Fig 1.
Establishment and characterization of in vitro SJSA-1 resistant sublines.
A, Protocol to obtain the CMIR resistant cell line by treating the SJSA-1 cells with a constant concentration of SAR405838. B, Protocol to establish the MIR1 and MIR2 cell lines by treating the SJSA-1 cells with stepwise increments of concentration of SAR405838. C, Cell growth inhibition of SAR405838 in SJSA-1 parental, three resistant and two control cell lines. Cell lines were treated with SAR405838 for 4 days and cell viability was determined by a WST assay. D, Induction of apoptosis by SAR405838 in three resistant and one control cell line. Cells were treated with SAR405838 for 48 h and stained with Annexin V/PI for flow cytometry. Data (mean ± SD) are from triplicates, including both early (Annexin V-positive/PI-negative) and late (Annexin V-positive/PI-positive) apoptotic cells. E, Cell cycle effect of SAR405838 in three resistant cell lines, one control cell line and the parental SJSA-1 cell line. Cells were treated with SAR405838 for 24 h for cell cycle analysis by flow cytometry with P.I. staining. F, Antitumor activity of SAR405838 on xenograft tumors established using the MIR2 cell line in mice. SCID mice bearing MIR2 xenograft tumors were treated with vehicle, 100 mg/kg or 200 mg/kg of SAR405838 by oral gavage for 14 days.
Fig 2.
Establishment and characterization of in vivo sublines established from SJSA-1 tumors treated with SAR405838 in mice.
A, SJSA-1 xenograft tumors were treated with vehicle or 100 mg/kg of SAR405838 orally for 14 days. When regressed SJSA-1 tumors treated with SAR405838 regrew, they were harvested and cultured to establish several in vivo sublines. One control subline was established from a tumor treated with vehicle. B, Inhibition of cell growth by SAR405838 in several in vivo SJSA-1 sublines (7.2–7.4, 7.6 and 7.7), one vehicle control subline (VH1) and the parental SJSA-1 cell line. Cells were treated with SAR405838 for 4 days and cell viability was determined by a WST assay. C, Examination of apoptosis induction and cell cycle arrest by SAR405838 in several in vivo SJSA-1 sublines, one vehicle control subline (VH1) and the parental SJSA-1 cell line. Cells were treated with SAR405838 for 48 h for apoptosis analysis by flow cytometry with Annexin V/PI double staining. The test was used to compare in vivo sublines to parental SJSA-1 cells. *, P < 0.05. Cells were treated with SAR405838 for 24 h for cell cycle analysis by flow cytometry with P.I. staining. D, Western blot analysis of p53, MDM2, p21 and GAPDH protein level in SJSA-1 parental, vehicle (VH1) and in vivo sublines (7.2, 7.3 & 7.7). Cells were treated with SAR405838 for 24 h at indicated concentrations.
Fig 3.
Establishment and characterization of sublines from treatment of 7.6 SJSA-1xenograft tumors with SAR405838 in mice.
A, 7.6 SJSA-1 xenograft tumors were treated with vehicle or 100 mg/kg of SAR405838 orally for 14 days, followed by 200 mg/kg SAR405838 for 8 days. When tumors treated with SAR405838 grew to 500 mm3, they were harvested and cultured to establish 7 sublines (G2M1-G2M2, G2M4-G2M8). Two sublines (G1M2 and G1M6) were also established by harvesting and culturing vehicle-treated 7.6 SJSA-1 tumors. B, Cell growth inhibition of SAR405838 in sublines obtained from 7.6 SJSA-1 tumors, the SJSA-1 parental cell line and a subline (VH1) established from a vehicle-treated SJSA-1 tumor from the experiment shown in Fig 2. Cells were treated with SAR405838 for 4 days and cell viability was evaluated by a WST assay. C, Analysis of apoptosis and cell cycle in G2M1-G2M2, G2M4-G2M8 sublines, the SJSA-1 parental cell line and VH1 cell line. Cells were treated with SAR405838 for 48 h for apoptosis analysis by flow cytometry with Annexin V/P.I. double staining. The test was used to compare sublines (G2M1, G2M2, G2M4-G2M8) to 7.6 subline. *, P < 0.05. Cells were treated with SAR405838 for 24 h for cell cycle analysis by flow cytometry with P.I. staining. D, Western blot analysis of p53, MDM2, p21 and GAPDH protein level in G2M1, G2M6, G2M5 and G2M7 sublines, SJSA-1 parental cell line and 7.6 subline. Cells were treated for 24 h with SAR405838 at indicated concentrations of SAR405838 and individual protein was probed with a specific antibody.
Fig 4.
Establishment and characterization of 7.2 sublines from treatment of 7.2 SJSA-1xenograft tumors with SAR405838 in mice.
A, Xenograft tumors established from the 7.2 SJSA-1 subline were treated with vehicle, 100 mg/kg or 200 mg/kg of SAR405838 orally for 14 days. When tumors treated with 200 mg/kg of SAR405838 grew to 200 mm3, they were harvested and cultured to establish several 7.2 sublines (G3M2, G3M3, G3M5-G3M7). One subline (G1M4/7.2) was also established by harvesting and culturing one 7.2 xenograft tumor treated with vehicle control. B, Cell growth inhibition of SAR405838 in G3M2, G3M3, G3M5-G3M7 sublines. VH1 and SJSA-1 parental cell lines were included as control cell lines. Cells were treated with SAR405838 for 4 days and cell viability was evaluated by a WST assay. C, Analysis of apoptosis and cell cycle in G3M2, G3M3, G3M5-G3M7 sublines, 7.2 subline, SJSA-1 parental cell line and VH1 cell line. Cells were treated with SAR405838 for 48 h for apoptosis analysis by flow cytometry with Annexin V/P.I. double staining. The test was used to compare sublines (G2M1, G2M2, G2M4-G2M8) to 7.6 subline. *, P < 0.05. Cells were treated with SAR405838 for 24 h for cell cycle analysis by flow cytometry with P.I. staining. D, Western blot analysis of p53, MDM2, p21 and GAPDH protein level in SJSA-1 parental, 7.2 subline, G1M4/7.2, G3M2, G3M5, G3M6, and G3M7 sublines. Cells were treated for 24 hr with SAR405838 at indicated concentrations.
Fig 5.
Direct comparison of the activity of SAR405838 in SJSA-1 parental and representative sublines obtained from either in vitro or in vivo treatment with SAR405838.
A, Cell growth inhibition of SAR405838 in SJSA-1 parental and representative in vitro and in vivo sublines. Cells were treated with SAR405838 for 4 days and cell viability was evaluated by a WST assay. B, Analysis of apoptosis and cell cycle in SJSA-1 parental and representative in vitro and in vivo sublines. Cells were treated with SAR405838 for 48 h for apoptosis analysis by flow cytometry with Annexin V/P.I. double staining. The test was used to compare sublines (G2M1, G2M2, G2M4-G2M8) to 7.6 subline. *, P < 0.05. Cells were treated with SAR405838 for 24 h for cell cycle analysis by flow cytometry with P.I. staining. C, Western blot analysis of p53, MDM2, p21 and GAPDH protein level in SJSA-1 parental, and representative in vitro and in vivo sublines. Cells were treated for 24 hr with SAR405838 at indicated concentrations.
Fig 6.
Computational modeling of p53 tetrameric structure containing a single heterozygous C176F p53 mutation in complex with DNA.
A, The loop-sheet-helix interaction interface between two p53 molecules revealed by the crystal structure (PDB entry: 4HJE). The backbone of DNA is shown in dark grey, the zinc ion is purple and hydrogen bonds are depicted as red dashed lines. B, The additional interactions gained in p53 dimer contacting a wild-type (colored in pink) and a C176F mutant (colored in blue) p53 at the loop-sheet-helix interaction interface based on the final snapshot of conformation from the 6 ns MD simulation of the FCCF model with DNA (not shown). The interactions include hydrophobic interaction between C176F and two M243 residues and the cation-aromatic interaction between C176F and the zinc ion.