Isolation of genes that are amplified in tumor cells provides a promising approach to identification of previously unknown genes involved in carcinogenesis and tumor progression. The technique of in-gel DNA renaturation was developed for detection and cloning of amplified genes of unknown nature. A new method which permits detection of DNA sequences amplified as little as 7- to 8-fold has been developed on the basis of this procedure. Two new cases of gene amplification in tumor cell lines have already been found which this new technique. In the first case erbA1 was amplified in Calu-3 lung adenocarcinoma, and in the second case amplification of the c-myc gene was associated with selection of PC-3 prostatic carcinoma cells for growth in nude mice. To understand the significance of in vivo amplification of the c-myc gene, PC-3 cells will be analyzed to determine whether c-myc is rearranged or non-uniformly amplified in unselected cells, and whether increased c-myc amplification provides PC-3 cells with a selective advantage for in vivo growth. To find previously unknown amplified genes associated with carcinogenesis and tumor progression, a large number of tissue specimens from primary and metastatic carcinomas of the colon and the ovary will be screened for the presence of amplified DNA using the newly developed assay. A series of cell lines, derived from colon carcinomas and melanomas and passaged as primary or metastatic tumors in nude mice, will also be tested for the presence of amplified DNA sequences. Tumor samples found to contain amplified DNA sequences will be tested for amplification of known oncogenes, and oncogene amplification will be correlated with type and grade of the tumor. DNA preparations that contain amplified sequences unrelated to known oncogenes will be analyzed to identify restriction fragments that are likely to be derived from or to be adjacent to the essential amplified gene. Such fragments, containing transcribed DNA sequences, will be cloned. The resulting clones will be used as probes to assay for amplification and transcription of corresponding genes in various tumors. Expression of the cloned sequences will be correlated with the tumor type, the stage of tumor progression and clinical history. Full-length cDNA clones of the tumor- associated amplified genes will be isolated and sequenced. cDNA sequences will be analyzed for homology to other proteins and the presence of potential functional sites. The function of the cloned tumor-associated amplified genes will be analyzed by gene transfer assays.
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