Colin Collins, Soo-in Hwang, Johanna Rommens, David Kowbel, Christopher Martin, Michael Palazzolo, Gordon Hutchinson, Tony Godfrey and Joe W. Gray
Lawrence Berkeley National Laboratory, Berkeley, California.
In developed and several developing countries breast cancer is one of the most frequently diagnosed neoplasms and the leading cause of cancer related death amongst women. The mortality rate for breast cancer is approximately 27 per 100,000 women. Pangenomic surveys using comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) have revealed >20 regions of allelic imbalance suggesting the presence of numerous previously unrecognized tumor suppressor genes and oncogenes. Evidence is accumulating that allelic imbalance at these loci may play an important role in neoplastic transformation, progression and development of resistance to chemotherapeutic agents.
Chromosome 20 band ql3.2 is amplified in 40% of breast cancer cell lines and 29% of primary breast tumors. Moreover, high level amplification (7% of primary tumors) has been shown to be associated with decreased disease-free survival and an increased S-phase fraction. We have cloned the 20ql3.2 amplicon as a 2 Mb sequence-ready P1 and BAC contig and localized the minimum common region of amplification to a ~600 kb interval by performing interphase FISH on primary tumors with 9 Pl probes spanning the contig. To identify genes in the amplicon that program the aggressive phenotype of these breast tumors we are: (1) applying exon trapping and cDNA direct selection to the P1 and BAC contig and (2) in collaboration with the LBNL Human Genome Center (HGC) sequencing the 600 kb amplicon.
Exon trapping has been performed on the P1 and BAC clones spanning the minimum common region of amplification by digesting the clones with Pstl and Sacl followed by subcloning into the pSPL3 exon trapping vector. To date, >30 exons have been isolated and sequenced from the 600 kb interval. Computational analysis using BLAST has revealed homologies to known genes, ESTs and S. cerevisiae chromosome XIV. cDNA direct selection has been performed with Pl and BAC clones spanning the amplicon using pooled cDNA synthesized from 9 tissues and cDNA from the breast cancer cell line BT474. Preliminary studies have localized four cDNAs to the core of the amplicon.
Directed genomic sequencing is being employed to sequence the minimum common region of amplification. Presently, three contiguous Pl clones spanning approximately 200 kb are being sequenced. Putative exons are being identified in the genomic sequence using the programs GRAIL2, XGRAIL, SORFIND and BLAST. Exons identified by exon trapping and genomic sequencing are expanded by performing RACE-PCR and analyzed for phylogenetic conservation by hybridization to zoo blots. Exons and cDNAs are being assessed for expression by performing RT-PCR and Northern hybridization using RNA isolated from appropriate breast cancer cell lines and primary tumors. It is expected that the combination of exon trapping, direct selection, and directed genomic sequencing will culminate in the complete molecular description of the 20ql3.2 amplicon resulting in the identification of the hypothesized oncogene(s), improved diagnosis and prognostication, molecular therapeutics and ultimately decreased mortality in breast cancer.
This work was supported by grants from US DOE contract DEAC0376SF00098, USPHS grants CA44768, CA45919, CA52807 and Vysis.SH is supported the Human Genome Distinguished Postdoctoral Fellowship from DOE/ORISE.
 Hospital for Sick Children, Toronto, Ontario.
 RabbitHutch Biotechnology, 100 Mile House, British Columbia.
 Division of Molecular Cytometry, University of California, San Francisco, California.
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