Integrated viral genomes can be lost from adenovirus type 12-induced hamster tumor cells in a clone-specific, multistep process with retention of theoncogenic phenotype

In adenovirus type 12 (Adl2)-induced tumor cells, in Adl2-transformed cells and in continuously passaged cell lines from these sources, the viral DNA is integrated in multiple copies, usually at a single chromosomal location. In different tumors or cell lines, the sites of integration of Ad12 DNA ar e all different. Rare exceptions exist. In most instances, the integrated v iral DNA resides very stably in, the host cell genomes. However, upon conti nuous serial passage of such cell lines, the integrated viral DNA can be de stabilized and lost. In two instances, i.e. in the Adl12-induced hamster tu mor cell lines H1111(1) and CLAC1, we have investigated the loss of integra ted viral DNA in detail. After extended serial passage, these two cell line s seemed to be devoid of Ad12 DNA sequences, as detectable by Southern blot hybridization, but continued to induce tumors after reinjection into hamst ers. Cells from these two cell lines were now recloned three times, and DNA s from cultures derived from several individual clones were reinvestigated for the presence of several parts of the viral genome by the polymerase cha in reaction (PCR). Some of the clones still carried parts of the Ad12 genom e. However, several clones were isolated that proved free of all parts of t he viral genome, except for minute segments from the right terminus of the Ad12 genome. Apparently, the loss of integrated viral DNA from these cell l ines proceeded as a continuous, gradual, multistep process whose pattern co uld differ from cell clone to cell clone, once destabilization had been ini tiated. The mechanism of destabilization is not understood. Cell population s of 2 x 10(6) to 3 x 10(7), and as low as 10(2), cells from the clones, th at contained only minimal remnants from the right viral DNA terminus, were reinjected into newborn or 13-20 day-old weanling Syrian hamsters (Mesocric etus auratus). Tumors developed within 5-17 days after injection. Tumor cel l clones also grew in soft agar. The injection of primary hamster skin fibr oblasts never elicited tumor formation. The tumor cells induced by this rei njection proved repeatedly free of Ad12 DNA both by Southern blot hybridiza tion and by PCR, except for those cell and tumor clones that contained smal l segments of the right terminal E4 region of the Ad12 genome. The tumor ce lls, however, retained their oncogenic phenotype. The results raise questio ns about the cell clone-specific excision patterns of integrated foreign DN A from the recipient genome and the possibility of a hit-and-run mechanism of adenoviral oncogenesis. (C) 1999 Elsevier Science B.V. All rights reserv ed.