Initiation of DNA replication in nuclei from quiescent cells requires permeabilization of the nuclear membrane.
We have investigated the replication capacity of intact nuclei from quiescent cells using Xenopus egg extract. Nuclei, with intact nuclear membranes, were isolated from both exponentially growing and contact-inhibited BALB/c 3T3 fibroblasts by treatment of the cells with streptolysin-O. Flow cytometry showed that > 90% of all contact-inhibited cells and approximately 50% of the exponential cells were in G0/G1-phase at the time of nuclear isolation. Intact nuclei were assayed for replication in the extract by incorporation of [alpha-32P]dATP or biotin-dUTP into nascent DNA. Most nuclei from exponential cells replicated in the egg extract, consistent with previous results showing that intact G1 nuclei from HeLa cells replicate in this system. In contrast, few nuclei from quiescent cells replicated in parallel incubations. However, when the nuclear membranes of these intact quiescent nuclei were permeabilized with lysophosphatidylcholine prior to addition to the extract, nearly all the nuclei replicated under complete cell cycle control in a subsequent incubation. The ability of LPC-treated quiescent nuclei to undergo DNA replication was reversed by resealing permeable nuclear membranes with Xenopus egg membranes prior to extract incubation demonstrating that the effect of LPC treatment is at the level of the nuclear membrane. These results indicate that nuclei from G1-phase cells lose their capacity to initiate DNA replication following density-dependent growth arrest and suggest that changes in nuclear membrane permeability may be required for the initiation of replication upon re-entry of the quiescent cell into the cell cycle.
Leno
G H
GH
Department of Biochemistry, University of Mississippi Medical Center, Jackson, 39216-4505.
Munshi
R
R
D016475:3T3 Cells; D000818:Animals; D001426:Bacterial Proteins; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D016193:G1 Phase; D006367:HeLa Cells; D006801:Humans; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D051379:Mice; D009685:Nuclear Envelope / Q000187:drug effects / Q000502:physiology*; D010063:Ovum; D010539:Permeability; D013301:Streptolysins / Q000494:pharmacology; D014981:Xenopus
D001426:Bacterial Proteins; D008244:Lysophosphatidylcholines; D013301:Streptolysins; C025964:streptolysin O