Gene amplification is an important mechanism of oncogene activation in breast and other cancers. Characterization of amplified regions of the genome in breast cancer has led to the identification of important oncogenes including erbB-2/HER-2, C-MYC, and fibroblast growth factor receptor (FGFR) 2. Chromosome 8p11-p12 is amplified in 10-15% of human breast cancers. The putative oncogene FGFR1 localizes to this region; however, we show evidence that FGFR inhibition fails to slow growth of three breast cancer cell lines with 8p11-p12 amplification. We present a detailed analysis of this amplicon in three human breast cancer cell lines using comparative genomic hybridization, traditional Southern and Northern analysis, and chromosome 8 cDNA microarray expression profiling. This study has identified new candidate oncogenes within the 8p11-p12 region, supporting the hypothesis that genes other than FGFR1 may contribute to oncogenesis in breast cancers with proximal 8p amplification.

Despite dramatic advances in the identification of human expressed sequence tags (ESTs), techniques that facilitate isolation of chromosome or chromosome band-specific ESTs would be of considerable value. This report demonstrates the feasibility of identifying chromosome-specific ESTs following microdissection of a single-copy chromosome region. For this study, a reduced complexity cDNA library was linkered and hybridized to normal human metaphase chromosomes. After stringency washes, the entire long arm of chromosome 6 (6q) was microdissected. Following PCR amplification using linker-specific primers, captured cDNAs were subcloned and 187 individual clones picked at random. These 187 clones were then sorted by filter cross-hybridization into 34 unique groups. Of these 34 groups, 19 (56%) mapped to chromosome 6 by Southern blot. We identified three previously known genes, human cytovillin (ezrin) mapped previously to 6q25-26, human cardiac gap junction protein (connexin 43) mapped previously to 6q21-23.2 and prolyloligopeptidase, which had not been mapped previously. BLASTN identified three clone groups with homology to known ESTs and 12 representing novel cDNA sequences. Six of the groups were sublocalized to specific band regions of 6q using a chromosome 6 hybrid mapping panel, five representative clones were tested on Northern analysis to verify their expression, and finally, nine clones were mapped against the Gene bridge 4 reduction hybrid panel to confirm their genetic map location on 6q. These results demonstrate that microdissection of single-copy sequences has sufficient specificity for isolation of chromosome-specific cDNAs.

AIM1 is a novel gene whose expression is associated with the experimental reversal of tumorigenicity of human malignant melanoma. The predicted protein product of the major 4.1-kb transcript shows striking similarity to the betagamma-crystallin superfamily. All known members of this superfamily contain two or four characteristic motifs arranged as one or two symmetrical domains. AIM1, in contrast, contains 12 betagamma motifs, suggesting a 6-domain structure resembling a trimer of beta- or gamma-crystallin subunits. The structure of the AIM1 gene shows remarkable similarity to beta-crystallin genes, with homologous introns delineating equivalent protein structural units. AIM1 is the first mammalian member of the betagamma superfamily with a primarily non-lens role. Other parts of the predicted AIM1 protein sequence have weak similarity with filament or actin-binding proteins. AIM1 is a good candidate for the putative suppressor of malignant melanoma on chromosome 6, possibly exerting its effects through interactions with the cytoskeleton.

We have developed a general strategy to reverse monochromosome suppression of the malignant phenotypes by retroviral transduction. Our approach involved the introduction of a retroviral expression vector-carried cDNA library into a chromosome 6-suppressed melanoma subline UACC-903(+6) [J. M. Trent et al., Science (Washington DC), 247: 568-571, 1990]. The cDNA library was constructed from polyadenylated RNA isolated from the suppressed UACC-903(+6) cells, packaged into high-titer amphotropic retrovirus particles, and transduced into UACC-903(+6) cells. Revertant his(R) transductants were selected by isolating colony-forming cells in soft agar. A total of 121 large (> 150 microm) colonies was picked from soft agar culture with 18 of 121 (15%) established as permanent sublines. The revertant sublines demonstrated 7-58% cloning efficiency upon plating in agar, in contrast to <0.05% for the UACC-903(+6) subline. All 18 revertant sublines, termed SRS1-SRS18 (for "selection of revertants for suppression"), displayed a reduced population-doubling time, with 9 of 18 showing focus formation in monolayer similar to the parental (nonsuppressed) cell line. Preliminary evidence for reversion of the suppressed phenotype by injection of cells into athymic nude mice has been completed for one revertant subline. Southern analysis has demonstrated integration of the retroviral vector sequence in all 18 sublines. This approach should facilitate the identification of genes involved in the tumorigenic phenotype of malignant melanoma, and is readily adaptable to other model systems.

Melanocytic transformation is thought to occur by the sequential accumulation of genetic alterations. Evidence implicating human chromosomes as a site for a gene(s) involved in melanoma suppression comes from studies of LOH [loss of heterozygosity], cytogenetics and biologic reversion of tumorigenicity following the introduction of a normal chromosome 6 by microcell-mediated chromosome transfer (Trent et al., 1990). Using a tumorigenic melanoma cell line (UACC 903) and a chromosome-6 suppressed melanoma subline [UACC 903 (+6)], we have isolated a series of genes uniquely expressed in the suppressed subline. A modified PCR-based cDNA subtraction technique was used to generate subtracted cDNA sublibraries for both the parental and (+6) suppressed cells. A total of 32 randomly selected clones from the suppressed sublibrary were isolated and examined, with 24 detecting a transcript by Northern analysis. Of these 24 clones, 21 (88%) demonstrated elevated expressed by Northern analysis in the suppressed subline relative to the tumorigenic parental cell line. In 6/21 differentially expressed clones (29%), expression was exclusive to the suppressed subline. Partial sequence analysis and database searching of these clones indicated that 5/6 were novel with one representing a previously characterized gene. Chromosomal localization of the five novel clones was performed following PCR amplification of a human/rodent somatic cell hybrid mapping panel or fluorescent in situ hybridization. One cDNA (termed AIM1) was localized to a band-region of chromosome 6 frequently deleted in melanomas (6q21). This novel approach should facilitate the identification of genes whose expression is causally related to the suppressed phenotype.

Chromosome microdissection was utilised for the analysis of cytogenetic markers of gene amplification [homogeneously staining regions (hsrs) and double minutes (dmins)] in two doxorubicin-resistant cell lines, fibrosarcoma HT1080/DR4 and small-cell lung cancer H69AR. Microdissection products from the hsr(7)(p12p15) of HT1080/DR4 were amplified and used for fluorescent in situ hybridisation (micro-FISH) analysis of drug-sensitive HT1080, resistant HT1080/DR4 and normal lymphocytes. The results demonstrated that the hsr contains a domain of DNA amplification of complex origin including sequences derived from 16p11.2-16p13.1, 2q11.2, 7q32-7q34 and 10q22. The amplification was confirmed by converting the micro-dissected probe into a microclone library for probing HT1080 and HT1080/DR4 Southerns. A micro-FISH probe from normal band region 16p11-16p13 further demonstrated amplification of 16p sequences in both HT1080/DR4 and H69AR. During the course of this analysis, Cole et al. (1992) (Science, 258, 1650-1653) published the amplification of the MRP gene in H69AR cells, which maps to chromosome 16p13.1. Our results corroborate the finding of MRP amplification in these doxorubicin-resistant cell lines, but, importantly, they provide information on the composition of the complex amplicon contributions from four different chromosomes. This study demonstrates the potential utility of chromosome microdissection for the rapid recovery of sequences from amplified regions in drug-resistant cells.