Polyomavirus large T antigen-dependent DNA amplification.

DNA amplification is a readily measurable indicator for genome destabilization. Contrary to normal senescing cells, those of most immortal or transformed cell lines are karyotypically unstable and permissive for amplification. Permissivity for amplification can be generated by gene products of several DNA tumor viruses whereby their interaction with the tumorsuppressor protein p53 is important. p53 is the major protein involved in check point control of DNA damage. Polyomavirus large T antigen is also involved in immortalization and transformation of cells but it does not interact with p53. We, therefore, examined whether this protein could still make the non-permissive cell line REF52 permissive for gene amplification. To this end REF52 cell lines were constructed which conditionally expressed the wild type polyomavirus large T antigen or a mutant form unable to bind the retinoblastoma protein. Using the inhibitor of de novo pyrimidine biosynthesis, phosphonoacetyl-L-aspartate (PALA), as selective agent we found that PALA resistant cells arise with a frequency of about 5 x 10(-5) and that the interaction of polyomavirus large T protein with the retinoblastoma protein or another related pocket protein is important for this to occur. PALA resistant cells have an increased number of chromosomes and dicentric chromosomes which are considered as starting point for DNA structures characteristic for amplified DNA. Such structures were indeed found with the help of fluorescence in situ hybridization. PALA resistant cells appear normal with respect to p53. Our data indicate that PALA induces a G1 block which can be partially overcome by polyomavirus large T protein by its interaction with E2F-pocket protein complexes providing further evidence that these complexes are downstream targets of p53.