[CANCER RESEARCH 61, 6987– 6990, October 1, 2001] Advances in Brief Plasmids with a Mammalian Replication Origin and a Matrix Attachment Region Initiate the Event Similar to Gene Amplification 1 Noriaki Shimizu, 2 Yuri Miura, Yu Sakamoto, and Ken Tsutsui Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima, 739-8521 [N. S., Y. M., Y. S.], and Department of Molecular Biology, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558 [K. T.], Japan Abstract Gene amplification plays a crucial role in the development of many human malignancies. Amplified genes are frequently localized on double minutes (DMs). We show here that plasmids bearing both a mammalian replication origin and a nuclear matrix attachment region were able to integrate into DMs if transfected to cells having DMs (COLO 320DM). Furthermore, these plasmids triggered the events leading to the de novo formation of the structure similar to DMs if transfected to the cells without DMs (COLO 320HSR or HeLa). Autonomous replication of these plasmids was suggested to be a prerequisite for these events to occur, because the presence of the origin sequences in the plasmids was required. The presence of matrix attachment region in the plasmids is also required for these events to occur, suggesting that matrix attachment plays an indispensable role in extrachromosomal replication. This model system will allow us to investigate the mechanism of gene amplification as well as to analyze the autonomous replication of the plasmid with mammalian replication origins. Introduction Gene amplification is a major mechanism by which tumor cells acquire unrestricted growth or drug resistances (reviewed in Ref. 1). Cytogenetically, amplified genes are most frequently detected on extrachromosomal DMs 3 chromatin in vivo, whereas the long-term passage in vitro usually lead to the domination of cells having chro- mosomal HSR. We and others have shown that elimination of the amplified genes on DMs from tumor cells leads to the reversion of their tumor phenotype and cellular differentiation (2– 4). Such elim- ination is mediated by the selective incorporation of DMs into micro- nuclei that may be expelled from the dividing cell (3, 5, 6). This micronucleation process is tightly related to the intracellular behavior of DMs during the cell cycle (7). DMs are composed of acentric circular DNA of various sizes (8). Despite their acentricity, DMs are stably segregated into daughter cells by their tethering to the mitotic chromosomes (7–9). Importantly, a recent study revealed that several viral nuclear plasmids including bovine papilloma virus (10), EBV (11), Kaposi’s sarcoma-associated herpes virus (12), and SV40 (13) also use the similar mechanism during segregation. Interestingly, a recent study showed that a plasmid with EBV replicon was able to integrate into DMs in tumor cells (14). In regard to plasmids carrying the SV40 origin of replication, successful tethering requires that the viral large T antigen be expressed. However, successful segregation can also be achieved when the plasmid-encoded large T gene is substituted by the nuclear MAR derived from the cell (13). MAR is found frequently at sites close to the replication origin in the mam- malian genome, and matrix attachment is hypothesized to play a role in genome replication (15). These observations led us to investigate whether plasmids that bear a mammalian replication origin and a MAR might mimic the intracellular behavior of DMs. Materials and Methods Plasmids. The construction of plasmids used in this study was summa- rized in Fig. 1. The plasmids pEPBG (11.0 kbp) and pSFVdhfr (11.0 kbp) were a generous gift from Drs. John Kolman and Geoffrey M. Wahl (The Salk Institute, San Diego, CA). The former plasmid carries the EBV latent origin of replication (OriP) and EBNA-1, as well as the fusion gene green fluorescence protein and G-associated polypeptide (GFP-GAP). The latter plasmid bears a 4.6-kbp fragment containing the Ori  derived from the region that is 3'-downstream to DHFR (16). The pSFV-V plasmid (6.4 kbp), which lacks an origin, was constructed from pSFVdhfr by deleting the entire DHFR-derived sequence by NotI digestion. The pNeo.Myc-2.4 plasmid (9.0 kbp; Ref 17) was a generous gift from Dr. Michael Leffak (Wright State University, Dayton, OH). It contains a 2.4-kbp HindIII/XhoI fragment from the promoter region of c-myc. This plasmid was used to construct pNeo-V, which lacks an origin, by deleting almost all of the sequences derived from c-myc by NotI/HindIII double digestion. The pNeo.Myc SV plasmid, which lacks MAR, was constructed by deleting most of the SV40-derived sequence that exhibits MAR activity (18) by BamHI/BsmI double digestion. Intronic enhancer MAR in the Ig gene was first amplified by PCR using the pG19/45 plasmid, and the pAR1 plasmid was constructed from the amplified product (19). The pNeo.Myc SV AR1 plasmid was then generated by inserting the MAR sequence from pAR1 into pNeo.Myc SV, thus replacing the MAR-like SV40-derived sequence. Other Methods. The human colorectal COLO 320DM and COLO 320HSR tumor cell lines were obtained and maintained as described (5). The HeLa cell line was obtained from American Type Culture Collection (CCL-2). All of the plasmids were purified using the Qiagen plasmid purification kit (Qiagen Inc., Valencia, CA) and transfected into cells by the GenePorter 2 lipofection kit (Gene Therapy Systems, San Diego, CA). Blasticidine (10 g/ml; Funakoshi, Tokyo, Japan; for pEPBG, pSFVdhfr, and its derivative) or 500 g/ml of Neomycin (Life Technologies, Inc., Rockville, MD; for pNeo.Myc-2.4 and its derivatives) were used to select the transformants. A biotin-labeled micronuclei probe that detects the COLO 320 amplicons was prepared according to our published protocol (5). Metaphase spreading, DIG-labeled probe preparation and FISH were performed according to published protocols (20) as was the in vitro nuclear matrix binding assay (19). Results and Discussion Plasmid with EBV Replicon Integrates to DMs. Before exam- ining mammalian replicons, we examined the best-characterized epi- somal vector, namely that based on the EBV replicon (pEPBG). This plasmid replicates autonomously from its viral cis-acting OriP se- quence, and its tethering to the chromosomes before segregation requires the expression of viral EBNA-1 (11). We transfected human COLO 320DM tumor cells bearing multiple DMs with this plasmid Received 5/29/01; accepted 8/13/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by Grant 11440220 grant-in-aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to N. S.) 2 To whom requests for reprints should addressed, at Faculty of Integrated Arts and Sciences, Hiroshima University, 1-7-1 Kagamiyama Higashi-hiroshima, Hiroshima, 739- 8521. Phone: 81-824-24-6528; Fax: 81-824-24-0759; E-mail: shimizu@hiroshima-u.ac.jp. 3 The abbreviations used are: DM, double minute; HSR, homogeneously staining region; MAR, matrix attachment region; DIG, digoxygenin; FISH, fluorescence in situ hybridization; DHFR, dihydrofolate reductase. 6987 Research. on November 28, 2021. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from