Homozygous Deletions Within the 11q13 Cervical Cancer Tumor-Suppressor Locus in Radiation-Induced, Neoplastically Transformed Human Hybrid Cells Marc S. Mendonca, 1,2 * Daphne L. Farrington, 1 Brendan M. Mayhugh, 1 Yan Qin, 1 Toni Temples, 1 Kathleen Comerford, 1 Rita Chakrabarti, 3 Kayvan Zainabadi, 3 J. Leslie Redpath, 4 Eric J. Stanbridge, 5 and Eri S. Srivatsan 3 1 Department of Radiation Oncology, Radiation and Cancer Biology Laboratory, Indiana University School of Medicine, Indianapolis, IN 2 Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 3 Department of Surgery, VAGLAHS/David Geffen School of Medicine at UCLA, Los Angeles, CA 4 Department of Radiation Oncology, UC Irvine, California College of Medicine, Irvine, CA 5 Department and Molecular Genetics, UC Irvine, California College of Medicine, Irvine, CA Studies on nontumorigenic and tumorigenic human cell hybrids derived from the fusion of HeLa (a cervical cancer cell line) with GM00077 (a normal skin fibroblast cell line) have demonstrated “functional” tumor-suppressor activity on chromosome 11. It has been shown that several of the neoplastically transformed radiation-induced hybrid cells called GIMs (gamma ray induced mutants), isolated from the nontumorigenic CGL1 cells, have lost one copy of the fibroblast chromosome 11. We hypothesized, therefore, that the remaining copy of the gene might be mutated in the cytogenetically intact copy of fibroblast chromosome 11. Because a cervical cancer tumor suppressor locus has been localized to chromosome band 11q13, we performed deletion-mapping analysis of eight different GIMs using a total of 32 different polymorphic and microsatellite markers on the long arm (q arm) of chromosome 11. Four irradiated, nontumorigenic hybrid cell lines, called CONs, were also analyzed. Allelic deletion was ascertained by the loss of a fibroblast allele in the hybrid cell lines. The analysis confirmed the loss of a fibroblast chromosome 11 in five of the GIMs. Further, homozygous deletion (complete loss) of chromosome band 11q13 band sequences, including that of D11S913, was observed in two of the GIMs. Detailed mapping with genomic sequences localized the homozygous deletion to a 5.7-kb interval between EST AW167735 and EST F05086. Southern blot hybridization using genomic DNA probes from the D11S913 locus confirmed the existence of homozygous deletion in the two GIM cell lines. Additionally, PCR analysis showed a reduction in signal intensity for a marker mapped 31 kb centromeric of D11S913 in four other GIMs. Finally, Northern blot hybridization with the genomic probes revealed the presence of a novel 15-kb transcript in six of the GIMs. These transcripts were not observed in the nontumorigenic hybrid cell lines. Because the chromosome 11q13 band deletions in the tumorigenic hybrid cell lines overlapped with the minimal deletion in cervical cancer, the data suggest that the same gene may be involved in the development of cervical cancer and in radiation-induced carcinogenesis. We propose that a gene localized in proximity to the homozygous deletion is the candidate tumor-suppressor gene. © 2004 Wiley-Liss, Inc. INTRODUCTION Molecular analyses of human cancers have shown carcinogenesis as a multistep process involv- ing activation of oncogenes and inactivation of tu- mor-suppressor genes (Boyd and Barrett, 1990; Stanbridge, 1990; Bishop, 1991; Goyette et al., 1992; Weinberg, 1995; Kinzler and Vogelstein, 1996; Fearon, 1997). This suggests that induction of genomic instability is required for the neoplastic transformation of normal human cells (Loeb, 1991; Cheng and Loeb, 1993). In cervical cancer, malig- nant transformation is thought to involve infection with the high-risk human papilloma viruses HPV16 or HPV18, which are present in the majority of cervical cancers (zur Hausen, 1996; Press, 1998). The HPV viral proteins E6 and E7 inactivate the function of the TP53 and RB1 tumor-suppressor Supported by: American Cancer Society; Grant number: IRG-84- 002-14-IRG (to M.S. Mendonca); Ralph W. and Grace M. Showalter Research Trust (to M.S. Mendonca); Department of Radiation On- cology, IUSM (to M.S. Mendonca); VAGLAHS West Los Angeles Surgical Education and Research program (E.S. Srivatsan); UCLA Academic Senate (E.S. Srivatsan), VA merit review (E.S. Srivatsan), California Cancer Research Program. *Correspondence to: Marc S. Mendonca, Ph.D., Radiation and Cancer Biology Laboratory, Department of Radiation Oncology, Indiana University School of Medicine, 975 West Walnut St., IB- 346, Indianapolis, IN 46202. E-mail: mmendonc@iupui.edu Received 15 July 2003; Accepted 17 October 2003 DOI 10.1002/gcc.20007 GENES, CHROMOSOMES & CANCER 39:277–287 (2004) © 2004 Wiley-Liss, Inc.