The frequent occurrence of amplified genes in human tumors has been suggested by the observations of double minute chromosomes and homogeneously staining regions in various types of cancer. Studies utilizing cloned probes permitted identification of some of the genes which are amplified in tumor cells, including several cellular oncogenes. Other genes that may be involved at different stages of carcinogenesis and tumor progression are also likely to be amplified in human malignancies. Amplification of these unidentified genes can provide a unique opportunity for their cloning and analysis. The present proposal is based on the procedure for detection, comparison, and cloning of amplified genes by in-gel DNA renaturation, a technique which has been recently developed by the principal investigator. This technique detects amplified DNA sequences in the absence of any preliminary information about their sequence or location in the genome. This procedure will be optimized and used to screen a large number of DNA preparations from various human tumor tissues and short-term cultues of tumor cells for the presence of amplified DNA sequences. The amplified DNA from different tumors will be compared by in-gel hybridization and the DNA fragments that are amplified in two or more tumors will be identified. In addition, the transcribed regions of the amplified DNA will be detected by hybridization with cDNA probes corresponding to mRNA from tumor cells. Those DNA fragments that are amplified in two or more tumors and/or transcribed in the corresponding tissues will be cloned. The resulting clones will be used as probes to assay their amplification and transcription in various tumors. Expression of the cloned gene sequences will be correlated with the tumor type, the stage of tumor progression, and the history of treatment. The cloned DNA sequences will be inserted into expression vectors and expressed in bacteria and antibodies to the resulting protein products will be raised. These antibodies will be used for isolation of the proteins encoded by the amplified genes from tumor cells and for further analysis of the functions of these proteins. It is hoped that the proposed experiments will result in better understanding of the genetic and biochemical mechanisms of tumor progression, which may lead to the development of new approaches to cancer diagnostics and treatment. (I)
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