A Consistent Pattern of RIN1 Rearrangements in Oral Squamous Cell Carcinoma Cell Lines Supports a Breakage-Fusion-Bridge Cycle Model for 11q13 Amplification Michele I. Shuster, 1 Limin Han, 2 Michelle M. Le Beau, 3 Elizabeth Davis, 3 Mark Sawicki, 4 Christa M. Lese, 1 No-Hee Park, 5 John Colicelli, 2 and Susanne M. Gollin 1 * 1 Department of Human Genetics, University of Pittsburgh Graduate School of Public Health and the University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 2 Department of Biological Chemistry and Molecular Biology Institute, UCLA School of Medicine, Los Angeles, California 3 Section of Hematology/Oncology, University of Chicago, Chicago, Illinois 4 Department of Surgery, UCLA School of Medicine, Los Angeles, California 5 Department of Dentistry, UCLA School of Medicine, Los Angeles, California Gene amplification is a common feature of tumors. Overexpression of some amplified genes plays a role in tumor progression. Gene amplification can occur either extrachromosomally as double-minute chromosomes (dmin) or intrachromosomally in the form of homogeneously staining regions (hsrs). Approximately one-half of our oral squamous cell carcinomas (OSCCs) are characterized by amplification of band 11q13, usually as an hsr located entopically (occurring or situated at the normal chromosomal site, as opposed to ectopically). Using chromosomal fluorescence in situ hybridization (FISH), we confirmed the amplification of the cyclin D1 (CCND1/PRAD1) and fibroblast growth factor types 3 and 4 (FGF3/INT2 and FGF4/HSTF1) genes within the 11q13 amplicon in our series of primary OSCCs and derived cell lines. The human RIN1 gene was isolated as an RAS interaction/interference protein in a genetic selection in yeast and has been described as a putative effector of both the RAS and ABL oncogenes. We mapped RIN1 to 11q13.2. FISH analysis of 10 11q13-amplified OSCC cell lines revealed high-level RIN1 amplification in two cell lines. Three additional cell lines have what appear to be duplications and/or low-level amplification of RIN1, visible in both interphase and metaphase cells. The hybridization pattern of RIN1 on the metaphase chromosomes is particularly revealing; RIN1 signals flank the 11q13 hsr, possibly as a result of an inverted duplication. The gene amplification model of Coquelle et al. (1997) predicted that gene amplification occurs by breakage-fusion-bridge (BFB) cycles involving fragile sites. Our data suggest that the pattern of gene amplification at 11q13 in OSCC cell lines is consistent with a BFB model. RIN1 appears to be a valuable probe for investigating the process of gene amplification in general and, specifically, 11q13 amplification in oral cancer. Genes Chromosomes Cancer 28:153–163, 2000. © 2000 Wiley-Liss, Inc. INTRODUCTION Gene amplification is a common finding in many human cancers. The amplification and subsequent overexpression of critical genes may play an impor- tant role in the loss of cell cycle control in cancer. Amplification of chromosomal band 11q13 is fre- quently observed in head and neck squamous cell carcinoma (HNSCC), its subset, oral squamous cell carcinoma (OSCC), other aerodigestive tract can- cers, and breast carcinomas (Berenson et al., 1989; Roelofs et al., 1993; Williams et al., 1993; Jin et al., 1995, 1998; Lese et al., 1995; Rubin et al., 1995; Speicher et al., 1995; Åkervall et al., 1997; Muller et al., 1997; Bekri et al., 1998). A number of genes have been shown to be amplified in this region, including CCND1 (encoding cyclin D1), EMS1 (en- coding human cortactin, an actin-binding protein possibly involved in the organization of the cy- toskeleton and cell adhesion structures), and FGF3 and FGF4 (fibroblast growth factors 3 and 4, also called INT2 and HSTF1; Fig. 1) (Schuuring et al., 1998). A proximal gene, VEGFB, was not found to be amplified in mammary carcinoma cell lines (Paavonen et al., 1996). However, more distal This study was carried out in part in the University of Pittsburgh Cancer Institute Cytogenetics Facility. Christa M. Lese’s current address is Department of Human Ge- netics, University of Chicago, Chicago, Illinois. Michele I. Shuster’s current address is Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania. Supported by: NIH; Grant numbers: F32DE05707 (to MS), RO1DE10513 and RO1DE12008 (to SMG), RO1CA56301 (to JC), DE10598 (to N-HP), and P30CA47049019 and S10RR11879; Smokeless Tobacco Research Council; Grant numbers: 0466, 0660 (to SMG); PHS; Grant number: CA40046 (to MML). *Correspondence to: Dr. Susanne M. Gollin, Department of Hu- man Genetics, University of Pittsburgh Graduate School of Public Health, 130 De Soto Street, Pittsburgh, PA 15261. E-mail: sgollin@helix.hgen.pitt.edu Received 25 August 1999; Accepted 4 November 1999 GENES, CHROMOSOMES & CANCER 28:153–163 (2000) © 2000 Wiley-Liss, Inc.