p53 wild type human cancer cell lines  (ATCC)

Journal: Oncogene

Article Title: Direct relationship between the level of p53 stabilization induced by rRNA synthesis-inhibiting drugs and the cell ribosome biogenesis rate.

doi: 10.1038/onc.2015.147

Figure Lengend Snippet: Figure 1. The level of p53 stabilization induced by ActD is directly related to the cell rRNA transcription rate. HCT116, HepG2, U2OS, MCF7, LoVo and ZR-75-1 cell lines were used. (a) rRNA synthesis was measured by the evaluation of [3H]-uridine radioactivity incorporated in the total RNA fraction (dpm per μg DNA). (b) Visualization of rRNA synthesis. Cells were labeled with 5-fluorouridine for 20 min and 5-fluorouridine was revealed by specific fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody. DAPI (4,6-diamidino-2-phenylindole) counter staining. Note the higher labeling in HCT116, HepG2 and U2OS cells in comparison with that observed in MCF7, LoVo and ZR-75-1 cells. Scale bar: 10 μm. (c) Real-time RT–PCR analysis of 45S rRNA expression in cells treated with 8 nM ActD for 12 h. The reduction of 45S rRNA after ActD exposure is similar in all cell lines. (d) Representative western blot and densitometric analysis of p53 expression in control and ActD-treated cells. The level of p53 stabilization after ActD treatment is higher in HCT116, HepG2 and U2OS cells than in MCF7, LoVo and ZR-75-1 cells. (e) Real-time RT–PCR analysis of TP53 mRNA expression in the six cell lines. Histograms show the values (mean ± s.d.) of three experiments.

Article Snippet: Cell lines, culture conditions and chemical treatments Six p53 wild-type human cancer cell lines (HCT116, HepG2, U2OS, MCF7, LoVo and ZR-75-1) were obtained from the American Type Culture Collection.

Techniques: Radioactivity, Labeling, Staining, Comparison, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: Oncogene

Article Title: Direct relationship between the level of p53 stabilization induced by rRNA synthesis-inhibiting drugs and the cell ribosome biogenesis rate.

doi: 10.1038/onc.2015.147

Figure Lengend Snippet: Figure 2. Downregulation of rRNA synthesis by serum starvation reduces p53 stabilization after ActD treatment. (a) Real-time RT–PCR analysis of 45S rRNA expression in serum-starved and control HCT116 and HepG2 cells. In order to downregulate rRNA synthesis, the cells were cultured at low serum concentration for 24 h. The cells were exposed to 8 nM ActD for 12 h. The 45S rRNA expression is lower in serum-starved cells than in control cells. ActD exposure causes a comparable reduction of 45S rRNA synthesis in serum-starved and control cells. (b) Representative western blot and densitometric analysis of p53 expression in serum-starved and control HCT116 and HepG2 cells treated with 8 nM ActD for 12 h. The level of p53 stabilization is significantly higher in control than in serum-starved cells. Histograms show the values (mean ± s.d.) of three experiments. *Po0.05; **Po0.01; ***Po0.001.

Article Snippet: Cell lines, culture conditions and chemical treatments Six p53 wild-type human cancer cell lines (HCT116, HepG2, U2OS, MCF7, LoVo and ZR-75-1) were obtained from the American Type Culture Collection.

Techniques: Quantitative RT-PCR, Expressing, Control, Cell Culture, Concentration Assay, Western Blot

Journal: Oncogene

Article Title: Direct relationship between the level of p53 stabilization induced by rRNA synthesis-inhibiting drugs and the cell ribosome biogenesis rate.

doi: 10.1038/onc.2015.147

Figure Lengend Snippet: Figure 3. The different levels of p53 stabilization after inhibition of rRNA transcription are related to different amounts of RPL11 bound to MDM2. (a) Representative western blot evaluation of p53 expression in control and RPL11-silenced HCT116 and LoVo cells. The cells were interfered with scramble or RPL11 small interfering RNA (siRNA) for 48 h and treated with 8 nM ActD for 12 h after the end of the silencing procedure, and the level of nuclear RPL11 analyzed by western blotting. The amount of RPL11 is reduced in both cell lines. No p53 stabilization is observed in RPL11-silenced HCT116 and LoVo cells after treatment with ActD, which in fact exhibit quite similar, low p53 values. (b) Representative western blot of the amount of RPL11 bound to MDM2 in HCT116 and LoVo cells after treatment with 8 nM ActD for 12 h. After immunoprecipitation with anti-MDM2 polyclonal antibody, the amount of RPL11 bound to MDM2 (IP:MDM2) is higher in HCT116 cells than in LoVo cells. The amount of p53 in the NO-IP fraction is higher in HCT116 cells than in LoVo cells. (c) Western blot and densitometric analysis of p53 protein expression in LoVo and HCT116 cells treated with 8 nM ActD for 6 h and exposed to the proteasomal inhibitor MG-132 at the concentration of 10 μM for 2 h. Histograms show the values (mean ± s.d.) of three experiments.

Article Snippet: Cell lines, culture conditions and chemical treatments Six p53 wild-type human cancer cell lines (HCT116, HepG2, U2OS, MCF7, LoVo and ZR-75-1) were obtained from the American Type Culture Collection.

Techniques: Inhibition, Western Blot, Expressing, Control, Small Interfering RNA, Immunoprecipitation, Concentration Assay

Journal: Oncogene

Article Title: Direct relationship between the level of p53 stabilization induced by rRNA synthesis-inhibiting drugs and the cell ribosome biogenesis rate.

doi: 10.1038/onc.2015.147

Figure Lengend Snippet: Figure 4. The level of p53 stabilization induced by doxorubicin, 5-FU and CX-5461 is directly related to the cell rRNA transcription rate. (a) Representative western blot evaluation and densitometric analysis of p53 expression in control and doxorubicin (Doxo)-treated HCT116, HepG2, MCF7 and LoVo cells. Cells were exposed to 10 μM Doxo for 12 h. (b) Representative western blot evaluation and densitormetric analysis of p53 expression in control and 5-FU-treated HCT116, HepG2, MCF7 and LoVo cells. Cells were exposed to 5-FU at the concentration of 20 μg/ml for 12 h. (c) Representative western blot evaluation and densitormetric analysis of p53 expression in control and CX-5461 (CX)- treated HCT116, HepG2, MCF7 and LoVo cells. Cells were exposed to 1 μM CX-5461 for 12 h. Histograms show the values (mean ± s.d.) of three experiments.

Article Snippet: Cell lines, culture conditions and chemical treatments Six p53 wild-type human cancer cell lines (HCT116, HepG2, U2OS, MCF7, LoVo and ZR-75-1) were obtained from the American Type Culture Collection.

Techniques: Western Blot, Expressing, Control, Concentration Assay

Journal: Oncogene

Article Title: Direct relationship between the level of p53 stabilization induced by rRNA synthesis-inhibiting drugs and the cell ribosome biogenesis rate.

doi: 10.1038/onc.2015.147

Figure Lengend Snippet: Figure 6. The level of p53 stabilization induced by drugs that do not hinder ribosome biogenesis is not related to the cell rRNA transcription rate. (a) Representative western blot evaluation and densitometric analysis of p53 expression in HCT116, HepG2, MCF7 and LoVo cells after HU treatment for 12 h at the dose of 3.4 mM. The amount of p53 stabilization after HU exposure is quite similar in all cell lines. (b) Representative western blot and densitometric analysis of p53 expression in serum-starved and control HCT116 and HepG2 cells. For rRNA synthesis downregulation, HCT116 and HepG2 cells were cultured at low serum concentration for 24 h. Cells were treated with 3.4 mM HU for 12 h. The amount of p53 stabilization after HU exposure is similar in serum-starved and control cells. (c) Representative western blot evaluation of p53 protein expression in serum-starved and control HCT116 and HepG2 cells treated with 10 μM Nutlin-3 (Nut) for 12 h. Histograms show the values (mean ± s.d.) of three experiments.

Article Snippet: Cell lines, culture conditions and chemical treatments Six p53 wild-type human cancer cell lines (HCT116, HepG2, U2OS, MCF7, LoVo and ZR-75-1) were obtained from the American Type Culture Collection.

Techniques: Western Blot, Expressing, Control, Cell Culture, Concentration Assay

Journal: Oncogene

Article Title: Direct relationship between the level of p53 stabilization induced by rRNA synthesis-inhibiting drugs and the cell ribosome biogenesis rate.

doi: 10.1038/onc.2015.147

Figure Lengend Snippet: Figure 7. Drugs not acting on ribosome biogenesis may cooperate with inhibitors of rRNA synthesis in stabilizing p53. (a) Representative western blot analysis of p53 expression, cleaved PARP-1 and cleaved caspase-3 in MCF7 and LoVo cells. Cells were treated with either 8 nM ActD or 3.4 mM HU, separately or with the two drugs together for 12 h. The simultaneous treatment with HU and ActD causes a major p53 stabilization compared with single-drug exposure and induces an increased PARP-1 cleavage in both cell lines. A greater amount of caspase-3 is observed in LoVo cells after double treatment with ActD and HU in comparison with cells treated with single drugs. Densitometric analysis of the cleaved portion of PARP-1 and p53 expression is reported. (b) Representative western blot and densitometric analysis of PARP-1 cleavage and p53 expression in HCT116 and HepG2 cells. Cells were treated with either 8 nM ActD or 3.4 mM HU, separately or with the two drugs together for 12 h. The contemporary treatment with HU and ActD does not increase the amount of p53 stabilization and PARP-1 cleavage in both cell lines, as compared with that obtained after the exposure to ActD alone. Histograms show the values (mean ± s.d.) of three experiments.

Article Snippet: Cell lines, culture conditions and chemical treatments Six p53 wild-type human cancer cell lines (HCT116, HepG2, U2OS, MCF7, LoVo and ZR-75-1) were obtained from the American Type Culture Collection.

Techniques: Western Blot, Expressing, Comparison

Journal: Oncogene

Article Title: Direct relationship between the level of p53 stabilization induced by rRNA synthesis-inhibiting drugs and the cell ribosome biogenesis rate.

doi: 10.1038/onc.2015.147

Figure Lengend Snippet: Figure 8. Schematic representation of the proposed relationship between the rate of the cell rRNA synthesis and the level of p53 stabilization induced by inhibitors of ribosome biogenesis, alone or in combination with drugs not acting on ribosome biogenesis. (a) In cells characterized by a high synthesis of rRNA, a high production of ribosomal proteins (RPs) also occurs. (b) After inhibition of ribosome biogenesis, a large amount of RPs, no longer used for ribosome building, binds to a large number of MDM2 molecules and neutralizes their ubiquitin ligase activity toward p53, thus inducing a strong p53 stabilization. This is not the case for cells with low ribosome biogenesis (c and d), in which the production of RPs is low and after the inhibition of rRNA synthesis a low amount of RPs is left free to bind to a limited number of MDM2 molecules, thus only partially inactivating the MDM2-mediated p53 digestion. (e) In cells with low ribosome biogenesis rates, the combination of rRNA synthesis inhibitors, which stabilize p53 through the RP-MDM2 pathway, with drugs that stabilize p53 through p53 phosphorylation (such as, for example, HU) may induce a p53 stabilization greater than that obtained using each type of drug alone.

Article Snippet: Cell lines, culture conditions and chemical treatments Six p53 wild-type human cancer cell lines (HCT116, HepG2, U2OS, MCF7, LoVo and ZR-75-1) were obtained from the American Type Culture Collection.

Techniques: Inhibition, Ubiquitin Proteomics, Activity Assay, Phospho-proteomics

Journal: Cancer Science

Article Title: Hedgehog G li3 activator signal augments tumorigenicity of colorectal cancer via upregulation of adherence‐related genes

doi: 10.1111/cas.12073

Figure Lengend Snippet: Lists of GO and pathway identified by KEGG classification associated with the genes which were upregulated by Gli3‐ FL transfection

Article Snippet: Cell lines Human colorectal cancer cell lines p53‐wild type HCT116 and p53‐mutant HT29, SW480 and DLD‐1 were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 mediated with 10% FBS (Sigma–Aldrich, Hokkaido, Japan) at 37°C in 5% CO 2 humidified incubator.

Techniques: Significance Assay

Journal: Cancer Science

Article Title: Hedgehog G li3 activator signal augments tumorigenicity of colorectal cancer via upregulation of adherence‐related genes

doi: 10.1111/cas.12073

Figure Lengend Snippet: Lists of GO and pathway identified by KEGG classification associated with the genes which were upregulated by Gli3‐ FL transfection

Article Snippet: Human colorectal cancer cell lines p53‐wild type HCT116 and p53‐mutant HT29, SW480 and DLD‐1 were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 mediated with 10% FBS (Sigma–Aldrich, Hokkaido, Japan) at 37°C in 5% CO 2 humidified incubator.

Techniques: Significance Assay