Article Figures & Data
Figures
- Figure 1.
Evaluating drug responses in genetically modified human colorectal cancer cell lines. A, ATR can be activated during S-phase in the presence of upstream Cdk2. The contributions of individual components of the Cdk2/ATR/Chk1 pathway were assessed with the use of isogenic lines harboring knockout and knockin alleles, as indicated. See text for additional details. B, clonogenic survival of DLD-1 and ATRS/S, CHK1+/− and CHK1S317A/− derivative cells after treatment with the indicated doses of drugs. Survival is plotted on log scale as a proportion of untreated controls.
- Figure 2.
Inhibition of cell growth after treatment with anticancer agents. DLD-1 cells were compared with their ATRS/S derivative in a high-throughput growth assay (see Materials and Methods). The growth of ATRS/S cells was plotted as the proportional growth of wild-type DLD-1 cells treated with the same drugs.
- Figure 3.
Inhibition of cell survival after treatment with selected anticancer agents. DLD-1 cells were compared with their isogenic derivatives in a clonogenic survival assay. Drug concentrations used were bortezomib (30 nmol/L), thiotepa (1 μmol/L), triethylenemelamine (1 μmol/L), dactinomycin (30 nmol/L), teniposide (300 nmol/L), and 5-fluorouracil (50 μmol/L). Cell lines were treated with drugs for 24 hours before replating.
- Figure 4.
Phosphorylation of Chk1 in response to genotype-selective chemotherapeutics. A, levels of total Chk1 protein and Chk1 S317-P phosphoprotein in drug-treated DLD-1 cells were determined by immunoblotting. Drug concentrations used were bortezomib (300 nmol/L), paclitaxel (300 nmol/L), temozolomide (1 μmol/L), triethylenemelamine (1 μmol/L), and dactinomycin (300 nmol/L). Lysates were harvested after 24 hours of drug treatment. B, DLD-1 cells were treated with 300 nmol/L bortezomib or 300 nmol/L dactinomycin for 24 hours. The localization and distribution of γH2AX were assessed by immunofluorescence. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI).
- Figure 5.
The role of Cdk2 in the cellular responses to ATR genotype–selective agents. A, HCT116 and isogenic CDK2−/− cells were treated with the indicated chemotherapeutic agents and harvested at 10 or 24 hours. Time “0” indicates no treatment. Levels of the Chk1 phosphoproteins Chk1S345-P and Chk1S317-P and total Chk1 were determined by immunoblotting. α-Tubulin was assessed as a loading control. B, clonogenic survival was measured in wild-type HCT116 and CDK2−/− cells treated with gemcitabine, hydroxyurea, or MMC for 24 hours or cisplatin for 48 hours, as indicated.
- Figure 6.
Effects of a Chk1 inhibitor and cell ploidy. A, wild-type HCT116 and CDK2−/− cells were treated for 24 hours with 100 nmol/L UCN-01 alone or in combination with 20 nmol/L gemcitabine (GCB; left) or 150 nmol/L MMC (right). B, chemical structures of gemcitabine, MMC, UCN-01, and nitrogen mustard. C, CHK1+/− cells that were primarily diploid were compared with wild-type DLD-1 and an isolated subclone of CHK1+/− tetraploid cells. All cells were treated with the alkylating agents MMC (1 μmol/L) or nitrogen mustard (1 μmol/L) for 24 hours. Clonogenic survival is expressed as proportional survival compared with untreated controls of the same genotype.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure 1 - PDF file - 74K, Effect of CDK2 on DNA replication fork dynamics. Replicating DNA was sequentially labeled with CIdU (red) and IdU (green), and the length of double-labeled DNA fibers were measured via confocal microscopy. (A) Distribution of fork rates in unperturbed HCT116 cells and the CDK2-/- derivative cell line. (B) Average fork rates in HCT116 parental and CDK2-/- cells. ** denotes significance (One-tailed paired Student's t-test, p<009). (C) Proportion of labeled fibers that represent replication origins and terminations. Colored arrows represent orientation of replicons.
Volume 11, Issue 1
