Elsevier

Experimental Cell Research

Volume 273, Issue 1, 1 February 2002, Pages 54-64
Experimental Cell Research

Regular Article
Sensitivity of the Origin Decision Point to Specific Inhibitors of Cellular Signaling and Metabolism

https://doi.org/10.1006/excr.2001.5421Get rights and content

Abstract

Chinese hamster ovary (CHO) cells become committed to initiate DNA replication at specific sites within the dihydrofolate reductase (DHFR) locus at a discrete point during G1 phase, the origin decision point (ODP). To better understand the requirements for passage through the ODP, we evaluated the ability of various inhibitors of G1-phase progression to prevent passage through the ODP. Of several protein kinase inhibitors tested, only inhibitors of cyclin-dependent kinase (cdk) activity (roscovitine, olomoucine) prevented passage through the ODP. Inhibitors of MAP kinase (PD98059), PKA (KT5720), PKG (KT5823), as well as inhibition of integrin-mediated signaling by preventing cell adhesion, all arrested cells in the post-ODP stages of G1 phase. Intriguingly, inhibitors of proteasome-dependent proteolysis (MG132, ALLN, lactacystin) and transcription (DRB, α-amanitin, actinomycin D) also inhibited passage through the ODP, whereas inhibition of protein synthesis (cycloheximide) had no effect on the ODP. Cross-checking each inhibitor for its affect on transcription revealed that the ODP could be uncoupled from transcription; MG132 and lactacystin did not inhibit transcription, and KT5720 was a potent inhibitor of transcription. Importantly, cells that were arrested upstream of the ODP with either roscovitine or lactacystin contained functional prereplication complexes (pre-RCs), supporting previous findings that pre-RC formation is not sufficient for origin specification. These results demonstrate that specification of the DHFR origin is independent of growth signaling mechanisms and does not require G1-phase synthesis of a protein regulator such as a cyclin or Dbf4/ASK1, positioning the ODP after pre-RC formation but prior to the activation of the known S-phase promoting kinases.

References (70)

  • D.H. Song et al.

    Endogenous protein kinase CK2 participates in Wnt signaling in mammary epithelial cells

    J. Biol. Chem.

    (2000)
  • K.K. Wary et al.

    A requirement for caveolin-1 and associated kinase Fyn in integrin signaling and anchorage-dependent cell growth

    Cell

    (1998)
  • D.M. Koepp et al.

    How the cyclin became a cyclin: Regulated proteolysis in the cell cycle

    Cell

    (1999)
  • S.J. Elledge et al.

    The role of protein stability in the cell cycle and cancer

    Biochim. Biophys. Acta

    (1998)
  • D.H. Lee et al.

    Proteasome inhibitors: Valuable new tools for cell biologists

    Trends Cell Biol.

    (1998)
  • A. Craiu et al.

    Lactacystin and clasto-lactacystin beta-lactone modify multiple proteasome beta-subunits and inhibit intracellular protein degradation and major histocompatibility complex class I antigen presentation

    J. Biol. Chem.

    (1997)
  • V.J. Palombella et al.

    The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B

    Cell

    (1994)
  • K.L. Rock et al.

    Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules

    Cell

    (1994)
  • J.R. Wu et al.

    Lovastatin arrests CHO cells between the origin decision point and the restriction point

    FEBS Lett.

    (2000)
  • T.J. McGarry et al.

    Geminin, an inhibitor of DNA replication, is degraded during mitosis

    Cell

    (1998)
  • N.P. Pavletich

    Mechanisms of cyclin-dependent kinase regulation: structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors

    J. Mol. Biol.

    (1999)
  • M.F. Dubois et al.

    Inhibitors of transcription such as 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole and isoquinoline sulfonamide derivatives (H-8 and H-7) promote dephosphorylation of the carboxyl-terminal domain of RNA polymerase II largest subunit

    J. Biol. Chem.

    (1994)
  • R. Zandomeni et al.

    Casein kinase type II is involved in the inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole of specific RNA polymerase II transcription

    J. Biol. Chem.

    (1986)
  • F. Grosse et al.

    The primase activity of DNA polymerase alpha from calf thymus

    J. Biol. Chem.

    (1985)
  • A. Dutta et al.

    Initiation of DNA replication in eukaryotic cells

    Annu. Rev. Cell Dev. Biol.

    (1997)
  • T.J. Kelly et al.

    Regulation of chromosome replication

    Annu. Rev. Biochem.

    (2000)
  • D.M. Gilbert

    Making sense of eukaryotic DNA replication origins

    Science

    (2001)
  • G. Abdurashidova et al.

    Start sites of bidirectional DNA synthesis at the human lamin B2 origin

    Science

    (2000)
  • D.M. Cimbora et al.

    Long-distance control of origin choice and replication timing in the human beta-globin locus are independent of the locus control region

    Mol. Cell Biol.

    (2000)
  • M.I. Aladjem et al.

    Genetic dissection of a mammalian replicator in the human beta-globin locus [see comments]

    Science

    (1998)
  • F. Toledo et al.

    oriGNAI3: A narrow zone of preferential replication initiation in mammalian cells identified by 2D gel and competitive PCR replicon mapping techniques

    Nucleic Acids Res.

    (1998)
  • R.D. Little et al.

    Initiation and termination of DNA replication in human rRNA genes

    Mol. Cell Biol.

    (1993)
  • S. Ina et al.

    A broad replication origin of Drosophila melanogaster, oriDalpha, consists of AT-rich multiple discrete initiation sites

    Chromosoma

    (2001)
  • P.A. Dijkwel et al.

    The chinese hamster dihidrofolate reductase origin consists of multiple potential nascent-strand start sites

    Mol. Cell Biol.

    (1995)
  • Cited by (18)

    • Development of quantitative and high-throughput assays of polyomavirus and papillomavirus DNA replication

      2010, Virology
      Citation Excerpt :

      We found that both compounds could prevent viral DNA replication (geldanamycin: IC50 = 0.025 and 0.05 μM for SV40 and HPV; 17-AAG: IC50 = 0.5 μM for both SV40 and HPV), with less of an effect on expression of CMV-Fluc. Finally, we investigated the effect of the proteasome inhibitor lactacystin, which was also reported to block S-phase entry by arresting cells either in G1 or G2/M phase (Katagiri et al., 1995; Keezer and Gilbert, 2002). Like the two HSP90 inhibitors, lactacystin inhibited viral DNA replication (IC50 = 1.25 μM for both SV40 and HPV).

    • 11 Analysis of centrosome amplification in cancer

      2005, Handbook of Immunohistochemistry and in Situ Hybridization of Human Carcinomas
    View all citing articles on Scopus
    1

    To whom reprint requests should be addressed. Fax: (315) 464-8750. E-mail: [email protected].

    View full text