This proposal seeks to identify and characterize mRNA's associated with human colorectal liver metastasis by using human colon cancer cell lines and fresh human tumor tissues in conjunction with a new, highly efficient molecular technique called differential display. This polymerase chain reaction-based methodology permits the side-by-side comparison of mRNA's derived from more than one source with the selection of mRNA's that are either over- or under-expressed for further study. This proposal is divided into two phases in order to permit adequate time for further training and didactics in molecular biology during Phase I: Phase I: (Years 1-2) 1. Identify up to four metastasis-associated cDNA probes using side-by-side comparisons of human colon cancer tumor cells characterized for low versus high metastatic potential in the nude mouse model of experimental liver metastasis using the differential display method. 2. Identify up to four metastasis-associated cDNA probes using fresh, human, synchronously-occurring primary (localized) and metastatic colorectal tumors--with each set of paired tumors derived from the same patients. Phase II: (Years 3-5) l. Construct and screen cDNA libraries to isolate full-length cDNA's associated with the cDNA probes for metastasis identified by differential display. 2. Sequence metastasis-associated, full-length cDNA's and perform computer searches for known homologies. 3. Use eucaryotic expression vectors to produce novel proteins from novel cDNA's. 4. Produce monoclonal and polyclonal antibodies against metastasis-associated proteins for use in screening frozen tissue tumor specimens for metastatic potential. Our long range goal will be to perform experiments that can document an integral function for metastasis marker proteins in the metastatic process by transfecting cDNA's linked to the metastatic process into non-metastatic cells or by using antisense oligonucleotide treatment with subsequent testing in the nude mouse model.
