Mesenchymal stem cells (MSCs) are of particular interest because they are being tested using cell and gene therapies for a number of human diseases. MSCs represent a rare population in tissues. Therefore, it is essential to grow MSCs in vitro before putting them into therapeutic use. This is compromised by senescence, limiting the proliferative capacity of MSCs. We analyzed the in vitro senescence of rat MSCs, because this animal is a widespread model for preclinical cell therapy studies. After initial expansion, MSCs showed an increased growth doubling time, lost telomerase activity, and expressed senescence-associated beta-galactosidase. Senescence was accompanied by downregulation of several genes involved in stem cell self-renewal. Of interest, several genes involved in DNA repair also showed a significant downregulation. Entry into senescence occurred with characteristic changes in Retinoblastoma (RB) expression patterns. Rb1 and p107 genes expression decreased during in vitro cultivation. In contrast, pRb2/p130 became the prominent RB protein. This suggests that RB2/P130 could be a marker of senescence or that it even plays a role in triggering the process in MSCs.
The different pocket proteins are established as negative cell cycle regulators. With regard to the repressor functions of pocket proteins in cellular senescence, studies so far have mainly focused on pRb/p105. Here, we show that in a broad range of wild-type p53-expressing human tumor cells, and in human diploid fibroblasts, Rb2/p130 is the dominating pocket protein in replicative and in accelerated senescence. Senescent cells are arrested at the transition from late G1-to early S-phase, as indicated by the absence of S-and G2-phase cyclins A and B. Expression of cyclin A and entry into S-phase resumed after RNA interference-mediated knockdown of Rb2/ p130. Activation of different upstream pathways by overexpression of either p21 or p16 converged on Rb2/ p130 accumulation and induced senescence. In contrast, p53-or p21-negative cells treated with DNA-damaging agents failed to accumulate Rb2/p130 and to enter senescence. Our data suggest that Rb2/p130 is a member of the p53-p21 DNA damage signaling cascade, and represents the essential pocket protein family member needed for the induction of any type of senescence.
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