Journal: Oncotarget

Article Title: Osteoactivin (GPNMB) ectodomain protein promotes growth and invasive behavior of human lung cancer cells

doi: 10.18632/oncotarget.7323

Figure Lengend Snippet: ( A ) GPNMB/OA mRNA levels (mean ± SD; n = 4) in lung cancer cell lines as determined by qPCR (*** p < 0.001 for SK-MES-1 and A549 cells versus calu-6). ( B ) The extent of GPNMB/OA ECD protein shedding (24 hr) into the conditioned media by SK-MES-1, A549 and calu-6 cells as determined by ELISA (mean ± SD; n = 6). The extent of GPNMB/OA ECD protein shedding in SK-MES-1 and A549 cells was significantly higher than calu-6 cells (*** p < 0.001). ( C ) Knockdown of GPNMB/OA expression (mean ± SD; n = 4) resulted in a significant reduction (*** p < 0.001) in GPNMB/OA ECD in conditioned media of SK-MES-1. # p > 0.05 when comparing scrambled siRNA-transfected cells versus control cells (−) GPNMB/OA-siRNA.

Article Snippet: Tumors were developed in female athymic nu/nu mice (6–8 weeks old; Charles River Laboratories, Wilmington, MA) by subcutaneous administration of calu-6 cells as we have previously reported [ ].

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Transfection

Journal: Oncotarget

Article Title: Osteoactivin (GPNMB) ectodomain protein promotes growth and invasive behavior of human lung cancer cells

doi: 10.18632/oncotarget.7323

Figure Lengend Snippet: ( A ) The percent healing rates (mean ± SD; n = 5–6) for SK-MES-1 and calu-6 cells at 24 hr after inflicting wound into the cell monolayers (*** p < 0.001 versus calu-6 cells). ( B ) The number of migrated cells (24 hr, mean ± SD; n = 5–6) for SK-MES-1 and calu-6 cell lines using Transwell migration assay. (*** p < 0.001 for SK-MES-1 cells versus calu-6 cells). ( C ) The number of migrated cells (24 hr, mean ± SD; n = 5–6) in transwell insert for calu-6 cells that were seeded with or without rOA (50 ng/mL). Calu-6 cells that were seeded with rOA showed a higher number of migrated cells versus control calu-6 cells (* p < 0.05). ( D ) The number of migrated cells (mean ± SD; n = 3–4) in transwell for SK-MES-1 with siRNA-mediated knockdown of GPNMB/OA was significantly reduced (* p < 0.05) compared to control cells with endogenous GPNMB/OA expression.

Article Snippet: Tumors were developed in female athymic nu/nu mice (6–8 weeks old; Charles River Laboratories, Wilmington, MA) by subcutaneous administration of calu-6 cells as we have previously reported [ ].

Techniques: Transwell Migration Assay, Expressing

Journal: Oncotarget

Article Title: Osteoactivin (GPNMB) ectodomain protein promotes growth and invasive behavior of human lung cancer cells

doi: 10.18632/oncotarget.7323

Figure Lengend Snippet: ( A ) Adhesion of SK-MES-1 and calu-6 cells (mean ± SD; n = 4–5) to fibronectin-coated plates at 37°C (*** p < 0.001 for SK-MES-1 versus calu-6 cells). The extent of cell adhesion expressed as absorbance values was quantified using MTT dye. ( B ) Adhesion (mean ± SD; n = 6) of calu-6 cells to fibronectin-coated plates with or without rOA protein (50 ng/mL) treatment. (*** p < 0.001; for calu-6 cells that were seeded with rOA versus control calu-6 cells. The extent of cell adhesion expressed as absorbance values was quantified using MTT dye. ( C ) Characterization (mean ± SD; n = 6) of calu-6 cell adhesion to culture plates coated with 1% BSA (negative control), fibronectin (8 μg/mL, positive control) and rOA (8 μg/mL, with or without RGD selective peptide). Cell adhesion to BSA-coated plates was significantly less than both fibronectin and rOA-coated plates. Cell treatment with a selective RGD peptide resulted in a significant reduction in cell adhesion to rOA-coated plates (* p < 0.05). The extent of cell adhesion expressed as fluorescence intensity was quantified by CyQuant assay.

Article Snippet: Tumors were developed in female athymic nu/nu mice (6–8 weeks old; Charles River Laboratories, Wilmington, MA) by subcutaneous administration of calu-6 cells as we have previously reported [ ].

Techniques: Negative Control, Positive Control, Fluorescence, CyQUANT Assay

Journal: Oncotarget

Article Title: Osteoactivin (GPNMB) ectodomain protein promotes growth and invasive behavior of human lung cancer cells

doi: 10.18632/oncotarget.7323

Figure Lengend Snippet: ( A ) Calu-6 tumor growth in athymic (nu/nu) mice as expressed by relative tumor volume (mean ± SD; n = 4–5 mice). Relative tumor volumes for calu-6 tumors with rOA were significantly larger than calu-6 cells developed with PBS (calu-6; ** p < 0.01). ( B ) Tumor weights (mean ± SD; n = 4–5 mice) measured on day 34 post-tumor implantation in athymic (nu/nu) mice. Calu-6 tumors developed with rOA supplementation resulted in significantly bigger tumors (* p < 0.05) compared to calu-6 tumors without rOA.

Article Snippet: Tumors were developed in female athymic nu/nu mice (6–8 weeks old; Charles River Laboratories, Wilmington, MA) by subcutaneous administration of calu-6 cells as we have previously reported [ ].

Techniques: Tumor Implantation

Journal: Oncotarget

Article Title: Osteoactivin (GPNMB) ectodomain protein promotes growth and invasive behavior of human lung cancer cells

doi: 10.18632/oncotarget.7323

Figure Lengend Snippet: ( A – B ) Representative tumor sections stained for cell proliferation based on for ki67 expression on day 34 post-tumor implantation in athymic nu/nu mice. The extent of ki67 staining was processed by ImmunoRatio for control calu-6 tumors (without exogenous rOA, with PBS) (A); and calu-6 tumors that received exogenous rOA supplementation (B). Top panel: original image. Bottom panel: original image with segmented staining components for quantification of Ki67 staining. ( C ) Quantification of Ki67 staining of tumor tissues based on percentage of positively stained nuclear area as processed with ImmunoRatio. *( p < 0.05, mean ± SD; n = 8 images) for calu-6 cells that were treated with exogenous rOA versus control calu-6 tumors.

Article Snippet: Tumors were developed in female athymic nu/nu mice (6–8 weeks old; Charles River Laboratories, Wilmington, MA) by subcutaneous administration of calu-6 cells as we have previously reported [ ].

Techniques: Staining, Expressing, Tumor Implantation

Journal: Oncotarget

Article Title: Osteoactivin (GPNMB) ectodomain protein promotes growth and invasive behavior of human lung cancer cells

doi: 10.18632/oncotarget.7323

Figure Lengend Snippet: ( A ) Representative tumor sections stained for apoptosis using TUNEL assay on day 34 post-tumor implantation in athymic nu/nu mice. Arrows indicate TUNEL positive cells for control calu-6 tumors (PBS-treated) (A) and calu-6 tumors that received exogenous rOA supplementation ( B ). ( C ) Quantification of TUNEL staining of tumor tissues excised on day 34 from calu-6 tumor-bearing athymic (nu/nu) mice. Data-points are plotted as the percentage of apoptotic (TUNEL positive) cells (mean ± SD; n = 6 images). *( p < 0.05) for calu-6 cells that were treated with exogenous rOA versus control calu-6 tumors.

Article Snippet: Tumors were developed in female athymic nu/nu mice (6–8 weeks old; Charles River Laboratories, Wilmington, MA) by subcutaneous administration of calu-6 cells as we have previously reported [ ].

Techniques: Staining, TUNEL Assay, Tumor Implantation

Journal: The Journal of Biological Chemistry

Article Title: The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53

doi: 10.1074/jbc.RA117.000950

Figure Lengend Snippet: HO-3867 exhibits differential cytotoxicity to cancer cells with p53MT compared with healthy (noncancerous) cells. a, clinically relevant models were used to analyze the safety of HO-3867 toward normal body cells while simultaneously observing its anticancer efficiency in human cancer-derived cell populations. The graphical representation shows the isolation of heterogeneous cell populations from breast, colon, and liver cancer samples. In addition, noncancerous healthy cells such as fibroblasts from stromal tissue adjacent to breast cancer and radio- and chemosensitive cells from lymphoid and GI tract tissue were used. All of these cells were treated with HO-3867, cisplatin, or vehicle alone. b, a cancer-specific pro-apoptotic effect of HO-3867 was observed in all cells depicted in panel a. Cells were treated with HO-3867 (10 μm), cisplatin (10 μm), or vehicle (DMSO) alone, and apoptosis was measured using annexin V flow cytometry. HO-3867 selectively induced apoptosis in tumor-derived cells and minimal apoptosis in primary culture from normal tissue of different origins as well as tumor-adjacent stroma-derived fibroblasts and radiosensitive lymphoid and GI tract tissue. Cisplatin nonspecifically killed a significantly higher percentage of cells derived from normal tissues (n = 3 for all experiments; p values are as indicated, ANOVA was used for p value calculations, and error bars indicate standard deviation). c, p53 mutational analysis of breast, colon, and liver cancer samples used for the cell cultures shown in b confirms the presence of DNA-binding domain mutations. The exact nucleotide sequence point mutations and resulting amino acid sequence changes are depicted. d, the ability of HO-3867 to induce apoptosis in cancer cells with a p53MT genotype was determined using annexin V flow cytometry. p53MT cells (A431, MDA-MB-468, WRO, and DU-145) and p53−/− cells (MCF-7p53−/− and HCT7p53−/−) were used in the analysis. Cellular apoptosis was not observed in untreated p53MT and p53−/− cells (bars 1–6). shRNA-mediated p53 knockdown and the exogenous addition of p53MT cDNA were used as controls in untreated cells (bars 7–18). In the experimental set, all cell lines were treated with HO-3867, and p53MT cells showed a significant increase in cellular apoptosis (bars 19–22). HO-3867–treated p53null cells did not show a marked increase in apoptosis (bars 23–24). shRNA-mediated p53MT knockdown abolished the HO-3867–induced increase in apoptosis (bars 25–30). The exogenous addition of p53MT cDNA alongside HO-3867 treatment significantly increased apoptosis in both p53MT and p53−/− cells (bars 31–36) (n = 3; mean ± S.D. shown). p values are shown on the figure; standard ANOVA test). Inset, the efficiency of lentiviral particles coding for p53MT cDNA or p53 shRNA was demonstrated using immunoblotting of MCF-7 p53−/− or MCF-7 cells. Untreated MCF-7 p53−/− samples showed no expression of p53 (lane 1). Overexpression of increasing amounts of p53MT cDNA led to increased p53 protein levels. p53 shRNA treatment showed effective knockdown of p53 expression (a representative image from n = 3 replicates is shown).

Article Snippet: p53 MT cells (HEC-1-A, CCRF-CEM, KLE, T47D, SW837, MDA-MB-468, SK-UT-1, SK-LMS-1, SKLU1, Calu-6, SNU-16, DMS-53, SW1271, BT-20, BT-549, MDA-MB-231, BT-474, HOS, DLD-1, MOLT-4, WiDr, PSN-1, MC116, ST486, P3HR-1, NCI-H23, HT-3, NCI-H1882, WRO, HCT-15, A-431, and DU-145), p53 WT (MCF-7 and HCT116), and 293T cells (for lentiviral production) were procured from ATCC (Manassas, VA). p53 −/− cells were derived from p53 WT (MCF-7 and HCT116) as described previously ( 9 ).

Techniques: Derivative Assay, Isolation, Flow Cytometry, Standard Deviation, Binding Assay, Sequencing, shRNA, Knockdown, Western Blot, Expressing, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53

doi: 10.1074/jbc.RA117.000950

Figure Lengend Snippet: HO-3867 covalently binds to p53MT in the DNA-binding domain. a, 1H-15N HSQC NMR spectrum of the p53MT-Y220C core domain (75 μm) after incubation with (green) or without (red) 1000 μm HO-3867 for 70 min. Chemical shift perturbations were observed for residues in proximity to the solvent-exposed cysteine 277. b, an NMR-generated model depicting the putative sites of interaction between p53MT and HO-3867. c, 1H-15N-HSQC NMR spectrum of the p53MT-Y220C core domain (75 μm) after incubating with (green) or without (red) 430 μm HO-3867 for 20 or 150 min. Chemical shift perturbations were time-dependent, suggesting a covalent binding mode. d, ESI (ES+) mass spectra of the p53MT-Y220C core domain (50 μm) after incubation without (left) or with HO-3867 (right) for 4 h at room temperature. HO-3867 treatment led to a mass increase of 713 or 1426 Da, confirming covalent binding to the p53 core domain by HO-3867. e, ESI (ES+) mass spectra of p53 DBD (50 μm) C182S/C277S, C124S/C277S, and C124S/C182S mutants after incubation without (left) or with HO-3867 (right) for 4 h at room temperature. HO-3867 treatment led to a mass increase of 713 Da only for the C124S/C277S and C124S/C182S mutants, confirming selective covalent modification of Cys-277 and Cys-182 by HO-3867.

Article Snippet: p53 MT cells (HEC-1-A, CCRF-CEM, KLE, T47D, SW837, MDA-MB-468, SK-UT-1, SK-LMS-1, SKLU1, Calu-6, SNU-16, DMS-53, SW1271, BT-20, BT-549, MDA-MB-231, BT-474, HOS, DLD-1, MOLT-4, WiDr, PSN-1, MC116, ST486, P3HR-1, NCI-H23, HT-3, NCI-H1882, WRO, HCT-15, A-431, and DU-145), p53 WT (MCF-7 and HCT116), and 293T cells (for lentiviral production) were procured from ATCC (Manassas, VA). p53 −/− cells were derived from p53 WT (MCF-7 and HCT116) as described previously ( 9 ).

Techniques: Binding Assay, Incubation, Solvent, Generated, Modification

Journal: The Journal of Biological Chemistry

Article Title: The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53

doi: 10.1074/jbc.RA117.000950

Figure Lengend Snippet: HO-3867 shows anticancer efficacy in both p53MT and p53WT tumor xenografts by inducing p53MT–RE interaction and induces expression of p53 downstream effectors. a, the anticancer effect of HO-3867 on genetically tractable tumor xenografts of p53WT (MCF-7), p53MT (A-431), and p53−/− (MCF7 p53−/−) cells was observed (n = 3). In row 1, the excised tumors for untreated p53WT, p53MT, and p53−/− xenografts after 4 weeks are shown. In row 2, all of the tumors were treated with HO-3867 along with lentivirus-assisted overexpression of p53WT. A reduction in the tumor volumes of all tumor types was observed in row 2 when compared with the control (row 1). In row 3, tumors were treated with vehicle (DMSO) and lentiviral transfections. The tumor volumes in the vehicle-treated group remained unaltered. In row 4, all tumors were treated with lentivirus coding for p53 shRNA. In row 5, all tumors were treated with HO-3867, and p53WT tumors and p53MT tumors showed a decrease in tumor volume for all biological replicates. Interestingly, in p53 knockdown tumors, HO-3867 did not exhibit very high anticancer efficacy. These data suggest a role for p53 in HO-3867-mediated anticancer activity that appears to be independent of p53 mutational status. In row 6, p53WT and p53MT tumors were treated with HO-3867 along with lentiviral particles coding for p53 shRNA. p53 knockdown in these tumors reversed the anticancer effect of HO-3867, and all biological replicates in both experimental groups showed larger tumor volumes. In rows 6 and 7, p53 null tumor xenografts were treated with HO-3867 and lentiviral particles coding for p53MT cDNA (p53R175H (row 6); p53R273H (row 7)). Interestingly, HO-3867 reduced tumor growth in the presence of p53MT cDNA (compare tumor volumes in row 5 with rows 6 and 7) (n = 3) (HO-3867 treatment started at week 0 in the plot). b, tumor growth curves showing the volume of MCF-7 p53WT, A-431 p53MT, and MCF-7 p53−/− tumors in the eight treatment groups over the course of 4 weeks. In both MCF-7 p53WT and A-431 p53MT tumors, treatment with HO-3867 and HO-3867+ p53WT cDNA led to the greatest reduction in tumor volume. Treatment of MCF-7 p53−/− tumors with HO-3867, HO-3867+p53R175H cDNA, and HO-3867+p53R273H cDNA led to a significant reduction in tumor volume compared with control. In the insets, the efficiency of lentiviral particles coding for p53 shRNA, p53WT cDNA, or p53MT cDNA was demonstrated in MCF-7 p53WT or A-431 p53MT cells using immunoblotting with the indicated antibodies. MCF-7 p53WT and A-431 p53MT samples treated with p53 shRNA showed no expression of p53 (lane 2). p53 shRNA showed effective knockdown of p53 expression. MCF-7 p53−/− cells were treated with p53WT, p53R175H, and p53R273H cDNA and blotted with anti-p53 antibody or anti-GAPDH antibody (loading control). Overexpression of p53WT cDNA or p53MT cDNA led to increased p53 protein expression in MCF-7 p53−/− cells (HO-3867 treatment started at week 0 in the plot. n = 3 for all experiments; p values are labeled on the figure, and two-factor ANOVA with repeated measures was performed for p value calculations).

Article Snippet: p53 MT cells (HEC-1-A, CCRF-CEM, KLE, T47D, SW837, MDA-MB-468, SK-UT-1, SK-LMS-1, SKLU1, Calu-6, SNU-16, DMS-53, SW1271, BT-20, BT-549, MDA-MB-231, BT-474, HOS, DLD-1, MOLT-4, WiDr, PSN-1, MC116, ST486, P3HR-1, NCI-H23, HT-3, NCI-H1882, WRO, HCT-15, A-431, and DU-145), p53 WT (MCF-7 and HCT116), and 293T cells (for lentiviral production) were procured from ATCC (Manassas, VA). p53 −/− cells were derived from p53 WT (MCF-7 and HCT116) as described previously ( 9 ).

Techniques: Expressing, Over Expression, Control, Transfection, shRNA, Knockdown, Activity Assay, Western Blot, Labeling

Journal: The Journal of Biological Chemistry

Article Title: The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53

doi: 10.1074/jbc.RA117.000950

Figure Lengend Snippet: HO-3867 converts mutant p53 conformation to its wildtype form. a, model depicting sites of mutagenesis in the p53 gene in a panel of 29 cell lines. All mutations are present in the p53 DNA-binding domain. b, a Fluidigm digital qPCR-based gene expression analysis of a panel of 14 genes (Fig. S3) was conducted in a panel of 29 control and HO-3867–treated cell lines. Consistent with qChIP analysis, p53-regulated genes were overexpressed in all HO-3867–treated p53MT cell lines; this effect was reversed upon p53 shRNA treatment. Cisplatin (10 μm) was used as a positive control for p53 activation. (n = 5 for all experiments; p values are labeled on the figure, and ANOVA was performed for p value calculations). c, ChIP analysis was conducted in a genetically tractable system of p53MT (A-431) and p53−/− (MCF-7p53−/−) cell lines to measure the binding of p53MT to its REs at the bax (left) and p21 (right) promoters. The results were analyzed using the QIAxcel advanced instrument platform (Qiagen). Input (lane 1), no antibody (lane 2), actin antibody (lane 3), and p53 shRNA (lanes 5 and 11) were used as controls. The data show the presence of p53 on the bax and p21 promoters in HO-3867–treated p53WT and p53MT cell lines but not p53−/− cell lines (lane 8). Exogenous addition of either p53WT (lane 12) or p53MT (lane 13) cDNA resulted in significant binding of p53 at its respective REs in HO-3867–treated p53MT and p53−/− cell lines. d, up-regulation of two important p53 target genes, p21 and Noxa, was confirmed at the protein level by Western blotting. A genetically tractable system of p53WT (MCF-7), p53MT (A-431), and p53−/− (MCF-7 p53−/−) cells was used to study the effect of HO-3867 treatment (10 μm) in p53MT cells (lanes 1–6). Lane 7, both p21 and Noxa Western blotting show less expression in MCF-7 p53−/− cells transfected with p53MT cDNA. However, the same combination in the presence of HO-3867 significantly increases p21 and Noxa expression (lane 8).

Article Snippet: p53 MT cells (HEC-1-A, CCRF-CEM, KLE, T47D, SW837, MDA-MB-468, SK-UT-1, SK-LMS-1, SKLU1, Calu-6, SNU-16, DMS-53, SW1271, BT-20, BT-549, MDA-MB-231, BT-474, HOS, DLD-1, MOLT-4, WiDr, PSN-1, MC116, ST486, P3HR-1, NCI-H23, HT-3, NCI-H1882, WRO, HCT-15, A-431, and DU-145), p53 WT (MCF-7 and HCT116), and 293T cells (for lentiviral production) were procured from ATCC (Manassas, VA). p53 −/− cells were derived from p53 WT (MCF-7 and HCT116) as described previously ( 9 ).

Techniques: Mutagenesis, Binding Assay, Gene Expression, Control, shRNA, Positive Control, Activation Assay, Labeling, Western Blot, Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53

doi: 10.1074/jbc.RA117.000950

Figure Lengend Snippet: HO-3867 converts mutant p53 conformation to its wildtype form. a, the p53MT and p53WT forms were immunoprecipitated using Ab 240 or Ab 1620, respectively, and immunoblotted using a polyclonal anti-p53 antibody (FL393) in p53MT (A-431), p53WT (MCF-7), and MCF-7p53−/− tumors. Input (lane 1), actin antibody (lane 2), and p53 shRNA (lanes 5 and 6) were used as controls for all tumors. In untreated MCF-7 tumors, p53 was recognized by Ab 1620 (lane 3) and to a minor extent by Ab 240 (lane 4). In untreated A-431 tumors, p53 was exclusively recognized by Ab 240 (lane 4). No signal was detected in MCF-7p53−/− tumors (third row). Overexpression of p53WT and p53MT cDNA in all three tumors resulted in a strong signal for Ab 1620 (lane 7) and Ab 240 (lane 10), respectively. HO-3867 treatment in MCF-7 tumors significantly increased detection by Ab 1620 (compare lanes 3 with 11). HO-3867 treatment in A-431 tumors resulted in a change in the p53 conformation from an Ab 1620–recognized form to an Ab 1620–recognized form (compare lanes 4 and 11). HO-3867 had no effect on MCF-7p53−/− tumors. Exogenous addition of p53WT cDNA in HO-3867–treated A-431, MCF-7, and MCF-7p53−/− tumors showed the strong presence of p53 in the Ab 1620–recognized form (lanes 15 and 16). Exogenous addition of p53MT cDNA in HO-3867–treated A-431, MCF-7, and MCF-7p53−/− tumors again showed the strong presence of p53 in the Ab 1620–recognized form (lanes 17 and 18) (n = 3). b, wildtype and mutant forms of p53 were immunoprecipitated using Ab 1620 and Ab 240, respectively, and immunoblotted for p53 protein (FL393) in p53WT (MCF-7 and HCT) or p53MT (A-431, DU-145, and MDA-MB-231) cell lines. Input (lane 1) and actin antibody (lane 2) were used as controls. In untreated p53WT cells, p53 was recognized by Ab 1620 (lane 3, rows 1 and 3). p53−/− (MCF-7p53−/− and HCTp53−/−) cells served as negative controls and showed no p53 signal (rows 2 and 4). In untreated p53MT cells, p53 existed exclusively in an Ab 240–recognized form (lane 4, rows 5–7), which upon HO-3867 treatment converted to an Ab 1620–recognized form (compare conversion from 240 to 1620 form, lanes 4 and 5) (n = 3). c, graphical representation of the experimental design for conducting in vitro transcription assays (top). The synthetic DNA template consisted of a poly(6)-p53 DNA-binding site followed by an adenovirus major late core promoter, a transcription start site, a G-less cassette as the coding region, and a poly(A) tail coding region (for qPCR-based detection) followed by a CCT stop signal. Nuclear extracts from p53null (H1299) cells were the source of the RNA polymerase machinery. Lack of reverse transcriptase to convert synthetic transcripts to a qPCR-detectable form in the reaction mix served as a negative control (No RT, bars 1 and 7). p53 immunoprecipitated from untreated MCF-7 cells in combination with H1299 nuclear extracts showed basal transcript synthesis (second bar). p53 from p53MT cell lines in combination with H1299 nuclear extract resulted in minimal transcript synthesis (bars 3–6). p53 immunoprecipitated from HO-3867–treated p53WT and p53MT cell lines in combination with H1299 nuclear extracts successfully generated RNA transcripts from the synthetic DNA template (blue) (n = 3 for all experiments; p values are labeled on the figure, and ANOVA was performed for p value calculations). d, luciferase-based reporter transcription assay (Cignal) was used to analyze p53-dependent transcription in HO-3867–treated p53MT cell lines in vivo. Empty vector (bars 1 and 7) was used as a negative control. Standard p53-dependent transcription was observed in p53WT MCF-7 cells. Results showed minimal p53-dependent transcription in a variety of p53MT cell lines. The effect of HO-3867 on p53-induced transcription was observed in treated p53WT and p53MT cells (n = 3 for all experiments; p values are labeled on the figure, and ANOVA was performed for p value calculations).

Article Snippet: p53 MT cells (HEC-1-A, CCRF-CEM, KLE, T47D, SW837, MDA-MB-468, SK-UT-1, SK-LMS-1, SKLU1, Calu-6, SNU-16, DMS-53, SW1271, BT-20, BT-549, MDA-MB-231, BT-474, HOS, DLD-1, MOLT-4, WiDr, PSN-1, MC116, ST486, P3HR-1, NCI-H23, HT-3, NCI-H1882, WRO, HCT-15, A-431, and DU-145), p53 WT (MCF-7 and HCT116), and 293T cells (for lentiviral production) were procured from ATCC (Manassas, VA). p53 −/− cells were derived from p53 WT (MCF-7 and HCT116) as described previously ( 9 ).

Techniques: Mutagenesis, Immunoprecipitation, shRNA, Over Expression, In Vitro, Binding Assay, Reverse Transcription, Negative Control, Generated, Labeling, Luciferase, Transcription Assay, In Vivo, Plasmid Preparation

Journal: PLoS Computational Biology

Article Title: Oxygen-Driven Tumour Growth Model: A Pathology-Relevant Mathematical Approach

doi: 10.1371/journal.pcbi.1004550

Figure Lengend Snippet: Panels (A-B) show fits of the model for Calu6 and Colo205. The plots also include simulation of hypoxia and necrosis. (A) is a faster proliferating tumour model (Calu6) and (B) grows slightly slower (Colo205). (C-D) CD31 IHC staining in Calu6 and Colo205 respectively. (E) summary data of CD31 for both tumour models.

Article Snippet: We stained tissue sections of the following tumours: Calu6 (n = 8), Calu3 (n = 7), one squamous cell lung cancer explant (n = 28) and squamous cell lung clinical cancers (n = 12) from AstraZeneca databanks.

Techniques: Immunohistochemistry

Journal: PLoS Computational Biology

Article Title: Oxygen-Driven Tumour Growth Model: A Pathology-Relevant Mathematical Approach

doi: 10.1371/journal.pcbi.1004550

Figure Lengend Snippet: (A) Growth curve fit for 4 explant models, 2 for squamous lung carcinoma and 2 for colorectal carcinoma. The xenografted cell line Calu3 shows very similar behaviour to explant models. (B) Comparison between Calu6 and Calu3 lung cell lines; a squamous lung cancer explant; and clinical tumour material analyses. The bar chart shows the proportions of microvessel density (MVD) in area (quantified from CD31), necrosis (quantified from Hematoxylin & Eosin staining) and stroma (alpha smooth muscle actin (αSMA) positive staining). (C) Images of the different tumour models stained for αSMA and counter-stained with hematoxylin.

Article Snippet: We stained tissue sections of the following tumours: Calu6 (n = 8), Calu3 (n = 7), one squamous cell lung cancer explant (n = 28) and squamous cell lung clinical cancers (n = 12) from AstraZeneca databanks.

Techniques: Comparison, Staining

Journal: PLoS Computational Biology

Article Title: Oxygen-Driven Tumour Growth Model: A Pathology-Relevant Mathematical Approach

doi: 10.1371/journal.pcbi.1004550

Figure Lengend Snippet: Parameter results ( k p and k R ′ ) for the ODM model in xenografts. Confidence intervals for each parameter are specified as well as the rank of the FIM (as a measure of the number of identifiable parameters), the collinearity index (Identifiable if γ <10), the condition number (Indentifiable if κ <1000) and the normalised residual. Cell lines denoted by * are unidentifiable and cell lines denoted by # are arguably identifiable.

Article Snippet: We stained tissue sections of the following tumours: Calu6 (n = 8), Calu3 (n = 7), one squamous cell lung cancer explant (n = 28) and squamous cell lung clinical cancers (n = 12) from AstraZeneca databanks.

Techniques: