Localization of Tumor Suppressor Activity Important in Nonsmall Cell Lung Carcinoma on Chromosome 11q
Loss of heterozygosity on chromosome 11q23 is observed at high frequency in human nonsmall cell lung carcinomas (NSCLCs), suggesting the presence of a tumor suppressor gene. Previous analysis of DNA from 79 patients identified a commonly deleted segment of 5 centimorgans. Complementation analysis wa...
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Published in | Proceedings of the National Academy of Sciences - PNAS Vol. 95; no. 14; pp. 8153 - 8158 |
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Main Authors | , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
National Academy of Sciences of the United States of America
07.07.1998
National Acad Sciences National Academy of Sciences The National Academy of Sciences |
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Abstract | Loss of heterozygosity on chromosome 11q23 is observed at high frequency in human nonsmall cell lung carcinomas (NSCLCs), suggesting the presence of a tumor suppressor gene. Previous analysis of DNA from 79 patients identified a commonly deleted segment of 5 centimorgans. Complementation analysis was used to further localize a putative tumor suppressor gene. Three yeast artificial chromosome (YAC) clones spanning the minimal loss of heterozygosity region were modified, and spheroplast fusion was used to transfer them into human A549 NSCLC or murine Lewis lung carcinoma (LLC) cell lines. The resulting yeast × human hybrid cell lines containing an intact copy of a 1.6-Mb YAC, 939b12, showed reduced growth in vitro. Injection of parental A549 cells into athymic (nu/nu) mice resulted in tumor formation at 27 of 28 injection sites. In contrast, two independent 939b12-containing cell lines formed tumors at only 3 of 20 injection sites. 939b12 also suppressed tumor formation by LLC NSCLC cells in nude mice, but YACs 785e12 and 911f2, which flank 939b12, had no suppressor activity. Further localization of tumor suppression activity on 939b12 was accomplished by introduction of defined fragmentation derivatives into A549 cells and by analysis of YACs that were broken on transfer into LLC cells. This complementation approach localized tumor suppression activity to the central 700 kb of 939b12 and provides a functional assay for positional cloning of this tumor suppressor gene. |
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AbstractList | Loss of heterozygosity on chromosome 11q23 is observed at high frequency in human nonsmall cell lung carcinomas (NSCLCs), suggesting the presence of a tumor suppressor gene. Previous analysis of DNA from 79 patients identified a commonly deleted segment of 5 centimorgans. Complementation analysis was used to further localize a putative tumor suppressor gene. Three yeast artificial chromosome (YAC) clones spanning the minimal loss of heterozygosity region were modified, and spheroplast fusion was used to transfer them into human A549 NSCLC or murine Lewis lung carcinoma (LLC) cell lines. The resulting yeast x human hybrid cell lines containing an intact copy of a 1.6-Mb YAC, 939b12, showed reduced growth in vitro. Injection of parental A549 cells into athymic (nu/nu) mice resulted in tumor formation at 27 of 28 injection sites. In contrast, two independent 939b12-containing cell lines formed tumors at only 3 of 20 injection sites. 939b12 also suppressed tumor formation by LLC NSCLC cells in nude mice, but YACs 785e12 and 911f2, which flank 939b12, had no suppressor activity. Further localization of tumor suppression activity on 939b12 was accomplished by introduction of defined fragmentation derivatives into A549 cells and by analysis of YACs that were broken on transfer into LLC cells. This complementation approach localized tumor suppression activity to the central 700 kb of 939b12 and provides a functional assay for positional cloning of this tumor suppressor gene. Loss of heterozygosity on chromosome 11q23 is observed at high frequency in human nonsmall cell lung carcinomas (NSCLCs), suggesting the presence of a tumor suppressor gene. Previous analysis of DNA from 79 patients identified a commonly deleted segment of 5 centimorgans. Complementation analysis was used to further localize a putative tumor suppressor gene. Three yeast artificial chromosome (YAC) clones spanning the minimal loss of heterozygosity region were modified, and spheroplast fusion was used to transfer them into human A549 NSCLC or murine Lewis lung carcinoma (LLC) cell lines. The resulting yeast × human hybrid cell lines containing an intact copy of a 1.6-Mb YAC, 939b12, showed reduced growth in vitro . Injection of parental A549 cells into athymic ( nu/nu ) mice resulted in tumor formation at 27 of 28 injection sites. In contrast, two independent 939b12-containing cell lines formed tumors at only 3 of 20 injection sites. 939b12 also suppressed tumor formation by LLC NSCLC cells in nude mice, but YACs 785e12 and 911f2, which flank 939b12, had no suppressor activity. Further localization of tumor suppression activity on 939b12 was accomplished by introduction of defined fragmentation derivatives into A549 cells and by analysis of YACs that were broken on transfer into LLC cells. This complementation approach localized tumor suppression activity to the central 700 kb of 939b12 and provides a functional assay for positional cloning of this tumor suppressor gene. Loss of heterozygosity on chromosome 11q23 is observed at high frequency in human nonsmall cell lung carcinomas (NSCLCs), suggesting the presence of a tumor suppressor gene. Previous analysis of DNA from 79 patients identified a commonly deleted segment of 5 centimorgans. Complementation analysis was used to further localize a putative tumor suppressor gene. Three yeast artificial chromosome (YAC) clones spanning the minimal loss of heterozygosity region were modified, and spheroplast fusion was used to transfer them into human A549 NSCLC or murine Lewis lung carcinoma (LLC) cell lines. The resulting yeast super(.) human hybrid cell lines containing an intact copy of a 1.6-Mb YAC, 939b12, showed reduced growth in vitro. Injection of parental A549 cells into athymic (nu/nu) mice resulted in tumor formation at 27 of 28 injection sites. In contrast, two independent 939b12-containing cell lines formed tumors at only 3 of 20 injection sites. 939b12 also suppressed tumor formation by LLC NSCLC cells in nude mice, but YACs 785e12 and 911f2, which flank 939b12, had no suppressor activity. Further localization of tumor suppression activity on 939b12 was accomplished by introduction of defined fragmentation derivatives into A549 cells and by analysis of YACs that were broken on transfer into LLC cells. This complementation approach localized tumor suppression activity to the central 700 kb of 939b12 and provides a functional assay for positional cloning of this tumor suppressor gene. Loss of heterozygosity on chromosome 11q23 is observed at high frequency in human nonsmall cell lung carcinomas (NSCLCs), suggesting the presence of a tumor suppressor gene. Previous analysis of DNA from 79 patients identified a commonly deleted segment of 5 centimorgans. Loss of heterozygosity on chromosome 11q23 is observed at high frequency in human nonsmall cell lung carcinomas (NSCLCs), suggesting the presence of a tumor suppressor gene. Previous analysis of DNA from 79 patients identified a commonly deleted segment of 5 centimorgans. Complementation analysis was used to further localize a putative tumor suppressor gene. Three yeast artificial chromosome (YAC) clones spanning the minimal loss of heterozygosity region were modified, and spheroplast fusion was used to transfer them into human A549 NSCLC or murine Lewis lung carcinoma (LLC) cell lines. The resulting yeast × human hybrid cell lines containing an intact copy of a 1.6-Mb YAC, 939b12, showed reduced growth in vitro . Injection of parental A549 cells into athymic ( nu/nu ) mice resulted in tumor formation at 27 of 28 injection sites. In contrast, two independent 939b12-containing cell lines formed tumors at only 3 of 20 injection sites. 939b12 also suppressed tumor formation by LLC NSCLC cells in nude mice, but YACs 785e12 and 911f2, which flank 939b12, had no suppressor activity. Further localization of tumor suppression activity on 939b12 was accomplished by introduction of defined fragmentation derivatives into A549 cells and by analysis of YACs that were broken on transfer into LLC cells. This complementation approach localized tumor suppression activity to the central 700 kb of 939b12 and provides a functional assay for positional cloning of this tumor suppressor gene. |
Author | Ohki, Misao Arai, Yasuhito Hunt, Piper Frischmeyer, Pam Tanigami, Akira Gomyou, Hiroki Sekiya, Takao Maruyama, Tomoko Nobukuni, Takahiro Tamura, Kenji Murakami, Yoshinori Cabin, Deborah Reeves, Roger H. |
AuthorAffiliation | Oncogene Division and ‡ Radiobiology Division, National Cancer Center Research Institute, Tokyo 104-0045, Japan; and § Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 |
AuthorAffiliation_xml | – name: Oncogene Division and ‡ Radiobiology Division, National Cancer Center Research Institute, Tokyo 104-0045, Japan; and § Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 |
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BackLink | https://www.ncbi.nlm.nih.gov/pubmed/9653156$$D View this record in MEDLINE/PubMed |
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Notes | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 Communicated by John W. Littlefield, The Johns Hopkins University School of Medicine, Baltimore, MD To whom reprint requests should be addressed at: Oncogene Division, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104, Japan. e-mail: ymurakam@ncc.go.jp. |
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Snippet | Loss of heterozygosity on chromosome 11q23 is observed at high frequency in human nonsmall cell lung carcinomas (NSCLCs), suggesting the presence of a tumor... |
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SubjectTerms | Animals Biological Sciences Carcinoma, Non-Small-Cell Lung - genetics Cell lines Chromosome Mapping Chromosomes Chromosomes, Artificial, Yeast Chromosomes, Human, Pair 11 Genes, Tumor Suppressor Genetics Humans Injection sites Loss of heterozygosity Lung cancer Lung Neoplasms - genetics Mice Mice, Nude Mutation Neoplasms, Experimental - genetics Neoplasms, Experimental - pathology Oncogenes Spheroplasts Tumor cell line Tumor suppressor genes Tumors Yeasts |
Title | Localization of Tumor Suppressor Activity Important in Nonsmall Cell Lung Carcinoma on Chromosome 11q |
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