Sensitivity of the origin decision point to specific inhibitors of cellular signaling and metabolism

Exp Cell Res. 2002 Feb 1;273(1):54-64. doi: 10.1006/excr.2001.5421.

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, alpha-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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Blotting, Western
  • Bromodeoxyuridine
  • CHO Cells
  • Cricetinae
  • Cyclins / metabolism
  • DNA Replication / physiology*
  • Enzyme Inhibitors / pharmacology*
  • G1 Phase / drug effects
  • G1 Phase / physiology
  • Mitogens / pharmacology
  • Protein Kinase Inhibitors*
  • Replication Origin / physiology*
  • S Phase / drug effects
  • S Phase / physiology
  • Signal Transduction / drug effects*
  • Tetrahydrofolate Dehydrogenase / metabolism
  • Transcription, Genetic
  • Xenopus

Substances

  • Cyclins
  • Enzyme Inhibitors
  • Mitogens
  • Protein Kinase Inhibitors
  • Tetrahydrofolate Dehydrogenase
  • Bromodeoxyuridine