The major goals of this project are to define the role of a retrovirus in human testicular germ cell cancer and, perhaps, other malignancies, and to determine if the virus is endogenous to humans. A retrovirus is produced by cells of all available human embryonal cell carcinoma (EC) cell lines and two mixed choriocarcinoma-EC lines. Virus production is stimulated after the cells are induced with 5-iodo-2'-deoxyuridine and dexamethasone. The possibility this virus may be passed vertically and the significant increase in incidence of testicular germ cell cancer are issues of utmost importance in human biology, especially since a family of exogenous retroviruses is known to be associated with malignancies and other diseases of the human immune system. Our approach to this project will integrate biological, molecular, and immunological methods with cell lines and reagents currently available and other reagents to be developed.
The first aim i s to establish conditions for maximum virus production, including tests of different growth conditions and induction procedures. These tests will emphasize clonal lines derived from the mixed choriocarcinoma-EC lines, which are known to produce more virus particles than the EC lines.
The second aim i s to obtain molecular clones and determine the nucleotide sequence of the retrovirus. Clones will be derived from (a) double-stranded cDNA of virion-associated RNA isolated from virions concentrated and purified from supernatant fluids of induced cultures and (b) the integrated proviral genome selected from a human DNA library prepared from these cells. The nucleotide sequence of the retrovirus will be compared to the nucleotide sequence of previously described human defective endogenous retroviral sequences and other mammalian retroviruses. Third, antisera will be prepared in rabbits to synthetic peptides based on selected sequences of (a) gag and env genes of the retrovirus sequences cloned in aim 2 and (b) gag and env genes of previously described human defective endogenous retroviruses.
The fourth aim i s to use the cDNa probes derived in aim 2 and the antisera to synthetic peptides obtained in aim 3 to screen (a) cell lines derived from human testicular germ cell tumors and other malignancies, (b) differentiated populations of EC cells, and (c) human normal and malignant tissues for viral-specific mRNA, proviral DNA, and proteins related to human retroviruses. This project will provide essential information on whether the virus is endogenous or exogenous to humans and the possible role of the virus in human normal or abnormal biology.
