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Antiproliferative Activity of G-rich Oligonucleotides Correlates with Protein Binding*

  • Paula J. Bates
    Affiliations
    Department of Medicine, Division of Hematology/Oncology, University of Alabama at Birmingham, Birmingham, Alabama, 35294-3300
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  • Jasbir B. Kahlon
    Affiliations
    Department of Medicine, Division of Hematology/Oncology, University of Alabama at Birmingham, Birmingham, Alabama, 35294-3300
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  • Shelia D. Thomas
    Affiliations
    Department of Medicine, Division of Hematology/Oncology, University of Alabama at Birmingham, Birmingham, Alabama, 35294-3300
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  • John O. Trent
    Affiliations
    Department of Medicine, Division of Hematology/Oncology, University of Alabama at Birmingham, Birmingham, Alabama, 35294-3300
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  • Donald M. Miller
    Correspondence
    To whom correspondence should be addressed: 520 Wallace Tumor Institute, 1824 Sixth Ave. South, Birmingham, AL 35294-3300.
    Affiliations
    Department of Medicine, Division of Hematology/Oncology, University of Alabama at Birmingham, Birmingham, Alabama, 35294-3300
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  • Author Footnotes
    * This work was supported in part by the U. S. Public Health Service, the Veterans' Administration, and the U. S. Army.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:September 10, 1999DOI:https://doi.org/10.1074/jbc.274.37.26369
      Oligonucleotides have been extensively studied as antisense or antigene agents that can potentially modulate the expression of specific genes. These strategies rely on sequence-specific hybridization of the oligonucleotide to mRNA or genomic DNA. Recently, it has become clear that oligonucleotides often have biological activities that cannot be attributed to their sequence-specific interactions with nucleic acids. Here we describe a series of guanosine-rich phosphodiester oligodeoxynucleotides that strongly inhibit proliferation in a number of human tumor cell lines. The presence of G-quartets in the active oligonucleotides is demonstrated using an UV melting technique. We show that G-rich oligonucleotides bind to a specific cellular protein and that the biological activity of the oligonucleotides correlates with binding to this protein. The G-rich oligonucleotide-binding protein was detected in both nuclear and cytoplasmic extracts and in proteins derived from the plasma membrane of cells. We present strong evidence that this protein is nucleolin, a multifunctional phosphoprotein whose levels are related to the rate of cell proliferation. Our results indicate that binding of G-rich oligonucleotides to nucleolin may be responsible for their non-sequence-specific effects. Furthermore, these oligonucleotides represent a new class of potentially therapeutic agents with a novel mechanism of action.
      PBS
      phosphate-buffered saline
      EMSA
      electrophoretic mobility shift assay
      GRO
      guanosine-rich oligonucleotide
      MTT
      3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
      NDM
      nonfat dried milk
      PVDF
      polyvinylidene difluoride
      CRO
      C-rich oligonucleotide
      Oligonucleotides have the potential to recognize unique sequences of DNA or RNA with a remarkable degree of specificity. For this reason they have been considered as promising candidates to realize gene-specific therapies for the treatment of malignant, viral, and inflammatory diseases. Two major strategies of oligonucleotide-mediated therapeutic intervention have been developed, namely the antisense and antigene approaches. The antisense strategy aims to down-regulate expression of a specific gene by hybridization of the oligonucleotide to the specific mRNA, resulting in inhibition of translation (
      • Gewirtz A.M.
      • Sokol D.L.
      • Ratajczak M.Z.
      ,
      • Crooke S.T.
      ,
      • Branch A.D.
      ,
      • Agrawal S.
      • Zhao Q.
      ). The antigene strategy proposes to inhibit transcription of a target gene by means of triple helix formation between the oligonucleotide and specific sequences in the double-stranded genomic DNA (
      • Helene C.
      • Giovannangeli C.
      • Guieysse-Peugeot A.L.
      • Praseuth D.
      ). Clinical trials based on the antisense approach are now showing that oligonucleotides can be administered in a clinically relevant way and have few toxic side effects (
      • Gewirtz A.M.
      • Sokol D.L.
      • Ratajczak M.Z.
      ,
      • Agrawal S.
      • Zhao Q.
      ).
      Whereas both the antisense and antigene strategies have met with some success, it has become clear in recent years that the interactions of oligonucleotides with the components of a living organism go far beyond sequence-specific hybridization with the target nucleic acid. Recent studies and reexamination of early antisense data have suggested that some of the observed biological effects of antisense oligonucleotides cannot be due entirely to Watson-Crick hybridization with the target mRNA. In some cases, the expected biological effect (e.g. inhibition of cell growth or apoptosis) was achieved, but this was not accompanied by a down-regulation of the target protein and was thus unlikely to be a true antisense effect (
      • White J.R.
      • Gordon-Smith E.C.
      • Rutherford T.R.
      ,
      • Dryden S.
      • Pickavance L.
      • Tidd D.
      • Williams G.
      ). In many cases, it was demonstrated that other non-sequence-specific oligonucleotides could exert biological effects that equaled or exceeded the antisense sequence (
      • Barton C.M.
      • Lemoine N.R.
      ,
      • Burgess T.L.
      • Fisher E.F.
      • Ross S.L.
      • Bready J.V.
      • Qian Y.X.
      • Bayewitch L.A.
      • Cohen A.M.
      • Herrera C.J.
      • Hu S.S.
      • Kramer T.B.
      • Lott F.D.
      • Martin F.H.
      • Pierce G.F.
      • Simonet L.
      • Farrell C.L.
      ,
      • Benimetskaya L.
      • Berton M.
      • Kolbanovsky A.
      • Benimetsky S.
      • Stein C.A.
      ). Although there is currently a high awareness among antisense investigators of the importance of appropriate control oligonucleotides, and the necessity of demonstrating inhibition of target protein production (
      • Stein C.A.
      ), the mechanism of non-antisense effects is poorly understood.
      In particular, phosphodiester and phosphorothioate oligodeoxynucleotides containing contiguous guanosines (G) have been repeatedly found to have non-antisense effects on the growth of cells in culture (
      • Burgess T.L.
      • Fisher E.F.
      • Ross S.L.
      • Bready J.V.
      • Qian Y.X.
      • Bayewitch L.A.
      • Cohen A.M.
      • Herrera C.J.
      • Hu S.S.
      • Kramer T.B.
      • Lott F.D.
      • Martin F.H.
      • Pierce G.F.
      • Simonet L.
      • Farrell C.L.
      ,
      • Benimetskaya L.
      • Berton M.
      • Kolbanovsky A.
      • Benimetsky S.
      • Stein C.A.
      ,
      • Saijo Y.
      • Uchiyama B.
      • Abe T.
      • Satoh K.
      • Nukiwa T.
      ). There is evidence that this activity is related to the ability of these oligonucleotides to form stable structures involving intramolecular or intermolecular G-quartets (
      • Burgess T.L.
      • Fisher E.F.
      • Ross S.L.
      • Bready J.V.
      • Qian Y.X.
      • Bayewitch L.A.
      • Cohen A.M.
      • Herrera C.J.
      • Hu S.S.
      • Kramer T.B.
      • Lott F.D.
      • Martin F.H.
      • Pierce G.F.
      • Simonet L.
      • Farrell C.L.
      ,
      • Benimetskaya L.
      • Berton M.
      • Kolbanovsky A.
      • Benimetsky S.
      • Stein C.A.
      ). These are square planar arrangements of four hydrogen-bonded guanines that are stabilized by monovalent cations. Such structures are thought to play an important role in vivo, and putative quartet-forming sequences have been identified in telomeric DNA (
      • Sundquist W.I.
      • Klug A.
      ), immunoglobulin switch region sequences (
      • Sen D.
      • Gilbert W.
      ), human immunodeficiency virus, type I, RNA (
      • Sundquist W.I.
      • Heaphy S.
      ), the fragile X repeat sequences (
      • Fry M.
      • Loeb L.A.
      ), and the retinoblastoma gene (
      • Murchie A.I.
      • Lilley D.M.
      ).
      It has been suggested that non-antisense effects may be due to sequestration of intracellular or surface proteins by the oligonucleotide (
      • Gold L.
      • Polisky B.
      • Uhlenbeck O.
      • Yarus M.
      ,
      • Stein C.A.
      ). For G-rich oligonucleotides that can form folded or G-quartet-containing structures, this binding is not mediated by recognition of the primary sequence of the oligonucleotides but rather of their unique three-dimensional shape. However, the protein targets of these oligonucleotides have not been well characterized.
      Here we identify a G-rich oligonucleotide-binding protein, and we show that the ability of G-rich oligonucleotides to bind to this protein is correlated with their propensity to form G-quartets, and with their ability to inhibit the growth of tumor cells.

      DISCUSSION

      Oligonucleotides are polyanionic species that are internalized in cells, probably by receptor-mediated endocytosis (
      • Vlassov V.V.
      • Balakireva L.A.
      • Yabukov L.A.
      ). They are likely to interact with many biomolecules within the cell and also in the extracellular membrane by virtue of both their charge and their shape, as well as sequence-specific interactions with nucleic acids. The proteins that bind to oligonucleotides and mediate non-antisense effects have not yet been unequivocally identified.
      We have described G-rich oligonucleotides that have potent growth inhibitory effects that are unrelated to any expected antisense or antigene activity. Although we have not yet delineated the mechanism of these effects, we have demonstrated that the antiproliferative effects of these oligonucleotides are related to their ability to bind to a specific cellular protein. Because the GRO-binding protein is also recognized by anti-nucleolin antibodies, we conclude that this protein is either nucleolin itself or a protein of a similar size that shares immunogenic similarities with nucleolin.
      Nucleolin is an abundant multifunctional 110-kDa phosphoprotein, thought to be located predominantly in the nucleolus of proliferating cells (for reviews, see Refs.
      • Tuteja R.
      • Tuteja N.
      and
      • Ginisty H.
      • Sicard H.
      • Roger B.
      • Bouvet P.
      ). It has been implicated in many aspects of ribosome biogenesis including the control of rDNA transcription, pre-ribosome packaging, and organization of nucleolar chromatin (
      • Tuteja R.
      • Tuteja N.
      ,
      • Ginisty H.
      • Sicard H.
      • Roger B.
      • Bouvet P.
      ,
      • Ginisty H.
      • Amalric F.
      • Bouvet P.
      ). Another emerging role for nucleolin is as a shuttle protein that transports viral and cellular proteins between the cytoplasm and nucleus/nucleolus of the cell (
      • Kibbey M.C.
      • Johnson B.
      • Petryshyn R.
      • Jucker M.
      • Kleinman H.K.
      ,
      • Lee C.H.
      • Chang S.C.
      • Chen C.J.
      • Chang M.F.
      ,
      • Waggoner S.
      • Sarnow P.
      ). Nucleolin is also implicated, directly or indirectly, in other roles including nuclear matrix structure (
      • Gotzmann J.
      • Eger A.
      • Meissner M.
      • Grimm R.
      • Gerner C.
      • Sauermann G.
      • Foisner R.
      ), cytokinesis, and nuclear division (
      • Léger-Silvestre I.
      • Gulli M.P.
      • Noaillac-Depeyre J.
      • Faubladier M.
      • Sicard H.
      • Caizergues-Ferrer M.
      • Gas N.
      ) and as an RNA and DNA helicase (
      • Tuteja N.
      • Huang N.W.
      • Skopac D.
      • Tuteja R.
      • Hrvatic S.
      • Zhang J.
      • Pongor S.
      • Joseph G.
      • Faucher C.
      • Almaric F.
      • Falasci A.
      ). Its multifunctional nature is reflected in its multidomain structure, consisting of a histone-like N terminus, a central domain containing RNA recognition motifs, and a glycine- and arginine-rich C terminus (
      • Lapeyre B.
      • Bourbon H.
      • Amalric F.
      ). Levels of nucleolin are known to relate to the rate of cellular proliferation (
      • Derenzini M.
      • Sirri V.
      • Trere D.
      • Ochs R.L.
      ,
      • Roussel P.
      • Hernandez-Verdun D.
      ), being elevated in rapidly proliferating cells, such as malignant cells, and lower in more slowly dividing cells. For this reason, nucleolin may be an attractive therapeutic target for the treatment of malignant disease.
      Although considered a predominantly nucleolar protein, our finding that nucleolin was present in the plasma membrane is consistent with several reports identifying cell surface nucleolin and suggesting its role as a cell surface receptor (
      • Larrucea S.
      • Gonzalez-Rubio C.
      • Cambronero R.
      • Ballou B.
      • Bonay P.
      • Lopez-Granados E.
      • Bouvet P.
      • Fontan G.
      • Fresno M.
      • Lopez-Trascas M.
      ,
      • Callebout C.
      • Blanco J.
      • Benkirane N.
      • Krust B.
      • Jacotot E.
      • Guichard G.
      • Seddiki N.
      • Svab J.
      • Dam E.
      • Muller S.
      • Briand J.-P.
      • Hovanessian A.G.
      ,
      • Semenkovich C.F.
      • Ostlund R.E.
      • Olson M.O.
      • Wang J.W.
      ,
      • Jordan P.
      • Heid H.
      • Kinzel V.
      • Kubler D.
      ).
      Previously, several mechanisms have been proposed to explain the non-sequence-specific effects of oligonucleotides. These include binding to cellular receptors (
      • Rockwell P.
      • O'Connor W.J.
      • Goldstein N.I.
      • Zhang L.M.
      • Stein C.A.
      ,
      • Coulson J.M.
      • Poyner D.R.
      • Chantry A.
      • Irwin W.J.
      • Akhtar S.
      ), modulation of cytokine or growth factor activity (
      • Hartmann G.
      • Krug A.
      • Waller-Fontaine K.
      • Endres S.
      ,
      • Sonehara K.
      • Saito H.
      • Kuramoto E.
      • Yamamoto S.
      • Yamamoto T.
      • Tokunaga T.
      ,
      • Fennewald S.M.
      • Rando R.F.
      ,
      • Guvakova M.A.
      • Yabukov L.A.
      • Vlodavsky I.
      • Tonkinson J.L.
      • Stein C.A.
      ,
      • Ramanathan M.
      • Lantz M.
      • MacGregor R.D.
      • Garovoy M.R.
      • Hunt C.A.
      ), inhibition of cell cycle progression (
      • Burgess T.L.
      • Fisher E.F.
      • Ross S.L.
      • Bready J.V.
      • Qian Y.X.
      • Bayewitch L.A.
      • Cohen A.M.
      • Herrera C.J.
      • Hu S.S.
      • Kramer T.B.
      • Lott F.D.
      • Martin F.H.
      • Pierce G.F.
      • Simonet L.
      • Farrell C.L.
      ), changes in cell adhesion (
      • Saijo Y.
      • Uchiyama B.
      • Abe T.
      • Satoh K.
      • Nukiwa T.
      ), and binding to an uncharacterized 45-kDa protein (
      • Scaggiante B.
      • Morassutti C.
      • Dapas B.
      • Tolazzi G.
      • Ustulin F.
      • Quadrofoglio F.
      ).
      In this present report, we have identified nucleolin (or a nucleolin-like protein) as a G-rich oligonucleotide-binding protein, and we have shown a strong correlation between binding to this protein and antiproliferative activity for a series of G-rich oligonucleotides. We believe that these findings strongly suggest a mechanistic role for nucleolin in non-antisense inhibition of cell growth by G-rich oligonucleotides. This belief has been strengthened by our recent immunofluorescence experiments that show significant differences in nucleolin levels between cells treated with GRO29A and untreated cells.
      P. J. Bates, N. Vigneswaren, S. D. Thomas, and D. M. Miller, manuscript in preparation.
      The relationship between nucleolin binding and antiproliferative activity for other, non-G-rich, oligonucleotides has not yet been fully evaluated. One mixed sequence oligonucleotide (MIX1) was found to bind to nucleolin, although it had no growth inhibitory effect. Nucleolin contains RNA binding domains that can recognize specific sequences of RNA or single-stranded DNA (
      • Dickinson L.A.
      • Kohwi-Shigematsu T.
      ,
      • Ghisolfi L.
      • Joseph G.
      • Puvion-Dutilleul F.
      • Almaric F.
      • Bouvet P.
      ). It is possible that this particular oligonucleotide contains a sequence or structure that resembles such a recognition element.
      In support of our findings that nucleolin binds selectively to G-rich oligonucleotides that form stable G-quartet structures, Maizels et al. (
      • Hanakahi L.A.
      • Sun H.
      • Maizels N.
      ,
      • Dempsey L.A.
      • Sun H.
      • Hanakahi L.A.
      • Maizels N.
      ) have recently demonstrated binding of purified nucleolin to G-quartet forming DNA sequences from immunoglobulin switch regions and ribosomal DNA. It is likely that nucleolin has currently undefined functions in vivo that depend on recognition of G-quartet forming sequences in ribosomal DNA, switch region sequences, or telomeres.
      It is our hypothesis that nucleolin contains a specific binding site that recognizes certain G-quartet structures and that binding at this site by a G-rich oligonucleotide inhibits one or more of the normal functions of nucleolin. The consequences of nucleolin inhibition on the growth of cells have not been well studied, but it is easy to envisage that inhibition of a protein whose functions include ribosome production, nuclear transport, and cell entry could have profound effects on the growth of cells.

      ACKNOWLEDGEMENT

      We thank Dr. Marie W. Wooten (Auburn University, AL) for the gift of nucleolin antiserum.

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