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Inhibition of DNA Replication and Induction of S Phase Cell Cycle Arrest by G-rich Oligonucleotides*

Open AccessPublished:November 16, 2001DOI:https://doi.org/10.1074/jbc.M104446200
      The discovery of G-rich oligonucleotides (GROs) that have non-antisense antiproliferative activity against a number of cancer cell lines has been recently described. This biological activity of GROs was found to be associated with their ability to form stable G-quartet-containing structures and their binding to a specific cellular protein, most likely nucleolin (Bates, P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O., and Miller, D. M. (1999) J. Biol. Chem. 274, 26369–26377). In this report, we further investigate the novel mechanism of GRO activity by examining their effects on cell cycle progression and on nucleic acid and protein biosynthesis. Cell cycle analysis of several tumor cell lines showed that cells accumulate in S phase in response to treatment with an active GRO. Analysis of 5-bromodeoxyuridine incorporation by these cells indicated the absence of de novo DNA synthesis, suggesting an arrest of the cell cycle predominantly in S phase. At the same time point, RNA and protein synthesis were found to be ongoing, indicating that arrest of DNA replication is a primary event in GRO-mediated inhibition of proliferation. This specific blockade of DNA replication eventually resulted in altered cell morphology and induction of apoptosis. To characterize further GRO-mediated inhibition of DNA replication, we used an in vitro assay based on replication of SV40 DNA. GROs were found to be capable of inhibiting DNA replication in the in vitro assay, and this activity was correlated to their antiproliferative effects. Furthermore, the effect of GROs on DNA replication in this assay was related to their inhibition of SV40 large T antigen helicase activity. The data presented suggest that the antiproliferative activity of GROs is a direct result of their inhibition of DNA replication, which may result from modulation of a replicative helicase activity.
      GROs
      G-rich oligonucleotides
      BrU
      5-bromouridine
      BrdUrd
      5-bromo-2′-deoxyuridine
      DMEM
      Dulbecco's modification of Eagle's medium
      PBS
      phosphate-buffered saline
      FCS
      fetal calf serum
      MTT
      3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
      RPA
      replication protein A
      TUNEL
      terminal transferase dUTP nick-end label
      NS
      nonspecific oligonucleotide
      Oligonucleotides can recognize both nucleic acids and proteins with a high degree of specificity. This is a major reason why they have been widely investigated as potential therapeutic agents for cancer, viral infections, and inflammatory diseases. Oligonucleotides can achieve target recognition by sequence-specific interactions with nucleic acids or proteins such as in the antisense, antigene, or decoy approaches (
      • Gewirtz A.M.
      • Sokol D.L.
      • Ratajczak M.Z.
      ,
      • Crooke S.T.
      ,
      • Praseuth D.
      • Guieysse A.L.
      • Helene C.
      ,
      • Mann M.J.
      • Dzau V.J.
      ). Alternatively, target recognition can be due to the specific three-dimensional structure of an oligonucleotide, as in the aptamer approach (
      • Gold L.
      ,
      • Hermann T.
      • Patel D.J.
      ). These aptameric oligonucleotides often contain secondary structure elements such as hairpins or G-quartets. The formation of G-quartet structures is also thought to contribute to non-antisense growth inhibitory effects of G-rich phosphodiester and phosphorothioate oligonucleotides (
      • 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.
      ,
      • Bates P.J.
      • Kahlon J.B.
      • Thomas S.D.
      • Trent J.O.
      • Miller D.M.
      ).
      Recently, we reported (
      • Bates P.J.
      • Kahlon J.B.
      • Thomas S.D.
      • Trent J.O.
      • Miller D.M.
      ) on a novel class of phosphodiester G-rich oligonucleotides (GROs)1 that could strongly inhibit the in vitro proliferation of tumor cells derived from prostate, breast, and cervical carcinomas. The antiproliferative GROs were able to form stable secondary structures consistent with G-quartet formation. It was determined that these GROs bound to a specific nuclear protein and, furthermore, that the growth inhibitory activity of the GROs was positively correlated with their ability to bind to this protein. The specific GRO-binding protein was captured using biotinylated GROs and was identified by polyclonal and monoclonal antibodies to nucleolin. Therefore, we concluded that these potentially therapeutic oligonucleotides worked by a novel mechanism that involved binding to nucleolin or a nucleolin-like protein. Our hypothesis was that binding of GROs causes inhibition of nucleolin function(s) that results in an arrest of proliferation.
      Nucleolin is an abundant 110-kDa phosphoprotein, thought to be located predominantly in the nucleolus of proliferating cells. Levels of nucleolin are known to relate to the rate of cellular proliferation (
      • Derenzini M.
      • Sirri V.
      • Trere D.
      • Ochs R.L.
      ,
      • Sirri V.
      • Roussel P.
      • Hernandez-Verdun D.
      ), being elevated in rapidly dividing cells such as malignant cells. Therefore, nucleolin may be an attractive molecular target for cancer therapy. The remarkable multifunctionality of nucleolin and its role in cell growth and proliferation have been highlighted in recent reviews (
      • Tuteja R.
      • Tuteja N.
      ,
      • Ginisty H.
      • Sicard H.
      • Roger B.
      • Bouvet P.
      ,
      • Srivastava M.
      • Pollard H.B.
      ). The most studied aspects of nucleolin function are its roles in ribosome biogenesis, which include the control of rDNA transcription, pre-ribosome packaging, and organization of nucleolar chromatin (
      • Tuteja R.
      • Tuteja N.
      ,
      • Ginisty H.
      • Amalric F.
      • Bouvet P.
      ). It is also thought that nucleolin can act 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.
      ). In addition, nucleolin has been 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.
      ), DNA replication (
      • Daniely Y.
      • Borowiec J.A.
      ), cytokinesis and nuclear division (
      • Léger-Silvestre I.
      • Gulli M.P.
      • Noaillac-Depeyre J.
      • Faubladier M.
      • Sicard H.
      • Caizergues-Ferrer M.
      • Gas N.
      ), and as a nucleic acid helicase (
      • Tuteja R.
      • Tuteja N.
      ,
      • Tuteja N.
      • Huang N.W.
      • Skopac D.
      • Tuteja R.
      • Hrvatic S.
      • Zhang J.
      • Pongor S.
      • Joseph G.
      • Faucher C.
      • Almaric F.
      • Falasci A.
      ). There have been numerous reports describing the presence of nucleolin in the plasma membrane of cells (
      • Bates P.J.
      • Kahlon J.B.
      • Thomas S.D.
      • Trent J.O.
      • Miller D.M.
      ,
      • Larrucea S.
      • Gonzalez-Rubio C.
      • Cambronero R.
      • Ballou B.
      • Bonay P.
      • Lopez-Granados E.
      • Bouvet P.
      • Fontan M.F.
      • 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.
      ,
      • Hovanessian A.G.
      • Puvion-Dutilleul F.
      • Nisole S.
      • Svab J.
      • Perret E.
      • Deng J.S.
      • Krust B.
      ,
      • Dumler I.
      • Stepanova V.
      • Jerke U.
      • Mayboroda O.A.
      • Vogel F.
      • Bouvet P.
      • Tkachuk V.
      • Haller H.
      • Gulba D.C.
      ,
      • Sorokina E.A.
      • Kleinman J.G.
      ), suggesting a further function of nucleolin as a cell surface receptor. Clearly, inhibition of nucleolin function, which we propose is an effect of GRO binding, would likely result in inhibition of cell proliferation and/or cell death.
      To elucidate further the mechanism of the GRO antiproliferative activity, we decided to study the effects of GROs on cellular processes, such as nucleic acid and protein synthesis, and cell cycle progression. We report our findings that GROs specifically inhibit DNA replication, and we discuss the implications of these results in terms of potential mechanisms for GRO activity.

      DISCUSSION

      GROs are a new type of antiproliferative oligonucleotide with considerable potential as therapeutic agents for cancer. Although the activity of GROs is known to correlate with their ability to bind to nucleolin protein, the precise mechanism by which they exert their antiproliferative effects is unknown. Because nucleolin is involved in many aspects of cell growth, proliferation, and apoptosis, knowledge of a putative target protein does not necessarily identify the processes that are affected by GROs. Further information regarding the pathways affected by GROs would facilitate the design of agents that act by a similar mechanism that may be even more active or have improved pharmacological features, compared with oligonucleotides. Therefore, in this study we proceeded to explore the mechanism of GROs by examining their effects on cellular processes.
      The conclusions of our studies are that treatment of cells with antiproliferative GRO29A causes an arrest of cell cycle progression in S phase and an inhibition of DNA replication. Because DNA synthesis is affected before RNA and protein synthesis, we have concluded that this is a primary cause of proliferation inhibition. Furthermore, because GROs can also inhibit DNA replication in a cell-free assay, we infer that the action of the GROs results directly from an effect on a protein (or proteins) involved in DNA replication. To investigate the hypothesis that GRO effects are mediated by an inhibition of DNA synthesis, we have used an in vitro DNA replication assay. This assay is based on the replication of a 7.4-kilobase pair circular genome that contains an SV40 origin of replication (ori), originally described by Li and Kelly (
      • Li J.J.
      • Kelly T.J.
      ,
      • Li J.J.
      • Kelly T.J.
      ) and modified by Roberts and Kunkel (
      • Roberts J.D.
      • Kunkel T.A.
      ). The reaction is initiated by SV40 large T antigen, which is required to recognize and unwind the ori. Proteins in human cell extracts carry out all subsequent steps. Inhibition of replication by GRO29A could be mediated either by damaging the template or by modulating the activity of a protein (or proteins) that is required for replication. The former mechanism is unlikely, because the replication products of damaged templates show severe inhibition of form I DNA but relatively greater amounts of nicked and linear DNA (
      • McGregor W.G.
      • Wei D.
      • Maher V.M.
      • McCormick J.J.
      ,
      • Thomas D.C.
      • Kunkel T.A.
      ). The GRO29A examined in this study severely inhibited the synthesis of all forms of DNA, suggesting interference with the proper function of the complex that carries out DNA replication (known as the replisome or synthesome). Therefore, we investigated the effects of GRO29A on the replication-associated proteins most likely to be modulated. These were the SV40 large T antigen, which is known to bind to and unwind G-quartet structures (
      • Baran N.
      • Pucshansky L.
      • Marco Y.
      • Benjamin S.
      • Manor H.
      ), and RPA, which is known to bind to nucleolin (
      • Daniely Y.
      • Borowiec J.A.
      ), the putative GRO-binding protein (
      • Bates P.J.
      • Kahlon J.B.
      • Thomas S.D.
      • Trent J.O.
      • Miller D.M.
      ). Although we could observe the interaction between nucleolin and RPA, we found that this was not significantly affected by the presence of GROs. On the other hand, the ability of T antigen to unwind a synthetic substrate representing a replication fork was strongly inhibited by GRO29A. Moreover, for a series of six oligonucleotides, the relative activity in inhibiting T antigen helicase mirrored the relative activity in inhibiting DNA replication in vitro and also in inhibiting tumor cell proliferation. Therefore, it would appear that the antiproliferative effects of GROs on cancer cells are mediated by inhibition of DNA replication, which in turn may be related to inhibition of helicase activity. Of course, SV40 T antigen is not normally present in human cells, but GRO29A could also be an inhibitor of a human replicative helicase, which will most likely share similar features with the viral T antigen. The identity of the human replicative helicase is still not certain, but a hexameric complex of proteins known as minichromosome maintenance has been reported to have helicase activity in vitro and is generally thought to be a good candidate (
      • Tye B.K.
      ,
      • Labib K.
      • Diffley J.F.
      ). The ability of this complex to bind to or unwind G-quartets has not yet been reported.
      G-quartet unwinding has been described previously (
      • Sun H.
      • Karow J.K.
      • Hickson I.D.
      • Maizels N.
      ,
      • Sun H.
      • Bennett R.J.
      • Maizels N.
      ,
      • Fry M.
      • Loeb L.A.
      ) for a number of helicases, but to our knowledge, this is the first report that the presence of G-quartet structures can prevent a replicative helicase unwinding its double-stranded substrate. Inhibition of helicase activity has been reported for several DNA-binding agents, including many antitumor antibiotics such as anthracyclines (
      • Bachur N.R.
      • Lun L.
      • Sun P.M.
      • Trubey C.M.
      • Elliott E.E.
      • Egorin M.J.
      • Malkas L.
      • Hickey R.
      ,
      • Bachur N.R., Yu, F.
      • Johnson R.
      • Hickey R.
      • Wu Y.
      • Malkas L.
      ,
      • Tuteja N.
      • Phan T.N.
      ,
      • Tuteja N.
      • Phan T.N.
      • Tuteja R.
      • Ochem A.
      • Falaschi A.
      ,
      • George J.W.
      • Ghate S.
      • Matson S.W.
      • Besterman J.M.
      ,
      • Maine I.P.
      • Sun D.
      • Hurley L.H.
      • Kodadek T.
      ,
      • Chino M.
      • Nishikawa K.
      • Yamada A.
      • Ohsono M.
      • Sawa T.
      • Hanaoka F.
      • Ishizuka M.
      • Takeuchi T.
      ,
      • Zhu K.
      • Henning D.
      • Iwakuma T.
      • Valdez B.C.
      • Busch H.
      ). In these cases, it is most likely that inhibition is caused by the formation of a strong complex between the DNA-binding ligand and the template DNA, which impede the action of the helicase (
      • Bachur N.R.
      • Lun L.
      • Sun P.M.
      • Trubey C.M.
      • Elliott E.E.
      • Egorin M.J.
      • Malkas L.
      • Hickey R.
      ). There is considerable evidence that inhibition of helicase activity by such compounds may play some role in their anticancer activity (
      • Gewirtz D.A.
      ), but the effect of helicase inhibition in cancer cells has not been extensively studied.
      Nucleolin has been identified by us as a GRO-binding protein (
      • Bates P.J.
      • Kahlon J.B.
      • Thomas S.D.
      • Trent J.O.
      • Miller D.M.
      ), and by several other groups (
      • Hanakahi L.A.
      • Sun H.
      • Maizels N.
      ,
      • Dempsey L.A.
      • Sun H.
      • Hanakahi L.A.
      • Maizels N.
      ,
      • Dickinson L.A.
      • Kohwi-Shigematsu T.
      ,
      • Ishikawa F.
      • Matunis M.J.
      • Dreyfuss G.
      • Cech T.R.
      ) as a G-quartet-binding protein. Although the results in this paper do not clearly define a link between the molecular effects of GROs and their binding to nucleolin, they point to an effect by inhibition of helicase activity and DNA replication. Therefore, it is interesting to note that nucleolin itself has been reported to have helicase activity and is also known as DNA helicase IV (
      • Tuteja R.
      • Tuteja N.
      ,
      • Tuteja N.
      • Huang N.W.
      • Skopac D.
      • Tuteja R.
      • Hrvatic S.
      • Zhang J.
      • Pongor S.
      • Joseph G.
      • Faucher C.
      • Almaric F.
      • Falasci A.
      ). In addition, nucleolin has been shown to interact with at least three components of the DNA replisome complex, namely RPA (
      • Daniely Y.
      • Borowiec J.A.
      ), topoisomerase I (
      • Bharti A.K.
      • Olson M.O.
      • Kufe D.W.
      • Rubin E.H.
      ), and poly(ADP-ribose) polymerase (
      • Borggrefe T.
      • Wabl M.
      • Akhmedov A.T.
      • Jessberger R.
      ).
      Our future studies will focus on identification of cellular helicases that may be inhibited by GRO29A, as well as clarification of the role of nucleolin in GRO activity. Such studies could lead to valuable insights into the role of nucleolin in DNA replication, as well as further elucidation of the molecular mechanisms of GRO effects.

      Acknowledgments

      We thank Virna Dapic for assistance with flow cytometry.

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