A similar shift was detected in G0/G1-arrested cells but not in cells that had been released from such an arrest into S phase by the readdition of serum (Fig

A similar shift was detected in G0/G1-arrested cells but not in cells that had been released from such an arrest into S phase by the readdition of serum (Fig. CP-409092 hydrochloride The latter phenotype suggested that CDK1 is required to prevent the initiation of S phase before G2 and mitosis MCM5 have been completed, a surveillance mechanism first explained for fission yeast (18). The present study arose from our observation that a very similar rereplicative phenotype is usually observed in parental HT1080 cells following DNA damage, consistent with the notion that CDK1 activity is usually downregulated in response to DNA damage. In confirming and characterizing such downregulation, we appear to have identified a general DNA damage-induced signaling pathway in human cells, culminating in the continued transcriptional repression of a CP-409092 hydrochloride family of genes that are normally derepressed as cells progress from G1 into S and G2. MATERIALS AND METHODS Cell culture and irradiation. CP-409092 hydrochloride HT1080 (fibrosarcoma), HT2-19, and HeLa (cervical carcinoma) cells were used as previously explained (25, 45). HCT116 (colon carcinoma) and WI-38 (main fibroblasts) cells were obtained from the American Type Culture Collection. Cells were produced in Dulbecco’s altered essential medium (GIBCO) supplemented with 10% fetal calf serum, penicillin (80 U/ml), and streptomycin (80 mg/ml) and managed at 37C in a humidified atmosphere of 5% CO2. Cells were irradiated, after having been allowed to attach and grow overnight, in an IBL 637 137Cs irradiator (CIS BIO International) delivering 1.85 to 20 Gy/min, and samples were harvested at various times after irradiation. A dose of 6 Gy was utilized for all cells except HeLa cells, because this dose gave an accessible populace of G2/M-arrested or rereplicating cells; while 10% of HT1080 (43, 62), HCT116 (54), or WI-38 (38) cells recover from this dose, sufficient G2/M-arrested cells remain attached to the plate for analysis. A lower dose (3 Gy) was required for HeLa cells because at 6 Gy, most cells become detached and are lost, presumably by apoptosis, within 1 to 2 2 days. To arrest WI-38 cells in G0/G1, the medium of confluent plates was replaced with serum-free medium for 7 days. Release from G0/G1 arrest was achieved by trypsinizing cells and replating them at a low density in medium with 20% fetal calf serum. Circulation cytometry. Propidium iodide (PI)-stained nuclei were prepared by modification of a previously described method (25, 40). Briefly, about 5 105 cells were centrifuged, and the pellet was resuspended in 1 ml of answer I (10 mM NaCl, 1 mg of trisodium citrate per ml, 0.06% [vol/vol] Nonidet P-40, 25 g of PI per ml, 10 g of RNase A per ml) and incubated at room temperature for 30 min. One milliliter of answer II (1.5% [wt/vol] citric acid, 0.25 M sucrose, 40 g of PI per ml) was added. The nuclear suspension was agitated and stored at 4C overnight before circulation cytometric analysis on a Becton Dickinson FACScan. CellQuest software (Becton Dickinson) was utilized for acquisition and manipulation of the data. Plasmids and in CP-409092 hydrochloride vitro mutagenesis. Plasmid pWTCDK1-LUC contains 3 kb of the promoter of the human gene upstream of a luciferase reporter gene and the and genes as selection markers for use, respectively, in bacteria and mammalian cells. To make pWTCDK1-LUC, the CDK1 cDNA was removed from pCDC/gpt (25) by digestion with promoter. Briefly, a 225-bp fragment of the promoter made up of the CDE and CHR elements was cloned into pBluescript II KS(+) (Promega) for mutagenesis. The following primers, and complementary primers, were used to produce mutations 1 to 5 (CDE and CHR are shown in italic type, and the mutations.

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