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Localization of tumor suppressor activity important in nonsmall cell lung carcinoma on chromosome 11q
Y Murakami1, T Nobukuni, K Tamura
1Oncogene Division, National Cancer Center Research Institute, Tokyo 104-0045, Japan. ymurakam@ncc.go.jp
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 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.
Insights
Loss of heterozygosity in chromosome 11q23 regions suggests a tumor suppressor gene in nonsmall cell lung cancer (NSCLC). A specific yeast artificial chromosome (YAC) suppressed tumor growth, localizing the gene to a 700 kb region.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Nonsmall cell lung carcinoma (NSCLC) frequently exhibits loss of heterozygosity on chromosome 11q23, indicating a potential tumor suppressor gene.
- Previous studies identified a commonly deleted 5-centimorgan segment in this region.
- The precise location and function of this putative tumor suppressor gene remain to be fully elucidated.
Purpose of the Study:
- To localize a putative tumor suppressor gene within the 11q23 region of NSCLC.
- To develop a functional assay for identifying and characterizing the tumor suppressor gene.
- To investigate the tumor suppressive activity of specific DNA fragments in lung carcinoma cell lines.
Main Methods:
- Complementation analysis using yeast artificial chromosomes (YACs) transferred into human (A549) and murine (LLC) NSCLC cell lines.
- Spheroplast fusion technique for introducing YACs into carcinoma cells.
- In vitro growth assays and in vivo tumor formation studies in athymic mice.
Main Results:
- A 1.6-Mb YAC (939b12) containing the minimal deleted region suppressed tumor formation in both A549 and LLC NSCLC cells in vivo.
- Flanking YACs (785e12 and 911f2) did not exhibit suppressor activity, confirming the location of the suppressor on 939b12.
- Further fragmentation and analysis localized the tumor suppression activity to the central 700 kb of the 939b12 YAC.
Conclusions:
- The 11q23 region harbors a tumor suppressor gene crucial for controlling NSCLC growth.
- The identified 700 kb region on YAC 939b12 contains the critical tumor suppressor locus.
- This study provides a functional assay and a refined region for the positional cloning of the NSCLC tumor suppressor gene.