The p16INK4a protein regulates cell cycle progression mainly by inhibiting the activity of G1-phase cyclin-dependent kinases (CDKs) 4 and 6, the subsequent retinoblastoma protein (pRb) phosphorylation and E2F transcription factor release. The p16INK4a protein can also repress the activity of other transcription factors, such as c-myc, nuclear factorkappaB and c-Jun/AP1. Here, we report that, in two p16WT and p53 WT cell lines (MCF7 and U87), p16INK4a overexpression induces a dramatic decrease in CDK1 protein expression. In response to p16
INK4a, the decreased rate of CDK1 protein synthesis, its unchanged protein half-life, unreduced CDK1 mRNA steady-state levels and mRNA half-life allow us to hypothesize that p16INK4a could regulate CDK1 expression at the post-transcriptional level. This CDK1 downregulation is mediated by the 3 0 -untranslated region (3 0 UTR) of CDK1 mRNA as shown by translational inhibition in luciferase assays and is associated with a modified expression balance of microRNAs (miRNAs) that potentially regulate CDK1, analyzed by TaqMan Human microRNA Array. The p16
INK4a-induced expression of two miRNAs (miR-410 and miR-650 chosen as an example) in MCF7 cells is confirmed by individual reverse transcriptionqPCR. Furthermore, we show the interaction of miR-410 or miR-650 with CDK1-3 0 UTR by luciferase assays. Endogenous CDK1 expression decreases upon both miRNA overexpression and increases with their simultaneous inhibition. The induction of miR-410, but not miR-650 could be related to the pRb/E2F pathway. These results demonstrate the post-transcriptional inhibition of CDK1 by p16 INK4a . We suggest that p16INK4a may regulate gene expression by modifying the functional equilibrium of transcription factors and consequently the expression balance of miRNAs.
SummaryNormal lymphocytes represent examples of somatic cells that are able to induce telomerase activity when stimulated. As previously reported, we showed that, during lymphocyte long-term culture and repeated stimulations, the appearance of senescent cells is associated with telomere shortening and a progressive drop in telomerase activity. We further showed that this shortening preferentially occured at long telomeres and was interrupted at each stimulation by a transitory increase in telomere length. In agreement with the fact that telomere uncapping triggers lymphocyte senescence, we observed an increase in γ γ γ γ -H2AX and 53BP1 foci as well as in the percentage of cells exhibiting DNA damage foci in telomeres. Such a DNA damage response may be related to the continuous increase of p16 ink4a upon cell stimulation and cell aging. Remarkably, at each stimulation, the expression of shelterin genes, such as hTRF1 , hTANK1 , hTIN2 , hPOT1 and hRAP1 , was decreased. We propose that telomere dysfunction during lymphocyte senescence caused by iterative stimulations does not only result from an excessive telomere shortening, but also from a decrease in shelterin content. These observations may be relevant for T-cell biology and aging.
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