Article Figures & Data
Figures
- Figure 1.
The influence of MK-1775 on radiation-induced G2 checkpoint activation. T98G (A), NHAs (C), and the GNS (D) cells were treated with MK-1775 (250 nmol/L, unless otherwise noted) or vehicle control 6 hours before irradiation (RT; 6 Gy) and collected at specified time points for analysis of cell-cycle phase distribution by flow cytometry. Mean ± SE of 3 independent experiments. B, mitotic ratio in T98G cells was determined using dual propidium iodide and phospho-histone H3 staining and calculated by the percentage of cells in M phase pre-/post-radiation. Results are representative of 3 independent experiments; mean ± SE.
- Figure 2.
MK-1775 attenuates radiation-induced CDC2 phosphorylation. Tumor cell lysates from T98G cells exposed to vehicle control (C), MK-1175 (MK) alone (250 nmol/L, 16 hours), radiation alone (RT, 6 Gy), or combined with MK-1775 (250 nmol/L, 6 hours before RT) at the indicated times were resolved in SDS-PAGE and probed with specific antibodies against the indicated proteins. Results are representative of 2 independent experiments.
- Figure 3.
The influence of MK-1775 on radiosensitivity in glioblastoma cell lines. Glioblastoma cell lines T98G (A; p53 mutant), U251 (B; p53 mutant), U87 (C; p53 wild-type), and GNS cell line G179 (D; p53 mutant) were plated, allowed to attach, and treated with either MK-1775 (100 nmol/L unless otherwise noted) or vehicle control for 6 hours pre-RT. Plates were replaced with fresh culture media after 24 hours, and surviving fractions were calculated 10 to 14 days following treatment, normalizing for the independent cytotoxicity of MK-1775. Mean ± SE of 3 independent experiments.
- Figure 4.
MK-1775 attenuates recovery during fractionated radiation. T98G cells were plated, allowed to attach, and exposed to a single fraction of radiation (RT) or fractionated RT (2 Gy every 24 hours) at the stated doses in the presence of MK-1775 (100 nmol/L) or vehicle control. Plates were replaced with fresh culture media after 24 hours (single fraction RT) and 48 and 72 hours for plates treated with 4 and 6 Gy fractionated RT, respectively. Colony survival was determined 10 to 14 days following treatment, normalizing for the independent cytotoxicity of MK-1775. Recovery rates (RR) during fractionated RT were calculated by dividing the percentage of cell survival following fractionated RT by the percentage of cell survival following an equivalent dose of radiation delivered as a single fraction. Mean ± SE of 3 independent experiments.
- Figure 5.
MK-1775 augments radiation-induced mitotic catastrophe and γH2AX expression. A, T98G cells growing in chamber slides were exposed to MK-1775 (250 nmol/L) or vehicle control for 6 hours, irradiated (RT, 6 Gy), and fixed at 24 hours post-irradiation for immunocytochemical analysis of mitotic catastrophe. Nuclear fragmentation (defined as the presence of 2 or more distinct lobes within a single cell) was evaluated in 100 cells per treatment per experiment. Mean ± SE (*, P = 0.03; **, P = 0.001). B, T98G cells were treated in the above described conditions, and cell lysates obtained at the indicated times were resolved in SDS-PAGE and probed with specific antibodies against the indicated proteins.
- Figure 6.
MK-1775 enhances radiation-induced tumor growth delay and attenuates the radiation-induced G2 checkpoint arrest in vivo. A, U251 cells were injected subcutaneously in a mouse flank model. When tumors reached about 150 mm3 in size, mice were randomized into 4 groups: vehicle control, MK-1775 (60 mg/kg twice daily × 3 days), irradiation (2 Gy × 3 days; RT), or MK-1775 + irradiation. To obtain a tumor growth curve, perpendicular diameter measurements of each tumor were measured with digital calipers, and volumes were calculated using formula (L × W × W)/2. Each group contained 7 mice (*, RT vs. RT + MK-1775: P = 0.04; **, MK-1775 vs. RT + MK-1775: P = 0.001). Mean ± SE. B, U251 cells were injected subcutaneously in a mouse flank model. When tumors reached about 500 mm3 in size, mice were randomized into 3 groups: vehicle control, irradiation (6 Gy; RT), or MK-1775 (60 mg/kg twice daily, beginning 2 hours before RT) + irradiation (6 Gy). At the stated times, animals were sacrificed and tumors were removed, formalin fixed, and paraffin embedded. Immunohistochemical staining was conducted to determine the mitotic ratio by immunostaining for phospho-histone H3 expression. The entire section was then quantified using an automated system and mitotic ratio calculated, which equaled the percentage of mitotic cells in the treatment group divided by the control group.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figures 1-8 - PDF file - 977K
Volume 10, Issue 12
