Cell extracts were prepared from cultures treated with control or Chk1 siRNAs for 24 h in the presence or absence of thymidine and analyzed by Western blotting. not require p53. Thus, Chk1 plays a primary role in the protection of cells from death induced by replication fork stress, whereas p21 mediates through its role in regulating access into S phase. These findings are of potential importance to malignancy therapy because we demonstrate that this efficacy of clinically relevant brokers can be enhanced by manipulation of these signaling pathways. INTRODUCTION Cells respond to DNA damage by triggering cell cycle arrest, DNA repair, or death. DNA damage response pathways are frequently disrupted during tumor development, leading to genetic instability, loss of cell cycle checkpoint controls, and defects in the induction of apoptosis (Kastan and Bartek, 2004 ). The related PIKK kinases Unfixed cells were assayed for active caspase-3 immediately after treatment using the CaspGLOW fluorescein active caspase-3 staining kit according to the manufacturer’s instructions (MBL, Woburn, MA). Cells were then resuspended in PBS made up of 50 mg/ml PI, 100 mg/ml RNAse A, and 0.1% (vol/vol) Triton X-100. After a 30-min incubation the cell suspensions were analyzed by circulation cytometry for active caspase-3 and DNA content simultaneously. Mitochondrial membrane potential was assessed with tetramethyl Repaglinide rhodamine (TMRM) ethyl ester (Rasola and Geuna, 2001 ; Invitrogen). Working answer (20 M) was prepared in mitochondrial buffer (80 mM KCl, 10 mM Tris-HCl, 3 mM MgCl2, 1 mM EDTA, 5 mM KH2PO4, and 10 mM sodium succinate, pH 7,4). Cells were harvested, washed in PBS, and incubated in mitochondrial buffer with digitonin (at the ratio digitonin:protein, 0.12) for 5 Repaglinide min at room temperature. Then, cells were incubated with 200 nM TMRM, 0.5 g/ml 7-anime-actinomycin D (7AAD; Sigma-Aldrich) and 100 g/ml RNAse for 15 min. The TMRM and 7AAD signals (excitation, 488 nm; emission, 585 nm) were analyzed by circulation cytometry. Detection of Phosphorylated Histone H3 Treated cells were fixed with 70% ice-cold ethanol and stored at -20C. Fixed cells were washed twice with PBS and incubated for 15 min in PBS, 0.1% bovine serum albumin (BSA), and 0.25% Triton X-100. After centrifugation, the cell pellet was suspended in 100 l of PBS made up of 1% BSA and 0.75 g of a polyclonal antibody recognizing the phosphorylated form of histone H3 (Upstate Biotechnology, Lake Placid, NY) and incubated for 3 h at room temperature. The cells were rinsed with PBS made up of 1% BSA and incubated with FITC-conjugated goat anti-rabbit immunoglobulin antibody (DakoCytomation Denmark, Glostrup, Denmark) diluted at a ratio of 1 1:30 in PBS made up of 1% BSA. After a 30-min incubation at room temperature in the dark, the cells were stained with PI answer (PBS with 5 g/ml PI and 100 g/ml RNAse A), and cellular fluorescence was measured by a circulation cytometer. Western Blotting Cell extracts were prepared as explained previously (Bolderson Apoptosisa Cell collection MMR status P53 status Control TdR 2 mM TdR 20 mM CPT 20 nM CPT 1 M SW480 Proficient Mutant 1.9 1.7 1.3 0.7 2.7 0.25 1.2 0.6 4.7 2.9 HCT 116 hMLH1? Wild type 3.5 1.9 2.9 0.8 5.0 0.2 3.5 0.6 33.6 1.4 RKO hMLH1? Wild type 2.9 1.0 5.8 3.0 3.6 2.7 36.7 7.2 DLD-1 hMSH6? Mutant 1.8 0.5 2.8 0.6 2.8 3.2 0.5 3.8 SKUT-1 hMSH2? Mutant 1.4 1.4 1.4 1.6 1.6 HEC-A1 hPMS2? ND 5.3 6.0 5.3 4.8 9.7 Open in a separate window ND, not decided. aPercentage of Annexin V+/PI? cells (media and SD) after 24-h treatment Repaglinide with the indicated brokers. We compared the response of these cell lines to thymidine with that induced by CPT, which induces DSBs at DNA replication forks (Avemann in HCT 116 cells transfected with control or Chk1 siRNAs and treated with thymidine as explained above. p21 expression was quantified and expressed as fold induction relative to the control. -Actin was used to normalize the amount of loaded RNA. Control and Chk1 depleted cultures of the p53-/- and p21-/- derivatives of HCT116 were obtained after a 24-h siRNA treatment as explained above (Physique 7B). After exposure to thymidine for 48 h, cells were fixed and analyzed by circulation cytometry. p53-/- HCT116 cells responded similarly to the parental Rabbit Polyclonal to CtBP1 strain with respect to the depletion of S-phase cells after thymidine treatment and the accumulation of cells with a sub-G1 DNA content (Physique 7C). In contrast, p21-/- cells showed a further significant increase.