Clonal origin of adenovirus type 12-induced hamster tumors: nonspecific chromosomal integration sites of viral DNA

Abstract

The mechanism of tumor induction by human adenovirus type 12 (Ad12) in newborn Syrian hamsters (Mesocricetus auratus) has been investigated further. Tumors were produced in newborn hamsters by the s.c. injection of CsCl-purified Ad12. In 60-70% of the surviving animals, tumors have been observed between 33 and 47 days after injection. In 60 independently elicited tumors, the patterns of Ad12 DNA integration have been studied by restriction enzyme analyses and Southern blot hybridization using Ad12 DNA or its terminal DNA fragments as hybridization probes. Moreover, the integrated viral genomes have been localized on different hamster chromosomes by the fluorescence in situ hybridization technique using either nonradioactively labeled digoxigenin probes and fluorescent antibodies or biotinylated probes and fluorescent avidin. In all of the tumors, 20 and more copies of viral DNA have been found covalently linked to cellular DNA, as apparent from the off-size restriction fragments that do not comigrate during electrophoreses with any of the known virion DNA fragments. In Ad12-induced tumors or Ad12-transformed hamster cell lines, there is no evidence for the persistence of nonintegrated, free viral DNA copies. In general, the multiple copies of Ad12 DNA are inserted into a single chromosomal cellular site, which is different for each tumor. Only in one tumor cell line have the integrated Ad12 DNA copies been localized on two different chromosomes. The off-size fragment patterns generated by restriction and Southern blotting experiments are also unique and different for each tumor. These off-size fragments represent the sites of linkage between viral and cellular DNA but may also contain rearranged viral DNA sequences. We conclude that, upon Ad12 tumor induction in hamsters, Ad12 DNA does not integrate at specific insertion sites or nucleotide sequences into the cellular genome. It is still possible that selective elements exist at the sites of viral DNA insertion, e.g., specific chromatin structures due to transcriptional activity. The data presented also demonstrate (a) that each tumor cell carries Ad12 DNA; (b) that the insertion site appears to be the same in each tumor cell in a given tumor; (c) that, upon continued passage of the tumor cells in culture, the chromosomal site of Ad12 DNA insertion does not change, at least up to 25-32 passages, which correspond to about 75-96 cell generations beyond the tumor stage; and (d) that the integrated Ad12 DNA is localized on different chromosomes in individual tumors. Hence, the Ad12-induced tumors are of clonal origin. Another peculiarity of this viral tumor system is the frequent occurrence of more than one tumor in one animal. By the criteria established above, each individual tumor is characterized by its specific chromosomal integration site and restriction pattern. Thus, multiple induced tumors in one animal exhibit separate and individual clonality. During the time of maximally 7 weeks of observation of Ad12-induced tumors in the animals, metastases into different organ systems have not been observed.