPH2, pΔRPH1+2 and pΔC-term according to the manufacturer's protocol. Five fig of total RNA from HO-1 cells were incubated with 5 μl of each in vitro translated protein at 37° C. from 0.5 to 2 h. The RNA was repurified using the Qiagen RNeasy mini kit (Qiagen) and Northern blotting was performed.
CDK Activity Assay: Cells were harvested in RIPA buffer and 500 μg of protein were incubated overnight at 4° C. with anti-CDK2 antibody and then with protein A agarose for 1 h at 4° C. The agarose beads were spun down at 10,000 g for 5 min, washed three times in RIPA buffer and once in kinase buffer. The immunoprecipitated material was employed for kinase assays using Histone H1 (Upstate Biotechnology) as substrate and [γ-32P]ATP (Amersham, Piscataway, N.J.) in a kinase buffer containing 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 18 mM MgCl2 and 1 mM DTT for 30 min at 30° C. Following the reaction the samples were subjected to 15% SDS-PAGE, the gel was dried, exposed to x-ray film and densitometric analysis was performed.
Statistical analysis: Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Fisher's protected least significant difference analysis.
7.3 Results
Polynucleotide phosphorylases are highly conserved across species ranging from bacteria, plants to mammals (74). They have a conventional structure containing RNAse PH domains and RNA binding domains (88, 89).
In order to comprehend the involvement of these domains in mediating the hPNPaseold-35-induced senescent phenotype, a number of deletion mutants were created and replication-incompetent adenoviruses (Ad) expressing these deletion mutants were generated (
It was considered important to initially determine if the level of expression of hPNPaseold-35 resulting from adenoviral delivery of this gene was comparable to the level of endogenous protein induced following IFN-β treatment. To address this issue, HO-1 cells were infected with either Ad.vec (control empty Ad) or with Ad.hPNPaseold-35 at an m.o.i. of 50 pfu/cell or treated with 1000 units/ml of IFN-βand the expression of hPNPaseOLD-35 was analyzed two days later by Western blot analysis using anti-hPNPaseold-35 antibody (82). Treatment with IFN-β resulted in marked induction of hPNPaseOLD-35 and the level of the protein generated upon Ad.hPNPaseold-35 infection was ˜2-fold more than that with IFN-β treatment (
To analyze the involvement of the different domains in hPNPaseold-35-induced growth inhibition and senescence, HO-1 human melanoma cells were infected with the different Ad at an m.o.i. of 10, 20 and 50 pfu/cell and colony formation assays were performed. As a control, cells were either uninfected or infected with Ad.vec at an m.o.i. of 50 pfu/cell. As shown in
To determine the senescence-inducing properties of these deletion mutants, HO-1 cells were infected with the different Ads and monitored for changes in cell morphology and senescence-associated β-galactosidase (SA-β-Gal) activity, a characteristic marker of senescence (61). Infection with Ad.hPNPaseold-35, Ad.ΔRPH1, Ad.ΔRPH2 and Ad.ΔC-term, but not Ad.vec and Ad.ΔRPH1+2, resulted in typical morphological changes in HO-1 cells (
Senescence is associated with arrest of cell cycle especially at the G1 phase, with a decrease in the S phase indicative of inhibition of DNA synthesis (86). Cell cycle analysis was performed in HO-1 cells infected with the different Ads. A time-dependent change in the cell cycle pattern was observed (
We next investigated the expressions of proteins that regulate the progression of the cell cycle beyond the G1 phase by Western blot analysis. There was a significant increase in p27KIP1 and a decrease in p21CIP1/WAF-1/MDA-6 upon infection with Ad.hPNPaseold-35, Ad.ΔRPH1, Ad.ΔRPH2 and Ad.ΔC-term, but not with Ad.vec and Ad.ΔRPH1+2 (
It was documented previously that downregulation of c-myc plays a significant role in mediating hPNPaseold-35-induced growth inhibition (83). Considering this possibility, the involvement of the domains of hPNPaseold-35 in regulating c-myc expression was analyzed. Infection with Ad.hPNPaseold-35, Ad.ΔRPH1, Ad.ΔRPH2 and Ad.ΔC-term, but not with Ad.vec and Ad.ΔRPH1+2, resulted in ˜50% reduction in c-myc mRNA level 3 days post-infection (
To support the observations of these expression studies, c-myc was overexpressed in HO-1 cells and these cells were infected with the different Ads and colony-forming ability was determined. Infection with Ad.hPNPaseold-35, Ad.ΔRPH1, Ad.ΔRPH2 and Ad.ΔC-term decreased colony formation by 75%, 73%, 79% and 74%, respectively in comparison to infection with Ad.vec and Ad.ΔRPH1+2 (
hPNPaseold-35 is a 3′, 5′ exoribonuclease (75), so we investigated whether the downregulation of c-myc is a consequence of direct degradation of c-myc mRNA by hPNPaseold-35. hPNPaseOLD-35 and its different deletion mutants were in vitro translated and the proteins were used for RNA degradation assays. As shown in
To establish a direct correlation between cell cycle arrest in the G1 phase and c-myc downregulation by hPNPaseOLD-35 HO-1 cells were sorted following Ad.hPNPaseold-35 infection and the expressions of hPNPaseOLD-35 and Myc were analyzed in the different phases of the cell cycle (
The presence of the mitochondrial localization signal indicates that hPNPaseOLD-35 is a predominantly mitochondrial protein (79, 82). The question naturally arises as to how a mitochondrial protein might degrade a cytoplasmic mRNA, like c-myc. To address this question, cells infected with different Ad were fractionated into mitochondrial and cytoplasmic fractions and the expressions of the proteins were analyzed by Western blotting using anti-HA antibody. As a control for the quality of the purification, membranes were reprobed with anti-actin antibody (for cytoplasmic fraction) and anti-cytochrome C antibody (for mitochondria). hPNPaseOLD-35, ΔRPH2 and ΔC-term showed predominantly mitochondrial expression resulting from the presence of the mitochondrial localization signal in these constructs (
The fractionation results were confirmed by immunofluorescence studies using anti-HA antibody (green) and MitoTracker (red) to determine the localization of hPNPaseOLD-35 and its deletion mutants (
7.4 Discussion
In this study, the importance of the RPH domains in mediating the characteristic phenotypic changes induced by hPNPaseold-35 is firmly established. We observed that presence of at least one RPH domain is required for the functional activity of the protein and either of the domains is as potent as the full-length molecule. This contrasts with bacterial PNPase in which mutation in the key residues in either of the RPH domains inhibits catalytic activity (70). However, our result is comparable to chloroplast PNPase in which the first RPH domain alone (comparable to our ΔRPH2 construct) is highly active enzymatically (91). Although the second RPH domain (our ΔRPH1) of chloroplast has low RNA degradation activity of non-polyadenylated RNA it has high activity for polyadenylated RNA (91) which explains the efficiency of the ΔRPH1 construct in degrading human polyadenylated mRNAs. The RPH domains themselves can bind to RNA and the PNPase domain is also involved in RNA binding which explains the preservation of the RNA degradation activity of the AC-term construct that lacks the KH and S1 RNA binding domains. Current studies are determining the effects of mutation in specific important and evolutionary conserved amino acid residues of hPNPaseold-35, especially those surrounding the tungsten binding region implicated in the enzymatic activity of the bacterial protein (70).
A unique and potentially significant finding is that hPNPaseOLD-35 displays specific degradation activity for c-myc mRNA. The 3′ stem-loop structure of a RNA species determines the enzymatic activity of PNPases (91). It is of significant interest to see whether there exists a difference between the 3′ secondary structure of c-myc and other mRNAs, such as GAPDH and GADD34, that facilitates its degradation by hPNPaseold-35. PNPases function as a homotrimer (53) and our findings suggest that hPNPaseOLD-35 also multimerizes. However, the deletion mutants, that do not multimerize, still retain their specific mRNA degradation activity. We can rule out the involvement of a c-myc specific RNA binding protein regulating the activity of hPNPaseOLD-35, since our in vitro RNA degradation assays, which contained a single protein, could effectively achieve RNA degradation. Elucidation and comparison of three-dimensional models of c-myc and other mRNAs might provide insights into the specific RNA degradation activity of hPNPaseold-35.
Structure analysis and localization studies reveal that hPNPaseOLD-35 is a predominantly mitochondrial protein (79, 82), thereby provoking the obvious question as to how hPNPaseOLD-35 might degrade a cytoplasmic target mRNA, such as c-myc. By fractionation and immunofluorescence analyses, we demonstrate that although the primary site of localization of hPNPase OLD-3 is the mitochondria, a considerable amount of the protein is also present in the cytoplasmic fraction of the cell. Similarly, ΔRPH2 and ΔC-term, that retain the mitochondrial localization signal, are also mainly localized in mitochonidria, although low but detectable levels are still found in the cytoplasm. ΔRPH1 and ΔRPH1+2, lacking the mitochondrial localization signal, were located in the cytoplasm although a very low level of ΔRPH1 was also evident in the mitochondria. This is not because of cross-contamination which was confirmed by mutually exclusive expression of cytochrome c and actin in mitochondria and cytoplasmic fractions, respectively. These findings suggest the existence of a potential cytoplasmic-mitochondrial shuttling of the hPNPaseOLD-35 protein that might be regulated by chaperone proteins. Further studies designed to identify and define interacting partners of hPNPaseOLD-35 will help address this important question.
Adenovirus-mediated delivery usually results in robust transgene expression that raises the important question of whether the senescence-inducing effect of Ad.hPNPaseold-35 is a genuine physiological phenomenon. We have observed that there is a comparable level of hPNPaseOLD-35 protein upon IFN-β treatment and with Ad.hPNPaseold-35 infection suggesting that the senescent phenotype observed with hPNPaseOLD-35 is indeed a physiologically relevant event. Bacterial PNPase autocontrols its expression post-transcriptionally by degrading its own mRNA (71). Although not yet confirmed for hPNPaseold-35, this post-transcriptional control might explain the restricted expression level of hPNPaseOLD-35 even with adenovirus-mediated delivery of this enzyme.
CDKIs, especially p16INK4A and p21CIP1/WAF-1/MDA-6 are intimately involved in the process of cellular senescence (52, 87). hPNPaseold-35-induced senescence is associated with an increase in p27KIP1 and a decrease in p21CIP1/WAF-1/MDA-6, a phenomenon that is observed in two other models of senescence-like growth arrest, one resulting from iron chelation in hepatocytes, and the other a consequence of inhibition of the phosphoinositide-3-kinase pathway in mouse embryo fibroblasts (57, 92). The involvement of p16 INK4A is ruled out as an essential component of this process because HO-1 cells do not express the p16INK4A protein. The increase in p27KIP1 is most likely secondary to the decrease in c-myc that controls p27KIP1 expression at multiple levels, including repression of transcription and facilitation of ubiquitination and subsequent degradation (77, 78, 90). Myc plays an important role in controlling cell cycle (65). It promotes entry into the S phase and shortens the G1 phase. We observed that downreguation of c-myc by hPNPaseold-35 resulted in a corresponding increase in Mad1 and this particular shift from MYC-MAX transcriptional activator to MAD1-MAX transcriptional repressor might be important for mediating the plethora of effects promulgated by hPNPaseold-35. However, since overexpression of Myc could not provide complete protection against hPNPaseold-35-mediated growth inhibition, additional targets of hPNPaseold-35 are likely to exist that might be involved in mediating its effects. While the effect of hPNPaseold-35 on c-myc is direct, its effect on MAD1 is probably indirect via additional targets, downregulation of which might lead to derepression of MAD1 expression.
In summary, we now confirm the importance of the RPH domains in hPNPase in inducing senescence, an event that is unique in its molecular mechanism and is critical for regulating organismal homeostasis. These studies provide unique perspectives on the molecular mechanism of senescence and the structure-function relationship of the hPNPaseOLD-35 protein. Crystallization of the hPNPaseold-35 protein and comparison of its crystal structure to that of the PNPase proteins of other species will directly facilitate identification of important residues mediating catalytic and senescence-inducing activity. Of added significance, the development of an animal model conditionally overexpressing hPNPaseold-35 will provide valuable insights into the involvement of hPNPaseold-35 in in vivo senescence.
8.1 Materials and Methods
Cell lines and cell viability assays: FM516-SV (referred to as FM516) normal immortal human melanocyte, WM35 early radial growth phase (RGP) primary human melanomas, HO-1 and MeWo metastatic melanomas, 2fTGH human fibrosarcoma and its derivates U1A, U3A, U4A and U5A, HeLa human cervical carcinoma and 293 adenovirus transformed human embryonic kidney (HEK 293) cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and penicillin (100 U/ml) and streptomycin (100 μg/ml). 2fTGH cells are wild type in IFN signaling while its derivates U1A, U3A, U4A and U5A have defects in IFN signaling that could be complemented by expression of TYK2, STAT1, JAK1 or IFNAR2, respectively (112, 113). Cell growth and viable cell numbers were monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) staining as described (150).
Generation of lentivirus expressing siRNA for hPNPaseold-35: Using the software siRNA Target Finder (Ambion, Austin, Tex.) four potential siRNAs for hPNPaseold-35 were designed and the siRNAs were constructed by in vitro transcription using the Silencer siRNA construction kit (Ambion) according to the manufacturer's protocol. These siRNAs were transfected into HeLa cells using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) according to the manufacturer's protocol and the next day the cells were treated with 1000 U/ml IFN-β for 24 h. The expression of hPNPaseOLD-35 in the lysates of these cells was analyzed by Western blot analysis. The siRNA demonstrating the maximum inhibition of hPNPaseOLD-35 induction by IFN-β was selected for construction of the lentivirus. The hPNPaseold-35 and control siRNA sequences were 5′ AACAAAACCTTCCCCTTCCCA 3′ (SEQ ID NO:40) and 5′ AAGGGTCGTCTATAGGGATCGAT 3′ (SEQ ID NO:41), respectively. Lentiviruses expressing either control siRNA or hPNPaseold-35 siRNA were constructed using BLOCK-iT Lentiviral RNAi Expression System (Invitrogen) according to the manufacturer's protocol. The siRNA was first ligated into BLOCK-iT U6 RNAi Entry Vector that drives expression of the siRNA under control of the human U6 promoter. The siRNA expression cassette was transferred to pLenti6 BLOCK-iT-DEST lentiviral vector by the LR recombination reaction. The resultant construct was transfected into HEK293FT cells with Lipofectamine 2000 along with ViraPower lentiviral packaging mix that expresses the proteins required for lentivirus replication. The lentivirus was amplified and titered by standard plaque assay.
Generation of stable cell clones: Stable clones of HO-1 cells expressing either control siRNA or hPNPaseold-35 siRNA were generated by transducing the cells with lentiviruses expressing the corresponding siRNA and selecting clones for 2 weeks using 4 μg/ml blasticidin. Stable HO-1 clones expressing c-myc were generated by transfecting HO-1 cells with a c-myc expression vector and selecting the cells for 2 weeks with 100 μg/ml hygromycin. Transfecting the cells with empty vector and selecting with hygromycin generated corresponding control clones.
Cell Cycle Analysis: Cells were harvested, washed in PBS and fixed overnight at −20° C. in 70% ethanol. The cells were treated with RNase A (1 mg/ml) at 37° C. for 30 min and then with propidium iodide (50 μg/ml). Cell cycle was analyzed using a FACScan flow cytometer and data were analyzed using CellQuest software (Becton Dickinson, San Jose, Calif.).
Colony formation assays: One-thousand cells were plated in 6-cm dishes and then treated with different doses of IFN-β for 2 weeks at which point the colonies were fixed, stained with Giemsa and colonies≧50 cells were counted.
Transfection of siRNA: Cells (5×105) were plated in a 6 cm dish and the next day were transfected with 25 nM of either control siRNA or c-myc siRNA (Ambion; catalogue# 4250) using Lipofectamine 2000 (Invitrogen) according to the Manufacturer's protocol. After 24 h, the cells were trypsinized and seeded into 96-well plates for cell viability assays and 6-cm dishes for colony formation assays and cell cycle analyses as described above.
RNA Isolation and Northern Blot Analysis: Total RNA was extracted from cells using Qiagen RNeasy mini kit (Qiagen) according to the manufacturer's protocol and Northern blotting was performed as described (102). The cDNA probes used were a 400-bp fragment from human c-myc, a 500-bp fragment from hPNPaseold-35 and full-length human GAPDH. For analysis of half-life of c-myc mRNA, cells were either untreated or treated with IFN-β (1000 U/ml) for 24 h and then treated with Actinomycin D (5 μg/ml) for 0.5, 1, 2, 4 and 8 h following which the cells were harvested for total RNA extraction and Northern blot analysis.
Western Blot Analysis: Western blotting was performed as previously described (102). Briefly, cells were harvested in RIPA buffer containing protease inhibitor cocktail (Roche, Mannheim, Germany), 1 mM Na3VO4 and 50 mM NaF and centrifuged at 12,000 rpm for 10 min at 4° C. The supernatant was used as total cell lysate. Thirty μg of total cell lysate were used for SDS-PAGE and transferred to a nitrocellulose membrane. The primary antibodies included: Myc (1:200; mouse monoclonal; Santa Cruz biotechnology, Santa Cruz, Calif.), hPNPaseOLD-35 (1:10000; chicken polyclonal), MDA-5 (1:5000; rabbit polyclonal) and EF1α (1:1000; mouse monoclonal; Upstate Biotechnology, Waltham, Mass.).
Statistical analysis: Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Fisher's protected least significant difference analysis.
8.2 Results
Regulation of hPNPaseold-35 and c-myc mRNA expression by IFN-β: To define a potential correlation between the expression regulation of hPNPaseold-35 and c-myc by IFN-β, HO-1, WM35 and MeWo human melanoma cells and SV40 T/t Ag-immortalized human melanocytes (FM-516-SV, here forth indicated as FM-516) were treated with 1000 U/ml of IFN-β for different times ranging from 12 to 48 h and the expression of hPNPaseold-35 and c-myc mRNA was determined by Northern blot analysis (
The mRNA expression results were confirmned on a protein level by Western blot analysis (
A direct correlation in IFN-β-induced dose-dependent changes in hPNPaseOLD-35 and Myc proteins was also evident. In HO-1 and WM-35 cells, hPNPaseOLD-35 induction and Myc downregulation were detected with 100 and 1000 U/ml of IFN-β, but not with 1 or 10 U/ml (
The regulation of expression of hPNPaseOLD-35 and Myc by IFN-β was confirmed in 2fTGH human fibrosarcoma cells and in its four variants. U1A (Tyk2-), U3A (STAT1-), U4A (JAK1-) and U5A (IFNAR2-), that have mutations in different molecules involved in the IFN-signaling pathway (112, 113). As shown in
IFN-β its growth of melanoma cells and melanocytes: The effect of IFN-β on the growth of HO-1, WM35, MeWo and FM-516 cells were analyzed by standard MTT cell survival assays (
The results obtained using cell viability assays were confirmed by colony formation assays (
Cell cycle analysis was performed to characterize growth inhibition. IFN-β treatment (1000 U/ml) in HO-1, WM-35, MeWo and FM-516 cells resulted in initial (day 1) cell cycle arrest in the G1 phase of the cell cycle with a concomitant decrease in the DNA synthesis phase as substantiated by the reduction in S phase (
hPNPaseOLD-35 regulates IFN-β-mediated downregulation of Myc: Since hPNPaseOLD-35 is a 3′, 5′ exoribonuclease and one of its substrates is c-myc mRNA we tested whether hPNPaseOLD-35, induced by IFN-β, promotes Myc downregulation. We have identified siRNA active in downregulating hPNPaseold-35 and created a lentivirus expressing this siRNA. Stable clones in an HO-1 background expressing either control siRNA or hPNPaseold-35 siRNA were generated by selection with blasticidin. As shown in
The half-life of c-myc mRNA with or without IFN-β treatment was analyzed in HO-1 cells and control siRNA and hPNPaseold-35 siRNA expressing clones (
Resistance of hPNPaseold-35-siRNA clones to IFN-β-mediated growth inhibition: Overexpression of hPNPaseold-35 induces growth inhibition and apoptosis in melanoma cells and c-myc is a positive regulator of cell growth, allowing cells to traverse the G1 phase of the cell cycle. Based on these considerations, we tested whether the lack of these two events in hPNPaseold-35-siRNA expressing clones would render them resistant to IFN-β-mediated growth inhibition. As shown in
The results obtained from cell survival and colony formation assays were confirmed by cell cycle analysis using flow cytometry. As shown in
To confirm that the mechanism underlying the resistance of hPNPaseold-35-siRNA expressing clones to IFN-β is mediated by their inability to downregulate c-myc, HO-1 cells and control siRNA and hPNPaseold-35-siRNA expressing clones were transfected with either control or c-myc siRNA and treated with IFN-β and cell viability, colony formation and cell cycle analyses were performed. Transfection of c-myc siRNA resulted in marked downregulation of Myc protein (
Resistance of c-myc overexpressing clones to IFN-β-mediated growth inhibition: We next evaluated the involvement of c-myc downregulation in IFN-β-mediated growth inhibition. For this purpose, stable Myc overexpressing HO-1 clones (HO-1-Myc) were developed by transfection with a c-myc expression vector and selection with hygromycin. Control hygromycin-resistant clones (HO-1-Hygro) were similarly generated.
These interesting findings were corroborated by cell cycle analysis. Treatment with IFN-β induced an initial G1 arrest and eventually apoptosis in HO-1-Hygro clones (
8.3 Discussion
Microarray studies have revealed a plethora of genes that are modulated by IFN treatment (115) (http://bioinfo.cnio.es/data/oncochip/). IFNs can directly affect gene expression by ISRE and GAS sequences in the promoters of target genes (97). In addition, IFNs can also affect gene expression by their ability to induce proteins involved in RNA metabolism, such as 2′,5′-oligoadenylate synthetase/RNase L, double stranded RNA-dependent protein kinase (PKR), melanoma differentiation associated gene-5 (mda-5), retinoic acid inducible gene-I (RIG-I) and hPNPaseold-35 (97, 99, 114, 116). Inhibition of gene expression by IFNs at a post-transcriptional level has been described for the heavy chain of immunoglobulin mu (117), the IL-4 receptor (118) and c-myc (110, 111), the focus of the present studies.
The observation that type I IFN selectively reduces c-myc mRNA has been described in multiple studies using several model cell culture systems. Jonak and Knight first hypothesized that IFN-β mediated downregulation of c-myc mRNA might mediate growth inhibition in Daudi human lymphoblastoid cells (110). As a follow-up study, Dani et al. demonstrated in Daudi cells that IFN c/5 did not affect the transcription rate of c-myc mRNA, but rather reduced the half-life of this mRNA (111). A posttranscriptional destabilization of c-myc mRNA as a mechanism of type I IFN-mediated c-myc suppression has also been described in colon carcinoma cells (119). It was shown that during terminal differentiation of hematopoietic cells, autocrine IFN-β induces c-myc suppression and induces G0/G1 arrest in these cells (120). Additionally, previous studies from our laboratory documented that IFN-β and mezerein-induced terminal differentiation of human melanoma cells also correlated with downregulation of c-myc mRNA (121). Moreover, studies in different cell types consistently describe the ability of type I IFN to reduce c-myc expression. However, the effect of IFN-γ on c-myc expression varies in different cell contexts. In HeLa cells, treatment with IFN-α decreased while IFN-γ increased c-myc expression (122). In a murine myeloid cell line, IFN-γ inhibited c-myc gene expression by impairing the splicing process (123). Another report described the importance of Stat-1 in IFN-γ-mediated downregulation of c-myc. Studies employing wild type and Stat-1-null mouse embryonic fibroblasts identified a gamma activated sequence element in the c-myc promoter that was necessary, but not sufficient, to suppress c-myc expression in wild type cells (124).
Although a consensus exists that type I IFNs induce post-transcriptional modulation of c-myc mRNA, the molecular mechanism underlying this process is unclear. Different components of IFN-inducible RNA degradation machinery have been implicated in this action. In colon carcinoma cells, 2′,5′-oligoadenylate synthetase/RNase L system is believed to regulate IFN-β-mediated post-transcriptional processing of c-myc mRNA (119). In M1 murine myeloid leukemia cells, PKR has been shown to mediate type I IFN-induced c-myc suppression (125). A recent report indicates that in mouse monocyte/macrophage leukemia cells, IFN-β reduces steady state levels of Myc protein by increasing degradation through the 26S proteasome (126). In our previous studies, we revealed for the first time by employing recombinant hPNPaseOLD-35 protein in in vitro mRNA degradation assays that a type I IFN-inducible exoribonuclease, hPNPaseold-35, could selectively degrade c-myc mRNA (102, 107). In the present studies, we now confirm that hPNPaseOLD-35 is the enzyme responsible for IFN-β-mediated degradation of c-myc mRNA in human melanoma cells.
Myc is an important regulator of cell proliferation (127). Expression of exogenous Myc in cultured fibroblasts promotes S-phase entry and shortens the G1 phase of the cell cycle, while activation of a conditional Myc is sufficient to drive quiescent cells into the cell cycle (128, 129). An association between c-myc downregulation and IFN-α-mediated G0/G1 arrest in Daudi cells was demonstrated (130) and it was shown that IFN-α-induced G0/G1 arrest correlated with upregulation of cyclin-dependent kinase inhibitors (CDKI), such as p21 and p15 early in this process and p27 in the late stage of growth arrest (131). Type I IFN treatment of Daudi cells induced p21 expression and G1 arrest and these events were preceded by a strong reduction in c-myc levels (132). Myc can directly suppress the transcription of p21 and p27 and promote the ubiquitination of phosphorylated p27 (133-135). Our previous experiments confirm that overexpression of hPNPaseold-35 downregulates c-myc and upregulates p27 (102) and in the present study we document that inhibition of hPNPaseold-35 as well as overexpression of c-myc protects melanoma cells from IFN-β-mediated G1 arrest. These findings firmly establish functional and mechanistic links between hPNPaseold-35 induction by IFN-β, c-myc mRNA degradation by hPNPaseold-35 and IFN-β-induced cell cycle arrest and eventual apoptosis (
What is the practical significance of our observations? Myc is overexpressed in multiple tumor subtypes, including melanomas (127). The expression level of Myc inversely correlates with patient survival and thus may be used as a prognostic marker in cutaneous, subungual, acral lentigious, scalp, head and neck melanomas (136-141). Indeed, antisense inhibition of c-myc significantly inhibited the growth of melanoma cells in in vitro cultures (142) and improved the response to chemotherapy in human melanoma xenografts in nude mice (143). Type I IFNs have been used as adjuvant therapy for malignant melanoma with significant but limited success and high toxicity (144). Experimental overexpression of Myc in mouse fibroblasts and myeloblastic cells renders these cells resistant to cell cycle arrest by type I IFNs (145, 146). Uveal melanomas with high c-myc expression are also associated with IFN-α resistance (147). In these contexts and based on the poor survival of patients with malignant melanoma, improved therapies are mandated and cancer cell specific expression of hPNPaseold-35, by means of the telomerase or progression elevated gene-3 promoter (148, 149), might prove beneficial as an innovative adjuvant therapeutic approach that exploits the ability of hPNPaseold-35 to degrade c-myc mRNA, thus inducing target cancer cell-specific growth arrest culminating in apoptosis.
Various publications are cited herein, the contents of which are hereby incorporated by reference in their entireties.
This application claims priority to U.S. Provisional Patent Application No. 60/616,774 filed Oct. 7, 2004 which is incorporated by reference in its entirety herein.
Number | Date | Country | |
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60616774 | Oct 2004 | US |
Number | Date | Country | |
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Parent | PCT/US05/36409 | Oct 2005 | US |
Child | 11784096 | Apr 2007 | US |