Our primary goal is to isolate and molecularly characterize the gene(s) found to be amplified in extragonadal and metastatic human germ cell tumors (GCTs) and to determine the role of the gene(s) in normal germ cell development and in tumorigenesis. Male human germ cell tumors occur in the testis and at extragonadal sites (primarily at midline locations) with the latter being of either primary or metastatic origin. These tumors, arising from the transformation of germ cells, can take on the histological appearance of both pre- and post-zygotic tissues. Karyotypic analysis of GCTs has revealed the presence of nonrandom chromosome markers and provided evidence for gene amplification in GCTs at extragonadal sites. Gene amplification has been confirmed at the molecular level using the DNA in-gel renaturation assay; however, the identity of the gene(s) amplified remain unknown. It is possible that the gene product acts as a signal in early embryogenesis for primordial germ cells to settle in the midline during subsequent gonad development.
Our specific aims would involve the following approaches. 1. Isolation of gene(s) amplified in extragonadal and metastatic GCTs using the In-gel renaturation technique. Preparative in-gel renaturation assays will be. performed on DNA extracted from a GCT cell line with a homogeneously staining region (HSR). Amplified DNA will be isolated from the gel and subsequently cloned into vectors for further identification of sequences uniquely amplified in this cell line. 2. Isolation and characterization of complete gene sequences from human genomic and CDNA libraries. Genomic libraries of DNA from the cell line will be screened with amplified fragments to isolate overlapping gene sequences. Further characterization of clones will involve restriction mapping, identification of transcribed sequences, CDNA isolation, sequencing, and examination of sequences for possible coding regions of gene products that may be ultimately overexpressed. 3. Screening of GCTs with amplified gene(s) as probes. Southern hybridization analysis will be performed on DNA isolated from GCTs of all histologies using as probes the amplified DNA sequences isolated above. This will determine the prevalence of amplification of this gene(s) in GCTs of both gonadal and extragonadal presentation. Such a study has possible clinical implications if patterns of amplification are observed in tumors with poor clinical outcome. 4. Identification of the role of Isolated genes In normal cells and In tumorigenesis. Experimental studies will be undertaken to investigate the gene(s) structure and copy number (Southern hybridization analysis), MRNA levels (Northern hybridization analysis and Sl nuclease assays) and protein levels (Western blotting and immunohistochemical techniques) in a variety of human tissues,tumors and cell lines. Subcellular localization of the protein will be examined by immunofluorescence.
