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Accelerated proliferative senescence of rat embryo fibroblasts after stable transfection of multiple copies of the c-Myc DNA-binding sequence.

The protooncogene c-myc positively regulates cellular proliferation whereas it exhibits negative effects on both cellular senescence and differentiation. Ectopic overexpression of c-myc in transfection experiments or titration of the c-myc mRNA by antisense oligonucleotides has demonstrated that small changes of the concentration of cellular c-myc mRNA or protein levels can be crucial for these processes. In view of the role of c-Myc as a transcription factor, most of these effects may be mediated via its binding to specific DNA sequences. Here we studied the cellular reactions after manipulating the cellular concentration of c-Myc DNA-binding sites. Multiple copies of the c-Myc-binding sequence GACCACGTGGTC or, alternatively, the control sequence GACCAGCTGGTC that displays only a poor affinity for c-Myc were stably introduced into the genome of rat embryo fibroblasts. Transfection with the c-Myc-binding sequence yielded much lower clone numbers and sizes than transfection with the control sequence. After polyclonal selection and further subcultivation cells transfected with c-Myc-binding sequence exhibited a reduced growth rate and achieved less than two-thirds of the cumulative population doublings before becoming senescent and irreversibly growth arrested compared to the controls. Southern blot analysis demonstrated that 30 binding sequences on average were integrated into the cellular genome. Our results can be interpreted as competition of the ectopically introduced c-Myc-binding sequences with the functional genomic ones and assume that a fairly low number of the latter exist in the normal cellular genome. Hence, only a low copy number of introduced c-Myc-binding sequences is sufficient to cause signs of accelerated proliferative senescence.