Chromosomal Instability and Evolution of Transformed Phenotype in Cell Lines Selected from Senescent Rat Embryonic Fibroblasts with Rapamycin

Abstract

The karyotype and phenotype evolution in cell lines (Rapa) obtained by rapamycin selection from rat embryonic fibroblasts (REF) has been studied. Rapamycin, an inhibitor of mTORC1 kinase, prevents replicative aging of REF cells starting after seven to ten passages in vitro. The karyotype of the established cell lines undergoes an evolution upon their cultivation in vitro. On early passages, clonal chromosome rearrangements, more specifically trisomy of chromosome 7 and translocations t(2;7) or t(4;11), were observed. Chromosome rearrangements (CR) accumulated at late passages. Simultaneously, signs of the transformed phenotype appeared: capacity for clonal growth, a decrease in contact inhibition, and high proliferation rate in the monolayer at saturating density. The evolution of the transformed phenotype was accompanied by the loss of G₁/S and/or G₂/M checkpoint control. Rapa lines were characterized by accumulation of γH2AX and p53BP foci, which, however, did not colocalize. Thus indicates the accumulation of unrepaired double-strand DNA breaks (DSB). The Rapa lines are shown to express the pluripotency markers Nanog and Oct3/4. The causes of genetic and karyotype instability of Rapa lines during long-term cultivation have been discussed.