(F) Representative images showing telomere clustering. significantly greater quantity of fragile telomeres than the parental telomerase-positive cell collection, H1299 (Fig. 1B and ?andC).C). Treatment with the G-quadruplex (here referred to as G4) ligand PDS (pyridostatin) induced more fragile telomeres, as previously reported (17, 18). Consistent with the phenotypes in H1299 and H1299ALT, SW39ALT cells also harbor more fragile telomeres than the telomerase-positive SW39 cells (Fig. S1B and C). These observations can be interpreted to indicate that ALT cells harbor more DNA replication problems at telomeres than telomerase-positive cells. Open in a separate windowpane FIG 1 Mitotic DNA synthesis at telomeres in ALT cells is definitely induced by DNA replication stress. (A) Overview of H1299ALT cell generation from its parental telomerase-positive malignancy cell collection, H1299 (13). (B and C) Fragile telomere analysis of H1299 and H1299ALT with or without PDS (pyridostatin) treatment (10 M). (B) Representative images showing fragile telomeres on metaphase spreads. (C) Quantification of fragile telomeres as a percentage of fragile telomeres (results are from three self-employed experiments and are given as means standard errors of the means [SEM]). (D and E) Metaphase chromosome-telomere dysfunction-induced focus (Meta-TIF) analysis of H1299 and H1299ALT with or without PDS treatment. (D) Representative images showing gamma-H2AX-positive telomeres on metaphase spreads. (E) Quantification of Meta-TIFs as the number of gamma-H2AX-positive telomeres per metaphase spread (results are from two self-employed experiments and are given as means SEM). (F) Experimental plan for mitotic DNA synthesis analysis (remaining) and cell cycle analysis for the indicated time points (right). (G and H) Telomeric mitotic DNA PI4KIIIbeta-IN-9 synthesis (MiDAS) analysis of H1299 and H1299ALT with or without PDS treatment. (G) Representative images showing mitotic DNA synthesis at telomeres on metaphase spreads. (H) Quantification of telomeric MiDAS and quantity of EdU-positive telomeres per metaphase spread (results are from three self-employed experiments and are given as means SEM). *, < 0.05; **, < 0.01; ***, < 0.001; ****, < 0.0001 (unpaired Student's test). Spontaneous telomeric DNA damage reactions in metaphase cells have been more frequently observed in ALT cells than in telomerase-positive cells (19, 20). Exacerbated replication problems in ALT cells may lead to prolonged DNA damage reactions during mitosis, especially in p53-null cells or conditions under which cells do not have an intact G2/M checkpoint (20). Therefore, we next PI4KIIIbeta-IN-9 tested whether replication problems in ALT cells lead to prolonged DNA damage reactions at telomeres. We measured gamma-H2AX signals colocalized having a telomere peptide nucleic acid probe on metaphase chromosomes to investigate telomere-specific DNA damage responses (also called TIFs, for telomere-induced dysfunctional telomeres). H1299ALT cells experienced more gamma-H2AX-positive telomeres on metaphase chromosomes (Fig. 1D and ?andE),E), and their levels were increased under PDS-treated conditions. This demonstrates that telomeric DNA damage responses are improved in ALT cells, probably due to DNA replication problems. It was recently demonstrated that replication stress causes DNA synthesis in mitosis via a break-induced replication (BIR)-like process (21). Mitotic DNA synthesis (here referred to as MiDAS) happens predominantly in common fragile sites in response to replication stress (22). Therefore, we next tested whether telomeric replication problems result in MiDAS at telomeres. Cells were remaining untreated or were treated with the G4 ligand PDS in S phase, synchronized at late G2 phase using the CDK1 inhibitor RO3306, and then released into mitosis in the presence of 5-ethynyl-2-deoxyuridine (EdU) for 1 h (Fig. 1F). PI4KIIIbeta-IN-9 Mitotic cells were then collected by shake-off to analyze telomeric EdU incorporation on metaphase chromosomes. We found that EdU incorporation in the telomere region was regularly observed in H1299ALT cells, whereas it was very rare in H1299 telomerase-positive cells (Fig. 1G and ?andH).H). Treatment with the G4-stabilizing compound PDS improved MiDAS in both telomerase-positive H1299 and H1299ALT cells, demonstrating that telomeric MiDAS can be induced by replication stress, such as G4 formation. We also found that two clonal derivatives of SW39ALT cells displayed telomeric MiDAS that was only rarely observed in telomerase-positive SW39 cells from which the SW39ALT cells were derived (Fig. S1D and E). Collectively, we PI4KIIIbeta-IN-9 display the telomeres in ALT cells PI4KIIIbeta-IN-9 are prone to encounter DNA replication stress, leading to spontaneous DNA synthesis processes during mitosis. Telomeric MiDAS is definitely a traditional DNA synthesis mechanism mediated by break-induced Nr2f1 replication processes. When the DNA replication fork is definitely stalled or collapsed but not reinitiated during S/G2 phase, mitotic DNA synthesis can be initiated..