Article Figures & Data
Figures
- Figure 1.
CDKN1A mutations in bladder cancer. A, diagram of the structure of the CDKN1A encoded protein p21 with mutations identified by the TCGA in bladder cancer (19) indicated above. Red circles, indel frame-shift mutations; yellow circles, nonsense mutations; and green circles, missense mutations. Mutations identified in bladder cancer cell lines are shown below. B, comutation plot for TP53 and CDKN1A mutations in the TCGA bladder cancer dataset (from http://www.cbioportal.org; ref. 19). It can be seen that eight (44%) CDKN1A mutations occur in cancers with TP53 mutations, and 10 (56%) occur in cancers without TP53 mutations. C, p21 expression in 14 bladder cancer cell lines assessed by immunoblotting. Actin was used as loading control; *, p21-mutated or -deficient cell lines. Note that some cell lines are loaded twice, as controls. D, activation of Chk1 by phosphorylation at S345, and induction of p21 by treatment with gemcitabine at 500 nmol/L for 12 hours. Note that cell lines TCCSUP and HCV29 are wild-type for each of TP53/CDKN1A; J82 is TP53 mutant and CDKN1A wild-type; 97-1 is TP53 wild-type and p21-deficient; whereas 647V and RT-112 are mutant for each of TP53/CDKN1A. E, p21 induction and pChk1-S345 and pChk1-S317 levels are increased by gemcitabine treatment in the TP53/CDKN1A wild-type cell line HCV29.
- Figure 2.
Synergistic effect of combined Chk1 inhibition and gemcitabine treatment in bladder cancer cell lines. A, cell viability curves are shown for a fixed dose of PF-477736 (PF, 500 nmol/L) and variable dose of gemcitabine (Gem, x-axis). Cell counts were assessed by CellTiter-Glo assay. Note that the reduction in cell viability is much higher for the CDKN1A-mutant cell lines 647V and RT-112 (both also mutant for TP53) and 97-1. B, cell viability curves are shown for various doses of PF-477736 (PF, top) and AZD7762 (AZD, bottom; 0, 50, 100, 200, and 400 nmol/L for each), and variable doses of gemcitabine (Gem, 0–50 nmol/L, x-axis). Cell viability was determined after 2 days incubation using CellTiter-Glo.
- Figure 3.
Cell-cycle kinetics and apoptosis of bladder cancer cell lines treated with gemcitabine and Chk1 inhibitor. A, effects of no treatment, gemcitabine alone (200 nmol/L × 1 hour), and gemcitabine followed by PF-477736 (500 nmol/L × 23 hours) on cell-cycle distribution of HCV29, J82, 97-1, 647V, and RT-112 cell lines measured 23 hours following cytotoxic treatment. Cell-cycle distribution was assessed by PI-DNA staining and FACS assay. B, cell-cycle distribution in gemcitabine-treated bladder cancer cells (as in A). Histograms, for cell-cycle distribution, as assessed by PI-DNA staining and FACS assay. C, bladder cancer cell lines were exposed to gemcitabine or PF477736, as well as the combination of these two agents for 18 hours. Immunoblot analysis was used to examine levels of PARP1, p-Chk1-345, p-γH2A.X-S139, and β-actin. Note major increase in cleaved PARP1 (PARP1-CL) and p-γH2A.X-S139 in the 647V and RT-112 cell lines. PARP1-FL is full-length PARP1.
- Figure 4.
p21 expression is critical to the response to combination treatment in p21-deficient bladder cancer cell lines. A–C, left, cell viability curves for 97-1, 647V, and RT-112 cell lines expressing doxycycline (Doxy)-inducible p21. Cell number was assessed by CellTiter-Glo after treatment with or without doxycycline to induce p21Cip1 expression, and treatment with 500 nmol/L PF-477736 and variable doses of gemcitabine. Right, immunoblot analysis of these treated cells. Note the induction of p21 and reduction in cleaved PARP-1 and p-γH2A.X expression in cells exposed to doxycycline.
- Figure 5.
Chk1 inhibition synergizes with gemcitabine to reduce bladder cancer cell growth in a xenograft model. A, RT-112 (5 × 106) cells were injected into the flanks of CB17/SCID mice. After the subcutaneous tumors reached a size of 10 cm3, mice were randomized to treatment with vehicle, gemcitabine (50 mg/kg) every 3 days, PF-477736 (15 mg/kg) every 3 days, or both drugs every 3 days at the same dose. Mean ± SD of tumor volume is shown. B and C, tumor weight (B) and tumor images (C) 7 weeks after mice were injected s.c. with the RT-112 cell line, and treated with gemcitabine or PF-477736, or the combination, as in A; mean ± SD. D, weights of mice undergoing treatment with vehicle, gemcitabine, PF-477736, or the combination. Mean ± SD of the normalized weight from the time of treatment initiation is shown.
Volume 14, Issue 1
