FINE MAPPING OF THE 11Q22–23 TUMOR SUPPRESSIVE REGION AND INVOLVEMENT OF TSLC1 IN NASOPHARYNGEAL CARCINOMA Hong Lok LUNG 1 , Yue CHENG 1 , Mande Kuppusamy KUMARAN 2 , Edison Tak-Bun LIU 2 , Yoshinori MURAKAMI 3 , Cheuk Yu CHAN 1 , Wing Lung YAU 1 , Josephine Mun Yee KO 1 , Eric J. STANBRIDGE 4 and Maria Li LUNG 1 * 1 Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (SAR), People’s Republic of China 2 Genome Institute of Singapore, Biomedical Sciences Institutes, Singapore 3 Tumor Suppression and Functional Genomics Project, National Cancer Center Research Institute, Japan 4 Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA Previous studies transferring an intact chromosome 11 into HONE1 cells demonstrated the functional significance of chromosome regions, 11q13 and 11q22–23, in nasopharyn- geal carcinoma (NPC) development. In our study the 11q22–23 region was comprehensively re-investigated by de- tailed microsatellite and single nucleotide polymorphism genotyping and by fluorescence in situ hybridization to map precisely the regions containing tumor suppressive activity. We observed 3 chromosomal intervals within 11q22–23 that were commonly lost in the tumor segregants derived from HONE1/chromosome 11 hybrids. One critical region of 0.36 Mb was mapped near the marker D11S2000 and a second 0.44 Mb region was located around the markers D11S1300 and D11S1391. In a third region high allelic loss was also observed at marker D11S4484, where a newly cloned tumor suppressor gene, TSLC1 (tumor suppressor in lung cancer 1), is located. The gene expression analysis showed absence or low expression levels of TSLC1 mRNA in 4 highly tumorigenic NPC cell lines. In addition, the methylation study results show that the TSLC1 promoter region was hypermethylated in all 4 NPC cell lines and re-expression of the gene occurs in HONE1 cells after 5-aza-2-deoxycytidine treatment. Hence, the mode of silencing of this candidate TSG in NPC may be attributed to promoter hypermethylation. We have ob- tained functional evidence for multiple critical tumor sup- pressive regions in 11q22–23 by fine deletion mapping and for inactivation of TSLC1 being one of these candidate TSGs in NPC development. © 2004 Wiley-Liss, Inc. Key words: chromosome 11q22–23; TSLC1; nasopharyngeal carci- noma; microcell hybrid; chromosome mapping Nasopharyngeal carcinoma (NPC) is a tumor with a striking geographical and ethnic distribution. Compared to other common human cancers, not much is known about the molecular basis for NPC development. Loss or inactivation of tumor suppressor genes (TSGs) is necessary for neoplastic initiation and tumor progres- sion. TSG activity at 11q22–23 has been observed in several cancers, including melanoma, 1 breast, 2 ovarian, 3 lung, 4 cervical, 5 bladder, 6 colorectal 7 and prostate cancers. 8 Molecular and cytoge- netic analyses also show extensive 11q22–24 losses in NPC 9 –10 and implicate putative TSGs as having a critical role in NPC development. A novel TSG, TSLC1 (tumor suppressor in lung cancer 1), has been identified recently on chromosome 11q23.2–3 in non-small cell lung cancer by functional complementation. 11 Previous studies demonstrate that the parental HONE1 cells are 100% tumorigenic in nude mice. The suppression of tumorigenic- ity was observed with all chromosome 11-injected HONE1 micro- cell hybrid (MCH) cell lines. NPC tumor segregants derived from HONE1/intact chromosome 11 MCHs exhibited a delayed lag period in tumor formation compared to that of the parental HONE1 cells. By polymerase chain reaction (PCR)-microsatellite typing, 2 commonly deleted regions at 11q13 and 11q22–23 were identified in NPC tumor segregants derived from HONE1/intact chromo- some 11 MCHs. 12 The delayed tumor appearance may be associ- ated with loss or inactivation of wild-type alleles from the normal donor chromosome 11 detected by the in vivo assay. Based on this hypothesis, we have successfully refined a TS region to 1.8 Mb at chromosome band 11q13. 13 The other critical region for the tumor suppression was localized to be within or near the D11S2000 locus in 11q22–23. In our present study, a more detailed microsatellite analysis and single nucleotide polymorphism (SNP) genotyping were carried out in the region surrounding the D11S2000 locus to identify the candidate TSG region(s) critically lost in the NPC tumor segregants as compared to the HONE1/chromosome 11 hybrid cells. Fluorescence in situ hybridization (FISH) was also carried out with bacterial artificial chromosome (BAC) probes to confirm the genotyping data. MATERIAL AND METHODS Cell lines and culture conditions The recipient NPC HONE1 cells, 14 donor chromosome 11 mouse hybrid cell MCH 556.15 and 5 HONE1/chromosome 11 MCH cell lines (HK11.1, HK11.8, HK11.12, HK11.13 and HK11.19) were maintained as described previously. 12 Each of the 5 chromosome 11 MCHs contained an extra intact chromosome 11 transferred by microcell-mediated chromosome transfer (MMCT) from MCH 556.15 to the recipient HONE1 cell. In a previous study, 12 we described how each of these MCH cell lines were subsequently injected into nude mice and exhibited a delayed latent period before tumor formation; these tumors were excised subsequently and reconstituted into tissue culture. These tumor segregants were grown in DMEM supplemented with 10% FCS Abbreviations: BAC, bacterial artificial chromosome; FISH, fluores- cence in situ hybridization; MCH, microcell hybrids; MMCT, microcell- mediated chromosome transfer; NPC, nasopharyngeal carcinoma; TSG, tumor suppressor gene; TSLC1, tumor suppressor in lung cancer 1. Grant sponsor: Research Grants Council of the Hong Kong Special Administrative Region, People’s Republic of China; Grant number: HKUST 6113/01M, CA99/00.SC02, CA03/04.SC01; Grant sponsor: Agency for Science Technology and Research, Singapore. Dr. Y. Cheng’s current address is Genetics Branch, National Cancer Institute, National Naval Medical Center, Bethesda, MD 20889-5105. Dr. M.K. Kumaran’s current address is Physiological and Genomics Medicine Group, Imperial College, London W12 ONN, UK. *Correspondence to: Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. Fax: +852-23581559. E-mail: bomaria@ust.hk Received 7 December 2003; Accepted 28 April 2004 DOI 10.1002/ijc.20454 Published online 8 July 2004 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 112, 628 – 635 (2004) © 2004 Wiley-Liss, Inc. Publication of the International Union Against Cancer