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© The Rockefeller University Press, 0021-9525/1998//1307 $5.00
The Journal of Cell Biology, Volume 140, Number 6, , 1998 1307-1320

Article

Selective Entrapment of Extrachromosomally Amplified DNA by Nuclear Budding and Micronucleation during S Phase



Noriaki Shimizu*, Nobuo Itoh*, Hiroyasu Utiyama*, and Geoffrey M. Wahl{ddagger}

* Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 724, Japan; and {ddagger} Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037

Acentric, autonomously replicating extrachromosomal structures called double-minute chromosomes (DMs) frequently mediate oncogene amplification in human tumors. We show that DMs can be removed from the nucleus by a novel micronucleation mechanism that is initiated by budding of the nuclear membrane during S phase. DMs containing c-myc oncogenes in a colon cancer cell line localized to and replicated at the nuclear periphery. Replication inhibitors increased micronucleation; cell synchronization and bromodeoxyuridine–pulse labeling demonstrated de novo formation of buds and micronuclei during S phase. The frequencies of S-phase nuclear budding and micronucleation were increased dramatically in normal human cells by inactivating p53, suggesting that an S-phase function of p53 minimizes the probability of producing the broken chromosome fragments that induce budding and micronucleation. These data have implications for understanding the behavior of acentric DNA in interphase nuclei and for developing chemotherapeutic strategies based on this new mechanism for DM elimination.

Abbreviations used in this paper: BrdU, 5-bromo-2'-deoxyuridine; DAPI, 4'-6-diamidino-2-phenylindole; DM, double-minute chromosome(s); FISH, fluorescence in situ hybridization; HU, hydroxyurea; PALA, N-phosphoracetyl-L-aspartate; PFA, paraformaldehyde; PI, propidium iodide; RPE-h, normal human retinal pigmented epithelial cells.

We would like to acknowledge S.P. Linke (National Cancer Institute, Bethesda, MD) for kindly providing cell lines, D. Peterson (Salk Institute, La Jolla, CA) for his kind help on the operation of confocal microscopy that appeared in Fig. 6, and D. Von Hoff and K. Davidson (both from Institute of Drug Development, University of Texas Health Science Center, San Antonio, TX) for allowing us to cite their unpublished results. We thank T. Shimura and N. Kumon (both from Hiroshima University, Higashi-Hiroshima, Japan) for their technical help. T. Paulson, T. Kanda, S. O'Gorman, S. Pfaff, G. Karpen, O. Vafa, and L.-c. Huang (all from Salk Institute except Paulson [Fred Hutchinson Cancer Research Center, Seattle, WA] and Huang [UBI, Menlo Park, CA]) provided helpful discussions concerning experimental procedures and topics presented in this manuscript.

This work was in part supported by a grant from the United States Department of Army, (grant number DAMD17-94-J4359) and the International Collaboration Grant from Japanese Ministry of Education (grant number 07044271), and the G. Harold and Leila Y. Mathers Charitable Foundation.

Address all correspondence to Geoffrey Wahl, Gene Expression Laboratory, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037. Tel: (619) 453-4100. Fax: (619) 552-8285. E-mail: wahl{at}salk.edu


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