Localization of tumor suppressor activity important in nonsmall cell lung carcinoma on chromosome 11q

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.

Figure 1

Physical mapping of the minimal NSCLC region of LOH on 11q23. Five YACs span the LOH region defined by D11S939 and D11S938 (12). Orientation relative to the Chr 11q centromere (CEN) and telomere (TEL) is indicated. The central 1,600-kb YAC, y939b12, was typed for 14 markers, including the vector-insert junction fragments, and subjected to fragmentation analysis; nine marker clusters (including the fixed positions of the vector-junction fragments) were identified. Three markers mapping within 330 kb of the centric end of YAC 939b12 were found on the YAC 785e12, which extends further toward the Chr 11q telomere; only the overlapping portion is diagrammed here. Five markers that are polymorphic between the insert of y939b12 and A549 are underlined. All deletion derivatives are products of acentric fragmentation and thus retain the centric YAC vector arm (adjacent to junction fragment ye939b12L).

Figure 2

Assessment of YACs in ybrid cell lines carrying full-length or fragmented YACs. PCR reactions were for 86CA and D11S1256, two of five markers that were found to be polymorphic between the A549 (human) cell line and human DNA cloned in YAC y939b12 (y939).

Figure 3

Localization of tumor suppressor activity on YAC 939b12 demonstrates that the distal 1.1 Mb is sufficient to suppress tumorigenesis of A549 or LLC cells injected in nu/nu mice. Number and percentage of sites forming tumors and the average latency are shown for parental lines A549 and LLC, for two independent ybrids with each parental line containing full-length YAC 939b12 (i.e., AY4-1, AY6-3, LLCY13-1 and LLCY13-2), and for ybrids containing fragmented or broken YACs retained in either parental cell line. Data for AY115 represents the aggregate tumor incidence from three independently derived ybrids containing the 1,010-kb fragmentation derivative, all of which showed ca. 70% tumor incidence with prolonged latency. The name of the ybrid line includes the name of ybrids made with fragmented YACs includes the name of the fragmentation derivative, e.g., AY115 results from a fusion with fragmentation derivative y939-115 (Fig. 1). Ybrids containing the overlapping YACs 911f02 or y785e12 showed no suppression, forming tumors at all injection sites. Phenotype designations are: T, tumorigenic; S, suppressed; PS, partial suppression of tumorigenesis. •, Markers present in hybrid cells; ○, markers absent in hybrid cells.