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 wh ereas it exhibits negative effects on both cellular senescence and dif ferentiation. Ectopic overexpression of c-myc in transfection experime nts or titration of the c-myc mRNA by antisense oligonucleotides has d emonstrated 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 studie d the cellular reactions after manipulating the cellular concentration of c-Myc DNA-binding sites. Multiple copies of the c-Myc-binding sequ ence GACCA<(CG)under bar>TGGTC or, alternatively, the control sequence GACCA<(GC)under bar>TGGTC that displays only a poor affinity for c-My c were stably introduced into the genome of rat embryo fibroblasts. Tr ansfection with the c-Myc-binding sequence yielded much lower clone nu mbers and sizes than transfection with the control sequence. After pol yclonal 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 becomin g senescent and irreversibly growth arrested compared to the controls. Southern blot analysis demonstrated that 30 binding sequences on aver age were integrated into the cellular genome. Our results can be inter preted as competition of the ectopically introduced c-Myc-binding sequ ences with the functional genomic ones and assume that a fairly low nu mber 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. (C) 1998 Academi c Press.