Article Figures & Data
Figures
- Figure 1.
CDK4 copy number and expression of cell-cycle regulatory proteins in liposarcoma cells. A, CDK4 copy number in liposarcoma cells and a patient-derived xenograft (PDX) was determined by qRT-PCR with the Rnase P gene as an internal normalization reference and human normal genomic DNA as a calibrator sample. Values represent mean ± SD (n ≥ 3) B, protein expression was analyzed by immunoblot analyses. PA, preadipocytes; A, adipocytes.
- Figure 2.
Effects of siRNA-mediated knockdown of CDK4 on RB phosphorylation and cell growth in liposarcoma cells. A, effects of CDK4 knockdown on RB phosphorylation were examined by immunoblot analysis at 40 hours in LP6 liposarcoma cells. B, effects of CDK4 knockdown on growth of LP6 cells were monitored daily by cell counting.
- Figure 3.
Effects of CDK4/6 inhibitor LEE011 on RB phosphorylation and cell growth in liposarcoma cells. A and B, effects of LEE011 on RB phosphorylation in LP6 (A) and other liposarcoma cells (B) were evaluated by immunoblot analysis at 24 hours. C, growth curves of LP6 cells treated with vehicle or LEE011 at indicated concentrations. Values represent mean ± SEM (n = 2). D, response of liposarcoma cells to 3 days treatment of LEE011. Values represent mean ± SD (n = 2). E and F, effects of LEE011 on cell-cycle distribution of liposarcoma cells at 24 hours. In E, LP6 cells were used; in F, values represent mean ± SD (n = 2).
- Figure 4.
siRNA-mediated knockdown of RB rescues the inhibitory effects of LEE011 in liposarcoma cells. A, siRNA-mediated knockdown of RB expression at 48 hours. B, LP6 cells were transfected with RB siRNA, control siRNA, or buffer only. LEE011 was added 48 hours after transfection and its effects on cell-cycle distribution were examined by flow-cytometric analysis at 24 hours. Data are representative of three independent experiments.
- Figure 5.
Effects of LEE011 treatment on RB phosphorylation, BrdUrd incorporation, tumor FDG uptake, and tumor growth in vivo. A and B, after three doses of LEE011 (250 mg/kg/d) or vehicle control, tumor samples were analyzed for in vivo BrdUrd incorporation. A, protein expression and phosphorylation in frozen LP6 tumor specimens were evaluated by immunoblot analyses. B, representative examples of IHC staining for BrdUrd and phospho-RB (Ser780) in LP6 tumor xenografts. Original magnification, ×200. C and D, FDG-PET response after three doses of LEE011 (250 mg/kg/d) or vehicle control. C, change in 18F-FDG SUVmax of LP6 tumors. Each bar represents a tumor lesion. D, representative PET imaging of LP6 xenografts. Small arrows, the anatomical location of tumor xenograft. Other areas of 18F-FDG signal represent the brain, heart, and bladder. E, established LP6 tumors were treated with 250 mg/kg LEE011 or with vehicle alone daily for 21 days by oral gavage. Tumor size was measured by caliper every 3 to 6 days. Mice were sacrificed when the tumor diameter reached 2 cm. Values represent mean volume ± SEM (n ≥ 8); *, P < 0.05; **, P < 0.01; ***, P < 0.001; compared with respective control group treated with vehicle. F, primary human liposarcoma xenograft LPS3 was treated with 250 mg/kg LEE011 or with vehicle alone by oral gavage following a 5 days on/2 days off schedule for 3 weeks. Tumor size was measured by caliper every 3 to 4 days. Values represent mean volume ± SEM (n = 6); *, P < 0.05; compared with respective control group treated with vehicle. G, primary human liposarcoma xenograft HSAX2655 was treated with 250 mg/kg LEE011 or with vehicle alone daily by oral gavage beginning 35 days after implantation and continued for 80 days. Tumor size was measured by caliper twice weekly. Values represent mean volume ± SEM (n = 4 in control group, n = 12 in LEE011 group).
- Figure 6.
Continuous exposure to LEE011 leads to enhanced RB phosphorylation and reversible cell-cycle re-entry. A and B, LP6 cells were continuously exposed to LEE011 for the indicated time and concentrations. RB phosphorylation (A, top) and the expression of cyclin D (B) were examined by immunoblot analyses 24 hours after the last dose, and cell-cycle distribution was determined by flow cytometry analysis (A, bottom). C and D, LP6 cells with or without pretreatment were exposed to LEE011 for 24 hours. RB phosphorylation (C, top) and the expression of cyclin D2 and CDK4 (D) were examined by immunoblot analyses. Cell-cycle distribution was determined by flow-cytometric analysis (C, bottom).
Additional Files
Supplementary Figures 1 through 3
Files in this Data Supplement:
- Data Supplement - Structure of NVP-LEE011
- Data Supplement - LEE011 reduced RB phosphorylation in a time-dependent manner.
- Data Supplement - Effects of LEE011 treatment on mice body weight
Volume 13, Issue 9
