Musacchio, Dr. mitosis. Despite their lack of mitosis, the cells showed APC/Cdh1-dependent degradation of the replication inhibitor geminin, followed by build up of Cdt1, which is required for source licensing. Cells then came into a second S phase resulting in whole-genome reduplication and tetraploidy. Upon repair of telomere safety, these tetraploid cells resumed cell division cycles and proliferated. These observations suggest a general mechanism for the induction of tetraploidization in the early phases of tumorigenesis when telomere dysfunction can result from excessive telomere shortening. Keywords: telomere, POT1, tumor, tetraploidy, aneuploidy Intro Aneuploidy is definitely a hallmark of solid human being cancers. Diploid cells can acquire an aneuploid chromosome match through repeated chromosome non-disjunction events (examined in (Kops et al., 2005)). However, as many solid tumors have sub-tetraploid karyotypes and supernumerary centrosomes, it is likely that the initial step toward aneuploidy is definitely tetraploidization (examined in (Storchova and Kuffer, 2008)). Tetraploid cells, most likely because they can form multipolar spindles, have a high rate of chromosome missegregation, explaining how tumors attain subtetraploid karyotypes in which some chromosomes are present at 4 copies whereas additional chromosomes have a lower copy number. Tetraploidization has been observed in the early stages of colon cancer (Danes, 1978; Levine et al., 1991), Barretts esophagus (Galipeau et al., 1996; Rabinovitch et al., 1989), breast tumor (Dutrillaux et al., 1991), and cervical malignancy (Olaharski et al., 2006). Three main mechanisms for tetraploidization in the context of human tumor have been proposed: cell fusion, a failure to total mitosis (mitotic slippage), and a failure to total cytokinesis (examined in (Ganem et al., 2007)). Cutamesine Here we display that tetraploidization can occur in response to the loss of telomere safety, which is thought to be a common event in human being tumorigenesis (examined in (Maser and DePinho, 2002)). The telomeres of most human being somatic cells undergo progressive telomere shortening due to the repression of telomerase. This process eventually limits cellular proliferation through the induction of apoptosis or senescence and is thought to symbolize a tumor suppressor mechanism that can be subverted from the activation of telomerase (Kim et al., 1994; Bodnar et al., 1998). In agreement, intense telomere shortening is definitely observed in the early phases of tumorigenesis before telomerase is definitely triggered (Chin et al., 2004). Furthermore, most clinically-relevant human being tumors, including telomerase-positive tumors, have short telomeres that carry witness to the telomere shortening in their proliferative history (de Lange et al., 1990; Hastie et al., 1990). Therefore, many human being cancers might encounter an episode of diminished chromosome end safety during their development. Telomeres that have become dysfunctional after considerable shortening activate the canonical DNA damage signaling pathways, mediated from the Cutamesine ATM and ATR kinases (d’Adda di Fagagna et al., 2003). At practical telomeres, ATM signaling is definitely repressed from the shelterin component TRF2, whereas the single-stranded telomeric DNA binding protein POT1 blocks the activation of the ATR kinase (Lazzerini Denchi and de Lange, 2007). In the current study, telomere dysfunction is definitely experimentally induced in mouse embryo fibroblasts (MEFs) that contain floxed alleles of the two mouse POT1 genes, POT1a and POT1b (Hockemeyer et al., 2006). Depletion of POT1a/b induces an ATR kinase response that leads to build up of DNA damage factors at chromosome ends and activation of the effector kinases Chk1 and Chk2 (Lazzerini Denchi and de Lange, 2007). This DNA damage response is prolonged because the restoration of the damaged telomeres by NHEJ is definitely repressed by TRF2, which remains associated with telomeres despite the removal of POT1a and Cb (Hockemeyer et al., 2006). When Cutamesine POT1a/b are erased from MEFs that lack a functional p53 pathway, the cells undergo polyploidization, resulting in 4N, 8N, and 16N DNA content material (Hockemeyer et al., 2006). The FACS profile of these cultures shows discrete peaks suggesting that the whole genome is definitely duplicated. Furthermore, the occasional metaphase spreads from POT1a/b DKO cultures display diplo- and quadruplochromosomes indicating that these cells have undergone two or three rounds of DNA replication without resolution of Gpr20 the centromeric cohesin. Here we document the consequences of telomere dysfunction with this establishing and demonstrate that by-pass of mitosis and endoreduplication are induced from the prolonged DNA damage transmission emanating from permanently damaged telomeres. RESULTS Polyploidization induced by prolonged DNA damage signaling The two mouse POT1 proteins were.