INITIATION OF DNA-REPLICATION IN NUCLEI FROM QUIESCENT CELLS REQUIRESPERMEABILIZATION OF THE NUCLEAR-MEMBRANE

We have investigated the replication capacity of intact nuclei from qu iescent cells using Xenopus egg extract. Nuclei, with intact nuclear m embranes, were isolated from both exponentially growing and contact-in hibited BALB/c 3T3 fibroblasts by treatment of the cells with streptol ysin-O. Flow cytometry showed that >90% of all contact-inhibited cells and similar to 50% of the exponential cells were in G0/G1-phase at th e time of nuclear isolation. Intact nuclei were assayed for replicatio n in the extract by incorporation of [alpha-P-32]dATP or biotin-dUTP i nto nascent DNA. Most nuclei from exponential cells replicated in the egg extract, consistent with previous results showing that intact G1 n uclei from HeLa cells replicate in this system. In contrast, few nucle i from quiescent cells replicated in parallel incubations. However, wh en the nuclear membranes of these intact quiescent nuclei were permeab ilized 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 t o undergo DNA replication was reversed by resealing permeable nuclear membranes with Xenopus egg membranes prior to extract incubation demon strating that the effect of LPC treatment is at the level of the nucle ar membrane. These results indicate that nuclei from G1-phase cells lo se their capacity to initiate DNA replication following density-depend ent growth arrest and suggest that changes in nuclear membrane permeab ility may be required for the initiation of replication upon re-entry of the quiescent cell into the cell cycle.